Understanding the role of L-type lectin receptor kinases

Understanding the role of L-type lectin receptor

kinases in Phytophthora resistance

Yan Wang

Thesis committee

Promotor

Prof. Dr Francine P.M. Govers

Personal chair at the Laboratory of Phytopathology

Wageningen University

Co-promotors

Prof. Dr Weixing Shan

Professor of Plant Protection

Northwest A&F University, Yangling, Shaanxi, China

Dr Klaas Bouwmeester

Research fellow, Laboratory of Phytopathology

Wageningen University

Other members

Prof. Dr M. Eric Schranz, Wageningen University

Dr A.F.J.M. (Guido) van den Ackerveken, Utrecht University

Dr Karin I. Posthuma, Enza Zaden R&D BV, Enkhuizen

Dr Vivianne G.A.A. Vleeshouwers, Wageningen University

This research was conducted under the auspices of the Graduate School of

Experimental Plant Sciences

Understanding the role of L-type lectin receptor

kinases in Phytophthora resistance

Yan Wang

Thesis

submitted in fulfilment of the requirements for the degree of doctor

at Wageningen University

by the authority of the Rector Magnificus

Prof. Dr M.J. Kropff,

in the presence of the

Thesis Committee appointed by the Academic Board

to be defended in public

on Monday 24 November 2014

at 4 p.m. in the Aula.

Yan Wang

Understanding the role of L-type lectin receptor kinases in Phytophthora resistance,

216 pages.

PhD thesis, Wageningen University, Wageningen, NL (2014)

With references, with summaries in English and Dutch

ISBN: 978-94-6257-132-7

Contents

Chapter I General introduction 7

Chapter II Characterization of the Phytophthora capsici-Arabidopsis pathosystem

IIa A novel Arabidopsis-oomycete pathosystem; differential interactions with Phytophthora capsici reveal a role for camalexin, indole glucosinolates and

salicylic acid in defence 28

IIb Induced expression of defence-related genes in Arabidopsis upon infection

with Phytophthora capsici 56

Chapter III Phenotypic analyses of Arabidopsis T-DNA insertion lines and expression profiling reveal that multiple L-type lectin receptor kinases are involved in plant immunity 63

Chapter IV Phytophthora resistance conferred by Arabidopsis L-type lectin receptor

kinases LecRK-IX.1 and LecRK-IX.2 is not associated with activation of

plant cell death 97

Chapter V Ectopic expression of Arabidopsis L-type lectin receptor kinase genes

LecRK-I.9 and LecRK-IX.1 confers Phytophthora resistance in

Nicotiana benthamiana 139

Chapter VI L-type lectin receptor kinases in Nicotiana benthamiana and tomato

and their role in Phytophthora resistance 155

Chapter VII General discussion 183

Summary 203

Samenvatting 206

Acknowledgements 209

Curriculum vitae 211

Publications 212

Education statement of the Graduate School 213

Chapter I

General introduction

Chapter I

8

INTRODUCTION

Plants are constantly attacked by a wide variety of microbes. Natural selection

shapes plants resistant to the majority of microbes, and thus plant diseases are

usually an exception rather than a rule. Plant diseases are often a major constraint to

crop production and those that destroy important staple crops are of great threat to

food security (Strange and Scott, 2005; Fisher et al., 2012). Several catastrophic

plant diseases have exerted profound socioeconomic impact on human history. For

example, the Great Irish Famine in the 1840s was due to the emergence of potato

late blight, and led to over a million people dying of starvation. The Great Bengal

Famine in 1943 is another disaster which caused starvation of over two million

people because of the failure of rice production after attack by the fungal pathogen

Cochliobolus miyabeanus (Strange and Scott, 2005; Vurro et al., 2010). Integrated

disease management is of vital importance to reduce losses caused by plant

pathogens, but yet a continuous challenge. Improvement of plant resistance by

breeding is hitherto considered as one of the most effective strategies to protect

crops from diseases. A major challenge for breeders is to obtain “durable resistance”,

and therefore a continuous introgression of novel resistance genes in breeding

programs is required. Exploring versatile disease resistance components in plants as

well as integrated deployment of these resistance components could be the key

leading to durable and broad-spectrum plant resistance.

Arms race between plants and pathogens

Disease resistance in plants is mediated by both preformed and induced defence.

Preformed defence, such as those conferred by cell walls and toxic secondary

metabolites, provides physical and chemical barriers to impede microbial penetration

(Huckelhoven, 2007; Underwood, 2012). However, pathogens developed strategies

to overcome these barriers, for example by secreting hydrolytic enzymes to

decompose plant cell walls, thereby facilitating pathogen invasion (Nuhse, 2012).

Once the layer of preformed plant defence has been breached, pathogens are then

confronted with another barrier raised by various inducible plant defence responses

(Jones and Dangl, 2006).

Unlike animals, plants lack adaptive immunity. Nevertheless, plants have

developed a sophisticated innate immune system to defend themselves against

potential pathogens. Recognition of conserved microbial structures known as

microbe-associated molecular patterns (MAMPs) by cell surface receptors

constitutes the first layer of innate immunity (Jones and Dangl, 2006; Zipfel, 2009).

MAMPs are not only present in pathogenic microbes but also in non-pathogenic or

even beneficial microbes (Mackey and McFall, 2006; Boller and Felix, 2009). MAMPs


9

are in general pivotal for the survival of microbes, and therefore they are unlikely to

be modified by microbes to evade plant recognition because of severe fitness

penalties (Naito et al., 2008; Zhang and Zhou, 2010; Thomma et al., 2011). MAMPs

are perceived as “non-self” molecules by plant pattern recognition receptors (PRRs)

(Dubery et al., 2012). Besides, host-derived compounds released during pathogen

infection or wounding, termed damage-associated molecular patterns (DAMPs), can

also be recognized by PRRs, as “modified-self” molecules (Boller and Felix, 2009).

Thus far, most of the identified PRRs are plasma membrane-localized receptor-like

kinases (RLKs) or receptor-like proteins (RLPs) (Schwessinger and Ronald, 2012;

Zipfel, 2014). Recognition of MAMPs/DAMPs leads to activation of PRR-mediated

downstream signal transduction that ultimately results in basal resistance, referred to

as pattern-triggered immunity (PTI). The PTI-associated immune responses include

reactive oxygen species (ROS) bursts, ion fluxes, callose deposition, defence-related

gene expression and secondary metabolite production (Boller and Felix, 2009). This

layer of defence is considered to play a major role in circumventing microbial

invasions in most cases (Boller and Felix, 2009; Dubery et al., 2012).

To achieve successful infection, pathogens acquired the capacity to deliver

effectors into plants to suppress PTI. Effectors interfere with plant defence by

sequestering MAMPs, by blocking or degrading PRRs or by hijacking key

components in PRR-mediated signaling (Boller and He, 2009; de Jonge et al., 2010;

Dou and Zhou, 2012; Giraldo and Valent, 2013; Pel and Pieterse, 2013). In turn,

plants are able to recognize specific pathogen effectors by an additional class of

immune receptors, known as resistance (R) proteins (Jones and Dangl, 2006).

Recognition occurs either directly through the interaction between R proteins and

matching effectors, or indirectly through the perception of modifications in the host

targets of effectors by R proteins (van der Hoorn and Kamoun, 2008; Krasileva et al.,

2010). Defence responses triggered upon recognition of pathogen effectors by R

proteins are similar to those associated with PTI, but differ in timing and magnitude

(Dubery et al., 2012). R protein-mediated recognition triggers stronger host defence

responses, often culminating in a hypersensitive response (HR), a form of

programmed cell death (Chisholm et al., 2006). This type of resistance is termed

effector-triggered immunity (ETI) and plays a dominant role in plant resistance (Jones

and Dangl, 2006). However, ETI only occurs when a matching R/effector pair is

present. Lacking either of these two components will lead to plant susceptibility.

Therefore, ETI is also known as race- or cultivar-specific resistance. Recognition by

R proteins has been a driving force for the evolution of pathogen effectors (Trivedi

and Wang, 2014). To counteract ETI, pathogens developed several strategies, for

example generating effector variants by mutations, silencing effector genes or

secreting additional virulence factors to suppress R-gene function (Chisholm et al.,

Chapter I

10

2006; Jones and Dangl, 2006; van Poppel et al., 2008; Takken and Rep, 2010;

Kasuga and Gijzen, 2013).

Phytophthora species: notorious plant pathogens

The “fungus-like” Phytophthora pathogens belong to the oomycetes, filamentous

organisms which are evolutionally distant from fungi and more closely related to

photosynthetic brown algae and diatoms (Baldauf, 2003). The first Phytophthora

species described in 1876 was Phytophthora infestans. Many other Phytophthora

species have been identified thereafter (Erwin and Ribeiro, 1996). In the last

decades, large scale surveys and advanced strategies for species delimitation have

resulted in a significant increase in the number of described Phytophthora species

(Cooke et al., 2007; Kroon et al., 2012; Martin et al., 2012; Martin et al., 2014). To

date, over 120 species are known which are divided into ten clades based on

combined phylogenetic analysis of mitochondrial and nuclear multilocus traits (Martin

et al., 2014) (Fig. 1).

All identified Phytophthora species are destructive plant pathogens (Kroon et

al., 2012). Most of them are hemibiotrophic; they behave like biotrophic pathogens in

the early stage of infection but act as necrotrophs in a late stage (Bouwmeester et

al., 2009). The most notorious species is P. infestans, the causal agent of potato and

tomato late blight, which causes billions of dollars loss annually through damage in

potato and tomato production (Fry, 2008; Nowicki et al., 2011). Infection on tomato

and potato by P. infestans occurs on leaves, stems and fruits/tubers at any stage of

development, even after harvest (Fig. 2, A and B). Infection causes dark necrotic

lesions that expand quickly and is accompanied by the production of massive

numbers of sporangia that are spread by wind and water (Fry, 2008). Subsequently,

sporangia will germinate directly or indirectly to start new infection cycles (Judelson

and Blanco, 2005). Phytophthora sojae is another economically important species

causing disease mainly on soybean (Tyler, 2007). It causes seed decay, “damping

off” of seedlings and root and stem rot of mature plants (Fig. 2C). While P. infestans

and P. sojae are species with narrow host ranges, others are capable of infecting

many plants across different families. For example, Phytophthora capsici, a highly

dynamic species, causes rot on roots, foliage and fruits of a number of important

vegetables belonging to the Solanaceae and Cucurbitaceae families (Lamour et al.,

2012a; Lamour et al., 2012b) (Fig. 2D). In addition to damaging crops, many

Phytophthora pathogens cause devastating diseases on woody plants (Hansen et al.,

2012). For example, the invasive species, Phytophthora ramorum, is the causal

agent of sudden oak death and ramorum blight on trees and woody ornamentals

(Fig. 2E). P. ramorum is a species with a wide-host-range that can infect over

hundred plant species (Grünwald et al., 2008). The economic impact caused by


11

Figure 1. Phylogeny of Phytophthora species.

The tree on the left is reproduced from Martin et al. (2014). Phytophthora species mentioned in this

chapter are listed on the right. Black arrowheads point to the position in the phylogenetic tree. Species

marked by ♦ are reported capable to infect Arabidopsis.

Chapter I

12

P. ramorum was estimated to be in the tens of millions of dollars in the USA

(Grünwald et al., 2008). Due to the substantial economic and ecological impact,

P. ramorum is often highlighted in the same rank as P. infestans in case of destructive

potential (Hansen et al., 2012).

Figure 2. Disease symptoms caused by different Phytophthora species.

A-B, Late blight symptoms caused by P. infestans on tomato (A) and potato (B). C, Stem and root rot

of soybean caused by P. sojae. D, Disease caused by P. capsici on pepper and tomato. E, Sudden

oak death caused by P. ramorum. Pictures are courtesy of Klaas Bouwmeester and Francine Govers

(panels A, B and E) and Anne E. Dorrance (panel C).

During interaction with plants, Phytophthora pathogens secrete effectors to

modulate plant defence and facilitate infection (Kamoun, 2006). Genome sequence

analysis revealed that Phytophthora genomes encode a large repertoire of effectors

that can be divided into two categories, i.e. apoplastic and intracellular effectors

(Tyler et al., 2006; Jiang et al., 2008; Haas et al., 2009). Apoplastic effectors are

instrumental in protecting pathogens against apoplastic defence and in mediating

invasion (Stassen and Van den Ackerveken, 2011). For example, some apoplastic

effectors inhibit host hydrolytic enzymes while others disrupt plant cell wall integrity to

facilitate plant infection (Tian et al., 2007; Kaschani et al., 2010; Bouwmeester et al.,

2011; Stassen and Van den Ackerveken, 2011). Intracellular effectors are currently

classified into two types, i.e. RXLR-effectors and Crinklers (CRN). RXLR-effectors

are named after a conserved four amino acid (R: Arginine, X: any amino acid, L:


13

Arginine) motif in the N-terminus that is considered to be important for effector

translocation into hosts, while CRN effectors contain yet another conserved motif,

namely LXLFLAK (L: Leucine, X: any amino acid, F: Phe, A: Ala, K: Lys) in the N-

terminus also with a potential translocation function (Dou et al., 2008a; Govers and

Bouwmeester, 2008). The genome of P. infestans contains 563 genes encoding

RXLR effectors and 196 genes encoding CRN effectors (Haas et al., 2009), whereas

the P. capsici genome contains 357 RXLR effector genes and 84 CRN genes

(Lamour et al., 2012b; Stam et al., 2013). P. sojae and P. ramorum have a similar

number of RXLR effector genes, but less CRN genes (Tyler et al., 2006; Jiang et al.,

2008). There is now ample evidence showing that multiple RXLR effectors as well as

several CRN effectors are capable of suppressing host innate immunity (Bos et al.,

2006; Dou et al., 2008b; Liu et al., 2011; Stassen and Van den Ackerveken, 2011;

Qiao et al., 2013; Zheng et al., 2014). In this respect, the large amount of effectors

may account for the rapid adaptation of Phytophthora pathogens.

Plant resistance against Phytophthora pathogens

Through co-evolution with pathogens, plants developed immune receptors that

monitor the presence of pathogens and mount effective innate immune responses.

So far, most of the identified immune receptors against Phytophthora pathogens

belong to the class of intracellular nucleotide-binding leucine-rich repeat (NLR)

proteins, termed R proteins. These proteins are characterized by the fact that they

recognize RXLR effectors in a “gene-for-gene” manner (Tyler, 2007). Therefore, R

proteins mediate resistance specifically to the Phytophthora races that secrete

matching RXLR effectors. Based on this specialized recognition, the implementation

of effectoromics and comparative genomics approaches accelerated R gene cloning

and specificity profiling (Vleeshouwers et al., 2008). Up to now, at least 23 and 17 R

genes have been cloned from Solanum species and soybean, respectively (Tyler,

2002; Li et al., 2011; Vleeshouwers et al., 2011; Lin et al., 2013; Zhang et al., 2013;

Sun et al., 2014). Unfortunately, race-specific resistance conferred by most of the

identified R genes proved to be not durable due to rapid adaptability of Phytophthora

pathogens (Fry, 2008; Vleeshouwers et al., 2011). Nevertheless, there are several

resistance genes, such as Rpi-blb1 isolated from Solanum bulbocastanum that

confer resistance to a broad spectrum of P. infestans isolates (Song et al., 2003; van

der Vossen et al., 2003). This could be due to the fact that the matching effector of

Rpi-blb1, i.e. IPI-O1 or its functional variants is present in all P. infestans isolates

(Champouret et al., 2009). Thus, R genes which recognize highly conserved effectors

are therefore more promising to confer broad-spectrum and durable resistance in

crops. Since the benefit of exploiting single R gene has been limited due to extremely

diversified Phytophthora field populations (Fry, 2008), stacking of multiple R genes is

Chapter I

14

anticipated to confer durable resistance to a broad set of isolates (Zhu et al., 2012).

Apart from recognizing effectors delivered into plant cells, plants are also

capable to recognize apoplastic effectors or Phytophthora cell wall components to

initiate defence responses (Tyler, 2002). For example, perception of INF1, the major

secreted elicitin of P. infestans, by N. benthamiana induces plant cell death and

compromises P. infestans infection (Kamoun et al., 1998). Plasma membrane-

localized receptors are considered as potential candidates to perceive pathogen-

secreted elicitors and activate plant defence (Dubery et al., 2012). Plant genomes

encode a large number of receptors, including RLPs and RLKs. Both RLPs and RLKs

contain diverse extracellular domains which define ligand specificity (Shiu and

Bleecker, 2003). RLKs contain a cytosolic kinase domain that can transduce signals

via phosphorylation of signaling partners. Given the lack of an obvious signaling

domain in the short cytoplasmic tail, RLPs are more likely to recruit (a) co-receptor(s)

for downstream signal transduction, as has been found for tomato protein Cf4, one of

the RLPs that function in resistance against the leaf mold fungus Cladosporium

fulvum (Liebrand et al., 2014). Another RLP from the wild potato species Solanum

microdontum, termed ELR, has recently been found to recognize INF1 and other

Phytophthora elicitins and seems to play a role in resistance against P. infestans (Du,

2014). The importance of RLKs in Phytophthora resistance has also been

demonstrated. For example, BAK1/SERK3, a RLK with leucine-rich repeats (LRR) in

the extracellular domain, acts as a central modulator of plant defence (Chinchilla et

al., 2009; Liebrand et al., 2014). BAK1/SERK3 plays an important role in transducing

signals upon recognition of elicitors secreted by Phytophthora pathogens. For

example, it is required for INF1-induced cell death (Chaparro-Garcia et al., 2011).

N. benthamiana plants silenced for NbSerk3 showed compromised INF1-induced cell

death and increased susceptibility to P. infestans. BAK1 is also implicated in defence

responses elicited by the cellulose binding elicitor lectin (CBEL), a glycoprotein

secreted by Phytophthora parasitica, and plays a role in Arabidopsis resistance to

P. parasitica (Larroque et al., 2013). Another RLK implicated in Phytophthora

resistance is the L-type lectin receptor kinase (LecRK) LecRK-I.9 in Arabidopsis

(Bouwmeester et al., 2011). Since this thesis focuses on LecRKs, LecRK-I.9 will be

described in more detail later.

Efforts have also focused on partial resistance to Phytophthora pathogens.

Several quantitative trait loci (QTL) have been identified conferring partial resistance,

for example in potato and soybean (Odeny et al., 2010; Johnson et al., 2012; Wang

et al., 2012; Mallard et al., 2013; Lee et al., 2014; Naegele et al., 2014). However, the

key genes governing QTL-mediated resistance and the molecular mechanisms

underlying the activation of partial Phytophthora resistance are largely unknown.

Phytophthora infection often triggers a subset of defence responses. ROS


15

production is one of the early defence responses in plants upon pathogen infection

(Boller and Felix, 2009). The role of ROS in plant resistance against Phytophthora

pathogens was supported by the finding that silencing the genes encoding the

plasma membrane-bound NADPH oxidases RbohA and RbohB compromised

P. infestans induced H2O2 accumulation as well as Phytophthora resistance in

N. benthamiana (Yoshioka et al., 2003). Secondary metabolites and related signaling

cascades are also involved in plant resistance against Phytophthora pathogens. For

example, both salicylic acid (SA) and ethylene (ET) pathways are essential for

N. benthamiana resistance to P. infestans (Shibata et al., 2010).

Reducing host susceptibility by mutation of susceptibility genes provides

another avenue to improve plant resistance (Lapin and Van den Ackerveken, 2013).

Screening of Arabidopsis EMS mutants led to the identification of six independent

loci, termed dmr1 to dmr6, that showed increased resistance to the downy mildew

pathogen Hyaloperonospora arabidopsidis, a species that similar to Phytophthora

species belongs to the oomycete (van Damme et al., 2005). Mapped-based cloning

revealed that increased resistance of the dmr1 mutant is determined by mutations in

a single gene that encodes a homoserine kinase which is required for metabolizing

homoserine. Point mutations affected the enzyme activity of DMR1 and led to

increased accumulation of homoserine which is capable to induce Arabidopsis

resistance to H. arabidopsidis (van Damme et al., 2009). Similarly, increased

resistance of dmr6 is determined by mutations in a single gene that encodes 2-

oxoglutarate (2OG)-Fe(II)-dependent oxygenase (van Damme et al., 2008).

Microarray analysis revealed that DMR6 negatively regulates defence-related gene

expression. A role of the substrate of the DMR6 encoded 2OG-Fe(II) oxygenase in

pathogen inhibition was proposed but this needs to be confirmed. A study focused on

plant susceptibility genes to Phytophthora pathogens was also carried out. By mutant

screening, several Arabidopsis mutants have been found that show increased

resistance to Phytophthora parasitica (Shan, W., personal communication). Detailed

functional analysis of the corresponding genes is in progress and will help to uncover

how plants host Phytophthora pathogens.

Arabidopsis, a model to study plant-Phytophthora interaction

Arabidopsis has been used extensively as a model plant to dissect plant-pathogen

interactions. As a favourite model plant, Arabidopsis has many advantages such as

complete genome sequences, feasible molecular modification, large sets of gene

expression data and extensive collections of natural accessions and mutant lines

(Koornneef and Meinke, 2010). Although natural infection of Arabidopsis by

Phytophthora has not been documented, laboratory infection assays have shown that

several Phytophthora species are capable to infect Arabidopsis. In the case of

Chapter I

16

Phytophthora brassicae, Phytophthora cinnamomi and Phytophthora parasitica,

successful infection was observed, as well as interaction specificity among different

Phytophthora isolates and Arabidopsis accessions (Roetschi et al., 2001; Robinson

and Cahill, 2003; Attard et al., 2010; Wang et al., 2011). In contrast, P. infestans and

P. sojae appeared to be non-pathogenic on Arabidopsis (Huitema et al., 2003; Sumit

et al., 2012). Penetration of P. infestans induces non-host defence responses such as

cell death and induced defence-related gene expression (Huitema et al., 2003). To

decipher plant resistance mechanisms against Phytophthora pathogens, Arabidopsis

mutant resources have proven to be instrumental. For example, non-host resistance

to P. infestans was compromised in pen2 (Lipka et al., 2005), a mutant of a

peroxisome-associated myrosinase that hydrolyses the toxic plant secondary

metabolite indole glucosinolate (iGS). In pen2 or other iGS mutants, Arabidopsis

resistance to another Phytophthora species, i.e. P. brassicae, is also slightly

attenuated (Schlaeppi et al., 2010; Schlaeppi and Mauch, 2010). The role of iGS in

Arabidopsis resistance to P. brassicae became evident when introducing a PAD3

mutation into pen2 since the resistance of the pen2pad3 double mutant to

P. brassicae was significantly reduced (Schlaeppi et al., 2010; Schlaeppi and Mauch,

2010). PAD3 encodes a cytochrome P450 monooxygenase that is required for

camalexin biosynthesis (Schuhegger et al., 2006). Interestingly, the pad3 mutant

itself or other mutants defective in camalexin biosynthesis did not show significant

disease susceptibility to P. brassicae (Schlaeppi et al., 2010; Schlaeppi and Mauch,

2010). In this way, the combined effect of camalexin and iGS on Arabidopsis

resistance become evident since blocking biosynthesis of either compound is not

sufficient to render Arabidopsis susceptibility (Schlaeppi and Mauch, 2010). However,

Arabidopsis mutants do not always respond similarly to different Phytophthora

species or even different isolates of a certain species. For example, the ethylene-

insensitive mutant ein2 showed increased susceptibility to P. parasitica but not to

P. brassicae (Roetschi et al., 2001; Attard et al., 2010). This indicates that

Arabidopsis employs sophisticated strategies to orchestrate resistance against

different Phytophthora species. In the Phytophthora genus, the aforementioned

species are scattered over different clades (Fig. 1). Comparative analysis of

interactions of Arabidopsis with these diverse Phytophthora species will likely lead to

new insights into host determinants underpinning interactions with pathogens.

Lectin receptor kinases in plants

Lectin receptor kinases are typical RLKs that are composed of an extracellular lectin

domain, a transmembrane domain and an intracellular kinase domain. Based on the

lectin domain, three types of lectin receptor kinases are found in plants, namely G, C

and L types (Bouwmeester and Govers, 2009). G-type lectin receptor kinases, also


17

known as S-locus RLKs contain a so-called Galanthus nivalis agglutinin (GNA)-

related lectin domain (Shiu and Bleecker, 2001). Members of this family have been

shown to be involved in plant self-recognition and plant defence (Sherman-Broyles et

al., 2007; Boggs et al., 2009; Gilardoni et al., 2011; Cheng et al., 2013). C-type lectin

receptor kinases contain a calcium-dependent lectin domain. They are rare in plants

and only one has been found in the Arabidopsis genome with yet unknown function

(Bouwmeester and Govers, 2009). The third type of lectin receptor kinases, defined

as LecRKs, contain a lectin domain that structurally resembles soluble legume lectins

(Barre et al., 2002). LecRK-I.9, one of the Arabidopsis LecRKs, was identified as a

potential host target of the P. infestans RXLR effector IPI-O and is involved in

mediating plasma membrane-cell wall adhesions (Gouget et al., 2006). Mutating

LecRK-I.9 reduces cell wall-plasma membrane adhesion and renders Arabidopsis

susceptible to non-adapted P. brassicae isolates. LecRK-I.9 is also implicated in

mediating plant responses upon PAMP perception. For example, flg22-triggered

callose deposition is reduced in LecRK-I.9 mutants (Bouwmeester et al., 2011).

LecRK-I.9 is not only functional as a resistance component in Arabidopsis, but also

confers resistance to P. infestans when transferred into potato (Bouwmeester et al.,

2014). Recently, LecRK-I.9 was identified as the first extracellular ATP (eATP)

receptor in plants and was found to be required for eATP-mediated responses,

including Ca2+ fluxes, mitogen-activated protein kinase activation and gene

expression (Choi et al., 2014). However, whether LecRK-I.9-mediated Phytophthora

resistance is dependent on eATP recognition remains elusive. LecRK-I.9 belongs to a

multigene family consisting of 45 members in Arabidopsis (Bouwmeester and

Govers, 2009). Several of these LecRKs are induced upon exposure to pathogens or

pathogen-derived elicitors, indicating involvement of LecRKs in plant defence.

OUTLINE OF THIS THESIS

The research described in this thesis focuses on the role of LecRKs in plant

resistance to Phytophthora pathogens and defence signaling cascades downstream

of LecRKs. The function of LecRKs in different plant species was analysed and the

potential of LecRKs to confer Phytophthora resistance when transferred to other

plant species was also addressed.

In Chapter II, we describe a novel Phytophthora-Arabidopsis pathosystem.

We monitored the ability of Phytophthora capsici to infect Arabidopsis and analysed

the interaction specificity in different P. capsici isolate-Arabidopsis accession

combinations. We assessed the role of secondary metabolite-based defence in

Arabidopsis resistance to P. capsici by performing infection assays on mutants

Chapter I

18

(Chapter IIa). Involvement of secondary metabolites and associated pathways in

Arabidopsis-P. capsici interactions was further studied by monitoring expression

patterns of corresponding marker genes (Chapter IIb).

In Chapter III, we present an inventory of the function of LecRKs in

Arabidopsis. All the publicly available LecRK T-DNA insertion mutants were

assembled and challenged with various pathogens and abiotic stresses. The

obtained phenotypic data were combined with in silico LecRK expression data

derived from public databases to investigate correlations between LecRK expression

and biological function. For one particular LecRK that functions in plant defence, we

also tested overexpression lines to further confirm its function.

Chapter IV focuses on two closely-related Arabidopsis LecRKs, namely

LecRK-IX.1 and LecRK-IX.2. We performed an in-depth functional analysis on their

role in Phytophthora resistance. We monitored basal defence responses in

Arabidopsis LecRK mutants and analysed the contribution of the lectin domain and

kinase activity through domain deletion and point mutation. In addition, we transiently

expressed both LecRKs in the Solanaceous plant Nicotiana benthamiana to study

LecRK-mediated defence responses and downstream signaling components. Finally,

a potential LecRK-interacting protein was identified using in planta co-

immunoprecipitation and mass spectrometry, and its role in Phytophthora resistance

was further characterized.

Chapter V describes the generation of transgenic N. benthamiana plants that

constitutively express Arabidopsis LecRK-I.9 and LecRK-IX.1. Transgenic lines were

subjected to molecular characterization and infection assays with P. capsici and

P. infestans.

Chapter VI focuses on Solanaceous LecRKs. By in silico screening of protein

sequences, genome sequences, ESTs and RNA-seq data, we identified LecRKs in

N. benthamiana and Solanum lycopersicum. These obtained LecRKs were subjected

to phylogenetic analysis together with Arabidopsis LecRKs to explore evolutionary

relationships. The role of Solanaceous LecRKs in Phytophthora resistance was

analysed by TRV-mediated gene silencing and infection assays with Phytophthora

pathogens.

In Chapter VII, the results presented in this thesis are discussed in a broader

perspective to formulate the role of LecRKs in plant immunity. The potentials and

obstacles in exploiting LecRKs for disease resistance in crops are also discussed.


19

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Chapter II

Characterization of the

Phytophthora capsici-Arabidopsis pathosystem

IIa

A novel Arabidopsis-oomycete pathosystem;

differential interactions with Phytophthora capsici

reveal a role for camalexin, indole glucosinolates

and salicylic acid in defence

Yan Wang, Klaas Bouwmeester, Judith E. van de Mortel, Weixing Shan♯, Francine

Govers

This chapter has been published in: Plant, Cell & Environment (2013); 36, 1192-1203.

In collaboration with: ♯ College of Plant Protection, Northwest A&F University, Yangling, Shaanxi, China

Arabidopsis-Phytophthora capsici interaction

29

ABSTRACT

Phytophthora capsici causes devastating diseases on a broad range of plant species.

To better understand the interaction with its host plants, knowledge obtained from a

model pathosystem can be instrumental. Here we describe the interaction between

P. capsici and Arabidopsis and the exploitation of this novel pathosystem to assign

metabolic pathways involved in defence against P. capsici. Inoculation assays on

Arabidopsis accessions with different P. capsici isolates revealed interaction

specificity among accession-isolate combinations. In a compatible interaction,

appressorium-mediated penetration was followed by the formation of invasive

hyphae, haustoria and sporangia in leaves and roots. In contrast, in an incompatible

interaction, P. capsici infection elicited callose deposition, accumulation of active

oxygen species and cell death, resulting in early pathogen encasement in leaves.

Moreover, Arabidopsis mutants with defects in salicylic acid signaling, camalexin or

indole glucosinolates biosynthesis pathways displayed severely compromised

resistance to P. capsici. It is anticipated that this model pathosystem will facilitate the

genetic dissection of complex traits responsible for resistance against P. capsici.

Chapter IIa

30

INTRODUCTION

In nature, plants are being endangered by a broad range of microbial pathogens.

Development of durable strategies to control plant diseases is therefore a major

challenge when striving for successful plant protection. To this end, increased

knowledge on the mechanisms underlying plant-pathogen interactions would be

helpful and this can greatly benefit from information obtained from model

pathosystems. Arabidopsis thaliana has been put forward as a model plant to study

plant-pathogen interactions due to its unique attributes such as the enriched genetic

and genomic resources (Schlaich 2011). New insights gained from Arabidopsis

research can be exploited to generate pathogen resistance in crop plants (Lacombe

et al., 2010; Zhang et al., 2010).

Successful defence in plants against pathogen attack relies on the initiation of

a range of inducible responses, including cell wall reinforcement, accumulation of

reactive oxygen species, programmed cell death, transcriptional activation of

defence-related genes and synthesis of biologically active secondary metabolites.

The plant hormones salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) play

important roles in resistance to various pathogens as induction of defence responses

in many plant species is coordinated by SA-, JA- and ET-dependent signaling

pathways (Robert-Seilaniantz et a., 2011). In the case of Arabidopsis and other

Brassicaceae species, accumulation of indolic secondary metabolites, such as

camalexin and indole glucosinolates (iGS), is also of central importance to limit

pathogen infection (Thomma et al., 1999; Ferrari et al., 2003; Schlaeppi et al., 2010).

Defence strategies, however, vary between host-pathogen interactions, as was

shown in a number of studies on Arabidopsis, in which mutants impaired in defence-

related pathways and collections of accessions were tested for differential responses

to various pathogens (Thomma et al., 2001; Kover and Schaal 2002; Bohman et al.,

2004; Glazebrook 2005; Spoel et al., 2007). For example, disease resistance in

Arabidopsis to the fungal pathogen Alternaria brassicicola requires camalexin and

JA-dependent pathways (Van Wees et al., 2003), while the bacterial pathogen

Pseudomonas syringae and the oomycete pathogen Hyaloperonospora arabidopsidis

can be inhibited by SA-dependent defence responses (Liu et al., 2010). Moreover,

closely related pathogens not necessarily activate the same defence signaling

pathways. This is, for example, illustrated in studies on interactions of Arabidopsis

with Phytophthora. In the three Phytophthora-Arabidopsis pathosytems that have

been described so far, the role of canonical defence signaling pathways has been

investigated and there seems to be no overlap (Roetschi et al., 2001; Rookes et al.,

2008; Attard et al., 2010; Schlaeppi et al., 2010). In this study, we describe a novel


31

Phytophthora-Arabidopsis pathosystem in which yet another distinct defence network

is activated in order to prevent pathogen invasion.

The novel pathosystem presented here is the interaction between Arabidopsis

and Phytophthora capsici, a renowned plant pathogen that causes severe diseases

on a broad range of economically important crops such as tomato, eggplant,

cucurbits and pepper and is responsible for $1 billion loss in worldwide vegetable

production every year (Lamour et al., 2012a). The P. capsici-Arabidopsis system has

attractive features that make it an ideal model system for dissecting broad-host-range

oomycete-plant interactions, as it not only benefits from the genetic and genomic

resources and tools available for the host, but also those available for the pathogen.

Compared to the other Phytophthora species that interact with Arabidopsis,

P. capsici has several advantages that will greatly facilitate the functional analyses of

candidate effectors and pathogenicity factors. It is one of the few (heterothallic)

species for which genetic crosses and backcrosses can be generated fairly easy,

molecular markers (including numerous single nucleotide variations) and high density

genetic linkage maps are available, it is amenable to DNA transformation, and its

genome has been sequenced (Huitema et al., 2011; Lamour et al., 2012a; Lamour et

al., 2012b).

The aims of this study were to analyse whether or not Arabidopsis can

function as a host for P. capsici and to gain more insight into the defence pathways

that are activated in P. capsici-Arabidopsis interactions. First, we inoculated different

Arabidopsis accessions with a set of P. capsici isolates in order to analyse natural

variation in resistance towards P. capsici. Subsequently, we analysed the cellular

defence responses in Arabidopsis by microscopy during compatible and incompatible

interactions. Finally, we tested mutants with specific defects in defence for enhanced

susceptibility to P. capsici and found that in Arabidopsis SA signaling, as well as

camalexin and iGS are required for resistance to P. capsici.

RESULTS AND DISCUSSION

Arabidopsis is a host for Phytophthora capsici

To determine whether P. capsici is capable to infect Arabidopsis, eight P. capsici

isolates (Table 1) were tested using mycelial plugs or zoospores on mature Col-0

leaves. In most cases, infection was observed, but disease development varied from

complete wilting to no visible symptoms three days post inoculation (dpi) (Fig. 1A).

Based on these observations, a disease severity index (DSI) was adopted to

evaluate disease development at 3 dpi, in which the lowest score (‘0’) represents no

infection and the highest score (‘4’) represents collapse of the inoculated leaf (Fig.

Chapter IIa

32

1B). As shown in Fig. 1C, plug inoculation revealed that five of eight isolates (i.e.

LT263, LT3241, LT3112, LT51 and LT3239) were capable to produce rapid

spreading lesions on Col-0, and more than 90% of the inoculated leaves showed a

DSI ≥ 3 at 3 dpi, resulting in a mean DSI ≥ 3.6. Isolate LT3145 was less virulent on

Col-0 as the proportion of infected leaves with a DSI ≥ 3 was approximately 60%,

with a mean DSI of 3.0. In contrast, isolates LT123 and LT62 were not virulent on

Col-0 as they produced no visible disease symptoms (mean DSI = 0.2 and 0,

respectively). Quantification of the P. capsici biomass in Col-0 leaves at 3 dpi by

quantitative(q) PCR revealed similar levels in leaves inoculated with LT263, LT3241,

LT3112, LT51 and LT3239 whereas P. capsici was hardly detectable in leaves

inoculated with isolates LT123 and LT62 (Supplemental Fig. S1). These results show

that disease severity is highly correlated with pathogen development. Upon zoospore

inoculation, disease symptoms on Col-0 developed at a slower rate when compared

with the plug inoculation, but the outcomes were similar. At 7 dpi, the mean DSI of

LT123 and LT3145 infected leaves was 0 and 3, respectively, whereas the DSI of

leaves infected by the remaining isolates, with the exception of LT62, was ≥ 3. LT62

was not included in this assay because this isolate could not produce enough

zoospores for inoculation.

Table 1. Phytophthora capsici isolates used in this study.

Origin

Isolate Year Country/state Collected from MT

LT51 1997 USA, Michigan cucumber A1a

LT62 1998 USA, Michigan squash A2b

LT123 1998 USA, Michigan cucumber A1a

LT263 2004 USA, Tennessee pumpkin A2a

LT3112 2006 USA, Tennessee pumpkin A1 a

LT3145 2006 USA, Tennessee pumpkin A2b

LT3239 2006 USA, Tennessee pumpkin A2b

LT3241 2006 USA, Tennessee pumpkin A2a a As previously reported (Donahoo and Lamour, 2008). b Determined as described by Harutyunyan et al., (2008).

Since P. capsici is known as a soil-borne pathogen, inoculation assays with

various isolates were also performed on Arabidopsis roots by dipping roots of 16-

day-old seedlings into zoospore suspensions. Consistent with the leaf inoculation

assays, disease phenotypes to individual isolates varied from no symptoms to 100%

mortality of the inoculated seedlings. Inoculation of Col-0 roots with the six isolates

that were found to infect Col-0 leaves, resulted in wilting of the lower leaves within 3


33

dpi, and a complete collapse of the seedlings at 4 dpi (Fig. 1D). In contrast, no

apparent disease symptoms were observed on seedlings inoculated with LT123 (Fig.

1D).

Figure 1. P. capsici isolates vary in virulence on Arabidopsis Col-0.

A, Lesions on plug-inoculated Arabidopsis Col-0 leaves three days post inoculation (dpi) with three

isolates of P. capsici. White arrowheads point to the inoculated leaf. B, Disease severity index (DSI)

presented by numbers from ‘0’ to ‘4’ and increasing levels of gray shading. ‘0’ no infection; ‘1’ lesion

diameter less than 0.5 cm; ‘2’ lesion covering less than 50% of the leaf; ‘3’ lesion covering between 50%

and 75% of the leaf; ‘4’ collapse of the inoculated leaf. C, Disease severity on leaves of Arabidopsis

Col-0 inoculated with eight P. capsici isolates at 3 dpi. Each circle represents the DSI (shown from

light to dark gray as in b) caused by the indicated P. capsici isolate on 40 leaves collected in three

independent experiments. D, Disease symptoms on Col-0 seedlings at 4 dpi. Roots of 16-day-old

seedlings were dipped into water (mock) or P. capsici zoospore suspensions (105 zoospores ml-1)

(LT263; LT123).

To know whether the behavior of P. capsici isolates on Arabidopsis is

comparable to that on host plants of P. capsici, all eight isolates were tested for their

ability to infect tomato. Notably, isolates LT123 and LT62 that were not capable of

infecting Arabidopsis Col-0 could infect tomato leaves (Supplemental Fig. S2). Both

Chapter IIa

34

isolates caused lesions that were visible at 3 dpi but, as on Arabidopsis Col-0, the

lesions caused by the other six isolates were much larger.

In summary, pathogenicity tests on Arabidopsis and tomato revealed striking

differences among P. capsici isolates. Since five out of eight isolates (i.e. LT263,

LT3241, LT3112, LT51 and LT3239) behaved similarly on Arabidopsis and tomato,

isolate LT263 was selected for further analysis, together with isolate LT123 which

caused no disease symptoms on Arabidopsis Col-0.

Natural variation of Arabidopsis accessions in response to P. capsici

Different responses of Arabidopsis Col-0 to P. capsici isolates point to interaction

specificity between Arabidopsis and P. capsici. To get a further understanding of the

interaction specificity, another 34 Arabidopsis accessions originating from different

geographic locations were analysed for their response to P. capsici isolates LT263

and LT123 (Fig. 2).

Figure 2. Disease severity on Arabidopsis accessions inoculated with P. capsici.

Disease severity was assessed at 3 dpi according to the DSI shown in Fig.1B. Each circle represents

the DSI (from light to dark gray as shown at the bottom on the right) caused by the indicated P. capsici

isolate on at least 30 leaves collected from three independent experiments. ***: accessions that show

significant susceptibility to isolate LT123 compared with Col-0 based on a t test (p < 0.001). **:

accessions that show significant resistance to isolate LT263 compared with Col-0 (p < 0.05).


35

All accessions could be infected by isolate LT263, but the disease severity on

the accessions varied significantly (Fig. 2). Of the 35 accessions, 29 including Col-0

were classified as susceptible. On these accessions, lesions extended quickly across

the leaves, resulting in a mean DSI ≥ 2.5 at 3 dpi. The remaining six accessions (i.e.

An-1, Can-0, Est-0, Mt-0, Rsch-4 and Ty-0) were moderately tolerant to LT263; there

was restricted lesion development, with around 70% of the inoculated leaves

showing a DSI ≤ 2. Furthermore, the variations in disease severity were consistent

with the level of colonization by P. capsici and the amount of sporangia present on

the lesions (data not shown).

After inoculation with LT123, only two of the tested accessions, Wei-0 and Ler-

0 developed obvious disease symptoms at 3 dpi (Fig. 2). Lesions spreading from the

inoculation spots as well as formation of sporangia confirmed the susceptibility of

Wei-0 and Ler-0. Nevertheless, infection of Wei-0 and Ler-0 leaves by LT123

resulted in lesions with a mean DSI of 2.2 and 2.1 at 3 dpi, respectively. This was not

comparable to the lesions caused by isolate LT263, with a mean DSI of 4.0 and 3.9,

respectively.

Compatible interaction between Arabidopsis and P. capsici

To further confirm the compatible interaction between Arabidopsis and P. capsici, we

analysed the infection process of Arabidopsis Col-0 with isolate LT263 by microscopy.

Col-0 plants were inoculated by applying LT263 zoospores on the upper side of

leaves. Initially, zoospores attached to the leaf surface and readily germinated, and

approximately 96% of cysts developed appressoria at the tips of germ tubes within 3

h (Fig. 3A). Three hours post inoculation (hpi), penetration pegs emerged beneath

the appressoria. Unlike Phytophthora brassicae (Roetschi et al., 2001) and

Phytophthora parasitica (Attard et al., 2010; Wang et al., 2011), P. capsici did not

only enter the leaf tissue at the junction between epidermal cell walls (Fig. 3B), but

was occasionally observed penetrating epidermal cells directly (Fig. 3C), or via

stomatal cavities (Fig. 3D). Upon penetration, haustoria differentiated from the

penetration hyphae adjacent to the epidermal cell. Subsequently, the infection

hyphae progressed to the adjacent epidermal cells or mesophyll cell layers and more

haustoria were observed. From 1 dpi onwards, intercellular hyphae with haustoria

increased markedly and massive ramifying mycelia were evident in the leaf at 2 dpi

(Fig. 3E). After that, hyphae started to emerge from the leaf through stomatal

openings or via the intercellular space between epidermal cells (Fig. 3, F and G) and

eventually developed sporangia on the leaf surface (Fig. 3H).

To evaluate cellular responses of Arabidopsis upon infection, aniline blue

staining was used to detect the callose deposition, while DAB and NBT were used as

marker dyes for H2O2 and O2- production, respectively. The first day after inoculation,

Chapter IIa

36

neither microscopic detectable callose deposition nor NBT-stained cells were

observed. Only some faint brown-colored cells were found at a few infection sites

after DAB staining (data not shown). Furthermore, infected cells did not show

recognizable trypan blue staining within 2 dpi. This indicates that Arabidopsis cells

remain viable after penetration which is likely a prerequisite for the initial biotrophic

relationship between P. capsici and Arabidopsis.

Figure 3. Cytology of a compatible P. capsici–Arabidopsis interaction.

Arabidopsis Col-0 leaves and roots inoculated with P. capsici LT263 zoospores (105 zoospores ml-1)

were harvested and stained with trypan blue. A, Zoospores attached to the leaf surface, germinated

and formed appressoria within 3 hpi. B-D, Appressorium-mediated penetration of the leaf through the

anticlinal cell wall junction (B), directly through the epidermis (► in C) or via stomata (D). E, Massive

invasive hyphae with haustoria in leaf tissue at 2 dpi. F-G, Infection hyphae emerging from leaves via

stomata (F) or the space between epidermal anticlinal cell walls (G). H, Sporangia development on the

leaf surface. I, Invasive hyphae with haustoria in root cells at 1 dpi. J, Sporangia on the root surface at

2 dpi. ap: appressorium; aw: anticlinal cell wall junction; cy: cyst; gt: germ tube; ha: haustorium; sp:

sporangium; st: stomata. Bars represent 20 µm.


37

P. capsici development in Col-0 roots followed a pattern similar to that

observed in leaves. After penetration into the roots, invading hyphae progressed

rapidly along cortex cells with hardly any branches but formed haustoria within 6 hpi.

Hereafter, infection hyphae started to branch, developed abundant haustoria and

perturbed into the vascular system (Fig. 3I). Sporangia were observed at the root

surface within 2 dpi (Fig. 3J). No recognizable host responses were observed during

the course of infection, i.e. no callose deposition or DAB- and NBT-stained cells (data

not shown).

Incompatible interaction between Arabidopsis and P. capsici

For microscopic analysis of an incompatible interaction between Arabidopsis and

P. capsici, Col-0 leaves were drop-inoculated with LT123 zoospore suspensions.

Approximately 90% of the LT123 zoospores germinated and formed appressoria on

the leaf surface within 6 hpi, but were unable to successfully penetrate plant cells. In

most cases, failure of the first penetration led to the formation of secondary germ

tubes and appressoria (Fig. 4A). However, this did not lead to successful infection as

cell death response, visualized by trypan blue-stained zones, was observed at each

penetration site (Fig. 4B). In addition, infection induced the accumulation of H2O2 and

O2-, which was revealed by the DAB- and NBT-stained cells, respectively, at every

attempted infection site (Fig. 4, C and D). Local accumulation of reactive oxygen

species (ROS), i.e. H2O2 and O2-, is often linked to cell wall-based defence responses,

such as the production of callose, a phenomenon considered to be an active

resistance response to invading pathogens (Hückelhoven, 2007). Consistent with this,

localized callose deposition as shown in Fig. 4E, was detected at nearly every

attempted penetration site during the early phase of infection. In a few instances,

invasive hyphae were detected in the mesophyll cell layer at the inoculation sites in

samples collected after 1 dpi; however, infection hyphae were accompanied by

extensive cell death which was observed not only in mesophyll cells in direct contact

with the infection hyphae but also in the neighboring mesophyll cells (Fig. 4G). No

haustoria or sporangia were detected in inoculated Col-0 leaves. Apparently, during

an incompatible interaction, ROS burst, callose deposition and cell death,

phenomena not commonly observed in the compatible interaction, contributed to

restricting the infection.

In order to examine whether the resistance phenotype observed on Arabidopsis

Col-0 leaves upon dip-inoculation of roots with LT123 zoospores (Fig. 1D, right panel)

is due to failed infection of root tissue, we cytologically examined these inoculated

roots. LT123 zoospores encysted and germinated, and infection of the root tissue

resulted in proliferation of infection hyphae and sporulation (Fig. 4, I and J). This

disease development in Col-0 roots was similar to that observed in roots in the

Chapter IIa

38

Figure 4. Cytology of an incompatible P. capsici–Arabidopsis interaction.

Arabidopsis Col-0 leaves and roots were inoculated with P. capsici LT123 zoospores. A, Abortive

penetration by LT123 led to the formation of secondary germ tubes and appressoria on the leaf

surface as was observed at 6 hpi. B, Cell death of epidermal cells indicated by the dark blue stained

Arabidopsis cells. C-D, DAB-reacting H2O2 accumulation (C) and NBT staining of O2- (D) in

Arabidopsis epidermal cells at 12 hpi. E, Cell wall deposition at the penetration site along the junction

between epidermal cell walls, revealed by aniline blue staining under UV light; F, Bright field image of

(E) showing the germinated cyst at the penetration site; G, Invasive hyphae surrounded by extensive

trypan blue-stained plant cells observed at 1 dpi. H-J, Arabidopsis roots were dipped in water (H) or in

a zoospore suspension of LT123 (I-J) and stained with trypan blue. Invasive hyphae of LT123

colonizing Col-0 roots (I) and sporulation on the root surface (J). cy: cyst; hy: invasive hypha; sa:

secondary appressorium; sg: secondary germ tube; sp: sporangium. Bars represent 20 µm.

compatible interaction between Col-0 and LT263. However, root colonization by

LT123 did not lead to systemic invasion as no classical disease symptoms were

observed on the aerial parts of the seedlings (Fig. 1D). This tolerance response was

also observed on Arabidopsis Col-0 when challenged with Phytophthora cinnamomi

(Rookes et al., 2008) and on cucurbits inoculated with a pathogenicity mutant of the

anthracnose fungus Colletotrichum magna (Freeman and Rodriguez, 1993).

Apparently, in these pathosystems, the ability to establish infection and the ability to


39

further colonize host tissues are separate events.

Phenotypic analysis of JA and ET signaling mutants

To further investigate the relevance of various defence signaling pathways and

secondary metabolites in resistance of Arabidopsis to P. capsici, a set of Col-0

mutants defective in SA, JA, and ET signaling pathways, or in camalexin and iGS

biosynthesis was selected (Fig. 5; Supplemental Table S2) and screened for loss of

resistance to P. capsici LT123, one of the isolates that is unable to infect Col-0 (Fig.

1). Disease severity on the selected mutants was determined according to the

disease severity index (DSI) as described above and is shown by circles in Fig. 5.

Quantitative values are included in Supplemental Table S2.

The analysis included seven mutants that are positioned in JA/ET pathways.

Of these seven, four retained Col-0-like resistance towards LT123, i.e., jin1 and jar1

in which JA signaling is blocked (Berger et al.,1996; Devoto and Turner, 2003), and

ein2 and ein3 which are mutated in ET signaling (Chao et al., 1997; Alonso et al.,

1999). In contrast, resistance was attenuated in the JA-insensitive mutants mpk4 and

eds8-1 (Petersen et al., 2000; Brodersen et al., 2006) and in etr1-1 that has a

mutation in the ET receptor gene ETR1 (Bleecker et al., 1988). These three mutants

all exhibited severe disease symptoms at 3 dpi with 90% of the inoculated leaves

having a DSI ≥ 2. The JA pathway mutants jin1 and jar1 are known to be relatively

weak alleles (Lorenzo et al., 2004) which may explain the difference in phenotype

with mpk4 and eds8-1. Because of the discrepancy in the four JA signaling mutants a

role for JA in P. capsici resistance cannot be ruled out. In the case of ET signaling

mutants differences in disease susceptibility have been reported before, for example

by Geraats et al. (2002), who tested susceptibility of these mutants to various

Pythium species, and by Johansson et al. (2006) and Pantelides et al. (2010), who

analysed the response to Verticillium dahliae. EIN2 and EIN3 act downstream of

ETR1 and the ein2-1 mutant has a stronger ethylene-insensitive phenotype than

etr1-1 (Geraats et al., 2002). Nevertheless, upon P. capsici inoculation etr1-1 is much

more susceptible than ein2-1 and hence there is no positive correlation between the

P. capsici susceptibility phenotype and the ethylene insensitive phenotype. Since

ein3-1 shows a slight gain of susceptibility compared to ein2-1 and Col-0, we cannot

rule out that ET has a role in resistance to P. capsici. However, since etr1-1 is not

only affected in the sensitivity to ET but also in hydrogen peroxide signaling and C6-

aldehyde-induced defence responses (Desikan et al., 2005; Kishimoto et al., 2006),

we speculate that ETR1 is involved in an ethylene independent pathway that affects

disease resistance to P. capsici.

Chapter IIa

40

Figure 5. Disease severity on Arabidopsis mutants impaired in defence related pathways after

inoculation with P. capsici.

A schematic model illustrating the network of defence related signaling pathways and secondary

metabolic biosynthesis routes, and the genes mutated in the selected Arabidopsis mutants

(Supplemental Table S2). Note that NahG is not a mutant but a transgenic line producing an enzyme

that breaks down SA. In the network the undotted arrows represent established relationships between

substrates, the enzymes encoded by the genes and the products; whereas dotted arrows represent

relationships that are deduced from genetic studies and mutant analysis. The disease severity on the

inoculated mutants at 3 dpi with P. capsici isolate LT123 is shown by circles. Each circle represents

the DSI (from light to dark gray as shown on the right) on 30 to 40 leaves collected in three

independent experiments. Mutants that, according to a t test, show significant gain of susceptibility

when compared with Col-0 are marked by *** (P < 0.001) or * (P < 0.05). Quantitative values are

included in Supplemental Table S2. SA: salicylic acid; JA: jasmonic acid; ET: ethylene; iGS: indole

glucosinolates; aGS: aliphatic glucosinolates; Trp: tryptophan; IAOx: indole-3-acetaldoxime; IAN:

indole-3-acetonitrile; DHCA: dihydrocamalexic acid; I3M: indole-3-yl-methyl glucosinolates; 4HO-I3M:

4-hydroxy-indole-3-yl-methyl glucosinolate.


41

SA, camalexin or iGS deficiency compromises resistance of Arabidopsis to

P. capsici

In the SA signaling pathway seven mutants were tested as well as the SA-deficient

line NahG (Delaney et al., 1994). NahG, pad4, eds1-2 and ndr1 (Century et al., 1997;

Zhou et al., 1998; Falk et al., 1999) displayed enhanced susceptibility towards

P. capsici LT123 with fast spreading lesions accompanied by sporulation at 3 dpi,

and resulting in mean DSIs significantly higher than 3. In contrast, inoculation of sid2-

2, npr1-1, npr1-3 and eds5 (Cao et al., 1994; Cao et al., 1997; Glazebrook et al.,

1996; Nawrath and Métraux, 1999; Ton et al., 2002) resulted in visible lesions with

mean DSIs smaller than 2. The difference in susceptibility of, on the one hand the

NahG, eds1-2, pad4 and ndr1 and on the other hand sid2-2, npr1-1, npr1-3 and eds5,

could be due to the functional redundancy of genes involved in the SA pathway; it is

known that SA synthesis is not solely dependent on isochorismate synthase (ICS/SID)

activity, but also on phenylalanine ammonium lyase (PAL) (Wildermuth et al., 2001;

Ferrari et al., 2003). Alternatively, the reduced resistance anticipated in the SA

signaling mutants sid2-2 and eds5 might be recovered by the enhanced

accumulation of camalexin observed in these mutants (Nawrath and Métraux, 1999;

Nawrath et al., 2002). Last but not least, mutations in EDS1, PAD4, and NDR1 not

only suppress SA-mediated defence responses; they can also suppress resistance

controlled by a subset of resistance (R) genes (Coppinger et al., 2004; Zhu et al.,

2011). Thus the enhanced susceptibility of these mutants could also point to a

potential role of R genes in the interaction between Col-0 and LT123. Because of the

pleiotropic effects encountered in the SA signaling mutants, the results on the role of

SA were not conclusive. To address the question whether loss of resistance to P.

capsici in the NahG plants is due to deficiency of SA, we tested the effect of

exogenous application of SA on lesion development. On NahG plants sprayed with

2.5 mM SA one day prior to inoculation with P. capsici LT123, lesions were smaller

than on NahG plants sprayed with water (Supplemental Fig. S3), confirming that SA

plays a role in defence against P. capsici.

In the pathways leading to the production of indolic secondary metabolites,

nine mutants were considered including pad4 mentioned above. The phytoalexin-

deficient mutants pad3 (cyp71b15) and pad4 (Glazebrook and Ausubel, 1994;

Glazebrook et al., 1997) displayed a high level of susceptibility to P. capsici LT123

(Fig. 5). Lesions became clearly visible as early as 2 dpi, and these fast advancing

lesions led to a mean DSI of 3.3 at 3 dpi. CYP71B15 (PAD3) catalyzes the last step

of camalexin biosynthesis (Schuhegger et al., 2006); PAD4 is a lipase-like protein

with a role in camalexin biosynthesis as well as SA/R gene-mediated defence

(Glazebrook et al., 1997; Zhou et al., 1998; Zhu et al., 2011). Unlike pad4, the mutant

pad3 has no other deficiencies apart from camalexin synthesis, and therefore the

Chapter IIa

42

observed susceptibility of pad3 plants to LT123 revealed an important role for

camalexin in disease resistance against P. capsici. Compared to pad3, the cyp71a13

mutant showed much smaller lesion sizes. This difference could be due to different

levels of camalexin. In pad3, camalexin production is completely abolished; even

upon pathogen attack or during abiotic stress there is no camalexin accumulation

(Glazebrook and Ausubel, 1994; Schuhegger et al., 2006). In contrast, in cyp71a13,

which has a mutation in the pathway upstream of CYP71B15, camalexin is still

produced albeit at a reduced level (Nafisi et al., 2007). Moreover, the cytochrome

P450 enzyme CYP71A12, which shows 89% identity with CYP71A13 at the amino

acid level, was reported to function in the camalexin biosynthesis pathway (Nafisi et

al., 2007; Millet et al., 2010). Hence, in the cyp71a13 mutant the absence of

CYP71A13 might be compensated by the activity of CYP71A12. Different

phenotypes for cyp71a13 and pad3 were also reported by Van de Mortel et al. (2012)

who tested the response to non-pathogenic, root-colonizing Pseudomonas

fluorescens bacteria.

PAD2 is a γ-glutamylcysteine synthetase that is required for synthesis of

glutathione (Parisy et al., 2007). The pad2 mutant, which is deficient in both iGS and

camalexin showed increased susceptibility to insect herbivory and P. brassicae

(Schlaeppi et al., 2008; Schlaeppi et al., 2010). Consistently, in our study, pad2 was

found to be very susceptible to P. capsici LT123 with a mean DSI of 3.9 at 3 dpi, thus

indicating that disease resistance to P. capsici is also largely dependent on

camalexin and iGS. Furthermore, the double mutant cyp79b2cyp79b3, which fails to

convert tryptophan into indole-3-acetaldoxime (IAOx), the precursor of camalexin and

iGS (Zhao et al., 2002), showed a similar high DSI (i.e. 4) as pad2. Schlaeppi et al.

(2010) reported that the mutation in CYP79B2 and CYP79B3 does not affect the

level of other defence-associated responses, and hence the susceptibility of the

double mutant cyp79b2cyp79b3 to P. capsici could be attributed to the deficiency of

iGS and camalexin.

To further confirm the potential function of iGS in disease resistance, the iGS-

related mutants pen2, myb51 and cyp81f2 were included in the screening. In the iGS

metabolic pathway, MYB51 is a positive regulator of indole-3-yl-methyl glucosinolate

(I3M) biosynthesis (Gigolashvili et al., 2007). I3M can be converted to 4-hydroxy-

indole-3-yl-methyl glucosinolate (4HO-I3M) by the cytochrome P450 monooxygenase

CYP81F2 (Pfalz et al., 2009). Subsequently, PEN2 myrosinase catalyzes the

hydrolysis of both I3M and 4HO-I3M (Bednarek et al., 2009). Consequently, the

compromised resistance in myb51, cyp81f2 and pen2 indicates that iGS, as well as

iGS hydrolysis products, are important for P. capsici resistance in Arabidopsis.

Arabidopsis produces besides iGS, also aliphatic glucosinolates (aGS), which have

been shown to function in defence towards pest insects (Beekweelder et al., 2008).


43

Biosynthesis of aGS is dependent on the transcription factors MYB28 and MYB29,

and the synthesis of aGS is completely abolished in the double mutant myb28myb29

(Beekweelder et al., 2008). Inoculation of myb28myb29 with P. capsici did not result

in any changes in disease susceptibility when compared with Col-0, indicating that

blocking aGS synthesis by mutation in MYB28 and MYB29 is not sufficient to confer

Arabidopsis susceptibility to P. capsici.

Arabidopsis exploits a different basal defence network for each Phytophthora

species

Collectively, the results described here indicate that basal resistance to P. capsici in

Arabidopsis involves the activation of SA signaling and the biosynthesis of camalexin

and iGS. Blocking the accumulation of SA, camalexin or iGS results in susceptibility

to P. capsici. In comparison, resistance to P. parasitica depends on JA/ET signaling

in addition to SA signaling, as the mutants etr1-3, ein2-1 and jar1-1 were found to be

more susceptible to this pathogen (Attard et al., 2010). In contrast, Arabidopsis

resistance to P. brassicae was shown to be independent of SA and JA/ET signaling

pathways; NahG plants, nor etr1, ein2 and jar1 mutants showed a change in

phenotype demonstrating that blocking SA, or JA/ET signaling had no significant

effect on resistance (Roetschi et al., 2001). Moreover, mutants with defects in the

accumulation of either camalexin or iGS, like pad3 or myb51, showed no significant

change in resistance to P. brassicae (Schlaeppi et al., 2010). However, the combined

deficiency of iGS and camalexin as manifested in either pad2 or cyp79b2cyp79b3,

resulted in susceptibility, demonstrating that the resistance to P. brassicae requires

the sequential action of iGS and camalexin (Schlaeppi et al., 2010). In the case of

P. cinnamomi, resistance in Arabidopsis seems to be independent of any of above

mentioned pathways. All mutants including pad2, pad3, pad4, ein2 and jar1 as well

as NahG plants tested by Rookes et al. (2008) retained wild-type resistance. Taken

together, it is evident that every attacking pathogen elicits the activation of specific

signaling networks and that Phytophthora species vary in their ability to tolerate

different defence components.

CONCLUSIONS

In this study, we demonstrated that P. capsici is capable to infect Arabidopsis and

that there is natural variation among Arabidopsis accessions in response to different

P. capsici isolates. Cell death, callose deposition and ROS accumulation were

induced in Arabidopsis during the early infection stages in an incompatible interaction.

Blocking SA signaling, camalexin or iGS biosynthesis pathways in Arabidopsis,

Chapter IIa

44

conferred susceptibility to P. capsici demonstrating that SA signaling and

biosynthesis of camalexin and iGS are essential for basal resistance to this

Phytophthora species. This newly established pathosystem will facilitate the

identification of novel traits responsible for resistance to the broad-host-range

oomycete pathogen P. capsici.

MATERIALS AND METHODS

P. capsici culture conditions and inoculum preparation

P. capsici isolates (Table 1) were routinely cultured in the dark at 25°C on 20% (v/v)

V8 juice agar plates (Erwin and Ribeiro, 1996). Zoospores for plant infection assays

were obtained by incubating mycelial plugs (Ø 1.0 cm) from the margin of a four-day

old growing colony in 10% (v/v) cleared V8 broth in the dark at 25°C for two days.

Hereafter, the V8 medium was replaced with sterilized mineral solution (Wang et al.,

2011) and refreshed once every hour up to four times. After two days of incubation

under continuous light, numerous sporangia developed. Zoospore release was

induced with a cold shock by flooding the plates with cold water (4°C) for 30 min.

Zoospores were filtered through one layer of Miracloth (Merck) and adjusted to the

desired concentrations.

Plant material and growth conditions

Arabidopsis seeds were sown on soil, or in vitro on 0.5 MS medium (Murashige and

Skoog, Duchefa) supplemented with 1% (w/v) sucrose and 1.2% (w/v) plant agar

(Duchefa), and were stratified by placing them in the dark at 4°C for three days.

Subsequently, Arabidopsis was grown in a conditioned growth chamber at 19-21°C,

with a 16 h photoperiod and a relative humidity (RH) of 75-80%. Tomato plants of

cultivar Moneymaker were grown under standard greenhouse conditions.

Plant inoculation procedures and SA treatment

Four-week-old Arabidopsis or tomato leaves were sprayed with water, and

subsequently inoculated on the abaxial leaf side with fresh mycelial plugs (Ø 0.5 cm).

Mock-inoculation was conducted with blank V8 plugs. Mycelial plugs were removed

from Arabidopsis leaves two days post inoculation (dpi) and from tomato leaves 1 dpi.

Zoospore inoculation of Arabidopsis leaves was performed by inoculating 10 μl

droplets containing 105 zoospores ml-1 on each leaf. For root inoculation, roots of 16-

day-old Arabidopsis seedlings were dipped in water or a zoospore suspension (105

zoospores ml-1) for 5 seconds, and then transplanted to soil. During the first 24 h

after inoculation, plants were kept in the dark at 22°C in trays covered with lids to


45

maintain a high humidity. Subsequently, plants were incubated at a 75-80% RH and

a 10 h photoperiod.

For SA treatment, plants were exposed to SA (Merck Schuchardt) 1 d prior to

inoculation with P. capsici by spraying the leaves with a solution containing 2.5 mM

SA, 0.1 % ethanol (v/v) and 0.02% Silwet L-77 (v/v). Control plants were sprayed

with water supplemented with 0.1 % ethanol (v/v) and 0.02% Silwet L-77 (v/v).

Disease severity index

Disease development on Arabidopsis leaves was evaluated using a disease severity

index (DSI) on a scale of 0 to 4. A score of ‘0’ indicates no visible disease symptoms

or small necrotic flecks; ‘1’, a lesion diameter less than 0.5 cm; ‘2’, a lesion covering

less than 50% of the leaf surface; ‘3’, a lesion covering 50-75% of the leaf surface,

and a score of ‘4’ refers to 75-100% collapse of the leaf. The mean DSI was

calculated according to the equation:

MeanDSI∑ . .

Tomato disease severity was monitored by measuring lesion sizes at 3 dpi. All

experiments were repeated at least three times.

Quantification of P. capsici biomass in infected Arabidopsis leaves

Six leaves inoculated with P. capsici collected at 3 dpi were used for genomic DNA

isolation with a DNeasy Plant Mini Kit (Qiagen). qPCR was performed for quantifying

the P. capsici biomass using an ABI 7300 PCR machine (Applied Biosystems). The

Arabidopsis Rubisco primer pair AtRub-F4/AtRub-R4 was used as endogenous

control, while the primer pair CAP-Fw/CAP-Rv1 was used to target P. capsici internal

transcribed spacer regions (Silvar et al., 2005; Yadeta et al., 2011; Supplemental

Table S1). The ratio of P. capsici genomic DNA to Arabidopsis DNA was calculated

using a 2−∆∆CT method. Two biological replicates were analysed, each with a

technical triplicate.

Histological staining and microscopy

Staining with trypan blue (Sigma) visualizing infection hyphae and plant cell death,

and aniline blue (Acros Organics) visualizing callose deposition, was carried out as

described by Bouwmeester et al. (2011). Detection of H2O2 and O2- was performed

as described by Thordal-Christensen et al. (1997) and Dunand et al. (2007).

Microscopy was performed with a Nikon 90i microscope (Nikon, Amstelveen, The

Netherlands) using differential interference contrast and epifluorescence settings (i.e.

DAPI filter, EX 340-380, DM 400, BA 435-4850).

Chapter IIa

46

ACKNOWLEDGEMENTS

We thank Kurt Lamour for providing Phytophthora capsici isolates, and Bart P.H.J.

Thomma and Joost Keurentjes for Arabidopsis accessions and mutant lines. This

project was financially supported by a Wageningen University sandwich PhD

fellowship (YW) and a Huygens scholarship (YW), by the Centre for BioSystems

Genomics (CBSG) (KB, FG) and by EcoLinc (JEvdM). CBSG and EcoLinc are

centres of the Netherlands Genomics Initiative that is affiliated with the Netherlands

Organisation for Scientific Research.

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o

r m

etab

olit

esb

Pat

ho

gen

s R

efer

ence

s

L

T12

3 (↓

: d

ecre

ased

; ↑:

incr

ease

d)

eds1

-2

3.4

± 0.

3***

S

A↓;

R

-gen

e-m

edia

ted

resi

stan

ce↓

Pse

udom

onas

syr

inga

e

Gla

zebr

ook

et a

l., 1

996;

Fal

k et

al.,

199

9;

F

eys

et a

l., 2

001;

Ven

ugop

al e

t al.,

200

9

eds5

1.

4 ±

0.4*

S

A↓;

cam

alex

in↑

P

seud

omon

as s

yrin

gae

G

laze

broo

k et

al.,

199

6; N

awra

th e

t al.,

199

9;

N

awra

th e

t al.,

200

2; T

on e

t al.,

200

2;

Hec

k et

al.,

200

3

ndr1

3.

4 ±

0.2*

**

SA↓;

R

-gen

e-m

edia

ted

resi

stan

ce↓

Pse

udom

onas

syr

inga

e

Cen

tury

et a

l., 1

997;

Sha

piro

and

Zha

ng 2

001

Nah

G

3.8

± 0.

3***

S

A↓;

cam

alex

in↓

Pse

udom

onas

syr

inga

e

Naw

rath

and

Mét

raux

199

9; H

eck

et a

l., 2

003

sid2

-2

1.6

± 0.

4*

SA↓;

cam

alex

in↑

Pse

udom

onas

syr

inga

e

Naw

rath

and

Mét

raux

199

9; H

eck

et a

l., 2

003

npr1

-3

1.5

± 0.

2*

SA↓

Pse

udom

onas

syr

inga

e

Cao

et a

l., 1

997;

Don

g, 2

004

npr1

-1

1.8

± 0.

3*

SA↓;

JA↓;

ET↓

Pse

udom

onas

syr

inga

e

A

ltern

aria

bra

ssic

icol

a C

ao e

t al.,

199

4; P

iete

rse

et a

l., 1

998;

Leon

-Rey

es e

t al.,

200

9

jar1

0.

6 ±

0.3

Inse

nsiti

ve to

JA

Dev

oto

and

Tur

ner,

200

3

jin1

0.7

± 0.

4 S

A↑;

inse

nsiti

ve to

JA

P

seud

omon

as s

yrin

gae

B

erge

r et

al.,

199

6; N

icks

tadt

et a

l., 2

004

mpk

4 2.

1 ±

0.2*

**

SA↑;

JA↓;

ET↓

Alte

rnar

ia b

rass

icic

ola

Pet

erse

n et

al.,

200

0; B

rode

rsen

et a

l., 2

006

eds8

-1

3.2

± 0.

4***

In

sens

itive

to J

A

Pse

udom

onas

syr

inga

e

Ton

et a

l., 2

002

Chapter IIa

52

Ara

bido

psis

-Phy

toph

thor

a ca

psic

i int

erac

tion

53

ein2

-1

0.1

± 0.

1 JA↑;

inse

nsiti

ve to

ET

A

ltern

aria

bra

ssic

icol

a

B

otry

tis c

iner

ea

Alo

nso

et a

l., 1

999;

Tho

mm

a et

al.,

199

9;

P

enni

nckx

et a

l., 1

998

ein3

-1

0.9

± 0.

4 In

sens

itive

to E

T

C

hao

et a

l., 1

997

etr1

-1

2.5

± 0.

2***

In

sens

itive

to E

T; H

2O2↓

; C

6-al

dehy

de-in

duce

d re

sist

anc

e↓

B

leec

ker

et a

l., 1

988;

Kis

him

oto

et a

l., 2

006

pad2

3.

9 ±

0.2*

**

SA↓;

cam

alex

in↓;

iGS↓

P

hyto

phth

ora

bras

sica

e

Gla

zebr

ook

and

Aus

ubel

199

4; R

oets

chi e

t al.,

20

01; S

chla

eppi

et a

l., 2

010

pad3

(cy

p71b

15)

3.3

± 0.

4***

C

amal

exin↓

Pse

udom

onas

syr

inga

e P

hyto

phth

ora

bras

sica

e

Gla

zebr

ook

and

Aus

ubel

199

4;

S

chuh

egge

r et

al.,

200

6

pad4

3.

3 ±

0.3*

**

SA↓;

cam

alex

in↓;

R

-gen

e-m

edia

ted

resi

stan

ce↓

H

yalo

pero

nosp

ora

para

sitic

a

Pse

udom

onas

syr

inga

e

Zho

u et

al.,

1998

; Fey

s et

al.,

200

1;

M

ert-

Tur

k et

al.,

200

3; B

arts

ch e

t al.,

200

6;

Ven

ugop

al e

t al.,

200

9

cyp7

1a13

1.

4 ±

0.2*

C

amal

exin↓

Alte

rnar

ia b

rass

icic

ola

Pse

udom

onas

syr

inga

e N

afis

i et a

l., 2

007

cyp7

9b2c

yp79

b3

4.0

± 0.

1***

C

amal

exin↓;

iGS↓

Alte

rnar

ia b

rass

icic

ola

P

seud

omon

as s

yrin

gae

B

lum

eria

gra

min

is

Naf

isi e

t al.,

200

7; B

edna

rek

et a

l., 2

009

cyp8

1f2-

2 2.

5 ±

0.2*

**

iGS↓

Blu

mer

ia g

ram

inis

P

falz

et a

l., 2

009;

Bed

nare

k et

al.,

200

9

myb

51

2.6

± 0.

4***

iG

S↓

G

igol

ashv

ili e

t al.,

200

7

pen2

3.

6 ±

0.3*

**

iGS↓

Blu

mer

ia g

ram

inis

B

edna

rek

et a

l., 2

009

myb

28m

yb29

0.

1 ±

0.1

aGS↓

Mam

estr

a br

assi

cae

Bee

kwee

lder

et a

l., 2

008

a Val

ues

repr

esen

t m

ean

DS

I (±

SD

) of

at

leas

t th

ree

inde

pend

ent

expe

rimen

ts e

ach

with

mor

e th

an 1

0 in

ocul

ated

leav

es.

Val

ues

that

ref

lect

a s

igni

fican

t

gain

of

susc

eptib

ility

com

pare

d w

ith C

ol-0

acc

ordi

ng t

o a

t te

st a

re m

arke

d w

ith *

(P

<0.

05)

or ***

(P

<0.

001)

. b ↓

: de

crea

sed;

↑:

incr

ease

d; S

A:

salic

ylic

acid

; JA

: jas

mon

ic a

cid;

ET

: eth

ylen

e; iG

S: i

ndol

e gl

ucos

inol

ates

; aG

S: a

lipha

tic g

luco

sino

late

s.

53


Chapter IIa Arabidopsis-Phytophthora capsici interaction

54

Table S1. Primers used in this study.

Primersa 5'-3'

AtRub-F4 GCAAGTGTTGGGTTCAAAGCTGGTG

AtRub-R4 CCAGGTTGAGGAGTTACTCGGAATGCTG

CAP-Fw TTTAGTTGGGGGTCTTGTACC

CAP-Rv1 CCTCCACAACCAGCAACA a Previously reported (Silvar et al., 2005; Yadeta et al., 2011).

Figure S1. Relative quantification (RQ) of P. capsici biomass in Arabidopsis Col-0 leaves at 3

dpi by qPCR. Bars represent mean values (± SD) relative to that of LT123-inoculated leaves.

Experiments were repeated twice with similar results.

0

40

80

120

RQ


55

Figure S2. Disease severity on tomato leaves inoculated with different P. capsici isolates.

Four-week-old tomato leaves were plug-inoculated with P. capsici. Lesion diameters were measured

at 3 dpi (two per lesion perpendicular to each other, assigned as diameter a and b) and lesion sizes

were calculated with the formula: lesionsize . Values represent mean lesion size (± SD) from

three independent experiments each with 16 inoculated leaves.

Figure S3. SA treatment of Arabidopsis NahG plants decreases susceptibility to P. capsici

isolate LT123.

NahG plants were sprayed with (A) water supplemented with 0.1% ethanol and 0.02% Silwet L-77 or

(B) a solution containing 2.5mM SA, 0.1 % ethanol and 0.02% Silwet L-77 one day before inoculation.

Photographs were taken at 3 dpi. Disease severity index of leaves treated with water and SA was 3.4

and 2.1, respectively and was determined based on 20 leaves collected in two independent

experiments.

0

2

4

6

8

10

Lesi

on s

ize

(cm

2)

IIb

Induced expression of defence-related genes in

Arabidopsis upon infection with

Phytophthora capsici

Yan Wang, Klaas Bouwmeester, Judith E. van de Mortel, Weixing Shan♯, Francine

Govers

This chapter has been published in: Plant Signaling & Behavior (2013); 8, e24618.


Defence-related gene expression in Arabidopsis upon P. capsici infection

57

ABSTRACT

Recognition of pathogens by plants initiates defence responses including activation

of defence-related genes and production of antimicrobial compounds. Recently, we

reported that Phytophthora capsici can successfully infect Arabidopsis and revealed

interaction specificity among various accession-isolate combinations. We used this

novel pathosystem to demonstrate that camalexin, indole glucosinolates (iGS) and

salicylic acid (SA) have a role in defence against P. capsici. To further investigate the

role of camalexin-, iGS- and SA-related pathways in the differential interaction

between Arabidopsis and P. capsici, we monitored expression of marker genes over

time during infection. In both compatible and incompatible interactions, induction of

expression was detected, but in compatible interactions transcript levels of camalexin

and iGS marker genes were higher.

Chapter IIb

58

INTRODUCTION Phytophthora capsici is a pathogen with a broad host range and is particularly

devastating in vegetable crops worldwide. Recently, we showed that P. capsici can

also infect Arabidopsis and we anticipate that such a model pathosystem will

facilitate the genetic dissection of complex traits responsible for P. capsici resistance

in crops (Wang et al., 2013). Microscopic investigation of the infection process

revealed that inoculation of Arabidopsis Col-0 with zoospores of the highly virulent P.

capsici isolate LT263 resulted in colonization of leaf and root tissue and formation of

haustoria followed by sporulation. In contrast, inoculation of Col-0 with zoospores of

the intermediate virulent isolate LT123 resulted in an incompatible interaction

characterized by callose deposition, accumulation of active oxygen species and cell

death (Wang et al., 2013). We also observed natural variation among Arabidopsis

accessions in response to the two isolates; LT263 caused severe disease symptoms

on 29 out of 35 Arabidopsis accessions tested, while LT123 could infect only 2 out of

the 35, i.e. Ler-0 and Wei-0. Moreover, Arabidopsis mutants deficient in SA signaling

and the biosynthesis of camalexin and iGS showed compromised resistance against

P. capsici, indicating a role for these components in defence. The aim of this study

was to find additional support for the involvement of these secondary metabolites in

defence against P. capsici, and therefore we monitored the expression of several

defence-related genes in Arabidopsis upon inoculation with P. capsici over time using

real-time PCR. For inoculation we used mycelial plugs instead of zoospores; the

outcome is similar but disease development is faster thus shortening the time course.

Based on previous work, we selected two P. capsici isolates and two Arabidopsis

accessions. This allowed us to compare an incompatible and compatible interaction

on either the same accession (Col-0) inoculated with different isolates (LT123 and

LT263, respectively), or different accessions (Col-0 and Ler-0, respectively)

inoculated with the same isolate (LT123). In these interactions the disease severity

index (DSI) as defined previously, ranged from 0.2 with hardly any visible symptoms,

to 3.9 showing complete collapse of the inoculated leaves (Fig. 1).

RESULTS AND DISCUSSION The cytochrome P450 monooxygenase (CYP) encoding genes CYP71B15 (PAD3)

and CYP71A13 were selected as marker genes for the biosynthesis of camalexin

(Nafisi et al., 2007; Schuhegger et al., 2006). Another CYP gene, CYP81F2, and the

gene encoding the transcription factor MYB51 were used as markers for the iGS

biosynthesis pathway (Gigolashvili et al., 2007; Pfalz et al., 2009). In Col-0,


59

Figure 1. Symptoms on Arabidopsis accessions Col-0 and Ler-0 three days after plug-

inoculation with P. capsici isolate LT263 or LT123.

The disease severity index (DSI) determined according to Wang et al. (2013) is indicated. White

arrowheads point to inoculated leaves.

inoculation with either P. capsici LT123 or LT263 leads to an increase in transcript

levels of these four genes over time with a peak at 36 hours post inoculation (hpi),

regardless of the compatible and incompatible nature of the interaction. In particular

for CYP71B15 and CYP71A13, drastic increases in transcript levels were observed

(Fig. 2) whereas this increase did not occur in mock-inoculated leaves. As shown

previously by Wang et al. (2013), loss-of-function mutants of these four genes

showed gain of susceptibility upon infection with P. capsici LT123, suggesting a role

in resistance and thus in incompatible interactions. Remarkably, at all time points the

transcript levels were significantly higher in the compatible interaction than in the

incompatible interaction (Fig. 2). This might be due to the fact that in LT263-infected

Col-0 leaves, many more cells are colonized thus resulting in a stronger response to

infection, especially for the camalexin-related marker genes. In several pathosystems,

camalexin accumulation as well as transcripts of relevant biosynthesis genes were

found to be strictly limited to the infection sites and correlated with the spatial

expansion of the pathogen (Glawischnig et al., 2004; Schuhegger et al., 2007). To

investigate if this holds for the P. capsici-Arabidopis pathosystem, we checked

expression of these marker genes during infection of Arabidopsis Ler-0. This

accession is susceptible to LT123 (DSI 2.1), but even more susceptible to LT263

(DSI 3.9). In these compatible interactions transcript levels of CYP71A13 were higher

than in the incompatible interaction of Col-0 with LT123 (DSI 0.2) but overall lower

than in the compatible interaction of Col-0 with LT263 (DSI 3.7). Remarkably, in Ler-0

basal expression of CYP71B15 is extremely low when compared to Col-0.

Nevertheless, also in infected Ler-0 leaves the CYP71B15 transcript levels increased

dramatically (more than 100-fold after 24 hpi), as shown in the inset in Fig.2. During

time, the peak in expression in LT263-infected Ler-0 leaves (with a higher DSI) was

at 24 hpi, so slightly earlier than in LT123-infected Ler-0 leaves (with a lower DSI)

Chapter IIb

60

and in Col-0, but overall the transcript changes followed a similar pattern in all

interactions. Taken together we found no evidence for a strict correlation between

transcript levels and DSI. We do however, observe a consistent pattern for the two

camalexin marker genes, namely induction of expression upon inoculation and

overall higher transcript levels in compatible compared to incompatible interactions. A

similar strong increase in the compatible interaction was found for one of the iGS

biosynthesis marker genes, i.e. CYP81F2. For the other one, MYB51, the differences

between transcript levels in the Col-0 compatible and incompatible interaction were

less pronounced and in Ler-0, MYB51 transcript levels in leaves with the lower DSI

(2.1) were overall higher than in those with a higher DSI (3.9).

Figure 2. Transcript levels of CYP71B15, CYP71A13, CYP82F2, MYB51, PR1 and PDF1.2 genes

during compatible and incompatible Arabidopsis-P. capsici interactions.

Four to five week-old Arabidopsis leaves were plug-inoculated with P. capsici LT123 (gray bars) and

LT263 (black bars). Six circular leaf discs (Ø 1 cm), with the inoculation spot in the center, were

collected from three plants at the time points indicated on the X-axes (hours post inoculation).

Subsequently, total RNA was isolated and used as template for cDNA synthesis. Transcript levels

were quantified by real-time PCR using gene-specific primers of which the sequences are shown in

the figure and were normalized with Arabidopsis Actin2 (F: 5’- TAACTCTCCCGCTATGTATGTC GC-

3’; R: 5’- GAGAGAAACCCTCGTAGATTGGC -3’). Values on the Y-axes are expressed as mean fold

changes (± SD) relative to the transcript level in mock-inoculated Col-0 leaves (white bars) at time

point 0 which was arbitrarily set as 1. Experiments were repeated twice with similar results.


61

PR1, the gene that encodes a pathogenesis-related protein, was selected as

marker gene for the SA pathway (Leon-Reyes et al., 2010). In Col-0, PR1 expression

was found to be induced during the incompatible interaction with P. capsici isolate

LT123 with a maximum at 36 hpi followed by a rapid decline to the basal level. In the

compatible interaction with isolate LT263, PR1 transcript accumulation was triggered

faster, but to a lesser extent. Also in the compatible interactions of Ler-0 with LT123

and LT263, PR1 expression was triggered rather fast and the fold increase

approached the levels observed in the incompatible interaction in Col-0. As a marker

gene for the JA/ET-pathway we used PDF1.2 (Leon-Reyes et al., 2010). In Col-0, the

PDF1.2 transcript levels started to increase from 12 hpi, reaching a peak at 24 hpi

and then decreasing steadily in both the compatible and incompatible interaction.

Transcript levels in the compatible interaction, however, are significantly higher than

in the incompatible interaction. Also in Ler-0 PDF1.2 transcript levels increase upon

inoculation but to a much lesser extent than in Col-0.

CONCLUSIONS

In summary and in line with our recent report in which we screened the response of

Arabidopsis mutants to P. capsici and revealed requirement of SA, camalexin and

iGS for resistance to this pathogen, we found induced expression of relevant marker

genes in Arabidopsis challenged with P. capsici. Variations in expression levels were

detected in different accession-isolate combinations, and for nearly all marker genes

expression was higher in compatible interactions as compared to incompatible

interactions. The expression profiles, however, do not reveal any obvious correlation

between expression levels and disease phenotype. These results raise the question

why in the absence of proteins encoded by the camalexin and iGS marker genes, so

in the mutants, the plants are susceptible and can no longer stop the pathogen while

in wild-type plants these genes are highly induced upon successful infection. When

assuming that expression indeed leads to protein production, one would expect that

in the compatible interaction camalexin and iGS are produced but apparently not in

time or at sufficient levels to block disease development.

ACKNOWLEDGEMENTS

This project was financially supported by a Wageningen University sandwich PhD

fellowship (YW) and a Huygens scholarship (YW) and by the Centre for BioSystems

Chapter IIb

62

Genomics (CBSG) (KB, FG), which is part of the Netherlands Genomics Initiative/

Netherlands Organisation for Scientific Research.

REFERENCES

Gigolashvili T., Berger B., Mock H.P., Müller C., Weisshaar B. and Flügge U.I. (2007). The

transcription factor HIG1/MYB51 regulates indolic glucosinolate biosynthesis in Arabidopsis

thaliana. The Plant Journal, 50, 886-901.

Glawischnig E, Hansen B.G., Olsen C.E. and Halkier B.A. (2004). Camalexin is synthesized from

indole-3-acetaidoxime, a key branching point between primary and secondary metabolism in

Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America,

101, 8245-8250.

Leon-Reyes, A., Van der Does, D., De Lange, E.S., Delker, C., Wasternack, C.,Van Wees, S.C.M.,

Ritsema, T. and Pieterse, C.M.J. (2010). Salicylate-mediated suppression of jasmonate-

responsive gene expression in Arabidopsis is targeted downstream of the jasmonate biosynthesis

pathway. Planta, 232, 1423-1432.

Nafisi M., Goregaoker S., Botanga C.J., Glawischnig E., Olsen C.E., Halkier B.A. and Glazebrook J.

(2007). Arabidopsis cytochrome P450 monooxygenase 71A13 catalyzes the conversion of indole-

3-acetaldoxime in camalexin synthesis. Plant Cell, 19, 2039-2052.

Pfalz M., Vogel H. and Kroymann J. (2009). The gene controlling the Indole Glucosinolate Modifier1

quantitative trait locus alters indole glucosinolate structures and aphid resistance in Arabidopsis.

Plant Cell, 21, 985-999.

Schuhegger R., Nafisi M., Mansourova M., Petersen B.L., Olsen C.E., Svatoš A., Halkier B.A. and

Glawischnig E. (2006). CYP71B15 (PAD3) catalyzes the final step in camalexin biosynthesis.


Schuhegger R., Rauhut T. and Glawischnig E. (2007). Regulatory variability of camalexin biosynthesis.


Wang Y., Bouwmeester K., van de Mortel J.E., Shan W. and Govers F. (2013). A novel Arabidopsis-

oomycete pathosystem; differential interactions with Phytophthora capsici reveal a role for

camalexin, indole glucosinolates and salicylic acid in defense. Plant, Cell & Environment, 36,

1192-1203.

Chapter III

Phenotypic analyses of Arabidopsis T-DNA insertion

lines and expression profiling reveal that multiple

L-type lectin receptor kinases are involved in

plant immunity

Yan Wang, Klaas Bouwmeester, Patrick Beseh, Weixing Shan♯, Francine Govers

This chapter has been published in: Molecular Plant-Microbe Interactions (2014); 27,

1390-1402.


Chapter III

64

ABSTRACT

L-type lectin receptor kinases (LecRKs) are membrane-spanning receptor-like

kinases with putative roles in biotic and abiotic stress responses and in plant

development. In Arabidopsis, 45 LecRKs were identified but their functions are

largely unknown. Here, a systematic functional analysis was carried out by evaluating

phenotypic changes of Arabidopsis LecRK T-DNA insertion lines in plant

development and upon exposure to various external stimuli. None of the LecRK T-

DNA insertion lines showed clear developmental changes, neither under normal

conditions nor upon abiotic stress treatment. However, many of the T-DNA insertion

lines showed altered resistance to Phytophthora brassicae, Phytophthora capsici,

Pseudomonas syringae or Alternaria brassicicola. One mutant defective in LecRK-

V.5 expression was compromised in resistance to two Phytophthora spp. but showed

enhanced resistance to P. syringae. LecRK-V.5 overexpression confirmed its dual

role in resistance and susceptibility depending on the pathogen. Combined analysis

of these phenotypic data and LecRK expression profiles retrieved from public

datasets revealed that LecRKs which are hardly induced upon infection or even

suppressed are also involved in pathogen resistance. Computed co-expression

analysis revealed that LecRKs with similar function displayed diverse expression

patterns. Since LecRKs are widespread in plants, the results presented here provide

invaluable information for exploring the potential of LecRKs as novel sources of

resistance in crops.

Function of Arabidopsis LecRKs in resistance

65

INTRODUCTION

Plasma membrane-localized receptor-like kinases (RLKs) are critical for plants to

perceive and process environmental stimuli. In Arabidopsis, there are over 400

plasma membrane-localized RLKs with a conserved cytosolic kinase domain and

divergent extracellular domains (Shiu and Bleecker, 2003). Several of these RLKs

have been analysed in detail and demonstrated to play essential roles in plant

development and/or plant immunity. For example, the RLK CLAVATA1 controls

meristem cell proliferation and differentiation (Dievart et al., 2003), and RLKs such as

FLS2, EFR and CERK1 recognize microbe-associated molecular patterns (MAMPs)

to initiate MAMP-triggered immunity (Boller and Felix, 2009). Other RLKs, such as

BAK1/SERK3 and SOBIR1, function as central components in regulation of various

receptor-mediated signaling processes (Chinchilla et al., 2009; Liebrand et al., 2013).

More insight into the function of a wider and more diverse range of RLKs would help

to better understand how plants interact with their environment.

Based on the divergent extracellular domains, RLKs can be divided into

different classes. One of the largest RLK classes in plants comprises the L-type lectin

receptor kinases, abbreviated as LecRKs. LecRKs are characterized by an

extracellular lectin domain structurally resembling soluble lectins of leguminous

plants (Hervé et al., 1996). In Arabidopsis, 45 LecRKs have been identified that were

divided into nine clades (i.e. Clade I to IX) and seven singletons (Bouwmeester and

Govers, 2009). Given this large number, LecRKs are expected to play diverse roles

in plant development and defence. Thus far, functional characterization has been

limited to a few Arabidopsis LecRKs. For example, LecRK-IV.2 (alias SGC lectin RLK)

was suggested to be essential for proper pollen development since a T-DNA

insertion mutant showed deformed and collapsed pollen grains (Wan et al., 2008).

LecRK-V.2 (alias LecRK-b2) and three LecRKs from clade VI (previously named

LecRK4.1, LecRK4.2 and LecRK4.3) were shown to be involved in regulating

abscisic acid (ABA) response during seed germination (Deng et al., 2009; Xin et al.,

2009). Later studies also demonstrated a role for LecRKs in plant immunity. LecRK-

I.9, a mediator of plant cell wall-plasma membrane integrity (Gouget et al., 2006),

was shown to be crucial for Phytophthora resistance in Arabidopsis (Bouwmeester et

al., 2011). More recently the same LecRK was identified as the first ATP receptor in

plants and shown to be required for ATP-mediated responses (Choi et al., 2014). As

yet it is not clear if the function of LecRK-I.9 as Phytophthora resistance component

is linked to its function as ATP receptor. Two other LecRKs, LecRK-V.5 and LecRK-

VI.2, were shown to be involved in bacterial resistance by regulating stomatal

immunity (Desclos-Theveniau et al., 2012; Singh et al., 2012). The latter was found

to associate with the bacterial flagellin-receptor FLS2 and to be a positive regulator of

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the flagellin-mediated defence responses (Huang et al., 2014). LecRK-IV.3 (alias

AtLPK1) might have a role in resistance to fungal pathogens because LecRK-IV.3

overexpression in Arabidopsis resulted in increased resistance to Botrytis cinerea

(Huang et al., 2013). For several other LecRKs, a function in plant growth and/or

stress response was anticipated based on in silico expression analysis

(Bouwmeester and Govers, 2009), but as yet experimental evidence for this is

lacking.

The objective of the current work was to elucidate the biological function of

individual LecRKs in Arabidopsis. LecRK T-DNA insertion lines were obtained and

analysed for phenotypic changes (i) in plant development under normal conditions or

abiotic stress and (ii) upon exposure to pathogens. The phenotypic data were then

integrated with in silico expression data with the aim to uncover potential

associations between LecRK gene expression and biological function. In addition,

LecRK-V.5, of which a mutant showed altered disease phenotypes, was selected for

more detailed study to validate its function.

RESULTS

Assembling the collection of Arabidopsis LecRK T-DNA insertion lines

The T-DNA Express database of the SALK Institute Genome Analysis Laboratory

(SIGnAL; http://signal.salk.edu) was used to search for T-DNA insertion lines for all

45 LecRKs (Table 1). Preferably, homozygous lines bearing T-DNA insertions within

the coding regions were selected to enhance the possibility of complete disruption of

gene function. If not available, homozygous lines with insertions in the promoter (5

lines), 5’UTR (14) or 3’UTR (2) or segregating lines were obtained. In total we

obtained 62 T-DNA insertion lines covering 41 LecRKs, all in a Col-0 background.

The seeds of the only T-DNA insertion line available for LecRK-IV.3 (i.e.

SAIL_529_D07) did not geminate, thereby reducing the number of lines to 61

covering 40 LecRKs. Of these 61, 52 were indicated as homozygous lines and 9 as

segregating lines. To confirm this, we performed PCR analysis and found that 42 out

of the 52 lines were indeed homozygous with correct T-DNA insertion sites. Of the

remaining ten, eight were found to be segregating at the site of the T-DNA insertion,

and two (i.e. SALK_053703C and SALK_019496C) did not show a T-DNA insertion

in the anticipated target gene (i.e. LecRK-IV.1) indicating that these lines are either

untransformed Col-0 or contain a T-DNA insertion elsewhere in the genome. So in

total we ended up with 17 segregating lines. These were propagated and the

offspring was genotyped to select the homozygous ones. For five lines however, we

were not able to detect any homozygous offspring among over 60 plants tested for


67

each individual line. This was the case for the single T-DNA insertion line that was

available for LecRK-II.2 as well as for the two lines mutated in LecRK-V.6 and the

two in LecRK-S.5. The consistency among the two independent LecRK-V.6 lines and

the two independent LecRK-S.5 lines suggests that a complete knockout of these

genes is lethal raising the possibility that these LecRKs have a crucial role in

development. In total 54 lines representing 36 individual LecRKs were confirmed as

homozygous T-DNA insertion lines and used for further analysis (Table 1).

Table 1. Overview of the clades and members of LecRK gene family in Arabidopsis, the T-DNA

insertion lines used in this study and their phenotypes in response to pathogen infection and

abiotic stress. The response to P. brassicae and P. capsici is depicted by bars that show the DSI

obtained from 20 to 40 inoculated leaves in three independent experiments. The DSI scale ranges

from 0 (light grey) to 4 (dark grey) as shown below the table. The response to P. capsici, P. parasitica,

A. brassicicola, Pst DC3000 and to abiotic stress is depicted by a ● when there is no difference

compared with Col-0, and by S (Susceptible), R (Resistant), EN (Enhanced necrotic symptoms but not

increased bacterial proliferation) or ES (Enhanced necrotic symptoms and increased bacterial

proliferation) to indicate the disease phenotype. Infection assays with P. capsici LT62 were performed

once while infection assays with all other pathogens were repeated at least three times.

Footnotes to Table 1 (see next page) a For alternative gene names and alternative mutant names see Table S1. b No T-DNA insertion lines

available. c The obtained SAIL_529_D07 line did not germinate. d SALK_053703C and

SALK_019496C were indicated as lines with T-DNA insertions in At2g37710 but in our analysis this

could not be confirmed. e >< : homozygous; >: segregating; /: no insertion in the gene under study. f

With 10-fold higher inoculation densities lecrk-I.9-1 and lecrk-I.9-2 show enhanced susceptibility to Pst

DC3000 as reported by Bouwmeester et al. (2011). g Similar results were obtained as described by

Desclos-Theveniau et al. (2012) and Singh et al. (2012). * T-DNA insertion lines showing significantly

higher or lower DSI when compared with Col-0 based on a t test, p < 0.05.

Chapter III

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69

Arabidopsis LecRK T-DNA insertion lines showed no phenotypic changes in

growth and development

To determine whether LecRKs are involved in plant growth and development, LecRK

T-DNA insertion lines were grown in soil and analysed for phenotypic changes in

rosette size, inflorescence emergence, and in flower and silique morphology for up to

8 weeks. In addition, seeds were grown in vitro on vertical 0.5 MS plates to monitor

shoot and root growth. Under these conditions, no consistent growth alterations were

observed in comparison to Col-0 (Table 1). Previously, Wan et al. (2008) described

that a homozygous mutant with two insertions in the region encoding the lectin

domain of LecRK-IV.2 developed abnormal siliques and they concluded that LecRK-

IV.2 is required for proper pollen development. The T-DNA insertion line lecrk-IV.2

that we analysed harbours a T-DNA insertion in the 5’UTR, at ~70 bp before the start

codon. Since this line showed no defects in silique development we analysed the

LecRK-IV.2 expression by qRT-PCR and found that the transcript level is only slightly

reduced when compared with Col-0 (approximately 40%) (Supplemental Fig. S1, A

and B). This reduction is apparently not sufficient to disturb normal silique

development (Supplemental Fig. S1C). Unfortunately the mutant analysed by Wan et

al. (2008) is no longer available in public resources thus hampering further functional

analysis of LecRK-IV.2.

Response of LecRK T-DNA insertion lines to abiotic stress

To evaluate the role of LecRKs in resistance to abiotic stresses, the LecRK T-DNA

insertion lines were grown on 0.5 MS medium supplemented with 100 mM NaCl or

200 mM mannitol. Plants were checked for changes in rosette size and growth of

shoots and roots. No consistent differences were observed between any of the

LecRK T-DNA insertion lines and Col-0. Previously, Deng et al. (2009) observed that

a line with a T-DNA insertion in LecRK-V.2 showed enhanced tolerance to NaCl and

mannitol and developed longer primary roots than Col-0 and concluded that LecRK-

V.2 is involved in salt and osmotic stress. In our study, lecrk-V.2, a T-DNA insertion

line with an insertion in the 3’UTR, ~60 bp after the stop codon showed similar

growth as Col-0 upon treatment with NaCl or mannitol, also when treated using

exactly the same conditions as described by Deng et al. (2009) (Table 1;

Supplemental Fig. S2, A and B). To find a reason for this discrepancy, we

determined the LecRK-V.2 expression in lecrk-V.2 and Col-0 by qRT-PCR.

Remarkably, the transcript level in lecrk-V.2 was higher than in Col-0, instead of

lower (Supplemental Fig. S2C). Possibly the T-DNA insertion in the 3’UTR in one

way or another stabilizes the mRNA and this may explain why we did not find defects

in salt and osmotic stress responses. T-DNA insertion line lecrk-V.2 was excluded

from further analysis.

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70

Response of LecRK T-DNA insertion lines to Phytophthora pathogens

Previously, LecRK-I.9 was reported as a novel resistance component against

Phytophthora brassicae in Arabidopsis (Bouwmeester et al., 2011). To determine the

involvement of other LecRKs in Phytophthora resistance, we performed infection

assays on the LecRK T-DNA insertion lines with P. brassicae, Phytophthora capsici

and Phytophthora parasitica. Recently it was reported that latter two are able to infect

Arabidopsis (Wang et al., 2011; Wang et al., 2013).

In order to identify LecRKs contributing to Phytophthora resistance, infection

assays were performed with isolates that are not able to infect Arabidopsis accession

Col-0 i.e. P. brassicae HH and P. capsici LT123. As shown by Bouwmeester et al.

(2011), isolate HH can successful infect LecRK-I.9 mutants. Inoculation of lecrk-I.9-1

and lecrk-I.9-2 with P. brassicae HH resulted in water-soaked lesions at four days

post inoculation (dpi) with a mean DSI of 2.2 and 2.4, respectively (Supplemental Fig.

S3A). P. capsici LT123 caused similar disease symptoms on these mutants and at 4

dpi the mean DSI was 2.0 and 1.9, respectively (Supplemental Fig. S3B). This shows

that LecRK-I.9 also contributes to resistance against P. capsici.

Inoculation of all available LecRK T-DNA insertion lines with P. brassicae HH

and P. capsici LT123 resulted in evident disease symptoms on several lines. For

example, lecrk-S.1, which contains a T-DNA insertion in the region encoding the

extracellular lectin domain and has a 90% reduction in LecRK-S.1 expression level

(Fig. 1, A and B), showed a mean DSI of 2.5 and 2.1 with HH and LT123,

respectively (Fig. 1C). In total 19 T-DNA insertion lines representing 13 LecRKs from

different clades showed a significantly higher DSI at 4 dpi than Col-0 (Table 1). From

these 13 LecRKs, three are from clade V, two from clade IX and four are singletons.

The four remaining ones are from clade I, III, IV and VIII, respectively. For four out of

13, two T-DNA insertion lines are available which both showed enhanced disease

severity when challenged with Phytophthora, with at least one of the two having an

insertion in the coding region (Table 1). For two other LecRKs for which two or more

T-DNA insertion lines are available, T-DNA insertion lines with different insertion sites

showed discrepancy in susceptibility. In the case of LecRK-VIII.2 with three lines

having insertions in the coding region, only lecrk-VIII.2-3 developed rapid spreading

lesions, resulting in a DSI ≥ 2 on approximately 80% of the inoculated leaves at 4 dpi.

The other two lines, lecrk-VIII.2-1 and lecrk-VIII.2-2 also showed disease symptoms

but the disease severity was less pronounced (Table 1). In the case of LecRK-S.7,

the two T-DNA insertion lines showed opposite responses. lecrk-S.7-1 is fully

resistant to both P. brassicae HH and P. capsici LT123, while lecrk-S.7-2 showed

clear disease symptoms with a mean DSI of 2.0 or 1.9, respectively (Table 1). Both

lines have the T-DNA insertion in the 5’UTR but at different positions. Expression

analysis revealed that the difference in phenotype between lecrk-S.7-1 and lecrk-S.7-


71

Figure 1. Response of Arabidopsis LecRK T-DNA insertion lines to infection by Phytophthora

spp.

A, Schematic model representing LecRK-S.1. The black arrowhead points to the T-DNA insertion site

in mutant lecrk-S.1 while the head-to-head arrow pairs indicate the position of the primers (S.1-RT)

used for qRT-PCR. B, Relative quantification of LecRK-S.1 transcript levels in Col-0 and lecrk-S.1 by

qRT-PCR. C, Disease symptoms on Col-0 and lecrk-S.1 at four days after plug-inoculation (dpi) with

P. brassicae HH and P. capsici LT123. White arrowheads point to inoculated leaves. Each bar

represents the DSI obtained from at least 30 leaves in three independent experiments with the number

showing the average DSI and a * a significant difference (p < 0.05) according to a t test. D, Disease

symptoms on T-DNA insertion line lecrk-I.7 and Col-0 inoculated with P. brassicae CBS686.95 and P.

capsici LT263 at 3 dpi. * indicates significant difference in DSI (p < 0.05) according to a t test.

Chapter III

72

2 correlates with LecRK-S.7 expression levels. In lecrk-S.7-2 expression is reduced

compared with Col-0 whereas in lecrk-S.7-1 it is increased (Supplemental Fig. S4B).

For the seven remaining LecRKs of which T-DNA insertion lines showed a change in

phenotype in the Phytophthora disease assays, only a single T-DNA insertion line

was available with six (i.e. lecrk-III.2, lecrk-V.4, lecrk-V.7, lecrk-IX.2, lecrk-S.1 and

lecrk-S.6) having a T-DNA insertion in the coding region and one (i.e. lecrk-V.5-3) in

the 5’UTR (Table 1).

When challenging the LecRK T-DNA insertion lines with Phytophthora isolates

that are less virulent, in this case P. capsici LT62 and P. parasitica Pp009, none of

the LecRK T-DNA insertion lines showed lesions (Table 1). They remained resistant

similar to Col-0. This suggests that these LecRKs are not essential to stop less

virulent isolates but might function as extra barrier for more virulent isolates.

To investigate the potential role of LecRKs in suppressing Phytophthora

resistance, LecRK T-DNA insertion lines were challenged with Phytophthora isolates

that are capable of infecting Col-0, i.e. P. brassicae CBS686.95 and P. capsici LT263.

Out of all tested LecRK T-DNA insertion lines, only lecrk-I.7, with an insertion in the

region encoding the kinase, showed slightly smaller lesions when compared with Col-

0 (Fig. 1D). All the other T-DNA insertion lines showed a comparable or slightly

higher disease severity than Col-0 (Table 1).

Response of LecRK T-DNA insertion lines to Pseudomonas syringae and

Alternaria brassicicola

Previously, LecRK-VI.2 was reported to play a role in resistance to bacterial

pathogens (Singh et al., 2012). To determine the involvement of other LecRKs in

bacterial resistance, we performed infection assays with Pseudomonas syringae

(Pst) DC3000. Upon spray inoculation, T-DNA insertion lines of eight LecRKs

showed enhanced yellowing and necrosis at 4 dpi when compared with Col-0 (Table

1; Fig. 2A). For three of these, two T-DNA insertion lines are available which both

showed enhanced symptoms when challenged with Pst DC3000, with at least one of

the two having an insertion in the coding region (Table 1). To determine to what

extent the visible symptoms are correlated with enhanced Arabidopsis susceptibility,

bacterial colonization was quantified in rosette leaves. In line with Singh et al., (2012),

we detected significantly higher bacterial titers in lecrk-VI.2-2 in comparison to Col-0

(Fig. 2B). Also in lesions on the two T-DNA insertion lines from LecRK-IV.4, the one

from LecRK-S.1 and two from LecRK-S.4, significantly higher bacterial titers were

detected and this was reproducible (Fig. 2B). Thus, these T-DNA insertion lines were

scored as ES (enhanced susceptibility). The phenotype of the remaining four T-DNA

insertion lines (i.e. lecrk-IV.2, lecrk-V.4, lecrk-V.7 and lecrk-S.6) was scored as EN

(enhanced yellowing and necrosis) rather than ES (Table 1). This is because the


73

bacterial titers were not consistently higher in all assays despite the fact that the

enhanced susceptibility phenotypes were reproducible.

Figure 2. Response of Arabidopsis LecRK T-DNA insertion lines to infection by Pseudomonas

syringae and Alternaria brassicicola.

A, Disease symptoms on Col-0, lecrk-S.1 and lecrk-VI.2-2 four days after inoculation with Pst DC3000

(108 cfu ml-1). B, Bacterial growth in Col-0 and various LecRK T-DNA insertion lines at 3 dpi. Bars

depict mean bacterial titers of four plants (± SD). * indicates p < 0.05 according to a t test for

comparisons between Col-0 and LecRK T-DNA insertion lines. Experiments were repeated three

times with similar results. C, Disease symptoms on Col-0, pad3 and various LecRK T-DNA insertion

lines five days after inoculation with A. brassicicola MUCL20297.

To determine the potential involvement of LecRKs in non-host resistance, we

tested the response of the LecRK T-DNA insertion lines to Alternaria brassicicola, a

fungal pathogen that is not pathogenic on Col-0 but is capable to cause disease

symptoms on the camalexin deficient mutant pad3 (van Wees et al., 2003). The

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majority of the LecRK T-DNA insertion lines showed no visible symptoms up to 5 dpi.

Five T-DNA insertion lines however, representing four clade I LecRKs, showed a

slight gain of susceptibility (Table 1; Fig. 2C). Two of three LecRK-I.3 T-DNA

insertion lines showed the phenotype, one with an insertion in the exon (lecrk-I.3-3)

and the other in the 5’UTR (lecrk-I.3-1). The third one (i.e. lecrk-I.3-2) also with a

5’UTR insertion was not infected. Also in the case of LecRK-I.8 the exon mutant

(lecrk-I.8-2) showed gain of susceptibility but not the T-DNA insertion line with a

5’UTR insertion (lecrk-I.8-1).

LecRK-V.5 plays a dual role in resistance to Pseudomonas syringae and

Phytophthora pathogens

Our infection assays revealed that the mutant lecRK-V.5-3 showed gain of

susceptibility to P. brassicae HH and P. capsici LT123 (Table 1; Fig. 3, A-C). In

contrast, Desclos-Theveniau et al. (2012) reported that mutants of LecRK-V.5

showed enhanced resistance towards Pst DC3000. Because of these opposite

phenotypes with respect to different pathogens we decided to investigate the function

of LecRK-V.5 in more detail.

Gain of Phytophthora susceptibility in lecrk-V.5-3 points to a role of LecRK-V.5

in Phytophthora resistance. To confirm this, we generated transgenic Arabidopsis

lines overexpressing LecRK-V.5 in a Col-0 background. Two lines (i.e. 35S-LecRK-

V.5-1/2) with constitutive and strongly enhanced LecRK-V.5 expression were

selected (Fig. 3B). Neither of the 35S-LecRK-V.5 lines showed any visible

developmental defects. Inoculation of Col-0 with P. brassicae CBS686.95 and

P. capsici LT263 resulted in almost fully colonized leaves at 3 dpi (Fig. 3D). In

contrast, the two 35S-LecRK-V.5 lines showed reduced lesion sizes with a DSI of

approximately 2.0 (Fig. 3D). To determine if the reduced disease severity on the

LecRK-V.5 overexpression lines is indeed correlated with enhanced resistance,

Phytophthora biomass was quantified by qRT-PCR in leaves inoculated with

P. capsici LT263 at 3 dpi. This showed that the P. capsici biomass in the 35S-LecRK-

V.5 lines was reduced by at least 50% compared with Col-0 (Fig. 3E).

To confirm the role of LecRK-V.5 in resistance to Pst DC3000 as postulated

by Desclos-Theveniau et al. (2012), we compared the phenotype of lecrk-V.5-3 with

the phenotypes of the previously analysed mutants lecrk-V.5-1 and lecrk-V.5-2.

Similar to lecrk-V.5-1 and lecrk-V.5-2, lecrk-V.5-3 showed less necrotic symptoms

and reduced bacterial proliferation compared with Col-0 (Fig. 3, F and G). Moreover,

both 35S-LecRK-V.5 lines showed enhanced susceptibility to Pst DC3000 with

increased necrosis and higher bacterial titers than Col-0 (Fig. 3, F and G). Taken

together, these results demonstrate that LecRK-V.5 has a dual role in regulating

defence to different pathogens.


75

Chapter III

76

Figure 3. LecRK-V.5 plays opposite roles in resistance to Phytophthora and Pseudomonas

syringae.

A, Schematic model representing LecRK-V.5. The black arrowhead points to the T-DNA insertion site

in lecrk-V.5-3 while the head-to-head arrow pairs indicate the position of the primers (V.5-RT) used for

qRT-PCR. B, qRT-PCR analysis of LecRK-V.5 transcript levels in Col-0, lecrk-V.5-3 and two

overexpression lines. * a significant difference (p < 0.05) according to a t test. C, Disease symptoms

on Col-0 and lecrk-V.5-3 four days after inoculation with P. brassicae HH and P. capsici LT123. White

arrowheads point to inoculated leaves. Each bar represents the DSI obtained from at least 30 leaves

in three independent experiments with the number showing the average DSI and a * a significant

difference (p < 0.05) according to a t test. D, Disease symptoms on Col-0 and two LecRK-V.5

overexpression lines three days after inoculation with P. brassicae CBS686.95 and P. capsici LT263.

E, Relative quantification of P. capsici biomass (RQ) in Col-0 and overexpression lines at 3 dpi by

qPCR. Bars represent mean values (± SD) relative to that of infected Col-0 leaves. * indicates

significantly less biomass (p < 0.05) according to a t test. Experiments were repeated twice with

similar results. F, Disease symptoms on Col-0, lecrk-V.5-3 and two overexpression lines four days

after inoculation with Pst DC3000. G, Bacterial growth in Col-0, lecrk-V.5-3 and two overexpression

lines at 3 dpi. Bars shown are mean values (± SD). * indicates significantly lower or higher bacterial

titers (p < 0.05) according to a t test; Experiments were repeated twice with similar results.

Are there associations between LecRK mutant phenotypes and LecRK

expression profiles?

Expression profiling analysis has been widely used to predict potential biological

function of genes (Li et al., 2006; Fukushima et al., 2012). Here, we explored the

transcriptome-to-phenotype relations by comparing LecRK expression profiles

retrieved from publicly available microarray data, with the mutant phenotypes


77

obtained in this study (Fig. 4). Since there is no genome-wide expression data

available from Arabidopsis inoculated with P. brassicae or P. capsici, we used the

expression data derived from Arabidopsis leaves inoculated with

Phytophthora infestans, a Phytophthora species that is not capable of infecting

Arabidopsis (Huitema et al., 2003). The 13 LecRKs, of which T-DNA insertion lines

showed compromised resistance to P. brassicae HH and P. capsici LT123, were

found to be differentially expressed during P. infestans infection (Fig. 4A). LecRK-

IX.2 was highly induced and the expression level was increased up to 50-fold (log2

value of 5.7) at 6 hpi. LecRK-I.9, LecRK-V.5 and LecRK-V.7 showed induced

expression with a maximum of four-fold-increase, whereas the other nine LecRKs are

either hardly expressed or even down-regulated during infection process. The same

holds for LecRK-I.7, the only LecRK of which the mutant showed slightly enhanced

Phytophthora resistance.

The nine LecRKs of which T-DNA insertion lines showed altered phenotypes

in response to Pst DC3000 (EN, ES or R) were all down-regulated at one or more

time points after inoculation with Pst DC3000 (Fig. 4A). The five LecRKs with T-DNA

insertion lines showing significantly altered bacterial colonization (ES or R) were

down-regulated at 12 hpi with log2 values ranging from -0.7 to -1.4.

Despite the fact that several LecRKs were differently expressed in various

tissues and in response to NaCl or mannitol treatment, none of the tested T-DNA

insertion lines showed consistent phenotypic changes during growth and

development or when exposed to abiotic stress. Hence, it is difficult to reconcile the

biological function of these LecRKs with expression profiles. However, lack of

phenotypic changes in mutants does not exclude that certain LecRKs have a function

in these processes. It could well be that functional redundancy masks the

phenotypes caused by a single gene disruption.

Genes that exhibit similar expression patterns are often expected to be

involved in the same biological processes (Eisen et al., 1998; Fukushima et al., 2012).

We computed co-expression profiles of all LecRKs in leaves inoculated with

P. infestans and Pst DC3000 over time. In order to easily visualize LecRK expression

patterns, expression levels (log2 values) were normalized by Z-score transformation

(Cheadle et al., 2003). LecRKs with similar expression patterns were grouped

together using hierarchical clustering with Pearson’s correlation coefficient (r) and

average linkage. Pearson’s r is a metric to evaluate the strength of the correlation

between variables, ranging from -1 to 1 (Usadel et al., 2009) and therefore we used

the cut-off value of r ≥ 0.6 for clustering which, in general, indicates strong positive

correlation (Aoki et al., 2007; Fukushima et al., 2012). With this criterion, LecRKs

could be grouped into five clusters based on their expression patterns upon infection

Chapter III

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Figure 4. Compilation of expression profiles of LecRKs and phenotypes of LecRK T-DNA

insertion lines.


79

A, The heat map in the left panel shows LecRK expression levels in different plant tissues and in

response to various biotic and abiotic stresses. Signal intensity log2 ratios are color-coded in the range

of -3 (purple) to 6 (green) as indicated in the scale bar, and represent the relative expression levels

compared with corresponding controls. The table on the right shows the phenotypes of the LecRK T-

DNA insertion lines compiled from the results presented in Table 1 with ‘R’ standing for ‘resistant’, ‘S’

for ‘susceptible’, ‘EN’ for ‘enhanced necrotic symptoms but no increased bacterial proliferation’ and

‘ES’ for ‘enhanced necrotic symptoms and increased bacterial proliferation. ● depicts ‘no consistent

phenotypic changes’ and ─ ‘not tested’. B-D, Hierarchical clustering of LecRKs based on expression

patterns at 6, 12 and 24 hours after inoculation with P. infestans (C), 2, 6 and 24 hours after

inoculation with Pst DC3000 (D) or combined (B) using Pearson’s correlation coefficient (r) of per

gene Z-score transformed log2 values (sample relative to control). Clusters were created using a cut-

off value of r ≥ 0.6. In (C) and (D) clusters are indicated by vertical colored bars connected to panels

showing LecRK expression patterns in each cluster. The lengths of the branches (da) are the scaled

(black lines) and unscaled (grey lines) average Pearson correlation distances (dp = 2da = 1 - r).

Normalized expression data are color-coded to indicate higher (yellow, saturated at 1.2) and lower

(blue, saturated at -1.2) expression relative to the respective controls. œ On the microarray the same

probes represent LecRK-V.7 and LecRK-V.8 and thus the expression values are identical. In (A-D)

LecRKs involved in Phytophthora resistance (S or R) are depicted in green and those involved in

Pst DC3000 resistance (ES or R) in dark red. LecRKs involved in both are depicted in combined

green-red.

with P. infestans (Fig. 4C) and another five clusters upon infection with Pst DC3000

(Fig. 4D). Of the 14 LecRKs of which T-DNA insertion lines showed altered

susceptibility to Phytophthora spp., five were grouped in cluster II and showed a

slight reduction in expression between 6 and 24 hpi. The four LecRKs that grouped in

cluster V show an opposite expression pattern compared with LecRKs in cluster II. Of

the remaining ones, two are in cluster I, i.e. LecRK-I.9 and LecRK-S.1, two in cluster

III, i.e. LecRK-S.4 and LecRK-S.7 and one in cluster IV, i.e. LecRK-III.2. Similar

transcriptome-to-phenotype patterns were observed upon challenging with

Pst DC3000 (Fig. 4D). Of the five LecRKs of which T-DNA insertion lines showed

altered susceptibility to Pst DC3000, LecRK-V.5 and LecRK-VI.2 are combined in

cluster I, LecRK-S.1 and LecRK-S.4 in cluster II while LecRK-IV.4 was allocated to

cluster III. When combining the expression patterns of the two time courses obtained

from leaves inoculated with the two different pathogens, 27 of the 45 LecRKs

grouped in clusters, 11 in total, while the remaining 18 did not share their expression

pattern with any other LecRK (Fig. 4B). Although the four LecRKs that are implicated

in resistance to both Phytophthora pathogens and Pst DC3000, do not group in one

cluster and are far apart in the dendrogram, the clustering of two, respectively three

relevant LecRKs is maintained, i.e. LecRK-V.4 and LecRK-VIII.2 and the combination

of LecRK-V.5, LecRK-VI.2 and LecRK-IX.2.

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DISCUSSION

Most of the plant immune receptors that have been identified to date are either

plasma membrane-localized receptors including RLKs and receptor-like proteins

(RLPs) or intracellular nucleotide binding-leucine-rich repeat proteins (NLRs) (Spoel

and Dong, 2012). The majority was discovered based on their ability to activate

defence responses upon recognition of MAMPs or pathogen effectors. For example,

the RLK gene encoding the MAMP receptor FLS2 was isolated by a forward genetics

approach based on screening random mutants for flagellin-insensitive mutants,

followed by map-based cloning of the locus harbouring the mutation (Gómez-Gómez

and Boller, 2000). Also the isolation of several NLR genes that function as major R-

gene in ‘gene-for-gene’ interactions was the result of combining phenotype (i.e.

pathogen recognition) and map based cloning, in this case facilitated by linkage

mapping using segregating progenies of resistant and susceptible parents (Marone

et al., 2013; Rodewald and Trognitz, 2013). Genome sequencing has revealed that

defence-related RLKs and NLRs often have many homologs of which the function is

unknown. They are grouped into families and sub-families based on sequence

similarity and shared domain architecture.

In this study we focused on one such RLK sub-family in Arabidopsis, namely

the LecRK family of which three out of 45 members were previously identified as

critical players in Arabidopsis innate immunity. Two of the three were selected for

functional studies based on their increased expression in plants treated with priming

agent BABA (LecRK-VI.2; Singh et al., 2012) or oligogalacturonic acid (LecRK-V.5;

Desclos-Theveniau et al., 2012). The third one was selected via a phage display

aimed at identifying RGD-binding proteins in plants (Gouget et al., 2006) and

appeared to be a potential host target of a P. infestans RXLR effector (LecRK-I.9;

Bouwmeester et al., 2011). We anticipated that functional screening of other

members of the LecRK family would reveal several novel candidates with a role in

disease resistance. In an unbiased strategy we monitored all available LecRK T-DNA

insertion lines and indeed, besides LecRK-I.9, LecRK-V.5 and LecRK-VI.2 mentioned

above, we found 16 additional LecRKs of which mutants showed phenotypic changes

in response to pathogens. T-DNA insertion lines are effective tools for determining

gene function and for linking genotype to phenotype (O’Malley and Ecker, 2010;

Lloyd and Meinke, 2012). In other studies similar unbiased screenings of T-DNA

insertion line collections resulted in novel insight in gene function. An example is the

study of ten Hove et al. (2011) who performed large scale screening of T-DNA

insertion lines of root-expressed leucine-rich repeat (LRR) RLKs and identified LRR-

RLKs implicated in hormone and abiotic stress responses. In a similar way Wang et

al. (2008) performed a screening of a broad range of T-DNA insertion lines in 56


81

genes encoding RLPs and assigned functions to these RLPs in plant development

and resistance. It should be noted that the success rate in these two studies was

relatively low; only few T-DNA insertion lines were found that could be linked to the

phenotype of interest. This could well be due to redundancy in function among

members of these sub-families. When one gene is inactivated because of the T-DNA

insertion another family member might take over its function resulting in the wild-type

phenotype. For example, SOMATIC EMBRYOGENESIS RECEPTOR KINASE1

(SERK1) and the closest homolog SERK2 were concluded to function redundantly in

controlling anther development and male gametophyte maturation based on the

observation that the single mutants serk1 and serk2 did not show any developmental

defects whereas serk1serk2 double mutants were defective in pollen development

(Colcombet et al., 2005). This implies that such screenings, including ours, are by

definition not conclusive for all members of the family; the ones of which mutants

don’t show a phenotype could still have an important role. Another issue to keep in

mind is the variation in knock-out efficiency among mutants with a T-DNA insertion in

the same gene. According to a survey on 1084 published Arabidopsis insertion lines,

an insertion in the coding region leads to inactivation of the target gene in over 98%

of the cases; in mutants with an insertion before the start codon or after the stop

codon, this percentage decreases but is still high (86% and 75%, respectively)

(Wang, 2008). Variation in knock-out efficiency might explain why in some cases we

found differences in phenotype among T-DNA insertion lines of the same gene like

for example, the three LecRK-I.3 lines. In one case where we analysed expression of

the target gene, i.e. LecRK-IV.2, we indeed detected residual expression in a mutant

that contains a T-DNA insertion in the 5’UTR, and no change in phenotype, whereas

a line with insertions in the exon of that same gene showed a phenotype

(Supplemental Fig. S1; Wan et al., 2008). In the case of LecRK-V.2 we even found

that a T-DNA insertion in the 3’UTR led to increased transcript levels of the target

gene (Supplemental Fig. S2), a phenomenon that was also noted in the survey of

Wang (2008). It is thus not surprising that we could not confirm the phenotype of a

LecRK-V.2 mutant observed by Deng et al. (2009). Since we have not systematically

checked all T-DNA insertion lines for expression there is a chance that some of them

are not true mutants in the sense that they lost gene function. This implies that

LecRKs with T-DNA insertion lines showing no phenotype might still be relevant for

pathogen resistance.

In the unbiased strategy that we used here successful identification of mutants

is highly dependent on the design and the methodology of the phenotypic screening.

In our screening none of the LecRK T-DNA insertion lines showed phenotypic

changes upon exposure to mannitol or NaCl despite the fact that multiple LecRKs are

induced during abiotic stress. It could well be that the conditions that we used for

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82

screening were not optimal. We only tested one concentration of mannitol or one of

NaCl instead of a whole range of concentrations. When screening for disease

resistance the choice of pathogen isolates is extremely important. In the large scale

screening performed by Wang et al. (2008), none of the four RLP30 T-DNA insertion

lines showed altered susceptibility towards the necrotrophic fungal pathogens

Sclerotinia sclerotiorum or Botrytis cinerea. Recently, however, it was found that two

of these RLP30 T-DNA insertion lines showed increased susceptibility to S.

sclerotiorum and B. cinerea which is likely due to the fact that Zhang et al. (2013)

used other isolates that are more virulent. In our screening we choose to use

different Phytophthora species and different isolates next to each other. The P.

capsici and P. brassicae isolates were selected based on their differential behavior

on Arabidopsis (Roetschi et al., 2001; Wang et al., 2013). We confirmed the role of

LecRK-I.9 in resistance to P. brassicae and showed that this LecRK also inhibits P.

capsici. In addition, we found several T-DNA insertion lines representing 12 LecRKs

with the same phenotype as lecrk-I.9-1 and lecrk-I.9-2, i.e. compromised resistance

to both Phytophthora spp. For four of these LecRKs (i.e. LecRK-IV.4, LecRK-VIII.2,

LecRK-IX.1 and LecRK-S.4) two or more independent T-DNA insertion lines showed

consistent gain of susceptibility demonstrating that they function as Phytophthora

resistance component. For another one, i.e. LecRK-V.5, we confirmed the mutant

phenotype by showing that overexpression of LecRK-V.5 enhances Phytophthora

resistance. For the remaining ones we have to be more cautious; in the phenotypic

analysis only a single T-DNA insertion line was included, the only one available or, in

the case of LecRK-S.7, only one of the two lines was a reliable mutant. Therefore, for

these LecRKs only a potential role in resistance against Phytophthora pathogens can

be suggested. With respect to P. brassicae and P. capsici, LecRK T-DNA insertion

lines showed no specificity suggesting that LecRKs do not discriminate between

Phytophthora species. Overall the T-DNA insertion lines are more susceptible to

P. brassicae HH than to P. capsici LT123 and less susceptible to P. brassicae

CBS686.95 than to P. capsici LT263. None of the LecRK T-DNA insertion lines was

infected by the less virulent isolate P. capsici LT62 and P. parasitica Pp009. These

two isolates are probably too weak to reach the stage where LecRKs come into play

as barrier for infection.

Four of the LecRKs that have a function in Phytophthora resistance were also

found to have a role in the interaction of Arabidopsis with the bacterial pathogen

Pst DC3000. Three of these promote resistance suggesting that they can monitor

different types of pathogens and potentially confer a broad-spectrum-resistance. The

fourth one however, LecRK-V.5, was found to have opposite roles in these two

pathosystems, i.e. a resistance component against Phytophthora pathogens but a

susceptibility factor for Pst DC3000. One explanation for this dual role is that


83

Phytophthora and bacteria have different modes to enter host tissues. Bacteria

usually penetrate via natural opening such as stomata, and stomatal closure can

essentially restrict bacterial invasion (Melotto et al., 2006). Phytophthora pathogens

can also penetrate via stomata but instead, they prefer to penetrate at the junctions

between epidermal cells (Hardham, 2007; Wang et al., 2011). Therefore, the function

of LecRK-V.5 in suppressing stomatal immunity is dispensable for preventing

Phytophthora penetration. In addition, LecRK-V.5 was found negatively regulating the

ABA response in Arabidopsis (Desclos-Theveniau et al., 2012). ABA plays a

multifaceted role in regulating plant resistance (Ton et al., 2009). On the one hand,

ABA enhances resistance against bacterial pathogens by regulating stomatal closure;

on the other hand, ABA compromises defence to various pathogens via suppression

of salicylic acid-signaling (Cao et al., 2011; Mohr and Cahill, 2007; Sanchez-Vallet et

al., 2012), a process that in Arabidopsis is essential for resistance to P. capsici

(Wang et al., 2013). Hence, the gain of susceptibility found in lecrk-V.5-3 upon

Phytophthora infection might be linked to ABA signaling.

Previously LecRK-VI.2 was found to be required for resistance to various

bacterial pathogens but not to the fungal pathogen Botrytis cinerea (Singh et al.,

2013; Huang et al., 2014). In our study, we confirmed the role of LecRK-VI.2 in

bacterial resistance and found that it is neither required for resistance to

Phytophthora pathogens nor the fungal pathogen A. brassicicola. This specificity

could be explained by the recent finding that LecRK-VI.2 associates with FLS2 and

participates in flg22-mediated defence (Huang et al., 2014), which is essential for

bacterial pathogen resistance but might be dispensable for resistance to

Phytophthora or fungal pathogens.

Considering the fact that the primary sequence of a protein is a prime

determinant of biological function, LecRKs with high sequence similarity might share

similar biological function. This might be the case for the four LecRKs in clade I of the

T-DNA insertion lines showed compromised resistance to A. brassicicola. This

clustering was not observed for LecRKs involved in Phytophthora or Pst DC3000

resistance. They are dispersed over different clades and thus sequence similarity as

such does not account for a similar function. Moreover, the computed expression

profiling revealed that LecRKs with similar function displayed rather different

expression patterns upon pathogen infection and therefore, expression is also not a

determinant of the functional similarity among LecRKs.

In summary, phenotypic analysis of a large set of T-DNA insertion lines

uncovered multiple Arabidopsis LecRKs with so far unknown function that contribute

to defence against pathogens, thus pointing to LecRKs as potential immune

receptors. The incentive for this screening was the finding that LecRK-I.9 is a

Phytophthora resistance component and, as importantly, that transgenic potato lines

Chapter III

84

expressing Arabidopsis LecRK-I.9 are more tolerant to P. infestans, the causal agent

of potato late blight (Bouwmeester et al., 2014). This suggests that LecRKs can be

exploited to confer resistance in crops. Since LecRKs are widespread in the plant

kingdom, understanding the function of LecRKs in Arabidopsis will facilitate the

functional analysis of LecRKs in crop species and this will be helpful to explore novel

resistance sources against damaging plant pathogens. Further studies should reveal

how the different LecRKs are activated, what their ligands are, and how they feed

into downstream signaling and activate defence. The overall importance of LecRKs in

plants is demonstrated by the recent finding that LecRK-I.9 (alias DORN1) is capable

to bind extracellular ATP (Choi et al., 2014). It is the first eATP receptor identified in

plants and has a completely different structure than eATP receptors in animals. The

collection of LecRK T-DNA insertion lines described in this study will be instrumental

to further explore the role of LecRKs as eATP receptors.

MATERIALS AND METHODS Analysis of T-DNA insertion lines

LecRK T-DNA insertion lines were obtained from the European Arabidopsis Stock

Center (http://arabidopsis.info; Alonso et al., 2003). T-DNA insertion sites and

homozygosity were checked by PCR using T-DNA primers LBb1.3 (SALK lines), LB1

(SAIL lines) or p745 (WiscDsLox lines) in combination with gene-specific primers

(Supplemental Table S2).

Plant growth conditions

Arabidopsis seeds were sown on soil or in vitro on 0.5 MS medium, stratified in the

dark at 4°C for three days and subsequently placed in a growth chamber at 19-21°C

with 75-78% relative humidity and a 12 h photoperiod. Alternatively, seeds were

grown on vertically orientated 0.5 MS plates or 0.5 MS plates supplemented with 100

mM NaCl or 200 mM mannitol or under the same conditions as described previously

(Deng et al., 2009). Seedling growth was evaluated from the 7th-to-16th day after

seed-sowing. Each treatment included at least three replicates and the experiment

was repeated four times.

Pathogen growth and infection assays

P. brassciae, P. capsici and P. parasitica were grown in the dark on 20% (v/v) V8

agar plates at appropriate temperatures. Infection assays on Arabidopsis with fresh

mycelial plugs were carried out as described previously (Bouwmeester et al., 2011).

Disease symptoms scoring using a disease severity index (DSI) and the


85

Phytophthora biomass quantification were conducted as described by (Wang et al.,

2013).

Pst DC3000 was grown on King's B medium supplemented with 50 µg ml-1

rifampicin. Arabidopsis plants were spray-inoculated with bacterial suspensions with

a concentration of 108 colony forming units (cfu) ml-1 in 10 mM MgSO4, 0.02% (v/v)

Silwet-L77, or mock-treated with 10 mM MgSO4, 0.02% (v/v) Silwet-L77. Bacterial

growth was determined at 3 dpi. Whole rosettes were collected and measured for

fresh weight and subsequently homogenized in 10 mM MgSO4. Homogenates were

serially diluted in 10 mM MgSO4 and were plated on Pseudomonas agar (BD)

containing 50 µg ml-1 rifampicin. Bacterial growth was evaluated by counting bacterial

cfu two days after incubation at 28°C and normalized as cfu/g using the total weight

of the inoculated rosette leaves. This experiment was repeated three times, each

with at least three replicates.

A. brassicicola MUCL20297 was cultured in dark at 22°C on potato dextrose

agar. Four to five-week-old Arabidopsis rosette leaves were drop-inoculated with 5 µl

of spore suspensions (106 spores ml-1) or mock-inoculated with 5 µl water. Plants

were placed in trays covered with lids and kept in the dark during the first 24 h.

Thereafter, they were moved to a climate chamber with aforementioned settings.

Disease symptoms were checked from 3 to 5 dpi.

Plasmid construction and plant transformation

The coding sequence of LecRK-V.5 was amplified by PCR using Pfu DNA

polymerase (Promega) with primers listed in Supplemental Table S2. Purified PCR

products were cloned into the vector pENTR™/D-TOPO® (Invitrogen). Plasmids with

correct sequence were recombined by LR reaction into the binary vector pGWB2.

The resulting vector was transformed into the Agrobacterium tumefaciens GV3101.

Arabidopsis Col-0 plants were transformed using the floral dip method as previously

described (Zhang et al., 2006).

RNA isolation and qRT-PCR

Total RNA was isolated from four-week-old Arabidopsis leaves with a NucleoSpin

RNA plant Kit (Macherey-Nagel) and thereafter used as the template for cDNA

synthesis with an oligo-dT primer using a M-MLV reverse transcriptase kit (Promega)

(Supplemental Table S2). qRT-PCR was performed using an ABI7300 Real-Time

PCR Systems (Applied Biosystems). Data were analysed using a 2−∆∆CT method

(Livak and Schmittgen, 2001). Transcript levels of LecRKs were normalized with

Arabidopsis Actin2 and expressed as mean fold changes (± SD) relative to that in

Col-0 leaves which was arbitrarily set as 1.

Chapter III

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Expression data analyses

LecRK expression data during plant growth or upon treatment with different stimuli

were obtained from the public microarray datasets using eFP-Browser at the Bio-

Array Resource (http://www.bar.utoronto.ca) (Winter et al., 2007). Signal intensity

log2 ratios of the samples relative to respective controls were used as a measure for

LecRK expression. Of the datasets sampled from Arabidopsis in different growth

stages and exposed to abiotic stresses, the maximal (highest or lowest) signal

intensity relative to the mean throughout all arrays in the database was retrieved.

Expression data of Arabidopsis treated with biotic stresses were derived from five-

week-old Arabidopsis Col-0 rosette leaves inoculated with Phytophthora infestans or

infiltrated with Pst DC3000. LecRK-V.7 (At3g59740) and LecRK-V.8 (At3g59750)

share the same probe-set and obtained the same values. For expression pattern

analysis, per gene log2 values were normalized by the Z-score method according to

Cheadle et al. (2003). Co-expression analysis of LecRKs was conducted using

hierarchical clustering with Pearson’s correlation coefficient (r) and average linkage

(da) as implemented in MultiExperiment Viewer (MeV4.0).

ACKNOWLEDGEMENTS We thank Laurent Zimmerli (National Taiwan University) for providing LecRK-VI.2

mutants, Michael F. Seidl (Wageningen University) for discussion and Kolawole

Oluwaseun (University of Ibadan) for technical assistance. This project was

financially supported by a Wageningen University sandwich-PhD fellowship (YW), a

Huygens scholarship (YW), a VENI grant (KB) from the Netherlands Organization for

Scientific Research (Technology Foundation NWO-STW) and the Food-for-Thought

campaign of the Wageningen University Fund.

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SUPPLEMENTAL INFORMATION Table S1. Summary of the available LecRK T-DNA insertion lines.

a No T-DNA insertion lines available. b SALK_053703C, SALK_019496C are listed in the NASC database as mutants with insertions in At2g37710, but in our analysis this

could not be confirmed. c The obtained SAIL_529_D07 could not germinate. d Mutant in Ler-0 background. e T-DNA insertion line show increased LecRK mRNA levels.


91


HM: homozygosity test. RT: primers used for q-RT-PCR. FL: primers for full length LecRK-V.5 cloning.

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Figure S1. SALK_119402C with a T-DNA insertion in the 5’UTR of LecRK-IV.2 does not show

defects in growth and development.

A, Schematic model representing LecRK-IV.2. The black arrowhead points to the T-DNA insertion site

in lecrk-IV.2 (i.e. SALK_119402C) and the head-to-head arrow pairs indicate the position of the

LecRK-IV.2 specific primers (IV.2-RT) used for qRT-PCR analysis. B, Relative quantification of

LecRK-IV.2 transcript levels in Col-0 and lecrk-IV.2. Transcript levels were normalized with

Arabidopsis Actin2 and expressed as mean fold changes (± SD) relative to the transcript level in Col-0

leaves which was arbitrarily set as 1. C, Silique morphology of seven-week-old lecrk-IV.2 and Col-0

plants.


93

Figure S2. SALK_014678C with a T-DNA insertion in the 3’UTR of LecRK-V.2 does not show an

altered response to NaCl and mannitol.

A, Schematic model representing LecRK-V.2. The black arrowhead points to the T-DNA insertion site

in lecrk-V.2 (i.e. SALK_014678C) and the head-to-head arrow pairs indicate the position of the LecRK-

V.2 specific primers (V.2-RT) used for qRT-PCR analysis. B, 16-day-old seedlings of Col-0 and lecrk-

V.2 grown on 0.5 MS plate or on 0.5 MS supplemented with either 100 mM NaCl or 200 mM mannitol.

C, Relative quantification of LecRK-V.2 transcript levels in Col-0 and lecrk-V.2. Transcript levels were

normalized with Arabidopsis Actin2 and expressed as mean fold changes (± SD) relative to the

transcript level in Col-0 leaves which was arbitrarily set as 1.

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Figure S3. Disease symptoms on LecRK-I.9 mutants upon inoculation with Phytophthora

pathogens.

A-B, Disease symptoms on lecrk-I.9-1 and lecrk-I.9-2 four days after plug-inoculation with

P. brassicae HH (A) and P. capsici LT123 (B). White arrowheads point to inoculated leaves.


95

Figure S4. Expression analysis of LecRK-S.7 in the T-DNA insertion lines lecrk-S.7-1 and lecrk-

S.7-2.

A, Schematic model representing LecRK-S.7. Black arrowheads point to the T-DNA insertion sites in

lecrk-S.7-1 (i.e. SALK_140480C) and lecrk-S.7-2 (SALK_008479C) and the head-to-head arrow pairs

indicate the position of the LecRK-S.7 specific primers (V.7-RT) used for qRT-PCR analysis. B,

Relative quantification of LecRK-S.7 transcript levels in Col-0, lecrk-S.7-1 and lecrk-S.7-2. Transcript

levels were normalized with Arabidopsis Actin2 and expressed as mean fold changes (± SD) relative

to the transcript level in Col-0 leaves which was arbitrarily set as 1.

Chapter IV

Phytophthora resistance conferred by Arabidopsis

L-type lectin receptor kinases LecRK-IX.1 and

LecRK-IX.2 is not associated with activation of

plant cell death

Yan Wang, Klaas Bouwmeester, Jan H. G. Cordewenerⱡ, Antoine H.P. Americaⱡ,

Weixing Shan♯, Francine Govers

In collaboration with: ⱡ Plant Research International, Wageningen UR, Wageningen, The Netherlands. ♯ College of Plant Protection, Northwest A&F University, Yangling, Shaanxi, China

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ABSTRACT

Plasma membrane-localized L-type lectin receptor kinases (LecRKs) are considered

as potential immune receptors. In this study, we characterized the two closely-related

Arabidopsis LecRKs, i.e. LecRK-IX.1 and LecRK-IX.2. For both LecRKs, T-DNA

insertion mutants showed compromised Phytophthora resistance as well as reduced

defence-related gene expression. In comparison, the double mutants showed additive

susceptibility. Overexpression of each of the two LecRKs in Arabidopsis increased

resistance to Phytophthora pathogens but also induced cell death. For their function

as Phytophthora resistance component, both the lectin domain and kinase activity are

required, while the ability to induce cell death is determined by the kinase activity but

not the lectin domain. Transient expression of both Arabidopsis LecRKs in

N. benthamiana induces cell death, with LecRK-IX.2 showing a stronger capacity to

induce cell death than LecRK-IX.1. Silencing of NbSIPK/NTF4 in N. benthamiana

completely abolished LecRK-IX.1-induced cell death but not Phytophthora resistance.

In planta co-immunoprecipitation with LecRK-IX.1 and LecRK-IX.2 as baits followed

by LC-MS analysis resulted in the identification of the N. benthamiana ABC

transporter NbPDR1 that interacts with both LecRK-IX.1 and LecRK-IX.2. Also its

closest Arabidopsis homolog ABCG40 interacts with LecRK-IX.1 and LecRK-IX.2 in

planta. In both mutants lecrk-IX.1 and lecrk-IX.2, expression of ABCG40 is reduced

upon Phytophthora infection. Similar to the LecRK mutants, ABCG40 mutants showed

compromised Phytophthora disease resistance. This study demonstrates that

LecRK-IX.1 and LecRK-IX.2 function as Phytophthora resistance components. Both

as well induce cell death when overexpressed, but the Phytophthora resistance

function is independent of cell death. Since both LecRK-IX.1 and LecRK-IX.2 interact

with the same ABC transporters, this suggests that they share similar signaling

components. Nevertheless, they were found to act independently to confer resistance

to Phytophthora pathogens.

LecRK-IX.1 and LecRK-IX.2 function in Phytophthora resistance and cell death induction

99

INTRODUCTION

Plants are constantly threatened by microbes and have evolved strategies to detect

microbial infection via various cell surface and intracellular receptors (Spoel and Dong,

2012). Defence responses initiated by pathogen perception through cell surface

pattern recognition receptors (PRRs) provide the first line of innate immunity and

collectively suppress the majority of invading microbes (Boller and Felix, 2009). PRRs

rapidly form complexes via association with other components to initiate signal

transduction cascades upon recognition of conserved microbial molecules, the so

called microbe-associated molecular patterns (MAMPs), or endogenous elicitors

released from plants, known as damage-associated molecular patterns (DAMPs)

(Sun et al., 2013; Liebrand et al., 2014; Macho and Zipfel, 2014). As a result, various

cellular responses are initiated, including production of reactive oxygen species

(ROS), activation of mitogen-activated protein kinase (MAPK) cascades, secretion of

antimicrobial compounds and induction of defence-related gene expression (Boller

and Felix, 2009). In some cases, activation of cell surface receptors also leads to

plant cell death (Gao et al., 2009).

Plant genomes encode a large number of cell surface receptors with an

intracellular kinase domain. These so-called receptor-like kinases (RLKs) not only act

as PRRs to perceive pathogens, but also can function as signaling partners

participating in PRR-mediated downstream signaling (Chinchilla et al., 2009; Liebrand

et al., 2014). RLKs are classified based on their versatile extracellular domains, which

are considered to determine ligand perception (Shiu and Bleecker, 2001). L-type lectin

receptor kinases (LecRKs) are a group of RLKs with an extracellular legume-like

lectin domain (Herve et al., 1999; Bouwmeester and Govers, 2009). Previously, we

found that one of the LecRKs in Arabidopsis, i.e. LecRK-I.9 plays a crucial role in

resistance to Phytophthora brassicae and acts as a mediator of plant cell wall-plasma

membrane adhesion (Bouwmeester et al., 2011). More recently, the same LecRK was

identified as the first plant receptor for extracellular ATP (eATP) and found to be

required for eATP-mediated downstream signaling (Choi et al., 2014). LecRK-I.9 is a

member of a family consisting of 45 LecRKs in Arabidopsis. Based on sequence

similarity and phylogeny, these 45 LecRKs were divided into nine clades (clade I to

clade IX) and seven singletons (Bouwmeester and Govers, 2009). In recent years,

several Arabidopsis LecRKs other than LecRK-I.9 have been found to be implicated in

plant immunity. For example, LecRK-VI.2, which is required for priming BABA-induced

resistance, associates with the flagellin receptor FLS2 and positively regulates

MAMP-mediated plant defence (Singh et al., 2012; Huang et al., 2014). LecRK-V.5

was found to play a dual role in plant resistance against different pathogens. It

negatively regulates plant stomatal immunity against bacterial pathogens, but

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positively contributes to Phytophthora resistance (Desclos-Theveniau et al., 2012;

Wang et al., 2014/Chapter III). A systematic screen of Arabidopsis LecRK T-DNA

insertion lines identified several additional LecRKs that are involved in resistance

against either Alternaria brassicicola, Phytophthora or Pseudomonas pathogens

(Wang et al., 2014/Chapter III). Among these are LecRK-IX.1 and LecRK-IX.2, the

only two members in clade IX (Bouwmeester and Govers, 2009).

In this study, we performed more detailed analyses on the role of Arabidopsis

LecRK-IX.1 and LecRK-IX.2 in Phytophthora resistance. We compared the

susceptibility of the null mutants lecrk-IX.1 and lecrk-IX.2 with double mutants and

generated overexpression lines to confirm the role of LecRK-IX.1 and LecRK-IX.2 in

Phytophthora resistance. Subsequently, we assayed the response of transgenic

Arabidopsis plants expressing mutated versions of these two LecRKs and pinpointed

the domains required for resistance and cell death responses. In addition, we

transiently expressed the two LecRKs in the Solanaceous plant Nicotiana

benthamiana to monitor induced plant responses. Finally, by means of in planta

co-immunoprecipitation and LC-MS, we identified an interacting protein that seems to

be required for Phytophthora resistance.

RESULTS

Arabidopsis mutants lecrk-IX.1 and lecrk-IX.2 show compromised Phytophthora

resistance

Previously, LecRK-IX.1 and LecRK-IX.2 were identified as potential resistance

components based on the finding that the Arabidopsis mutants lecrk-IX.1 and

lecrk-IX.2 showed gain of susceptibility to the non-adapted isolates

Phytophthora brassicae HH and Phytophthora capsici LT123. In contrast, the

response to the fungal pathogen Alternaria brassicicola MUCL20297 or the bacterial

pathogen Pseudomonas syringae was not changed in these mutants (Wang et al.,

2014/Chapter III; Supplemental Fig. S1). In Arabidopsis, secondary metabolites such

as salicylic acid (SA), jasmonic acid (JA), ethylene (ET), camalexin and indole

glucosinolates (iGS) have been found to be required for resistance to Phytophthora

pathogens, and expression of marker genes indicative for the related pathways was

found to be induced upon inoculation with P. brassicae or P. capsici (Roetschi et al.,

2001; Schlaeppi et al., 2010; Wang et al., 2013a; Wang et al., 2013b). To determine

whether these pathways are implicated in resistance mediated by LecRK-IX.1 and

LecRK-IX.2, we compared the expression levels of these marker genes in lecrk-IX.1,

lecrk-IX.2 and Col-0 during infection by P. capsici LT123. As shown in Fig. 1A,

induction of all selected marker genes is less pronounced in lecrk-IX.1 and lecrk-IX.2


101

Figure 1. Mutants lecrk-IX.1 and lecrk-IX.2 show defects in defence against Phytophthora

pathogens.

A, Expression of defence-related genes in lecrk-IX.1, lecrk-IX.2 and Col-0 upon inoculation with

P. capsici LT123. Arabidopsis leaves plug-inoculated with P. capsici isolate LT123 were harvested at 12

and 24 hours post inoculation (hpi). Relative transcript levels were normalized to Arabidopsis Actin2.

Values are expressed as mean fold changes (± SD) relative to the transcript level in mock-inoculated

leaves. B, Schematic presentation of LecRK-IX.1 and LecRK-IX.2. Domains were predicted by SMART.

Black arrowheads point to the T-DNA insertion sites and primers used for semi-qRT-PCR are indicated.

TM, transmembrane domain; STK, kinase domain. C, Transcript levels of LecRK-IX.1 and LecRK-IX.2

in Col-0, lecrk-IX.1, lecrk-IX.2 and double mutants (i.e. lecrk-IX.1/2 and lecrkK-IX.2/1) determined by

semi-qRT-PCR. Actin2 was used as control. D, Disease symptoms observed on Col-0, lecrk-IX.1,

lecrk-IX.2 and double mutants four days post inoculation (dpi) with P. capsici LT123. White arrowheads

point to the inoculated leaves.

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when compared to Col-0, in particular for PR1, a SA marker gene and CYP71B15,

which is required for camalexin biosynthesis. Compared to lecrk-IX.1, overall lower

expression levels of PR1 and CYP71B15 were found in lecrk-IX.2. In addition, PDF1.2,

a JA/ET marker gene also showed a lower expression in lecrk-IX.2, whereas the

expression of CYP81F2, a marker gene for iGS was comparable in the two mutants.

To determine whether LecRK-IX.1 and LecRK-IX.2 are functionally redundant, two

homozygous double mutants, termed lecrk-IX.1/2 and lecrk-IX.2/1 were generated

with T-DNA insertions in both LecRK-IX.1 and LecRK-IX.2. Absence of full length

mRNA of both LecRK-IX.1 and LecRK-IX.2 was confirmed in the double mutants

(Fig.1C). Compared to Col-0 and the parental null mutants, the double mutants

showed no visible growth alterations or developmental defects. Upon inoculation with

P. capsici LT123, the double mutants showed increased susceptibility compared to the

single mutants (Fig. 1D). These results indicate that LecRK-IX.1 and LecRK-IX.2

function as independent Phytophthora resistance components.

Overexpression of LecRK-IX.1 or LecRK-IX.2 in Arabidopsis induces cell death

and Phytophthora resistance

To further confirm the role of LecRK-IX.1 and LecRK-IX.2 in Phytophthora resistance,

we generated Arabidopsis transgenic lines expressing LecRK-IX.1 or LecRK-IX.2

under control of the CaMV 35S promoter, named 35S-IX.1 and 35S-IX.2, respectively

(Fig. 2). During the first two weeks of growth, the 35S-IX.1 and 35S-IX.2 lines were

morphologically indistinguishable from the recipient Col-0 plants or Col-0 transformed

with a GFP construct (35S-GFP). Thereafter, part of the 35S-IX.1 and 35S-IX.2 lines

displayed spontaneous necrosis and retarded growth in comparison to Col-0 or

35S-GFP (Fig. 2C). When examined under the microscope, these plants showed

intensive staining with trypan blue indicative of cell death (Fig. 2C). When transgene

expression levels were monitored by qRT-PCR, it appeared that transgenic lines with

a cell death phenotype showed higher transgene expression levels than those without

(Fig. 2B). Apparently, the induced cell death correlates with transgene expression

levels.

To determine whether overexpression of LecRK-IX.1 or LecRK-IX.2 increases

Arabidopsis resistance to Phytophthora pathogens, transgenic lines with a similar

growth phenotype as Col-0 plants, i.e. showing neither macroscopically nor

microscopically visible cell death, were selected for infection assays with P. capsici

LT263. Col-0 and 35S-GFP lines developed severe disease symptoms three days

post inoculation (dpi). In comparison, the 35S-IX.1 and 35S-IX.2 lines showed less

severe disease symptoms and less P. capsici biomass (Fig. 3, A and D; Supplemental

Fig. S2). These lines also showed enhanced resistance to P. brassicae CBS686.95


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Figure 2. Phenotypic changes of Arabidopsis transgenic lines expressing LecRK-IX.1,

LecRK-IX.2 or derivatives lacking the lectin domain or mutated in the kinase domain.

A, Schematic representation of LecRK-IX.1, LecRK-IX.2 and their derivatives used in this study.

B, Correlation of plant cell death phenotype and expression levels of LecRK-IX.1, LecRK-IX.2 or

derivatives in different transgenic lines. Relative transcript levels were normalized to Arabidopsis Actin2

and values are expressed as mean fold changes (± SD) relative to the transcript level in Col-0 plants

which was arbitrarily set as 1. ‘+’ / ‘-’ indicates transgenic lines with or without a cell death phenotype.

C, Growth of transgenic lines constitutively expressing GFP, LecRK-IX.1, LecRK-IX.2 or their

derivatives. Inset figures are microscopic images showing leaf pieces upon trypan blue staining. Scale

bars represent 200 µm.

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(Supplemental Fig. S3), demonstrating that expression of LecRK-IX.1 and LecRK-IX.2

can inhibit infection by different Phytophthora species. To determine whether

overexpression of LecRK-IX.1 and LecRK-IX.2 causes increased plant cell death

during P. capsici infection, inoculated leaves were stained with trypan blue and

investigated by microscopy two days after drop-inoculation with LT263 zoospores.

Preliminary data indicate that overexpression of LecRK-IX.1 or LecRK-IX.2 blocks the

early steps of infection given the fact that only a few penetration events were

observed on 35S-IX.1 and 35S-IX.2 lines. In the mesophyll cell layers of Col-0 and

35S-GFP-1, expanding hyphae with haustoria were observed at 2 dpi. In contrast,

only penetration pegs or short primary infection hyphae were observed in the leaves

of 35S-IX.1-1 and 35S-IX.2-1 (Fig. 3B). Nevertheless, there was no increase in cell

death upon P. capsici penetration in the 35S-IX.1-1 or 35S-IX.2-1 leaves when

compared to Col-0 (Fig. 3B).

Collectively, these results indicate that both LecRK-IX.1 and LecRK-IX.2 function

in plant cell death induction and Phytophthora resistance. The induced cell death,

however, is not a prerequisite for Phytophthora resistance.

Contribution of the lectin domain and kinase activity to LecRK-mediated cell

death and Phytophthora resistance in Arabidopsis

The extracellular domains and a functional kinase domain have been shown to be

essential for the biological function of several RLKs (Gomez-Gomez et al., 2001;

Schwessinger and Ronald, 2012). Hence, we investigated whether both domains are

required for LecRK-IX.1 and LecRK-IX.2 to mediate plant cell death and Phytophthora

resistance. Truncated constructs lacking the lectin domain (i.e. LecRK-IX.1-∆lectin

and LecRK-IX.2-∆lectin) were generated and transformed to Col-0 (Fig. 2A). Similar to

35S-IX.1 and 35S-IX.2 lines, plants expressing either LecRK-IX.1-∆lectin or

LecRK-IX.2-∆lectin (35S-IX.1-∆lectin and 35S-IX.2-∆lectin lines, respectively) varied

in cell death phenotype (Fig. 2C). Transgenic lines with relatively high transgene

expression levels showed cell death (Fig. 2, B and C). To assess the importance of

the kinase domain, kinase-dead mutants were generated by substitution of the

essential RD residues in the catalytic loop to RN or AA (Fig. 2A). In the transgenic

lines, − i.e. 35S-IX.1-RN/AA and 35S-IX.2-RN/AA, respectively − different transgene

expression levels were detected (Fig. 2B), but none of these lines displayed cell death

or any other apparent phenotypic change compared to Col-0 (Fig. 2, B and C). Overall,

these results indicate that the cell death phenotype triggered by overexpression of

LecRK-IX.1 or LecRK-IX.2 is dependent on the kinase activity, but not on the lectin

domain.


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Figure 3. Infection of P. capsici on Col-0 and transgenic lines expressing LecRK-IX.1,

LecRK-IX.2 or derivatives lacking the lectin domain or mutated in the kinase domain.

A, Disease symptoms on Col-0 and transgenic lines 35S-GFP, 35S-IX.1-1 and 35S-IX.2-1 three days

after plug-inoculation with P. capsici LT263. White arrowheads point to the inoculated leaves. B,

Microscopic investigation of P. capsici infection on Col-0 and transgenic lines 35S-GFP, 35S-IX.1-1 and

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106

35S-IX.2-1. Arabidopsis leaves inoculated with P. capsici LT263 zoospores (105 zoospores ml-1) were

harvested at 2 dpi and stained with trypan blue. White arrowheads point to the penetration sites. ha,

haustorium; hy, invasive hyphae. Scale bars represent 20 µm. C, Disease symptoms on Col-0 and

different transgenic lines at 3 dpi. White arrowheads point to the inoculated leaves. D, Relative

quantification of P. capsici biomass in Col-0 and different transgenic lines using qPCR. Bars represent

mean values (± SD) relative to that of infected Col-0 leaves. * indicates significant difference in

biomass (p < 0.05) according to a t test. Experiments were repeated twice with similar results.

Infection assays were performed on transgenic lines that are morphologically

undistinguishable from Col-0. These transgenic lines 35S-IX.1-∆lectin and

35S-IX.2-∆lectin showed similar disease symptoms as Col-0 or 35S-GFP upon

inoculation with P. capsici LT263 (Fig. 3C; Supplemental Fig. S2A). Similar

susceptibility was also observed on the transgenic lines expressing the kinase-dead

mutants (Fig. 3C; Supplemental Fig. S2). In line with these phenotypes, P. capsici

biomass was only reduced in 35S-IX.1 and 35S-IX.2 lines when compared to that in

Col-0, but not in the transgenic lines expressing lectin-deletion or kinase-dead

mutants (Fig. 3D; Supplemental Fig. S2B). Therefore, we conclude that both the lectin

domain and the kinase activity of LecRK-IX.1 and LecRK-IX.2 are essential to

mediate Phytophthora resistance.

Cell death induced by overexpression of LecRK-IX.1 and LecRK-IX.2 is not

correlated with leaf senescence

In silico gene expression analysis using publicly available microarray data indicated

that LecRK-IX.2 but not LecRK-IX.1 is highly induced in senescent Arabidopsis leaves

(Fig. 4A). This was verified by monitoring the transcript levels of LecRK-IX.1 and

LecRK-IX.2 by qRT-PCR in Col-0 leaves undergoing senescence (Fig. 4B).

LecRK-IX.1 showed no significant change in expression but LecRK-IX.2 expression

levels increased in leaves with more severe symptoms of senescence (Fig. 4B). This

expression pattern was comparable to that of SAG12, a gene encoding a cysteine

protease that has been widely used as a reliable senescence marker gene. SAG12 is

only expressed in senescing tissues but not in stress- or hypersensitive

response-related cell death (Noh and Amasino, 1999; Pontier et al., 1999; Zhou et al.,

2009). To determine whether cell death induced by overexpression of LecRK-IX.2 is

correlated with plant senescence, we compared the expression levels of SAG12 in

different LecRK-IX.2 transgenic lines with or without cell death phenotype (Fig. 4C).

No correlation was found between SAG12 expression and LecRK-IX.2 expression

levels or plant phenotypic changes (Fig. 2B and Fig. 4C). This also holds for the

LecRK-IX.1 transgenic lines (Fig. 2B and Fig. 4C). Apparently, the cell death

phenotype induced by overexpression of LecRK-IX.1 or LecRK-IX.2 is not linked to


107

plant senescence.

Figure 4. Cell death induced by overexpression of LecRK-IX.1 or LecRK-IX.2 is not correlated

with leaf senescence.

A, Expression profiles of LecRK-IX.1 and LecRK-IX.2 in different Arabidopsis tissues. Signal intensity

log2 ratios are color-coded in a range of -2 (blue) to 6 (yellow) as indicated in the scale bar. B,

Expression levels of LecRK-IX.1, LecRK-IX.2 and SAG12 in Col-0 leaves in different extent of

senescence quantified by qRT-PCR. Relative transcript levels were normalized to Arabidopsis Actin2

and values are expressed as mean fold changes (± SD) relative to that in Col-0 leaves at the

senescence stage 1 (S1) which was arbitrarily set as 1. C, Expression levels of SAG12 in Col-0 and the

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108

presented transgenic lines. Total RNA was isolated from the whole rosette and used for qRT-PCR.

Relative transcript levels were normalized to Arabidopsis Actin2 and values are expressed as mean

fold changes (± SD) relative to the transcript levels in Col-0 plants which was arbitrarily set as 1.

Transient expression of LecRK-IX.1 or LecRK-IX.2 induces H2O2 production and

cell death in N. benthamiana

To further confirm the role of LecRK-IX.1 and LecRK-IX.2 in Phytophthora resistance

and cell death induction, C-terminal GFP-tagged LecRK-IX.1 and LecRK-IX.2 were

expressed in N. benthamiana using Agrobacterium-mediated transient gene

expression and induced defence responses were analysed.

Accumulation of LecRK-IX.1-eGFP and LecRK-IX.2-eGFP was determined in the

agro-infiltrated N. benthamiana leaves. Total protein was isolated, immunopurified

using GFP-trap_A® beads and detected by western blot with anti-GFP. Both fusion

proteins could be detected, but the sizes were slightly larger than that of the predicted

monomers (Fig. 5A). Since both LecRKs contain putative N-glycosylation sites

(Supplemental Fig. S4), the discrepancy in size could be partially due to glycosylation.

This type of post-translational modification has been shown to be important for some

plant receptors in mediating immune responses (Haweker et al., 2010; Sun et al.,

2012). To test this hypothesis, we investigated whether the two LecRKs are

glycosylated using anti-horseradish peroxidase (HRP). Purified LecRK-IX.1-eGFP

and LecRK-IX.2-eGFP detected with anti-HRP had the similar size as that detected

with anti-GFP (Fig. 5A), indicating that both LecRK-IX.1 and LecRK-IX.2 are

N-glycosylated. Also, the kinase-dead mutants could be detected with both anti-GFP

and anti-HRP and showed similar sizes as the corresponding full length LecRKs (Fig.

5A). It should be noted that in all cases the detected LecRK-IX.2 protein level was less

than LecRK-IX.1. Also, accumulation of LecRK-IX.1 and LecRK-IX.2 protein was

lower when compared to the kinase-dead mutants, indicating that the latter are more

stable or show lower turnover rate.

A hallmark of defence following activation of receptors is the rapid production of

ROS (Boller and Felix, 2009). Hence, we monitored H2O2 production using DAB ((3,

3’-diaminobenzidine)) staining in N. benthamiana upon transient expression of

Arabidopsis LecRK-IX.1 and LecRK-IX.2. Agro-infiltrated leaves expressing GFP did

not show any distinguishable DAB staining whereas the other half of the leaves

expressing LecRK-IX.1 or LecRK-IX.2 clearly showed DAB staining visualized as a


109

Figure 5. H2O2 accumulation and cell death induced by transient expression of LecRK-IX.1,

LecRK-IX.2 and derivatives in N. benthamiana.

A, LecRK-IX.1-eGFP, LecRK-IX.2-eGFP and kinase-dead mutants are expressed and glycosylated in

N. benthamiana. eGPF-tagged LecRKs and kinase-dead mutants were co-expressed with P19 in

N. benthamiana by Agrobacterium-mediated transformation. Total protein was isolated and subjected

to immunopurification (IP) using GFP-trap_A® beads. The immunopurified proteins were detected by

western blot (IB) with anti-GFP or anti-HRP. Coomassie staining shows the 50 KDa Rubisco band

present in the total protein extract to indicate equal loading of protein in each lane. B, H2O2

accumulation in N. benthamiana leaves expressing GFP, LecRK-IX.1-eGFP, LecRK-IX.2-eGFP and

LecRK-S.4-eGFP with (+P19) or without P19 (-P19). Leaves were collected two days after

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agro-infiltration (dpa), stained with DAB solution and destained with ethanol. C, Cell death on

N. benthamiana leaves expressing GFP, LecRK-IX.1-eGFP and LecRK-IX.2-eGFP with (+P19) or

without P19 (-P19) at 3 dpa. D, Quantification of cell death induced by expression of LecRK-IX.1-eGFP

and LecRK-IX.2-eGFP in N. benthamiana by ion leakage measurement. Six leaf discs (Ø 1cm)

collected from infiltration zones were incubated in deionized water for 2 h with 200 rpm shaking.

Supernatants were collected for measuring ion leakage. Bars represent mean values (± SE) of six

replicates from each experiment. * indicates significant difference (p < 0.05) according to a t test. This

experiment was repeated three times with similar results. E, H2O2 accumulation in N. benthamiana

leaves expressing LecRK-IX.1-eGFP, LecRK-IX.2-eGFP and derivatives with P19. Leaves collected at

1.5 dpa were stained with DAB and destained with ethanol. F, Cell death development in

N. benthamiana leaves expressing LecRK-IX.1-eGFP, LecRK-IX.2-eGFP and derivatives with P19 at 3

dpa.

brown precipitate (Fig. 5B). Co-expression with the silencing suppressor P19 resulted

in more intense DAB staining demonstrating significantly enhanced H2O2 production

(Fig. 5B). Irrespective of P19, DAB staining was always stronger in leaves expressing

LecRK-IX.2 than those expressing LecRK-IX.1, indicating that the presence of

LecRK-IX.2 causes a higher H2O2 production (Fig. 5B). In contrast, DAB staining was

not observed in leaves expressing another LecRK, i.e. LecRK-S.4, suggesting that

induction of H2O2 is typical for the two clade IX LecRKs. Also the spontaneous cell

death phenotype that was observed in leaves expressing LecRK-IX.2 was much

stronger than in leaves expressing LecRK-IX.1 (Fig. 5C). The extent of cell death was

quantified by measuring ion leakage and this showed that the conductivity was higher

in N. benthamiana leaves expressing LecRK-IX.2 than in those expressing

LecRK-IX.1 (Fig. 5D). Upon co-expression with P19, LecRK-IX.1 produced a slightly

delayed but eventually similar extent of cell death as LecRK-IX.2 (Fig. 5, C and D).

None of the leaves expressing GFP, with or without P19, showed any visible cell death

(Fig. 5, C and D).

Expression of lectin-deletion mutant of LecRK-IX.1 or LecRK-IX.2 in

N. benthamiana induced a similar extent of H2O2 production and cell death as

expression of the full length proteins (Fig. 5, E and F). In contrast, leaves expressing

kinase-dead mutants showed neither H2O2 production nor cell death. Collectively,

these results show that the kinase activity but not the lectin domain plays a role in

LecRK-IX.1- and LecRK-IX.2-mediated H2O2 production and cell death induction.


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Silencing of NbSIPK/NTF4 abolishes LecRK-IX.1-induced cell death in

N. benthamiana

To identify downstream signaling components required for LecRK-IX.1 and

LecRK-IX.2-induced cell death in N. benthamiana, we analysed the role of several

genes known to be involved in cell death induction (Rusterucci et al., 2001; Yoshioka

et al., 2003; Gabriëls et al., 2007; Takahashi et al., 2007). NbNPR1, NbEDS1,

NbMEK2, NbNRC1, NbRbohA/B and NbSIPK/NTF4 were individually silenced in

N. benthamiana using TRV-mediated virus induced gene silencing (VIGS).

TRV:GUS-treated plants were used as control. Three to four weeks after

TRV-inoculation, LecRK-IX.1-eGFP, LecRK-IX.2-eGFP and GFP were expressed in

leaves using agro-infiltration. In all cases, expression of LecRK-IX.1-eGFP or

LecRK-IX.2-eGFP resulted in cell death on TRV:GUS plants (Fig. 6A). Similarly, cell

death was observed in leaves silenced for NbEDS1, NbNRC1 and NbRbohA/B,

whereas it was attenuated in leaves silenced for NbMEK2 or NbNPR1 (Supplemental

Fig. S5). These results indicate that LecRK-mediated cell death is not dependent on

NbEDS1, NbNRC1 or NbRbohA/B, but partially dependent on NbMEK2 and NbNPR1.

In contrast, cell death induced by LecRK-IX.1-eGFP was completely abolished in

leaves silenced for NbSIPK and its closest homolog NbNTF4 (Fig. 6A). The efficiency

of silencing was checked and shown to be close to 100% (Fig. 6B). Loss of

LecRK-IX.1-induced cell death in NbSIPK/NTF4-silenced leaves was confirmed by

staining with trypan blue (Fig. 6C). In addition, cell death induced by

LecRK-IX.2-eGFP was also remarkably compromised in the NbSIPK/NTF4-silenced

leaves (Fig. 6 A and C). But under the same conditions, cell death elicited by the

Phytophthora infestans elicitor NPP1 was not altered. Compared to TRV:GUS-treated

leaves, leaves silenced for NbSIPK/NTF4 showed a slight delay in cell death

development, but eventually the extent of cell death induced by NPP1 was

comparable (Fig. 6A). In line with this, NbSIPK/NTF4-silenced leaves expressing

LecRK-IX.1-eGFP or LecRK-IX.2-eGFP showed a significant reduction in ion leakage

compared to TRV:GUS-treated leaves; but for leaves expressing NPP1, this was not

the case (Fig. 6D). To determine whether the loss of cell death observed in

NbSIPK/NTF4-silenced plants is due to reduced protein accumulation, GFP and

LecRK-IX.1-eGFP protein levels were determined but no differences were found when

compared to those detected in the TRV:GUS-treated plants (Fig. 6E), indicating that

inhibition of LecRK-IX.1-induced cell death in NbSIPK/NTF4-silenced leaves is not

due to reduced protein accumulation. In conclusion, NbSIPK and NbNTF4 are

indispensable downstream signaling components for LecRK-IX.1- and LecRK-IX.2-

mediated cell death.

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Figure 6. Silencing of NbSIPK/NTF4 suppresses LecRK-IX.1- and LecRK-IX.2-induced cell death

in N. benthamiana leaves.

A, Cell death induced by LecRK-IX.1-eGFP, LecRK-IX.2-eGFP and NPP1 on control (TRV:GUS) and

NbSIPK/NTF4-silenced N. benthamiana leaves. GFP, NPP1, LecRK-IX.1-eGFP and LecRK-IX.2-

eGFP were expressed by Agrobacterium-mediated transient expression. Pictures were taken at 3 dpa.

B, Relative quantification of NbSIPK and NbNTF4 transcript levels in N. benthamiana leaves three

weeks after inoculation with TRV:GUS or TRV:NbSIPK/NTF4 constructs. Transcript levels were

normalized using NbActin and expressed as mean fold changes (± SD) relative to the transcript level in

TRV:GUS-treated leaves which was arbitrarily set as 1. C, Microscopic investigation of cell death

induced by LecRK-IX.1-eGFP and LecRK-IX.2-eGFP in TRV:GUS- and TRV:NbSIPK/NTF4-treated

N. benthamiana leaves. Leaves were collected at 3 dpa and stained with trypan blue. Scale bars

represent 200 µm. D, Quantification of ion leakage in TRV:GUS- or TRV:NbSIPK/NTF4-treated

N. benthamiana leaves expressing GFP, NPP1, LecRK-IX.1-eGFP or LecRK-IX.2-eGFP. Quantified ion

leakage was relative to that in GFP expressing leaves which was arbitrarily set as 1. * indicates


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significant difference (p < 0.05) according to a t test. Similar results were obtained in three independent

experiments. E, Accumulation of GFP and LecRK-IX.1-eGFP in TRV:GUS- and

TRV:NbSIPK/NTF4-treated N. benthamiana leaves. Total protein was immunopurified by GFP-trap_A®

beads and immunodetected with anti-GFP. Coomassie staining indicates the amount of loading in each

lane.

Silencing of NbSIPK/NTF4 compromises LecRK-IX.1-mediated Phytophthora

resistance in N. benthamiana

To determine whether LecRK-IX.1 and LecRK-IX.2 are capable to enhance

Phytophthora resistance in N. benthamiana, we inoculated P. capsici on

N. benthamiana leaves with one half of the leaf expressing GFP and the other half

expressing LecRK-IX.1-eGFP or LecRK-IX.2-eGFP. Disease symptoms were

evaluated at 3 dpi. Leaves expressing LecRK-IX.1-eGFP showed significantly

reduced lesion sizes compared to those expressing GFP (Fig. 7A). Leaves expressing

LecRK-IX.2 showed severe cell death and were not included in the infection assays

(Fig. 5C).

Host cell death often associates with disease resistance, especially against

(hemi)biotrophic pathogens (Mur et al., 2008). Absence of LecRK-IX.1-induced cell

death in NbSIPK/NTF4-silenced plants raised the possibility that also the resistance

function of LecRK-IX.1 might be affected. Hence, we transiently expressed

LecRK-IX.1-eGFP and GFP in TRV:GUS- or TRV:NbSIPK/NTF4-treated plants and

inoculated these plants with P. capsici. Lesion sizes on TRV:GUS-treated leaves

expressing LecRK-IX.1-eGFP were significantly reduced compared to those

expressing GFP (Fig. 7, B and C). Similarly, TRV:NbSIPK/NTF4-treated leaves

expressing LecRK-IX.1-eGFP also showed reduced lesion sizes compared to those

expressing GFP. However, the reduction in lesion size was not as strong as on

TRV:GUS-treated leaves (Fig. 7, B and C), indicating that silencing of NbSIPK/NTF4

did not completely abolish the function of LecRK-IX.1 in Phytophthora disease

resistance. Since cell death was completely abolished in NbSIPK/NTF4-silenced

leaves, it can be concluded that LecRK-IX.1-mediated Phytophthora resistance is not

entirely a consequence of induced cell death. It is likely that additional components or

responses other than cell death play a role in inhibiting Phytophthora colonization.

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Figure 7. Phytophthora resistance conferred by transient expression of LecRK-IX.1 in

N. benthamiana.

A, Disease symptoms and lesion sizes on N. benthamiana leaves expressing LecRK-IX.1 or GFP three

days after plug-inoculation with P. capsici LT263. P. capsici was inoculated on N. benthamiana leaves

24 hours after agro-infiltration. Each experiment included at least 18 infiltrated N. benthamiana leaves.

Bars represent mean lesion sizes (± SE). * indicates significant difference in lesion size (p < 0.05)

according to a t test. This experiment was repeated three times with similar results. B-C, Disease

symptoms and quantified lesion sizes on TRV:GUS- and TRV:NbSIPK/NTF4-treated N. benthamiana

leaves expressing LecRK-IX.1-eGFP or GFP three days after inoculation with P. capsici LT263. The

infection assay included at least 12 infiltrated N. benthamiana leaves per treatment. Bars represent

mean lesion sizes (± SE). * indicates significant difference in lesion size (p < 0.05) according to a t test.

This experiment was repeated three times with similar results.

LecRK-IX.1 and LecRK-IX.2 interact with N. benthamiana NbPDR1 and

Arabidopsis ABCG40 in planta

To identify potential LecRK-interacting proteins, our idea was to co-immunoprecipitate

proteins that form a complex with the eGFP-tagged LecRK-IX.1 and eGFP-tagged

LecRK-IX.2 in the stable transgenic Arabidopsis lines and subsequently analyse these

co-immunoprecipitated proteins by liquid chromatography mass spectrometry

(LC-MS). However, we were unable to purify sufficient amounts of protein for LC-MS

analysis either because transgene expression was too low or because lines with a


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higher transgene expression showed severe cell death (Fig. 2). Since Arabidopsis

LecRK-IX.1 and LecRK-IX.2 retained their function when transiently expressed in

N. benthamiana, we anticipated also that the interaction partners might be conserved

in the two plant species. Hence, we transiently expressed LecRK-IX.1-eGFP,

LecRK-IX.2-eGFP and the control GFP in N. benthamiana and analysed the

(co-)immunoprecipitated proteins with LC-MS after trypsin digestion. The resulting

masses were used as queries to search in the protein database containing sequences

from N. benthamiana and the input LecRKs and GFP. A large number of peptides

matching LecRK-IX.1 and LecRK-IX.2 were detected, demonstrating successful

purification of the input LecRKs (Fig. 8A). Further analysis revealed several potential

candidates that were detected only in the LecRK samples but not in the control GFP

sample. One of these is an ATP-binding cassette (ABC) transporter. Multiple peptides

in both the LecRK-IX.1 and LecRK-IX.2 samples matched uniquely to NbPDR1

(NbS00038999g0004.1), a pleiotropic drug resistance-type ABC transporter (Fig. 8, A

and B; Supplemental Table S2). Apparently, both LecRK-IX.1 and LecRK-IX.2 interact

with NbPDR1 in planta.

We subsequently determined the interaction of both LecRKs with Arabidopsis

ABCG40 (alias PDR12), the closest homolog of NbPDR1 in Arabidopsis.

LecRK-IX.1-eGFP, LecRK-IX.2-eGFP or GFP were co-expressed with ABCG40-Myc

in N. benthamiana and immunoprecipitated using GFP-trap_A® beads. As shown by

western blots probed with Myc antibody, ABCG40-Myc co-immunoprecipitated with

both LecRK-IX.1-eGFP and LecRK-IX.2-eGFP, but not with GFP (Fig. 8C). Note that

two bands were detected, with the lower band closer to the predicted protein size of

ABCG40-Myc. Interaction was further confirmed by swapping the epitope tags.

Consistently, both LecRK-IX.1-Myc and LecRK-IX.2-Myc, but not GUS-Myc

co-immunoprecipitated with ABCG40-eGFP and again two bands were detected for

ABCG40-eGFP (Fig. 8C).

Since both LecRK-IX.1 and LecRK-IX.2 interact with NbPDR1 and ABCG40, we

checked whether LecRK-IX.1 associates with LecRK-IX.2 in planta, but no interaction

was detected (Fig. 8D). Also in preliminary experiments, no interaction was detected

between kinase-dead mutants of LecRK-IX.1 and LecRK-IX2. However, LecRK-IX.1

and LecRK-IX.2 were found to interact with themselves, indicating homodimer

formation. Similarly, the kinase-dead mutants of both LecRKs were also found to form

homodimers (Fig. 8D).

Collectively, these results indicate independent interaction of LecRK-IX.1 and

LecRK-IX.2 with ABCG40 in Arabidopsis and the ability to form homodimers.

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Figure 8. NbPDR1 and ABCG40 interact with LecRK-IX.1 and LecRK-IX.2 in planta.

A, The total number of peptides identified by LC-MS matching LecRK-IX.1, LecRK-IX.2 and the

potential interactor NbPDR1. B, Protein sequence of NbPDR1 with peptides detected by LC-MS


117

highlighted. Peptides detected only in LecRK-IX.1 sample are depicted as yellow, peptides detected in

both LecRK-IX.1 and LecRK-IX.2 samples are depicted as green while peptides that match uniquely to

NbPDR1 are underlined. C, ABCG40 interacts with LecRK-IX.1 and LecRK-IX.2 in planta. ABCG40

was co-expressed with LecRK-IX.1, LecRK-IX.2, GFP or GUS in N. benthamiana. Immunoprecipitation

was performed using anti-GFP Immunoblotting was performed using anti-GFP or anti-Myc. Coomassie

staining indicates equal amount of loading in each lane. D, Dimerization of LecRK-IX.1, LecRK-IX.2

and kinase-dead mutants in N. benthamiana. GPF- and Myc-tagged LecRKs were co-expressed with

P19 in N. benthamiana. Total protein was isolated, immunopurified by GFP-trap_A® beads and

detected with anti-GFP or anti-Myc. Coomassie staining indicates equal amount of loading in each

lane.

Arabidopsis ABCG40 is a potential Phytophthora resistance component

To determine the biological relevance of the interaction between ABCG40 and the two

LecRKs, we monitored the expression of ABCG40 in Col-0 and LecRK mutants during

infection with P. capsici LT123. In Col-0, expression of ABCG40 was increased

significantly from 12 to 24 hpi. In comparison, the expression levels were much lower

in both lecrk-IX.1 and lecrk-IX.2 (Fig. 9A), indicating that ABCG40 functions

downstream of the two LecRKs. To determine the role of ABCG40 in Phytophthora

resistance, two homozygous Arabidopsis mutants, i.e. abcg40-1 and abcg40-2 with

T-DNA insertions in the coding regions (Fig. 9B), were analysed in Phytophthora

infection assays. Absence of full-length ABCG40 mRNA in the mutants was confirmed

by semi-qRT-PCR (Fig. 9C). Upon inoculation with P. brassicae HH and P. capsici

LT123, both lines developed clear disease symptoms at 4 dpi, while Col-0 plants

remained fully resistant (Fig. 9D). This points to a function for ABCG40 in

Phytophthora resistance.

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Figure 9. Arabidopsis ABCG40 is a potential Phytophthora resistance component.

A, ABCG40 expression in Col-0, lecrk-IX.1 and lecrk-IX.2 upon inoculation with P. capsici LT123.

Relative transcript levels were normalized to Arabidopsis Actin2. Values are expressed as mean fold

changes (± SD) relative to the transcript levels in mock-inoculated leaves. B, Schematic representation

of ABCG40. Domains were predicted by SMART. Black arrowheads point to the T-DNA insertion sites in

mutants abcg40-1 and abcg40-2 while ABCG40-P1 indicates the primer pair used for semi-qRT-PCR.

C, Transcript levels of ABCG40 in Col-0, abcg40-1 and abcg40-2 detected by semi-qRT-PCR. Actin2

was used as control. D, Disease symptoms on Col-0, abcg40-1 and abcg40-2 four days after

inoculation with P. brassicae HH and P. capsici LT123. White arrowheads point to the inoculated

leaves.


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DISCUSSION

As one of the largest RLK subfamilies in Arabidopsis, LecRKs are considered to play

diverse roles in plant adaptation. In recent years, detailed studies on three members

of this subfamily, i.e. Arabidopsis LecRK-I.9, LecRK-V.5 and LecRK-IV.2, revealed

their importance in plant immunity (Bouwmeester et al., 2011; Desclos-Theveniau et

al., 2012; Singh et al., 2012). In this study, we focus on two Arabidopsis LecRKs

belonging to clade IX, namely LecRK-IX.1 and LecRK-IX.2 that were previously

identified as potential Phytophthora resistance components based on phenotypic

analyses of T-DNA insertion lines (Wang et al., 2014/Chapter III). We confirmed the

role of LecRK-IX.1 and LecRK-IX.2 in Phytophthora resistance and found that they act

as functional analogs. Knockout of either LecRK-IX.1 or LecRK-IX.2 in Arabidopsis

leads to a gain of susceptibility to Phytophthora pathogens and reduced expression of

defence-related genes upon infection. Overexpression of each of the two in

Arabidopsis increases Phytophthora resistance, but also induces spontaneous plant

cell death, in particular in transgenic lines with a relatively high transgene expression

level. In addition, LecRK-IX.1 retained its function as a Phytophthora resistance

component when transiently expressed in the Solanaceous plant N. benthamiana.

Both LecRKs as well induced cell death when expressed in N. benthamiana,

suggesting conservation of downstream components for LecRK-mediated signaling in

different plant species. Both LecRKs were found to associate with ABC transporters

independent of each other suggesting that they participate in the same signaling

network. In Arabidopsis, the ABC transporter ABCG40 turned out to be a potential

Phytophthora resistance component.

Programmed cell death often occurs in response to pathogen invasion (Coll et al.,

2011; Dickman and de Figueiredo, 2013; Dickman and Fluhr, 2013). The effect of cell

death on plant resistance depends largely on the lifestyle of the invading pathogen. In

general, activation of cell death at early infection stages often limits proliferation of

biotrophic and hemibiotrophic pathogens but promotes infection of necrotrophic

pathogens. However, several reports showed that plant cell death is not always

essential for resistance (Yu et al., 1998; Genger et al., 2008; Takahashi et al., 2012).

The cell death phenotype induced by overexpression of LecRK-IX.1 and LecRK-IX.2

raised the question whether cell death is involved in LecRK-mediated Phytophthora

resistance. Here, we provide three lines of evidence to show that LecRK-mediated

Phytophthora resistance is not due to induction of cell death. First of all, for both

LecRKs, increased Phytophthora resistance was detected in transgenic Arabidopsis

lines that did not show the cell death phenotype. The intensity of cell death was found

to be correlated with transgene expression levels and cell death was only observed in

the transgenic lines with relative high LecRK-IX.1 or LecRK-IX.2 expression levels;

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whereas LecRK-mediated Phytophthora resistance was also found in the lines with

relative low transgene expression. Microscopic analysis showed that the early stage

of Phytophthora infection was compromised on Arabidopsis LecRK overexpression

lines, but this was not correlated with increased cell death. Secondly, the lectin

domain was found to be required for LecRK-mediated Phytophthora resistance but

dispensable for cell death execution. Apparently, the extracellular lectin domain is

required for monitoring pathogen attack by recognizing either effectors/PAMPs of

Phytophthora or DAMPs released upon infection. This recognition could in turn lead to

activation of plant defence mediated by the kinase domain. The spontaneous cell

death phenotype may be a consequence of constitutive activation of pathogen-

independent responses, such as production of ROS that has been shown to play a

prominent role in the execution of plant cell death (van Breusegem and Dat, 2006).

Along with induced cell death, elevated H2O2 production was detected in

N. benthamiana expressing LecRK-IX.1 or LecRK-IX.2, either full length or truncated

versions lacking the lectin domain. This indicates that the H2O2 production induced by

expression of LecRK-IX.1 or LecRK-IX.2 is tightly correlated with cell death induction.

Nevertheless, there is still a possibility that both LecRKs are involved in regulating

plant cell death, similar to SOBIR1, an Arabidopsis RLK which also activates plant cell

death upon overexpression (Gao et al., 2009). The third line of evidence is that

LecRK-mediated cell death and Phytophthora resistance in N. benthamiana require

different downstream components. One of the important events leading to plant cell

death is the sequential activation of MAPK cascades (Meng and Zhang, 2013). By

virus-induced gene silencing in N. benthamiana, we identified NbSIPK/NbNTF4 as

being essential for LecRK-IX.1 and LecRK-IX.2-mediated cell death. Both NbSIPK

and NbNTF4 have been shown to be involved in defence responses downstream of

receptors and capable to induce cell death when expressed in N. benthamiana

(Zhang and Liu, 2001; Ren et al., 2006; Segonzac et al., 2011; Huang et al., 2014).

Silencing of NbSIPK/NbNTF4 completely abolished LecRK-IX.1-mediated cell death,

whereas Phytophthora resistance was decreased, but not completely lost. This

indicates that NbSIPK and NbNTF4 participate in, but not determine

LecRK-IX.1-mediated Phytophthora resistance in N. benthamiana.

For RLKs, the kinase domain was found to be crucial in initiating signal

transduction via phosphorylation of downstream substrates as point mutations often

abolish RLK-mediated functions (Gomez-Gomez et al., 2001; Morillo and Tax, 2006;

Schwessinger et al., 2011; Liebrand et al., 2013). Although it is very likely that the

kinase domain of the various LecRKs has a similar role in signal transduction, there is

as yet no proof that their kinase activity is essential for biological function. LecRK-VI.2

has been shown that is capable to auto-phosphorylate in vitro (Singh et al., 2013), but

whether this kinase activity is essential for its role in priming plant defence is unknown.


121

In this study, we analysed the relevance of the kinase activity of LecRK-IX.1 and

LecRK-IX.2 for mediating resistance and cell death induction. LecRKs belong to

RD-kinases with an aspartate (D) in the catalytic loop preceded by a conserved

arginine (R) (Nolen et al., 2004; Kornev et al., 2006). It has been demonstrated that

mutation of the conserved RD motif into RN or AA blocks kinase activity

(Schwessinger et al., 2011; van Damme et al., 2012). Upon mutation of the RD motif,

both LecRK-mediated Phytophthora resistance and cell death were completely

abolished. These results demonstrated that both LecRKs harbor an active kinase and

that kinase activity is indispensable for their function.

Plant receptors do not work alone but rather function as part of multi-protein

complexes (Liebrand et al., 2014; Macho and Zipfel, 2014). Recently, one of the

Arabidopsis LecRKs, namely LecRK-VI.2, was found to associate with the flagellin

receptor FLS2 and to prime flg22-mediated defence in Arabidopsis and

N. benthamiana (Huang et al., 2014). In our hands, the identification of potential

LecRK-IX.1- and LecRK-IX.2-interacting proteins in Arabidopsis was hampered by the

severe cell death phenotype. Due to the conserved function of the two LecRKs in

N. benthamiana, we exploited N. benthamiana as a convenient alternative. This

resulted in the identification of NbPDR1 and its closest homolog in Arabidopsis, i.e.

ABCG40, as potential LecRK-interacting proteins. NbPDR1 and ABCG40 belong to a

pleiotropic drug resistance transporter subfamily (van den Brule and Smart, 2002;

Rea, 2007). ABCG40 in Arabidopsis was previously reported to be involved in lead

detoxification and ABA transport and has also been implicated in transport of the

diterpenoid sclareol to restrict pathogen growth (Lee et al., 2005; Kang et al., 2010).

For example, inhibition of Ralstonia solanacearum in tobacco by ABCG40 and its

homolog NtPDR1 is associated with sclareol-related compounds (Seo et al., 2012).

Similarly, its close homolog in Nicotiana plumbaginifolia (i.e. NpPDR1) contributes to

transport of sclareol and resistance to Botrytis cinerea, Fusarium oxysporum,

Rhizoctonia solani and Phytophthora nicotianae (Stukkens et al., 2005; Bultreys et al.,

2009). In Arabidopsis, however, ABCG40 does not confer resistance to

Fusarium oxysporum, A. brassicicola and P. syringae, indicating that it is not a

universal resistance component (Campbell et al., 2003). In this study, the

compromised resistance of the two independent Arabidopsis ABCG40 mutants to

Phytophthora pathogens indicates that, similar to LecRK-IX.1 and LecRK-IX.2,

ABCG40 functions as a Phytophthora resistance component. It is not surprising that

both LecRK-IX.1 and LecRK-IX.2 interact with NbPDR1 and ABCG40 in planta since

they were also found to activate similar downstream defence responses. It is unlikely

though that LecRK-IX.1, LecRK-IX.2 and ABCG40 form a ternary complex because

LecRK-IX.1 and LecRK-IX.2 do not form a heterodimer. The fact that both clade IX

LecRKs interact with the same ABC transporter but yet function independently raises

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the possibility that their interaction with the ABC transporter is temporally and spatially

different. Since ABC transporters contribute to transport of a wide variety of

compounds, including ions, nucleic acids and anti-microbial compounds (Rea, 2007),

binding between ABCG40 and the clade IX LecRKs might cause rearrangement of

plant molecules and the subsequent activation of signal transduction culminating in

effective defence.


Plant material and growth conditions

Arabidopsis T-DNA insertion lines SALK_042414 (henceforth named as lecrk-IX.1),

SALK_111817 (lecrk-IX.2), SALK_148565.27.50 (abcg40-1) and SALK_005635.51.50

(abcg40-2) were obtained from the European Arabidopsis stock center NASC

(http://arabidopsis.info) and homozygosity of the T-DNA insertions was determined as

previously described (Wang et al., 2014/Chapter III). Arabidopsis Col-0 and derived

mutants or transgenic lines were grown on soil and maintained in a conditioned

growth chamber at 19-21°C with a 12 h photoperiod and a 75-80% relative humidity.

N. benthamiana plants were grown in soil in a greenhouse at 19-21°C with a

75-78% relative humidity and a 16/8 h photoperiod. Supplementary light (100 W m2-)

was applied when the light intensity dropped below 150 W m2-.

Pathogen maintenance and infection assays

Culturing of Phytophthora pathogens, Pst DC3000 and A. brassicicola, infection

assays on Arabidopsis with these pathogens and biomass measurements were

performed as previously described (Wang et al., 2013/Chapter IIa; Wang et al.,

2014/Chapter III).

Infection assays on N. benthamiana with P. capsici were performed by inoculating

fresh mycelial plugs (Ø 0.5 cm) on leaves of six-week-old plants one day after

agro-infiltration. Inoculated plants were kept in a climate chamber with

aforementioned conditions. Disease symptoms were evaluated by measuring lesion

sizes at 3 dpi as described by Vleeshouwers et al. (1999).

Gene expression analysis

Arabidopsis RNA was isolated from eight circular leaf discs (Ø 0.8 cm), each with the

inoculation spots in the center from four inoculated Arabidopsis plants or from rosette

leaves collected from transgenic plants. For N. benthamiana, six leaves from three

individual plants were pooled together and ground in liquid nitrogen. Total RNA

isolation and qRT-PCR analysis were carried out following description in Chapter III.


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First strand cDNA was synthesized using 1.5 µg RNA in a total volume of 25 µl and

the resultant cDNA was diluted 20-fold. Semi-quantative RT-PCR was performed

using 1 µl of diluted cDNA with gene-specific primers (Supplemental Table S1) in 45

cycles at 94°C for 30s, 55°C for 30s, and 72°C for 1 min. Actin2 was used as control

and amplified using the same PCR conditions in 30 cycles.

LecRK expression data were obtained from publicly available microarray datasets

using the eFP-Browser at the Bio-Array Resource website (http://www.bar.utoronto.ca;

Winter et al., 2007). Signal intensity log2 ratios of the samples relative to the mean

were used as a measure for LecRK expression. Of the datasets sampled from

Arabidopsis seeds, roots and flowers, the highest value throughout all the arrays was

retrieved.

Plasmid construction

The coding sequences of the full-length or truncated Arabidopsis LecRK-IX.1,

LecRK-IX.2, LecRK-S.4 and ABCG40 were amplified by PCR using Pfu DNA

polymerase (Promega) with primers listed in Supplemental Table S1. Point mutations

in the kinase domain were generated using overlap extension PCR with primers

shown in Supplemental Table S1. The purified PCR fragments were ligated into

pENTR/D-TOPO® vector (Invitrogen) for sequencing. Thereafter, the correct

sequences were introduced into binary vectors pSol2095 or pGWB20 using Gateway®

LR Clonase II® (Invitrogen). The resulting vectors were transformed into

Agrobacterium tumefaciens strain GV3101.

The fragment used for gene silencing of NbRbohA/B (Yoshioka et al., 2003) was

synthesized (Eurofins MWG Operon) while the fragment used for NbSIPK/NTF4

silencing was amplified using primers listed in Supplemental Table S1. Synthesized or

PCR-amplified fragments were cloned into the vector pTRV-RNA2 and transformed

into A. tumefaciens GV3101.

Generation of Arabidopsis transgenic lines and double mutants

Arabidopsis plants were transformed with A. tumefaciens GV3101 carrying the

aforementioned constructs using the floral dip method (Zhang et al., 2006).

Transformed seeds were selected on 0.5 MS (Murashige and Skoog, Duchefa) plates

containing 50 µg ml-1 kanamycin or 20 µg ml-1 hygromycin B. Transgene expression in

the different transgenic lines was detected by qRT-PCR using primers described in

Supplemental Table S1.

Double mutants were generated by reciprocally crossing mutant lecrk-IX.1 with

lecrk-IX.2. Homozygosity of the T-DNA insertion in both LecRK-IX.1 and LecRK-IX.2

in F2 progeny was confirmed by PCR-based genotyping. Expression levels of

LecRK-IX.1 and LecRK-IX.2 in the double mutants were analysed by semi-qRT-PCR

Chapter IV

124

with Actin2 as control.

A. tumefaciens-mediated transient expression in N. benthamiana leaves

A. tumefaciens strains carrying binary vectors were grown overnight at 28°C in Yeast

Extract Broth with appropriate antibiotics. A. tumefaciens cells were collected by

centrifugation, resuspended and incubated in MMA induction medium (10 mM MES,

10 mM MgCl2, 50 μM acetosyringone, pH 5.6) for 3 h. Thereafter, A. tumefaciens cells

were collected, resuspended in an infiltration medium (10 mM MES, 10 mM MgCl2,

150 μM acetosyringone, pH 5.6) and incubated for another 1 h.

For gene silencing, A. tumefaciens suspensions carrying pTRV-RNA2 constructs

and A. tumefaciens carrying pTRV1 were mixed at a ratio of 1:1 to a final OD600 of

1.0 before infiltration into cotyledons of two-week-old N. benthamiana plants. For

transient gene expression, A. tumefaciens suspensions with a final OD600 of 0.6 were

syringe-infiltrated into five-week-old N. benthamiana leaves or co-infiltrated with the

silencing suppressor P19 at a ratio of 1:1 to a final OD600 of 0.6.

Protein extraction, immunoprecipitation and western blotting

Agro-infiltrated N. benthamiana leaves were collected and gound in liquid nitrogen.

Total protein was extracted by incubating ground leaf samples for 30 min in an

extraction buffer containing 150 mM NaCl, 50 mM Tris-HCl pH 8.0, 1.0% IGEPAL®

CA-630 (Sigma) and one protease inhibitor cocktail tablet (Roche) per 50 ml. The

homogenate was centrifuged at 18,000 rpm for 20 min and the supernatant was

subsequently incubated with GFP-trap_A® beads (Chromotek) at 4°C for 1-2 h. After

incubation, GFP-beads were pelleted and washed with extraction buffer for 6 times.

Proteins were eluted from GFP-beads by boiling for 5 min, separated on a 8%

SDS-PAGE gel and electroblotted onto a PVDF membrane (Bio-Rad). Accumulation

of GFP-tagged protein was detected by immunoblotting with anti-GFP-HRP (Miltenyi

Biotec). Detection of Myc-tagged protein was performed by incubating the blots in

1:2,000 diluted anti-c-Myc (Santa Cruz Biotechnology) and subsequently 1:2,000

diluted anti-Mouse Ig-HRP (Amersham). Protein N-glycosylation was detected using

anti-HRP as previously described (Liebrand et al., 2012). Supersignal West Femto

Chemiluminescent Substrate (Thermo Scientific) was used for signal development.

Equal loading was demonstrated by staining the membrane with coomassie brilliant

blue.

Tryptic digestion of immunopurified proteins and mass spectrometry analysis

For mass spectrometry analysis of LecRK-interacting proteins, 5 g agro-infiltrated

N. benthamiana leaves were used for protein isolation and the resultant protein

extract was incubated with 60 μl GFP-trap_A® beads (Chromotek) at 4°C for 2 h. The


125

purified proteins on GFP-trap_A® beads were subjected to on-bead digestion followed

by LC-MS using both Data-Independent Acquisition (DIA/MSE) and Data-Dependent

Acquisition (DDA) as previously described (Liebrand et al., 2012).

Measurement of electrolyte leakage

Six N. benthamiana leaf discs (Ø 1.0 cm) collected three days after agro-infiltration

were floated on 4 ml of deionized water for 2 h under continuous shaking (200 rpm) at

room temperature. Conductivity was measured using a Mettler Toledo InLab®741 ISM

conductivity meter (Mettler Toledo, Tiel). Three biological repeats were performed

each with six replicates.

Trypan blue and DAB staining

Trypan blue staining was performed as previously described (Wang et al., 2011). For

DAB staining, agro-infiltrated N. benthamiana leaves were immersed into DAB

solution (1 mg ml-1 DAB-HCl, pH 3.7) for 8 h. Leaves were subsequently washed with

dH2O to remove the staining solution on the surface and destained with 96% ethanol.

Experiments were repeated three times each with at least six leaves from three

independent plants.

ACKNOWLEDGEMENTS

We thank Patrick Smit and Daniela Sueldo for providing the pSol2095 vector and the

GFP-HA construct, Henk Smid and Bert Essenstam from Unifarm for taking care of

the plants and Hagos M. Juhar for assistance. This project was financially supported

by a Wageningen University sandwich-PhD fellowship (YW), a Huygens scholarship

(YW), a VENI grant (KB) from the Netherlands Organization for Scientific Research

(Technology Foundation NWO-STW), CBSG2012/NPC2 proteomics hotel and the

Food-for-Thought campaign of the Wageningen University Fund.

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SUPPLEMENTAL INFORMATION Table S1. Primers used in this study.

Primer name 5'-3' Used to / for

IX.1-P1-F CACCATGGCCAACTCAATTCTG Detect T-DNA insertion in lecrk-IX.1

IX.1-P1-R TAACCAAATGTTCCTGCTAACC Detect T-DNA insertion in lecrk-IX.1

IX.1-P2-F CAAGGCGAGTAATGTGATGCT qRT-PCR

IX.1-P2-R TAACCAAATGTTCCTGCTAACC qRT-PCR

LecRK-IX.1-FL-F CACCATGGCCAACTCAATTCTG Clone Arabidopsis LecRK-IX.1

LecRK-IX.1-R ACGACCATGTTGAGCACTTG Clone Arabidopsis LecRK-IX.1

LecRK-IX.1(RD/RN)-R CGCCTTGATGTTTCTGTGTACAAC Generate Arabidopsis LecRK-IX.1 RD/RN mutant

LecRK-IX.1(RD/RN)-F GTACACAGAAACATCAAGGCG Generate Arabidopsis LecRK-IX.1 RD/RN mutant

LecRK-IX.1(RD/AA)-R CCTTGATTGCTGCGTGTACAAC Generate Arabidopsis LecRK-IX.1 RD/AA mutant

LecRK-IX.1(RD/AA)-F TGTACACGCAGCAATCAAGGC Generate Arabidopsis LecRK-IX.1 RD/AA mutant

LecRK-IX.1-SP(+TM)-R TCCAGGCTTGAGACAAAAG Generate LecRK-IX.1-∆lectin

LecRK-IX.1-(SP+)TM-F CTTTTGTCTCAAGCCTGGAGC Generate LecRK-IX.1-∆lectin

IX.2-P1-F TTGGAATGCAAGCTCTCACAG Detect T-DNA insertion in lecrk-IX.2

IX.2-P1-R GGCAGCTATAAACCCGAACAT Detect T-DNA insertion in lecrk-IX.2; qRT-PCR

IX.2-P2-F GGCTCGGCTAATGAACCATG qRT-PCR

IX.2-P2-R CGTTCCAGCGATTTTCTCCC qRT-PCR

LecRK-IX.2-FL-F CACCATGCTTTATTTCATTTTCTG Clone Arabidopsis LecRK-IX.2

LecRK-IX.2-R ACGACCATACTCGATGCCTGAG Clone Arabidopsis LecRK-IX.2

LecRK-IX.2(RD/RN)-R GCCTTGATGTTTCTGTGCAGT Generate Arabidopsis LecRK-IX.2 RD/RN mutant

LecRK-IX.2(RD/RN)-F ACTGCACAGAAACATCAAGGC Generate Arabidopsis LecRK-IX.2 RD/RN mutant

LecRK-IX.2(RD/AA)-R CTTGATTGCTGCGTGCAGTAC Generate Arabidopsis LecRK-IX.2 RD/AA mutant

LecRK-IX.2(RD/AA)-F ACTGCACGCAGCAATCAAGGC Generate Arabidopsis LecRK-IX.2 RD/AA mutant

LecRK-IX.2-SP(+TM)-R ACTTGAAACAACAAAAGGGAGG Generate LecRK-IX.2-∆lectin

LecRK-IX.2-(SP+)TM-F TTGTTGTTTCAAGTTTGGACAGCG Generate LecRK-IX.2-∆lectin

PDR12-FL-F CACCATGGAGGGAACTAGTTTTC Clone Arabidopsis ABCG40

PDR12-R TCGTTTTTGGAAATTGAAACTC Clone Arabidopsis ABCG40; semi-qRT-PCR

PDR12-RT-F GAAGCGGCTTTAGGAGTCGATTTCGC Arabidopsis ABCG40 qRT-PCR; semi-qRT-PCR

PDR12-RT-R CGTCCACTCGAATCCTATCATAGCG Arabidopsis ABCG40 qRT-PCR

Actin 2-RT-F TAACTCTCCCGCTATGTATGTCGC Arabidopsis Actin2 qRT-PCR; semi-qRT-PCR

Actin 2-RT-R GAGAGAAACCCTCGTAGATTGGC Arabidopsis Actin2 qRT-PCR

Actin 2-SemiRT-R GAGGGCTGGAACAAGACTTCTG Arabidopsis Actin2 semi-qRT-PCR

AtRub-F4 GCAAGTGTTGGGTTCAAAGCTGGTG Arabidopsis Rubisco qRT-PCR

AtRub-R4 CCAGGTTGAGGAGTTACTCGGAATGCTG Arabidopsis Rubisco qRT-PCR

Nbactin-RT-F TATGGAAACATTGTGCTCAGTGG N. benthamiana Actin qRT-PCR

Nbactin-RT-R CCAGATTCGTCATACTCTGCC N. benthamiana Actin qRT-PCR

NbSIPK1-VIGS-F GAATTCCAAGATTGATGCCAAGAGG Clone fragment for NbSIPK/NTF4 silencing


131

NbSIPK1-VIGS-R GAGCTCGCAATTATTCACATGTGCTGGT Clone fragment for NbSIPK/NTF4 silencing

NbSIPK1/NTF4-RT-F GGTGGCAGGTTCATTCAATACAAT NbSIPK/NTF4 qRT-PCR

NbSIPK1/NTF4-RT-R CTTCTCTCTGTGGTGGTGGTAT NbSIPK/NTF4 qRT-PCR

NbNTF4-RT-F CACAGCAACCAGCTCCTCCTC NbNTF4 qRT-PCR

NbNTF4-RT-R TGCCATAAGCTCCTTTACCG NbNTF4 qRT-PCR

NbSIPK1-RT-F GGAGCAGCCACCTCCGGCGG NbSIPK qRT-PCR

NbSIPK1-RT-R GTAAGCACCTCTACCAATAG NbSIPK qRT-PCR

CAP-Fw TTTAGTTGGGGGTCTTGTACC Detect P. capsici biomass

CAP-Rv1 CCTCCACAACCAGCAACA Detect P. capsici ITS amplification

Oligo-dT GACTCGAGTCGACATCGATTTTTTTTTTTTTTT cDNA synthesis

Chapter IV

132

Table S2. Peptides matching NbPDR1 that were identified using LC-MS.

Input Output

Annotation DDA MSE

peptidea No. peptidea No.

LecR

K-I

X.1

-GF

P

NbPDR1

TVVCTIHQPSIDIFEAFDELFLMK 1 AANIKPDADIDMFMK 1

TTLMDVLAGR 1 ALFMDEISTGLDSSTTYSIVNSLK 1

TTLLLALAGK 1 EIYVGPLGR 1

MMFVEEVMDLVELTPLR 1 EMTVR 1

AANIKPDADIDMFMK 1 FEHLNIDADAYVGSR 1

FIFTTFIALIFGTMFWDIGTK 1

GAADFLQEVTSK 1

GGQEIYVGPLGR 1

IGEMTVR 1

LLLALAGK 1

LMGVSGAGK 1

MPRDTAEDGGIYSGALFFVVIMIMFNGLSELPMTLYK 1

NGLSTEQR 1

SALVGLPGVNGLSTEQR 1

SGAFRPGVLTALMGVSGAGK 1

SVSGAFRPGVLTALMGVSGAGK 1

TATYISQHDLHIGEMTVR 2

TTLLLALAGK 1

TVVCTIHQPSIDIFEAFDELFLMK 1

VCTERELLLMQR 2

LecR

K-I

X.2

-GF

P

NbPDR1

TTLLLALAGK 1

TTLMDVLAGR 1

a Peptides that are not unique to NbPDR1 are in grey.


133

Figure S1. Disease symptoms on Arabidopsis LecRK mutants upon infection by various

pathogens.

A, Disease symptoms on Col-0, lecrk-IX.1 and lecrk-IX.2 upon inoculation with Phytophthora brassicae

HH and Phytophthora capsici LT123 four days post inoculation (dpi), Alternaria brassicicola

MUCL20297 at 5 dpi and Pseudomonas syringae (Pst) DC3000 at 4 dpi. White arrowheads point to

inoculated leaves. B, Bacterial growth in Col-0, lecrk-IX.1 and lecrk-IX.2 at 3 dpi. Bars depict mean

bacterial titers of four plants (± SE). Experiments were repeated three times with similar results.

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134

Figure S2. Infection assays with P. capsici LT263 on transgenic lines expressing LecRK-IX.1,

LecRK-IX.2 or derivatives.

A, Disease symptoms on Col-0 and different transgenic lines at 3 dpi. White arrowheads point to

inoculated leaves. B, Relative quantification of P. capsici biomass in Col-0 and different transgenic lines

using qPCR. Bars represent mean values (± SD) relative to that detected in infected Col-0 leaves. *

indicates significant difference in biomass (p < 0.05) compared to Col-0 according to a t test.

Experiments were repeated twice with similar results.


135

Figure S3. Disease symptoms on Col-0 and transgenic lines expressing GFP, LecRK-IX.1,

LecRK-IX.2 or derivatives upon inoculation with P. brassicae CBS686.95 at 3 dpi.

Chapter IV

136

Figure S4. Alignment of protein sequences of LecRK-IX.1 and LecRK-IX.2 using ClustalW.

Identical amino acids are shaded in black. The N-terminal lectin domain is depicted by a blue bar, the

predicated transmembrane domain by a yellow bar and the kinase domain by a green bar. The RD

motif in the catalytic loop is marked with asterisks while predicted N-glycosylation sites according to

NetNGlyc 1.0 Server are highlighted in red.


137

Figure S5. Cell death induced by LecRK-IX.1, LecRK-IX.2 and NPP1 on TRV-treated

N. benthamiana leaves.

N. benthamiana leaves treated with TRV:GUS TRV:NbEDS1, TRV:NbMEK2, TRV:NbNPR1,

TRV:NbNRC1, TRV:NbRbohA/B and TRV:NbSIPK/NTF4 were agro-infiltrated to express GFP, NPP1,

LecRK-IX.1 or LecRK-IX.2. Pictures were taken three days after agro-infiltration.

Chapter V

Ectopic expression of Arabidopsis L-type lectin

receptor kinase genes LecRK-I.9 and LecRK-IX.1

confers Phytophthora resistance in

Nicotiana benthamiana

Yan Wang, Klaas Bouwmeester, David L. Nsibo, Hagos M. Juhar, Francine Govers

Arabidopsis LecRK-I.9 and LecRK-IX.1 confer Phytophthora resistance in N. benthamiana

140

ABSTRACT

L-type lectin receptor kinases LecRK-I.9 and LecRK-IX.1 were previously found to

function as Phytophthora resistance components in Arabidopsis. In this study, we

checked the role of these two LecRKs in Phytophthora resistance when transferred

into the Solanaceous plant Nicotiana benthamiana. Multiple transgenic lines

expressing AtLecRK-I.9 or AtLecRK-IX.1 were generated. Molecular analyses

revealed that the obtained transgenic lines vary in transgene copy number, transgene

expression level and protein accumulation. Infection assays showed that transgenic

N. benthamiana plants expressing either AtLecRK-I.9 or AtLecRK-IX.1 are more

resistant to Phytophthora capsici and Phytophthora infestans. These results

demonstrate that Arabidopsis LecRK-I.9 and LecRK-IX.1 retained their Phytophthora

resistance function after transfer into N. benthamiana.

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INTRODUCTION

Plant diseases caused by Phytophthora species are widespread and cause enormous

economic losses on a large variety of crops (Tyler, 2007; Fry, 2008; Lamour et al.,

2012). Under favorable conditions, Phytophthora pathogens reproduce rapidly and

become epidemic within a short period of time. Phytophthora disease control is costly

and often depends on excessive application of preventive fungicides. Hence,

development of plant cultivars with durable resistance against different Phytophthora

species is under high demand. Currently, breeding programs are mainly focused on

the exploitation of cytoplasmic resistance (R) genes that encode intracellular

nucleotide-binding leucine-rich repeat (NLR) proteins to restrict Phytophthora

pathogens (Vleeshouwers et al., 2011). However, these attempts are often hampered

by the quick adaptation of Phytophthora pathogens that leads to evasion of the

R-gene mediated recognition (Fry, 2008; Vleeshouwers et al., 2011; Rodewald and

Trognitz, 2013).

Plants also respond to pathogens by activation of plasma membrane-localized

receptors (Zipfel, 2014). Recently, L-type lectin receptor kinases (LecRKs) have been

found to play a role in plant resistance to different Phytophthora pathogens. One of

the Arabidopsis LecRKs, i.e. LecRK-I.9, was identified as a potential host target of the

Phytophthora infestans RXLR effector IPI-O and function as a Phytophthora

resistance component (Bouwmeester et al., 2011). LecRK-I.9 belongs to a family of 45

members in Arabidopsis (Bouwmeester and Govers, 2009). To unravel the function of

other LecRKs, we previously carried out infection assays on Arabidopsis T-DNA

insertion mutants with different Phytophthora pathogens (Wang et al., 2014/Chapter

III). Mutants of 14 LecRKs showed compromised resistance, indicating that these

LecRKs are also implicated in Phytophthtora resistance. One of these LecRKs,

namely LecRK-IX.1, was analysed in more detail and was confirmed to be a

Phytophthora resistance component in Arabidopsis (Chapter IV).

Engineering plants via interfamily transfer of resistance components has the

potential to improve disease resistance in crop plants. A successful example is the

transfer of Arabidopsis EFR into Solanaceous plants (Lacombe et al., 2010). EFR is a

receptor of bacterial elongation factor EF-TU and is restricted to the Brassicaceae

family (Kunze et al., 2004; Zipfel et al., 2006). Nicotiana benthamiana and tomato

transgenic plants expressing EFR gained the capacity to respond to EF-Tu and

showed enhanced resistance to various bacterial pathogens (Lacombe et al., 2010).

In a similar way, Arabidopsis LecRK-I.9 was shown to confer Phytophthora resistance

in potato (Bouwmeester et al., 2014). Consistently, transient expression of LecRK-I.9

in N. benthamiana also increased resistance to P. infestans. Likewise, Arabidopsis

LecRK-VI.2 maintained its function in bacterial resistance when expressed in


142

N. benthamiana (Huang et al., 2014).

The objective of this work was to generate stable transgenic N. benthamiana lines

expressing Arabidopsis LecRK-I.9 or LecRK-IX.1 and to analyse their response to

Phytophthora infection. To this end, transgenic N. benthamiana plants were generated

using Agrobacterium-mediated transformation. The obtained transgenic lines were

subjected to molecular analyses to determine transgene copy number, transgene

expression level and protein accumulation. Thereafter, we monitored the phenotypic

changes of these transgenic N. benthamiana lines in growth and response to different

Phytophthora pathogens.

Figure 1. Flowchart of the generation, selection and characterization of transgenic

N. benthamiana lines harboring Arabidopsis LecRK-I.9 or LecRK-IX.1.


Generation of transgenic N. benthamiana ectopically expressing Arabidopsis

LecRK-I.9 or LecRK-IX.1

Agrobacterium-mediated transformation of N. benthamiana with the binary vectors

pBIN-KS-35S::AtLecRK-I.9-eGFP, pBIN-KS-35S::AtLecRK-IX.1-eGFP and pBIN61-

35S::GFP (Fig. 2A) resulted in 17, 12 and 4 putative T0 kanamycin-resistant lines,

respectively. These were named as I.9-OE-1 to 17, IX.1-OE-1 to 12 and EV-1 to 4

(Table 1). The flowchart in Fig. 1 gives an overview of the various steps in the process

of selection and analysis of the T0 and T1 transgenic lines. For the molecular

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143

characterization, we first determined whether AtLecRK-I.9 and AtLecRK-IX.1 were

successfully transferred into N. benthamiana by PCR using gene specific primers (Fig.

2A). Of the selected kanamycin-resistant plants, the four empty vector transgenic

lines (EV-1 to EV-4) and one LecRK-I.9 line, i.e. I.9-OE-7, gave no PCR product (Fig.

2B).

To determine transgene copy number, we performed qPCR analysis. For both

AtLecRK-I.9 and AtLecRK-IX.1, the copy number in transgenic lines ranges from 0 to

5 and this number was not always consistent with that of the calculated NPTII copy

number (Table 1). In the transgenic lines I.9-OE-7, I.9-OE-10, I.9-OE-11, IX.1-OE-2,

IX.1-OE-3, IX.1-OE-5 and IX.1-OE-6, more copies were detected for NPTII than for

LecRKs, indicating the presence of truncated T-DNA fragments. This likely explains

why the T0 line I.9-OE-7 lacks AtLecRK-I.9 but is still kanamycin-resistant.

Expression of AtLecRK-I.9 and AtLecRK-IX.1 in the T0 plants was determined by

quantifying mRNA levels using qRT-PCR. For both AtLecRK-I.9 and AtLecRK-IX.1,

the expression level varies among individual transgenic lines (Fig. 2C). Variations in

transgene expression level in stable transgenic lines have often been attributed to the

site(s) of transgene insertion and transgene copy number (Kole et al., 2010). In T0

plants, however, no correlation was found between copy number and transgene

expression level. Transgene expression level varies among individual transgenic lines

with same transgene copy number. Some of the transgenic lines with a single copy of

the transgene showed even higher expression levels than those with two or more

copies. For example, line I.9-OE-16 contains a single AtLecRK-I.9 copy but showed

the highest transgene expression level of all I.9-OE lines. Even transgenic lines with

the same transgene number, e.g. line IX.1-OE-3 and IX.1-OE-11, showed quite

different expression levels (Fig. 2C). In the two EV lines, different amounts of GFP

with the expected size around 28 kDa were detected using GFP antibody. To

determine whether the transgenic lines produce the LecRK proteins, a subset of

transgenic lines was selected for protein isolation. Among the seven tested I.9-OE

lines, variable amount of LecRK-I.9-eGFP was detected (Fig. 2D). Comparison with

the expression levels suggests that the accumulation of the LecRK-I.9 is correlated

with the transgene expression level in these lines. LecRK-IX.1-eGFP was only

detected in line IX.1-OE-10, the one with the highest transgene expression levels of

the three selected IX.1-OE lines (Fig. 2D).


144

Figure 2. Molecular characterization of transgenic N. benthamiana lines.

A, Schematic representation of T-DNA regions of the vectors used for N. benthamiana transformation.

PCR amplified fragments and position of the primers are indicated by the head-to-head arrow pairs.

The fragments F-I.9-1, F-I.9-2, F-IX.1-1, F-IX.1-2 and NPTII-RT were amplified to determine transgene

presence in transgenic lines, while fragments I.9-RT and IX.1-RT were amplified to monitor transgene

mRNA levels. B, Presence of AtLecRK-I.9 or AtLecRK-IX.1 in transgenic N. benthamiana lines.

Genomic DNA from each line was used as template for amplification with primers indicated in (A). C,

Relative quantification of transgene expression levels in transgenic N. benthamiana lines. Transcript

levels were normalized to NbActin and values are expressed as mean fold changes (± SD) relative to

Chapter V

145

the transcript level of AtLecRK-I.9 in I.9-OE-4 or the transcript level of AtLecRK-IX.1 in IX.1-OE-8 which

was arbitrarily set as 1. D, GFP, LecRK-I.9-eGFP and LecRK-IX.1-eGFP accumulation in transgenic

N. benthamiana lines. Proteins were immunodetected using anti-GFP-HRP. Coomassie staining was

used to indicate the amount of loading in each lane.

Table 1. Transgenic N. benthamiana lines vary in transgene copy number and growth.

Line No. Transgene copy number Morphology (transformants vs wild type)a Germination rateb (%)

AtLecRK-I.9/IX.1 NPTII T0/T1 plants T1 progeny

EV

-

1 0 1 − 100

2 0 2 − 100

3 0 5 − 100

4 0 2 − 100

I.9-O

E-

1 1 1 Slightly smaller 32

2 2 2 Smaller plants with curly leaves 60

3 3 3 Smaller plants with compact rosettes 40

4 3 3 − 60

5 1 1 − 100

6 4 4 − 55

7 0 1 − /a

8 1 1 − 60

9 1 1 Smaller 85

10 5 6 Smaller 95

11 4 6 Smaller 65

12 1 1 Smaller plants with compact rosettes, thick leaves 55

13 2 2 − 50

14 2 2 − 70

15 1 1 Smaller 60

16 1 1 Smaller plants with compact rosettes 35

17 3 3 Slightly smaller and curly leaves 85

IX.1

-OE

-

1 2 2 − 100

2 1 2 Smaller plants with curly round leaves 100

3 1 2 Smaller 100

4 2 2 − 100

5 2 3 − 100

6 1 2 − 100

7 1 1 Smaller 100

8 2 2 − 100

9 1 1 − 100

10 1 1 Smaller plants with old leaves showing necrosis 100

11 1 1 Smaller plants with old leaves showing necrosis 100

12 1 1 Smaller plants with old leaves showing necrosis /a a ‘─’ means no difference compared with the wild-type N. benthamiana, ‘/’ not tested. b Percentage of germinated seeds (n=18-24) three days after grown on MS. This experiment was only

performed once.


146

Morphology and growth alterations of I.9-OE and IX.1-OE lines

In a previous study, it was reported that overexpression of AtLecRK-I.9 in Arabidopsis

led to more compact rosettes with smaller and slightly wrinkled leaves (Bouwmeester

et al., 2011). In line with this, ectopic expression of AtLecRK-I.9 in potato also led to

developmental defects, such as aberrant leaf morphology (Bouwmeester et al., 2014).

In this study, the T0 transgenic N. benthamiana plants were monitored for growth and

developmental alterations starting from one-week after transfer into soil until seed set

(Table 1; Fig. 1). All the four EV lines showed normal development; i.e. leaf

morphology, branching and plant height were similar to untransformed

N. benthamiana. In contrast, several I.9-OE lines were smaller in size and displayed

more compact rosettes or curly leaves. These phenotypic changes, however, were not

correlated with AtLecRK-I.9 expression levels, but this could be due to a combined

effect of varying transgene copy numbers, transgene insertion sites and transgene

expression levels in the different transgenic lines. Three out of the twelve IX.1-OE

lines, namely IX.1-OE-10, IX.1-OE-11 and IX.1-OE-12, showed spontaneous cell

death in leaves of over five-week-old plants (Fig. 3A). Considering that these three

IX.1-OE lines showed much higher AtLecRK-IX.1 expression levels than the rest, the

cell death phenotype might be attributed to the elevated AtLecRK-IX.1 expression.

The cell death phenotype was also found when AtLecRK-IX.1 was overexpressed in

Arabidopsis and is correlated with AtLecRK-IX.1 expression levels (Chapter IV). To

determine whether the capacity of LecRK-IX.1 in inducing cell death can be

maintained in the offspring, line IX.1-OE-10 was propagated and the progeny plants

(T1) were assayed for both the presence of AtLecRK-IX.1 and development of

spontaneous cell death. Cell death was only found in the T1 plants harboring

AtLecRK-IX.1 (Fig. 3B). These plants also showed retarded growth when compared to

those without AtLecRK-IX.1 (Fig. 3C). Taken together, these results indicate that

AtLecRK-IX.1 plays a role in mediating plant cell death which could hamper plant

growth.

For all the EV and IX.1-OE lines, seeds harvested from T0 plants have a similar

germination efficiency as untransformed N. benthamiana when grown in soil or on MS

(Murashige and Skoog) medium (Table 1; Fig. 3D). However, ten out of the sixteen

I.9-OE lines showed severe defects in seed germination, with a germination rate of 60%

or lower (Table 1). Also here, the severity of the phenotype does not correlate with

AtLecRK-I.9 expression levels in the various transgenic lines.

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Figure 3. Morphology of transgenic N. benthamiana lines.

A, T0 N. benthamiana IX.1-OE-10 plant, but not the T0 EV-2 plant displayed cell death. Ten-week-old

plants were photographed. B, T1 N. benthamiana IX.1-OE-10 plant, but not EV-2 plant developed cell

death. Six-week-old plants were photographed. C, T1 progeny of the IX.1-OE-10 line harboring

AtLecRK-IX.1 (+) is smaller in size than the T1 progeny without AtLecRK-IX.1 (‒). Six-week-old plants

were photographed. D, Germination of seeds harvested from untransformed and transgenic

N. benthamiana plants. Six seeds were sown in each pot. Two-week-old seedlings were photographed.

I.9-OE and IX.1-OE lines show enhanced resistance to Phytophthora pathogens

Both LecRK-I.9 and LecRK-IX.1 were previously shown to function in Phytophthora

resistance in Arabidopsis (Bouwmeester et al., 2011; Chapter IV). In order to

determine whether both LecRKs retain their function in Phytophthora resistance, we

checked the response of N. benthamiana I.9-OE and IX.1-OE lines upon inoculation

with Phytophthora capsici and Phytophthora infestans. Lines I.9-OE-1, I.9-OE-8,

IX.1-OE-3 and IX.1-OE-10 were selected and leaves from T1 progenies harboring the

transgenes were used for infection assays. It has to be noted that for IX.1-OE-10, only

leaves without any visible cell death phenotype were used for infection assays.


148

Figure 4. Infection assays on transgenic N. benthamiana lines with different Phytophthora

pathogens.

A-C, Disease symptoms on transgenic N. benthamiana EV, I.9-OE and IX.1-OE lines three days after

plug-inoculation with P. capsici LT263 (A) or LT3239 (B), or six days after zoospore-inoculation with

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149

P. infestans 14-3-GFP (C). Lesions are indicated by black circles. D, Average lesion sizes on

N. benthamiana plants upon inoculation with Phytophthora pathogens. Each experiment included

12-20 inoculation sites. Bars represent the mean lesion sizes (± SE). * indicates significantly difference

in lesion sizes (p < 0.05) compared to the EV lines according to a t test. Infection assays were repeated

three times with both P. capsici isolates and twice with P. infestans with similar results.

Upon plug-inoculation with P. capsici LT263 or LT3239, smaller lesions were found on

the I.9-OE and IX.1-OE lines when compared to EV-1 and EV-2 (Fig. 4, A and B),

indicating that constitutive expression of AtLecRK-I.9 or AtLecRK-IX.1 in

N. benthamiana enhances resistance to different isolates of P. capsici. This increased

resistance was also found in I.9-OE and IX.1-OE lines when inoculated with

P. infestans (Fig. 4C). Irrespective of inoculation with either P. capsici or P. infestans,

lesions on IX.1-OE-10 plants with higher AtLecRK-IX.1 expression levels were

smaller than those on IX.1-OE-3 (Fig. 2C and Fig. 4D). However, this is not the case

for I.9-OE plants. Despite a higher LecRK-I.9 expression level, I.9-OE-8 plants

showed slightly larger lesions when compared to I.9-OE-1 upon inoculation with

P. infestans (Fig. 2C and Fig. 4D).

CONCLUSIONS

In this study, multiple transgenic N. benthamiana lines with constitutive expression of

Arabidopsis LecRK-I.9 or LecRK-IX.1 were obtained. Transgenic lines vary in

transgene copy number, transgene expression level and protein accumulation.

Ectopic expression of either AtLecRK-I.9 or AtLecRK-IX.1 in N. benthamiana

increased the resistance to different Phytophthora species. Our results suggest that

Arabidopsis LecRK-I.9 and LecRK-IX.1 maintained their function in Phytophthora

resistance when transferred into N. benthamiana, which is in line with the previous

study on the transfer of LecRK-I.9 into potato. These results also suggest that LecRKs

could be used as a complementary resistance resource next to R genes for

engineering broad-spectrum disease resistance to Phytophthora pathogens. Since

ectopic expression of both LecRKs also caused several adverse effects on plant

fitness, such as curly leaves, leaf necrosis or reduced plant sizes, further analysis is

needed to reduce these pleiotropic effects. These transgenic N. benthamiana lines

can be used as a valuable experimental tool for further analysis of the components

required for LecRK-mediated resistance, for example via the virus-induced gene

silencing or protein complex pull-down assays.


150


Plant growth conditions and infection assays

N. benthamiana seeds were surface-sterilized by 70% ethanol and 1% NaClO, and

grown on MS medium (4.4 g MS salt, 20 g sucrose and 8 g agar) or MS medium

supplemented with antibiotics in a conditioned growth chamber at 19-21°C, with a 16

h photoperiod and a relative humidity of 75-80%. Plants grown in soil were kept in a

greenhouse with similar settings. Supplementary light (100 W m2-) was applied when

the light intensity dropped below 150 W m2-.

P. capsici isolates LT263 and LT3239 were maintained in the dark on V8 plates at

25°C (Wang et al., 2013/Chapter IIa), and Phytophthora infestans isolate 14-3-GFP

on rye sucrose agar at 18°C (Bouwmeester et al., 2014). P. infestans zoospores were

obtained by treating sporulating mycelia with cold water for 3-4 h. For detached-leaf

assays, leaves from five-week-old N. benthamiana plants were collected and the

abaxial sides were inoculated with fresh P. capsici mycelial plugs (Ø 0.5 cm) or 10 µl

droplets of a P. infestans zoospore suspension with a concentration of 5*105

zoospores ml-1. Inoculated leaves were kept in transparent plastic boxes with high

humidity in the dark overnight and thereafter exposed to a condition with a 12 h

photoperiod and appropriate temperature settings. Disease severity was monitored by

measuring lesion sizes as previously described by Vleeshouwers et al. (1999) three

and six days after inoculation with P. capsici and P. infestans, respectively.

Agrobacterium-mediated transformation of N. benthamiana

A. tumefaciens GV3101 carrying the binary vectors pBIN-KS-35S::AtLecRK-I.9-

eGFP, pBIN-KS-35S::AtLecRK-IX.1-eGFP (Chapter IV) and pBIN61-35S::GFP (Fig.

2A) were grown overnight at 28°C in Yeast Extract Broth with appropriate antibiotics.

A. tumefaciens cells were pelleted, resuspended and incubated in MMA induction

medium (10 mM MES, 10 mM MgCl2, 50 μM acetosyringone, pH 5.6) for 3 h.

A. tumefaciens cells were collected by centrifugation and resuspended in MS broth

supplemented with 150 μM acetosyringone. Leaf pieces (2-3 cm2) were cut from

five-week-old N. benthamiana leaves and incubated with A. tumefaciens cells for 30

min. Thereafter, leaf discs were dried on filter paper to remove excess A. tumefaciens

and incubated on regeneration medium consisting of MS salt, 1 mg/L cytokinins

6-BAP (6-benzyl amino purine), 0.1 mg/L auxin NAA (1-naphthaleneacetic acid) and

0.8% agar for two days at 19-21°C. Leaf pieces were transferred every week to fresh

regeneration medium supplemented with 400 mg/L cefotaxime, 200 mg/L vancomycin

and 200 mg/L kanamycin until the appearance of plantlets. The generated plantlets

were transferred to MS medium containing 200 mg/L kanamycin to allow root

development. Upon root generation, plantlets were transferred into soil and kept in

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151

greenhouse for seed harvesting.

Transgene detection in transgenic N. benthamiana

Genomic DNA was isolated using CTAB buffer (0.02% CTAB, 100 mM Tris-HCl pH 8.0,

20 mM EDTA pH 8.0, 1.4 M NaCl and 1% PVP) followed by precipitation with

isopropanol. RNA was removed by RNaseA. Transgene presence was checked by

PCR using specific primers for AtLecRK-I.9 and AtLecRK-IX.1 and a primer matching

sequence in the binary vector (Fig. 2A; Supplemental Table S1).

Transgene copy number in T0 transgenic lines was determined according to

Honda et al. (2002). Briefly, qPCR was performed using genomic DNA as template

with specific primers for AtLecRK-I.9, AtLecRK-IX.1, neomycin phosphotransferase II

gene (NPTII) or Nbactin (NbS00011031g0002.1) (Table S1). The copy number was

calculated by normalizing the amplification of AtLecRK-I.9, AtLecRK-IX.1 or NPTII to

Nbactin.


Total RNA was isolated from leaves collected from six-week-old T0 transgenic

N. benthamiana plants with a NucleoSpin RNA plant Kit (Macherey-Nagel) and

thereafter used as template for cDNA synthesis with an oligo-dT primer and a M-MLV

reverse transcriptase kit (Promega). qRT-PCR was performed as previously

described (Wang et al., 2014/Chapter III). Transcript levels of AtLecRK-I.9 and

AtLecRK-IX.1 were normalized to Nbactin.

Protein extraction, immunoprecipitation and western blotting

Leaves collected from six-week-old T0 transgenic N. benthamiana plants were ground

in liquid nitrogen and subsequently incubated for 30 min in an extraction buffer

containing 150 mM NaCl, 50 mM Tris-HCl pH 8.0, 1.0% IGEPAL® CA-630 (Sigma)

and one protease inhibitor cocktail tablet per 50 ml (Roche). The homogenate was

centrifuged at 18,000 rpm for 20 min and the supernatant was incubated with

GFP-trap_A® beads (Chromotek) at 4°C for 1-2 h. After incubation, GFP-beads were

spinned down and washed six times with extraction buffer. Proteins were eluted from

GFP-trap_A® beads by boiling for 5 min, separated on a 8% SDS-PAGE gel and

electroblotted onto PVDF membrane (Bio-Rad). Accumulation of eGFP-tagged

LecRK-I.9 and LecRK-IX.1 was detected by immunoblotting using anti-GFP-HRP

(Miltenyi Biotec). Supersignal West Femto Chemiluminescent Substrate (Thermo

Scientific) was used for signal development. Coomassie brilliant blue staining was

used to indicate the amount of loading.


152

ACKNOWLEDGEMENTS

We thank Bert Essenstam from Unifarm for taking care of the transgenic plants. This

project was financially supported by a Wageningen University sandwich-PhD

fellowship (YW), a Huygens scholarship (YW), a VENI grant (KB) from the

Netherlands Organization for Scientific Research (Technology Foundation NWO-STW)

and the Food-for-Thought campaign of the Wageningen University fund.

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1192-1203.



kinases are involved in plant immunity. Molecular Plant-Microbe Interactions, 27, 1390-1402. Zipfel, C., Kunze, G., Chinchilla, D., Caniard, A., Jones, J.D.G., Boller, T. and Felix, G. (2006).

Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated

transformation. Cell, 125, 749-760.

Zipfel, C. (2014). Plant pattern-recognition receptors. Trends in Immunology, 35, 345-351.


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SUPPLEMENTAL INFORMATION

Table S1. Primers used in this study. Primer Sequence 5'-3' Used to / for

NPTII-RT-F GGAGAGGCTATTCGGCTATG Check the presence of NPTII

NPTII-RT-R TCGTCCTGCAGTTCATTCAG Check the presence of NPTII

Nbactin-F TATGGAAACATTGTGCTCAGTGG Endogenous control

Nbactin-R CCAGATTCGTCATACTCTGCC Endogenous control


pGRAB-F1 CCCACTATCCTTCGCAAGACCCTTCC Check the presence of T-DNA sequence

LecRK-IX.1-RT-F CAAGGCGAGTAATGTGATGCT Check presence of AtLecRK-IX.1; qRT-PCR

LecRK-IX.1-RT-R TAACCAAATGTTCCTGCTAACC qRT-PCR

LecRK-IX.1-F TCAAGCCTGGAGCTAATAG Check the presence of AtLecRK-IX.1

LecRK-IX.1-R ACGACCATGTTGAGCACTTG Check the presence of AtLecRK-IX.1

LecRK-I.9-RT-F TTTGCCAGATTTCTCACCATACAC qRT-PCR

LecRK-I.9-RT-R TCTGTTGACTGCCAAGCGTAAG qRT-PCR

LecRK-I.9-F ATGGCTCGTTGGTTGCTTCAG Check the presence of AtLecRK-I.9

LecRK-I.9-R GCTTTGACATCTCGGTGCAGAAC Check the presence of AtLecRK-I.9

Chapter VI

L-type lectin receptor kinases in

Nicotiana benthamiana and tomato and their role in

Phytophthora resistance

Yan Wang, Klaas Bouwmeester, Rob Weide, Francine Govers

Chapter VI

156

ABSTRACT

Membrane-bound receptors play crucial roles as sentinels of plant immunity against

a large variety of invading microbes. One class of receptors known to be involved in

self/non-self-surveillance and plant resistance comprises the L-type lectin receptor

kinases (LecRKs). Previously, we reported that several Arabidopsis LecRKs play a

role in Phytophthora resistance. In this study we determined whether homologs of

these LecRKs from the Solanaceous plants Nicotiana benthamiana and tomato

(Solanum lycopersicum) play similar roles in defence against Phytophthora. In

genome-wide screenings a total of 38 (Nb)LecRKs was identified in N. benthamiana

and 22 (Sl)LecRKs in tomato, each consisting of both a lectin and a kinase domain.

Phylogenetic analysis revealed that in contrast to Arabidopsis, which has a LecRK

family comprised of nine clades, Solanaceous species have just five of these nine

clades (i.e. IV, VI, VII, VIII and IX), plus one additional clade that lacks Arabidopsis

homologs. Subsequently, several Solanaceous LecRKs were selected for functional

analysis using virus-induced gene silencing. Infection assays with Phytophthora

capsici and Phytophthora infestans on LecRK-silenced plants revealed that

N. benthamiana and tomato homologs in clade IX play a role in Phytophthora

resistance similar to the two Arabidopsis LecRKs in this clade, hence suggesting

conserved functions of clade IX LecRKs across different plant families. This study

provides a first insight into the diversity of Solanaceous LecRKs and their role in plant

immunity, and shows the potential of LecRKs for Phytophthora resistance breeding.

Solanaceous LecRKs and their role in Phytophthora resistance

157

INTRODUCTION

Plant diseases caused by Phytophthora pathogens are a major constraint to the

production of a large variety of Solanaceous crops (Kroon et al., 2012). Renowned

are Phytophthora infestans, the causal agent of late blight disease on potato and

tomato, and Phytophthora capsici which is highly destructive to multiple Solanaceous

crops, including tomato, eggplant and pepper (Fry, 2008; Bouwmeester et al., 2009;

Lamour et al., 2012). Breeding for Phytophthora resistance has been focused largely

on the introgression of resistance (R) genes encoding the intracellular nucleotide-

binding leucine-rich repeat receptors (NLRs), that mediate effector-triggered

immunity (ETI) upon recognition of cognate effectors (Vleeshouwers et al., 2011).

Since Phytophthora pathogens can quickly adapt, NLR-mediated resistance is often

not durable. In the pathogen population new races emerge that have inactivated or

modified effector genes and can thus circumvent R gene recognition (Vleeshouwers

et al., 2011; Kasuga and Gijzen, 2013).

Besides ETI, plants rely on defence mediated by plasma membrane-localized

receptor-like kinases (RLKs), which play pivotal roles in the surveillance of “non-self”

(e.g. microbe-associated molecular patterns; MAMPs) and “modified-self” (e.g.

damage-associated molecular patterns; DAMPs) molecules as exogenous stress

signals to initiate non-race-specific immunity. These so-called pattern recognition

receptors (PRRs) have been suggested to mediate basal defence, which is thought

to have a high potential to confer broad spectrum disease resistance in plants (Boller

and Felix, 2009). So far, PRRs have received limited attention in resistance breeding.

One class of RLKs that has been suggested to function as PRRs in recognition of

stress signals and subsequent initiation of plant defence, is comprised of the L-type

lectin receptor kinases (LecRKs). Arabidopsis has 45 LecRK genes that are

distributed over nine clades (i.e. clade I to IX) and seven singletons, with several

showing induced expression upon pathogen attack and in response to pathogen-

associated elicitors and MAMPs (Bouwmeester and Govers, 2009). One of these is

LecRK-I.9 that functions in maintaining cell wall integrity and plays a crucial role in

Phytophthora resistance in Arabidopsis (Gouget et al., 2006; Bouwmeester et al.,

2011). Interfamily gene transfer of Arabidopsis LecRK-I.9 to the Solanaceous plants

Nicotiana benthamiana and potato conferred enhanced resistance to P. infestans

(Bouwmeester et al., 2014; Chapter V), suggesting a conserved functionality in

stress signal recognition and immunity among plant species. Recently, it was shown

that LecRK-I.9 (alias DORN1) also functions as a receptor of extracellular ATP

(eATP) (Choi et al., 2014). Possibly, eATP is released upon pathogen attack or

wounding and as such may function as a DAMP (Choi et al., 2014). Two other

Arabidopsis LecRKs that have been studied for their roles in plant defence are

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LecRK-V.5 and LecRK-VI.2. Both were found to play a role in bacterial resistance by

mediating stomatal immunity (Desclos-Theveniau et al., 2012; Singh et al., 2012).

Ectopic expression of LecRK-VI.2 in N. benthamiana appears to prime MAMP-

mediated defence, and to increase resistance against various hemibiotrophic and

necrotrophic bacterial pathogens (Huang et al., 2014). Another example is

Arabidopsis LecRK-I.8, which is required for proper PR-1 induction upon treatment

with egg-derived elicitors of the cabbage butterfly Pieris brassicae (Gouhier-Darimont

et al, 2013). Recently, we published a study that was aimed at investigating the role

of all Arabidopsis LecRKs in defence against a variety of plant pathogens. This

revealed that next to LecRK-I.9, there are at least 14 other LecRKs that have a

putative role in resistance against Phytophthora pathogens in Arabidopsis (Wang et

al., 2014/Chapter III). Arabidopsis lines with T-DNA insertions in these 14 LecRK

genes showed altered susceptibility when challenged with P. capsici and

Phytophthora brassicae, suggesting that LecRK family members collectively build up

a basal level of Phytophthora resistance.

Analysis of LecRKs for a role in defence in plant species other than Arabidopsis

has so far been limited to one gene, i.e. NbLRK1 from Nicotiana benthamiana

(Kanzaki et al., 2008). NbLRK1 was reported to interact with INF1, an elicitin

secreted by P. infestans, and suggested to play a role in intensifying INF1-induced

cell death (Kanzaki et al., 2008). In addition, several LecRKs were indicated to be

involved in plant defence since they are induced upon treatment with pathogens or

pathogen-derived elicitors. Multiple cucumber (Cucumis sativus) LecRKs were found

to be induced upon infection by P. capsici and Phytophthora melonis (Wu et al.,

2014); and a cotton (Gossypium hirsutum) LecRK, i.e. GhLecRK-2 was shown to be

up-regulated upon treatment with a cell wall-derived fraction of the vascular wilt

fungus Verticillium dahliae (Phillips et al., 2013).

Characterization of defence mechanisms in model plants, such as Arabidopsis,

paves the way to study similar processes in crops (Koornneef and Meinke, 2010).

Disrupting gene homologs encoding proteins with a conserved physiological function

often results in similar phenotypes in different plant species. For example, the role of

the PRR FLS2 in the perception of bacterial flagellin was first discovered in

Arabidopsis, and thereafter found to be largely conserved in various plant lineages

(Gomez-Gomez and Boller, 2000; Hann and Rathjen, 2007; Robatzek et al., 2007;

Takai et al., 2008).

To determine whether LecRKs are functionally conserved in Solanaceous

species, we set out to investigate the function of Solanaceous LecRKs in

Phytophthora resistance. In this study, we performed a genome-wide identification of

LecRKs in N. benthamiana and tomato (Solanum lycopersicum) and analysed the

phylogenetic relationship of these LecRKs with Arabidopsis LecRKs. Subsequently,


159

several Solanaceous LecRKs were selected for functional analysis using virus-

induced gene silencing and infection assays to pinpoint their role in Phytophthora

resistance.


Identification of LecRKs in N. benthamiana and tomato

LecRKs in N. benthamiana and tomato were identified following the pipeline depicted

in Fig.1. Protein sequences of Arabidopsis LecRKs (AtLecRKs) were used as queries

for BLAST searches against the predicted protein databases of N. benthamiana and

tomato in the Sol Genomic Network (SGN). Presence of both a lectin domain and a

kinase domain was used as the major criterion in the selection of putative LecRKs.

Using reciprocal BLAST searches, we identified 37 (Nb)LecRKs and 22 (Sl)LecRKs

in N. benthamiana and tomato, respectively (Table 1). Corresponding coding DNA

(cDNA) sequences were obtained and in combination with EST data, RNA-seq data

and genomic DNA sequences used for gene model validation. Strikingly, over half of

the NbLecRK gene models are incomplete due to erroneous open reading frame

(ORF) prediction including erroneous prediction of start codon, intron or stop codon.

The coding sequence for NbLRK1, a N. benthamiana LecRK homologous to

AtLecRK-VIII.2 (deposited in GenBank as AB247455.1; Kanzaki et al., 2008), was

found to contain multiple SNPs when compared with RNA-seq data. Hence, we PCR-

amplified and sequenced the cDNA and it was found to match to

NbS00026192g0010.1. cDNA and protein sequences were therefore corrected

accordingly and used for further analysis (Table 1). Making use of the RNA-seq data,

we found another NbLecRK (i.e. NbS00021029g0001.1) of which the encoded

protein was annotated as missing the kinase domain in SGN (Table 1). For the

SlLecRKs, three were found to be incomplete and were corrected according to the

RNA-seq data (Table 1). The obtained LecRK protein sequences including the

revised ones are available upon request and will be included in a supplemental file

when this chapter is submitted for publication.

In Arabidopsis, only six out of the 45 LecRKs contain introns. Five of these

have one intron and the other one contains two introns. To determine whether

Solanaceous LecRKs have introns, cDNA sequences were compared to genomic

DNA sequences. In this way, 12 NbLecRKs were found to contain introns ranging

from one to four per gene (Table 1). By using RNA-seq data as extra check six

NbLecRKs were confirmed to contain one intron, while for the other six, introns could

not be validated due to lack of RNA-seq data. Three out of the 22 SlLecRKs were

confirmed to contain one intron by the RNA-seq data, whereas another three

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SlLecRKs were predicted to contain one, four and five introns, respectively (Table 1).

Figure 1. Pipeline depicting the procedures used for identification and analysis of LecRKs in

Nicotiana benthamiana and tomato.

Chromosomal location of tomato LecRKs

To investigate tandem duplication events, we examined the chromosomal distribution

of the obtained SlLecRKs in tomato. For N. benthamiana no such information is

available, so we were only able to summarize information obtained on SlLecRKs.

SlLecRKs are distributed over eight of the 12 tomato chromosomes, i.e. chromosome

1, 2, 3, 4, 5, 7, 9, 10, with the number of LecRKs ranging from 1 to 6 (Table 1). Most

of the SlLecRKs are located on chromosomes 9 and 10, with each chromosome

contains six SlLecRKs. In addition, two tandem duplicated SlLecRK gene clusters

were found on chromosome 9 and one on chromosome 10 based on the criterion

that tandem duplicated genes are located within 10 adjacent gene models (Shiu and

Bleecker, 2003). In comparison, LecRKs in Arabidopsis show a much higher tandem

duplication rate according to the cluster analysis by Bouwmeester and Govers

(2009). Here nine distinct clusters were found, each with two to six AtLecRKs.


161

Table 1. Inventory of LecRKs of Arabidopsis, Nicotiana benthamiana and tomato.

a,b,c Tandem duplicated LecRKs. d Gray blocks represent clades of Solanaceous LecRKs. e The

number of introns was predicted by comparing cDNA and genomic DNA. f Signal peptide prediction

based on SignalP 3.0. g +: with RD motif, ─: without RD motif due to lack of the VIb subdomain.

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Domain composition of NbLecRKs and SlLecRKs

Domain analysis performed using multiple bioinformatics webtools revealed that of

the 38 identified NbLecRKs, 32 contain the typical composition of a LecRK, i.e. a

putative signal peptide (SP), an extracellular L-type lectin domain, a single

transmembrane (TM) domain and a cytosolic serine/threonine kinase domain. Of the

remaining six, three have no clear TM domain, two have no SP and another one

lacks both the SP and the TM domain. Similarly, 21 of the 22 SlLecRKs contain all

representative LecRK features, whereas the other one was predicted to lack both the

SP and the TM domain (Table 1). In addition, the SlLecRK Solyc09g011060.2.1

contains an additional domain, namely a NTP-binding domain following the kinase

domain in the C-terminus. Although NbS00043874g0006.1, Solyc10g047680.1.1 and

Solyc10g047700.1.1 were predicted to contain a lectin signature, the domain

composition is incomplete (Table 1). Kinase (STK) domains of RLKs are in general

highly conserved and contain 12 subdomains which are essential for kinase activity

(Shiu and Bleecker, 2001; Hanks, 2003; Afzal et al., 2008). Alignment of the

predicted kinase domains revealed that six NbLecRKs and three SlLecRKs lack one

or more subdomains, which could impair kinase activity (Table 1). Protein kinases

that are activated by phosphorylation in the activation loop typically carry a

conserved arginine (R) that precedes the catalytic aspartate (D) in subdomain VIb

(Nolen et al., 2004; Kornev et al., 2006). They are therefore known as RD kinases

whereas kinases that lack the RD motif are collectively termed non-RD kinases. It

has been found that activation of non-RD kinases does not require phosphorylation

of the activation loop (Dardick and Ronald, 2006). Nearly all identified Solanaceous

LecRKs contain a RD motif with the exception of three NbLecRKs and two SlLecRKs.

NbC25369236g0004.1 and Solyc10g047700.1.1 do not contain a RD motif due to

absence of the kinase subdomain VIb, whereas NbS00029224g0003.1,

NbS00001395g0006.1 and Solyc03g080060.1.1 have an RD motif substituted by KN

(Table 1). This is also the case for some of the Arabidopsis LecRKs, i.e. AtLecRK-I.2

lacks the kinase subdomain VIb and AtLecRK-III.1 and AtLecRK-III.2 contain GN

residues instead of RD.

Phylogeny of LecRKs in Arabidopsis, N. benthamiana and tomato

In Arabidopsis, LecRKs were divided into nine clades based on the definition that a

clade contains at least two homologs with a minimum of 50% similarity in both the

DNA sequence and the amino acid sequence (Bouwmeester and Govers, 2009).

However, due to high sequence divergence this criterion is not applicable for the

Solanaceous LecRKs (data not shown). Hence, Solanaceous LecRKs were grouped

together if they share over 50% similarity at the amino acid level. In this way, twelve

clades were found by means of pairwise alignment (Table 1), ranging in size from


163

three to 10 members.

To evaluate the evolutionary relationship among LecRKs from different plant

species, an un-rooted phylogenetic tree containing 43 full length AtLecRKs, 38

NbLecRKs and 22 SlLecRKs was generated using the Neighbor-Joining algorithm

(Fig. 2). The reliability of the phylogram was determined by bootstrap analysis with

10,000 replicates. A comparable consensus tree was obtained using the Maximum-

Likelihood algorithm with the same bootstrap replicates (data not shown). In addition,

a similar phylogenetic analysis was performed using solely the extracellular lectin

domains or the intracellular kinase domains. The resulting tree topologies are overall

in agreement with the one shown in Fig. 2, which was constructed based on the full

length LecRKs (Supplemental Fig. S1).

In the phylogenetic tree, 38 of the 43 AtLecRKs fall into nine distinct clades (I-

IX) and the other five are singletons, which is in agreement with the previous study

by Bouwmeester and Govers (2009). In addition, the degree of similarity in LecRKs

was found to be indicative of the phylogenetic relationship among these three

species (Table 1; Fig. 2). In the tree, most of the NbLecRKs and SlLecRKs group

together with AtLecRKs in the clades IV, VI, VII, VIII.2 and IX and with the five

AtLecRK singletons. In those branches often the AtLecRK clade members or the

AtLecRK singletons group with one SlLecRK and two NbLecRKs in one sub-branch.

In such a sub-branch, the NbLecRKs and SlLecRK are often closer to each other

than to their Arabidopsis counterpart, as expected, and the duplicated number of

NbLecRKs is most probably due to the allo-tetraploid nature of N. benthamiana. In

addition, closely related sub-branches comprising only Solanaceous LecRKs are

observed in the tree, with up to eight SlLecRKs and NbLecRKs, often in a 1:2 ratio.

These sub-branches are most prominent next to those containing the singletons

AtLecRK-S.4 and AtLecRK-S.5 and the two clade IX AtLecRKs. In contrast to these

expansions in numbers on certain branches of the tree, none of the Solanaceous

LecRKs fall into clades I, II, III and V. Similarly, no LecRKs belonging to these four

clades were identified in cucumber (Wu et al., 2014). It should be noted that the

AtLecRKs in clade I, II, III and V exhibit high frequency of tandem duplication

(Bouwmeester and Govers, 2009). In addition, these four LecRK clades appear to be

conserved among plants of the Brassicales order (Nsibo et al., submitted).

Collectively, these indicate a lineage-specific expansion of LecRKs after the split of

the Brassicales. Several LecRKs belonging to these four Arabidopsis-specific clades

were found to be involved in resistance to Phytophthora, Alternaria brassicicola and

bacterial pathogens (Bouwmeester et al., 2011; Wang et al., 2014/Chapter III),

suggesting that this lineage-specific expansion of LecRKs might be important for

Arabidopsis adaptation. Next to the Arabidopsis-specific clades, the phylogenetic

analysis revealed one Solanaceous-specific (Sol-specific) clade and one NbLecRK

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that ends up as a singleton (Fig. 2). The three Solanaceous LecRKs carrying a

substitution in the RD motif form a well-supported clade separated from other

LecRKs. The singleton NbS00037263g0008.1 is on a branch in the vicinity of the

cluster comprising AtLecRK clade IX.

Figure 2. Phylogenetic tree of LecRKs from Arabidopsis, N. benthamiana and tomato.

Neighbor-Joining tree based on predicted full length amino acid sequences, including 43 AtLecRKs,

38 NbLecRKs and 22 SlLecRKs. Branches are colored according to the clades delineated by

Bouwmeester and Govers (2009). Numbers above the branches represent the level of clade support

inferred by 10,000 bootstrap replicates.


165

Arabidopsis clade IX LecRK homologs in N. benthamiana are functional in


To determine the role of NbLecRKs in Phytophthora resistance we selected

homologs of AtLecRKs that, in a previous study, were identified as putative

resistance components in Arabidopsis (Wang et al., 2014/Chapter III). That study

revealed 14 AtLecRKs implicated in Phytophthora resistance of which seven have

close homologs in N. benthamiana. TRV-based constructs were made to silence

NbLecRKs that are homologous to six of these AtLecRKs, i.e. AtLecRK-VIII.2,

AtLecRK-IX.1/LecRK-IX.2, AtLecRK-S.1, AtLecRK-S.6 and AtLecRK-S.7. Loss of

function of a single gene might fail to cause any phenotypic change as its function

can be either completely or partially complemented by the presence of homologous

genes. To avoid this problem, we made TRV-based constructs to simultaneously

silence several LecRKs with high sequence similarity. As shown in Fig. 3A, seven

TRV constructs were made, of these three were made to target different AtLecRK-

IX.1/LecRK-IX.2 homologs. In all cases, at least 60% reduction in transcript levels of

the target NbLecRKs was observed in the NbLecRK-silenced N. benthamiana plants

when compared to TRV:GUS-treated plants (Fig. 3B). NbLecRK silencing did not

affect plant growth and development as none of the NbLecRK-silenced plants

showed consistent phenotypic changes in terms of plant size, leaf color, leaf

morphology and shoot growth (Supplemental Fig. S2). Upon inoculation with

P. capsici LT263 or P. infestans 14-3-GFP, TRV:NbIX.1b and TRV:NbIX.1c-treated

N. benthamiana leaves showed significantly larger lesions compared to those

observed on TRV:GUS-treated plants (Fig. 3, C and D). The increased susceptibility

of TRV:NbIX.1b- and TRV:NbIX.1c-treated N. benthamiana plants indicates that the

silenced LecRKs are involved in Phytophthora resistance. In contrast, silencing of the

other two NbLecRKs (i.e. NbS00048421g0010.1 and NbS00051756g0005.1) that

group with clade IX but are relatively distant from AtLecRK-IX.1 and AtLecRK-IX.2

(Fig. 2), did not result in increased susceptibility to Phytophthora (Fig. 3C). In

addition, none of the plants treated by TRV:NbVIII.2, TRV:NbS.1 or TRV:NbS.7

showed significant differences in susceptibility to either P. capsici or P. infestans

when compared to TRV:GUS-treated plants (Fig. 3C). TRV:NbS.6-treated plants

though, showed increased susceptibility upon infection by P. infestans 14-3-GFP, but

not when inoculated with P. capsici LT263 (Fig. 3C). It is possible that the activity of

some of these NbLecRKs is simply too weak to prevent Phytophthora infection. This

is exemplified by the fact that TRV:NbS.6-treated plants allow P. infestans 14-3-GFP

to expand but not P. capsici LT263, an isolate with relatively higher virulence (Fig.

3D). Also the sequence divergence between the AtLecRKs and their N. benthamiana

homologs may contribute to functional divergence that we observed.

Chapter VI

166


167

Figure 3. Response of NbLecRK-silenced N. benthamiana to Phytophthora infection.

A, NbLecRKs targeted by different TRV constructs. The phylogenetic tree was made as described in

Figure 2. B, Relative NbLecRK transcript levels in TRV:GUS- and TRV:NbLecRK-treated

N. benthamiana leaves. Transcript levels were normalized with NbActin and expressed as mean fold

changes (± SD) relative to the transcript level in TRV:GUS-treated leaves which was arbitrarily set as

1. This experiment was repeated four times with similar results. C, Lesion sizes on TRV:GUS- and

TRV:NbLecRK-treated N. benthamiana leaves upon inoculation with Phytophthora capsici and

Phytophthora infestans three days post inoculation (dpi) and 6 dpi, respectively. Bars represent mean

lesion sizes (± SE) of over 20 inoculation sites. * indicates significant difference in lesion sizes (p <

0.05, t test) between TRV:GUS and TRV:NbLecRK-treated plants. This experiment was repeated four

times with similar results. D, Disease symptoms on TRV:GUS-, TRV:NbIX.1- and TRV:NbS.6-treated

N. benthamiana leaves after inoculation with P. capsici at 3 dpi and P. infestans at 6 dpi. Pictures

were taken under daylight and UV light. E-F, Disease symptoms (E) and quantified lesion sizes (F) on

N. benthamiana leaves transiently expressing GFP or NbLecRK-IX.1/2 after inoculation with P. capsici

and P. infestans. Bars represent the mean lesion sizes (± SE) of at least twenty infiltrated

N. benthamiana leaves. * indicates significant difference in lesion sizes (p < 0.05) according to a t test.

This experiment was repeated three times with similar results.

To further investigate the role of NbLecRK-IX.1 homologs in Phytophthora

resistance, we selected NbS00000562g0002.1 for further study and renamed it

NbLecRK-IX.1/2 to emphasize its relation with the two clade IX AtLecRKs. The full

length coding sequence of NbLecRK-IX.1/2 was PCR-amplified and cloned into a

binary vector enabling transient expression in N. benthamiana leaves. Upon

inoculation with P. capsici or P. infestans, N. benthamiana leaves transiently

expressing NbLecRK-IX.1/2 showed much smaller lesions than that growing on

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leaves expressing GFP (Fig. 3, E and F). This confirms that NbLecRK-IX.1/2 indeed

functions in resistance to Phytophthora. It should be noted that transient expression

of NbLecRK-IX.1/2 in N. benthamiana did not cause any visible cell death, a

phenomenon that was observed when transiently expressing AtLecRK-IX.1 or

AtLecRK-IX.2 in N. benthamiana (Chapter IV).

NbLecRK silencing does not alter elicitor-induced cell death in N. benthamiana

NbLRK1 was previously found to interact with the P. infestans elicitor INF1 via its

kinase domain (Kanzaki et al., 2008). Silencing of NbLRK1 in N. benthamiana was

reported to compromise INF1-induced cell death and it was proposed to be an

important component of a host receptor complex that recognizes INF1 (Kanzaki et

al., 2008). We found four LecRKs in N. benthamiana that are homologous to NbLRK1

(Table 1). Sequence alignment of the predicted cDNA sequences revealed that three

of these four are likely silenced by the fragment used by Kanzaki et al. (2008)

(termed NbVIII.2-2 in Supplemental Fig. S3) raising the question which of the three

genes is responsible for the compromised cell death that they observed. In this study,

we used another fragment named TRV:NbVIII.2 to simultaneously silence these three

NbLecRKs (Fig. 3A; Supplemental Fig. S3) and confirmed by qRT-PCR that

expression of the three genes is reduced (Fig. 3B). In contrast to Kanzaki et al.

(2008) we found no difference in the appearance of cell death upon transient

expression of inf1 between N. benthamiana plants treated with TRV:NbVIII.2 and

TRV:GUS (Fig. 4). This discrepancy could be due to the fact that Kanzaki et al.

(2008) infiltrated the leaves with INF1 protein whereas we expressed the inf1 gene in

planta by Agrobacterium-mediated transient expression. In the natural situation, INF1

is secreted by P. infestans into the plant apoplast and is assumed to remain in the

apoplastic space (Kamoun, 2006). Hence, it is puzzling how INF1 can interact with

the cytoplasmic kinase domain of NbLRK1 to mediate cell death induction. Kanzaki

et al. (2008) hypothesized that INF1 could be translocated into plant cells via

receptor-mediated endocytosis but they were not able to show interaction between

INF1 and NbLRK1 in planta.

We also investigated whether other NbLecRKs affect cell death triggered by

INF1 and whether NbLecRKs play a role in cell death triggered by two other

Phytophthora elicitors i.e. CRN2 (Crinkling and necrosis-inducing protein) and NPP1

(necrosis-inducing Phytophthora protein 1). Transient expression of inf1, crn2 and

npp1 in NbLecRK-silenced plants resulted in cell death in all cases and no visible

differences in the appearance of cell death were observed when compared with

TRV:GUS-treated leaves (Fig. 4). Apparently, none of the NbLecRKs tested here play

a role in cell death induced by these three elicitors.


169

Figure 4. Silencing of NbLecRKs does not affect cell death induced by Phytophthora elicitors.

Cell death in TRV:GUS- and TRV:NbLecRK-treated N. benthamiana leaves expressing inf1, crn2 and

npp1. Each experiment included at least six leaves per construct. This experiment was repeated three

times with similar results.

Silencing of the tomato LecRK homolog of AtLecRK-IX.1/LecRK-IX.2 reduces


To assess the role of tomato LecRKs in Phytophthora resistance, we used the same

strategy as for the NbLecRKs and silenced SlLecRKs that group with the six

AtLecRKs mentioned above. The TRV constructs were designed to silence a single

SlLecRK gene, with the exception of TRV:SlVIII.2 that targets two SlLecRKs

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simultaneously, i.e. Solyc10g084250.1.1 and Solyc09g007510.1.1 (Fig. 5A).

Quantitative analysis of transcript levels in TRV-treated plants revealed that

Solyc03g043710.1.1 and Solyc07g065610.1, targeted by TRV:SlIX.1 and TRV:SlS.7,

respectively, are reduced up to 70% in expression in comparison to TRV:GUS-treated

plants. Silencing of the other SlLecRKs was found to be less efficient (Fig. 5B).

Inoculation of TRV:SlIX.1-treated plants with P. capsici LT263 or P. infestans 14-3-

GFP, resulted in significantly larger lesions than those on inoculation of TRV:GUS-

treated plants (Fig. 5, C and D; Supplemental Fig. S4), indicating that the most

closely related tomato homolog of AtLecRK-IX.1 and AtLecRK-IX.2 plays a role in

Phytophthora resistance similar to NbLecRK-IX.1/2, the homolog in N. benthamiana.

In plants treated with silencing constructs targeting the other SlLecRKs, no increased

susceptibility was found, neither to P. infestans nor to P. capsici (Supplemental Fig.

S4).

CONCLUSIONS In total, 38 and 22 LecRKs were identified in the genomes of N. benthamiana and

tomato, respectively. Multiple deposited Solanaceous LecRK gene models were

found to be erroneous due to mis-prediction of ORF and these sequences were

corrected based on the transcriptome data. Domain composition analysis indicated

that most of the identified LecRKs have the typical LecRK structure, but there are

several LecRKs, especially the ones from N. benthamiana, that lack a signal peptide,

a TM domain or both. Phylogenetic analysis revealed that most of the Solanaceous

LecRKs group together with Arabidopsis LecRKs, whereas one clade containing two

NbLecRKs and one SlLecRK seems to be a Solanaceous-specific clade. Functional

analysis of Solanaceous LecRKs using TRV-mediated gene silencing and

Agrobacterium-mediated transient gene expression demonstrated that homologs of

AtLecRK-IX.1/LecRK-IX.2 in both N. benthamiana and tomato function in resistance

to different Phytophthora pathogens. These data demonstrated that the Phytophthora

resistance function of clade IX LecRKs is conserved across different plant species.


171

Figure 5. Silencing of SlLecRK-IX.1/LecRK-IX.2 compromises Phytophthora resistance in

tomato.

A, SlLecRKs targeted by different TRV constructs. The phylogenetic tree was made as described in

Figure 2. B, Relative SlLecRK transcript levels in TRV:GUS- and TRV:SlLecRK-treated tomato leaves.

Transcript levels were normalized with SlActin and expressed as mean fold changes (± SD) relative to

the transcript level in TRV:GUS-treated leaves which was arbitrarily set as 1. C-D, Disease symptoms

(C) and quantified lesion sizes (D) on TRV:GUS- and TRV:SlIX.1-treated tomato leaves inoculated

with P. capsici LT263 (1*105 zoospores ml-1) and P. infestans 14-3-GFP (5*105 zoospores ml-1). Each

experiment included at least 20 leaves treated for each construct. Bars represent the mean lesion

sizes (± SE). * indicates significant difference (p < 0.05) in lesion sizes between TRV:GUS and

TRV:SlIX.1-treated plants according to a t test. The infection assay with P. capsici was performed once

whereas infection assays with P. infestans were repeated three times with similar results.

Chapter VI

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MATERIAL AND METHODS

Sequence identification and gene analysis

Protein sequences of Arabidopsis LecRKs analysed by Bouwmeester and Govers

(2009) were retrieved from the TAIR website (http://www.arabidopsis.org). Protein

sequences were used as queries for reciprocal BLAST searches via the Sol Genomic

Network (SGN) website (http://solgenomics.net) against the genomic databases of

N. benthamiana and tomato. Obtained LecRK sequences were further analysed by

comparative analysis using publicly available ESTs, RNA-seq data derived from the

University of Sydney Nicotiana benthamiana Genome Page (http://sydney.edu.au

/science/molecularbioscience/sites/benthamiana/) (Nakasugi et al., 2012; Naim et al.,

2012) and RNA-seq data derived from tomato leaves (Faino, L., personal

communication). NbS00026192g0010.1 was verified by sequencing after

amplification of the entire coding sequence using Pfu DNA polymerase (Promega)

and gene specific primers (Supplemental Table S1). In addition, the retrieved cDNA

sequences were compared with the genomic DNA sequences, followed by manual

validation of the presence of introns and open reading frames. Amino acid sequences

were subjected to the protein domain and motif annotation webtools SMART

(http://smart.embl-heidelberg.de), SignalP 3.0 (http://www.cbs.dtu.dk/services/

SignalP-3.0) and TMHMM 2.0 Server (http://www.cbs.dtu.dk/services/TMHMM).

Predicted kinase domain sequences were aligned by ClustalW and manually

checked for sub-domains according to those defined based on LecRK-VI.2 and

NtCPK5 (Wang et al., 2005; Singh et al., 2013).

Multiple sequence alignment and phylogenetic analysis

Full length protein sequences and sequences comprising the lectin domain or the

kinase domain were aligned with ClustalW using the protein weight matrix GONNET

with a penalty gap opening of 10 and a gap extension of 0.1. The obtained sequence

alignment was subsequently used as input to construct a Neighbor-Joining tree and a

Maximum Likelihood tree with 10,000 bootstrap replicates using the Jones-Taylor-

Thornton substitution model (Hall, 2013) in MEGA 5.1. Branches corresponding to

partitions reproduced in <60% of bootstrap replicates were collapsed in the

phylogenetic trees.

Plasmid construction

The coding sequence of NbS00000562g0002.1 was amplified by PCR using Phusion

DNA polymerase (Thermo) and primers listed in Supplemental Table S1. Purified

PCR fragments were cloned into the pENTR/D-TOPO® vector (Invitrogen) and

verified by sequencing. The correct PCR fragments were subcloned into the binary


173

vector pSol2095 via LR reaction using Gateway® LR ClonaseTM II (Invitrogen).

Resulting plasmids were transformed into Agrobacterium tumefaciens strain GV3101.

Gene fragments used to generate silencing constructs were synthesized by Eurofins

Genomics (Supplemental file S1), and were subsequently cloned into the gene-

silencing vector pTRV-RNA2. pTRV-RNA2 derivatives and pTRV-RNA1 vectors were

transformed into A. tumefaciens strain GV3101.

Plant growth conditions

N. benthamiana and tomato (cultivar Moneymaker) were grown in soil in a

conditioned greenhouse at 19-21°C with a 16 h photoperiod and at a relative

humidity of 75-78%. Supplementary light (100 W m-2) was applied when the light

intensity dropped below 150 W m-2.

Agro-infiltration and TRV-mediated silencing assays

A. tumefaciens strains carrying binary vectors were grown overnight at 28°C in Yeast

Extract Broth with appropriate antibiotics. A. tumefaciens cells were pelleted,

resuspended and incubated in an induction medium (10 mM MES, 10 mM MgCl2,

50 μM acetosyringone, pH 5.6) for 3-4 h, and thereafter for 1 h in infiltration medium

(10 mM MES, 10 mM MgCl2, 200 μM acetosyringone, pH 5.6). For gene silencing,

A. tumefaciens cultures carrying pTRV-RNA2 constructs and A. tumefaciens carrying

pTRV1 were mixed at a ratio of 1:1 to a final OD600 of 1.0 and 2.0 before infiltration

into cotyledons of two-week-old N. benthamiana and ten-day-old tomato seedlings,

respectively. TRV:PDS and TRV:GUS were used as controls. For transient gene

expression, A. tumefaciens suspensions with appropriate concentrations were

syringe-infiltrated into N. benthamiana leaves. Co-infiltration with the silencing

suppressor P19 was performed at a ratio of 1:1.


For each silencing construct, six leaves (the fifth and sixth true leaves) from three

individual plants were harvested and ground in liquid nitrogen. Total RNA was

isolated from 100 mg ground sample with the NucleoSpin RNA Plant kit (Macherey-

Nagel), and subsequently used as template for first strand cDNA synthesis using a

M-MLV reverse transcriptase kit (Promega) with an oligo-dT primer (Supplemental

Table S1). qRT-PCR was performed as described by Wang et al. (2014) using Actin

as endogenous control (Supplemental Table S1). Relative expression levels reflect

fold-changes compared to control.

Phytophthora cultivation and infection assays

Culturing of P. capsici isolate LT263 and P. infestans isolate 14-3-GFP and

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production of zoospores were performed as previously described (Champouret et al.,

2009; Wang et al., 2013; Bouwmeester et al., 2014).

N. benthamiana and tomato leaves were collected three-to-four weeks after TRV-

treatment or two days after agro-infiltration, and placed in water-saturated floral foam

in trays as described by Vleeshouwers et al. (1999). Leaves were inoculated on the

abaxial sides with fresh mycelial plugs (Ø 0.5 cm), or 10 µl droplets containing 1*105

ml-1 P. capsici zoospores or 5*105 ml-1 P. infestans zoospores. Inoculated leaves

were kept in transparent plastic boxes to maintain high humidity and placed in a

climate chamber with a 12 h photoperiod and appropriate temperature settings.

Boxes were kept the first 24 h in the dark. Lesion diameters were measured for

P. capsici at 3 dpi and for P. infestans at 4 or 6 dpi. Lesion sizes were calculated as

previously described (Vleeshouwers et al., 1999).

ACKNOWLEDGEMENTS

We thank Harold Meijer and Luigi Faino for discussion, Michael Seidl, Huchen Li and

David Nsibo for assistance, Patrick Smit and Daniela Sueldo for providing the

pSol2095 vector and GFP-HA construct, and Henk Smid and Bert Essenstam at

Unifarm for excellent plant care. This research was supported by a Wageningen

University sandwich-PhD fellowship (YW), a Huygens scholarship (YW), a VENI

grant (KB) from The Netherlands Organization for Scientific Research (Technology

Foundation NWO-STW) and the Food-for-Thought campaign of the Wageningen

University Fund.

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177

SUPPLEMENTAL INFORMATION


Primers 5'-3' Used to

NbS00026192g0010.1-F TCTCCCCTTAGTCTGTCAGTT Amplify the coding region of

NbS00026192g0010.1 and sequencing NbS00026192g0010.1-R ACAATCCACCGAACAAAACCA

NbLecRK-VIII.2-RT-F CTGACCCTATGGTTAGACCAACAATG Check the silencing efficiency of TRV:NbVIII.2

NbLecRK-VIII.2-RT-R GAAGGTCTAGTTCTTGGGACAATTGG

NbLecRK-IX.1a-RT-F TGGTGGTTGTGTTTTGGTTTTTGTATGTG Check the silencing efficiency of TRV:NbIX.1a

NbLecRK-IX.1a-RT-R GTAAAGGAACTTCTTTGGTCCTGTGC

NbLecRK-IX.1/2-RT-F CAAATGGAGCGTTTAATGGTTCTCG Check the expression of NbS00000562g0002.1

NbLecRK-IX.1/2-RT-R TCAGAATTAAGAACATGGATTGCTTGC

NbLecRK-IX.1c-RT-F TGATCTTGAAAAGGGAACAGGACCG Check the silencing efficiency of TRV:NbIX.1c

NbLecRK-IX.1c-RT-R TTGTAAACTTCTCCAAATCCTCCTTCG

NbLecRK-S.1-RT-F GTTCTCTAAGGATGAGCTACTTGGG Check the silencing efficiency of the TRV:NbS.1

NbLecRK-S.1-RT-R GAGTCATGGTTCACACATTTCACTGC

NbLecRK-S.6-RT-F CGTCTTTTCTGGCGTTCCAACTG Check the silencing efficiency of TRV:NbS.6

NbLecRK-S.6-RT-R CCACATTCATCGATAGGCTTTCTCC

NbLecRK-S.7-RT-F ATGGGATACCTTGCTCCTGAGTACC Check the silencing efficiency of TRV:NbS.7

NbLecRK-S.7-RT-R CCCACAAGCCACTTCTAGTATAACCAC

Nb00562-FL-F CACCATGGTTTTCAGCAAAAT Amplify the coding region of NbLecRK-IX.1/2

Nb00562-R CAAAAGTGATTTTGTTGACG

SolyLecRK-VIII.2-RT-F CTAGCTTGTTCACATCCCGACC Check the silencing efficiency of TRV:SlVIII.2

SolyLecRK-VIII.2-RT-R GAAGGTCTAGTTCTTGGGACAATTGG

SolyLecRK-IX.1-RT-F TCAGAGGAACGAAAATTAGGTGAAGG Check the silencing efficiency of TRV:SlX.1

SolyLecRK-IX.1-RT-R TCCCCTGCTTAGACCCTCTCG

SolyLecRK-S.1-RT-F GGGTTCTCTAAAGATGAGCTACTTGG Check the silencing efficiency of TRV:SolyS.1

SolyLecRK-S.1-RT-R GAGTCATGGTTCACACATTTCACTGC

SolyLecRK-S.6-RT-F GCTGGAACAATGGGATATTTTGCACC Check the silencing efficiency of TRV:SlS.6

SolyLecRK-S.6-RT-R GATGCCACTTCCAGTACTACAACACC

SolyLecRK-S.7-RT-F AGACCAATTGAAGGAGAAGGTACTGG Check the silencing efficiency of TRV:SlS.7

SolyLecRK-S.7-RT-R GCCTCTTGTCTGCTGCATCG


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Figure S1. Neighbor-Joining trees constructed based on lectin domain (A) and kinase domain

(B) of 43 AtLecRKs, 38 NbLecRKs and 22 SlLecRKs.

Nbc25369236g0004.1 was excluded in (A). Branches of the phylogenetic trees were colored

according to the clades delineated by Bouwmeester and Govers (2009). Numbers above branches

represent the level of clade support inferred by 10,000 bootstrap replicates.


179

Figure S2. Morphology of N. benthamiana plants treated by TRV:NbLecRKs, TRV:PDS and

TRV:GUS.

Plants were photographed three weeks after inoculation with the silencing constructs.

Chapter VI

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NbS00026192g0010.1 1315 CTTATGCCTAATGGGAGTCTTGATAAGGCATTATTTGAATCAAGAATGATTCTACCTTGG NbS00026087g0010.1 1315 CTAATGCCTAATGGGAGTCTTGATAAGGCATTATTTGAATCAAGAATGATTCTACCTTGG NbVIII.2 1 --------------------GAATTCGGCATTATTTGAATCAAGAATGATTCTACCTTGG NbS00016101g0013.1 1318 TTGATGCCAAATGGTAGTCTTGATAAGGCATTATTTGAGTCAAGAATGGTACTCCCATGG

NbS00026192g0010.1 1375 GTACATAGGAGGAAAATTTTGCTAGGTGTTGCTTCAGCCTTGGCATATTTACATCAAGAA NbS00026087g0010.1 1375 CTACATAGGAGGAAAATTTTGCTAGGTGTTGCTTCAGCCTTGGCATATTTACATCAAGAA NbVIII.2 41 CTACATAGGAGGAAAATTTTGCTAGGTGTTGCTTCAGCCTTGGCATATTTACATCAAGAA NbS00016101g0013.1 1378 CTACATAGGAGGAAAATTTTGTTAGGTGTTGCTTCAGCCTTAGCATATTTACATCAAGAA NbS00026192g0010.1 1435 TGTGAAAACCAGGTGATTCACAGGGATATAAAGAGTAGTAACATTATGTTGGATGAAGGG NbS00026087g0010.1 1435 TGTGAAAATCAGGTGATTCATAGGGATATAAAGAGTAGTAACATTATGTTAGATGAAGGG NbVIII.2 101 TGTGAAAATCAGGTGATTCATAGGGATATAAAGAGTAGTAACATTATGTTAGATGAAGGG NbS00016101g0013.1 1438 TGTGAAAATCAAGTTATGTTAAGGGATATTAAGACTAGTAATATTATGTTGGATGAAGGG

NbS00026192g0010.1 1495 TTCAATGCAAGATTAGGTGATTTTGGATTAGCAAGACAAGTTGAACATGACAAGTCCCCC NbS00026087g0010.1 1495 TTCAATGCAAGATTAGGTGATTTTGGATTAGCAAGACAAGTTGAACATGACAAGTCCCCC NbVIII.2 161 TTCAATGCAAGATTAGGTGATTTTGGATTAGCAAGACAAGTTGAACATGACAAGTCCCCC NbS00016101g0013.1 1498 TTTAATGCAAGATTAGGTGATTTTGGGTTAGCAAAACAAATTGAACATGACAAATCTCCT

NbS00026192g0010.1 1555 GATGTAACAGTAGCAGCTGGGACAATGGGCTACTTGGCTCCTGAATACTTGTTAACCGGA NbS00026087g0010.1 1555 GATGCAACGGTAGCAGCCGGGACAATGGGCTACTTGGCTCCTGAATACTTGTTAACCGGA NbVIII.2 221 GATGCAACGGTAGCAGCCGGGACAATGGGCTACTTGGCTCCTGAATACTTGTTAACCGGA NbS00016101g0013.1 1558 GATGCGACAGTAGCAGCAGGAACAATGGGATACTTAGCACCTGAATATTTATTAACCGGT

NbS00026192g0010.1 1615 AGAGCAACCGAAAAAACTGATGTTTTTAGCTATGGAGCTGTGGTTCTTGAAGTGGCAAGT NbS00026087g0010.1 1615 AGAGCAACCGAAAAAACTGATGTTTTTAGCTATGGAGCAGTTGTTCTTGAAGTGGCAAGT NbVIII.2 281 AGAGCAACCGAAAAAACTGATGTTTTTAGCTATGGAGCAGTTGTTCTTGAAGTGGCAAGT NbS00016101g0013.1 1618 CGAGCAAGTGAAAAAACTGATGCTTTTAGCTATGGCGCAGTGGTTCTTGAAGTGGCAAGT

NbS00026192g0010.1 1675 GGAAGGAGGCCAATTGAGAGGGAAACAACAAGAGTTGAGAAAGTTGGAGTGAAGAGTAAC NbS00026087g0010.1 1675 GGAAGGAGGCCAATTGAGAGGGAAACAACAAGAGTTGAGAAAGTTGGAGTGAATAGTAAC NbVIII.2 341 GGAAGGAGGCCAATTGAGAGGGAAACAACAAGAGTTGAGAAAGTTGGAGTGAATAGTAAC NbS00016101g0013.1 1678 GGGAGAAGGCCAATTGAGAAAGAAACAAATGGGGTTGGGAAAGTTGGATTAAATAGCAAT

NbS00026192g0010.1 1735 TTAGTTGAATGGGTTTGGGGATTGCATAGAGAAGGGAATTTGCTAATGGCAGCAGATTCA NbS00026087g0010.1 1735 TTAGTTGAATGGGTTTGGGGATTGCATAGAGAAGGGAATTTGCTAATGGCAGCTGATTCA NbVIII.2 401 TTAGTTCCCGGG------------------------------------------------ NbS00016101g0013.1 1738 TTGGTGGAATGGGTTTGGGGATTACATAAAGAAGGGAGGTTATTAGCAGCAGCTGATTCA

NbS00026192g0010.1 1855 TGTTCACAACCTGACCCTATGGTTAGACCAACAATGAGAAGTGTGGTCCAAATGCTAGTA

NbS00026087g0010.1 1855 TGTTCACAACCTGACCCTATGGTTAGACCAACAATGAGAAGTGTGGTCCAAATGCTAGTA NbVIII.2-2 1 ------------GACCCTATGGTTAGACCAACAATGAGAAGTGTGGTCCAAATGCTAGTA NbS00016101g0013.1 1858 TGTTCACACCCTGACCCTATGGCTAGACCAACAATGAGAGGTGTGGTTCAAATGTTAGTT NbS00026192g0010.1 1915 GGTGAAGCTGAAGTTCCTATTGTCCCAAGAACTAAGCCTTGTATGAGTTTCAGCACATCA NbS00026087g0010.1 1915 GGTGAAGCTGAAGTTCCTATTGTCCCAAGAACTAAGCCTTCTATGAGTTTCAGCACATCA NbVIII.2-2 49 GGTGAAGCTGAAGTACCTATTGTCCCAAGAACTAAGCCTTCTATGAGTTTCAGCACATCA NbS00016101g0013.1 1918 GGTGAGGCTGAAGTTCCAATTGTCCCTAGAGCTAAGCCAACTATGAGTTTTAGCACATCT NbS00026192g0010.1 1975 CATCTTCTAATGACTTTGCAAGATAGTGTTTCTGATTTGAATGGTATGATCACACTTTCC NbS00026087g0010.1 1975 CATCTTCTAATGACTTTGCAAGATAGTGTTTCTGATTTGAATGGTATGATCACACTTTCA NbVIII.2-2 109 CATCTTCTAATGACTTTGCAAGATAGTGTTTCTGATTTGAATGGTATGATCACACTTTCC NbS00016101g0013.1 1978 CATCTGCTAATGACTTTGCAAGATAGTGTTTCTGACTTGAATGGTATGATCACACTTTCC NbS00026192g0010.1 2035 TCTTCATCATCTGAAAACAGTTTCACTGGTGTTGCCAATGGGATGGACTTGGTCTAA NbS00026087g0010.1 2035 ACTTCATCATCTGAAAACAGCTTCAATAGTGCTGGCAATGGCATGGACTTGGTCTAA NbVIII.2-2 169 ACTTCATCATCTGAAAACAGTTTCACTGGTGTTGCCCATGGGATGA---TGGA---- NbS00016101g0013.1 2038 ACGTCTTCATCTGAAAATAGCTTCAATGGTGGTGGTGTGGGTATGGACCTAGTCTAG

Figure S3. Sequence alignment of NbS00016101g0013.1, NbS00026192g0010.1, NbS00026087

g0010.1 and the fragments used for silencing.

NbVIII.2 represents the fragment used in this study while NbVIII.2-2 represents the fragment retrieved

according to Kanzaki et al. (2008).


181

Figure S4. Quantified lesion sizes on SlLecRK-silenced tomato leaves after inoculation with P. capsici

(mycelial plugs Ø 0.5 cm) or P. infestans (5*105 zoospores ml-1). Bars represent the mean lesion size

(± SE). Each experiment included at least twenty leaves silenced for each construct. * indicates

significant difference in lesion sizes (p < 0.05) between TRV:GUS and TRV:SlLecRK-IX.1-treated

plants according to a t test. This experiment was repeated twice, with similar outcome for P. capsici but

not for P. infestans.

Supplemental file 1. DNA sequences of the fragments used for TRV-mediated silencing in

N. benthamiana and tomato.

>TRV:NbVIII.2/TRV:SlVIII.2

gaattcggcattatttgaatcaagaatgattctaccttggctacataggaggaaaattttgctaggtgttgcttcagccttggcatatttacatcaaga

atgtgaaaatcaggtgattcatagggatataaagagtagtaacattatgttagatgaagggttcaatgcaagattaggtgattttggattagcaag

acaagttgaacatgacaagtcccccgatgcaacggtagcagccgggacaatgggctacttggctcctgaatacttgttaaccggaagagcaa

ccgaaaaaactgatgtttttagctatggagcagttgttcttgaagtggcaagtggaaggaggccaattgagagggaaacaacaagagttgaga

aagttggagtgaatagtaacttagttcccggg

>TRV:NbIX.1a

gaattctaattccttttgcgacttcgctgtccttcaatttcgatagttttagacccagcgatcaaaatgtaacatacgagagagatgcttatccagcaa

atggtgcaattcaacttaccacagaccttatcaatcgtgatataaacgcaactataggtcgagccacatattccaagcttctgcatctttgggaca

aggcctcaggaaatgtcacagatttcagtactcacttctcctttagcatcaattcacaaggcagaacaagatatggtgatggtcttgccttcctgag

aacacaaccagaggtggcagtcttggccatacaagtaatactcaacgactgaatacatcggccaatcattttgtagctgtggagtttgatacgta

ccagaacgtccagtatgatcccggg

>TRV:NbIX.1b

gaattcctcagcatcaactggacaattgtttcagaaaaacaatgtcaagtcttgggatttcaattcaagtatgagttttgatccaaacaaagttcctg

aacatgctgaaccgccgagtgcaaatcctcctattactcaagttactcctccttctaaccaagaaatcagcaccagaaaaaaaggaaataagg

gactagtcgttggatcgagcataggtttgcctattctggttatcgtattgattactggtagctgctttttatggaagaaaaagagtaaaaggaatgata

Chapter VI

182

aagaccatgtttttattgatctcagtatggacaatgaatttcaaaagggtactggccctaagaagttttcctatggtgaattagctcgtgcaacgaac

aattttgctgaggtacagcccggg

>TRV:NbIX.1c

gaattccgattcaagttttaatgtcaagctcggtgattttggcttagctagactaatggaccatgaattaggtcctcagactacagggttggctggaa

ctttaggttatttggctcctgaatacataaaaacaggccgagcaagtaaagagtcagacgtatacagcttcggaatagttgcacaagaaattgc

aaccggaagaaaatcagttgatccggggacggggaaatctgatgcagtgttagtggagtatgtttgggagctttataaaggacaacttctttctg

ctgttgatgagaaattaaacctggattttgacgcgaaacaagtagagcgattgatggctaccaggttatggtgtgctcatccggagagcaatctg

aggccatctataagacaagcaattcccggg

>TRV:SlIX.1

ggatccgacatggattacttacaatgctacaacaaataatcttagtgtcatttggaactatggaacaggtccgaattcgagcatgttttacataata

aacctcagagatgttctgcctccgtgggtcacaattggattctctgctgcaacaggactaaatgtcgagagacatacacttgaatcgtgggaattc

agctcgagtcttgttataacagagttaggtggaaatgatcgcgaaaagattggacttattgcagggctaacaacattaggtgggattttgttagtga

gcgcgattttggctttgatagttttgaggaaacgtagacgaaaggtgaagggaaatccagagacaataagcttaacatctttcaatgatgatcttg

aaaagggggcaggaccaagacccggg

>TRV:NbS.1/TRV:SlS.1

gaattcatggaagtctgaataaatggatatttgataagccagagaaggttatgaattggctagacaggaggaaggtcctaactgatgttgctgag

ggtttaaactatttacatcatggttgggaacaagtggttgtacatagggatattaaatctagcaatgtcttgttagattgtgaaatgagagggagact

gggagatttcgggctagcaaagttatacactcacggtggtgtaccaaatacaactagggtagtaggtacattagggtacttggcacctgaagttg

tgacaagggctacaccaactgcagctagtgacccggg

>TRV:NbS.6/TRV:SlS.6

gaattcttgtatatggtggcttagctgcagttgttacacttgttgttggtattctggctattgtgtctgtttttgtgttaaggagaaaaaaaagggatggta

gtagaggggaaaatgaaggtcaaatgtgtagattccaaggaaatagagtgcctcaaagattgtcattatctgaaataaaatcagccacagaa

ggatttaataatgaaaggataattggtgaaggaggatctgctgttgtatatgaaggagatattccttctaagggaactgttgctattaagagatttgtt

caagggactagattaggtccttcacatattccatttaacactgaatttgcttctatggttggctgcttaagacacaagaatttgattcagttccaagga

tggtgttgtgagagcccggg

>TRV:NbS.7

gaattcatttggagatggtttggcttttttcctttcacctgataatcagactttaggtagtccaggtggtttcttgggtctggttaattcatcccaattgacta

agaacaagtttgttgctgttgagttcgacacttggcaagatttgcactttaatgatcctgatgataaccatgtaggtcttgatatagatagccttatctc

aatcaagactgctaatgtaaagttggctggtgttgatctcaaaagtaggaatttgattacttcttggattgattacaagagtgaggaaaagcagttgt

ttgtgttcttgagttactcaagttccaagcctaaaaaaccactcttgactgtggatattgacctgtctgattatctaaaggagtttatgtacgtggggttt

gctcccggg

>TRV:SlS.7

gaattcaagacttcaaagaggaagagatgaaaaagttgctacttgttggactgagctgtgcaaatcctgatagcacagaaaggccttgtatga

ggagagtatttcagatactcaacaatgaggcggaacctatctttgttccgaaagtgaaaccaactctaactttctccactagcatcccgttcaacat

tgatgacattttctcagacagtgaagggagtgaggcaccagaacatgagctcgaaatcagaatacccggg

Chapter VII

General discussion

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184

INTRODUCTION Receptor-like kinases (RLKs) form one of the largest families in plants, with over 600

and 1100 members in Arabidopsis and rice, respectively (Shiu and Bleecker, 2001a;

Shiu et al., 2004). The RLK family is dramatically expanded in plants when compared

to other organisms, such as animal, fungi and protists (Shiu and Bleecker, 2001b;

Shiu and Bleecker, 2003). Within the Arabidopsis RLK family, over 400 members

contain a domain configuration resembling a transmembrane receptor composed of

an extracellular putative ligand binding domain and a cytosolic kinase domain which

transduces signals through phosphorylation of downstream signaling components

(Antolin-Llovera et al., 2012; Kadota et al., 2014; Li et al., 2014). Plant RLKs have

been implicated in many different processes, including development, growth,

hormone perception and response to abiotic and biotic stresses (De Smet et al.,

2009). One of the most important determinants for plant adaptation is the capacity to

perceive environmental stimuli and initiate defence responses accordingly. Several

RLKs are capable to recognize conserved microbial signatures, named microbe-

associated molecular patterns (MAMPs), or molecules derived from plants

themselves upon pathogen attack, the so-called damage-associated molecular

patterns (DAMPs). This type of RLKs is also known as pattern recognition receptors

(PRRs).

One example that has been well-characterized with respect to ligand

perception and defence activation is FLS2, a RLK with an extracellular domain

composed of 28 tandem leucine-rich repeats (LRRs). FLS2 plays an important role in

perception of bacterial pathogens by detecting a conserved epitope of flagellin,

referred to as flg22. Perception occurs via the direct binding of flg22 to the LRR

domain of FLS2 and this leads to the rapid recruitment of BAK1, another RLK which

acts as a co-receptor for FLS2 to bind flg22 (Sun et al., 2013). This intermolecular

interaction leads to the rapid phosphorylation of the cytoplasmic kinase BIK1, which

constitutively interacts with FLS2 and BAK1 (Lu et al., 2010). In turn, BIK1 is

released from the FLS2 complexes and directly binds and phosphorylates the

NADPH oxidase RbohD to activate rapid production of ROS (Kadota et al., 2014; Li

et al., 2014).

RLKs vary greatly in their extracellular domain. Based on the diverse

extracellular domains, RLKs were classified into different subfamilies (Shiu and

General discussion

185

Bleecker, 2001a). Both FLS2 and BAK1 belong to LRR-RLKs, the largest subfamily

which consists of 235 members in Arabidopsis with varying number of LRRs (Shiu

and Bleecker, 2001a). Thus far, four LRR-RLKs in Arabidopsis have been found to

function as PRRs, i.e. FLS2, EFR, PEPR1 and PEPR2 (Huffaker et al., 2006; Zipfel,

2006; Yamaguchi et al., 2010). Another two members, i.e. BAK1 and SOBIR1, act as

co-receptors of multiple RLKs or receptor-like proteins (RLPs) (Chinchilla et al., 2009;

Liebrand et al., 2014). Several other LRR-RLKs, such as ERECTA, are also involved

in plant immunity, but the exact activation mechanisms are yet unknown (Sanchez-

Rodriguez et al., 2009). Next to the LRR-RLK subfamily, a few other large subfamilies

have been implicated in plant defence. One such family comprises the L-type lectin

receptor kinases (LecRKs) which are characterized by an legume lectin-like

extracellular domain (Shiu and Bleecker, 2001a; Barre et al., 2002; Bouwmeester

and Govers, 2009). This chapter focuses on the role of LecRKs in plant defence and

their potential to be exploited for disease resistance management in crops.

LecRKs exhibit species-specific expansions in plants

LecRKs are present in many different plant species, but have so far not been found in

the genome sequences of yeast (Saccharomyces cerevisiae), human or green algae

(Shiu and Bleecker, 2001b; Navarro-Gochicoa et al., 2003; Lehti-Shiu et al., 2009).

The available plant genome sequences enable the identification of LecRKs on a

genome-wide scale. The Arabidopsis genome encodes 45 LecRKs with 41 containing

the typical LecRK composition and four either lacking the signal peptide, the lectin

domain or the transmembrane domain (Bouwmeester and Govers, 2009). A

comparable number of LecRKs, i.e. 38 LecRKs were identified in

Nicotiana benthamiana (Chapter VI) while only about half of that number was found

in the genome of cucumber (Cucumis sativus) and tomato (Solanum lycopersicum),

i.e. 25 and 22, respectively (Wu et al., 2014; Chapter VI). In the monocot rice (Oryza

sativa), in total 69 full length LecRKs were identified (Vaid et al., 2012). Also for a few

other plant species, LecRKs have been reported but not on a genome-wide scale,

such as Gossypium hirsutum (GhLecRK-2), Medicago truncatula (i.e. MtLecRK1;1,

MtLecRK7;1, MtLecRK7;2 and MtLecRK7;3), Populus nigra (PnLPK), Pisum sativum

(PsLecRLK), (Nishiguchi et al., 2002; Navarro-Gochicoa et al., 2003; Joshi et al.,

2010; Phillips et al., 2013).

In Arabidopsis, the 45 LecRKs were classified into nine clades (I to IX) and

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186

seven singletons based on sequence similarity and phylogenetic analyses

(Bouwmeester and Govers, 2009). Similar analyses were performed on LecRKs of

Arabidopsis and two Solanaceous plants, i.e. N. benthamiana and tomato, which

revealed that Solanaceous plants have no LecRKs belonging to clades I, II, III and V

but contain another clade that is absent in Arabidopsis (Chapter VI). To gain a better

insight into the divergence of LecRKs in plants, we compared LecRKs of the dicot

plants Arabidopsis (AtLecRKs), N. benthamiana (NbLecRKs) and tomato (SlLecRKs)

with those of the monocot rice (OsLecRKs) and constructed a phylogenetic tree (Fig.

1). Of the 69 OsLecRKs, the majority does not group into the clades defined based

on the Arabidopsis sequences. None of the OsLecRKs fall into the four Arabidopsis-

specific clades or the Solanaceous-specific clade. However, an extensively expanded

clade was identified with 36 OsLecRKs which is closest to Arabidopsis clade IV.

Similar to what has been found for Arabidopsis-specific clades (Bouwmeester and

Govers, 2009; Chapter VI), OsLecRKs in this clade exhibit high frequency of tandem

duplications based on the definition that tandem duplicated genes are those within 10

genes apart in genome (Lehti-Shiu et al., 2009). Hence, the expansion of this rice-

specific LecRK clade seems to depend largely on tandem duplication events. Another

large cluster of 14 OsLecRKs groups with clade IX indicating that clade IX is an

ancient clade that arose before the separation of monocots and dicots. Yet, the

increased number of clade IX LecRKs found in rice (14), N. benthamiana (6), and

tomato (5) compared to Arabidopsis (2) indicates independent gene loss or gain

events after the monocot-dicot split. This is also the case for the clade comprising the

Arabidopsis singleton LecRK-S.5 homologs, which includes seven OsLecRKs, six

NbLecRKs and three SlLecRKs. OsLecRKs are also present in the clades comprising

clade VI LecRKs, and the Arabidopsis singletons LecRK-S.6 and LecRK-S.7, as are

NbLecRKs and SlLecRKs. Hence these clades are also conserved in both monocot

and dicot plants, but as yet there are no indications for species-specific gene

expansions. Collectively, these data indicate that the LecRK family is widespread in

different plant species, and underwent species-specific expansions.

General discussion

187

Figure 1. Phylogenetic analysis of LecRKs belonging to four plant species.

Full-length protein sequences were aligned in ClustalW using the weight matrix GONNET with a

penalty gap opening of 10 and a gap extension of 0.1. The alignment was subsequently used to

construct a Neighbor-Joining tree with 1,000 bootstrap replicates using the Jones-Taylor-Thornton

substitution model (Hall, 2013) in MEGA 5.1. Branches are colored according to the clades delineated

by Bouwmeester and Govers (2009), and branches with less than 60% bootstrap support are

collapsed. Arabidopsis LecRKs (AtLecRKs) were downloaded via TAIR10 and N. benthamiana and

tomato LecRKs (i.e. NbLecRKs and SlLecRKs) from the Sol Genomics website (Chapter VI). The rice

LecRKs (OsLecRKs) are the ones defined by Vaid et al. (2012) and were downloaded from Rice

Genome Annotation Project website (http://rice.plantbiology.msu.edu/). The tree includes 43

AtLecRKs, 38 NbLecRKs, 22 SlLecRKs and 69 OsLecRKs.

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188

LecRKs function as PRRs

Emerging evidence indicates that the extracellular domains of RLKs determine ligand

binding specificity (Liu et al., 2012; Sun et al., 2013; Macho and Zipfel, 2014). The

extracellular lectin domains of LecRKs resemble the soluble carbohydrate-binding

legume lectins (Shiu and Bleecker, 2001a; Barre et al., 2002). Legume lectins are

metalloproteins with both a Ca2+ and a transition metal ion (usually Mn2+)

incorporated in their structure which are essential for the carbohydrate-binding (van

Damme et al., 2008; Etzler, 2009). Hence, the corresponding ion-binding residues

that form the carbohydrate-binding sites are extremely conserved among the legume

lectins (Etzler, 2009). However, structural analysis and molecular modelling revealed

that the lectin domains of Arabidopsis LecRKs are poorly conserved in the ion-

binding residues but contain a conserved hydrophobic cavity, which was proposed to

bind hydrophobic ligands, such as tryptophan and auxin (Barre et al., 2002). So far,

for only one LecRK, i.e. LecRK-I.9 from Arabidopsis, potential ligands that interact

with the extracellular lectin domain have been reported. Arabidopsis LecRK-I.9 was

first identified as a candidate host target of the Phytophthora infestans RXLR effector

IPI-O, and more recently as the first plant extracellular ATP (eATP) receptor (Gouget

et al., 2006; Bouwmeester et al., 2011a; Choi et al., 2014).

IPI-O belongs to a large family of oomycete effectors that shares the host cell

targeting motif RXLR (Champouret et al., 2009). It also contains an RGD (R:

arginine; G: glycine; D: aspartic acid) motif ‒ that partially overlaps with the RXLR

(RXLRGD) motif ‒which is well-known to interfere with cell adhesion in animals and

to disrupt cell wall (CW)-plasma membrane (PM) adhesions in plants (Ruoslahti,

1996; Mellersh and Heath, 2001; Senchou et al., 2004; Bouwmeester et al., 2011b).

Screening of a phage library for peptides binding to the RGD motif of IPI-O identified

two heptamers, i.e. IHQASYY and AAQPHPR which can inhibit the RGD-binding

activity of plasma membrane proteins (Gouget et al., 2006). Both heptamers were

then used to screen the Arabidopsis proteome for plasma membrane proteins with

potential RGD-binding activity. In this way, Arabidopsis LecRK-I.9 was identified as

one of the candidates (Gouget et al., 2006). The capacity of the LecRK-I.9 lectin

domain to bind IPI-O was further confirmed by binding and photoaffinity labelling

assays. The RGD-binding activity of LecRK-I.9 raised the question to what extent

LecRK-I.9 plays a role in mediating CW-PM adhesion. Bouwmeester et al. (2011a)

found that Arabidopsis LecRK-I.9 mutants show disrupted CW-PM integrity, a

General discussion

189

phenomenon that was also found in transgenic Arabidopsis plants with constitutive

expression of ipiO. Taken together, these results indicate that LecRK-I.9 protects the

CW-PM continuity via binding to the RGD-proteins.

Extracellular ATP is a well-known signaling agent that has been implicated in

diverse physiological processes in animals (Bours et al., 2006). Also in plants, eATP

was found to be an important signaling molecule that regulates various cellular

responses (Cao et al., 2014). For example, ATP treatment rapidly triggers elevation

of cytosolic Ca2+ concentration and production of reactive oxygen species in plants

(Demidchik et al., 2009). In animals, eATP signals through the plasma membrane-

localized purinoceptors, including ligand-gated nonselective cation channels (P2X

receptors) or G-protein-coupled receptors (P2Y receptors) (Ralevic and Burnstock,

1998). Identification of plant eATP receptors based on sequence homology to animal

purinoceptors failed to obtain any suitable candidates (Tang et al., 2003; Choi et al.,

2014). It is conceivable that eATP signals through different types of receptors in

plants. Using a forward genetic screening of Arabidopsis mutants that are defective in

response to ATP, Choi et al. (2014) identified LecRK-I.9 and demonstrated its

function as an eATP receptor. Arabidopsis LecRK-I.9 mutants show defects in Ca2+

response upon treatment with ATP or ADP, but not upon treatment with the pyrimidine

nucleotide CTP, biotic elicitors (i.e. flg22, chitin, elf26 and pep1) or abiotic stresses

(i.e. NaCl, mannitol and cold water) (Choi et al., 2014). In vitro binding assays

indicated that the extracellular lectin domain of LecRK-I.9 has a relative high binding

affinity for ATP (Kd ∼ 46 nM) (Choi et al., 2014). ATP can be released from plants

upon pathogen attack or exposure to other stress stimuli, in particular upon wounding

(Tanaka et al., 2010; Clark and Roux, 2011). It has been documented that the

released eATP in Arabidopsis upon wounding can reach a concentration up to 45 µM

(Song et al., 2006). Transcriptome analysis revealed that 60% of the genes induced

upon treatment with eATP were also induced by wounding (Choi et al., 2014). Based

on these findings, Choi et al. (2014) proposed a model in which eATP acts as a

damage-associated molecular pattern (DAMP) that is recognized by LecRK-I.9 to

initiate downstream responses, including increased cytosolic Ca2+ concentration

(Song et al., 2006; Choi et al., 2014). Given the fact that LecRK-I.9 is absent in

multiple plant species, its function in eATP perception may be taken over by other

LecRKs.

The fact that LecRK-I.9 binds, on the one hand, IPI-O and on the other hand

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eATP, raises many questions on how these two processes are regulated. For

example, what is the exact ATP-binding site(s) in the lectin domain? Is IPI-O

competing with eATP for LecRK-I.9 binding and in turn interfering with eATP-

mediated downstream signaling? In animals, the eATP binding sites on the P2X and

P2Y receptors have been well investigated (Chataigneau et al., 2013). However, this

information seems not helpful to understand the eATP binding in plants since LecRK-

I.9 is a different type of eATP receptor. It is expected that a combination of

crystallography and site-directed mutagenesis will lead to the identification of the

eATP binding site(s) in LecRK-I.9. This will unravel the mechanisms underpinning

eATP binding and activation of downstream signaling. The binding of IPI-O to LecRK-

I.9 is highly dependent on the RGD motif and it was found that mutation of RGD to

RGA abolished the binding (Gouget et al., 2006). Hence, eATP binding assays with

Arabidopsis protoplasts derived from transgenic Arabidopsis plants expressing ipiO

or ipiORGA may give some clues on whether IPI-O competes with eATP for LecRK-I.9

binding. In addition, by monitoring the response of ipiO/ipiORGA expressing

Arabidopsis lines upon ATP treatment may answer the question whether IPI-O affects

eATP-mediated signal transduction.

LecRKs function in defence against various biotic agents

Maintenance of CW-PM continuity has been found to be essential for plant defence

responses triggered by pathogens (Kiba et al., 1998; Mellersh and Heath, 2001). The

role of LecRK-I.9 in mediating CW-PM adhesions was an indication that LecRK-I.9 is

involved in plant defence. Indeed, Arabidopsis LecRK-I.9 mutants were found to be

compromised in cell-wall-associated defence response, i.e. reduced callose

deposition upon treatment with the Pseudomonas syringae (Pst) hrcC mutant or flg22

(Bouwmeester et al., 2011a). Moreover, LecRK-I.9 expression is induced upon

infection by Phytophthora brassicae. LecRK-I.9 mutants showed gain of susceptibility

to non-adapted P. brassicae isolates and accordingly overexpression lines showed

enhanced resistance to a virulent P. brassicae isolate CBS686.95 (Bouwmeester et

al., 2011a). Besides LecRK-I.9, several Arabidopsis LecRKs are differentially

expressed upon treatment with pathogens or elicitors according to in silico gene

expression analyses (Bouwmeester and Govers, 2009). Experimental evidence for a

function of other Arabidopsis LecRKs in defence was provided when Arabidopsis

LecRK T-DNA insertion lines showed compromised resistance to different pathogens

General discussion

191

(Wang et al., 2014/Chapter III). In this way, in total 14 LecRKs were found to have a

potential role in Phytophthora resistance. These 14 LecRKs fall into different clades

and display different expression patterns upon P. infestans infection. It should be

noted that in the screening of the T-DNA insertion lines, two phylogenetically distant

Phytophthora species were used for infection. None of the LecRKs mutants,

however, showed specificity to either species, suggesting that the 14 LecRKs confer

broad-spectrum Phytophthora resistance. Recognition of pathogens by receptors is

often correlated with diverse biochemical and cell biological changes (Boller and

Felix, 2009). It is difficult to figure out whether all these changes are involved in

limitation of pathogen colonization. For instance, overexpression of Arabidopsis

LecRK-IX.1 and LecRK-IX.2 induces plant cell death but also increases plant

resistance to Phytophthora pathogens (Chapter IV). Plant cell death could play an

essential role in arresting pathogen growth in early infection stages (Dickman and

Fluhr, 2013). However, Phytophthora resistance and cell death induction mediated by

both LecRK-IX.1 and LecRK-IX.2 appeared to be independent (Chapter IV). Both

LecRK-IX.1 and LecRK-IX.2 were found to interact with the ABC transporters

NbPDR1 in N. benthamiana and ABCG40 in Arabidopsis and the latter was found to

be a Phytophthora resistance component (Chapter IV). ABCG40 and its closest

homologs in Nicotiana plumbaginifolia and Nicotiana tabacum were found to be

required for transport of antimicrobial compounds such as ABA and diterpenes (Kang

et al., 2010; Seo et al., 2012). Hence, we speculate that LecRK-IX.1 and LecRK-IX.2

may interact with ABCG40 or NbPDR1 to activate the transport of antimicrobial

compounds that subsequently leads to Phytophthora resistance. The observation that

LecRK-IX.1 and LecRK-IX.2 are not interacting with each other reduces the chance

that LecRK-IX.1, LecRK-IX.2 and ABCG40 are present in one complex. Although

LecRK-IX.1 and LecRK-IX.2 function in a similar manner, knockout of both LecRKs in

Arabidopsis leads to increased susceptibility compared to the null mutants,

suggesting that these two LecRKs have an additive effect on Phytophthora

resistance (Chapter IV). Since clade IX is evolutionarily ancient, LecRKs belonging

to this clade seem to be important in plant adaptation. In line with this, the homologs

of LecRK-IX.1 and LecRK-IX.2 in N. benthamiana and tomato are also involved in

Phytophthora resistance, demonstrated a conserved biological function of clade IX

LecRKs across different plant species (Chapter VI).

Stomatal immunity plays an important role in limiting entry of bacterial

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192

pathogens into plants (Melotto et al., 2006). One of the Arabidopsis LecRKs, i.e.

LecRK-V.5, which is induced upon wounding and oligogalacturonic acid treatment,

was shown to negatively regulate plant stomatal immunity (Desclos-Theveniau et al.,

2012). LecRK-V.5 mutants are resistant to the hemibiotrophic bacterial pathogen Pst

DC3000, but only upon surface-inoculation, not by infiltration-inoculation (Desclos-

Theveniau et al., 2012). Mutation of LecRK-V.5 compromised stomatal reopening,

and accordingly overexpression of LecRK-V.5 resulted in increased stomatal

reopening when treated with Pst DC3000 or MAMPs (i.e. flg22, lipopolysaccharides

and the elongation factor epitope elf26). Upon MAMP treatment, LecRK-V.5 mutants

demonstrated normal apoplastic PTI responses, such as flg22-triggered ROS

production, bacteria-induced callose deposition and PTI-responsive marker gene

expression. However, LecRK-V.5 mutants showed constitutive increased ROS

production in the guard cells and this was found to be indispensable for the stomatal

closure (Desclos-Theveniau et al., 2012). Additionally, LecRK-V.5 was found to

negatively regulate stomatal closure mediated by abscisic acid (ABA) but not by

other hormones, such as salicylic acid or jasmonic acid (Desclos-Theveniau et al.,

2012). Collectively, these data support the negative role of LecRK-V.5 in mediating

stomatal immunity. In contrast, LecRK-V.5 was found to positively regulate

Arabidopsis resistance to Phytophthora pathogens (Wang et al., 2014/Chapter III).

The determinants for the multifaceted role of LecRK-V.5 in mediating resistance

against different pathogens is not yet known, but apparently LecRK-V.5-regulated

stomatal closure is not affecting Arabidopsis resistance to Phytophthora pathogens

since the penetration by Phytophthora pathogens into plants is not preferentially

occurring via the stomata (Roetschi et al., 2001; Wang et al., 2013). LecRK-VI.2,

which is required for BABA-primed defence, is another Arabidopsis LecRK found to

be important for bacterial resistance (Singh et al., 2012). In contrast to LecRK-V.5,

LecRK-VI.2 positively regulates stomatal closure upon MAMP treatment or bacterial

infection and this regulation is not related to ABA signalling. Upon bacterial infection

or treatment with MAMPs (i.e. flg22 or elf26), the LecRK-VI.2 mutant showed

compromised PTI-related responses, such as reduced callose deposition, defence-

related gene expression and reduced MPK3 and MPK6 activity (Singh et al., 2012).

Recently, LecRK-VI.2 was found to associate with FLS2 in a ligand independent

manner (Huang et al., 2014). However, mutation of LecRK-VI.2 in Arabidopsis did not

affect the flg22-induced early responses in plants, such as FLS2 and BAK1

General discussion

193

association, ROS production or BIK1 phosphorylation (Singh et al., 2012). Since

BIK1 was shown to be immediately activated upon recognition of flg22 (Lu et al.,

2010), these results suggest that either LecRK-VI.2 functions downstream of BIK1 or

that LecRK-VI.2-mediated defence responses signal through a BIK1-independent

pathway. In addition, LecRK-VI.2 is involved in mediating plant response to multiple

MAMPs (Singh et al., 2012), suggesting that LecRK-VI.2 is also involved in other

receptor complexes. Transcriptome profiling revealed that many defence-related

genes were up-regulated in Arabidopsis lines overexpressing LecRK-VI.2 (Singh et

al., 2012), however, LecRK-VI.2 likely functions specifically in resistance against

bacterial pathogens but not to others such as Phytophthora or fungal pathogens

(Singh et al., 2013; Huang et al., 2014; Wang et al., 2014/Chapter III). In addition to

the two aforementioned LecRKs, another three Arabidopsis LecRKs (i.e. LecRK-IV.4,

LecRK-S.1 and LecRK-S.4) were indicated to be involved in bacterial resistance

since the corresponding mutants showed increased susceptibility (Wang et al.,

2014/Chapter III).

LecRKs are also indicated to be involved in fungal pathogen resistance and

perception of insects. For example, overexpression of LecRK-IV.3 (alias AtLPK1)

increased Arabidopsis resistance to Botrytis cinerea (Huang et al., 2013). Infection

assays on Arabidopsis LecRK mutants with Alternaria brassicicola indicated that four

of the clade I LecRKs contribute to A. brassicicola resistance (Wang et al.,

2014/Chapter III). One of the four, i.e. LecRK-I.8, is also implicated in recognition of

eggs deposited by the cabbage butterfly Pieris brassicae (Gouhier-Darimont et al.,

2012). Expression of LecRK-I.8 is induced upon treatment with P. brassicae egg

extract, and the LecRK-I.8 mutant was shown to be defective in expression of the

defence-related gene PR1 upon treatment with egg extract.

Systematic analyses of LecRK function by monitoring response of Arabidopsis

LecRK mutants to different pathogens revealed function specificity of LecRKs, i.e.,

several LecRKs function in resistance against particular pathogens, while others are

implicated in resistance to multiple pathogens. Function specificity of LecRKs could

not be reconciled with the differences in gene expression profiles (Wang et al.,

2014/Chapter III), but may be determined by the recognition specificity of the

extracellular lectin domain. This idea was supported by the finding that the lectin

domain is indispensable for Phytophthora resistance mediated by the two clade IX

LecRKs (Chapter IV).

Chapter VII

194

Engineering LecRKs for broad-spectrum resistance: potentials and obstacles

LecRKs have several features that make them attractive as potential resistance

components. They are widespread in the plant kingdom, confer broad-spectrum

disease resistance and show conserved biological functions among different plant

species. A disadvantage, however, is that resistance conferred by LecRKs is

relatively mild when compared to the resistance mediated by intracellular nucleotide-

binding leucine-rich repeat proteins (NLRs), which is overall dominant and often

yields clear resistance phenotypes (van der Vossen et al., 2003; van der Vossen et

al., 2005). LecRK-mediated resistance often fails to arrest the infection by the

adapted pathogens. Hence, exploiting LecRKs for improving crop resistance is still a

major challenge. Since overexpression of LecRKs proved to be more effective

(Bouwmeester et al., 2011a; Singh et al., 2012; Chapter IV; Chapter VI), increased

resistance could be achieved by boosting the expression level of LecRKs.

Due to species-specific expansion of LecRKs, not all LecRKs which are

functional in plant defence have homologs present in different plant species (Chapter

VI). For example, LecRK-I.9 from the cruciferous plant Arabidopsis is absent in

N. benthamiana, tomato and rice. Therefore, transfer of LecRKs across plant families

provides a new avenue to increase disease resistance. Thus far, three Arabidopsis

LecRKs have been transferred into the Solanaceous plants and all of them proved to

maintain their function in plant defence. Ectopic expression of LecRK-I.9 in potato

(Solanum tuberosum) increases constitutive expression of protease inhibitor genes

and enhances resistance to Phytophthora infestans (Bouwmeester et al., 2014).

Similarly, ectopic expression of Arabidopsis LecRK-I.9 and LecRK-IX.1 in

N. benthamiana increases resistance to P. infestans (Chapter V). Although LecRK-I.9

and LecRK-IX.1 were identified for resistance against P. brassicae and P. capsici in

Arabidopsis, both confer resistance to P. infestans when transferred into

Solanaceous plants. P. infestans is phylogenetically distant from P. brassicae and

P. capsici and this again supports the idea that LecRKs do not discriminate between

Phytophthora species. N. benthamiana plants ectopically expressing Arabidopsis

LecRK-VI.2 showed increased response to flg22, − e.g. enhanced ROS production,

callose deposition, MAP kinase activity and defence-related gene expression − which

might account for the increased bacterial resistance detected in the transgenic lines

(Huang et al., 2014). The retained function of these LecRKs indicates that the

downstream signaling pathways are conserved between different plant families. As

General discussion

195

such, LecRKs have potential to be transferred into distantly related species to

increase disease resistance.

A disadvantage of ectopic/overexpression of defence-related genes is that it

often results in pleiotropic side effects that affect plant fitness (Gurr and Rushton,

2005). This is also the case for LecRKs. Overexpression of LecRK-I.9 in Arabidopsis

or in other plant species such as potato and N. benthamiana results in aberrant plant

development (Bouwmeester et al., 2011a; Bouwmeester et al., 2014; Chapter V).

The severity of morphological changes in transgenic potatoes was found to be

correlated with transgene expression levels. Similarly, expression of LecRK-IX.1 in

Arabidopsis or in N. benthamiana leads to plant cell death and this phenotype was

also found to be correlated with LecRK-IX.1 expression levels (Chapter IV; Chapter

V). To minimize side effects, LecRKs could be modified by using plant inducible

promoters that allow the fine-tuning of transgene expression (Gurr and Rushton,

2005). In the case of LecRK-IX.1 and LecRK-IX.2, their function in Phytophthora

disease resistance is independent of that in plant cell death induction (Chapter IV),

and therefore it should be possible to manipulate the signaling pathways to suppress

cell death induction without losing the LecRK-mediated disease resistance. Another

alternative is to identify the key residues in LecRKs controlling disease resistance

and cell death induction through random mutagenesis. Mutation of these key

residues is expected to improve the resistance and reduce the fitness costs

associated with expression of LecRKs.

Model pathosystems facilitate functional studies of LecRKs

Undoubtedly, functional analysis of LecRKs benefits from established model

pathosystems. Previously several pathosystems have been described in which the

interactions between Arabidopsis and different Phytophthora species were

characterized. In those studies, different Phytophthora isolates were tested on

Arabidopsis accessions to identify interaction specificity among accession-isolate

combinations. For Phytophthora parasitica and Phytophthora cinnamomi, the tested

isolates turned out to be either infectious on all the selected Arabidopsis accessions

or unable to infect any (Robinson and Cahill, 2003; Wang et al., 2011). In the case of

P. brassicae on Arabidopsis, various compatible and incompatible interactions with

Arabidopsis were detected among accession-isolate combinations (Roetschi et al.,

2001). Isolates with moderate virulence were identified that are virulent only on part

Chapter VII

196

of the selected accessions. For example, isolate HH is not capable to infect Col-0 but

causes disease on Ler-0. Moreover, it causes disease on Col-0 mutants defective in

biosynthesis of the anti-microbial secondary metabolites camalexin and indole

glucosinolates or on the LecRK-I.9 mutants (Schlaeppi et al., 2010; Bouwmeester et

al., 2011a). In Chapter II, we described a pathosystem between Arabidopsis and

P. capsici. We found an isolate, i.e. LT123, that behaves similarly as P. brassicae HH.

Although isolate LT123 is not capable to infect Col-0, it causes disease on a variety

of Col-0 mutants defective in defence-related signaling. Hence, P. capsici isolate

LT123 proved to be useful to distinguish essential components for plant resistance.

The prominent advantage of these model pathosystems is that we can make use of

the public Arabidopsis mutant resources. Screening of Arabidopsis T-DNA insertion

lines has been considered as an effective tool to link phenotype to gene function.

Disease phenotyping of a large set of LecRK T-DNA insertion lines using various

pathogens showing different levels of virulence identified several LecRKs that play a

role in resistance. In this way, we identified LecRKs with function specificity or varying

resistance capacity. Nevertheless, we still may have overlooked some LecRKs that

function redundantly with other genes or play a subtle role in plant defence.

Conclusions and prospective

This thesis describes our efforts to study LecRKs in different plant species to

understand their role in plant immunity. Although LecRKs are not as effective as the

classical NLR proteins that mediate effector-triggered immunity to stop pathogen

infection, the resistance provided by LecRKs was proved to be broad-spectrum

instead of race-specific. Therefore, LecRKs can provide an additional layer of

defence. Pyramiding LecRKs with classical R genes is expected to give a

quantitative synergistic effect that could lead to broad-spectrum disease resistance.

The first research article that made notice of a LecRK being a receptor for

monitoring the plant pathogen effector IPI-O dates from 2006 (Gouget et al., 2006).

Despite the increasing interest in LecRKs in recent years, our current understanding

on the molecular basis of LecRKs in plant defence is still in its infancy. To obtain a

comprehensive overview, many questions remain to be answered. For example, how

do these LecRKs fit into the defence signaling cascades? How are these LecRKs

activated to implement resistance function? Do other LecRKs besides LecRK-I.9

perceive eATP and are LecRKs receptors of DAMPs or MAMPs? Which components

General discussion

197

are required for LecRK-mediated signaling? Since interactions between plants and

pathogens involve bilateral communication, it is also necessary to understand how

pathogens respond to LecRK-mediated resistance and how pathogens with high

virulence suppress LecRK-mediated defence. It is anticipated that integration of

forward and reverse genetics, biochemistry approaches, bioinformatics and

pathogenomics will allow us to answer some the above-mentioned questions in the

near future.

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203

Summary

Phytophthora pathogens are notorious for causing severe damage to many

agriculturally and ornamentally important plants. Effective plant resistance depends

largely on the capacity to perceive pathogens and to activate rapid defence.

Cytoplasmic resistance (R) proteins are well-known for activation of plant immunity

upon recognition of matching effectors secreted by Phytophthora. However,

Phytophthora pathogens are notoriously difficult to control due to their rapid

adaptation to evade R protein-mediated recognition. Hence, exploring novel

resistance components is instrumental for developing durable resistance.

Receptor-like kinases (RLKs) function as important sentinels in sensing exogenous

and endogenous stimuli to initiate plant defence. One RLK that was previously

identified as a novel Phytophthora resistance component is the Arabidopsis L-type

lectin receptor kinase LecRK-I.9. This RLK belongs to a multigene family consisting of

45 members in Arabidopsis but whether or not the other members function in

Phytophthora resistance was thus far unknown. The research described in this thesis

was aimed at unravelling the role of LecRKs in plant immunity, in particular to

Phytophthora pathogens.

Chapter I describes various Phytophthora diseases and the current

understanding of the mechanisms underlying plant innate immunity with emphasis on

disease resistance to Phytophthora pathogens.

In Chapter II, we describe the development of a new Arabidopsis-Phytophthora

pathosystem. We demonstrated that Phytophthora capsici is capable to infect

Arabidopsis. Inoculation assays and cytological analysis revealed variations among

Arabidopsis accessions in response to different P. capsici isolates. Moreover, infection

assays on Arabidopsis mutants with specific defects in defence showed that salicylic

acid signaling, camalexin and indole glucosinolates biosynthesis pathways are

required for P. capsici resistance (Chapter IIa). The importance of these pathways in

Arabidopsis resistance was supported by the finding that the corresponding marker

genes are induced upon infection by P. capsici (Chapter IIb). This model pathosystem

can be used as an additional tool to pinpoint essential components of Phytophthora

resistance.

We then exploited Arabidopsis-Phytophthora pathosystems to uncover the role of

LecRKs in Phytophthora resistance. In Chapter III we describe a systematic

phenotypic characterization of a large set of Arabidopsis LecRK T-DNA insertion lines.

The T-DNA insertion lines were assembled and assayed for their response towards

different Phytophthora pathogens. This revealed that next to LecRK-I.9, several other

LecRKs function in Phytophthora resistance. We have also analysed whether the

Summary

204

LecRKs are involved in response to other biotic and abiotic stimuli. Several T-DNA

insertion lines showed altered responses to bacterial or fungal pathogens, but none of

the lines showed visible developmental changes under normal conditions or upon

abiotic stress treatment. Combining these phenotypic data with LecRK expression

profiles obtained from publicly available datasets revealed that LecRKs that are hardly

induced or even suppressed upon infection, might still have a function in pathogen

resistance. Computed co-expression analysis revealed that LecRKs with similar

function display diverse expression patterns.

Arabidopsis LecRK clade IX comprises two members. T-DNA insertion mutants of

both LecRK-IX.1 and LecRK-IX.2 showed gain of susceptibility to non-adapted

Phytophthora isolates and therefore the role of these two LecRKs in Phytophthora

resistance was further investigated. In Chapter IV we describe that overexpression of

either LecRK-IX.1 or LecRK-IX.2 in Arabidopsis resulted in increased resistance to

Phytophthora, but also induced plant cell death. A mutation in the kinase domain

abolished the ability of LecRK-IX.1 and LecRK-IX.2 to induce Phytophthora resistance

as did deletion of the lectin domain. Cell death induction however, only required the

kinase, not the lectin domain. Since transient expression of both LecRKs in

Nicotiana benthamiana also resulted in increased Phytophthora resistance and

induction of cell death, we used N. benthamiana to explore downstream components

required for LecRK-IX.1- and LecRK-IX.2-mediated Phytophthora resistance and cell

death. Virus-induced gene silencing of candidate signaling genes revealed that

NbSIPK1/NPT4 is essential for LecRK-IX.1-mediated cell death but not for

Phytophthora resistance. Collectively, these results illustrate that the Phytophthora

resistance mediated by LecRK-IX.1 and LecRK-IX.2 is independent of the cell death

phenotype. By co-immunoprecipitation we identified putative interacting proteins, one

of which was an ATP-binding cassette (ABC) transporter. A homolog in Arabidopsis,

the ABC transporter ABCG40, was found to interact in planta with both LecRK-IX.1

and LecRK-IX.2. Similar to the LecRK mutants, Arabidopsis ABCG40 mutants

showed compromised Phytophthora resistance, indicating that ABCG40 has a

function in Phytophthora resistance.

In Chapter V, we describe the generation of stable transgenic N. benthamiana

plants expressing Arabidopsis LecRK-I.9 or LecRK-IX.1. Multiple transgenic lines

were obtained varying in transgene copy number and transgene expression level.

Ectopic expression of LecRK-I.9 resulted in reduced plant sizes and aberrant leaf

morphology. In addition, expression of LecRK-IX.1 induced plant cell death.

Transgenic N. benthamiana lines expressing either LecRK-I.9 or LecRK-IX.1 showed

increased resistance towards P. capsici or Phytophthora infestans. This demonstrated

that Arabidopsis LecRK-I.9 and LecRK-IX.1 retained their role in Phytophthora

resistance upon interfamily transfer.

Summary

205

Based on the results obtained on Arabidopsis LecRKs, we speculated that

LecRKs in other plant species could play a similar role in Phytophthora resistance. In

Chapter VI, we focus on LecRKs in two Solanaceous plants, i.e. N. benthamiana and

tomato. By exploring genome databases, we identified 38 and 22 LecRKs in

N. benthamiana and tomato, respectively. Phylogenetic analysis revealed that both

N. benthamiana and tomato lack LecRKs homologous to Arabidopsis LecRKs of

clades I, II, III and V, but contain a Solanaceous-specific clade of LecRKs. Functional

analysis of various Solanaceous LecRKs using virus-induced gene silencing followed

by infection assays revealed that homologs of Arabidopsis LecRK-IX.1 and

LecRK-IX.2 in N. benthamiana and tomato are implicated in Phytophthora resistance.

These results indicate that the role of clade IX LecRKs in Phytophthora resistance is

conserved across plant species.

In Chapter VII, the experimental data presented in this thesis are summarized

and discussed in a broader context. We present an overview of the current

understanding of LecRKs in plant immunity and discuss how LecRKs can be exploited

to improve plant resistance.

206

Samenvatting

Plantenziekten veroorzaakt door Phytophthora pathogenen richten ernstige schade

aan in belangrijke land- en tuinbouwgewassen en in de sierteelt. Effectieve

ziekteresistentie in planten is grotendeels gebaseerd op een vroegtijdige herkenning

van pathogenen gevolgd door initiatie van afweer. Immuniteit wordt bewerkstelligd

door intracellulaire resistentie-eiwitten (R) die geactiveerd worden door herkenning

van Phytophthora effectoren. Phytophthora pathogenen zijn echter moeilijk te

bestrijden. Ze kunnen zich gemakkelijk aanpassen waardoor R-gen-afhankelijke

resistentie vaak snel wordt doorbroken. Voor het ontwikkelen van duurzame

resistentie is het daarom van belang om alternatieve resistentie-componenten te

identificeren. Receptor kinases spelen een cruciale rol in de herkenning van exogene

en endogene stimuli en in het activeren van afweer in de plant. Eerder werd

aangetoond dat LecRK-I.9, een Arabidopsis L-type lectine receptor kinase, fungeert

als een Phytophthora resistentie-component. LecRK-I.9 is een van de 45 leden van

de LecRK familie in Arabidopsis. Of, en hoe, andere LecRKs functioneren in

Phytophthora resistentie is tot dusver niet onderzocht. Het in dit proefschrift

beschreven onderzoek was gericht op het ontrafelen van een potentiële rol van

LecRKs in resistentie, in het bijzonder tegen Phytophthora pathogenen.

Hoofdstuk I beschrijft verschillende plantenziekten veroorzaakt door

Phytophthora en geeft een overzicht van de huidige kennis van immuniteit tegen

plantenpathogenen, met nadruk op resistentie tegen Phytophthora pathogenen.

In hoofdstuk II beschrijven we de ontwikkeling van een nieuw

Arabidopsis-Phytophthora pathosysteem. We toonden aan dat Phytophthora capsici

in staat is Arabidopsis te infecteren. Infectietoetsen en cytologische analyses

onthulden verschillen in afweer tussen Arabidopsis accessies in reactie op infectie

met verschillende P. capsici isolaten. Uit infectietoetsen op Arabidopsis mutanten met

verstoorde signaleringsroutes bleek dat salicylzuur-afhankelijke signalering, en

biosynthese van camalexin en indoolglucosinolaten nodig zijn voor een effectieve

resistentie tegen P. capsici (hoofdstuk IIa). Het belang van deze signaleringsroutes

voor afweer werd ondersteund door waarnemingen dat expressie van bijbehorende

markergenen wordt geïnduceerd na infectie met P. capsici (hoofdstuk IIb). Dit

pathosysteem kan dus gebruikt worden als model om essentiële componenten van

resistentie tegen Phytophthora te identificeren.

Vervolgens gebruikten we verschillende Arabidopsis-Phytophthora

pathosystemen om de potentiële rol van LecRKs in Phytophthora resistentie te

onderzoeken. Hoofdstuk III beschrijft een systematische fenotypische karakterisering

van Arabidopsis LecRK mutanten. In een collectie van T-DNA insertielijnen werd de

Samenvatting

207

respons op infectie met verschillende Phytophthora pathogenen onderzocht. Hieruit

bleek dat, naast LecRK-I.9, ook andere LecRKs een rol spelen in Phytophthora

resistentie. Daarnaast onderzochten we of LecRKs ook belangrijk zijn voor de

respons op andere pathogenen en op abiotische stressfactoren. Verscheidene T-DNA

insertielijnen vertoonden fenotypische verschillen in vatbaarheid voor pathogene

bacteriën en schimmels, maar geen van deze Arabidopsis lijnen vertoonden

afwijkingen in groei en ontwikkeling onder normale omstandigheden of na

blootstelling aan abiotische stress. Deze fenotypische gegevens werden gekoppeld

aan LecRK expressieprofielen beschikbaar in publiekelijk toegankelijke

databestanden. Hieruit bleek dat LecRKs waarvan de expressie na infectie niet of

nauwelijks wordt geïnduceerd, of zelfs wordt onderdrukt, mogelijk toch een functie

hebben in resistentie tegen pathogenen. Verder bleek uit deze co-expressie analyses

dat LecRKs die een vergelijkbare rol hebben kunnen verschillen in expressiepatroon.

LecRK clade IX in Arabidopsis bestaat uit slechts twee leden. T-DNA insertielijnen

van zowel LecRK-IX.1 als LecRK-IX.2 vertoonden een verhoogde vatbaarheid voor

Phytophthora, en daarom werden deze twee LecRKs in meer detail onderzocht. In

hoofdstuk IV beschrijven we dat verhoogde constitutieve expressie van LecRK-IX.1

of LecRK-IX.2 in Arabidopsis resulteerde in een verhoogde resistentie tegen

Phytophthora, maar tevens in spontane inductie van celdood. Door een mutatie in het

kinasedomein van LecRK-IX.1 of LecRK-IX.2, verdween niet alleen de verhoogde

Phytophthora resistentie, ook de spontane celdood trad niet meer op. Echter, deletie

van het lectine-domein van beide LecRKs had alleen een effect op de Phytophthora

resistentie; het spontane celdood fenotype bleef behouden en is dus alleen

afhankelijk van het kinase-domein, niet het lectine-domein. Transiënte expressie van

beide LecRK-IX genen in Nicotiana benthamiana resulteerde ook in een verhoogde

resistentie tegen Phytophthora en in spontane celdood. Dit bood de mogelijkheid om

N. benthamiana te gebruiken voor het in kaart te brengen van de benodigde

signaleringscomponenten voor LecRK-IX.1- en LecRK-IX.2-afhankelijke

Phytophthora resistentie en celdood. Met behulp van virus-geïnduceerde silencing

van genen betrokken bij verscheidende signaleringsroutes konden we aantonen dat

NbSIPK1/NPT4 essentieel is voor LecRK-IX.1-afhankelijke celdood, maar niet voor

Phytophthora resistentie. Uit deze resultaten blijkt dat Phytophthora resistentie

gebaseerd op LecRK-IX.1 of LecRK-IX.2 onafhankelijk is van het celdood fenotype.

Door middel van co-immunoprecipitatie werden eiwitten geïdentificeerd die

interacteren met de clade IX LecRKs. Dit resulteerde in de selectie van een ABC

transporter (ATP-binding cassette transporter), die sterke homologie vertoond met de

ABC transporter ABCG40 in Arabidopsis. ABCG40 bleek in planta te interacteren met

zowel LecRK-IX.1 als LecRK-IX.2 en, net als de LecRK-IX mutanten, vertoonden

Arabidopsis ABCG40 mutanten een verhoogde vatbaarheid voor Phytophthora. Deze

Samenvatting

208

resultaten suggereren dat ABCG40 een rol speelt in Phytophthora resistentie.

In hoofdstuk V beschrijven we het genereren van stabiele transgene N.

benthamiana planten die Arabidopsis LecRK-I.9 of LecRK-IX.1 constitutief tot

expressie brengen. Meerdere transgene lijnen werden verkregen met variaties in het

aantal transgen kopieën en in transgen expressieniveaus. Constitutieve expressie

van LecRK-I.9 resulteerde in kleinere planten met een afwijkende bladmorfologie

terwijl hoge constitutieve expressie van LecRK-IX.1 celdood veroorzaakte. De

transgene N. benthamiana lijnen met LecRK-I.9 of LecRK-IX.1 als transgen

vertoonden een verhoogde weerstand tegen P. capsici en Phytophthora infestans. Dit

wijst erop dat de Arabidopsis LecRK-I.9 en LecRK-IX.1 genen hun rol in Phytophthora

resistentie in een N. benthamiana achtergrond kunnen handhaven.

Op basis van de resultaten verkregen met de Arabidopsis LecRKs, speculeerden

we dat LecRKs in andere plantensoorten ook een rol hebben in Phytophthora

resistentie. Hoofdstuk VI is gericht op LecRKs in N. benthamiana en tomaat, twee

soorten in de nachtschadefamilie. Door het exploreren van genoomdatabanken

konden we 38 LecRKs in N. benthamiana en 22 LecRKs in tomaat identificeren. Uit

fylogenetische analyses bleek dat N. benthamiana en tomaat een

nachtschade-specifieke LecRK clade bevatten, maar geen homologen van

Arabidopsis LecRKs in clades I, II, III en V. Uit functionele analyse van LecRKs met

behulp van virus-geïnduceerde silencing van de betreffende genen gevolgd door

infectietoetsen, bleek dat homologen van Arabidopsis LecRK-IX.1 en LecRK-IX.2 in

zowel N. benthamiana als tomaat betrokken zijn bij Phytophthora resistentie. Deze

resultaten geven aan dat de rol van clade IX LecRKs in Phytophthora resistentie

geconserveerd is in verschillende plantensoorten.

In hoofdstuk VII, zijn de experimentele gegevens uit dit proefschrift samengevat

en besproken in een bredere context. Daarnaast geeft het een overzicht van de

huidige kennis van LecRKs in immuniteit, en bespreken we hoe LecRKs kunnen

worden benut om resistentie in planten te verbeteren.

209

Acknowledgements

Time flies. Four years of study in Wageningen almost comes to an end. I am very excited with the completion of my PhD thesis. However, this achievement would not have been possible without the help and support from my supervisors, friends and family. Here I would like to take this opportunity to thank all of them.

First of all, I would like to thank my supervisor Francine Govers for giving me the opportunity to perform my PhD in your group. I appreciate both your critical suggestions and words of encouragement on the research; I appreciate your supervision in making presentations, slides and writing the articles; I appreciate your patience in correcting spelling errors and grammar mistakes in my manuscripts ; and I also appreciate your encouragements to improve my social skills. Your serious attitude and passion towards science impressed me a lot and definitely you set a good example of being a scientist.

Special thanks to my supervisor Weixing Shan. I was so lucky to study under your supervision during my MSc training. I got the chance to learn many experimental techniques in your lab that were very helpful during my PhD study. I am impressed by your commitment to the research and your charming personality, which were important in shaping my view on science. Thank you for encouraging me to learn English, and stimulating me to go abroad for the PhD study. I appreciate your support and inspiration during my PhD study.

My sincere thanks go to my supervisor Klaas Bouwmeester. Thank you for picking me up at Schiphol in 2010 and your daily supervision. Instead of being a ‘real’ supervisor, you are more like a big funny brother. You are very positive, patient and helpful. You were always available when I asked you for a work discussion. I really appreciate your contributions to my PhD project.

My thanks extend to colleagues and students. I am so lucky to work together with friendly and helpful colleagues in Phytopathology. Thank you guys and girls for giving me a hand when I needed help. I want to thank Rob Weide, Harold Meijer, Chara Schoina, Johan van den Hoogen, Elysa Overdijk and Natalie Verbeek for your help in the lab and the great fun in Pinf-activities. Thanks to the MSc students Patrick Beseh, David Nsibo and Hagos Juhar for your hard work and contributions to my PhD project. Thanks to Judith van de Mortel for generating Arabidopsis iGS mutants and your suggestions on writing the manuscripts present in Chapter II. Thanks to Michael Seidl and Luigi Faino for advice related to bioinformatics and data mining. Thanks to Peter van Esse, Koste Yadeta, Emilie Fradin, Mireille van Damme, Patrick Smit, Daniela Sueldo and Thomas Liebrand for suggestions and encouragement. Thanks to Ali Ormel and Marion Rodenburg for administrative issues. Thanks to Jan Cordewener and Twan America for mass spectrometry analysis. Thanks to Henk Smid, Bert

Acknowledgements

210

Essenstam, Gerrit Stunnenberg and Taede Stoker from Unifarm for growing and taking care of the plants.

Living abroad is not an easy thing for me and I am so lucky to have supportive and kind-hearted friends around. Chenlei Hua, Lisha Zhang, Xu Cheng, Wei Qin, Chunxu Song and Freddy Yeo Kuok San, thank you for ‘taking care of me’ when I arrived in the Netherlands. Thank you for introducing me the nice places to visit and delicious Chinese food and learning me how to cook. Yu Du, we studied in the same university in China, and here we are working in the same group. We are like twins and I always feel very close to you. I am very grateful for your company. Thanks to Zhao Zhang, Miao Han, Guozhi Bi, Yin Song, Jinling Li, Xiaoqian Shi, Furong Liu, Xiaolin Wu, Huayi Li and Jinbin Wu for the funny jokes and nice dinners we had together. Thanks to Feng Zhu, Lisong Ma, Fang Xu, Ke Peng, Hieng Ming Ting, Yafen Lin, Tingting Xiao, Hunchen Li, Hanzi He, Yanxia Zhang, Hui Li, Qing Liu, Ting Yang, Bo Wang, Dong Xiao, Xuan Xu, Juan Du, Tao Li, Dongli Gao, Yunmeng Zhang, Chunzhao Zhao, Ying Zhang, Junyou Wang, Junli Guo, Suxian Zhu and Wei Song for your help in life and the pleasant time.

My sincere appreciations go to my family. I want to express my great gratitude to my parents and parents-in-law for always supporting my decisions and your endless love. Great thanks to my dear brothers(-in-law) Bo Wang, Guoshuai Qiu and sister-in-law Jingjing Sun. Thank you for taking care of our parents and your support to Yinyin and me. I also would like to thank my cute nephews Daofeng Wang, Hongen Qiu, Daoheng Wang and my niece Tian Qiu for the great fun when we talk on the telephone. To my dear husband Yinyin Sun (孙银银), thank you for your constant encouragement, support and endless love.

Yan 20th October 2014

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Publications Wang, Y., Bouwmeester, K., Cordewener, J.H.G., America, A.H.P., Shan, W. and

Govers, F. Arabidopsis lectin receptor kinases LecRK-IX.1 and LecRK-IX.2 are

functional analogs in regulating Phytophthora resistance and plant cell death.

Submitted.

Wang, Y., Weide, R., Govers, F. and Bouwmeester, K. L-type lectin receptor kinases

in Nicotiana benthamiana and tomato and their role in Phytophthora resistance.

Submitted.

Deng, F., Wang, Y., Jia, J., Li, J., Govers, F. and Shan, W. The Raf-like MAPKKK

gene Raf36 regulates Arabidopsis thaliana susceptibility to Phytophthora

parasitica and programmed cell death. In preparation.

Wang, Y., Bouwmeester, K., Beseh, P., Shan, W. and Govers, F. (2014). Phenotypic

analyses of Arabidopsis T-DNA insertion lines and expression profiling reveal that

multiple L-type lectin receptor kinases are involved in plant immunity. Molecular

Plant-Microbe Interactions, 27, 1390-1402.

Wang, Y., Bouwmeester, K., van de Mortel, J.E., Shan, W. and Govers, F. (2013). A

novel Arabidopsis-oomycete pathosystem: differential interactions with

Phytophthora capsici reveal a role for camalexin, indole glucosinolates and

salicylic acid in defence. Plant, Cell & Environment, 36, 1192-1203.

Wang, Y., Bouwmeester, K., van de Mortel, J.E., Shan, W. and Govers, F. (2013).

Induced expression of defence-related genes in Arabidopsis upon infection with

Phytophthora capsici. Plant Signaling & Behavior, 8, e24618.

Wang, Y., Meng, Y., Zhang, M., Tong, X., Wang, Q., Sun, Y., Quan, J., Govers, F. and

Shan, W. (2011). Infection of Arabidopsis thaliana by Phytophthora parasitica and

identification of variation in host specificity. Molecular Plant Pathology, 12, 187-

201.

213

Education Statement of the Graduate School

Experimental Plant Sciences

Issued to: Yan Wang Date: 24 November 2014 Group: Phytopathology University: Wageningen University & Research Centre

1) Start-up phase date

► First presentation of your project The role of lectin recptor kinases in resistance to Phytophthora Nov 10, 2010 ► Writing or rewriting a project proposal Huygens Scholarship Programme proposal: The role of lectin receptor kinases in disease resistance to Phytophthora pathogens' Dec 2010 ► Writing a review or book chapter ► MSc courses Plant-Microbe Interactions (PHP-30306) Mar-Apr, 2010 ► Laboratory use of isotopes

Subtotal Start-up Phase 5.5 credits*

2) Scientific Exposure date ► EPS PhD student days EPS PhD student day, Utrecht University Jun 01, 2010 2nd European Retreat for PhD Students in Plant Sciences, Cologne, Germany Apr 15-17, 2010 2nd PhD retreat in Northwest A&F University, Yangling Dec 04-05, 2010 4th Eurpean Retreat for PhD Students in Plant Sciences, Norwich, UK Aug 15-17, 2012

EPS PhD student day, University of Amsterdam Nov 30, 2012 ► EPS theme symposia

EPS theme 2 symposium 'Interactions between Plants and Biotic Agents, and Willie Commelin Scholten Day, Wageningen UR Feb 10, 2012

EPS theme 2 symposium 'Interactions between Plants and Biotic Agents, and Willie Commelin Scholten Day, Utrecht University Jan 24, 2013

EPS theme 2 symposium 'Interactions between Plants and Biotic Agents, and Willie Commelin Scholten Day, University of Amsterdam Feb 25, 2014

► NWO Lunteren days and other National Platforms NWO-ALW meeting 'Experimental Plant Sciences', Lunteren Apr 19-20, 2010 The Chinese Society for Plant Pathology, annual meeting Aug 19-21, 2011 NWO-ALW Platform Molecular Genetics Annual Meeting Oct 07, 2011 NWO-ALW meeting 'Experimental Plant Sciences', Lunteren Apr 03-04, 2012

NWO-ALW meeting 'Experimental Plant Sciences', Lunteren Apr 22-23, 2013

NWO-ALW meeting 'Experimental Plant Sciences', Lunteren Apr 14-15, 2014► Seminars (series), workshops and symposia CBSG2012 Summit 2010 Mar 16, 2010 Invited seminar Roeland Boer: 'Structural studies on proteins that prepare DNA for replication and conjugation' Apr 22, 2010

Invited seminar Matteo Brilli: 'Development of an integrated gene regulation-metabolism mathematical model of carbon utilization in E.coli’

Apr 29, 2010

Invited seminar Brigitte Mauch-Mani: 'Grapevine and downy mildew - Wine is not the only difference between grapevine and Arabidopsis'

May 31, 2010

Invited seminar Felix Mauch: 'Old fashioned secondary metabolites save Arabidopsis from Phytophthora brassicae' May 31, 2010

CBSG Seminar by H. Yang, S. Huang and R. Luo (from BGI and CAAS, China) Apr 01, 2010

CBSG workshop: Genome mining: taste the tomato genome Oct 27, 2011

Invited seminar David C. Baulcombe ''Plant versus virus: defense, counter defense, and counter counter defense'' Oct10, 2012

Invited seminar Ralph Panstruga: ‘Comparative pathogenomics of powdery mildew fungi: chasing the molecular secrets of obligate biotrophy and fungal pathogenesis’

Dec 04, 2012

Symposium: Intraspecific pathogen variation-implications and opportunities Jan 22, 2013

Invited seminar Detlef Weigel: “Arabidopsis thaliana as a model system for the study of evolutionary questions” Feb 27, 2013

Invited seminar Marc Boutry: ‘Plant drug smugglers’ about transport of secondary metabolites in plants Mar 13, 2013

Symposium: Food for all sustainable nutrition security, 95th Dies Natalis Mar 15, 2013

Invited seminar Howard S. Judelson 'Biology,pathology and evolution of Phytophthora infestans' May 07, 2013

Invited seminar Rays H.Y. Jiang 'integrative genomics of destructive pathogens from ommycete to malaria parasites' May 07, 2013

Invited seminar Brian Staskawicz:“Effector-Targeted Breeding for Durable Disease Control of Xanthomonas diseases in Tomato and Cassava”

May 21, 2013

Invited seminar Kuang Hanhui 'Using the Nicotiana-TMV system to study resistance gene evolution and plant genome stability' Sep 11, 2013

Invited seminar Jane Parker : 'Reprogramming cells for defence in plant innate immunity' Apr 09, 2014

Invited seminar Frank van Breusegem : 'Plant Metacaspases' Apr 09, 2014

► Seminar plus ► International symposia and congresses Annual Oomycete molecular genetics network meeting, Toulouse, France Jun 06-08, 2010

Annual Oomycete molecular genetics network meeting, Nanjing, China May 25-28, 2012

The 3rd International Conference on Biotic Plant Interactions, Yangling, Shaanxi, China Aug 18-21, 2013

10th International Congress of Plant Pathology, Beijing, China Aug 25-30, 2013

Annual Oomycete molecular genetics network meeting, Norwich, UK Jul 03-05, 2014

► Presentations Poster: Infection of Arabidopsis thaliana by Phytophthora parasitica Jun 06-08, 2010

Poster: Phytophthora capsici-Arabidopsis, a model pathosystem for studying the role of lectin receptor kinases in Phytophthora disease resistance

May 25-28, 2012

Poster: L-type lectin receptor kinases: novel Phytophthora resistance components Aug 18-21, 2013 Poster: L-type lectin receptor kinases: novel Phytophthora resistance components Apr 14-15, 2014 Oral: EPS Summer School Rhizosphere Signaling Aug 23-25, 2010 Oral: ALW meeting 'Experimental Plant Sciences', Lunteren Apr 03-05, 2012 Oral: CBSG Meeting Proteomics Hotel Projects Jan 18, 2013 Oral: EPS Theme 2 Symposium & WCS day Feb 25, 2014 ► IAB interview Meeting with a member of the International Advisory Board of the Graduate School EPS Nov 15, 2012

► Excursions Subtotal Scientific Exposure

23.9 credits*

214

3) In-Depth Studies date ► EPS courses or other PhD courses EPS PhD Summer School Rhizosphere Signaling Aug 23-25, 2010 EPS PhD Autumn School Host-Microbe Interactomics Nov 01-03, 2011 ► Journal club Literature discussion group at Pytopathology, Wageningen UR and at NWSUAF 2010-2014 ► Individual research training

Subtotal In-Depth Studies 4.8 credits*

4) Personal development date ► Skill training courses Information Literacy for PhD including EndNote Introduction Jun 15-16, 2010 Interactive workshop on presentation skills Nov 06, 2012 CBSG matchmaking event Oct 18, 2012 Techniques for Writing and Presenting a Scientific Paper Oct 16-19, 2013 Mobolizing your scientific network Mar 19 and 27, 2014 ► Organisation of PhD students day, course or conference ► Membership of Board, Committee or PhD council

Subtotal Personal Development 3.2 credits*

TOTAL NUMBER OF CREDIT POINTS* 37.4 Herewith the Graduate School declares that the PhD candidate has complied with the educational requirements set by the Educational Committee of EPS which comprises of a minimum total of 30 ECTS credits * A credit represents a normative study load of 28 hours of study.

This research was performed at the Laboratory of Phytopathology, Wageningen

University with financial support of a Wageningen University Sandwich-PhD fellowship,

a Huygens scholarship and the Food-for-Thought campaign of the Wageningen

University Fund.

Cover design and layout: Yan Wang Printed at Wöhrmann Print Service, Zutphen, The Netherlands.

Documents

Understanding the role of L-type lectin receptor kinases