IDENTIFICATION AND CHARACTERIZATION OF A NOVEL RECEPTOR-LIKE KINASE

INVOLVED IN THE INITIATION AND REGULATION OF ARABIDOPSIS INNATE

IMMUNITY.

by

XIN YANG

KATRINA M. RAMONELL, COMMITTEE CHAIR

JANIS M. O'DONNELLPERRY F. CHURCHILLMARGRET D. JOHNSON

AMY LITT

A DISSERTATION

Submitted in partial fulfillment of the requirementsfor the degree of Doctor of Philosophyin the Department of Biological Sciences

in the Graduate School ofThe University of Alabama

TUSCALOOSA, ALABAMA

2011

Copyright Xin Yang 2011ALL RIGHTS RESERVED

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ABSTRACT

Receptor-like kinases (RLKs) are known to be involved in the recognition of pathogen-

associated molecular patterns (PAMPs) and subsequently activate resistance pathways against

broad classes of pathogens. While initiation and maintenance of defense pathways is critical for

survival, mechanisms to damp down these responses are just as necessary though currently not as

well understood. We have identified CRLK1, an Arabidopsis RLK that is highly induced by

chitin at early time points and localizes to the plasma membrane. Knock-out mutants in crlk1 are

more susceptible to both biotrophic and necrotrophic fungal pathogens though the response of

the mutants to bacterial pathogens is unaffected. Interestingly expression ofMAPK3, an

important positive regulator of innate immunity, is increased in crlk1 mutants. Our data show

that CRLK1 is essential for the establishment of defense against biotrophic and necrotrophic

fungi and that the mutation in CRLK1 does not fully block chitin-enhanced Arabidopsis

resistance. We show that CRLK1 is a functional kinase in vitro and its kinase activity required

the presence of manganese. Overexpression of a 35S:CRLK1: GUS fusion protein in

Arabidopsis confers enhanced resistance to the powdery mildew pathogen Golovinomycetes

cichoracearum. In addition, CRLK1 induction by chitin is increased in mapk3 and several wrky

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mutants indicating that CRLK1 may be repressed by MAPK3 and WRKY transcription factors in

planta. The results presented provide important information about the function and regulation of

CRLK1 in Arabidopsis.

iv

DEDICATION

This dissertation is dedicated to everyone who had accompanied me and helped me

through the experiments and trials of creating this manuscript. In particular, my family and

colleagues whostood by me throughout the time taken to complete this masterpiece.

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LIST OF ABBREVIATIONS AND SYMBOLS

AA amino acid

ACC 1-aminocyclopropane 1-carboxylic acid

BiFC Bimolecular fluorescence complementation

BIK1 Botrytis-induced kinase1

CEBiP chitin oligosaccharide elicitor-binding protein

CERK1 Chitin Elicitor Receptor Kinase 1

CRLK1 chitin-induced receptor-like kinase 1

CRG chitin responsive genes

CSC crab shell chitin

DAB diamino-benzidine

DAMP danger-associated molecular patterns

EMS ethyl methanesulfonate

EIX Ethylene-induced xylanase

ET ethylene

ETI effectors-triggered immunity

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GFP green fluorescent protein

IP immunoprecipitation

JA jasmonic acid

OG oligogalacturonide

SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis

MAPK mitogen-activated protein kinase

MBP myelin basic protein

MS mass spectrometry

PAMP pathogen associated molecular pattern

PRR pattern recognition receptors

PTI PAMP-triggered immunity

qRT-PCR quantitative reverse transcriptase–polymerase chain reaction

RLK receptor-like kinase

RLP receptor-like protein

RLCK receptor-like cytoplasmic kinase

RNAi RNA interference

ROS reactive oxygen species

SA Salicylic acid

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TAP tandem affinity purification

TM transmembrane domain

WT wild type

viii

ACKNOWLEDGMENTS

I am pleased to dedicate this dissertation to all people who have helped and supported me

during the past five years. The faculty in Department of Biological Sciences provided unselfish

help to me. Dr. Katrina Ramonell, my mentor, recruited me as a graduate student from oversea

and mentored me in pursuing Ph.D. in the area of plant-microbe interactions. She instructed me

for not only techniques for experiments but also supported tremendously with her inspiration and

encouragement. I was supported through out five years with teaching/research assistantships and

Alabama EPSCoR Fellowship with the help of Dr. Ramonell. Dr. Margret Johnson taught me

how to critique and think in a way of scientists. Dr. Perry Churchill helped me with

biochemistry work in this dissertation and enlightened me to explore more information about my

CRLK1. Dr. Janis O'Donnell guided me to the world of genetics and modern molecular

techniques in studying model organism. It is my pleasure to have them all as my committee

members and I would like to thank them for their great support during the past five years. I

would also to thank my parents and my wife Ning for loving me and supporting my decision of

being a graduate student in Alabama. I also appreciate the support and concern by Dr. David

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Wang, Mr. Zaicun Zhang and Mrs. Huizhong Chen. Finally I want to give my thanks to my

labmates Mr. Jay Antico and Mrs. Fengyan Deng for their help and friendship.

x

CONTENTS

ABSTRACT................................................................................................ ii

DEDICATION............................................................................................iv

LIST OF ABBREVIATIONS AND SYMBOLS........................................ v

ACKNOWLEDGMENTS........................................................................viii

LIST OF TABLES.....................................................................................xii

LIST OF FIGURES..................................................................................xiii

CHAPTER ONE: AN INTRODUCTION TO RLKS/RLPS ANDCRLK1 INVOVLED IN PLANT INNATE IMMUNITY.......................... 1

CHAPTER TWO: CHARACTERIZATION OF A NOVELRECEPTOR-LIKE KINASE INVOLVED IN THE INITIATION ANDREGULATION OF ARABIDOPSIS INNATE IMMUNITY...................22

INTRODUCTION..................................................................................... 22

MATERIALS AND METHODS...............................................................24

RESULTS.................................................................................................. 29

DISCUSSION............................................................................................47

REFERENCES.......................................................................................... 51

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CHAPTER THREE: CRLK1 IS A FUNCTIONALMANGANESE-DEPENDENT RECEPTOR-LIKE KINASEINVOLVED IN DEFENSE AGAINST FUNGAL PATHOGENS................................................................................................................... 54

INTRODUCTION..................................................................................... 54

MATERIALS AND METHODS...............................................................56

RESULTS.................................................................................................. 63

DISCUSSION............................................................................................81

REFERENCES.......................................................................................... 86

CHAPTER FOUR: SUMMARY OF WORK ANDFUTURE DIRECTIONS .......................................................................... 89

REFERENCES.......................................................................................... 97

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LIST OF TABLES

Table 1.1 The list of RLKs and RLPs involved inplant innate immunity................................................................................ 17

Table 1.2 Gene name, locus and mutant accessionnumber of seven receptor-like kinases selectedfrom chitin-responsive genes.................................................................... 20

Table 3.1 Potential phosphorylation sites inphospho-peptide identified from MS/MS.................................................. 71

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LIST OF FIGURES

Figure 1.1 Average numbers of conidiophores per colony (c/c)on receptor-like kinase mutants, Col-0 wild type and NahG.....................21

Figure 2.1 Crlk1 is more susceptible to G. cichoracearum butexhibits normal ROS production............................................................... 32

Figure 2.2 Crlk1 is more susceptible to the necrotroph P. cucumerina....34

Figure 2.3 Expression of CRLK1 after chitin, ACC, MeJA andSA treatments.............................................................................................37

Figure 2.4 CRLK1 encodes a RLK that has multiple W-boxesin its promoter sequence and is localized to the plasma membrane.......... 40

Figure 2.5 Histochemical staining of GUS activity in transgenicArabidopsis harboring pCRLK1::GUS......................................................43

Figure 2.6 Expression ofMAPK3 andWRKY53 after chitintreatment in Col-0 wild type and crlk1...................................................... 46

Figure 2S1 Crlk1 is not compromised in its resistance tobacterial pathogens.....................................................................................35

Figure 2S2 Wound-induced GUS expression in pCRLK1::GUStransgenic Arabidopsis plants.................................................................... 44

Figure 3.1 A phylogenetic tree based on protein sequence alignmentof CRLK1 and seven related proteins........................................................64

Figure 3.2 Subdomains and predicted phosphorylation sitesof CRLK1.................................................................................................. 66

xiv

Figure 3.3 Purification and in vitro kinase assay of truncated CRLK1(ΔTM-CRLK1) in E.coli............................................................................69

Figure 3.4 Phenotypes of Arabidopsis CRLK1 mutants andoverexpression lines...................................................................................75

Figure 3.5 CRLK1 gene expression in Arabidopsis organs andunder various treatments/conditions.......................................................... 79

Figure 3.6 Expression of CRLK1 after chitin treatment in MAPKandWRKY mutants.................................................................................... 80

Figure 3S1 Prediction of CRLK1 transmembrane domain.......................65

Figure 3S2 Phospho-peptides identified from MS/MS for CRLK1......... 72

Figure 3S3 Sequence alignment of CRLK1 with Arabidopsiskinase (AT), Arachis kinase (AH) and conversed manganesedependent kinase domain (Accession number COG0515)........................ 83

Figure 4.1 Model of CRLK1 in chitin-mediated innate immunity............96

1

CHAPTERCHAPTERCHAPTERCHAPTERONEONEONEONE

ANANANAN INTRODUCTIONINTRODUCTIONINTRODUCTIONINTRODUCTION TOTOTOTO RECEPTOR-LIKERECEPTOR-LIKERECEPTOR-LIKERECEPTOR-LIKE KINASES/RECEPTOR-LIKEKINASES/RECEPTOR-LIKEKINASES/RECEPTOR-LIKEKINASES/RECEPTOR-LIKE PROTEINSPROTEINSPROTEINSPROTEINSANDANDANDAND CRLK1CRLK1CRLK1CRLK1 INVOVLEDINVOVLEDINVOVLEDINVOVLED ININININ PLANTPLANTPLANTPLANT INNATEINNATEINNATEINNATE IMMUNITYIMMUNITYIMMUNITYIMMUNITY

SignificanceSignificanceSignificanceSignificance ofofofof studyingstudyingstudyingstudying plantplantplantplant andandandand microbemicrobemicrobemicrobe interactionsinteractionsinteractionsinteractions

Plants constantly undergo pathogen attack during their life cycles. These pathogens

include a variety of fungi (ex. powdery mildew), bacteria, viruses, and nematodes. The ability to

recognize different classes of pathogens is critical for plants to initiate timely defense responses.

Plants have evolved complicated defense mechanisms to resist pathogens and plant pathogens

have also co-evolved with their hosts to suppress defense responses and favor infection.

Among the classes of pathogens, fungi are recognized as one of the major causes of crop

yield loss. For example, Magnaporthe grisea , an ascomycete fungus that infects rice, can cause

yield losses equaling 25% of rice production every year (Ribot et al. 2008). Fungal pathogens

also account for a 15% decrease in barley yield, which accounts for 7.5% of worldwide barley

production (Oerke et al 2004). Our work is focused on the fungal pathogen powdery mildew, a

disease of more than 9000 plant species caused by members of the Erysiphales. Every year

powdery mildew is responsible for large losses in yield of numerous agriculturally important

crops such as cereals, vegetables, and fruit trees. Tremendous effort has been focused on

developing methods that may be used to control the spread of powdery mildew on crops.

Current research in the Ramonell lab is concentrated on understanding broad spectrum

defense responses (innate immunity) in plants against the powdery mildew pathogen. In

particular we are interested in unraveling the signal transduction pathways that are triggered in

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plants by exposure to the fungal elicitor chitin. The innate immune response is an ancient form

of defense against pathogens that is shared by plants, insects and vertebrates (Nurnberger and

Scheel, 2001). It involves the recognition of general pathogen-associated elicitors followed by a

rapid induction of basal defense responses. An improved understanding of innate immunity will

be beneficial in the design of crops with increased natural resistance to fungal pathogens.

PlantPlantPlantPlant innateinnateinnateinnate immunityimmunityimmunityimmunity

Unlike animals, plants do not have an adaptive immune system, which could recognize

and respond to a vast number of pathogens. Plants have evolved multiple lines of defense to

protect themselves from pathogen attack. In order to activate these defense responses, plants

perceive and recognize pathogens through Pattern Recognition Receptors (PRRs). The plant

genome encodes a large number of PRRs that recognize conserved microbial signatures called

pathogen-associated molecular patterns (PAMPs). Once PRRs perceive pathogens via PAMPs,

downstream signaling pathways are activated to mount defense responses such as the

hypersensitive response (HR) and the production of antimicrobial secondary metabolites. To

suppress PAMP-triggered immunity (PTI), pathogens deliver effectors into plant cells that target

key components of plant immunity. For example, the bacterial effector AvrPtoB targets and

ubiquitinates the Arabidopsis receptor CERK1 for degradation (Gimenez-Ibanez et al. 2009)

overcoming CERK1-mediated defense responses. In turn, some plant species have also evolved

resistance (R) proteins that recognize pathogen effectors either directly or indirectly and initiate

effector-triggered immunity (ETI), also known as the gene-for-gene model. Recent studies have

shown that PTI and ETI share signaling pathways that overlap significantly (Tsuda et al. 2009)

and PRRs have been shown to physically associate with R proteins in vivo (Qi et al. 2011). Both

3

PTI and ETI are essential for plant immunity in order to restrict pathogen invasion. Due to the

importance of PRRs in recognizing microbial signatures, transgenic plants overexpressing

exogenous PRRs have been created to confer broad-spectrum resistance to bacterial pathogens

(Lacombe et al. 2010).

TheTheTheThe rolerolerolerole ofofofof receptor-likereceptor-likereceptor-likereceptor-like kinases/proteinskinases/proteinskinases/proteinskinases/proteins inininin plantplantplantplant innateinnateinnateinnate immunityimmunityimmunityimmunity

Receptor-like kinases (RLKs) and Receptor-like proteins (RLPs) in plants are involved in

many biological processes including development, innate immunity, cell differentiation and

patterning, nodulation and self-incompatibility. As more and more plant genome sequences have

become available, the number of genes annotated as RLKs or RLPs in plants has been growing.

The Arabidopsis genome contains more than 600 RLKs and 57 RLPs, accounting for almost

2.5% of the Arabidopsis genome (Shiu et al. 2001; Wang et al. 2008). The rice genome contains

2,210 RLKs and more than 443 of rice RLKs appear to share common ancestors with

Arabidopsis RLKs (Shiu et al. 2004). Ninety genes were predicted to be RLPs in the rice

genome and 73 of these are believed to be involved in pathogen defense (Fritz-Laylin et al.

2005). Additionally, over 650 RLKs were identified in the soybean genome by searching for

RLK homologs in an EST database (Liu et al. 2009). While the functions of most RLKs and

RLPs are unknown, increasing experimental data points to their importance in the plant.

A typical RLK contains an extracellular domain, a transmembrane domain (TM) and an

intracellular kinase domain. Some RLKs lack an extracellular domain and are designated as

receptor-like cytoplasmic kinases (RLCKs). RLPs are composed of an extracellular domain, a

transmembrane domain and a short cytoplasmic region and lack an associated kinase domain

(Wang et al. 2008). The extracellular domains of both RLKs and RLPs function primarily in

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recognition of either endogenous or exogenous molecular cues. For example, extracellular

leucine-rich repeat domains (eLRR) in RLKs are well characterized and have been shown to be

involved in recognition of general elicitors (FLS2, Gomez-Gomez and Boller 2000) and plant

hormones (BRI1, Li and Chory 1997). Some RLK/RLP extracellular domains have also been

shown to bind carbohydrate derivatives such as chitin (CERK1, Iizasa et al. 2010) and

oligogalacturonide (WAK1, Decreux and Messiaen 2005). The transmembrane domain (TM) is

critical for localization of RLKs and RLPs to the plasma membrane and deletion of the TM

domain results in cytoplasmic localization of an RLK (Bleckmann et al. 2010). Additionally,

transmembrane domains are known to play critical roles in many protein-protein interactions

(Reviewed by Senes et al. 2004). The intracellular kinase domain of RLKs is involved in

phosphorylation of other proteins to relay signals and initiates downstream signaling pathways.

Interestingly, a large number of RLKs are actually receptor-like cytoplasmic kinases (RLCKs).

It has been reported that there are 379 putative RLCKs in the rice genome (Vij et al. 2008) and

200 RLCKs in Arabidopsis (Jurca et al. 2008). Since they lack an extracellular domain, RLCKs

do not perceive a signal directly but interact with other receptors to form a complex that then

proceeds to amplify the downstream signal (Rowland et al. 2005; Veronese et al. 2006; Lu et al.

2010). In the last decade, many RLKs and RLPs have been characterized that function in plant-

pathogen interactions and have critical roles in the initiation and transduction of signals in major

plant defense pathways. In this chapter, we will summarize our current understanding of RLKs

and RLPs that are involved in plant innate immunity and what is known regarding their

mechanism of action in plant defense pathways. An overview of all RLKs and RLPs discussed in

this review is summarized in table 1.

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RLKsRLKsRLKsRLKs andandandand RLPsRLPsRLPsRLPs areareareare involvedinvolvedinvolvedinvolved inininin defensedefensedefensedefense againstagainstagainstagainst fungalfungalfungalfungal pathogenspathogenspathogenspathogens viaviaviavia PAMPPAMPPAMPPAMP andandandand DAMPDAMPDAMPDAMP

recognitionrecognitionrecognitionrecognition

Plants recognize and respond to pathogen attack by sensing pathogen-associated

molecular patterns (PAMPs), pathogen effectors and danger-associated molecular patterns

(DAMPs) (Reviewed by Postel and Kemmerling 2009). To date, many elicitors have been

identified that originate from either the pathogen; such as the fungal elicitors chitin and xylanase

and the bacterial elicitors flg22, EF-Tu and lipopolysacchride, or from plants themselves; such as

oligogalacturonide (OG) and the peptide signal Pep1 (Reviewed by Postel and Kemmerling

2009). However, only a few RLKs and RLPs have been identified that act as receptors for these

known elicitors.

For example, both the RLP Chitin oligosacchride elicitor-binding protein (CEBiP) and

RLK Chitin Elicitor Receptor Kinase 1 (CERK1) contain LysM domains that have been shown

to bind chitin and trigger chitin-mediated defense signaling (Kaku et al. 2006; Miya et al. 2007;

Wan et al. 2008; Shimizu et al. 2010). CEBiP was purified from suspension-cultured rice cells

and shown to bind chitin fragments. Microarray analysis showed that a majority of chitin-

responsive genes did not respond to chitin treatment in CEBiP-RNAi knockdown rice cells

(Kaku et al. 2006). Transgenic rice plants were also constructed to suppress the CEBiP

transcripts using RNA interference (RNAi). Data from these experiments showed that CEBiP

RNAi lines had more cells penetrated by the rice blast fungus Magnaporthe oryzae.

Overexpression of CEBiP in rice repressed M. oryzae infection to some extent and increased

levels of reactive oxygen species (ROS) produced (Kishimoto et al. 2010). The barley HvCEBiP

protein is an ortholog of CEBiP that shares 60% amino acid identity with the rice protein. When

barley HvCEBiP was silenced using virus-induced gene silencing (VIGS), the silenced plants

6

developed more severe symptoms compared to control plants inoculated with the fungal

pathogenM. oryzae mossd1,a mutant that fails to infect rice and barley (Tanaka et al. 2010). The

rice ortholog of CERK1, OsCERK1, was identified from a group of 10 rice LysM RLKs and

shown to interact with CEBiP (Shimizu et al. 2010) Similar to CEBiP, knockdowns of

OsCERK1 in RNAi rice cell lines also blocked the induction or repression of most chitin

responsive genes upon chitin treatment. ROS induced by chitin was also suppressed in

OsCERK1-RNAi cell lines (Shimizu et al. 2010). In Arabidopsis, CERK1 was shown to be the

major receptor that binds and perceives chitin elicitors (Wan et al. 2008; Miya et al. 2007; Iizasa

et al. 2010). Arabidopsis CERK1 knockout mutants exhibited impaired immunity to the

biotrophic fungus Golovinomycetes cichoracearum and the necrotrophic fungus Alternaria

brassicicola. Although three other CEBiP-like proteins were also identified in Arabidopsis, their

functions remain unknown (Wan et al. 2008).

Ethylene-induced xylanase (EIX) is a potent fungal elicitor that stimulates ethylene

production, the alkalinization response and necrosis when applied to tobacco and tomato leaves

(Enkerli et al. 1999). Two genes LeEix1 and LeEix2 were identified from tomato as LRR RLPs

potentially involved in the response to this elicitor (Ron and Avni 2004). EIX-induced cell death

was suppressed in LeEix1-RNAi transgenic Nicotianana tabacum cv Samsun plants that are

known to respond to EIX. Interactions between EIX and tobacco cells were not detected in

silenced lines. The study showed that while both LeEIX1 and LeEIX2 proteins were able to bind

EIX only LeEIX2 was capable of inducing the hypersensitive response (HR) (Ron and Avni

2004). Recent work by Bar et al. (2010) showed that LeEIX1 and LeEIX2 interact with each

other in tobacco cells upon EIX treatment.

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The function of LeEIX1 in tobacco appears to be to attenuate EIX-induced LeEIX2 endocytosis

and subsequent EIX-induced defense responses (Bar et al. 2010).

Elicitins are conserved extracellular proteins that are secreted by the fungal pathogen

Phytophthora infestans. Treatment of plants with elicitins triggers the hypersensitive response

(HR) and necrotic lesions in tobacco (Ricci et al. 1989). NbLRK1, a lectin RLK found in

Nicotiana benthamiana, was identified as an interactor of the protein INF1, an elicitin from P.

infestans (Kanzaki et al. 2008). Yeast two-hybrid experiments using a series of truncated

NbLRK1 proteins showed that INF1 interacted with the VIb subdomain of NbLRK1’s

intracellular kinase domain. In NbLRK1-silenced tobacco plants, INF1-induced H2O2 production

was inhibited and the HR response was delayed. Another RLK, NgRLK1, was discovered in

Nicotiana glutinosa and has been shown to interact directly with the fungal elicitin capsicein

from Phytophthora capsici (Kim et al. 2009). Both NbLRK1 and NgRLK1 are potential

candidate genes for elicitin-mediated immunity though no direct evidence for a role in innate

immunity has been shown to date.

Oligogalacturonide (OG) is known to trigger extensive gene expression in plants and is

classified as a DAMP generated from plant cell wall pectin (Denoux et al. 2008; Postel and

Kemmerling 2009). Arabidopsis Wall-Associated Kinase 1 (WAK1) is a receptor-like kinase

that interacts with pectin and OG in vitro (Decreux and Messiaen 2005; Decreux et al. 2006). In

a chimeric receptor study, the WAK1 extracellular domain was shown to interact with OGs and

activate downstream defense responses (Brutus et al. 2010). Arabidopsis plants over-expressing

WAK1 were more resistant to the necrotrophic fungi Botrytis cinerea (Brutus et al. 2010).

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OsWAK1, a homolog of WAK1 identified in rice, was induced significantly by M. oryzae

infection and over-expression of OsWAK1 in rice plants conferred increased resistance to M.

Oryzae (Li et al. 2009).

Another plant DAMP, Arabidopsis peptide 1 (AtPep1), is a 23 amino acid peptide

derived from a 92 aa precursor found in leaf tissue (Huffaker et al. 2006). Treatment of

Arabidopsis plants with AtPep1 induces expression of the defense marker gene PDF1.2 and

production of H2O2.Over-expression of AtPep1 in Arabidopsis confers increased resistance to

the oomycete Pythium irregulare (Huffaker et al. 2006). An ortholog of AtPep1, ZmPep1, was

identified in corn and pretreatment of maize plants with this peptide enhanced plant resistance to

the fungal pathogens Cochliobolis heterostrophus and Colletotrichum graminicola (Huffaker et

al. 2011). The receptor for AtPep1 (AtPepR1) was isolated from Arabidopsis suspension-

cultured cells and identified as an LRR RLK (Yamaguchi et al. 2006). Another protein, AtPepR2,

was first identified as a homolog of AtPepR1 in Arabidopsis but was subsequently found to

interact directly with AtPep1 (Yamaguchi et al. 2010). Plants with mutations in both proteins

(pepr1/ pepr2) were completely insensitive to AtPep1 treatment while single mutations in either

pepr1 or pepr2 showed only partial insensitivity to AtPep1 treatment (Krol et al.2010). However,

there was no significant difference in the response of wild type and pepr1, pepr2 and

pepr1/pepr2 mutants infected with the fungal pathogens P. irregulare and A. brassicicola

without AtPep1 pretreatment (Yamaguchi et al. 2010). Though compelling this data cannot

exclude the possibility that AtPepR1 and AtPepR2 function in AtPep1-mediated resistance

against fungal pathogens in some capacity.

OrphanOrphanOrphanOrphan RLKs/RLPsRLKs/RLPsRLKs/RLPsRLKs/RLPs involvedinvolvedinvolvedinvolved inininin defensedefensedefensedefense againstagainstagainstagainst fungalfungalfungalfungal pathogenspathogenspathogenspathogens

9

While ligands have been identified for a few RLKs/RLPs, the binding partners of most

RLKs/RLPs in the plant genome remain unknown. Several orphan RLKs/RLPs have been

shown to be important in plant innate immunity, though their mechanism of action was revealed

only by further study. Llorente et al. surveyed 75 Arabidopsis accessions and found that

Landsberg erecta (Ler-0) was highly susceptible to the necrotrophic fungi Plectosphaerella

cucumerina (Llorente et al. 2005). QTL analysis showed that the LRR RLK ERECTA was a

candidate gene for Ler-0’s resistance to P.cucumerina. Loss-of-function mutants in ERECTA

showed that both the receptor and kinase domains were required for resistance to P. cucumerina

(Llorente et al. 2005) and to the oomycete pathogen Pythium irregulare (Adie et al. 2007).

Further experimentation showed that ERECTA-mediated resistance was associated with cell wall

content alteration suggesting that ERECTA may also function as a sensor for cell wall integrity

in plants (Sanchez-Rodriguez et al. 2009).

In Arabidopsis, BOTRYTIS-INDUCED KINASE 1 (BIK1) is highly induced by

inoculation with the necrotrophic fungi Botrytis cinerea. T-DNA insertional mutants of bik1

displayed more severe disease symptoms than wild type plants inoculated with B.cinerea and A.

brassicicola. Interestingly, bik1 is more resistant to the bacterial pathogen P. syringae DC3000,

suggesting that BIK1 regulates basal resistance to pathogens instead of race-specific resistance

(Veronese et al. 2006). The tomato homolog of BIK1, TPK1b was shown to be induced by

various stimuli including infection with Botrytis cinerea and Pseudomonas syringae pv. tomato

DC3000 (Pst DC3000), wounding, and treatment with the herbicide paraquat. TPK1b RNAi

plants showed increased susceptibility to Botrytis and supported more fungal growth than wild

type plants. However, data showed that TPK1b was not required for resistance to the bacterial

pathogen Pst DC3000. Over-expression of TPK1b in Arabidopsis suppressed the susceptible

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phenotype of the bik1 mutant suggesting that TPK1b and BIK1 may have similar functions in

plant innate immunity (Abuqumar et al. 2008).

Another orphan RLK, OsBRR1 from rice, is highly induced by infection with the rice

blast fungi M. oryzae. Knockdowns of OsBRR1 in RNAi transgenic plants displayed increased

susceptibility to a weakly virulent isolate ofM. oryzae. OsBRR1 expression is not induced

significantly by ABA, SA, or JA suggesting that it is not involved in the defense pathways

mediated by these hormones (Peng et al. 2009). In wheat, TaRLK-R1, 2 and 3 were identified

and cloned as three receptor-like kinases that were induced by stripe rust infection. Virus-

induced gene silencing of TaRLK-R1, 2 and 3 transcripts resulted in more senescence-like

symptoms and the appearance of more rust sori on infected leaves (Zhou et al. 2007).

Two homologs of Arabidopsis BAK1/SERK3 were found in N. Benthamiana and

knockdowns of both genes using VIGS lines resulted in enhanced susceptibility to P. infestans

but not to P. mirabilis, an avirulent species. In tobacco plants NbSERK3A/B was shown to be

required for INF1-triggered innate immunity since silencing and NbSERK3A/B lead to a

significant reduction in cell death in INF1-treated tobacco (Chaparro-Garcia et al. 2011). The

oomycete pathogen Hyaloperonospora parasitica (Hp) Waco9 was able to infect NbSERK3-

silenced N. benthamiana but not wild type plants (Heese et al. 2007) suggesting a general role

for SERK3/BAK1 in plant innate immunity.

The BROAD-SPECTRUM RESISTANCE 1 (BSR1) protein was identified as a rice

receptor-like cytoplasmic kinase (RLCK) in a screen for Pst DC3000 resistant rice-FOX

Arabidopsis lines that over-express full-length rice cDNAs (Dubouzet et al. 2010). Over-

expression lines of BSR1 in Arabidopsis were also resistant to the hemitrophic fungal pathogen

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Colletotrichum higginsianum. Transgenic rice lines over-expressing BSR1 were more resistant to

the rice fungal pathogenMagnaporthe grisea.

The Ve1 gene has also been shown to be an RLK that plays an important role in

resistance against Verticillium wilt diseases (Fradin et al. 2009). Fradin et al (2009) compared

coding sequences of Ve1 among several resistant and susceptible tomato cultivars. A single

nucleotide deletion that resulted in a truncated Ve1 protein was found only in susceptible but not

in resistant cultivars of tomato. Silencing of Ve1 via VIGS compromised the resistance of

tomato plants to Verticillium dahliae. Meanwhile over-expression of Ve1 in susceptible tomato

cultivars enhanced plant resistance to V. dahliae and Verticillium albo-atrum.

The Apple Vf locus contains four orphan LRR RLPs (Vfa1, Vfa2, Vfa3 and Vfa4) that

confer resistance to the fungal pathogen Venturia inaequalis (Xu and Korban 2002).

Introduction of Vfa1 and Vfa2 into two apple cultivars (Galaxy and McIntosh) enhanced

resistance to Venturia inaequalis compared to non-transformed plants (Malnoy et al. 2008;

Belfanti et al. 2004). Interestingly, Vfa4 is a negative regulator of apple innate immunity as Vfa4

transformants are more susceptible to V. inaequalis.

NegativeNegativeNegativeNegative RegulatorsRegulatorsRegulatorsRegulators ofofofof PlantPlantPlantPlant InnateInnateInnateInnate ImmunityImmunityImmunityImmunity

Although many RLKs/RLPs are involved in positive regulation of defense against

pathogens, several have been identified that act as negative regulators of plant innate immunity.

For example, FERONIA (FER), an RLK controlling pollen tube reception, plays a critical role in

negatively regulating Arabidopsis defense against the powdery mildew Golovinomyces orontii

(Kessler et al. 2010). Due to the similarity between powdery mildew hyphal tip growth and plant

pollen tube reception, it is hypothesized that powdery mildew may produce ligands similar to

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that of plant pollen tube cells, which may cause FER-mediated susceptibility (Govers and

Angenent, 2010).

Another negative regulator in Arabidopsis innate immunity is the BAK1-interacting

receptor-like kinase (BIR1) (Gao et al. 2009). T-DNA insertional mutations in BIR1 cause over-

accumulation of H2O2 and SA. The bir1-1 mutant is highly resistant to the oomycete

Hyaloperonospora parasitica Noco2. Another protein, SOBIR1, (suppressor of BIR1), also

encodes an LRR RLK and suppressed the cell death and resistance phenotype observed in the

BIR1 mutant. The authors showed that SOBIR1 alone is not required for basal resistance to Pst

DC3000; however, over expression of SOBIR1 in Arabidopsis can induce cell death and enhance

resistance to bacterial pathogens (Gao et al. 2009).

Receptor-likeReceptor-likeReceptor-likeReceptor-like kinases/proteinskinases/proteinskinases/proteinskinases/proteins areareareare involvedinvolvedinvolvedinvolved inininin defensedefensedefensedefense againstagainstagainstagainst bacterialbacterialbacterialbacterial pathogenspathogenspathogenspathogensPositivePositivePositivePositive regulatorsregulatorsregulatorsregulators inininin defensedefensedefensedefense againstagainstagainstagainst bacteriabacteriabacteriabacteria

Many RLKs/RLPs are involved not only in resistance to fungal pathogens but also in

resistance to bacterial pathogens. For example, Arabidopsis CERK1/LysM RLK1 was reported

to be targeted and ubiquitinated for degradation by AvrPtoB, a type III effector of the bacterial

pathogen P. Syringae. In the absence of AvrPtoB, AtCERK1/LysM RLK1 plays a critical role in

restricting bacterial growth in Arabidopsis (Gimenez-Ibanez et al. 2009), suggesting that

AtCERK1/LysM RLK1 may bind an unknown PAMP in bacteria. The ERECTA RLK was also

shown to play a role in defense against Ralstonia solanacearum, the causal agent of bacterial wilt

(Godiard et al. 2003). Null mutations in SNC2 lead to impairment of basal resistance in

Arabidopsis resulting in more bacterial growth on the plant (Zhang et al. 2010). The rice

receptor-like cytoplasmic kinase BSR1 confers resistance to Pst DC3000 in Arabidopsis and to X.

oryzae pv. oryzae (Xoo) in rice (Dubouzet et al. 2010). Additionally, silencing of NbSERK3 in N.

13

Benthamiana enhances susceptibility to the bacterial pathogens P. syringae pv. tabaci 11528

(Pta 11528), P. syringae pv. Tomato DC3000 (Pto DC3000) and the nonpathogenic strain Pto

DC3000 hrcC (Heese et al. 2007).

Besides RLKs/RLPs listed above, many RLKs/RLPs have only been investigated for

their role in bacterial resistance. The Flagellin-sensitive 2 (FLS2) and EF-Tu (EFR) receptors

are LRR RLKs that bind the bacterial PAMPs flg22 and EF-Tu respectively (Gomez-Gomez and

Boller 2000; Zipfel et al. 2006). Null mutations in FLS2 or EFR render mutant plants insensitive

to their ligands (flg22 or elf18 respectively) resulting in susceptibility to bacterial pathogens

(Zipfel et al. 2004; Zipfel et al. 2006). Expression of EFR in N. benthamiana and tomato, which

are insensitive to elf18, causes ROS production and expression of defense-responsive genes upon

elf18 treatment (Lacombe et al. 2010). Transgenic N. Benthamiana and tomato plants expressing

EFR showed increased resistance to bacterial pathogens when compared to wild type plants.

Mutations in the RLKs AtPepR1 and AtPepR2 also impact resistance to bacterial pathogens.

Both pepr1 and pepr2 mutants displayed no difference in their resistance response compared to

wild type plants after inoculation with Pst DC3000 without AtPep1 pretreatment (Yamaguchi et

al. 2010). However upon pretreatment of wild type, pepr1 or pepr2 plants with AtPep1, there

was a marked reduction in the disease symptoms caused by infection with Pst DC3000. AtPep1

pretreated pepr1/pepr2 double mutants had a similar level of susceptibility to Pst DC3000 to the

untreated double mutant plants suggesting that both AtPepR1 and AtPepR2 are required for

initiating AtPep1-mediated defense responses against bacterial pathogens (Yamaguchi et al.

2010).

The rice RLK XA21 confers resistance to a broad spectrum of Xoo (Xanthomonas oryzae

pv. oryzae) races through the recognition of a sulfated peptide Ax21 (activator of XA21-

14

mediated immunity) (Lee et al. 2009). Transgenic plants carrying Xa21 are highly resistant to 29

of 32 Xoo isolates from eight countries (Wang et al. 1996). Xa21D, a natural variant of Xa21,

encodes a receptor-like protein that carries an LRR domain but lacks the transmembrane and

kinase domains. Plants carrying Xa21D also recognize pathogens carrying Ax21 and display

partial resistance to Xoo (Wang et al. 1998). Taken together these data indicate that Xa21 may

recognize Ax21 through its LRR domain. Another RLK/RLP in rice, Xa3/Xa26/Xa22(t), also

confers resistance to Xoo. Transgenic plants carrying Xa26 displayed high levels of resistance to

Xoo. Xa21 and Xa26 are the only two confirmed RLK/RLP resistance (R) genes and although

they both confer resistance to Xoo there are differences between the mechanisms the two genes

use to mediate immunity. Xa21-mediated resistance increases progressively from the susceptible

early seedling stage to full resistance at adult stage. In contrast, Xa26-mediated resistance can be

detected from the juvenile stage through the adult stage in rice.

In tomato, the cytosolic domain of Tomato Atypical Receptor-like kinase 1 (TARK1)

interacts with XopN a type III effector of the bacterial pathogen Xanthomonas campestris

pathovar vesicatoria (Xcv). During infection, XopN compromises tomato defense pathways by

suppressing callose deposition and expression of PTI marker genes such as PTI5, WRKY28,

LRR22, and GRAS2. Null mutations of XopN in Xcv resulted in reduced pathogenicity in tomato.

TARK1 RNAi tomato plants supported more Xcv ΔNopN growth than did wild type tomato

plants, indicating that TARK1 is a positive regulator of tomato basal innate immunity.

Interestingly, TARK1 has been shown to be an inactive kinase and it may function in innate

immunity by interacting with other primary receptors (Kim et al. 2009).

Several RLKs were found to negatively regulate plant defense responses to bacterial

pathogens. The RIN4-interacting receptor-like kinase (RIPK) was identified from the RIN4

15

protein complex in Arabidopsis expressing the bacterial effector avrRpm1. RIPK encodes a

receptor-like cytoplasmic receptor that negatively regulates plant innate immunity (Liu et al.

2011). T-DNA knockout mutants of RIPK were more resistant to Pst DC3000 after spray

inoculation. There was however no difference between ripk KO mutants and wild type plants

when inoculating Pst DC3000 on plants via syringe infiltration (Liu et al. 2011). These results

indicate that RIPK is capable of suppressing Arabidopsis defense at an early stage of infection.

An S locus RLK, CBRLK1, also acts as a negative regulator of Arabidopsis defense against

bacterial pathogens. Cbrlk1-1 mutants are resistant to Pst DC3000 and the mechanism of

resistance is most likely due to enhanced PR gene expression (Kim et al. 2009). Despite its role

in powdery mildew infection, FER also negatively regulates Arabidopsis innate immunity to the

bacterial pathogen PstDC3000 as FER protein levels are induced within 5 minutes of flg22

treatment (Keinath et al. 2010). Flg22-induced ROS levels were significantly higher and stomata

remained constantly closed in fer mutants, which may account for its resistance to P. syringae

infection.

CRLK1CRLK1CRLK1CRLK1 isisisis aaaa receptor-likereceptor-likereceptor-likereceptor-like kinasekinasekinasekinase involvedinvolvedinvolvedinvolved inininin ArabidopsisArabidopsisArabidopsisArabidopsis defensedefensedefensedefense againstagainstagainstagainst fungifungifungifungi

Chitin is a general elicitor that can trigger plant innate immunity and treatment of plants

with chitin can rapidly induce and suppress a large number of defense-related genes in rice and

Arabidopsis (Kaku et al. 2006; Ramonell et al. 2002). In Arabidopsis, many chitin-responsive

genes (CRG) have been shown to play important roles in plant defense (Ramonell 2005). In

order to identify genes that conferred resistance to pathogens, we used a forward genetics

approach to study classes of genes that responded to chitin treatment. My dissertation project

focused on identifying several receptor-like kinases involved in Arabidopsis defense against the

16

powdery mildew pathogen Golovinomycetes cichoracearum. Seven homozygous T-DNA

insertional mutants (Table 1.2) were obtained from the Arabidopsis Biological Resource Cencer

(ABRC) and challenged with G. cichoracearum (Refer to Materials and Methods, Chapter two).

A light infection assay (Refer to Materials and Methods, Chapter two) showed that mutants in

two RLKs, crlk1 and crlk7, were more susceptible to powdery mildew than Col-0 wild type,

which is only moderately susceptible to powdery mildew (Figure 1.1). The following chapters

detail my research describing the identification and characterization of one of these RLKs,

CRLK1, and our current understanding of its function in chitin-mediated defense against fungal

pathogens.

17

Table 1.1. The list of RLKs and RLPs involved in plant innate immunity.

Name ofRLK/RLP

Organism Type ofRLK/RLP

Resistant Susceptible References

BIK1 Arabidopsis RLCK B.cinereaA. brassicicola

Pst DC3000 Veronese etal. 2006

BIR1 Arabidopsis LRR RLK H.parasiticaNoco2

Gao et al.2009

SOBIR1 Arabidopsis LRR RLK Pst DC3000 Gao etal.2009

OSBRR1 Rice LRR RLK M. oryzae Peng et al.2009

BSR1 Arabidopsis RLCK C.higginsianumPst DC3000

Dubouzet etal. 2010

CERK1/LysMRLK1

Arabidopsis LysM RLK G.cichoracearumA. brassicicola

Wan et al.2008;Miya et al.2007;

OsCERK1 Rice LysM RLK Shimizu et al.2010

CEBiP Rice LysM RLP M.oryzae Kaku et al.2006Kishimoto etal. 2010

HvCEBiP Barley LysM RLP M. oryzae Tanaka et al.2010

LeEix1 and 2 Tobacco LRR RLP Ron andAvni, 2004

CBRLK1 Arabidopsis S-locusRLK

Pst DC3000 Kim et al.2009

EFR Arabidopsis LRR RLK A. tumefaciens Zipfel et al.2006

ERECTA Arabidopsis LRR RLK P. cucumerinaP. irregulare

Llorente etal. 2005Adie et al.2007

FLS2 Arabidopsis LRR RLK Pst DC3000 Gomez-Gomez andBoller 2000;Zipfel et al.2004

FER Arabidopsis G. orontiiPst DC3000

Kessler et al.2010

18

Keinath et al.2010

NbLRK1 N.benthamiana

Lectin-likeRLK

Kanzaki et al.2008

AtPepR1 and 2 Arabidopsis LRR RLK Yamaguchiet al. 2006;Krol etal.2010

NgRLK1 N.glutinosa B-lectin,S-locusglycoprotein

Kim et al.2010

TaRLK-R1,2,and3

Wheat Wheat striperust

Zhou et al.2007

AtRLP30 Arabidopsis LRR RLP P.syringae pvphaseolicola1448A

Wang et al.2008

RIPK Arabidopsis RLCK Pst DC3000 Liu etal.2011

NbSERK3/BAK1 N.benthamiana

LRR RLK Pta 11528PtoDC3000PtoDC3000hrcCP. infestans

Heese et al.2007Chaparro-Garcia et al.2011

SNC2 Arabidopsis LRR RLP Pst DC3000 Zhang et al.2010

SNC3 Arabidopsis LRR RLP Zhang et al.2010

TARK1 Tomato LRR RLK Xanthomonascampestrispathovarvesicatoria(Xcv)

Kim et al.2009

TPK1b Tomato RLCK B.cinereaTobaccohornworm(Manducasexta)

Abuqamar etal.2008

Ve1 Tomato LRR RLP V. DahliaeV. albo-atrum

Fradin et al.2009

Vfa4 Apple LRR RLP V.inaequalis

Malnoy et al.2008

Vfa1 and Vfa2 Apple LRR RLP V. inaequalis Malnoy et al.2008;Belfanti et al2004

19

WAK1 Arabidopsis EGF-likeRLK

B. cinerea Decreux andMessiaen2005;Decreux etal. 2006

OsWAK1 Rice EGF-likeRLK

M. Oryzae Li et al. 2009

XA21 Rice LRR RLK Xanthomonasoryzae pv.oryzae

Lee et al.2009

Xa3/Xa26 Rice RLK Xanthomonasoryzae pv.Oryzae

Sun et al.2004

Xa21D Rice RLP Xanthomonasoryzae pv.Oryzae

Wang et al.2004

20

Table 1.2 Gene name, locus and mutant accession number of seven receptor-like kinasesselected from chitin-responsive genes.

Gene Locus Mutant Accession NumberCRLK1 At5g46080 SAIL_801_H08; Salk_063626CRLK2 At4g25390 Salk_093369CRLK3 At1g53080 SAIL_897_G10CRLK4 At4g11470 Salk_106993CRLK5 At4g18250 Salk_036670CRLK6 At5g59680 Salk_001929CRLK7 At2g41890 Salk_147631

21

0

10

20

30

40

50

Col-0 NahG crlk1 crlk2 crlk4 crlk5 crlk6 crlk7

c/c

FigureFigureFigureFigure 1.11.11.11.1 AverageAverageAverageAverage numbersnumbersnumbersnumbers ofofofof conidiophoresconidiophoresconidiophoresconidiophores perperperper colonycolonycolonycolony (c/c)(c/c)(c/c)(c/c) onononon receptor-likereceptor-likereceptor-likereceptor-like kinasekinasekinasekinasemutants,mutants,mutants,mutants, Col-0Col-0Col-0Col-0 wildwildwildwild typetypetypetype andandandand NahG.NahG.NahG.NahG. Mutants crlk1 and crlk7 showed a higher average numberof conidiophores per colony than Col-0 wild type, indicating that CRLK1 and CRLK7 encodegenes positively regulate Arabidopsis innate immunity. Each bar represents average number ofconidiophores counted in 25 single colonies for each mutant. NahG is transgenic linesoverexpressing SA hydroxylase and highly susceptible to powdery mildew infection. Theconidiophores were counted at 5 days after infection.

22

CHAPTERCHAPTERCHAPTERCHAPTER TWOTWOTWOTWO

CHARACTERIZATIONCHARACTERIZATIONCHARACTERIZATIONCHARACTERIZATION OFOFOFOF CRLK1,CRLK1,CRLK1,CRLK1, AAAA NOVELNOVELNOVELNOVEL RECEPTOR-LIKERECEPTOR-LIKERECEPTOR-LIKERECEPTOR-LIKE KINASEKINASEKINASEKINASEINVOLVEDINVOLVEDINVOLVEDINVOLVED ININININ THETHETHETHE INITIATIONINITIATIONINITIATIONINITIATION ANDANDANDAND REGULATIONREGULATIONREGULATIONREGULATIONOFOFOFOF ARABIDOPSISARABIDOPSISARABIDOPSISARABIDOPSIS INNATEINNATEINNATEINNATE

IMMUNITYIMMUNITYIMMUNITYIMMUNITY

INTRODUCTIONINTRODUCTIONINTRODUCTIONINTRODUCTION

Organisms in the environment are under siege by a host of disease-causing organisms

including bacteria, fungi, viruses and nematodes. Removal of these extracellular threats is

paramount to the survival of multicellular organisms (Inohara et al. 2005). Both plants and

animals are capable of detecting pathogens using specific host pattern-recognition receptors

(PRRs) that recognize pathogen-associated molecular patterns (PAMPs) associated with broad

classes of pathogens. Interaction between PRR and PAMP leads to the induction of innate

immunity.

Arabidopsis is a powerful tool to study the interactions between powdery mildew and

plants. There are at least four compatible powdery mildew pathogens for Arabidopsis:

Golovinomyces cichoracearum (Gc), G. orontii (Go), Oidium neolycopersici (On) and Erysiphe

cruciferarum (Ec) (Reviewed by Lipka et al. 2008). An imcompatible powdery mildew such as

Blumeria graminis f.sp. hordei (Bgh) was also used to study the genes that critical for

compatibility (Stein et al. 2006). Receptor-like kinases have been shown to play a critical role in

powdery mildew-Arabidopsis interaction (Miya et al. 2007; Wan et al. 2008; Kessler et al. 2010).

23

In Arabidopsis, CERK1 is the major receptor-like kinase that binds chitin derived from fungal

pathogens and triggers Arabidopsis innate immunity (Miya et al. 2007; Wan et al. 2008).

CERK1 homologs in other plant species were also found to play a similar role in plant defense

against fungal pathogens (Shimizu et al. 2010). FERONIA (FER) is a receptor-like kinase that

negatively regulates plant innate immunity upon powdery mildew infection (Kessler et al. 2010).

Since FER also functions in Arabidopsis pollen tube reception, it is hypothesized that powdery

mildew hyphae may mimic Arabidopsis pollen tube and utilize FER to establish compatibility

(Kessler et al. 2010; Govers and Angenent 2010).

Since there are more than 600 RLKs in Arabidopsis genome, many RLKs were

upregulated or downregulated by chitin treatment (Shiu et al. 2001; Wan et al. 2008). We

hypothesized that some RLKs induced by chitin may play critical roles in Arabidopsis defense

against powdery mildew. In our preliminary study, we screened several RLK homozygous T-

DNA insertional mutants using G. cichoracearum (Chapter one). In this chapter, we describe a

novel RLK, Chitin-induced RLK1 (CRLK1) that is up regulated in response to chitin treatment

in Arabidopsis. Knock-out mutants in CRLK1 are more susceptible to the biotrophic pathogen

Golovinomycetes cichoracearum and the necrotrophic pathogen Plectosphaerella cucumerina.

CRLK1 localizes to the plasma membrane and is most highly expressed in the early stages of

chitin-induced immunity. CRLK1 expression is not induced by salicylic acid, jasmonic acid or

ethylene indicating that it is unique to chitin-induced signaling. Expression of MAPK3 was

increased in crlk1 mutants suggesting that CRLK1 may play a role in negative regulation of

chitin signaling.

24

MATERIALSMATERIALSMATERIALSMATERIALS ANDANDANDANDMETHODSMETHODSMETHODSMETHODS

BiologicalBiologicalBiologicalBiological materialsmaterialsmaterialsmaterials andandandand growthgrowthgrowthgrowth conditionsconditionsconditionsconditions

T-DNA insertional mutants of CRLK1 (At5g46080; Sail_801_H08; Salk_063626) and

NahG were obtained from the Arabidopsis Biological Resource Center (ABRC, Ohio State

University). Homozygous T-DNA mutants were identified using these primers: CRLK1: 5'-

TCCAAATCACCCTCCTTCC-3' (F); 5'-CTTCTTCTCATAATCTTCCTCTTC -3' (R). T-DNA

primer: 5'-TAGCATCTGAATTTCATAACCAATCTCGATACAC-3'. One backcross was

performed on all mutants with Col-0. Plants were grown under controlled conditions in a growth

chamber (Percival Scientific) at 22°C day/19°C night with 16 hrs of light per 24 hrs and 50% RH.

G. cichoracearum was cultured on squash and maintained at 22°C day/19°C night with 16 hrs of

light per 24 hrs and 85% RH. Fungal inoculums were prepared and inoculations performed as

described (Wilson et al., 2001). P. cucumerina was grown on potato dextrose agar at 28 °C, as

described (Sanchez-Vallet et al., 2010).

InfectionInfectionInfectionInfection assaysassaysassaysassays withwithwithwith G.G.G.G. cichoracearumcichoracearumcichoracearumcichoracearum

Heavy infection with G. cichoracearum was done by touching 21-day old Arabidopsis

rosettes with heavily infected squash leaves and scored for resistance phenotypes after 10 days

(Wilson et al. 2001). For determination of fungal biomass, plants were lightly infected as

described (Wilson et al., 2001). For H2O2 staining, leaves were harvested 5 dpi and stained in

1mg/ml DAB for 8 hrs and then cleared in 95% of ethanol overnight. Cleared leaves were

equilibrated in a 1:1:1 mixture of water: lactic acid: glycerol for 3 hrs and counter-stained in 200

mg/ml Trypan blue for 2 hrs. Conidiophores and H2O2 stained leaves were scored under 40X

25

magnification with a light microscope. Student’s t-test was performed using Excel to determine

SE with n=10.

P.P.P.P. cucumerinacucumerinacucumerinacucumerina BiomassBiomassBiomassBiomass DeterminationDeterminationDeterminationDetermination

Spores of P. cucumerina were sprayed onto 4-week old Arabidopsis plants at a

concentration of 2×106 spores/ml. To determine fungal biomass within leaf tissue, fungal DNA

was extracted from infected leaves 3 dpi. Relative fungal biomass was quantified using qRT-

PCR in a real-time PCR system (Roche), as described (Sanchez-Vallet et al. 2010).

DiseaseDiseaseDiseaseDisease assaysassaysassaysassays ofofofof P.P.P.P. syringaesyringaesyringaesyringae

P. syringae DC3000 virulent or avirulent strains (avrRpt2) were cultured on King's B

plates with kanamycin (50μg/ml) for 2 days. Strains were resuspended in 10mM MgCl2 and

grown to an OD600 reading of 0.05 (5×107 cells/ml). Cells were diluted 1:1000. Diluted strains

were used to inoculate Arabidopsis leaves using a 1ml syringe. Inoculated leaves were collected

48 and 96 hpi and pulverized in 1.5 ml tube containing 200 μl ice cold H2O. Resuspended

bacteria were plated in serial dilutions to determine levels of growth as described (Hinsch and

Staskawicz. 1996).

ChitinChitinChitinChitin andandandand hormonehormonehormonehormone treatmentstreatmentstreatmentstreatments

Seedlings were surface sterilized and grown in 10 ml of liquid MS medium supplemented

with Gambourg vitamins and 2% dextrose (Sigma-Aldrich) in 50 ml tubes. Seedlings in liquid

culture were grown under continuous light at room temperature as described (Zhang et al. 2002).

14 day old seedlings were treated with either a solution containing 1μg/ml hydrolyzed crab shell

26

chitin (CSC, Sigma-Aldrich) in ddH2O or ddH2O only for time points starting at 30 min and

ending at 24 hrs. Chitin solution was made by adding ddH2O to CSC and vortexing overnight.

For hormone treatments, 14 day old seedlings in liquid culture were treated with 5 mM SA, 100

µM methyl jasmonate or 0.5 mM ACC for 24 hrs before harvesting. Harvested seedlings were

frozen immediately on liquid nitrogen.

QuantitativeQuantitativeQuantitativeQuantitative realrealrealreal timetimetimetime PCRPCRPCRPCR

Total RNA was isolated from 14 day old seedlings using Trizol Reagent (Invitrogen) and

treated with DNase I (Ambion). 5 µg of total RNA was reverse transcribed to cDNA using a

SuperscriptTM II RT kit (Invitrogen). qRT-PCR was carried out using an Opticon2TM qRT-PCR

System (BioRad). Accession numbers for genes used are: CRLK1: At5g46080; MAPK3:

At3g45640;WRKY53: At4g23810; Actin-2: At3g18780. Primers for qRT-PCR were: CRLK1: 5-

'GGAGAAAGAATCGTCGTAAC-3' (F); 5'-GATGTGAATCTACCGTGAAC-3' (R); Actin-2:

5'-GGTATTCTTACCTTGAAGTATCCTA-3' (F); 5’-

TCATTGTAGAAAGTGTGATGCCAGATC-3' (R);MAPK3: 5'-

CTCACGGAGGACAGTTCATAAG-3' (F) 5'-GAGATCAGATTCTGTCGGTGTG-3' (R);

WRKY53: 5'-CCTACGAGAGATCTCTTCTTCTG-3' (F); 5'-

AGATCGGAGAACTCTCCACGTG (R)-3'. Each qRT-PCR reaction contained 12.5 µl IQ

SYBR Green Supermix (Bio-Rad), 8.5 µl DEPC-treated water (Ambion), 2 µl of cDNA

(10ng/µl), and 1 µl forward and reverse primer (12.5µM). PCR reaction conditions were: 50°C

for 2 min; 95°C for 10 min; 95°C for 15 s, 60°C for 30 s and 72°C for 30 s for 40 cycles. Raw

data were collected by Opticon monitor 3 software (Bio-Rad) and relative gene expression levels

were analyzed using LinregPCR software (Ramakers et al. 2003).

27

TransientTransientTransientTransient expressionexpressionexpressionexpression inininin tobaccotobaccotobaccotobacco

A fragment containing full length CRLK1 cDNA was generated by PCR. Primers for

35S:CRLK1:GFP construct were:

5'-GGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGACAAATCTGAAAAAATCGC- 3'

(F);

5'- GGGGACCACTTTGTACAAGAAAGCTGGGTCCCCTCCTTCCCTCTCCAAA-3' (R).

Forward primer for 35S:ΔTM CRLK1:GFP was:

5'GGGACAAGTTTGTACAAAAAAGCAGGCTTCAGAAAAAAACCAAACC-3' (F).

Reverse primer for 35S:ΔTM CRLK1:GFP was the same sequence as the 35S:CRLK:GFP

reverse primer.

Underlined sequences are CRLK1 specific. PCR product was cloned into pDONR221 using

Gateway technology (Invitrogen), subcloned into the binary vector pMDC83 (Curtis and

Grossniklaus. 2003) and transformed into Agrobacterium tumefaciens strain GV3010. Transient

expression was carried out by co-infiltrating tobacco leaves with an A. tumefaciens strain

containing free GFP or 35S:CRLK1:GFP fusion genes and another strain of A. tumefaciens

containing the plasma membrane marker 35S:AtPIP2:mCherry (Sparkes et al. 2006; Nelson et al.

2007). For plasmolysis experiment, 1 M of NaCl was infiltrated into tobacco leaves 10 minutes

before images were taken. Images were collected using a Leica TCS SP2 AOBS Confocal

Microscope (Leica Microsystems) 72 hrs after infiltration. GFP and mCherry were excited using

a 488 nm Ar/Kr laser and a 543 nm He/Ne laser respectively.

28

PredictionPredictionPredictionPrediction ofofofof W-boxesW-boxesW-boxesW-boxes

W boxes in the promoter region of CRLK1 were predicted by manually scanning for the

W-box sequence [(T)TGAC(C/T)] 1.5 kb upstream CRLK1 start codon.

ConstructionConstructionConstructionConstruction ofofofof pCRLK1::GUSpCRLK1::GUSpCRLK1::GUSpCRLK1::GUS vectorvectorvectorvector andandandand histochemicalhistochemicalhistochemicalhistochemical stainingstainingstainingstaining withwithwithwith X-glucX-glucX-glucX-gluc

A 2kb DNA fragment was amplified from Arabidopsis genomic DNA using Iproof High

Fidelity Polymerase (Bio-Rad Laboratories) using primers contain attB recombination site. The

sequences were as following:

Forward:

GGGGACAAGTTTGTACAAAAAAGCAGGCTTC CATGTTTGAACTGCATTATGA

Reverse:

GGGGACCACTTTGTACAAGAAAGCTGGGTCTTGATTTCAACAAAGAAAATAAAT

The underlined sequences are attB recombination site. The PCR product was introduced to entry

vector pDONR211 using BP clonase mix (Invitrogen). The resultant pDONR221 vector

harboured CRLK1 promoter was subcloned into pMDC162 (Curtis and Grossniklaus, 2003), a T-

DNA binary vector containing GUS gene downstream recombination sites. The pCRLK1：：

GUS vector was transformed to Agrobacterium tumefaciens strain GV3010 and then transformed

into Arabidopsis Col-0 via floral dip method (Zhang et al. 2006). The transformants were

selected on agar plates containing 50μg/ml of hygromycin B and full-strength of MS salt

supplemented with Gambourg vitamins (Sigma-Aldrich). Homozygous transgenic lines were

selected from T3 lines for histochemical staining. Tissues were stained in staining buffer

containing 0.5M sodium phosphate buffer (pH 7.2), 10% Triton X-100, 100mM potassium

ferrocyanide, 100mM potassium ferricyanide and 10mM X-Gluc. Wounding treatment was

29

carried out by stabbing transgenic plant leaves with needles. The leaves were stained

immediately after wound treatment. Cross-section of Arabidopsis roots were made as following

procedure: stained Arabidopsis seedlings as above, then destained in 70% EtOH overnight. The

seedlings were incubated in Glutaraldehyde for 1 hour. Then the seedlings were rinsed in sodium

phosphate buffer (PH=7.2) three times and then incubated in gradient EtOH (25%, 50%, 75%,

and 100%) for 30 minutes each step. Then the seedlings were incubated in resin (10g of ERL

4206, 4g of DER 736, 26g of NSA, and 0.5ml of DMAE): EtOH=2:1 for 1 hour, resin:EtOH=1:1

for 1 hour and resin overnight in 70°C. The seedlings embedded in resin were trimmed and

section using LKB ULTROTOME 4801A and then visualized under light compound microscope.

RESULTSRESULTSRESULTSRESULTS

IdentificationIdentificationIdentificationIdentification andandandand characterizationcharacterizationcharacterizationcharacterization ofofofof CRLK1CRLK1CRLK1CRLK1

We identified a group of LRR-RLKs that appeared to specifically respond to chitin by

analyzing publically available microarray data of Arabidopsis plants treated with three elicitors:

1) Flg22, the conserved domain of bacterial flagellin; 2) oligogalacturonides and 3) purified

chitin fragments. Focusing on genes involved in chitin perception, we identified genes that were

A) up-regulated by chitin; B) classified as RLKs by gene annotation and C) had not been

previously characterized in relation to defense responses and obtained T-DNA insertional

mutants in the selected genes from the Salk Institute Genomic Analysis Laboratory collection

(http://signal.salk.edu/cgi-bin/tdnaexpress). All lines were screened to identify homozygous

mutants and one backcross was performed on all insertional mutants with wild-type Columbia

(Col-0; Data not shown).

30

Since chitin fragments are one of the pathogen-associated molecular patterns that are

known to elicit strong defense responses in plants (Ramonell et al. 2002; Zhang et al. 2002) and

plants have evolved a mechanism to detect chitin fragments then one would predict that

mutations in chitin-responsive genes would result in an enhanced susceptibility to pathogens.

We inoculated the T-DNA mutants with the powdery mildew pathogen, G. cichoracearum, and

evaluated them for disease resistance. To evaluate susceptibility the T-DNA lines were

compared with powdery mildew inoculated Col-0 as the control and with the hyper-susceptible

transgenic plant NahG (Nawrath and Metraux 1999). At high density inoculation, one of the T-

DNA insertional mutants designated chitin receptor-like kinase 1 (crlk1; At5g46080) had more

severe macroscopic symptoms compared with Col-0 wild type plants (Figure 2.1A) though the

fungal growth was not as advanced as that on hyper-susceptible NahG plants. Independent

insertional mutants of CRLK1 also showed enhanced disease susceptibility when infected with G.

cichoracearum confirming that the altered disease phenotype can be attributed to the loss of

CRLK1 function and not to a secondary mutation (Data not shown). To more quantitatively

examine the susceptibility phenotype we inoculated crlk1, Col-0 and NahG at low density and

examined hyphal growth and the number of conidiophores produced per colony. Though visible

differences in the amount of hyphal growth on each were subtle (Fig. 2.1B), fungal colonies on

crlk1 mutant plants produced a higher density of conidiophores compared to Col-0 (Figure 2.1C).

However crlk1 mutants did not produce as many conidiophores as the NahG transgenic line

(Reuber et al. 1998).

A characteristic of the initial response to pathogens is ROS production (Mittler et al.

2004). Since crlk1 was more susceptible to powdery mildew we wanted to determine if

production of ROS in crlk1 was compromised. Leaves of crlk1, Col-0 and NahG were infected

31

at low density and stained with diamino-benzidine (DAB) to visualize H2O2 and with trypan blue

to observe the fungal hyphae. Both crlk1 and Col-0 exhibited a similar level and pattern of DAB

staining at sites of H2O2 production where the fungus was attempting to penetrate the leaf

epidermal cell (Figure 2.1D). In contrast, no DAB staining was observed in the NahG transgenic

line as it is compromised in ROS production.

32

FigureFigureFigureFigure 2.2.2.2.1111 crlk1crlk1crlk1crlk1 isisisis moremoremoremore susceptiblesusceptiblesusceptiblesusceptible totototo GGGG.... cichoracearumcichoracearumcichoracearumcichoracearum butbutbutbut exhibitsexhibitsexhibitsexhibits normalnormalnormalnormal ROSROSROSROSproduction.production.production.production. A.A.A.A.Whole plant visible phenotype of crlk1, Col-0 and NahG 10 dpi. B.B.B.B. Fungalhyphae and conidiophores of G. cichoracearum stained by trypan blue 5 dpi. Arrows indicateconidiophores. Bar = 0.2mm. C.C.C.C. Quantification of conidiophores per colony (c/c). Averagevalue and SE were calculated from n=25 colonies. D.D.D.D. DAB staining to determine H2O2

production at sites of infection in Col-0, crlk1and NahG. Arrows indicate points of H2O2 stainingat infection sites. Bar = 0.1mm.

33

CRLK1CRLK1CRLK1CRLK1 isisisis involvedinvolvedinvolvedinvolved inininin defensedefensedefensedefense againstagainstagainstagainst biotrophicbiotrophicbiotrophicbiotrophic andandandand necrotrophicnecrotrophicnecrotrophicnecrotrophic fungalfungalfungalfungal pathogenspathogenspathogenspathogens

Since mutations in CRLK1 render plants more susceptible to infection by the biotrophic

fungus G. cichoracearum (Figure 2.1A, 2.1B and 2.1C), we were interested in learning if CRLK1

participated in defense against other classes of fungi. To test this 3-week-old seedlings of crlk1

were inoculated with the necrotrophic fungus P. cucumerina and fungal biomass within the

leaves was quantified (Figure 2.2). To evaluate the levels of susceptibility crlk1 was compared

to Col-0, Arabidopsis G-protein beta subunit 1-1 (agb1-1) that is hyper-susceptible to P.

cucumerina and enhanced resistance to necrotrophs 1-1 (ern1-1) that is resistant to P.

cucumerina infection. At 3 dpi crlk1 had more than double the amount of fungal biomass in the

leaves compared with the Col-0 control plants (Figure 2.2). This level of fungal growth was

comparable to that of the hyper-susceptible agb1-1 mutant and much less than that in the

resistant ern1-1 leaves (Figure 2.2) indicating that CRLK1 function is needed for defense against

both biotrophic (Figure 2.1) and necrotrophic (Figure 2.2) pathogens.

To determine if CRLK1 was acting more broadly in plant basal defense responses against

other classes of pathogens we tested the resistance phenotype of crlk1 mutants infected with both

virulent (DC3000 vir) and avirulent (DC3000 avrRpt2) strains of the bacterial pathogen

Psuedomonas syringae pv tomato DC3000. Bacterial counts on infected leaves in both Col-0

and crlk1 mutants were the same regardless of strain or time after inoculation (Figure 2S1)

suggesting that CRLK1 acts only in defense against fungal pathogens.

34

FigureFigureFigureFigure 2222.2.2.2.2 crlk1crlk1crlk1crlk1 isisisis moremoremoremore susceptiblesusceptiblesusceptiblesusceptible totototo thethethethe necrotrophnecrotrophnecrotrophnecrotroph P.P.P.P. cucumerinacucumerinacucumerinacucumerina.... Fungal biomass inleaves was quantified by qRT-PCR 3 dpi. Data represents ratio of fungal biomass between wild-type Col-0 and each mutant (n = 3).

35

FigureFigureFigureFigure 2S12S12S12S1 crlk1crlk1crlk1crlk1 isisisis notnotnotnot compromisedcompromisedcompromisedcompromised inininin itsitsitsits resistanceresistanceresistanceresistance totototo bacterialbacterialbacterialbacterial pathogens.pathogens.pathogens.pathogens. AAAA.Quantitative analysis of growth of either P. syringae DC3000 vir or P. syringae avrRpt2 strainswithin leaves of Col-0 and crlk1 at 0 (white bar), 48 (black bar) and 96 (striped bar) hrs postinfection. BBBB. Visible phenotypes of Col-0 and crlk1 Arabidopsis leaves after inoculation witheither P. syringae DC3000 vir or P. syringae avrRpt2 strains. Black and red pen marks denotesite of inoculation with pathogen. Photos were taken at 96 hours post inoculation.

36

CRLK1CRLK1CRLK1CRLK1 isisisis inducedinducedinducedinduced earlyearlyearlyearly inininin thethethethe chitinchitinchitinchitin responseresponseresponseresponse andandandand isisisis notnotnotnot affectedaffectedaffectedaffected bybybyby otherotherotherother defense-relateddefense-relateddefense-relateddefense-relatedhormoneshormoneshormoneshormones

To determine the timing of CRLK1 expression after chitin treatment expression levels of

CRLK1 were monitored 30 min, 1hr, 1.5 hr, 2 hr, 4 hr and 24 hrs post chitin treatment. Analysis

of the gene expression revealed that CRLK1 is induced more than 10-fold within 1 hour of chitin

treatment (Figure 2.3A) compared with mock treated plants. CRLK1 expression then quickly

dropped back to negligible levels within 4 hours (Figure 2.3A). At 24 hours post-treatment,

CRLK1 expression appears to increase slightly though the level of induction is far below that of

earlier time points (Figure 2.3A).

Salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) are plant hormones that play

important roles in plant defense responses (Bari and Jones 2009). Since there is cross-talk

between many plant defense response pathways we were interested in determining if CRLK1

expression was influenced by SA, JA or ET. Col-0 seedlings were treated with either SA, Methyl

jasmonate (MeJA) or the ethylene precursor 1-aminocyclopropane 1-carboxylic acid (ACC) for

24 hours and expression of CRLK1 was analyzed. In all three hormone treatments CRLK1 was

repressed similarly (Figure 2.3B) compared to mock-treated plants. These data suggest that

CRLK1 is repressed by a general mechanism at work during SA-, JA- and ET-mediated

responses and is not specifically regulated by any of these pathways.

37

FigureFigureFigureFigure 2.32.32.32.3 ExpressionExpressionExpressionExpression ofofofof CRLK1CRLK1CRLK1CRLK1 afterafterafterafter chitin,chitin,chitin,chitin, ACC,ACC,ACC,ACC, MeJAMeJAMeJAMeJA andandandand SASASASA treatments.treatments.treatments.treatments. A.A.A.A. Inductionof CRLK1 transcripts by chitin at various time points. Col-0 seedlings were treated with chitin orddH2O (mock) in MS liquid medium for 0.5, 1, 1.5, 2, 4 and 24 hrs. Bar represents ratio betweenmock and chitin treated Col-0. B.B.B.B. Affects of ACC, MeJA and SA treatment on CRLK1expression. Data represents ratio of gene expression between ddH2O (mock) and hormonetreated Col-0 plants after 24 hrs treatment.

38

CRLK1CRLK1CRLK1CRLK1 encodesencodesencodesencodes aaaa receptor-likereceptor-likereceptor-likereceptor-like kinasekinasekinasekinase thatthatthatthat isisisis localizedlocalizedlocalizedlocalized totototo thethethethe plasmaplasmaplasmaplasma membranemembranemembranemembrane

Using protein prediction software we found that CRLK1 encoded a receptor-like kinase

with a short N-terminal domain (12 amino acids), a transmembrane domain (13-29 amino acids)

and a C-terminal kinase domain (data not shown). The N-terminal domain of CRLK1 does not

contain any known binding or interaction sites so its potential for interaction with other proteins

appears limited. To understand the regulation of CRLK1 we performed an analysis of the gene’s

upstream promoter region. Seven w-boxes were found in the CRLK1 promoter region within

2.5kb of the start site suggesting that WRKY transcription factors may play a role in its

regulation (Figure 2.4A).

To determine CRLK1’s location within the cell a construct containing a C-terminal

fusion of the green fluorescent protein (GFP) to CRLK1 under the control of the CaMV 35S

promoter (35S:CRLK1: GFP) was made and transiently expressed in tobacco epidermal cells.

Unlike free GFP (Figure 2.4B), 35S:CRLK1: GFP was clearly localized to the plasma membrane

in the tobacco cells (Figure 2.4C). We also performed a co-localization using our

35S:CRLK1:GFP construct and the plasma membrane marker AtPIP2A:mCherry (Cutler et al.

2000). 35S:CRLK1: GFP clearly co-localized with the plasma membrane marker (Figure 2.4C)

confirming its localization to the plasma membrane. To further rule out the possibility of

CRLK1 is associated with plant cell wall, tobacco leaves expressing 35S:CRLK1: GFP and

AtPIP2A:mCherry were infiltrated with 1M NaCl for plasmolysis. As figure 2.4D showed,

CRLK1:GFP were colocalized with AtPIP2A:mCherry on plasma membrane detached from cell

wall indicating CRLK1 was only expressed in plant plasma membrane. The first 30 amino acids

of CRLK1 were predicted to be transmembrane domain (TM). We hypothesized that the first 30

amino acids were critical for CRLK1 localization and function. To test this hypothesis, a

39

construct containing mutants CRLK1 with deletion of TM was generated. Upon visualization,

mutant CRLK1 (35S:ΔTM CRLK1:GFP) was predominantly delivered to cytoplasm (Figure

2.4E), suggesting a critical role of first 30 amino acids in CRLK1 targeting.

40

FigureFigureFigureFigure 2.42.42.42.4 CRLK1CRLK1CRLK1CRLK1 encodesencodesencodesencodes aaaa RLKRLKRLKRLK thatthatthatthat hashashashas multiplemultiplemultiplemultiple W-boxesW-boxesW-boxesW-boxes inininin itsitsitsits promoterpromoterpromoterpromoter sequencesequencesequencesequence andandandandisisisis localizedlocalizedlocalizedlocalized totototo thethethethe plasmaplasmaplasmaplasma membrane.membrane.membrane.membrane. A.A.A.A.Multiple W boxes found 1.5 kb upstream of theCRLK1 start site. The numbers indicate positions from start codon of CRLK1 transcript. ATG:start codon; TGA: stop codon; TM: transmembrane domain; TTGACC: consensus for W boxelement. B-E:B-E:B-E:B-E: CRLK1 localizes into plasma membrane in tobacco leaves. Panel B-Ecorrespond to tobacco cells expressing free 35S:GFP, 35S:CRLK1:GFP, 35S:CRLK1:GFPtreated with 1M NaCl and 35:ΔTM CRLK1:GFP respectively. Free GFP was as control andlocalized into plasma membrane and cytoplasm (B) and 35S:CRLK1:GFP fusion protein wasonly expressed in plasma membrane, as 35S:CRLK1:GFP colocalized with plasma membranemarker under both normal condition (C) and plasmolysis (D). Deletion of transmembranedomain in 35S:ΔTM CRLK1:GFP fusion protein targeted the fusion protein into plasmamembrane, cytoplasm and nucleus (E), as fusion protein in cytoplasm did not colocalize withplamsa membrane marker.Bar=50μm.GFP, free GFP or fusion GFP proteins; mCherry, plasma membrane marker AtPIP2:mCherry.

41

ExpressionExpressionExpressionExpression ofofofof CRLK1CRLK1CRLK1CRLK1 inininin ArabidopsisArabidopsisArabidopsisArabidopsis tissuestissuestissuestissues atatatat differentdifferentdifferentdifferent developementaldevelopementaldevelopementaldevelopemental stagesstagesstagesstages

In order to study the where CRLK1 localized in tissue level, transgenic Arabidopsis were

generated to express GUS under the control of a 2kb CRLK1 promoter region. Homozygous

transgenic lines were selected at T2 generation and subjected to GUS staining under 37°C for 3

hours (described under Materials and Methods). Intense GUS staining were detected in

cotyledons, hypocotyls and roots as early as 2 days (Figure 2.5A). As the seedlings grew, the

pCRLK1::GUS activity were still present in cotyledons, hypocotyls and roots, however, the

expression pattern in cotyledons changed from uniformly distributed (Figure 2.5A) to patch-like

(Figure 2.5B and 2.5C) and then to predominantly in vasculature (Figure 2.5D). In roots, GUS

activity was detected mainly in stele (ST) and endodermis (EN) . However, no activity was

found in root hair cells cortex (CO) and epidermis (EP) (Figure 2.5E and 2.5G). GUS is

continuously expressed along roots except the meristemaic zone (MZ) (Figure 2.5F). In 21-day

soil-grown plants, GUS was expressed predominantly in vasculature of mature rosette leaves

(Figure 2.5H). After transgenic plant developed reproductive organs, GUS was mainly

expressed in sepals, stigma and stamen of mature flowers (Figure 2.5I and 2.5J). No GUS

activity was detected in immature developing flower buds (Figure 2.5I) or mature flower anther

(Figure 2.5J). After plants developed siliques, pCRLK1::GUS was expressed in tips and

abscission zone of young developing siliques (Figure 2.5K). Interestingly, GUS expression was

not detected in mature siliques except petioles (Figure 2.5L). The histochemical analysis of

pCRLK1::GUS indicated that CRLK1 may be regulated temporally and spatially during

42

Arabidopsis development. CRLK1 strong expression in cotyledons and rosettes leaves suggested

roles of CRLK1 in sensing pathogens. Upon wounding treatment, pCRLK1::GUS in tissue

around the wound was rapidly induced (Supplemental figure 2S2 B and 2S2D) indicating

expression of CRLK1 may be regulated by other defense pathways.

43

FigureFigureFigureFigure 2.52.52.52.5 HistochemicalHistochemicalHistochemicalHistochemical stainingstainingstainingstaining ofofofof GUSGUSGUSGUS activityactivityactivityactivity inininin transgenictransgenictransgenictransgenic ArabidopsisArabidopsisArabidopsisArabidopsis harboringharboringharboringharboringpCRLK1::GUS.pCRLK1::GUS.pCRLK1::GUS.pCRLK1::GUS. Transgenic Arabidopsis seedlings were grown in 7.5% MS agar supplementedwith Gambourg vitamins and seedlings were stained in 10mM X-gluc for 3 hours in 37°C asdescribed in Material and Methods. pCRLK1::GUS activity was detected in cotyledons,hypocotyls and roots in 2-day old seedlings (A), 3-day old seedlings (B), 4-day old seedlings (C)and 5-day old seedlings (D). GUS was expressed in a similar pattern in hypocotyls and roots butdifferent in cotyledons during seedlings development. In 5-day old seedling roots, GUS wasexpressed in maturation zone and elongation zone of seedlings but no activity was detected inmeristemaic zone (MZ) (F). GUS activity was detected in stele (ST) and endodermis (EN) incross-section of 5-day seedling roots (E and G). In 21-day old Arabidopsis, GUS was expressedin vasculature of rosette leaves (H). After plant developed flowers, GUS was expressed ininflorescences such as sepals, stigma, and stamen ( I and J). In young siliques, GUS wasexpressed in silique tip and abscission zone (K). In mature siliques, GUS was expressed insilique petioles and GUS activity in siliques was undetectable (L).Scale bars represent for 1mm from A to D and 0.1mm from E to G.ST, stele; EP, Epidermis; EN, Endodermis; CO, Cortex.

44

Control Wound

FigureFigureFigureFigure 2S22S22S22S2 Wound-inducedWound-inducedWound-inducedWound-induced GUSGUSGUSGUS expressionexpressionexpressionexpression inininin pCRLK1::GUSpCRLK1::GUSpCRLK1::GUSpCRLK1::GUS transgenictransgenictransgenictransgenic ArabidopsisArabidopsisArabidopsisArabidopsisplants.plants.plants.plants. Arabidopsis leaves were wounded and immediately stained with X-gluc for 3 hoursunder 37 °C as described in Materials and Methods. A and C, mock; B and D, wound treatment.GUS is highly expressed peripherally in the mesophyll and vein cells around wound, as shown inD.

45

RoleRoleRoleRole ofofofof CRLK1CRLK1CRLK1CRLK1 inininin chitinchitinchitinchitin mediatedmediatedmediatedmediated signalingsignalingsignalingsignaling

Though several significant players in chitin mediated signaling have been identified we

are far from a comprehensive understanding of this signaling pathway (Wan et al. 2008;

Berrocal-Lobo et al. 2010; Kishi-Kaboshi et al. 2010; Shimizu et al. 2010). Since CRLK1 was

induced within 30 minutes of chitin treatment and its expression was not upregulated by other

defense related hormones we wanted to determine where CRLK1 might be acting within the

known chitin-mediated signaling pathway. To determine this we tested the expression levels of

two well-characterized marker genes, MAPK3 andWRKY53, that are strongly induced after

chitin treatment in the crlk1 mutant and Col-0 (Wan et al. 2004; Wan et al. 2008). Expression of

bothMAPK3 and WRKY53 was not blocked in crlk1 and expression levels of WRKY53 were not

significantly different between Col-0 and crlk1 (Figure 2.6A & 2.6B). Interestingly, MAPK3

expression was more than 2-fold higher in the crlk1 mutant compared with Col-0 wild type

plants (Figure 2.6A). Since MAPK3 is known to act fairly early in chitin-mediated responses

(Wan et al. 2004), this suggests that functional CRLK1 may be involved in repression or

modulation of upstream chitin signaling responses as fungal infection progresses.

46

FigureFigureFigureFigure 2.62.62.62.6 ExpressionExpressionExpressionExpression ofofofofMAPK3MAPK3MAPK3MAPK3 andandandandWRKY53WRKY53WRKY53WRKY53 afterafterafterafter chitinchitinchitinchitin treatmenttreatmenttreatmenttreatment inininin Col-0Col-0Col-0Col-0 wildwildwildwild typetypetypetypeandandandand crlk1crlk1crlk1crlk1.... Data represents the ratio of (A) MAPK3 or (B) WRKY53 expression after chitintreatment with mock (ddH2O) treated plants. Plants were treated with chitin for 30 min beforecollection for total RNA. Chitin treatments were as described in experimental methods.

47

DISCUSSIONDISCUSSIONDISCUSSIONDISCUSSION

Plants are capable of sensing general elicitors derived either from the invading pathogen

or the damaged plant itself to trigger broad-spectrum innate immune responses. In the current

study we identified a receptor-like kinase, CRLK1 that is involved in chitin-mediated defense

responses to fungal pathogens. Several RLKs are known to recognize PAMPs particularly those

involved in responses to bacterial pathogens. The overall picture for chitin-mediated signaling

against fungal pathogens is less clear. In rice chitin oligosaccharide elicitor-binding protein

(CEBiP) was shown to bind chitin and a knockdown of CEBiP mRNA levels in rice cells

resulted in a reduction in the number of chitin responsive genes expressed (Kaku et al. 2006).

Furthermore the rice chitin elicitor receptor kinase 1 (OsCERK1) was shown to form a protein

complex with CEBiP and both CEBiP and OsCERK1 were shown to be required for activation

of chitin-mediated signaling in rice (Shimizu et al. 2010). In Arabidopsis CERK1 was identified

as the major receptor that binds chitin and initiates chitin-mediated signaling (Miya et al. 2007)

though potential interacting partners remain to be identified. It is possible that CRLK1 may act in

concert with CERK1 in a complex similar to that of CEBiP/OsCERK1 to activate chitin-

mediated signaling events. Experiments to determine interacting partners of CRLK1 will be

critical to more completely understand its function within the known chitin-signaling cascade.

Unlike most RLKs CRLK1 does not contain an extracellular domain that is necessary to

recognize PAMPs. Therefore the function of CRLK1 in signaling is probably mediated through

interactions with other RLKs or RLPs. In bacteria the BIK1 kinase has been reported to associate

with both BAK1 and FLS2 and is required for flg22-triggered innate immunity (Lu et al. 2010).

BIK1 also lacks an extracellular domain and its phosphorylation upon flg22 binding is required

by BAK1 and FLS2. Similarly CRLK1 may function as a partner of other RLKs or proteins,

48

such as CERK1, via direct binding and phosphorylation. Our data suggest that CERK1 and

CRLK1 may act together either by direct interaction or through a complex since both cerlk1 and

crlk1 mutants were susceptible to the biotrophic fungus G. cichoracearum. Additionally in the

cerk1 knock-out mutant the induction of CRLK1 upon chitin treatment was blocked indicating

that transcriptional regulation of CRLK1 by chitin is dependent on CERK1.

We have shown that CRLK1 is essential for defense against the fungal pathogens G.

cichoracearum and P. cucumerina. Several other RLKs have been identified in plants that are

involved in defense against biotrophic or necrotrophic fungi. In Arabidopsis ERECTA (ER) was

found in a QTL analysis to play a role in defense against the necrotrophic fungi Plectosphaerella

cucumerina (Llorente et al. 2005). In tomato, a receptor-like cytoplasmic kinase Tomato Protein

Kinase 1b (TPK1b) was shown to play a critical role in defense against the necrotrophic fungus B.

cinerea but has no role in defense against the bacterial pathogen P. syringae (Abuqamar et al.

2008). In our study CRLK1 was also shown to have no effect on the defense response to P.

syringae (Figure 2S1). Though originally linked to elicitor-mediated defense against bacterial

pathogens, Arabidopsis T-DNA insertional mutants of BAK1 were shown to confer higher

susceptibility to two necrotrophic fungi B. cinerea and A. brassicicola (Kemmerling et al. 2007).

Another kinase, BOTRYTIS-INDUCED KINASE1 (BIK1), has also been shown to be required for

resistance to both B. cinerea and A. brassicicola (Veronese et al. 2006). While the data is far

from complete, it does appear that most RLKs linked to defense against fungal pathogens are

unique to fungal defense pathways and are not involved in defense against other classes of

pathogens.

Chitin has been shown to induce more than 800 genes in Arabidopsis within 30 minutes

of treatment (Wan et al. 2008). Many of these genes are induced temporarily by chitin and then

49

their transcripts decrease rapidly. In a time course study of chitin-induced gene expression,

AtMAPK3 expression was shown to be induced after as little as 5 minutes of chitin treatment. It

then reached a maximum expression level after 30 minutes and then decreased to 1-fold within 4

hours (Wan et al. 2004). Chitin-induced expression of CRLK1 resembled that of AtMAPK3

though CRLK1 is induced at a higher level than AtMAPK3. We surveyed 39 receptor-like

kinases from the 890 genes induced by chitin and found that most of the RLKs were induced

between 2-4 fold after 30 minutes of chitin treatment (data not shown). CRLK1 was highly

induced by chitin compared to most of the other RLKs, which may suggest that increased

amounts of CRLK1 are required to prolong and sustain chitin-mediated defense. The fact that

CRLK1 transcripts rapidly decreased after 1 hour of chitin treatment strongly suggest that

CRLK1 is essential in the early stages of defense against fungal pathogens.

We observed enhanced induction ofMAPK3 in crlk1 mutants after chitin treatment but

no effects were observed on WRKY53 expression. Based on these findings we propose a model

whereby chitin binding to CERK1 induces CRLK1 expression through MAPK3 and other

unidentified signaling molecules with CRLK1 expression reaching maximal levels within one

hour of chitin perception. CRLK1 and/or its interacting partners can then (1) go on to activate

other unknown molecules in fungal defense and (2) act to suppressMAPK3 expression in order

to lower or regulate the response over time in a negative feedback loop. Additional experiments

aimed at identifying CRLK1’s interacting partners and its precise role in modulating MAPK3

will be important in determining the precise role of CRLK1 in defense against fungal pathogens

and in elicitor-mediated defense.

50

Since CRLK1 is involved in Arabidopsis defense against fungal pathogens, we hypothesized that

CRLK1 was predominantly expressed in leaves in order to mediate the defense when fungal

pathogens invaded Arabidopsis leaves. We determined the expression pattern of GUS driven by

2kb CRLK1 promoter in Arabidopsis (Figure 2.5). Interestingly, GUS was expressed

predominantly in vascular tissues of Arabidopsis leaves, which indicated that CRLK1 may have

an unknown function in vascular tissue development. The CRLK1 expression in floral organs

suggested CRLK1 may also participate in flower development. However, there is no phenotype

in floral organs of crlk1 mutants, suggesting compensation by redundant RLKs in Arabidopsis.

Some plant resistance proteins were expressed in a vascular tissue-specific pattern and these

proteins were involved in vascular tissue development (Ramirez et al. 2011) or initiation of

vascular tissue defense responses (Liu and Ekramoddoullah 2011).

.

51

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Mittler, R., Vanderauwera, S., Gollery, M., and Van Breusegem, F. (2004). Reactive oxygengene network of plants. Trends Plant Sci. 9, 490-498.

Miya, A., Albert, P., Shinya, T., Desaki, Y., Ichimura, K., Shirasu, K., Narusaka, Y., Kawakami,N., Kaku, H., and Shibuya, N. (2007). CERK1, a LysM receptor kinase, is essential for chitinelicitor signaling in Arabidopsis. Proc. Natl. Acad. Sci. U. S. A. 104, 19613-19618.

Nawrath, C., and Metraux, J.P. (1999). Salicylic acid induction-deficient mutants of Arabidopsisexpress PR-2 and PR-5 and accumulate high levels of camalexin after pathogen inoculation.Plant Cell 11, 1393-1404.

Nelson, B.K., Cai, X., and Nebenfuhr, A. (2007). A multicolored set of in vivo organelle markersfor co-localization studies in Arabidopsis and other plants. Plant J. 51, 1126-1136.

Ramakers, C., Ruijter, J.M., Deprez, R.H., and Moorman, A.F. (2003). Assumption-free analysisof quantitative real-time polymerase chain reaction (PCR) data. Neurosci. Lett. 339, 62- 66.

Ramirez, V., Agorio, A., Coego, A., Garcia-Andrade, J., Hernandez, M.J., Balaguer, B.,Ouwerkerk, P.B., Zarra, I., and Vera, P. (2011). MYB46 modulates disease susceptibility toBotrytis cinerea in Arabidopsis. Plant Physiol. 155, 1920-1935.

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Reuber, T.L., Plotnikova, J.M., Dewdney, J., Rogers, E.E., Wood, W., and Ausubel, F.M.(1998).Correlation of defense gene induction defects with powdery mildew susceptibility in Arabidopsisenhanced disease susceptibility mutants. Plant J. 16, 473-485.

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Shimizu, T., Nakano, T., Takamizawa, D., Desaki, Y., Ishii-Minami, N., Nishizawa, Y., Minami,E., Okada, K., Yamane, H., Kaku, H., and Shibuya, N. (2010). Two LysM receptor molecules,CEBiP and OsCERK1, cooperatively regulate chitin elicitor signaling in rice. Plant J. 64, 204-214.

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CHAPTERCHAPTERCHAPTERCHAPTER THREETHREETHREETHREE

CRLK1CRLK1CRLK1CRLK1 ISISISIS AAAA FUNCTIONALFUNCTIONALFUNCTIONALFUNCTIONALMANGANESE-DEPENDENTMANGANESE-DEPENDENTMANGANESE-DEPENDENTMANGANESE-DEPENDENT RECEPTOR-LIKERECEPTOR-LIKERECEPTOR-LIKERECEPTOR-LIKE KINASEKINASEKINASEKINASEINVOLVEDINVOLVEDINVOLVEDINVOLVED ININININ DEFENSEDEFENSEDEFENSEDEFENSE AGAINSTAGAINSTAGAINSTAGAINST FUNGALFUNGALFUNGALFUNGAL PATHOGENSPATHOGENSPATHOGENSPATHOGENS

INTRODUCTIONINTRODUCTIONINTRODUCTIONINTRODUCTION

Plants have evolved a system of defense mechanisms to perceive the invasion of

pathogens through Pathogen-associated Molecular Patterns (PAMPs) or effectors. When

pathogens attack plants, conserved signature molecules from pathogens can be recognized by

plant pattern recognition receptors (PRR) to initiate a PAMP-triggered immunity (PTI) (Zipfel

2009). However, pathogens may also suppress the PTI through secreting effector proteins into

the plant cell cytosol and altering defense signaling pathways. To overcome the pathogen

effectors, plants have evolved a specialized system to recognize pathogen effectors and initiate

effector-triggered immunity (ETI) (Chisholm et al. 2006).

Receptor-like kinases (RLKs) are one of the largest gene families in Arabidopsis and

many RLKs have been shown to recognize PAMPs and activate PTI (Shiu et al. 2001). A few

RLKs have been intensively studied and their ligands and functions in plant innate immunity

have been identified. For example, CERK1/LysM RLK1was shown to bind the elicitor chitin

and initiate defense responses against fungal pathogens in both Arabidopsis and rice (Miya et al.

2007; Wan et al. 2008; Shimizu et al. 2010). A typical RLK contains an extracellular domain, a

transmembrane domain, and a functional cytoplasmic kinase domain. Although many RLKs do

not have a ligand binding domain, they may still activate signaling pathways through association

55

with other RLKs to form protein complexes (Lu et al. 2010). Several RLKs lacking the ligand

binding domain have been reported to play prominent roles in defense against fungal pathogens.

BIK1, an Arabidopsis RLCK, is induced by the necrotrophic fungus Botrytis cinerea and bik1

knockout mutants displayed severe disease symptoms after fungal infection (Veronese et al

2006). In tomato, TPK1b, a homolog of BIK1, also plays an important role in defense against

B.cinerea (Abuqamar et al. 2008). Overexpression of a rice RLCK, OsBSR1, in Arabidopsis

increased resistance to the fungal pathogen C. higginsianum. Overexpression of OsBSR1 in rice

also conferred resistance to bacterial and fungal pathogens (Dubouzet et al. 2011).

CRLK1 was identified in an Arabidopsis screen for chitin-upregulated genes using

microarray analysis (Ramonell et al. 2005). Previous experiments in this dissertation showed

that knockout mutants of crlk1 were more susceptible to G. cichoracearum and P. cucumerina,

suggesting that CRLK1 is involved in resistance to fungal pathogens (Chapter two). Upon chitin

treatment, CRLK1 is induced rapidly about 10 fold within 1 hour and its expression drops to

negligible levels within 4 hours. Transient expression of a CRLK1-GFP fusion protein showed

that CRLK1 localizes to the plasma membrane (PM) and its transmembrane domain is critical for

correct targeting. In this chapter we show that CRLK1 encodes a functional kinase and that Mn2+

is required for its kinase activity. Overexpression of a 35S:CRLK1:GUS fusion protein in stable

transgenic Arabidopsis plants leads to an increase in resistance against G. cichoracearum.

Chitin-induced CRLK1 expression was repressed in both mapk3 andWRKY transcription factor

mutants suggesting that CRLK1 may be negatively regulated by these proteins during the defense

response.

56

MATERIALSMATERIALSMATERIALSMATERIALS andandandandMETHODSMETHODSMETHODSMETHODS

BiologicalBiologicalBiologicalBiological materialsmaterialsmaterialsmaterials andandandand growthgrowthgrowthgrowth conditionsconditionsconditionsconditions

T-DNA insertional mutant crlk1-2 (Salk_063626) and allWRKY mutants were obtained

from the Arabidopsis Biological Resource Center (ABRC, Ohio State University). To identify

homozygous T-DNA mutants, PCR reactions were performed using the following primers:

crlk1-2 (Salk_063626):

5'-GATGAAACCGAGTTAATACGGC-3'(F); 5'-GGTCGGTTAGGAACGGTTTAC-3' (R);

wrky6 (Salk_012997):

5'-GAACGTATTAGCCAATCACGC-3'(F); 5'-TGTGGACGTGTCATAATTTGG-3'(R);

wrky72 (Salk_055293):

5'-GAGTGGAAGAGAGTGGCTGTG-3' (F); 5'-CAAAACATGGTTGATCATCCC-3' (R);

wrky17 (Salk_076337):

5'-TGGATTTTGGTTAAAGACCTTC-3' (F); 5'-AGCAAGAAAGATCGAAGAGCC-3' (R);

wrky48 (Salk_066438):

5'-TTACCGGTGACCAGTGTTTTC-3' (F); 5'--CTTTTTGGCCGTATTTTCTCC-3' (R);

wrky53 (Salk_034157):

5'-TCAGGCACGACTTAGAGAAGC-3' (F); 5'-GGGAAAGTTGTGTCAATCTCG-3' (R);

wrky46 (Salk_134310):

5'-TCTGTCGATTCCAACAAAACC-3' (F); 5'-AAGCCAATTTTTATCCATCGG-3' (R);

wrky18 (Salk_093916):

5'-CGATATTTGTCACCTTCATCG-3' (F); 5'-TCATTTCGATGCAAAGACATTC-3' (R);

T-DNA primer: 5'-TAGCATCTGAATTTCATAACCAATCTCGATACAC-3'. Both the mapk3

and mapk6 mutants were a kind gift from Dr. Brian Ellis.

57

Arabidopsis plants were grown in soil under controlled environmental conditions in a

Percival chamber (22°C day/19°C night with 16 hours light/8 hours dark and relative humidity of

50%). For 35S:CRLK1:GUS lines, Arabidopsis seeds were surface sterilized and grown on

Murashige and Skoog (MS) medium plates with 50mg/ml hygromycin B under the same

environmental conditions as soil grown plants. After 7 days healthy seedlings were selected and

transferred to soil to complete the growth cycle. G. cichoracearum was cultured on cucumber

leaves (Burpee Gardening) according to standard protocols before inoculation onto Arabidopsis.

InInInIn silicosilicosilicosilico analysisanalysisanalysisanalysis ofofofof CRLK1CRLK1CRLK1CRLK1

Sequence alignment and construction of a phylogenetic tree was performed using the

software program phylogeny.fr (http://www.phylogeny.fr/version2_cgi/index.cgi ) as described,

which is based onMUMUMUMUltiple SSSSequence CCCComparison by LLLLog- EEEExpectation (MUSCLE) (Dereeper

et al. 2008). Transmembrane domain and phosphorylation sites of CRLK1 were predicted by

TMpred (http://www.ch.embnet.org/software/TMPRED_form.html) and PhosPhAt

(http://phosphat.mpimp-golm.mpg.de/). CRLK1 catalytic subdomains were predicted by

aligning CRLK1 with AhSTYK (Rudrabhatla and Rajasekharan 2002) using MUSCLE

(http://www.ebi.ac.uk/Tools/msa/muscle/). Data regarding expression of CRLK1 in various

Arabidopsis organs and after treatment with various pathogens and elicitors were obtained using

Genevestigator (https://www.genevestigator.com/gv/).

ExpressionExpressionExpressionExpression andandandand PurificationPurificationPurificationPurification ofofofof ΔΔΔΔTM-CRLK1TM-CRLK1TM-CRLK1TM-CRLK1

58

The ΔTM-CRLK1 construct was amplified by PCR using Iproof high fidelity DNA

polymerase (BioRad Hercule, CA). The primers used for amplification were:

5'-GGATCCAGAAAAAAACCAAACCGGTCC-3' (F);

5'-AAGCTTTCACCCTCCTTCCCTCTCCAA-3' (R).

The PCR product was cloned into the pGEM-T vector (Promega, Madison, WI) and then

digested using BamHI and HindIII restriction enzymes (New England BioLab). The DNA

fragment containing the CRLK1 sequence was extracted from an agrose gel, purified and

religated into the pQE80L protein expression vector (Qiagen). The vector containing the ΔTM-

CRLK1 was transformed into E.coli. Transformed E.coli was grown in 1.2L of LB medium with

100ug/ml ampicillin overnight. CRLK1 protein expression was induced by the addition of 100

μM IPTG (100μM) to the growth medium. The culture was allowed to grow for 12 hours post

induction. The cell culture was pelleted by centrifugation at 5000 rpm for 5 minutes at 8 °C. The

pellet was homogenized in 50 mM phosphate buffer and 0.1 mM protease inhibitor (PMSF). The

resuspended E.coli was frozen, thawed and sonicated for 1 hour on ice and then centrifuged at

18000 rpm for 20 minutes at 8 °C. The transparent supernatant was recovered and subjected to

ultracentrifugation for 1 hour. The supernatant containing the CRLK1 protein was then applied

to a Ni2+ column and eluted in 300 mM imidazole according to the manufacturer protocol

(Sigma). The eluted protein solution was then subjected to dialysis at 4°C for overnight in buffer

containing 30% glycerol, 200 mM NaCl and then stored at -80°C.

InInInIn vitrovitrovitrovitro kinasekinasekinasekinase assayassayassayassay ofofofof ΔΔΔΔTM-CRLK1TM-CRLK1TM-CRLK1TM-CRLK1

CRLK1 kinase activity was determined in 10 μl buffer containing 1 μl of 500 mM Tris

HCl, 1 μl of 100 mM Hepes, 0.2 μl of 100 mM DTT, 0.5 μl of 1mg/ml CRLK1 protein (a final

59

concentration of 50μg/ml), 2 μl of 2 mg/ml myelin basic protein, 1 μl of a 100 mM solution of

either Mn2+, Ca2+ or Mg2+, 5mCi/ml [γ32-P] ATP (3000Ci mmol-1; PerkinElmer, Waltham, MA),

10M ATP and H2O to a final volume of 10 μl. The reaction was incubated at room temperature

for 1 hour and then terminated by adding equal amount of 1% SDS and boiling the solution for 5

minutes. The samples were then separated by SDS-PAGE, the gels dried and then exposed to X-

ray film for 2 days.

IdentificationIdentificationIdentificationIdentification ofofofof phosphorylationphosphorylationphosphorylationphosphorylation sitessitessitessites inininin CRLK1CRLK1CRLK1CRLK1

The CRLK1 autophosphorylation site analysis was performed by Applied Biomics, Inc

(Hayward, CA). The procedure is as follows: CRLK1 (31-332) was autophosphorylated in the

presence of 10nM Mn2+ for 2 hours at room temperature and then the reaction buffer was

changed to 25 mM ammonium bicarbonate (PH 8.0) followed by reduction, alkylation and

trypsin digestion. The digested CRLK1 phospho-peptides were enriched by using Supel-Tips

columns (Sigma-Aldrich, St. Louis. MO), desalted by Zip-tip C18 (Millipore, Billerica,MA) and

then eluted from the Zip-tip C18 column with matrix solution (α-cyano-4-hydroxycinnamic acid,

5 mg/ml in 50% acetonitrile, 0.1% trifluoroacetic acid, 25 mM ammonium bicarbonate). The

eluted phospho-peptides were spotted onto a matrix-assisted laser desorption/ionization (MALDI)

plate and MALDI-TOF analysis was performed using an AB Sciex Proteomics Analyzer (AB

Sciex). The top hits with high confidence were subjected to UCSF protein Prospector

(http://prospector.ucsf.edu/prospector/mshome.htm) for virtual digestion. The MS precursors

that matched the virtual digestion were then subsequently subjected to MS fragmentation. The

phospho-peptide mass and associated fragmentation spectra were then analyzed using the

MASCOT search engine (http://www.osc.edu/supercomputing/software/apps/mascot.shtml) to

60

search the NCBI non-redundant database (NCBInr). Candidates with a protein intensity score

higher than 95 were considered significant.

GenerationGenerationGenerationGeneration andandandand histochemicalhistochemicalhistochemicalhistochemical analysisanalysisanalysisanalysis ofofofof transgenictransgenictransgenictransgenic ArabidopsisArabidopsisArabidopsisArabidopsis 35S:35S:35S:35S:CRLK1CRLK1CRLK1CRLK1:::: GUSGUSGUSGUS

A fragment containing full length CRLK1 cDNA was generated by PCR. Primers for

35S:CRLK1: GUS construct were:

5'-GGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGACAAATCTGAAAAAATCGC- 3'

(F);

5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCCCCTCCTTCCCTCTCCAAA-3' (R).

The underlined sequence is CRLK1 specific. The resulting PCR product was introduced into the

pDONR221 vector using Gateway® BP Clonase II enzyme mix (Invitrogen), subcloned into

pMDC139 and then transformed into Agrobacterium tumefaciens strain GV3101. Transgenic

Arabidopsis lines were generated via Agrobacterium-mediated transformation using standard

protocols (Zhang et al. 2006). The T1 transformants were selected for on plates containing 50

μg/ml of hygromycin B and stained using standard GUS staining protocols to confirm GUS

expression. Stable lines expressing GUS were selected and grown to the T3 generation. The T3

homozygous transgenic lines were then used in the G. cichoracearum infection assays.

InfectionInfectionInfectionInfection assaysassaysassaysassays withwithwithwith G.G.G.G. cichoracearumcichoracearumcichoracearumcichoracearum

Inoculation of Arabidopsis transgenic lines was performed by dusting G. cichoracearum

fungal spores onto Arabidopsis rosette leaves as previously described (Wilson et al., 2001). At 5

dpi, infected Arabidopsis leaves were cleared in 70% ethanol overnight and then stained with

trypan blue solution (25μg/ml trypan blue dissolved in 1:1:1 of glycerol, lactic acid and H2O) for

61

2 hours. After destaining with a solution containing 1:1:1 of glycerol, lactic acid and water, the

numbers of conidiophores produced per colony were counted using at least 20 leaves per

genotype. For chitin pretreatment, 21-day old Arabidopsis plants were sprayed with 200μg/ml

crab shell chitin in water either 4 hours or 24 hours before inoculation with G. cichoracearum

(described by Wan et al. 2008).

ElicitorElicitorElicitorElicitor treatmenttreatmenttreatmenttreatment ofofofof ArabidopsisArabidopsisArabidopsisArabidopsis liquidliquidliquidliquid cultureculturecultureculture

Arabidopsis seeds were surface sterilized and grown in 10ml of liquid culture (2%

Dextrose, 1 package of Murashige and Skoog salts dissolved in H2O) in 50ml conical tubes

under continuous light for two weeks. Plants were treated with either 50μl of 20mg/ml chitin

(Sigma, St Louis, MO, USA) or 10μl of 10mM flg22 (Genscript, Piscataway, NJ) for 30 minutes.

After treatment, plants were harvested, frozen immediately in liquid nitrogen and stored at -80°C.

TotalTotalTotalTotal RNARNARNARNA extraction,extraction,extraction,extraction, reversereversereversereverse transcription,transcription,transcription,transcription, andandandand quantitativequantitativequantitativequantitative realrealrealreal timetimetimetime PCRPCRPCRPCR

Arabidopsis total RNA was isolated from frozen tissues using TRizol Reagent

(Invitrogen) in a protocol suggested by the manufacturer. The extracted RNA was treated with

RQ1 DNase (Promega, Madison, WI) and then purified with an E.Z.N.A. RNA cleanup kit

(Omega Bio-tek, Norcross, GA). The purified RNA was screened for genomic DNA

contamination by qRT-PCR using Actin-2 primers. Actin-2 primer sequence was:

5'-GGTATTCTTACCTTGAAGTATCCTA-3'(F); 5’-

TCATTGTAGAAAGTGTGATGCCAGATC-3' (R).

The total RNA was digested with DNase until no genomic DNA was detectable by qRT-PCR

using Actin-2 primers. First strand cDNA was synthesized using the RevertAid First Strand

62

cDNA Synthesis Kit (Fermentas) and random hexamers according to the manufacturer’s protocol.

qRT-PCR was performed using PerfeCTa SYBR Green SuperMix for iQ™ (Quanta Biosciences,

Gaithersburg, MD) with a final volume of 20μl per reaction. qRT-PCR reaction was performed

in Hard-Shell® 96-Well Skirted PCR Plates using a DNA Engine Opticon® 2 system (BioRad,

Hercules, CA). Gene primers were designed using Beacon DesignerTM software (Premier Biosoft,

Palo Alto, CA) to have annealing temperatures between 55°C to 65°C and an amplicon length of

less than 300 bp. The qRT-PCR program consisted of: 50°C for 2 min; 95°C for 10 min; 95°C

for 15 s, 60°C for 30 s and 72°C for 30 s for 40 cycles. The raw qRT-PCR data was analyzed

using the LinRegPCR software program (Ramakers et al. 2003) and cycle threshold (CT) values

with optimized amplification efficiency and relative gene expression was calculated using Gene

Expression MacroTM (BioRad, Hercule, CA) that is based on ΔΔCT method. The following gene

specific primers were used in qRT-PCR :

CRLK1 (At5g46080):

5-'GGAGAAAGAATCGTCGTAAC-3' (F);

5'-GATGTGAATCTACCGTGAAC-3' (R);

Three independent biological replicates were performed for each experiment.

AccessionAccessionAccessionAccession numbersnumbersnumbersnumbers ofofofof genesgenesgenesgenes usedusedusedused inininin thisthisthisthis studystudystudystudy

CRLK1, At5g46080;MAPK3, At3g45640;MAPK6, At2g43790;WRKY6, At1g62300;

WRKY72, At5g15130;WRKY17, At2g24570; WRKY48, At5g49520; WRKY53, At4g23810;

WRKY46, At2g46400;WRKY18, At4g31800; ACTIN-2, At3g18780.

63

RESULTSRESULTSRESULTSRESULTS

SequenceSequenceSequenceSequence andandandand phylogeneticphylogeneticphylogeneticphylogenetic analysisanalysisanalysisanalysis ofofofof CRLK1CRLK1CRLK1CRLK1

Chitin responsive receptor-like kinase 1 (CRLK1) has been shown to play an important

role in Arabidopsis defense against both biotrophic and necrotrophic fungal pathogens (Chapter

two). Using the CRLK1 deduced amino acid sequence and NCBI BLAST 7 protein sequences

with an identity greater than 50% to CRLK1 were chosen for alignment and construction of a

phylogenetic tree using the Phylogeny.fr software program (http://www.phylogeny.fr/).

Phylogenetic analysis showed that CRLK1 is most related to a putative serine and threonine

kinase (AlRLK1) from Arabidopsis lyrata (Figure 3.1). To analyze CRLK1 subdomains, the

deduced CRLK1 amino acid sequence was further analyzed using TMpred

(http://www.ch.embnet.org/software/TMPRED_form.html) and PhosPhAt

(http://phosphat.mpimp-golm.mpg.de/). The TMpred output suggested that CRLK1 contained a

single transmembrane domain (TM), which a length of 12 to 30 amino acids (Figure 3S1).

PhosPhAt analysis predicted that the juxtamembrane (JM) domain is from amino acid 31 to 65

and the catalytic kinase domains are from amino acid 66 to 324 (Figure 3.2). Phosphorylation

sites of CRLK1 were predicted by PhosPhAt, which suggested 4 potential sites within the

juxtamembrane domain and 7 within the catalytic kinase domain (Figure 3.2). CRLK1 contains

all 12 conserved kinase catalytic subdomains (I to XI) when aligned with AhSTYK, a kinase

with subdomains predicted by Rudrabhatla and Rajasekharan (2002).

64

FigureFigureFigureFigure 3.13.13.13.1 AAAA pppphylogenetichylogenetichylogenetichylogenetic treetreetreetree basedbasedbasedbased onononon proteinproteinproteinprotein sequencesequencesequencesequence alignmentalignmentalignmentalignment ofofofof CRLK1CRLK1CRLK1CRLK1 andandandand sevensevensevensevenrelatedrelatedrelatedrelated proteins.proteins.proteins.proteins. The genebank accession number of each protein is: CRLK1 (Arabidopsisthaliana), NP_199420; AlRLK1 (Arabidopsis lyrata), XP_002863430; MtPK1 (Medicagotruncatula), ABN08719; RcPK (Ricinus communis), XP_002509422; PtPK2 (Populustrichocarpa), XP_002305775; PtPK1 (Populus trichocarpa), XP_002329804; VvPK1 (Vitisvinifera), XP_002278731; VvPK2 (Vitis vinifera), CAN77470. Protein sequences were alignedwith multiple sequence comparison by log-expectation (MUSCLE). The phylogenetic tree wasgenerated using the Phylogeny.fr platform (http://www.phylogeny.fr/). Horizontal branch lengthsrepresent protein divergence. Bootstrap values are above branches.

65

FigureFigureFigureFigure 3S13S13S13S1 PredictionPredictionPredictionPrediction ofofofof CRLK1CRLK1CRLK1CRLK1 transmembranetransmembranetransmembranetransmembrane domaindomaindomaindomainssss.... Hydropathy analysis of CRLK1was performed using TMpred. The TMpred output suggests a transmembrane domain fromamino acids 12-30.

66

MDKSEKIAYASVLSLLSLSLLLLIIFLFLLCRKKPNRSDDEYLLPETKPATTTTMMMM **** **** JJJJMMMM

GLSYSLTELDSATDGFNQRRIIGSGRLGTVYAAIIPDHKNLVAVKRIHP**** **** **** IIII IIIIIIII

GLVLSKPGFGFSTVIKSLSSSHHPNVVSILGFSEAPGERIVVTEFVGEIIIIIIIIIIII IIIIVVVV **** VVVV

GKSLSDHLHGGSNSATAVEFGWKTRFKIAAGAARGLEYLHEIANPRIV**** VVVVIIII AAAA ****

HGRFTSSNVLVDEKSTAKICDYGFGFLIPIEKSGIFGYIEEGYCKESDVVVVVIIII BBBB VVVVIIIIIIII VVVVIIIIIIIIIIII

YGYGVVLMEILSGRRSENGLIVKWATPLIKEQRFAELLDPRIVVQSEIKIIIIXXXX XXXX **** XXXXIIIISLVIRLAKVALACVGNSRRSRPSISEVAAILNSLEREGG

****

FigureFigureFigureFigure 3.23.23.23.2 SubdomainsSubdomainsSubdomainsSubdomains andandandand predictedpredictedpredictedpredicted phosphorylationphosphorylationphosphorylationphosphorylation sitessitessitessites ofofofof CRLK1.CRLK1.CRLK1.CRLK1. The first 30 aminoacids (underlined) were predicted to be a transmembrane domain in TMpred (Hofmann andStoffel, 1993). The underlined protein sequences with roman numerals are canonical kinasecatalytic subdomains generated by aligning CRLK1 with the ahSTYK kinase (Rudrabhatla andRajasekharan 2002). Asterisks indicate phosphorylation sites predicted by PhosPhAt 3.0. TM,transmembrane domain; JM, juxtamembrane domain.

67

CRLK1CRLK1CRLK1CRLK1 isisisis aaaa functionalfunctionalfunctionalfunctional kinasekinasekinasekinase andandandand itsitsitsits activityactivityactivityactivity requirerequirerequirerequiressss MnMnMnMn2+2+2+2+

The CRLK1 amino acid sequence predicted that it contained a tyrosine/threonine/serine

intracellular kinase domain. To test whether CRLK1 was a functional kinase, we expressed the

intracellular kinase region of CRLK1 in E.coli. The full length CRLK1 was also expressed in

E.coli but was mainly expressed in the insoluble fraction of total E.coli proteins, suggesting that

CRLK1 may also localize into the membrane system of E.coli. The ΔTM-CRLK1 protein was

expressed as a fusion protein that contained six histidine residues on the truncated N-terminus.

The protein was purified by affinity chromatography and detected as a 32k Da protein by SDS-

PAGE (Figure 3.3 A). This finding was consistent with the prediction from the amino acid

sequence. The purified protein was incubated buffers containing [γ32-P]-ATP and myelin basic

protein (MBP) as an artificial substrate. The mixture was analyzed via SDS-PAGE and

autoradiographed for 48 hours. Results showed that the autophosphorylation of ΔTM-CRLK1

could only be detected in buffers containing Mn2+ (Data not shown). Thus, we predicted that

CRLK1 kinase activity was dependent on the presence of Mn2+. In order to test this hypothesis,

we incubated the CRLK1 protein with MBP as an artificial substrate in buffers containing

incrementally increasing concentrations of Mn2+. CRLK1 kinase activity was extremely low in

buffers containing less than 5 mM Mn2+ (Figure 3.3B). Both autophosphorylation of CRLK1 and

MBP phosphorylation reached a high level at 10 mM Mn2+ and a maximum level at 20 mMMn2+.

The CRLK1 kinase activity began to be inhibited at concentrations of 50 mM Mn2+.

In order to determine whether CRLK1 is also active in the presence of other divalent

metal ions, we incubated CRLK1 with MBP in buffers combining Ca2+, Mg2+ and Mn2+. Very

weak kinase activity of CRLK1 can be detected in the presence of Ca2+ but no activity was

68

detected when incubated with Mg2+ alone. When combining Ca2+ and Mg2+, CRLK1 activity

showed a similar intensity to that of Ca2+ alone (Figure 3.3C). In a buffer solution containing

Ca2+ and Mn2+ together, CRLK1 showed less activity than with Mn2+ alone, indicating that Ca2+

may compete with Mn2+ for kinase binding sites. Mg2+ and Mn2+ increased the

autophosphorylation activity of CRLK1, but decreased the MBP phosphorylation compared to

that of Mn2+ alone. In the presence of Ca2+, Mg2+ and Mn2+, the Mn2+ dependent kinase activity is

strongly inhibited, compared to the combinations of Ca2+/Mn2+ and Mg2+/Mn2+ (Figure 3.3C).

69

30kDa25kDa

17kDa

7kDa

46kDa58kDa80kDa

175kDa

ΔTM-CRLK1

AAAA

0 0.1 0.5 1 2 5 10 20 50 mM

MBP

Mn2++++[[[[CCCC]]]]BBBB

ΔTM-CRLK1

Ca2+Mg2+Mn2+

+ - - + + - +- + - + - + +- - + - + + +

MBP

CCCC

ΔTM-CRLK1

FigureFigureFigureFigure 3.33.33.33.3 PurificationPurificationPurificationPurification andandandand inininin vitrovitrovitrovitro kinasekinasekinasekinase assayassayassayassay ofofofof truncatedtruncatedtruncatedtruncated CRLK1CRLK1CRLK1CRLK1 ((((ΔΔΔΔTM-CRLK1)TM-CRLK1)TM-CRLK1)TM-CRLK1) ininininE.coliE.coliE.coliE.coli.... AAAA. The purified ΔTM-CRLK1 from E.coli total protein was analyzed by SDS-PAGE. BBBB.CRLK1 kinase activity increased in a gradient of MnCl2. CCCC. CRLK1 kinase activity incombination of bivalent ions Ca2+, Mg2+ and Mn2+. MBP, myelin basic protein; ΔTM-CRLK1,mutant CRLK1 protein with transmembrane domain deleted.

70

PhosphorylationPhosphorylationPhosphorylationPhosphorylation sitesitesitesite identificationidentificationidentificationidentification inininin thethethethe CRLK1CRLK1CRLK1CRLK1 proteinproteinproteinprotein usingusingusingusing MassMassMassMass SpectrometrySpectrometrySpectrometrySpectrometry ((((MS)MS)MS)MS)

In order to determine where CRLK1 is autophosphorylated in vitro, we determined the

phosphorylation sites of CRLK1 using Mass Spectrometry (MS). In brief, CRLK1 purified

protein was autophosphorylated in vitro and then subjected to trypsin digestion. The digested

CRLK1 phospho-peptides were enriched and then analyzed by MS. Two phospho-peptides were

identified in CRLK1’s cytoplasmic kinase domain region as potential phosphorylation sites

(Table 3.1). For the first phospho-peptide containing amino acids 241 to 258, two tyrosines were

phosphorylated in vitro. Two out of four serines in the second peptide (AA 150 to 170) may also

be autophosphorylated. Identification of the serine at amino acid 152 was consistent with the

predicted phosphorylation sites identifies using PhosPhat. The data suggest that CRLK1 is a

dual-specificity kinase that phosphorylates tyrosine and serine in vitro. The ion score and signal

count for the two phospho-peptides were low due to either low phosphorylation efficiency in the

sample or a low concentration of purified protein (Figure 3S2). It is possible that other

phosphorylation sites may be identified if phosphorylation efficiency can be increased or a

higher concentration of purified protein is used for the in vitro kinase assay.

71

Table 3.1 Potential phosphorylation sites in phospho-peptide identified from MS/MS

Observed Massa Start - Endb Phospho-Peptide Sequencec Loss of phosphated2066.7854 241-258 ESDVYGYGVVLMEILSGR 22519.8459 150-170 SLSDHLHGGSNSATAVEFGW

K2

a The Mass Value (Da) of phospho-peptide sequence.bPosition of phospho-peptide sequence in the complete amino acid sequence/cContains phosphorylated residue (tyrosine, threonine, or serine). The potential phosphorylationsites were underlined.dNumber of phosphorylated amino acids in the peptide.

72

A

Loss of Phosphate(2146 - 80 - 80 = 1986 Da)

ESDVYGYGVVLMEILSGR Phospho(Y)[5,7]Ion Score = 0

B

Loss of Phosphate2519 - 98 - 98 = 2323 Da

SLSDHLHGGSNSATAVEFGWK

Ion Score = 0

FigureFigureFigureFigure 3S3S3S3S2222 Phospho-peptidesPhospho-peptidesPhospho-peptidesPhospho-peptides identifiedidentifiedidentifiedidentified fromfromfromfromMS/MSMS/MSMS/MSMS/MS forforforfor CRLK1.CRLK1.CRLK1.CRLK1.CRLK1 purified protein was autophosphorylated for 2 hours at room temperature in the presenceof manganese. The phosphorylated protein was then digested by trypsin and enriched forphospho-peptides that were subjected to MS/MS for analysis. AAAA. A 2146 Da phospho-peptidecontaining two tyrosine residues that are potentially phosphorylated. BBBB. A 2519 Da phospho-peptide containing four serine residues. Two of the identified serine residues are potentiallyphosphorylated.

73

CRLK1CRLK1CRLK1CRLK1 participatesparticipatesparticipatesparticipates inininin plantplantplantplant defensedefensedefensedefense againstagainstagainstagainstG.G.G.G. cichoracearumcichoracearumcichoracearumcichoracearum andandandand ooooverexpressionverexpressionverexpressionverexpression ofofofofCRLK1CRLK1CRLK1CRLK1 enhanceenhanceenhanceenhancessss resistanceresistanceresistanceresistance totototo G.G.G.G. cichoracearumcichoracearumcichoracearumcichoracearum infection.infection.infection.infection.

We previously showed that a homozygous T-DNA insertional mutants in crlk1

(Sail_801_H08) display a higher susceptibility to the biotrophic fungal pathogen G.

cichoracearum and the necrotrophic fungus P. cucumerina compared to wild type plants

(Chapter two). In order to further confirm this result, transgenic Arabidopsis plants

overexpressing 35S:CRLK1:GUS fusion protein driven by CaMV 35S promoter was generated

via Agrobacterium-mediated transformation (Zhang et al. 2006). It has been shown that

pretreatment of Arabidopsis plants with crab shell chitin (CSC) can enhance plant resistance to

both fungal and bacterial pathogens (Wan et al. 2008). When mutants in the chitin receptor

CERK1/LysM RLK1 were pretreated with crab shell chitin (CSC), this failed to protect plants

from A. brassicicola and P. syringae pv tomato DC3000 infection since chitin-mediated

signaling is blocked (Wan et al. 2008) indicating that CERK1/LysM RLK1 is critical for chitin-

induced resistance. In order to dissect the function of CRLK1 in chitin signaling, we pretreated

two CRLK1 mutants (crlk1-1 and crlk1-2), CRLK1 overexpression lines (35S:CRLK1: GUS),

Col-0 wild type and the hypersusceptible transgenic plant NahG with crab shell chitin 4 hours

and 24 hours before inoculation of G. cichoracearum. At 5 days post-inoculation (dpi), the

susceptibility of each line was determined by quantifying the conidiophores per fungal colony on

infected Arabidopsis leaves. As Figure 3.4 shows, pretreatment of Col-0 wild type with crab

shell chitin (CSC) increased resistance against G. cichoracearum compared to control treated

plants. Without pretreatment, both T-DNA insertion mutants of CRLK1 (crlk1-1 and crlk1-2)

were significantly more susceptible to G. cichoracearum compared to Col-0 wild type (Figure

3.4 A, B and C). In addition, overexpression of the 35S:CRLK1: GUS fusion protein in

74

Arabidopsis significantly increased plant resistance to G. cichoracearum (Figure 3.4B). At 5

dpi, the number of fungal colonies on 35S:CRLK1: GUS leaves was much less than on Col-0

wild type (data not shown). At 10 dpi CRLK1 overexpression lines were still resistant to G.

cichoracearum infection (Figure 3.4C), indicating that CRLK1 is critical in activating plant

defense against fungal pathogens. Compared with untreated crlk1-1 and crlk1-2 mutants, both

chitin-pretreated crlk1-1 and crlk1-2 mutants showed an enhanced resistance to G.

cichoracearum, suggesting that mutations in CRLK1 do not fully block chitin-mediated signaling.

Pretreatment of 35S:CRLK1: GUS lines with Crab Shell Chitin (CSC) conferred a similar

resistance compared to untreated 35S:CRLK1: GUS plants (Figure 3.4B). Therefore the

protective effect of pre-treatment with chitin may be negligible in Arabidopsis due to the

constitutive activation of defenses by CRLK1 overexpression.

75

0

2

4

6

8

10

12

14

16

18

cccc////cccc* *

*

*

++

+

Contro l Chitin trea tm ent

Col-0 crlk1-1 crlk1-2 35S:CRLK1: GUS NahG

Col-0 crlk1-1 crlk1-2 35S:CRLK1: GUS NahGA

B

C

76

FigureFigureFigureFigure 3.43.43.43.4 PhenotypesPhenotypesPhenotypesPhenotypes ofofofof ArabidopsisArabidopsisArabidopsisArabidopsis CRLK1CRLK1CRLK1CRLK1 mutantsmutantsmutantsmutants andandandand overexpressionoverexpressionoverexpressionoverexpression lines.lines.lines.lines. A.A.A.A.Microscopic photographs were taken from infected leaves stained with trypan blue from control(panel a) and chitin pretreated (panel b) Arabidopsis at 5 dpi. Fungal conidiophores and hyphaewere stained by trypan blue and Arabidopsis leaves were cleared in 70% ethanol after staining.Scale bar represents 0.5mm. BBBB.... Quantification of the numbers of G. cichoracearumconidiophores on chitin or H2O (control) pretreated Arabidopsis rosette leaves. Each data pointrepresents the average number of conidiophores per colony (c/c) counted from 18 leaves of 6plants for each genotype. Error bar denotes standard error. The white and gray bars indicatecontrol and chitin treatment respectively. The asterisk indicates significant difference (p<0.05)between untreated Arabidopsis lines and Col-0 wild type plants. The plus symbol indicates thesignificant difference (p<0.05) between the chitin treated and control plants of the same genotype.The p-value is calculated based on a student's t-test. Each data set was repeated for at least twobiological replicates. C.C.C.C.Macroscopic photographs were taken at 10 dpi from Arabidopsis leavesinoculated at high density with spores of G. cichoracearum. Leaves of the T-DNA insertionalmutants crlk1-1 (SAIL_801_H08) and crlk1-2 (Salk_063626) developed a heavier fungalcoverage on the leaves and displayed more disease symptoms than Col-0 wild type. Leaves fromCRLK1 overexpression lines (35S:CRLK1: GUS) were highly resistant to G. cichoracearuminfection.

77

CRLK1CRLK1CRLK1CRLK1 expressionexpressionexpressionexpression isisisis inducedinducedinducedinduced uponuponuponupon infectioninfectioninfectioninfection withwithwithwith variousvariousvariousvarious pathogenspathogenspathogenspathogens andandandand treatmenttreatmenttreatmenttreatment withwithwithwithotherotherotherother elicitorselicitorselicitorselicitors

We showed that CRLK1 is highly induced by chitin within 1 hour and that the induction

dropped to a negligible level within 4 hours (Chapter two). However, whether CRLK1 responds

to other pathogens or elicitors is unknown. To investigate the expression of CRLK1 in different

tissues / development stages in Arabidopsis, after infection with other pathogens and after

treatment with other elicitors, we obtained expression data on CRLK1 from Genevestigator, a

database containing publicly available microarray data (Zimmermann et al. 2005). Based on this

microarray data, CRLK1 is highly expressed in both young and developed flowers (Figure 3.5A)

which is consistent with the expression pattern of pCRLK1::GUS in plants (Chapter two).

Overall, the expression levels of CRLK1 in other Arabidopsis tissues/organs were very low

throughout all other developmental stages (Figure 3.5A). Upon stimuli, CRLK1 is induced by

many pathogens including Botrytis cinerea (necrotrophic fungi), Blumeria graminis (biotrophic

fungi), Bemisia tabaci type B (Insect), Golovinomyces cichoracearum (biotrophic fungi) and

Pseudomonas syringae (bacterium). Aside from chitin, CRLK1 is also induced by bacterial

elicitors flg22, hrpZ, EF-Tu, LPS, and Oligogalacturonide (OG), a plant danger-associated

molecular pattern (DAMP) (Figure 3.5 B). The induction of CRLK1 by flg22 was confirmed by

qRT-PCR (Figure 3.5 C).

FLS2 and CERK1/LysM RLK1 are the Arabidopsis receptors for flg22 and chitin

respectively (Gomez-Gomez and Boller 2000; Miya et al. 2007; Wan et al. 2008). In order to

test whether mutations in FLS2 and CERK1 can affect induction of CRLK1 upon chitin or flg22

treatment, CRLK1 expression was detected in the Arabidopsis Ws-0 ecotype (FLS2 is mutated in

78

Ws-0) treated with chitin or cerk1 mutants treated with flg22. As figure 3.5C showed, CRLK1

expression in the flg22-treated cerk1 mutant remained similar to wild type levels. Chitin treated

Ws-0 mutants had a higher induction of CRLK1 than that of wild type plants, indicating that

FLS2 may repress CERK1-mediated chitin signaling.

Induction of most of chitin responsive genes including CRLK1 was blocked in the cerk1

knockout mutant (Wan et al. 2008). One model suggests that CERK1/LysM RLK1 may operate

through induction of a MAPK cascade and transcription factors such as those in the WRKY

family (Wan et al. 2008). MAPK3 and MAPK6 are induced upon chitin treatment (Wan et al.

2004) and are known to play important roles in plant innate immunity (Rodriguez et al. 2010).

WRKY proteins are a large family of transcription factors that have been shown to positively and

negatively regulate Arabidopsis defense pathways (Pandey and Somssich 2009). WRKY

transcription factors bind to W boxes (core sequence of TGAC) in the promoter region of many

RLKs (Du and Chen 2000). There are seven W boxes in the promoter region of CRLK1

suggesting CRLK1 may also be regulated by WRKY transcription factors (Chapter two). In

order to test whether MAPK3/6 and WRKY TFs regulate CRLK1, induction of CRLK1 by chitin

was determined in mapk3, mapk6 and several wrky mutants. As shown in Figure 3.6A-C,

CRLK1 induction by chitin was increased in mapk3, wrky6, wrky17, wrky48, wrky53 and wrky46

mutants, suggesting that CRLK1 is negatively regulated by MAPK3 and WRKY transcription

factors during chitin-mediated defense.

79

0

100

200

300

400

500

600

700

800

900

germinatedseeds

seedling

young rosette

developedrosette

bolting

young flower

developedflower

flowersand siliques

mature siliques

Signalintensity

AAAA

0

2

4

6

8

10

12

Foldchange

B.cinerea

B.graminis

G.cichoracearum

B.tabacitype B

P.syringae chi

tinEF-Tu

flg22

HrpZ(1h)

LPS (1h)

OG(1h)

GST-NPP1

BBBB

0

5

10

15

20

25

30

35

Col-0 Col-0Col-0 Ws-0 cerk1

chitin flg22 chitin+flg22 chitin flg22Treatment

Background

Foldchange

CCCC

FigureFigureFigureFigure 3.53.53.53.5 CRLK1CRLK1CRLK1CRLK1 genegenegenegene expressionsexpressionsexpressionsexpressions inininin ArabidopsisArabidopsisArabidopsisArabidopsis organsorgansorgansorgans andandandand underunderunderunder variousvariousvariousvarioustreatments/conditions.treatments/conditions.treatments/conditions.treatments/conditions. AAAA.... CRLK1 expression in Arabidopsis organs were analyzed using themicroarray database software Genevestigator. B.B.B.B. CRLK1 expression is induced by pathogensand general elicitors. Expression data from Genevestigator was obtained from microarray rawdata using the MASS algorithm. C.C.C.C. Expression of CRLK1 was analyzed after 30 minutetreatments with either flg22 or chitin in Col-0, Ws-0 and cerk1 (At3g21630) by qRT-PCR. Datarepresents ratio of expression between mock (H2O) and treated plants in three independentbiological replicates. Error bars represents standard error.

80

Foldchange

051015202530354045

Col-0 mapk3 mapk6

AAAA

0

10

20

30

40

05101520253035

Foldchange

Foldchange

Col-0 wrky6 wrky72 wrky17

wrky48 wrky53 wrky46 wrky18Col-0

BBBB

CCCC

FigureFigureFigureFigure 3.63.63.63.6 ExpressionExpressionExpressionExpression ofofofof CRLK1CRLK1CRLK1CRLK1 afterafterafterafter chitinchitinchitinchitin treatmenttreatmenttreatmenttreatment ininininMAPKMAPKMAPKMAPK andandandandWRKYWRKYWRKYWRKYmutants.mutants.mutants.mutants. A.A.A.A.Induction of CRLK1 in Col-0 wild type, mapk3 and mapk6 after CSC treatment for 30 minutes. BBBBand C.C.C.C. Induction of CRLK1 in Col-0 wild type, wrky6, wrky72, wrky17, wrky48, wrky53, wrky46,and wrky18 mutants after chitin treatment (30 minutes). Data represent ratio between mock andtreated plants of three independent biological replicates. Error bars indicate standard error.

81

DISCUSSIONDISCUSSIONDISCUSSIONDISCUSSION

CRLK1CRLK1CRLK1CRLK1 isisisis distinctdistinctdistinctdistinct fromfromfromfrom otherotherotherother RLKsRLKsRLKsRLKs inininin ArabidopsisArabidopsisArabidopsisArabidopsis andandandand otherotherotherother plantplantplantplant speciesspeciesspeciesspecies

In Arabidopsis thaliana, CRLK1 is in the CR4-L clade but is markedly distinct from other

RLK members in the CR4-L group (Shiu and Bleecker 2001). When comparing the RLK protein

sequences from Arabidopsis thaliana and rice, CRLK1 is closely related to the rice RLK

Osi007326.1 and these two RLKs form their own group RLCK-XIV (Shiu et al. 2004). These

data indicate CRLK1 is quite distinct from other RLK family members in Arabidopsis. In order

to study whether CRLK1 is closely related to any RLK’s in other plant species, we compared the

CRLK1 protein sequence with 7 other RLKs that had an identity score higher than 50% from

various plant species (Figure 3.1). We found that CRLK1 is most closely related to AlRLK

(XP_002863430) in Arabidopsis lyrata, a close relative species of Arabidopsis thaliana. CRLK1

and AlRLK1 form a clade that is sister to another clade composed of MtPK1, RcPK, PtPK1,

PtPK2, VvPK1 and VvPK2. Both clades had 100% bootstrap support. Taken together, CRLK1

is distinct among RLK members found both in Arabidopsis thaliana and in other plant species.

CRLK1CRLK1CRLK1CRLK1 encodeencodeencodeencodessss aaaa functionalfunctionalfunctionalfunctional MnMnMnMn2+2+2+2+ -dependent-dependent-dependent-dependent kinasekinasekinasekinase

To date, many RLKs have been reported to be involved in plant defense against

pathogens. However, only a few RLKs have actually been expressed and purified in order to

determine their ability to phosphorylate themselves or other substrates. It is assumed that RLKs

function in plant signaling pathways through phosphorylation of various substrates. However, a

RLK called Atypical Receptor-like kinase 1 (TARK1) from tomato was found to be exception

because its cytoplasmic kinase domain is inactive (Kim et al. 2009). TARK1 may function in

defense responses by interacting with other primary receptors (Kim et al. 2009). Nemoto et al.

82

(2011) studied serine/threonine autophosphorylation in 759 Arabidopsis kinases and found that

179 of these protein kinases demonstrated autophosphorylation activity. CRLK1

autophosphorylation was not analyzed in those experiments (Nemoto et al. 2011). In the current

work, we have shown that CRLK1 is capable if both autophosphorylation and phosphorylation of

an artificial substrate, myelin basic protein (MBP) (Figure 3.3B). To determine the effects of

divalent metal ions on CRLK1 kinase activity, we showed that CRLK1 has its highest kinase

activity in the presence of Mn2+ (Figure 3.3C). Many studies have shown that Mn2+ is required or

preferred for activating many plant RLK. For example, Arabidopsis CRINKLY4 (ACR4), an

RLK close to CRLK1 in the Arabidopsis RLK phylogenetic tree (Shiu and Bleecker 2001), has

been shown to prefer Mn2+ to Mg2+ for its own autophosphorylation (Meyer et al. 2011). In

addition, a pepper receptor-like kinase, CaRLK1, exhibits kinase activity only in the presence of

Mn2+ (Yi et al. 2010). Alignment of the Mn2+ dependent kinase AtSTYPK(AT) with another

known Mg2+ dependent kinase AhSTYPK(AH) and the conserved sequence for Mn2+dependent

kinase domains suggested that a conserved histidine at amino acid 248 in AtSTYPK might be

critical for Mn2+ dependent kinase activity (Reddy and Rajasekharan 2006). Mutation of the

conserved histidine to an alanine residue in AtSTYPK leads to a loss of kinase activity (Reddy

and Rajasekharan 2006). Alignment of CRLK1 with AtSTYPK revealed that CRLK1 also

contains a conserved histidine that is critical for kinase activity. However, the conserved

histidine was not found in AhSTYPK, a Mg2+ -dependent kinase (Figure 3S3).

83

AT LVNYEEWTIDLRKLHMGPAFAQGAFGKLYRGTYNGEDVAIKLLERSDSNPEKAQALEQQF 177AH LENFDEWTIDLRKLNMGEAFAQGAFGKLYRGTYNGEDVAIKILERPENELSKAQLMEQQF 135CRLK1 SYSLTELDSATDGFNQRRIIGSGRLGTVYAAIIPDHKNLVAVKRIHPGLVLSKPGFG--F 110Mn -----------------RLLSRGAEGDIYLTEWGSRAAVLKIRRARGYRNAD-------L 36

:. * * :* .. : : . . :AT QQEVSMLAFLKHPNIVRFIGACIKPMVWCIVTEYAKGG-SVRQFLTKRQNRAVPLKLAVM 236AH QQEVMMLATLKHPNIVRFIGACRKPMVWCIVTEYAKGG-SVRQSLMKRQNRSVPLKLAVK 194CRLK1 STVIKSLSSSHHPNVVSILGFSEAPGERIVVTEFVGEGKSLSDHLHGGSNSATAVEFGWK 170Mn DARLRKRRTVREAEIIRQARSAGVPVPVVFFVDTVECS-ITMQHVRGRPVSSFSGAALVR 95

. : :..::: . * ...: . . : : : .AT QALDVAR----GMAYVHERN---FIHRDLKSDNLLISADRSIKIADFGVARIEVQTEGMT 289AH QALDVAR----GMAYVPWLG---LIHRDLKSDNLLIFGAKSIKIADFGVAGIEVQTEGMT 247CRLK1 TRFKIAAGAARGLEYLHEIANPRIVHGRFTSSNVLVDEKSTAKICDYGFG-FLIPIE--- 226Mn LAEQIGR----MAGTLHKNG---IMHGDLTTSNFIRSGG-TLYAIDFGLS---------- 137

.:. : ::* :.:.*.: : *:*..AT PETGTYRWMAPEMIQHRPYTQKVDVYSFGIVLWELITGLLPFQNMTAVQAAFAVVNRGVR 349AH PETGTYRWMAPEMIQHRPYTQKVDVYSFGIVLWELIPGMLPFQNMPAVQAAFAVVTKNVR 307CRLK1 -KSGIFGYIEEG------YCKESDVYGYGVVLMEILSGRR---------SENGLIVKWAT 270Mn -----------------ARTDKPEDHAVDLRLFKEI-----------------LNSAHVR 163

.: : :. .: * : : : .

FigureFigureFigureFigure 3S3S3S3S3333 SequenceSequenceSequenceSequence alignmentalignmentalignmentalignment ofofofof CRLK1CRLK1CRLK1CRLK1 withwithwithwith ArabidopsisArabidopsisArabidopsisArabidopsis kinasekinasekinasekinase (AT),(AT),(AT),(AT), ArachisArachisArachisArachis kinasekinasekinasekinase(AH)(AH)(AH)(AH) andandandand thethethethe conservedconservedconservedconserved manganesemanganesemanganesemanganese----dependentdependentdependentdependent kinasekinasekinasekinase domaindomaindomaindomain (Accession(Accession(Accession(Accession numbernumbernumbernumberCOG0515).COG0515).COG0515).COG0515). Accession numbers for kinases are as follows: AT, NP_565568; AH, AY027437.Amino acids marked in red indicate the conserved histidine residues for Mn2+ dependent kinases.Asterisks indicate conserved amino acids among all kinase domains.

84

CRLK1CRLK1CRLK1CRLK1 andandandand itsitsitsits signalingsignalingsignalingsignaling pathwaypathwaypathwaypathway

It is clear that CRLK1 acts downstream of CERK1/LysM RLK1 in the chitin signaling

pathway since CRLK1 induction by chitin is blocked in cerk1 mutants (Wan et al. 2008).

Interestingly, CRLK1 can also be induced by the bacterial elicitor flg22 in Col-0 wild type

(Figure 3.5C). Zipfel et al. showed that induction of almost all flg22-responsive genes was

blocked in an fls2-17 mutant treated with flg22 (Zipfel et al. 2004). CRLK1 was induced 2.7 fold

and 1.1 fold in the Arabidopsis ecotype Landsberg erecta Ler-0 and fls2-17 mutants respectively

suggesting that CRLK1 also operates downstream of FLS2 in the flagellin-induced signaling

pathway (Zipfel et al. 2004). Both MAPK3 and MAPK6 have been intensively studied regarding

their function in pathogen-triggered plant defense signaling and it is well known that they can be

stimulated by various PAMPs (Reviewed by Tena et al. 2011). In Arabidopsis treated with

chitin,MAPK3 mRNA transcripts were induced by chitin and MAPK3 and MAPK6 kinase

activity was strongly increased upon chitin treatment (Wan et al. 2004). Our data shows that

CRLK1 induction upon chitin treatment is significantly increased in mapk3 mutants, suggesting

thatMAPK3 may be a negative regulator of CRLK1 expression.

WRKY transcription factors have been shown to regulate plant defense both positively

and negatively. For example, AtWRKY48 is a negative regulator of Arabidopsis defense against

the virulent bacterial pathogen P. Syringaepv. tomato DC3000 (Xing et al. 2008). WRKY18 was

found to be partially redundant with WRKY40 and WRKY60 in the negative regulation of

defense responses against P. syringae pv. tomato DC3000 (Xu et al. 2006) and the biotrophic

fungal pathogen Golovinomyces orontii (Shen et al. 2007). In contrast, wrky18/40 and

wrky18/60 double mutants conferred higher susceptibility to the necrotrophic fungal pathogen B.

cinerea (Xu et al. 2006). WRKY53 also displayed opposing roles in defense signaling when

85

Arabidopsis was challenged by different pathogens (Pandey and Somssich 2009). In the current

study, we show that CRLK1 induction by chitin was significantly higher in wrky6, wrky17,

wrky48, wrky46, wrky53 than in Col-0 wild type (Figure 3.6C and D). While it is possible that

these WRKY transcription factors negatively regulate CRLK1 in chitin signaling further study is

needed to investigate whether these WRKY transcription factors can bind directly to the CRLK1

promoter and regulate its expression.

In conclusion, we have shown that CRLK1 is a functional receptor-like kinase involved in

defense against fungal pathogens and that Mn2+ is critical for its kinase activity in vitro.

Furthermore, we show that CRLK1 may be negatively regulated by MAPK3 and severalWRKY

transcription factors during chitin-mediated defense responses (Figure 3.6A-C). The

characterization of CRLK1 provides important information on both its function and regulation in

chitin-mediated signaling. This work establishes a foundation for further studies to identify

CRLK1’s interactor(s) and its mechanism of regulation during defense responses.

86

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CHAPTERCHAPTERCHAPTERCHAPTER FOURFOURFOURFOUR

SUMMARYSUMMARYSUMMARYSUMMARYOFOFOFOFWORKWORKWORKWORKANDANDANDAND FUTUREFUTUREFUTUREFUTURE DIRECTIONSDIRECTIONSDIRECTIONSDIRECTIONS

SUMMARYSUMMARYSUMMARYSUMMARYOFOFOFOFWORKWORKWORKWORK

In this dissertation, we report the identification and characterization of CRLK1 a

receptor-like kinase that is involved in Arabidopsis innate immunity. We have shown that

CRLK1 is critical in Arabidopsis defense against the biotrophic fungi G. cichoracearum and the

necrotrophic fungal pathogen P. cucumerina. Mutations in crlk1 result in plants that are more

susceptible to infections and that support significantly more conidiophores of G. cichoracearum

and leaf biomass of P. cucumerina than Col-0 wild type plants (Figure 2.1 and 2.2).

Overexpression of CRLK1 in Arabidopsis results in plants that are highly resistant to G.

cichoracearum (Figure 3.4). However, our data indicate that CRLK1 is not involved resistance

to bacterial pathogens since there is no significant difference between the response of crlk1

mutants and Col-0 wild type plants when inoculated with Pst DC3000 (Figure 2S1). Expression

of CRLK1 can be induced by infection with various pathogens and by treatment with several

general defense elicitors such as chitin, flg22 and OG (Figure 3.5). Additionally, CRLK1

appears to be involved in early defense signaling as induction of CRLK1 is highest one hour post

chitin treatment and then drops rapidly to baseline levels after 4 hours (Figure 2.3A). We also

show that pretreatment of plants with chitin enhances the resistance to G. cichoracearum (Figure

3.4). Unlike CERK1, crlk1 mutants still displayed enhanced resistance after chitin pretreatment

indicating that mutation in CRLK1 does not fully block chitin-triggered signaling cascades. The

induction of CRLK1 transcripts after chitin treatment was increased in the mpk3 mutant and vice

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versa suggesting that CRLK1 and MPK3 may suppress each other after chitin treatment (Figure

2.6 and 3.6). This may function as a backup mechanism if another defense pathway is blocked

by secreted pathogen effectors in order to make up for the lost pathway. The CRLK1 promoter

region contains 7 W-boxes that serve as binding sites for WRKY transcription factors, a class of

TF known to play important roles in regulating defense gene expression. CRLK1 induction by

chitin was higher in wrky6, -17, -48, -53 and -46 mutants suggesting that these WRKY

transcription factors may act as negative regulators for CRLK1 expression (Figure 3.6C). An in

vitro kinase assay showed that CRLK1 is a functional kinase and that its kinase activity is

dependent on Mn2+ (Figure 3.3B). CRLK1 is able to both autophosphorylate itself and to

phosphorylate the artificial substrate myelin basic protein in vitro (Figure 3.3B). In planta,

CRLK1 is localized to the plasma membrane (PM) and the first 30 amino acids are critical for

correct targeting to the PM (Figure 2.4 B-E). Bimolecular fluorescence complementation (BiFC)

showed that CRLK1 did not interact with either the chitin receptor CERK1 or with itself to form

homodimers (Data not shown). However, the data does not rule out the possibility that CRLK1

may interact with other RLKs or RLPs to activate downstream signaling pathways. Expression

of GUS driven by CRLK1’s native promoter showed that CRLK1 is mainly expressed in the

vasculature and flowers of Arabidopsis (Figure 2.5).

Based on the results above, we propose a model for CRLK1 activity in chitin-mediated

signaling and defense. As shown in figure 4.1, upon chitin treatment or fungal pathogen infection

CERK1 binds chitin oligomers and is phosphorylated in order to activate multiple downstream

signaling pathways (Petutschnig et al. 2010; Figure 4.1 Step 1). One of the activated pathways

operates throughMPK3 and WRKY transcription factors, which then feedback to repress further

induction of CRLK1 after chitin treatment (Figure 4.1 Step 2). Another possibility is that an

91

unknown pathway operating through unknown kinases and transcription factors activates the

expression of CRLK1 (Figure 4.1 Step 3). After the CRLK1 protein is synthesized, it is targeted

to the plasma membrane, where it may interact with unidentified receptor-like proteins or kinases.

CRLK1 may undergo autophosphorylation or be phosphorylated by other kinases for activation

and it then goes on to trigger defense signaling leading to defense gene expression (Figure 4.1

Step 4).

FUTUREFUTUREFUTUREFUTURE DIRECTIONSDIRECTIONSDIRECTIONSDIRECTIONS

DissectingDissectingDissectingDissecting CRLK1CRLK1CRLK1CRLK1 functionfunctionfunctionfunction viaviaviavia aaaa seriesseriesseriesseries ofofofof allelicallelicallelicallelic mutantsmutantsmutantsmutants

Although two different T-DNA insertional mutants in CRLK1 have been tested for their

susceptibility to powdery mildew, more crlk1 mutants are needed for more complete dissection

of CRLK1 function. Crlk1-1 and crlk1-2 are homozygous mutants with T-DNA insertions in

only one exon of CRLK1 locus that both generate a null mutation. In order to further explore

CRLK1 function, a series of point mutations can be generated in the CRLK1 exon via Targeting

Induced Local Lesions IN Genomes (TILLING; Bush and Krysan 2010). Custom TILLING in

genes of interest is commercially available through the Seattle Arabidopsis TILLING Project and

one can get mutants within 4 months (http://tilling.fhcrc.org/). With a series of CRLK1 mutant

alleles, one could challenge the mutants with powdery mildew to determine the susceptibility and

resistance phenotypes compared to Col-0 wild type and the original crlk1 knockout mutants.

Four phenotypes can be anticipated: highly susceptible, partial susceptibility, wild-type

phenotype (weak susceptibility) and resistant. After correlating the phenotypes with the

particular amino acid mutated, one can study the roles of specific amino acids in CRLK1

function. For example, mutations in critical phosphorylation sites (Serine, Tyrosine and

92

Threonine) in CRLK1 may render plants very susceptible when infected by powdery mildew. In

contrast, gain-of-function mutations in certain amino acids may create a constitutively activated

kinase. For example, a mutation in SNC2-1D (suppressor of npr1-1, constitutive 2)

transmembrane domain resulted in semi-dominant mutants that constantly activate Arabidopsis

defense responses (Zhang et al. 2010). Changing an alanine to a threonine in the SNC4-1D

(suppressor of npr1-1, constitutive4-1D) kinase domain also caused constitutive activation of the

receptor-like kinase (Bi et al. 2010). To dissect whether there is cross-talk between CRLK1 and

other defense pathways, double mutants could also be created by crossing the crlk1 TILLING

mutants and mutants in other well-defined defense pathways such as npr1 and pad4 (Salicylic

acid-mediated defense), etr1 (Ethylene-mediated defense) and jar1 (Jasmonic acid-mediated

defense) and then challenging them with powdery mildew to determine resistance phenotypes.

EMSEMSEMSEMS ScreenScreenScreenScreen andandandandMap-basedMap-basedMap-basedMap-based cloningcloningcloningcloning totototo identifyidentifyidentifyidentify suppressorssuppressorssuppressorssuppressors ofofofof crlk1crlk1crlk1crlk1

In Chapter two, CRLK1 was shown to play an important role in defense against

biotrophic and necrotrophic fungal pathogens. However, nothing is known about genes that may

regulate CRLK1 during defense responses. Plant defense responses are fine-tuned by both

positive and negative regulators to restrict pathogen attack. In order to identify genes that

potentially regulate CRLK1, crlk1 mutants seeds can be treated with ethyl methanesulfonate

(EMS) and progeny screened for secondary mutations that reverse the original phenotype

(suppressor screening). These secondary mutations can be identified via map-based cloning

within one year based on current protocols (Jander et al. 2002). After suppressors of crlk1

(scrlk1 for short) are identified, experiments can be carried out to elucidate the functions of the

new genes such as:

93

1. T-DNA mutants can be obtained for each new gene identified and tested for their

resistance to powdery mildew. Double mutants can then be made by crossing crlk1 and scrlk1

and a subsequent round of testing with powdery mildew to confirm whether scrlk1 can reverse

the original crlk1 phenotype.

2. SCRLK1 candidates can be characterized in many ways. Bimolecular fluorescence

complementation and co-immunoprecipitation can be used to determine whether CRLK1 and

SCRLK1 interact with each other directly. To determine whether SCRLK1 is co-expressed with

CRLK1 after chitin treatment, SCRLK1 gene expression can be monitored by quantitative real-

time PCR in a series of time-course experiments after chitin treatment. The expression of

SCRLK1 in Arabidopsis can also be studied via generating transgenic Arabidopsis lines that

express pSCRLK1::GUS.

DetermineDetermineDetermineDetermine thethethethe phosphorylationphosphorylationphosphorylationphosphorylation statusstatusstatusstatus andandandand sitessitessitessites inininin vivovivovivovivo afterafterafterafter treatmenttreatmenttreatmenttreatment withwithwithwith chitinchitinchitinchitin ororororinfectioninfectioninfectioninfection withwithwithwith powderypowderypowderypowdery mildewmildewmildewmildew

I have shown that CRLK1 is induced early in the chitin-mediated defense response in a

time-course experiment (Chapter two). However, little is known about the expression levels of

the CRLK1 protein after chitin treatment or powdery mildew infection. Previously, I generated

an antibody against CRLK1’s cytoplasmic kinase domain. Western blot assays can be used to

quantify CRLK1 protein levels post chitin treatment and powdery mildew inoculation. To study

phosphorylation status and sites after treatment or infection, CRLK1 can be immunoprecipitated

from Arabidopsis liquid cell culture after chitin treatment. The CRLK1 protein eluted can be

analyzed by SDS-PAGE, excised and then digested with trypsin. After enrichment of the

phospho-peptide(s), samples could then be subjected to mass spectrometry for analysis as

described by Wang et al. (2005). To study the effects of critical amino acids on CRLK1

94

autophosphorylation, a series of mutant proteins could be generated via site-directed mutagenesis

and then analyzed for kinase activity using in vitro kinase assays (Chapter three). Combined

with allelic mutants generated by TILLING, critical amino acids could be identified and their

effects on kinase activity and plant immunity elucidated.

CreateCreateCreateCreate powderypowderypowderypowdery mildewmildewmildewmildew resistantresistantresistantresistant transgenictransgenictransgenictransgenic cropscropscropscrops bybybyby exogenouslyexogenouslyexogenouslyexogenously overexpressingoverexpressingoverexpressingoverexpressing CRLK1CRLK1CRLK1CRLK1

Powdery mildew severely affects yield of many crops such as tomato, soybean, potato

and numerous cereal crops. Lacombe et al (2010) reported that transferring a PRR from

Arabidopsis to tomato and Nicotiana benthamiana generated broad-spectrum defense to a

pathogenic bacterium (Lacombe et al. 2010). Similarly, exogenous overexpression of CRLK1 in

crop plants may increase plant defense and generate broad-spectrum resistance to not only

powdery mildew but other fungal pathogens. In Chapter three, I constructed CRLK1

overexpression plants in a T-DNA binary vector that expressed a 35S:CRLK1: GUS fusion

protein. The same or similar vector could be used for transformation of cereal crops, maize and

soybean via Agrobacterium tumefaciens (Shrawat and Good 2011; Ishida et al. 2007; Ko et al.

2006) and selection of transformants would be relatively easy by screening for GUS activity in

the plants. The transgenic crops generated could then be tested for their resistance to powdery

mildew and their fungal pathogens.

IdentificationIdentificationIdentificationIdentification ofofofof CRLK1CRLK1CRLK1CRLK1 interactorsinteractorsinteractorsinteractors viaviaviavia yeastyeastyeastyeast twotwotwotwo hybridhybridhybridhybrid andandandandMassMassMassMass SpectrometrySpectrometrySpectrometrySpectrometry

Receptor-like kinases do not act alone in the activation of plant defense pathways. Many

proteins may be involved and/or associated with RLKs before or after perception of the pathogen.

An example is that of the Arabidopsis BAK1 kinase that can associate with many RLKs

95

including FLS2, BRI1, PEPR1 and PEPR2 (reviewed by Postel et al. 2010). Thus identification

of interactors is an important way to elucidate the precise mechanism of CRLK1 activity. There

are two methods that might be used to identify CRLK1 associated protein(s).

1. Yeast two hybrid analysis can be used to screen a large number of genes that may

interact with CRLK1. Due to the limitations of this technique, a high number of false positives

are to be expected. I have cloned CRLK1’s cytoplasmic kinase domain into the yeast-two-hybrid

bait vector pAS1 and the vector is ready for yeast transformation. Co-immunoprecipitation

experiments (Co-IP) can be carried out to confirm the interactions in planta.

2. In order to elucidate CRLK1 interactors in Arabidopsis, tandem affinity purification

(TAP) and MS would be suitable to study dynamic protein-protein interaction in time course

experiments after chitin treatment (Leene et al. 2011). In these experiments, CRLK1 will be

cloned into the Gateway compatible vector pKNGSTAP, which contains GS tag (tag based on

protein G with streptavidin-binding peptide) fused to a target open reading frame (ORF) (Van

Leene et al. 2011). Stable transgenic Arabidopsis expressing CRLK1-GS will be generated via

Agrobacterium-mediated transformation (Zhang et al. 2006). Arabidopsis liquid cell culture can

then be treated with chitin and then extracted for total protein that is then subjected to tandem

affinity purification. Protein complexes are concentrated and then resolved on NuPAGE gels

(Invitrogen) to separate proteins from complexes. Each protein band can then be excised and

subjected to matrix-assisted laser desorption/ionization (MALDI) tandem MS as described by

Van Leene et al (2011).

96

Chitin

CERK1 PPPP

LysM domain

kinase domain

CRLK1

?

TFs

? MPK (eg., MPK3)

WRKY TFs

?

Expression ofdefense genes

�

�

� �

unknown RLK?

Defense responses

MPKKK (eg., MEKK1)

MPKK (eg., MKK4/5)

CRLK1 gene expression

FigureFigureFigureFigure 4.14.14.14.1.... ModelModelModelModel ofofofof CRLK1CRLK1CRLK1CRLK1 inininin chitin-mediatedchitin-mediatedchitin-mediatedchitin-mediated innateinnateinnateinnate immunity.immunity.immunity.immunity. 1. CERK1 isphosphorylated upon chitin treatment. 2. Activated CERK1 triggers a MPK signaling pathway.3. CERK1 may activate an unknown signaling pathway that induces expression of CRLK1. 4.CRLK1 activates defense responses or expression of defense genes through as yetuncharacterized signaling partners.

97

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