UNIVERSITE DE GENEVE

Département de botanique

et de biologie végétale

FACULTE DES SCIENCES

Professeur W. J. Broughton

Dr. W. J. Deakin

Regulation and Effects of the Type-three Secretion System

of Rhizobium species NGR234

THESE

Présentée à la Faculté des Sciences de l’Université de Genève

pour obtenir le grade de Docteur ès sciences, mention biologie

par

Kumiko KAMBARA

de

Toyohashi (Japon)

Thèse N° 3967

Genève

2008

2

Contents

French summary of the thesis – Résumé en français de la thèse 3

Chapter 1: General introduction

1. Symbiosis

Rhizobia

Arbuscular mycorrhizae

8

2. Symbiotic signal transduction within plants

Plant perception of Nod and Myc factors

Common symbiosis pathway

Downstream of the common signalling pathway

– specificity of the symbiosis

Common symbiosis pathway

Nod factor response factors

12

3. Symbiotic signals produced by rhizobia

a) Regulation overview – the role of NodD proteins

How many nod-boxes does a rhizobial strain need?

NodD proteins control Nod-factor synthesis via NB

NodD proteins initiate a signalling cascade

TtsI and tts-boxes

b) The roles of rhizobial surface polysaccharides in symbiosis

c) T3SS and its secretion protein

17

Summary of NGR234 symbiotic signals 29

Chapter 2: Do NodV & NodW regulate symbiotic signal production in NGR234? 31

Chapter 3: Characterisation of NopM and the role in symbiosis of NGR234 effector proteins

66

Chapter 4: Functions of Nops in eukaryotic cells 87

Chapter 5: Perspectives 105

References list 112

Publications list 128

Acknowledgemnts 130

3

Résumé en français de la thèse

L'interaction symbiotique entre les plantes légumineuses et les bactéries du sol a

été étudiée car ces symbioses présentent un avantage agricole. Les Rhizobia envahissent

les racines de la plante légumineuse et forment un organe très spécialisé appelé nodule.

Les bactéries symbiotiques ont une capacité de fixer l'azote atmosphérique et de le

convertir en ammoniac assimilable par la plante. Rhizobium sp. NGR234 a un large

spectre d'hôte, et peut noduler plus de 112 genres de légumineuses aussi bien que la non-

légumineuse (Pueppke and Broughton, 1999). La nodulation commence par un échange

de signaux moléculaires spécifiques entre la plante hôte et la bactérie (Long, 1996; Roche

et al., 1996; Spaink, 2000). La plante produit un cocktail de molécules composé de

flavonoides qui sont liberés des racines de la légumineuse pour attirer le rhizobia à la

racine et induire la cascade de régulation qui comprend des composés tels que les facteurs

du Nod, le système de sécrétion de type III (T3SS) et des polysaccharide de surface

(Figure 1).

Flavonoids

Nops

TtsI

nodA nodB nodC

ttsI

NodCNodA

NodB

nop

NB8

NB18 TB8

TtsI

RhcQ RhcN

NopA

NopX

NopBNopC

ATP ADP

NopJ

NopMNopP

NopTNopL

NopJ

NopP

NopL

Rhizobium sp. NGR234

Determination of symbiotic compatibility

HMW-EPS

KPS LPS

TB2

TtsI

TtsI

Nod-factors

Induction of nodulemorphogenesis

LMW-EPS

Rhamnose rich LPS

rmlB rmlD rmlA wbgA

NodD1

NodD1

NodD1

Figure 1. Modèle pour l'interaction entre les legumineuses et NGR234. Les flavonoid sécrétés par la racine de la légumineuse induisent l'expression de gènes symbiotiques dans la bactérie et la synthèse d'un deuxième signal bactérien tels que les facteurs du Nod via NodD1, le T3SS et les polysaccharides de surface.

4

Vraisemblablement, les flavonoids diffusent dans les bactéries où ils réagissent

réciproquement avec la protéine NodD appartenant à la famille de régulateurs

transcriptionels du type LysR, et déclenche une cascade de transduction du signal qui

contrôle le processus de l'infection (Broughton et al., 2000; Perret et al., 2000). Dans

NGR234, NodD1 est l'activateur principal (Relić et al., 1993), et dirige l'expression de 18

des 19 cis-élément conservés, appelées nod-boxes en liant (Kobayashi et al., 2004). En

plus de la protéine NodD, dans Bradyrhizobium japonicum, NodV et NodW membres de

famille des régulateurs à deux-composants, sont aussi impliqués dans la modulation de

l'expression des gènes de nodulation par des isoflavonoids via une série d’étapes de

phosphorylation (Göttfert et al., 1990; Loh et al., 1997; Sanjuan et al., 1992). En outre,

NodW de B. japonicum active ttsI qui possède en amont un promoteur nod box

dépendant, ainsi que la région du gène nodD1nodD2nolA (Krause et al., 2002). Dans

NGR234, les deux ORFs (ngr159 and ngr160) qui codent pour des membres putatifs de

la famille des régulateurs à deux composants que NodV-NodW ont été localisés sur le

megaplasmid (Streit et al., 2004). Nous avons étudié le rôle que jouent NodVW dans la

cascade régulatrice, basé sur le modèle de NodVW dans B. japonicum.

Un mutant polaire, NGRΩnodVW a été produit, l'effet de NodV et NodW a été

testé sur la cascade de régulation flavonoïde-dépendante tel que le modèle des protéines

de sécrétion, des polysaccharides. Le phénotype symbiotique a été testé sur plusieurs

plantes hôte. Cependant, aucun effet considérable de NodVW n'a put être observé Des

résultats d'EMSA montrant que NodW se lie seulement à NB18, plusieurs activités des

promoteurs ont été testées et comparées. L'analyse initiale a montré que les mutants de

nodVW ont réduit légèrement ou considérablement les activités des promoteurs des NBs

(NB8 et NB18) et TBs (TB2 et TB8) après induction par le flavonoïde. NodVW affectent

la cascade régulatrice, mais ils n'abolissent pas complètement l'expression du promoteur.

D’autres analyses des promoteurs pourraient révéler un effet distinct de NodVW sur la

région des promoteurs de nodD1 et NB19 qui paraissent être la fonction directe de

NodVW. Les effets de NodVW sur ces promoteurs ont déjà été observés sans induction

de flavonoïde. Ils suggèrent que la fonction de NodVW de NGR234 est le plus

probablement la répression de NodD1 et SyrM2 en absence de flavonoïde pour maintenir

5

un niveau d'expression bas de NodD1 et de la cascade régulatrice de la symbiose. Alors

une fois que l'inducteur stimule la cascade de la symbiose, NodVW active NodD2 qui est

le répresseur de NodD1. Par conséquent, l’abscence de NodVW, change la

synchronisation de la cascade régulatrice de la symbiose (Figure 2). En dépit de

l'homologie partagée, NodVW de NGR234 n'a pas montré la même fonction que NodVW

de B. japonicum. NodVW dans NGR234 pourrait être un régulateur plus global qui agit

plutôt sur les autres régulateurs clés que TtsI spécifiquement. Des études supplémentaires

détermineront les fonctions du NodV et NodW.

Flavonoids

Tim

e

NodV

NodW

NB3

NodD1

SyrM2

NodD2

NB19

SB2

Figure 2. Modèle de fonctionnement de NodVW dans la cascade régulatrice de NGR234. NodVW est un répresseur de NodD1 et SyrM2 en absence de flavonoïde pour maintenir un niveau d’expression bas de NodD1 et donc de la cascade régulatrice de la symbiose. Alors une fois que l'inducteur stimule la cascade de la symbiose, NodVW active NodD2 qui est le répresseur de NodD1. Par conséquent, un manque de NodVW change la synchronisation de la cascade régulatrice de la symbiose.

Un système de sécrétion de type III (T3SS) est aussi l’un des composants du

symbiote important qui détermine la gamme d'hôte. Cette machine injecte un cocktail de

protéines, appelées Nops (nodulation outer proteins) dans les cellules de l'hôte, et change

le fonctionnement normal de la cellule de l'eucaryote (Galan and Collmer, 1999).

NGR234 sécrète au moins neuf protéines, qui sont classées en deux groupes, les protéines

de translocation qui sont des composants de la machine de sécrétion et les protéines

effectrices qui sont injectées dans le cytoplasme de l’hôte via le T3SS (Figure 3). Par

exemple, NopA, NopB, NopC et NopX sont nécessaires pour le passage des protéines de

la bactéries vers le cytoplasme cellulaire de la plante (Ausmees et al., 2004; Deakin et al.,

2005; Marie et al., 2003; Saad et al., 2005). NopL et NopP ont été caractérisées comme

6

étant des protéines effectrices du T3SS rhizobia-spécifique et qui peuvent être

phosphorylées par kinases de la plante (Ausmees et al., 2004; Bartsev et al., 2003;

Bartsev et al., 2004; Skorpil et al., 2005).

NopJ

NopM NopP

NopTNopL

RhcQ RhcN

NopA

NopX

NopB

NopC

ATP ADP

Plant cell cytoplasm

Plant plasma membrane

Plant cell wall

Bacterial outer membrane

Bacterial cytoplasm

Bacterial inner membraneRhcT RhcVRhcR

RhcC2RhcC1

RhcU

RhcJ

RhcS

Figure 3. Modèle proposé du Type III sécrétion système de NGR234, adapté et modifié de Viprey et associés (Viprey et al., 1998). Les composants conservés du T3SS (protéines Rhc) constituent le canal à travers les membranes internes et externes bactériennes. Les protéines de translocation, NopA, NopB et NopC composent le pili, NopX forme un pore dans la membrane plasmique de la cellule hôte. Les protéines effectrices : NopJ, NopL, NopM, NopP et NopT sont injectées dans cytoplasme de l’hôte, et perturbent son métabolisme.

Des protéines effectrices supplémentaires, NopM, NopJ, et NopT (autrefois y4fR,

y4lO, et y4zC, respectivement) ont été suggéré à partir de leur homologie avec les

facteurs de la virulence (Freiberg et al., 1997; Marie et al., 2001). Le sérum anti-NopM a

détecté dans le surnageant de NGR234 une protéine d’approximativement 60 kDa mais

pas dans le mutant T3SS ou le mutant de suppression de nopM.

Le mutant de nopM a révélé que NopM peut agir comme un effecteur positif (par

exemple sur L. purpureus) ou comme un effecteur négatif (par exemple sur P. tuberosus)

selon les espèces de la plante. NopM est la première protéine de la sécrétion qui a été

observée ayant un effet positif ou négatif selon la plante hôte. Cependant, le phénotype

du mutant de nopM n'était pas équivalent à celui du mutant T3SS nul, c’est ce qui nous a

mené à construire des mutants multiples et d’émettre alors l'hypothèse que chaque Nop

7

dans le mélange de Nops sécrété par NGR234 est reconnu différemment et la capacité

globale de la nodulation de NGR234 est l'effet net de ceci. Le L. purpureus pourrait

expliquer la différence de phénotype entre NGR234 et NGRΩrhcN qui sont le résultat de

la somme de l’effecteur positif et négatif (Figure 4).

0

5

10

15

20

25

30

35

0

1

2

3

4

5

6

7

8

9

NGR234 NGRΩrhcN NGR∆nopM NGRΩnopJ

Nod

ule

num

ber

NGRΩnopL NGR∆nopP NGR∆nopT

Pla

ntw

eig

ht(g

)

0

5

10

15

20

25

30

35

0

1

2

3

4

5

6

7

8

9

NGR234 NGRΩrhcN NGR∆nopM NGRΩnopJ

Nod

ule

num

ber

NGRΩnopL NGR∆nopP NGR∆nopT

Pla

ntw

eig

ht(g

)

Figure 4. Phénotype symbiotique de Nops sur Lablab purpureus. Les mutants Nops de cinq effecteurs différents ont été inoculés sur L. purpureus et les phénotypes ont été comparés avec NGR234 et NGRΩrhcN. Chaque barre indique le nombre moyen de nodules fixateurs d'azote par plante, et les erreurs types des moyennes sont indiquées sur la barre.

Dans cette étude, le nombre d’effecteurs Rhizobiens du T3SS connus ont été

augmenté et ont montré leur interaction complexe sur plusieurs légumineuses. Nous

suggérons que ces réponses multiples sont selon la reconnaissance des plantes hôte par

rapport à chaque Nops. Peut-être que les effecteurs positifs aident dans le processus de

nodulation en modulant la voie de signalisation de l’hôte. Au contaire, l'effecteur négatif

doit être reconnu comme facteurs de l'avirulence et mène à une réaction de défense. Cela

aidera à comprendre le mécanisme de la symbiose en prouvant la fonction de chaque

protéine effectrice de NGR234.

Pour déterminer la fonction des Nops, nous avons utilisé comme modèle

eucaryote Saccharomyces cerevisiae. La protéine NopM fusionnée à la GFP a été

localisée dans noyau 3 h après induction, cependant cette protéine de fusion était instable.

L’augmentation de NopL est toxique pour la cellule de la levure. NopT est clivable lui-

même après avoir été exprimé dans cellule de la levure, le mutant ponctuel de NopT sur

la C93S a perdu cette activité enzymatique.

8

Chapter 1: General introduction

1. Symbiosis

A symbiotic interaction between two organisms, which are widely separated

phylogenetically, is an intimate association and can be prolonged or temporary. In a

symbiosis, growth, survival and/or reproduction of both the organisms are benefited

(Odum and Smalley, 1959). However, symbiotic interactions also include commensalism,

amensalism and parasitism. For plants, associations between fungi and bacteria are

thought to have been key innovations in the colonization of land and of subsequent

specific habitats. Plant-associated microbes act as metabolic partners accessing limiting

nutrients and also as protectors, producing toxins that ward off herbivores or pathogens.

Similar associations have arisen with animals, allowing colonization of diverse niches,

such as specialized feeding on plant or animal tissues. The organisms involved in a

symbiosis may be sufficiently fused that they cannot live apart or be recognized as

distinct entities without close scrutiny. The symbiotic interactions of legumes and

rhizobia, as well as the widespread mutualistic symbiosis between arbuscular mycorrhizal

fungi and vascular flowering plants, have been extensively studied as these symbioses

contribute a significant agricultural benefit.

Rhizobia

Soil bacteria belonging to Azorhizobium, Bradyrhizobium, Mesorhizobium,

Rhizobium and Sinorhizobium genera of the order Rhizobiales (collectively called

rhizobia) are able to have a symbiotic interaction with the plant family Leguminosae.

Rhizobia invade legume roots (or occasionally shoots) which form a highly specialized

organ, the nodule. Rhizobia have the ability to fix atmospheric nitrogen to ammonia

(Mylona et al., 1995). In nitrogen scarce environments, this is important for biological

productivity and soil fertility and thus for agriculture. Within plant cells of nodules are

bacteroids, a differentiated form of rhizobia able to fix nitrogen and supply it to the host

legume plant. In return, rhizobia obtain photosynthetic products and other nutrients from

their hosts. Infection of legumes by rhizobia and thus nodule development is highly

9

restricted in a process termed host specificity. Host plants only interact with a particular

species or strains of rhizobia (Dénarié et al., 1992). Host specificity is variable and can

depend on the rhizobia strain as well. Some strains have a very narrow host range, such

as Sinorhizobium meliloti or Rhizobium leguminosarum biovar trifolii, which nodulate

only a few legume genera. Whereas, other rhizobia have a broad host range as

exemplified by Rhizobium sp. NGR234 (hereafter NGR234), which can nodulate more

than 112 genera of legume as well as the non-legume Parasponia andersonii (Pueppke

and Broughton, 1999; Trinick, 1980).

OHOOH

OOH

OH

OOH

OOH

H

flavonols flavones

isoflavonesflavanones

OHOH

OH

HO

HOHO

HO O O

OO

Figure 1. Chemical structures of different groups of flavonoid compounds.

Nodulation begins with an exchange of specific molecular signals between the

host plant and rhizobia (Dénarié et al., 1996; Ehrhardt et al., 1996; Spaink, 2000). The

cocktail of plant produced flavonoid (2-phenyl-1,4-benzopyrone derivatives) compounds

(Figure 1) (Reddy et al., 2007) are released from legume roots attracting rhizobia to the

root and induce the expression of rhizobial nodulation-related (nod) genes. Some of the

nod gene products synthesize and secrete specific chitin-like lipochitooligosaccharides,

known as Nod factors, from the bacteria (Figure 2). Nod factors have 3 to 5 N-

acetylglucosamine residues attached to an unsaturated fatty acid at the non-reducing end,

10

and contain various chemical modifications (Lopez-Lara et al., 1995; Schultze et al.,

1992). These modifications are dependent on the rhizobial strain and confer host

specificity (Roche et al., 1996). Recognition of Nod factors by the plant causes a series of

host responses, including the activation of host gene expression, calcium spiking, root

hair deformation and curling, as well as the replication of root cortical cells (Downie and

Walker, 1999; Geurts et al., 2005; Oldroyd and Downie, 2004; Oldroyd and Downie,

2006). These physiological and morphological changes ultimately lead to the formation

of the nodule, in which rhizobia find an ideal environment to fix atmospheric nitrogen.

Thus, Nod factors play a key role during initiation of nodule development and bacterial

invasion (Broughton et al., 2000; Perret et al., 2000).

Figure 2. General structure of Nod factors produced by rhizobia, adapted from D'Haeze and Holsters (D'Haeze and Holsters, 2002). The substitutions (R1–R10) and the oligomerization degree (n) are dependent on bacterial species and strains.

In response to Nod factor secretion, root hairs are stimulated and cell wall growth

reoriented (Smit et al., 1992), resulting in curled root hairs (Figure 3). Nod factors also

promote the formation of infection threads, which are plant-derived tubular structures.

Thus rhizobia enter a pocket within a curled root hair, from which they are taken up into

a developing infection thread and begin to travel towards the root cortex where the

nodule primordium is developing (Cullimore et al., 2001; Parniske, 2000). At the tip of

the infection thread, rhizobia are released into the cytosol of a subset of nodule

primordium cells and enveloped in a plant-derived membrane, to form a symbiosome.

Subsequent cell divisions and rhizobial differentiation into bacteroids leads to the

formation of fully functional nitrogen-fixing root nodules (Oldroyd et al., 2005).

11

Infection thread

C

Cortical cell sionsdivi

D

Root hair curling

B

D

Rhizobia

Root hair

Flavonoids Nod factors

A

D E

Nodule formation

E

Nodule formation

E

Nodule formation

Infection thread

C

Cortical cell sionsdivi

D

Root hair curling

B

D

Rhizobia

Root hair

Flavonoids Nod factors

A

D E

Nodule formation

E

Nodule formation

E

Nodule formation

Figure 3. Invasion of legume root hairs by Rhizobium. A: Rhizobia naturally colonize the rhizosphere metabolizing organic compounds secreted by root cells. Flavonoids released by host legume roots further attract rhizobia leading to their attachment to root hairs. B: Elevated flavonoid concentrations at close proximity trigger the synthesis of Nod factors by rhizobia which induce root hair curling and bacterial penetration at the centre of infection pocket. C: Infection threads develop within the root hair towards the cortical cells of the root. D: A developing infection thread ramifies near the nodule primordia formed by dividing cortical cells and rhizobia are released from the infection thread to form symbiosomes within nodule cells (shown in pink). E: Numerous release events and subsequent cortical cell divisions lead to the development of the new root organ, the nodule.

Arbuscular mycorrhizae

Arbuscular mycorrhizae (AM) form a symbiotic association with the plant roots

supporting vascular plant development under nutrient-limiting and various stress

conditions (Graham and Miller, 2005). The AM-root interaction is an ancient symbiosis,

fossil evidence shows that it has existed in the roots of the earliest land plants for at least

460 million years (Remy et al., 1994) and may have played a key role in facilitating the

movement of plants onto land (Heckman et al., 2001; Redecker et al., 2000; Remy et al.,

1994). Within angiosperms, more than 80 % of species are able to form AM symbioses.

12

To initiate an AM symbiosis, following spore germination the hyphal germ tube

grows through the soil in search of a host root. Once contact between the symbionts has

been established, the fungus forms an appressorium on the root surface through which it

enters the root (Strack et al., 2003). Then, inside the root, fungal hyphae continue

growing until they penetrate the cell wall of an inner cortical cell, where highly ramified

fungal hyphae form tree-like structures, termed arbuscules (Harrison, 1997). At the same

time, AM also develop extensive hyphae outside of plant root, and this extraradical

hyphal development allows the fungus to supply important nutrients, including phosphate

from the a greater area of the soil to the plant, whilst in return AM receive carbohydrates

from the plant (Shachar-Hill et al., 1995; Smith et al., 2001). The AM symbiosis also

confers resistance to the plant against biotic and abiotic stresses.

The molecular signalling mechanisms between AM and host plants is not as well

understood as for the legume/rhizobia symbiosis. Although a recent study discovered that

the strigolactone 5-deoxystrigol is a signal factor in root exudates of Lotus japonicus

responsible for the induction of hyphal branching in germinating mycorrhizal spores

(Akiyama et al., 2005). Prior to this work, strigolactones had only been known as

germination inducers of seeds of the parasitic plants Striga and Orobanche. Whether

plants produce further molecules to trigger AM spore germination, attract hyphae or to

induce AM root colonisation is not known. The identity of any molecular signals

emanating from AM that signal to the host plant have also not been identified to date,

although the presence of a so-called Myc factors has been postulated (Genre et al., 2005;

Harrison, 2005). Myc factors have a function conceptually analogous to those of rhizobial

Nod factors i.e. to be essential symbiotic signals that activate the host plant's symbiotic

program.

2. Symbiotic signal transduction within plants

Genes required for the development of the host plant’s symbiotic program have

been identified by screening mutants of the model legumes L. japonicus and Medicago

truncatula unable to establish an efficient symbiosis. Mutants defective in one type of

symbiosis are subsequently checked for their ability to form the other symbiosis and

13

whether the biochemical signal of calcium spiking (see below) can be observed. This has

led to the establishment of a signalling cascade of legume genes with several genes

required for both types of symbiosis, but the initial detection events and subsequent root

re-development stages branching as specific plant genes are required (Figure 4). Despite

the extensive morphological differences between the rhizobial and AM symbioses, it is

remarkable that they share a number of common signalling components in legumes

(Figure 4). Several host genes are essential for both the rhizobial and AM symbioses, as

shown using several legume mutants which are not only defective for nodulation but also

for the AM interaction (Albrecht et al., 1999; Hirsch et al., 2001; Kistner and Parniske,

2002). These so called common symbiosis (SYM) genes (Kistner et al., 2005) are also

universally conserved in other legumes and in non-legumes (Zhu et al., 2006). These

observations supports the hypothesis that the rhizobial symbiosis in legumes may have

evolved from the more ancient AM symbiosis (Gianinazzi-Pearson, 1996).

Plant perception of Nod and Myc factors

Potential Nod factor receptor mutants should be blocked at all stages of Nod

factor signalling, i.e. Nod factor-induced root hair deformation, calcium influx and

spiking and nodule formation but should potentially still be capable of mycorrhization

(Amor et al., 2003; Miwa et al., 2006). Using these phenotypic criteria in mutant screens,

led to the identification of putative Nod factor receptor mutants and thus genes in L.

japonicus (Lj-NFR1 and Lj-NFR5) (Madsen et al., 2003; Radutoiu et al., 2003), Pisum

sativum (Ps-SYM10) (Geurts et al., 1997; Walker et al., 2000), and M. truncatula (Mt-

NFP) (Amor et al., 2003). Sequence comparisons of Lj-NFR5, Ps-SYM10 and Mt-NFP,

show they are orthologues and encode LysM-type receptor kinases (LysM-RKs), and

could be located in the plasma membrane. Previously LysM domains have been found in

proteins that bind peptidoglycans (Bateman and Bycroft, 2000) which are not structurally

dissimilar to Nod factors. Therefore these LysM-RKs are good candidates to bind to Nod

factors, however direct binding evidence is still lacking. Another potential receptor is Ps-

SYM2 from P. sativum, mutation of which has been shown to be deficient in the

perception of specific chemical modifications to Nod factors (Geurts et al., 1997). There

are other candidate receptor genes in M. truncatula, the two LysM-RKs, Mt-LYK3 and

14

Mt-LYK4, which have homology to Lj-NFR1 and Ps-SYM2, and these mediate Nod

factor-induced infection (Limpens et al., 2003).

For AM symbioses, as for the hypothetical Myc factors no potential host receptors

have been identified as yet. Furthermore to date, no AM mutants have been identified

blocked in the calcium signalling response, which would be indicative of an upstream

function (Figure 4).

Common symbiosis pathway

Genes common to both symbioses have been identified in M. sativa, P. sativum, L.

japonicus, M. truncatula, Phaseolus vulgaris, Vicia faba, and Melilotus alba. Mutants of

these genes are blocked at an early stage of both the rhizobial- and fungal-plant symbiotic

interactions. Examples from L. japonicus include the symbiosis receptor kinase, SYMRK

(Stracke et al., 2002), two transmembrane ion channel-like proteins CASTOR & POLLUX

(Imaizumi-Anraku et al., 2005; Kawaguchi et al., 2002; Schauser et al., 1998;

Szczyglowski et al., 1998), the nucleoporin NUP133 (Kanamori et al., 2006) and SYM24

(Miwa et al., 2006). As well as being incapable of an AM interaction, mutants of these

genes still exhibit root hair deformation in response to Nod factors, but subsequent root

hair curling, infection thread formation and calcium spiking are abolished (Imaizumi-

Anraku et al., 2005). Therefore, these genes act between downstream of NFR1 and NFR5

and upstream of intracellular calcium spiking (Figure 4). Lj-SYMRK orthologues were

found in M. truncatula (DMI2), M. sativa (NORK), and P. sativum (SYM19) (Endre et al.,

2002; Stracke et al., 2002). The M. truncatula DMI1 gene (Ane et al., 2004) is a

POLLUX orthologue, and as expected mutants of Mt-DMI1 and Mt-DMI2 block calcium

spiking but not root hair deformation (Catoira et al., 2000; Miwa et al., 2006; Shaw and

Long, 2003), indicating that they act upstream of calcium spiking, at early stage of both

symbiotic interactions.

Although the M. truncatula Mt-DMI3 mutant is also blocked at early stage of both

symbiotic interactions (Catoira et al., 2000) its phenotype is subtly different as it is still

capable of calcium spiking. Mt-DMI3 encodes a calcium and calmodulin-dependent

15

protein kinase (CCaMK) (Levy et al., 2004; Mitra et al., 2004a), and has been placed

downstream of calcium spiking response (Oldroyd and Downie, 2004). Similarly the Lj-

CCaMK, Lj-SYM6 (Harris et al., 2003; Kistner et al., 2005; Schauser et al., 1998) and Ps-

SYM9 (Levy et al., 2004; Mitra et al., 2004a), are Mt-DMI3 orthologues, and their

mutants cause similar phenotypes as the Mt-DMI3 mutant (Schneider et al., 2002).

Downstream of the common signalling pathway – specificity of the symbiosis

Downstream of the common signalling pathway, there must be a divergence in the

signalling cascade to initiate the transcriptional changes required for the distinct

morphological and developmental changes in each symbiosis. Genes involved at this

stage were identified from screens of legume mutants, still able to perform the early

signalling steps such as root hair deformation and calcium spiking, but unable to form

nodules or induce nodulin expression (Figure 4). In M. truncatula, mutants in two genes,

Mt-NSP1 and Mt-NSP2 (Catoira et al., 2000; Oldroyd and Long, 2003) showed that they

were required for nodule morphogenesis but acted downstream of Mt-DMI3 (Levy et al.,

2004; Mitra et al., 2004a). These mutants, Mt-NSP1 or Mt-NSP2 exhibit root hair

deformation (Catoira et al., 2000; Kalo et al., 2005; Smit et al., 2005) a normal Nod

factor-induced calcium influx and spiking (Oldroyd and Long, 2003), however they

completely lack infection threads, any sign of cortical cell division and there is no

induction of nodulin genes (Catoira et al., 2000; Mitra et al., 2004b; Oldroyd and Long,

2003). NSP1 and NSP2 are predicted to be GRAS-domain transcriptional regulators

(Heckmann et al., 2006). The mutant phenotypes and the similarity to GRAS domain

proteins suggests that they could be Nod factor-activated transcription regulators possibly

controlling key genes in nodule development (Kalo et al., 2005; Smit et al., 2005).

Homologues are also present in L. japonicus, Lj-NSP1 and Lj-NSP2, are also predicted

GRAS domain transcriptional regulators (Heckmann et al., 2006). Another potential

transcriptional regulator has been identified in L. japonicus Lj-NIN (orthologous to Ps-

SYM35) encodes a transmembrane protein with a potential nuclear localization signal and

a predicted DNA-binding domain and may also mediate symbiotic gene expression. Lj-

NIN is thought to act downstream of calcium spiking and is not required for

mycorrhization (Borisov et al., 2003; Schauser et al., 1998).

16

Flavonoids

Nod factor Myc factor?

Nod factor receptorLysM domein protein kinases

(e.g. Lj-NFR1/ Mt-LYK3,4/ Ps-SYM2Lj-NFR5/ Mt-NFP/ Ps-SYM10)

Myc-receptor?

Lj-NSP1, 2/ Mt-NSP1, 2Lj-NIN/ Ps-SYM35

Lj-SYMRK/ / Lj-SYM24Lj-CASTOR/ Lj-POLLUX/ Mt-DMI1/ Lj-NUP133

Mt-DMI2/ Ps-SYM19/ Ms-NORK

Calcium spiking

Lj-CCaMK/ / Mt-DMI3/ Ps-SYM9Lj-SYM6

Cortical cell divisions

Plant component(s)?

MycorrhizationNodulation

?

Gene expressionNodulins ?

Commonsignallingpathway

?

Figure 4. The nodulation and endomycorrhization signalling pathways. Specific components are shown in blue (nodulation) and pink (mycorrhization). Nod factor production is induced by plant produced flavonoids and then perceived by plant LysM receptor kinases. Putative Myc factors are also proposed to be perceived by unknown specific receptors. After the initial recognition events, a common signalling pathway (genes in green) is mediated by at least: seven loci in L. japonicus (SYMRK, CASTOR, POLLUX, NUP133, SYM24, SYM6, and CCaMK); three loci in M. truncatula (DMI1, DMI2, and DMI3); two loci in P. sativum (SYM19 and SYM9) and one loci in M. alba (NORK), these are required for both nodulation and mycorrhization. Downstream of the common signalling pathway are specific regulators and gene expression for each type of symbiosis.

17

3. Symbiotic signals produced by rhizobia

The majority of the experimental work in this thesis concerns the molecular

signals produced by rhizobia to enable nodule formation; particularly their regulation and

actions. Thus the next three sections (a-c) will give an introduction to these signals and

what is known of their functions, particularly focusing on NGR234. The first two

sections on regulation and the diversity of symbiotic signals will be discussed further in

chapter 2 in light of new results, and the final section on rhizobial type III secretion

systems will be expanded upon in chapters 3 & 4.

a) Regulation overview – the role of NodD proteins

The rhizobial regulation cascade which is induced by plant-produced flavonoids is

intricate and many factors participate (Broughton et al., 2000; Perret et al., 2000). The

initial signals of nodulation, flavonoids, accumulate in the cytoplasmic membrane of

rhizobia (Hubac et al., 1993) and interact with NodD proteins, members of the LysR

family of transcriptional regulators. NodD binds to highly conserved DNA sequences,

cis-regulatory elements, called nod-boxes (NB) found in the promoter regions of most

(nodulation) nod-genes, inducing a bend in the DNA at the binding site (Fisher and Long,

1993). The chaperonins GroESL modulate the binding activity of NodD and are known to

be necessary for the correct folding of NodD in S. meliloti (Fisher and Long, 1989; Yeh

et al., 2002). There is no direct evidence for a direct interaction between NodD proteins

and flavonoid yet, however it has been suggested that a NodD-flavonoid complex is

formed at the NB (Peck et al., 2006). Even in the absence of flavonoids, binding of NodD

to NB can occur (Feng et al., 2003) regardless of whether the actual flavonoid can

actually activate the downstream nod-loci (Fisher and Long, 1993). Interactions between

flavonoids and NodD proteins do not always lead to transcription, several flavonoids can

bind to NodD1 from S. meliloti, but only luteolin was capable of activating nod gene

expression (Peck et al., 2006). NodD proteins from different rhizobia respond to different

classes of flavonoids, and the spectrum of flavonoids secreted by a legume is considered

a determinant of host specificity. Conversely at the rhizobial level, although broad-host-

range rhizobia, such as NGR234 can be responsive to a wide range of flavonoid inducers,

18

other rhizobia with limited host ranges, such as R. leguminosarum bv. viciae, can still

respond to many flavonoids.

Rhizobia usually possess between one and five nodD homologues depending on

the species, for example: R. leguminosarum bv. viciae has one copy; Bradyrhizobium

japonicum USDA110 (hereafter USDA110) and NGR234 have two nodD genes, nodD1

and nodD2 (Fellay et al., 1998; Garcia et al., 1996; Göttfert et al., 1992); whilst S.

meliloti contains three, nodD1, nodD2 and nodD3 (Honma et al., 1990). In addition to

activation of genes preceded by NB, some NodD proteins repress the expression of

promoters containing NB (see below). In USDA110 and NGR234, NodD2 is known as a

repressor of nod-genes (Fellay et al., 1998; Garcia et al., 1996; Göttfert et al., 1992).

Whereas the single nodD gene from R. leguminosarum bv. viciae is auto-repressed by its

own product (Hu et al., 2000). Besides the NodD transcriptional regulators, USDA110

also possesses a two-component sensor-regulator system, NodV and NodW, responsive

to plant-produced isoflavone signals which functions as an independent regulator of nod

genes (Göttfert et al., 1990; Sanjuan et al., 1994) (see chapter 2).

How many nod-boxes does a rhizobial strain need?

Genomic sequence has revealed that rhizobia have multiple NB: S. meliloti 1021

has seven; M. loti MAFF303099 nine and USDA110 seven (Galibert et al., 2001; Kaneko

et al., 2000a; Kaneko et al., 2002). On the symbiotic plasmid of Rhizobium etli CFN42,

fifteen putative NBs were identified (Gonzalez et al., 2003). The symbiotic plasmid

pNGR234a of NGR234 carries nineteen NBs (Freiberg et al., 1997; Perret et al., 1999).

NBs do not only control Nod-factor synthesis (see below) as they are found in the

promoter regions of a variety of genes. For example, in B. japonicum only two NBs

regulate Nod factor production, upstream of nodY (Wang and Stacey, 1991) and nolYZ

(Dockendorff et al., 1994). In NGR234, although fourteen genes are specifically required

for Nod factor synthesis (Freiberg et al., 1997) they are distributed in five operons with

each controlled by a NB (Kobayashi et al., 2004). The other fourteen NBs are located in

promoter regions of genes/operons unconnected to Nod factor synthesis, such as a type

19

III protein secretion system (T3SS), modification of extracellular polysaccharides and

synthesis of indole acetic acid (IAA) (Kobayashi et al., 2004).

NodD proteins control Nod-factor synthesis via NB

As described earlier, Nod-factors are the first rhizobial signal molecule produced

and essential for nodule formation (Downie, 1998; Lerouge et al., 1990; Smit et al., 2005).

Nod factors consist of a β-1,4-linked N-acetyl-d-glucosamine backbone of three to five

residues of which the non-reducing terminal residue is substituted at the C2 position with

an acyl chain. The structure of acyl chain can vary depending on the rhizobial species.

The structural variation of a given rhizobial Nod factor determines its host specificity.

Nod factors are synthesized and exported from the bacteria by the products of nod genes.

The common nodulation genes nodABC are found in all bacteria that form nitrogen-fixing

nodule (Moulin et al., 2001), and they are required for basic Nod factor synthesis. The

only known exception was recently reported in two group II photosynthetic

Bradyrhizobium strains, BTAi1 and ORS278, which lacked any nod gene homologues

(Giraud et al., 2007). These enzymes are encoded by the nodABC genes link the

individual N-acetylglucosamine together, and attach an acyl group to them (Atkinson et

al., 1994; Geremia et al., 1994; John et al., 1993; Kafetzopoulos et al., 1993; Rohrig et al.,

1994; Spaink et al., 1994). In addition, a given rhizobial species will possess species-

specific nod genes, which modify the basic Nod factor. These host-specific modifications

include the addition of sulphuryl, methyl, carbamoyl, acetyl, fucosyl, arabinosyl and

other groups to different positions on the backbone, as well as modifications to the

structure of the acyl chain. As an example, the nodSU genes control the ability of

NGR234 to nodulate Leucaena leucocephala through N-methylation and 6-O-

carbamoylation of the non-reducing terminus of its Nod factors (Jabbouri et al., 1995).

NodD proteins initiate a signalling cascade

NBs are also found upstream of genes encoding other transcriptional regulators.

Some rhizobial strains possess one or two copies of another LysR-type regulator syrM

(for symbiotic regulator) (Fellay et al., 1998; Hanin et al., 1998; Michiels et al., 1993;

20

Mulligan and Long, 1989; Swanson et al., 1993). SyrM proteins are NodD homologues

and can also act as activators of nod and nif genes. SyrM from S. meliloti which is

regulated by a NB, binds to the promoter regions of nodD3 and syrA (encoding another

regulatory protein) to activate their transcription (Barnett et al., 1996; Maillet et al., 1990).

Interestingly, NodD3 can then activate transcription of syrM forming a self-amplifying

loop that does not require flavonoids (Swanson et al., 1993). In NGR234, there are two

copies of syrM, syrM1 and syrM2, found on pNGR234a (Freiberg et al., 1997). SyrM1 is

involved in activation of a number of genes and controls the level of sulphated Nod

factors (Hanin et al., 1998). Transcription of syrM2 (unlike syrM1) is under the control of

a NB and is necessary for the expression of nodD2 (Kobayashi et al., 2004). In S. meliloti

SyrM proteins are thought to activate genes by binding to another cis-element, the SyrM-

motif (syr-box or SB), found upstream of nodD3 and syrA (Barnett et al., 1996; Barnett et

al., 1998; Perret et al., 1999; Xiao et al., 1998). A putative syr-box was found in the

promoter region of nodD2 (and another hypothetical gene, y4xD) of pNGR234a

suggesting a similar regulatory mechanism may exist (Kobayashi et al., 2004).

Certain rhizobia possess another, NB controlled transcriptional activator, ttsI. TtsI

has homology to the regulator proteins of the two-component sensor-regulator family

(Krause et al., 2002; Marie et al., 2004; Marie et al., 2003; Viprey et al., 1998). TtsI

activates genes by binding to conserved cis-elements, termed tts-boxes (TB) (Krause et

al., 2002; Marie et al., 2004; Wassem et al., 2008). TtsI controls a number of host specific

symbiotic signals thought to be required for nodule formation later than Nod factors, in

NGR234 examples include the T3SS and modifications to lipopolysaccharide structure

(see below).

Undoubtedly the flavonoid-induced and NodD-dependent regulation of symbiotic

signal synthesis has to be carefully controlled. In NGR234 NodD1 heads a signalling

pathway composed of several regulators to ensure a temporal gradation in symbiotic

signal production and also its own down regulation (Figure 5). In this way the expression

of a symbiotic gene can be coincided with the requirement of its product at a particular

stage of root infection or nodule development (Kobayashi et al., 2004).

21

NodD1

SyrM2

NodD2

TtsI

NB8

NB6

NB18NB19

NB3

Nod-factors

Type III secretion

Rhamnose-rich LPS

Flavonoids

Tim

e

TB8

TB2SB2

Figure 5. Proposed model for the flavonoid- and NodD1-dependent regulatory cascade in NGR234, modified from Kobayashi and associates (Kobayashi et al., 2004). In this model, flavonoids interact with NodD1 and trigger the regulatory cascade. Activation is shown with solid black arrows, whereas repression by NodD2 of NodD1-expression is marked with a dashed line. Following flavonoid induction, NodD1 rapidly activates the transcription of operons responsible for the synthesis of Nod-factors. NodD1 also activates synthesis of TtsI and SyrM2 via NB18 and NB19 respectively. This triggers additional functions that are probably required when more intimate contact between the bacteria and their hosts has occurred. In turn, SyrM2 activates transcription of nodD2. At this third regulatory level, NodD2 triggers late flavonoid-inducible loci such as fixF (controlled by NB6), which is involved in the synthesis of a rhamnose-rich LPS. TtsI also activates the synthesis of rhamnose-rich LPS as well as T3SS via TB. Concomitantly, NodD2 also represses the expression of nodD1.

TtsI and tts-boxes

Although TtsI has homology to transcriptional activators of the two component

sensor-regulator family, such regulatory systems usually consist of a sensor histidine

protein kinase and a response regulator protein (TtsI). The sensor kinase auto-

phosphorylates at a histidine residue upon detection of an external stimuli and

subsequently the phosphate group is transferred to an aspartate residue in the response

regulator leading to its activation (Stock et al., 2000). No partner sensor has been found

for TtsI, however, which poses the question as to how TtsI is activated. Notably, all he

TtsI homologues so far identified contain a glutamate residue instead of the conserved

aspartate residue (Marie et al., 2004). In other bacteria such an exchange from aspartate

to glutamate leads to the constitutive activation in the response regulator (Klose et al.,

22

1993; Lan and Igo, 1998), thus TtsI may not require phosphorylation step and thus a

sensor kinase partner to be functional. Instead only the expression of TtsI is required,

which is known to be dependent on flavonoids and NodD (Kobayashi et al., 2004; Marie

et al., 2004).

TtsI homologues have been found in several bacteria, such as USDA110 (Göttfert

et al., 2001; Krause et al., 2002), M. loti MAFF303099 (Hubber et al., 2004; Kaneko et

al., 2000a), S. fredii USDA257 (Krishnan et al., 2003) and NGR234 (Freiberg et al.,

1997; Viprey et al., 1998) which also all possess a T3SS. The rhizobial T3SS machine is

composed of the Rhc proteins, secretes a number of proteins called Nops (nodulation

outer proteins) and is an important host range determinant (see below). The promoter

regions of the nop and rhc genes, all contain the specific cis-element, the tts-box or TB.

Bioinformatic searches have revealed that numerous TB are present in T3SS-possessing

rhizobial genomes, for example in USDA110, up to 30 TB were found, suggesting the

TtsI-regulon encompasses more than just activation of the T3SS (Suss et al., 2006).

USDA110 is also noteworthy as ttsI is under the control of the NodV & NodW regulatory

proteins, as well as NodD1 (see chapter 2).

In NGR234, sequence analysis revealed the presence of 11 TB elements on

pNGR234a (Marie et al., 2004). The majority of the TBs were found upstream of genes

encoding the T3SS machine or possible secreted proteins (Figure 6). Two TBs were

located in a cluster of genes involved in rhamnose synthesis, one of which, TB2 activates

genes essential for the production of a rhamnose-rich lipopolysaccharide (LPS) known to

be important for successful nodulation (Broughton et al., 2006; Marie et al., 2004; Reuhs

et al., 2005). Thus, TtsI regulates not only T3SS but also other symbiosis factors.

Recently, transcriptional assays have shown that the expression of 10 of the 11 TBs was

flavonoid- and TtsI-dependent and that TtsI can bind to TB-containing promoters in vitro

(Wassem et al., 2008).

23

no

pT

nopM

nopJ

wbgATB1

TB4 TB5

TB6

TB2 TB3

TB8TB7 TB9 TB10

TB11

rmlB

rmlD

rmlA

gJ

mE

mF

fl5

xK xM xN xO xP yA yB yJ yQ ySnopL

nopX

nopP

nopB

nopC

nopA

rhcC

2tt

sI

rhcC

1

rhcJ

nolU

rhcN

rhcQ

rhcR

rhcS

rhcT

nolV

rhcU

rhcV

2 kb

Figure 6. Genetic organization of loci controlled by tts-boxes in pNGR234a, adapted and modified from Marie and associates (Marie et al., 2004). Genes and gene-fragments are represented by arrows showing the direction of transcription. The position of ttsI (preceded by a NB) is shown in red. Eleven TBs were identified in the promoter regions of genes encoding proteins with the following functions: green, the type III secretion machine (rhc); yellow, nop genes; blue, synthesis of rhamnose-rich LPS; and open other or unknown function. The positions and orientations of the TBs (labelled TB1 to TB11) are marked with black arrows.

b) The roles of rhizobial surface polysaccharides in symbiosis

Surface polysaccharides or SPS which include extracellular polysaccharides

(EPS), lipopolysaccharides (LPS), capsular polysaccharides (K-antigens and KPS) and

cyclic glucans are important bacterial extracellular components usually produced to

protect the cells from environmental stress. Studies with rhizobial SPS mutants have

shown they can be very important for a successful symbiosis (Fraysse et al., 2003). SPS

contribute to various stages of symbiotic development such as root colonization, host

recognition, infection thread formation, nodule invasion and host specificity although

they are not normally under the control of NodD proteins as for other symbiotic

signalling molecules (Becker et al., 2005; Spaink, 2000).

EPS has a role for the early stage of symbiosis, in establishing and extending the

infection thread. The major symbiotically active form of EPS in S. meliloti is

24

succinoglycan, mutations in genes required for its synthesis cannot fully invade the root

to establish infection threads, and lead to the formation of empty nodules (Finan et al.,

1985; Leigh et al., 1985). Succinoglycan is produced in two major forms reflecting

different degrees of subunit polymerization: the HMW (High Molecular Weight)

succinoglycan (consisting of hundreds to thousands of repeating units) which is

representative of typical bacterial EPS and also a symbiotically active form of LMW

(Low Molecular Weight) succinoglycan of monomers, dimers and trimers produced by

digestion of the HMW form by extracellular glycanases (Gonzalez et al., 1996; Wang et

al., 1999). NGR234 synthesises a HMW form of EPS similar in structure to that of S.

meliloti and is a known host range determinant. An EPS mutant cannot induce the

nitrogen-fixing nodules on Leucaena leucocephala. A number of genes involved in

synthesis of EPS have been identified in an exo cluster on the pNGR234b megaplasmid

(Streit et al., 2004). Although it was clearly shown that it was the (HMW EPS derived)

LMW EPS produced after glycanase action that were the actual critical factors (Staehelin

et al., 2006).

KPS are tightly associated with the rhizobial outer membrane, and often play a

role in the early stage of symbiosis. Rhizobial KPSs are strain-specific antigens, with

structures analogous to the group II K-antigens found in Escherichia coli. S. meliloti

Rm41 produces form of K antigen that is symbiotically active but only when EPS is

absent (Reuhs et al., 1993). In NGR234, by identifying and deleting the genes responsible

for the synthesis of KPS, the resulting mutant had a reduced ability to initiate symbiotic

infection (Le Quéré et al., 2006). However, the precise role of KPS in symbiotic infection

and the regulation of KPS expression remain unclear.

LPS are major components of the outer membrane of Gram negative bacteria and

are generally thought of as protective molecules. Rhizobial LPS can also play various

roles at different stages of the symbiosis such as in the initial recognition, infection thread

development, root tissue invasion, bacterial release into plant cells and even formation of

symbiosomes. LPS molecules consist of a lipid A anchor which maintains the molecule

in the hydrophobic outer membrane. Lipid A is associated with a core polysaccharide,

which can be substituted by an O-antigen domain. LPS are attached to the membrane by

25

the lipidic part being inserted into the bacterial phospholipid monolayer and thus the

saccharidic part is oriented to the exterior of the cell (Carlson et al., 1999; Noel and

Duelli, 2000; Price, 1999). Two forms of LPS are often synthesized, rough LPS (R-LPS)

and smooth LPS (S-LPS). The low molecular weight form of LPS (R-LPS) contains only

the lipid A and core oligosaccharide, whereas high molecular weight form of LPS (S-

LPS) includes an additional O antigen (Reuhs et al., 1998). LPS core oligosaccharides

were isolated and partially or fully characterized in R. leguminosarum bv. phaseoli (Bhat

et al., 1994; Bhat et al., 1991; Carlson et al., 1989), R. trifolii ANU843 (Carlson et al.,

1988), R. etli (Forsberg and Carlson, 1998), S. fredii and NGR234 (Reuhs et al., 1998).

Additionally, several metabolic steps in the biosynthesis of these molecules have been

elucidated, although mutant analysis can be complicated by either pleiotropic or lethal

phenotypes. In NGR234 a symbiotic form of S-LPS is produced in presence of flavonoids,

and its absence adversely affected nodulation of several host legume species (Marie et al.,

2004). This S-LPS molecule is noteworthy as it is predominantly composed of rhamnose

residues, and the biosynthetic enzymes required are under the control of TtsI (Broughton

et al., 2006).

c) T3SS and its secretion protein

T3SS are highly conserved multi-protein complexes, and important virulence

factors in pathogen-eukaryote interactions. T3SS were found in many Gram-negative

bacteria infecting humans, animals, and plants (Hueck, 1998). Thus T3SS had previously

been thought to be unique to pathogenic bacteria, however, these systems have now been

identified in rhizobia (Marie et al., 2001) such as: NGR234 (Freiberg et al., 1997), M. loti

MAFF303099 (Kaneko et al., 2000b), B. japonicum USDA110 (Göttfert et al., 2001), S.

fredii USDA257 (Meinhardt et al., 1993), USDA191 (Bellato et al., 1997), and HH103

(Bellato et al., 1997; Marie et al., 2001). In contrast, S. meliloti 1021 (Galibert et al.,

2001) and M. loti R7A (Sullivan et al., 2002) do not contain T3SSs. In these strains, a

type IV secretion system may serve a similar function (Hubber et al., 2004). Thus, T3SS

are present in some but not in all rhizobia.

26

The T3SS injects a cocktail of proteins (called effectors) into eukaryotic cells, to

change normal functioning of eukaryotic cell (Galan and Collmer, 1999). Understanding

the specific functions of these effectors has become a top priority for the rhizobial-plant

interaction. Rhizobial T3SS secrete nodulation outer proteins (Nops), some of which may

be transported into host plant cells via a pili structure and thus be considered as effectors

(Marie et al., 2001).

NGR234 secretes at least nine Nops, which are classified into two groups,

translocatory proteins that are external components of the secretion machine and effector

proteins injected into host cytoplasm through the T3SS (Figure 6). For example, NopA,

NopB, NopC and NopX are required for the transit of proteins from bacteria to the plant

cell cytoplasm (Ausmees et al., 2004; Deakin et al., 2005; Marie et al., 2003; Saad et al.,

2005). In NGR234, NopL and NopP have been characterized as rhizobial-specific

effector proteins which can be phosphorylated by plant kinases (Ausmees et al., 2004;

Bartsev et al., 2003; Bartsev et al., 2004; Skorpil et al., 2005). A double mutant of

NGRΩnopL∆nopP was shown to have a more pronounced phenotype than either single

mutant, suggesting that the effector Nops may also function cooperatively (Skorpil et al.,

2005). Additional effector proteins on pNGR234a were identified from homology

searches (Freiberg et al., 1997; Marie et al., 2001) nopM, nopJ, and nopT (formerly y4fR,

y4lO, and y4zC, respectively) that also containing TBs in their promoter regions (see

chapter 3 for more detail) (Kambara et al., 2008).

27

NopJ

NopM NopP

NopTNopL

RhcQ RhcN

NopA

NopX

NopB

NopC

ATP ADP

Plant cell cytoplasm

Plant plasma membrane

Plant cell wall

Bacterial outer membrane

Bacterial cytoplasm

Bacterial inner membraneRhcT RhcVRhcR

RhcC2RhcC1

RhcU

RhcJ

RhcS

Figure 6. Proposed model for the type III secretion system of NGR234, adapted and modified from Viprey and associates (Viprey et al., 1998). The conserved components of the T3SS (Rhc proteins) form a channel through the bacterial inner and outer membranes. The translocatory proteins, NopA, NopB and NopC are components of pili and required for the transit of Nops from bacteria to plant cell cytoplasm. NopX forms a pore in the plant cell plasma membrane. The effector proteins, NopJ, NopL, NopM, NopP and NopT are injected into the plant cell cytoplasm, and are thought to interfere with host metabolism.

Mutations in the T3SS machinery that abolish Nop secretion cause symbiotic

phenotypes dependent on the host plant (Ausmees et al., 2004; Krause et al., 2002;

Krishnan et al., 2003; Lorio et al., 2004; Marie et al., 2003; Viprey et al., 1998). For

instance, in NGR234 a functional T3SS is required for efficient nodulation of some plant

species, such as Tephrosia vogelii, Flemingia congesta and Lablab purpureus, however

for Pachyrhizus tuberosus and Crotalaria juncea the T3SS appear to be extremely

deleterious (Ausmees et al., 2004; Marie et al., 2003). Mutations within genes that encode

for effector Nops cause different symbiotic phenotypes depending on the host plants, see

28

chapter 3 (Ausmees et al., 2004; Bartsev et al., 2003; Kambara et al., 2008; Skorpil et al.,

2005).

S. fredii strains USDA257 and HH103 possess very similar T3SS to NGR234,

which have been shown to secrete Nops. In USDA257, NopX, NopB, Nop38, and Nop7

were revealed as Nops, they associated with the pili (Krishnan, 2002; Krishnan et al.,

2003; Lorio et al., 2004). In HH103, NopA, NopC, NopL, NopP, and NopX were

confirmed to be T3SS secreted proteins (Rodrigues et al., 2007). Furthermore, NopM and

NopD were also identified as putative secreted effector proteins (Rodrigues et al., 2007),

these Nops are homologous to NopM of NGR234 and XopD of X. campestris pv.

vesicatoria, respectively (Hotson et al., 2003). XopD in X. campestris pv. vesicatoria

targets SUMO (small ubiquitin-like modifier) conjugated proteins in planta, suggesting

that the XopD protease mimics a host protease that removes SUMO modifications. This

proteolysis could alter host cell signalling events for the pathogen.

In B. japonicum USDA110, genes with homology to nopA, nopB, nopL and nopP

were found in the tts gene cluster (Krause et al., 2002). However, nopX is not present in

the genome. A conserved TB motif was found in the putative promoter region of six other

genes encoding possible secreted proteins (Suss et al., 2006). Using mass spectrometry

techniques, eight different genistein-inducible secreted protein spots were identified. One

of the proteins, Blr1752, has similarity to NopP from NGR234 (Suss et al., 2006).

Mutation of the S. fredii and B. japonicum T3SS also leads to host plant specific

phenotypes and a similar result was observed after mutation of the T3SS of M. loti

MAFF303099. Although protein secretion was not observed by the T3SS of

MAFF303099, its mutation enabled nodulation of Leucaena leucocephala (Hubber et al.,

2004). Fascinatingly mutation of the T4SS of M. loti R7A, the genes (virB1 to B11 and

virD4) had a similar phenotype. This T4SS is very similar to the Agrobacterium

tumefaciens vir T4SS that transfers T-DNA and several proteins to plants (Christie and

Cascales, 2005; Sullivan et al., 2002). The expression of the M. loti T4SS depends on

flavonoids, which activate NodD1 and subsequently activate the VirA/VirG two-

component regulatory system (Leroux et al., 1987; Winans et al., 1986). A putative NB

29

was found 851 bp upstream of virA (Hubber et al., 2004; Sullivan et al., 2002),

suggesting that the vir genes are under the control of NodD (Hubber et al., 2007).

Previously, it was reported the presence of two vir-boxes consensus nucleotide repeats in

the M. loti R7A symbiosis island, one in the promoter region of the msi061 gene which

encodes effector proteins and the other in the intergenic region between the divergently

transcribed virB1 and virG genes (Hubber et al., 2004). Most probably, VirG binds at vir

boxes to induce their transcription (Gao et al., 2006; Jin et al., 1990; Pazour and Das,

1990; Powell et al., 1989). Mutations of the M. loti vir genes have host-dependent

phenotype (Hubber et al., 2004). It was suggested that the host-dependent symbiotic

phenotypes are due to the same effector proteins, after secretion by either a T3SS or a

T4SS.

Summary of NGR234 symbiotic signals

As described above, in response to the presence of flavonoids released by

potential host-plants, rhizobia have evolved a complex regulatory network for successful

symbiosis. Recent studies have focused not only on the effects of Nod factors but also on

the numerous other (secondary) symbiotic signals, such as surface polysaccharides or

secreted proteins. In the case of NGR234 there appear to be numerous secondary

symbiotic signalling systems (Figure 7), the regulation and function of which will be

investigated in chapters 2-4.

30

Flavonoids

Nops

TtsI

nodA nodB nodC

ttsI

NodCNodA

NodB

nop

NB8

NB18 TB8

TtsI

RhcQ RhcN

NopA

NopX

NopBNopC

ATP ADP

NopJ

NopMNopP

NopTNopL

NopJ

NopP

NopL

Rhizobium sp. NGR234

Determination of symbiotic compatibility

HMW-EPS

KPS LPS

TB2

TtsI

TtsI

Nod-factors

Induction of nodulemorphogenesis

LMW-EPS

Rhamnose rich LPS

rmlB rmlD rmlA wbgA

NodD1

NodD1

NodD1

Figure 7. Summary of the symbiotic signal molecules produced by NGR234. Flavonoid compounds from legume roots trigger gene expression and synthesis of signals by NodD1 binding to NBs. The primary symbiotic signals, the Nod factors are secreted and allow bacterial entry into root hairs and initiate plant programs for rhizobial infection and root nodule development. NodD1 consequently activates TtsI which activates T3SS and synthesis of rhamnose rich LPS by binding to TBs. LMW EPS, KPS, rhamnose-rich LPS and Nops can all be considered as secondary symbiotic signals, although not essential, they can be extremely important for the nodulation of certain legumes.

31

Chapter 2: Do NodV & NodW regulate symbiotic signal production in NGR234?

Introduction

NodD proteins are undoubtedly the main transcriptional regulators of symbiotic

signals in rhizobia (Göttfert et al., 1992; Schlaman et al., 1992). In Bradyrhizobium

japonicum, however, other regulators (NodV&W and NolA) also have critical roles in

modulating expression of nodulation genes. NodV and NodW belong to the two-

component regulatory family and activate expression of the nodulation genes by

isoflavonoids via a series of phosphorylation steps (Göttfert et al., 1990; Loh et al., 1997;

Sanjuan et al., 1992). As described earlier, members of this family can be grouped into

two subclasses, the sensor class and the regulator class. As result of conserved domain

searches, NodV was shown to be the sensor and to have a histidine kinase-like ATPases

domain auto-phosphorylation so that could occur. NodW is the regulator, activated by

phosphorylation from the NodV sensor partner, it possesses a helix-turn-helix domain to

bind to DNA and initiate transcription. Phosphorylation of NodW is known to be required

for efficient nod gene expression (Sanjuan et al., 1994) and a nodW mutant showed

reduced levels of nod gene expression (Loh et al., 1997). NodVW, however, are required

for nodulation of certain host plants of B. japonicum, i.e. for cowpea (Vigna unguiculata),

mung bean (Vigna radiata), and siratro (Macroptilium atropurpureum), but not for

soybean (Glycine max) (Göttfert et al., 1990). Instead NodD1 is absolutely required for B.

japonicum nodulation of soybean. The host-specific requirement of NodVW suggests that

soybean plants produce a cocktail of isoflavones that interact with NodD1 but are not

sensed by NodVW. In contrust cowpea, siratro and mung bean plants produce inducers

which interact specifically with NodVW and not NodD1. The NodVW flavonoid sensing

system has also been shown in previous studies to be required for expression of ttsI and

thus regulates the T3SS of USDA110 (Figure 1) (Krause et al., 2002).

32

Figure 1. Model of the regulatory cascade controlling expression of the T3SS gene cluster, adapted from Krause and associates (Krause et al., 2002). Open reading frames (ORFs) unique to Bradyrhizobium japonicum are in black, and nod genes are shown in white. ORFs with homologues in T3SS gene clusters of other rhizobia are gray. The gray arrowhead indicates the position of the nod box. Positions of tts boxes are marked by black arrowheads.

In NGR234, two ORFs (ngr159 and ngr160) encoding homologues of NodV &

NodW were found on the megaplasmid (Streit et al., 2004). Downstream of nodVW and

possibly co-transcribed are two further ORFs (ngr158 and ngr157) with homology to

nodW (Table 1) (Figure 2).

Table 1. Characteristics of the NGR234 NodV and NodW homologues. Homology (similarity and identity) was identified by BLAST-P search. The number of amino acids in a parenthesis indicates identity and similarity over what number of amino acids to NGR234.

Amino acid Size Similarity to

NGR234 Identity to NGR234

NodV Rhizobium sp. NGR234 1333 aa 148.8 kDa - -

NodV Bradyrhizobium japonicum 889 aa 99 kDa 44 % (760 aa) 27 %

NodW Rhizobium sp. NGR234 216 aa 23.6 kDa - -

ngr158 Rhizobium sp. NGR234 120 aa 12.9 kDa 55 % (114 aa) 37 %

ngr157 Rhizobium sp. NGR234 217 aa 23.5 kDa 53 % (198 aa) 67 %

NodW Bradyrhizobium japonicum 227 aa 25.2 kDa 72 % (208 aa) 49 %

NwsB Bradyrhizobium japonicum 221 aa 23.9 kDa 71 % (203aa) 53 %

33

Figure 2. The nodVW region of pNGR234b, adapted from Streit and associates (Streit et al., 2004). ORFs were named genes when BLAST-P searches of the National Center for Biotechnology Information database indicated an identity of <E−80.

The potential presence of multiple forms of NodW in NGR234 is interesting as

B. japonicum in addition to NodW, possesses a second response regulator NwsB, which

shares 65 % amino acid with NodW. Although NwsB was shown to control expression of

nodulation genes, an nwsB mutant could still nodulate cowpea, siratro, and mungbean

(Grob et al., 1993). A more detailed analysis of NGR157 and NGR158 (Figure 3) showed

that NGR157 has homology over its entire length to NodW, possessing both the signal

receiver domain where phosphorylation occurs and also the DNA binding helix-turn-

helix domain. NGR158 however lacked the DNA binding domain (Figure 3). The

observation that NodW of NGR234 contains the conserved aspartate residue, suggests

that phosphorylation is necessary for functional NodW. Unlike TtsI of NGR234 (which is

also homologous to response regulator proteins) but contains a glutamate residue instead

of the conserved aspartate residue possibly rendering TtsI constitutively active (as

discussed earlier).

....|....| ....|....| ....|....| ....| ....| ....|....| ....|....| ....|....| ....|....| 10 20 30 40 50 60 70 80

MT------SD DHVVFIVDDD ERIREALSDL LDSHGIRAIA FGSA GEYVSA DKPDVPACLI LDVELPDING LDLQRQIADV MSPQLGSEED EPLVIIVDDD ASVRAALSEL ILSAGFRPVS FAST RELLDA DTLDAPGCLI LDVRMPGESG LHLQRHLADN MN------KT RHVVAIVDDD ARLLESVSDL LESAGYVARS FPSA GSLLAS G-LSDLDVLI TDIGMPGMDG LELRDRVKKS

....|....| ....|....| ....|....| ....| ....| ....|....| ....|....| ....|....| ....|....| 90 100 110 120 130 140 150 160

DHP-PIVFIT GHGDIPSSVR AIKHGAVDFL TKPFSDADLM AAIG AAIAED RVKRAARAEL SMLGQRYREL TPREREVLPL GNPKPIIFLT GHGDIPMTVE AMKAGAVDFL TKPVRDQTLL DAVTAGIAMD AERRAEAAIS RLNIERLETL TQREREVLYE RPELPVFLIT GRHEIADQGR AQ--GNSGFF RKPFDAQALL AAIA NALDK- ---------- ---------- ----------

....|....| ....|....| ....|....| ....| ....| ....|....| ....|....| ... 170 180 190 200 210 220

VVSGLLNKQA AAELGISEVT LQIHRRNVMH KMAADSLADL VRIAERLEIP ITHSRRVGGN DHE VARGRLNKQI AFDLGISEVT VKAHRSSVMH KMGAASVGEL IRAFETLP-- -AQMRQAGAR --- ---------- ---------- ---------- ---------- ---- ------ ---------- —

NodW ngr157 ngr158

NodW ngr157 ngr158

NodW ngr157 ngr158

: signal receiver domain

: helix-turn-helix

: conserved Asp residuce

Figure 3. Amino acid alignment of NodW and the two proteins encoded by downstream ORFs in the nodVW cluster. The predicted amino acid sequences were shown to have conserved domains by CD-BLAST searches and then aligned. Pink and blue highlighting indicates the signal receiver domain and helix-turn-helix DNA binding domain, respectively. The green highlight indicates the conserved aspartate residue phosphorylated by the sensor histidine protein kinase.

34

In NGR234, it is known that NodD1 is the main activator of loci whose

expression is flavonoid-dependent (Relić et al., 1993), and a nodD1 mutant is unable to

nodulate any host plant. Despite this genes on the symbiotic plasmid were shown to be

induced in a nodD1 mutant after flavonoid treatment (Fellay et al., 1995). This

observation suggests the presence of other flavonoid-sensing regulators, and NodVW are

possible candidates. Furthermore the role of NodVW in the regulation of the USDA110

T3SS prompted the question of whether a similar regulation might occur in NGR234. If

NodVW did control other flavonoid-inducible genes in NGR234 would this influence

nodulation of certain host plants as seen in B. japonicum? Thus initially as a model to test

we applied the known role of NodVW in B. japonicum in the induction of its T3SS, to the

regulatory cascade of NGR234 (Figure 4).

TtsI Type III secretion

Rhamnose-rich LPS

Flavonoids

Tim

e

NodV

NodW

TB8

TB2

NB18

Figure 4. Model of the regulatory cascade of NGR234, adapted from Kobayashi and associates (Kobayashi et al., 2004). Based upon NodVW function in B. japonicum, a potential role for NodVW from NGR234 was added to this model. Red arrows indicate the hypothetical target of NodVW.

35

Materials and methods

Microbiological techniques

Escherichia coli strains were grown in Luria-Bertani (LB) media at 37 °C

(Sambrook et al., 1989). Rhizobium strains were grown at 27 °C in either complete (TY)

(Beringer, 1974) media or minimal media containing succinate as the carbon source

(RMS) (Broughton et al., 1986). Antibiotics were added to the media at the following

final concentrations; ampicillin (Ap), 50 µg ml-1; gentamycin (Gm), 10 µg ml-1 (for E.

coli), 20 µg ml-1 (for Rhizobium); kanamycin (Kn), 50 µg ml-1; rifampicin (Rif), 50 µg

ml-1; spectinomycin (Sp), 50 µg ml-1; tetracycline (Tet), 15 µg ml-1; and chloramphenicol

(Cm), 15 µg ml-1. Flavonoids were added to the media at 2 × 10-7 M final concentrations.

Mutagenesis of nodV

To obtain NGRΩnodVW, nodV was amplified by PCR from genomic DNA of

NGR234 using the following primer pairs: A (5’-AGCGGCCGGTTTACGAAGTG-3’)

and B (5’-CCATGTCGACGAGTTGCGAG-3’). The 1.7 kb fragment containing nodV

was first cloned into the EcoRV site of pBluescript KS+ and fidelity of the PCR verified

by sequencing. An omega interposon conferring resistance to spectinomycin (Fellay et al.,

1987) was inserted into an internal EcoRV site located 1520 bp from the ATG of nodV.

ApaI and SacI were used to excise a 3690 bp fragment of this mutated nodV which was

then subcloned into the suicide vector pJQ200SK (Quandt and Hynes, 1993). Triparental

matings were used to transfer the resulting plasmid into NGR234. Double recombination

was selected by plating bacteria onto TY plates containing 5 % sucrose and appropriate

antibiotics. Putative mutants were confirmed by PCR and Southern blots of restricted

genomic DNA using standard procedures (Chen and Kuo, 1993; Sambrook et al., 1989).

The double mutant NGRΩnodD1ΩnodVW and NGRΩnodD2ΩnodVW were generated

using a similar approach, but an omega interposon conferring resistance to Kanamycin

(Fellay et al., 1987) was substituted for spectinomycin resistance. Triparental matings

were used to transfer the resulting plasmid into NGRΩnodD1 (Relić et al., 1993). To

36

create NGRΩnodD2ΩnodVW, pJQΩnodVW with resistance to spectinomycin was

transferred into NGRΩnodD2 (Fellay et al., 1995).

Analysis of secreted proteins and polysaccharides

Secreted proteins were purified and analyzed as described in (Marie et al., 2003).

Rhizobium strains were grown in 100 ml of RMS for 40 h in the presence of daidzein or

genistein. Secreted proteins present in the supernatants of cultures were precipitated using

ammonium sulphate (60 %, wt/vol) and subsequently desalted using Sephadex G25-

containing columns (Amersham Biosciences, Uppsala, Sweden). Then proteins were

separated by 15 % SDS-polyacrylamide gel electrophoresis, followed by silver nitrate

staining or transfer to PVDF membranes for immunoblotting.

To extract polysaccharides, rhizobial cells obtained by centrifuging 1.5 ml liquid

cultures of RMS were lysed using 100 µl of lysis buffer as described previously

(Hitchcock and Brown, 1983). Two volumes of sample buffer (120 mM Tris, pH 6.8, 3 %

(w/v) SDS, 9 % (v/v) β-mercaptoethanol, 30 % (v/v) glycerol, 0.03 % (w/v) bromphenol

blue) were then added. The final mixtures were separated by DOC (deoxycholic acid

sodium salt) - PAGE (18 % acrylamide) using 0.375 M Tris (pH 8.8) and 0.5 % DOC. As

the anode buffer comprised 0.1 M Tris, 0.1 M gricine, and 0.25 % (w/v) DOC, at 20 mA.

Gels were stained specifically for LPS using periodate oxidation-silver (Tsai and Frasch,

1982), and KPS were visualized by sequential staining with alcian blue-silver (Corzo et

al., 1991; Reuhs et al., 1998), with omission of the periodate treatment so that LPS was

not detected. These procedures readily distinguish the KPS from LPS; the alcian blue

pretreatment is required for KPS visualization, and periodate oxidation is required for

LPS staining.

β-Galactosidase assay

Flavonoid induction and assays for β-Galactosidase activity were performed as

described in Kobayashi and associates (Kobayashi et al., 2004). Rhizobial cultures grown

to a density of 1cmOD600 of 0.5-0.6 were diluted to 1cmOD600 of 0.1 in RMS medium and

induced with daidzein and genistein. β-Galactosidase activity was assayed according to

37

Miller (Miller, 1972). The results reported represent the means of at least three

independent experiments.

Promoter activity assay with GFP

Promoter region of nopB, nopJ, fixF, rkpL, rkpY and exoK were cloned into pGT-

GFP (Le Quéré et al., 2008). Triparental matings were used to transfer the resulting

plasmids into NGR234 and NGRΩnodVW. Rhizobial cultures were inoculated to RMS

liquid media from RMS plates then induced with apigenin, daidzein, genistein,

kaempfenrol or naringenin. The GFP fluorescence was measured by Plate

CHAMELEON (HIDEX) every 24 h during up to 96 h after flavonoid induction. The

promoter activities were calculated as below: for each sample the GFP fluorescence level

and the density of OD600 were measured and corrected with the GFP fluorescence level

and density of OD600 of free media respectively. Then the corrected OD600 was

normalized by dividing with the average of each time point OD600. Final GFP

fluorescence levels were computed from the average of normalized GFP fluorescence at

least five independent experiments.

NodW overexpression

To obtain pETBlue-nodW, nodW was amplified by PCR from genomic DNA of

NGR234 using the following primer pairs: A (5’-CATGACGAGCGATGATCATG-3’)

and B (5’-TTCATGGTCATTCCCTCCCAC-3’). The 653 bp fragment containing full-

length of nodW was cloned into the EcoRV and PvuII site of pETBlue-2 (Novagen) and

sequenced. To overexpress nodW in E. coli Tuner (DE3) pLacI (Novagen) derivative

strain was used as a host. Cells contain the in-frame six-histidine tag fusion at amino-

terminal of nodW, pETBlue-nodW were grown in LB containing Ap and Cm. Protein

expression was induced at 1cmOD600 of 0.5 by adding 100 µM isopropyl-β-D-

thiogalactopyranoside (IPTG), and incubation was continued for an additional 6 h at

37 °C. Cells were harvested and resuspended in buffer A (50 mM NaPO4 pH 7.7, 10 %

glycerol, 0.5 M NaCl, 0.1 % Triton). The cell lysate was sonicated for 40 min on ice and

then centrifuged at 10,000 x g for 30 min at 4 °C to pellet the cellular debris. On 12 %

38

SDS-PAGE, the supernatant contained a major polypeptide of approximately 27 kDa that

was not present in the un-induced sample.

NodW purification

Immobilized metal affinity chromatography (IMAC) columns were used for

purification of His-tagged NodW. To remove non-specific binding peptides, the column

was washed with 0.1 M EDTA. Excess EDTA was removed by rinsing with water. The

column was then activated with ammonium acetate pH 4.0. His-tagged NodW

supernatant was loaded onto the IMAC column at a rate of 1 ml/min. Then, buffer A (50

mM NaPO4 pH 7.4, 500 mM NaCl, 0.5 % Triton and 10 % glycerol) which was mixed

with different concentration of imidazole (20 to 500 mM) were added to the IMAC

column continually. Eluted samples were collected and separated by 15 % SDS-PAGE

gel and stained with coomassie blue. Imidazole was removed by dialysis in 50 mM Tris

(pH 8.0), 150 mM NaCl, 0.1 mM EDTA, 0.1 mM DTT, 50 % glycerol for 15 to 20 h at

4 °C. To obtain pure NodW protein, the sample were washed and eluted with a Heparin

column with different concentrations of NaCl (0.1 mM to 1.2 mM) in the heparin buffer

(10 mM Tris-Cl pH 8.0, 0.1 mM EDTA, 0.1 mM DTT, 10 mM MgCl2 and 5 % glycerol).

Total protein concentration of the purified NodW containing was determined to be 16

mM using a protein assay (BIO-RAD).

Electrophoresis mobility shift assay and DNase I footprinting

The DNA fragments (NB and TB) used for gel mobility shift assays were

amplified by PCR using the following primer pairs: NB3 (5’-ACAAAGCCTGTTCTTCA

GGA-3’ and 5’-GCCGTGAGTTTGCGTTCGGT-3’), NB6 (5’-GTTTGAACGGGTCTC

GGGA-3’ and 5’-TATGGATTATCCTGAGAGCGA-3’), NB8 (5’-ACCTTCCCGTATC

ACTCGCA-3’ and 5’-CCAACTTCCTTGTTTTCGAGAG-3’), NB18 (5’-TAACGGCA

AAGCAGTGTGCGA-3’ and 5’-CACCTGAGATTTCTCTGCGCA-3’), TB2 (5’-AAGC

ACCCCGAAAACTACCT-3’ and 5’-GCATTGCGAAATTTGGATGGA-3’), TB8 (5’-

CTCGTCTTGATAAACCAAATCTGAA-3’ and 5’-GGACTCGATTACTTAACTCTTT

GAC-3’). The DNA fragments were 32P-labelled at the 5′ end using T4 polynucleotide

39

kinase. Binding reaction mixtures contained the labelled DNA fragments, 5 µg/ml

competitor oligonucleotide, and different concentrations of NodW in Gel Shift Binding

Buffer (50 mM Tris-HCl [pH 7.6], 8 mM Mg acetate, 27 mM NH4 acetate, 100 mM K

acetate, 1 mM DTT). The reaction mixtures were incubated for 30 min at room

temperature and reaction were stopped by the addition of 1 µl of loading buffer (0.1 %

bromophenol, 0.5 mM DTT and 50 % glycerol). The samples were loaded on a 4 %

native Tris–glycine Gel and dried on a Gel Dryer (Miliam). The gels were scanned using

a CycloneTM phosphoimager (Packard), and the intensities of the bands were determined

using OptiQuant software.

DNase I footprinting reactions were also performed using NodW. NB18 PCR

product was 32P-labelled at the 5’ and 3’ends using T4 polynucleotide kinase. The

radiolabelled NB18 DNA fragment and difference concentrations of NodW were mixed

and incubated at room temperature for 30 min in a 20 µl volume, then 1 µl of DNase I

(0.1 units/µl; Invitrogen) was added to digest for 2 min. The reaction was terminated, and

then extracted with phenol–chloroform. The aqueous phase was precipitated with ethanol,

and the samples were analyzed on 6 % denaturing polyacrylamide gel. The gels were

dried on a Gel Dryer (Miliam) and scanned using a cycloneTM phosphoimager (Packard),

and the intensities of the bands were determined using OptiQuant software.

RNA extraction and Quantification RT-PCR

NGR234 and NGRΩnodVW strains were grown in RMS medium in the presence

of daidzein or genistein. The 5 x 1010 cells were collected at 1, 6 and 24 h after flavonoid

induction then frozen at -70 °C. RNA was extracted from the pellets via modified phenol-

chloroform extraction (SIGMA). Isolated total RNA were treated with DNase I

(QIAGENE) then used for templates for the synthesis of cDNA (iScript Select cDNA

Synthesis Kit; Bio-Rad). One microgram of total RNA were reverse transcribed to cDNA.

Quantification real-time RT-PCR was carried out as described by Pfaffl (Pfaffl,

2001). Oligonucleotide primers specific for nodV (5’-AATGGTACGCGGTTGCT

ATCGAA-3’ and 5-CCTACGTCCAGACCGAAGAA-3’), rpsL (5’-GCTTCGAAGTGA

40

TCGGCTAC-3’ and 5’-ACACCCTGCGTATCGAGAAC- 3’) and 16S (5’-AGCCACAT

TGGGACTGAGAC-3’ and 5’-ACCCTAGGGCCTTCATCACT-3’) were generated

(SIGMA-GENOSYS) and optimized to equal annealing temperatures of 60 °C. For RT-

PCR reaction a mastermix of the following reaction components was prepared to the

indicated end-concentration: 3 µl water, 1 µl forward primer (10 µM), 1 µl reverse primer

(10 µM) and 10 µl SYBR Green (iQ SYBR Green Supermix; Bio-Rad). Mastermix (15

µl) was filled in 96 wells plate and mixed with 5 µl cDNA (100 ng). The thermal cycler

program was as follows; denaturation program (95 °C for 3 min), amplification and

quantification program repeated 40 times (95 °C for 30 sec, 60 °C for 1 min), melting

curve program (60–95 °C with a heating rate of 0.1 °C per second and a continuous

fluorescence measurement) and finally a cooling step to 40 °C.

Plant material and nodulation assays

Seed source are listed in Pueppke and Broughton (Pueppke and Broughton, 1999).

Plant seeds were sterilized as described below; Vigna unguiculata and Vigna radiate:

concentrated sulphuric acid 10 minutes, 5 % (v/v) H2O2 5 minutes and 70 % (v/v) ethanol

5 minutes; Lenceana leucocephala: concentrated sulphuric acid 20 minutes and 70 %

(v/v) ethanol 5 minutes; Crotalaria juncea and Macroptilium atropurpureum:

concentrated sulphuric acid 10 minutes, 0.1 % (v/v) Tween 20 5 minuites and 5 % (v/v)

H2O2 5 minutes; Lablab purpureus and Tephrosia vogelii: concentrated sulphuric acid 10

minutes, 0.1 % (v/v) Tween 20 10 minuites and 5 % (v/v) H2O2 5 minutes; Pachyrhizus

tuberosus: concentrated sulphuric acid 25 minutes, 0.1 % (v/v) Tween 20 10 minuites and

5 % (v/v) H2O2 5 minutes. Nodulation tests were performed in Magenta jars as described

by Vipray and associates (Viprey et al., 1998). All plants were grown at a day

temperature of 28 °C, a night temperature of 18 °C, and a photoperiod of 16 h. Each plant

was inoculated with 107 bacteria and harvested five to seven weeks after inoculation.

41

Table 2. Strains and plasmids used in this study

Strain Relevant characteristics Reference

Escherichia coli DH5α supE44 ∆lacY169 ( 80lacZ∆M15) hsdR17 recA1 endA1 gyrA96 thi-1

relA1 BRL, Bethesdda, MD, U.S.A.

Tuner (DE3) pLacI F−ompT hsdSB (rB−mB

−) gal dcm lacY1 (DE3) Novagen

Rhizobium strains

NGR234 Broad host-range bacterium isolated from nodules of Lablab purpureus, Rifr

(Lewin et al., 1990)

NGRΩnodD1 NGR234 derivative containing an Ω insertion in the BamHI site of nodD1, Rifr Spr

(Relić et al., 1993)

NGRΩnodD2 NGR234 derivative containing an Ω insertion in the BamHI site of nodD2, Rifr Knr

(Fellay et al., 1995)

NGR∆ttsI NGR234 derivative with ttsI deleted, Rifr (Wassem et al., 2008)

NGR∆rmlB-wbgA NGR234 derivative in which 3.7 kb EcoRV fragment containing rmlB-wbgA was replaced by an Ω cassette, Rifr Knr

(Broughton et al., 2006)

NGRΩnodVW NGR234 derivative containing an Ω insertion in nodVW, Rifr Spr This work

NGRΩnodD1ΩnodVW NGRΩnodD1 derivative containing an Ω insertion in nodVW , Rifr Spr

Knr This work

NGRΩnodD2ΩnodVW NGRΩnodD2 derivative containing an Ω insertion in nodVW , Rifr Spr Knr

This work

Plasmids

pBluescript II KS+ High copy number ColE1-based phagemid, Apr Straragene, La Jolla,CA

pJQ200SK Suicide vector used for directed mutagenesis, Gmr (Quandt and Hynes, 1993)

pJQ-ΩnodVW pJQ200SK derivative construct in which 3.7 kb fragment containing an Ω insertion in nodVW was inserted into the ApaI and SacI site, Spr Gmr

This work

pJQ-ΩnodVW pJQ200SK derivative construct in which 3.7 kb fragment containing an Ω insertion in nodVW was inserted into the ApaI and SacI site, Knr Gmr

This work

pttsI pLAFR-6 derivative containing a 1.5 kb fragment with the ttsI gene under the control of nod-box 18

(Marie et al., 2004)

pRK2013 Tra+ helper plasmid (Figurski and Helinski, 1979)

pMP220 IncP expression vector containing a promoterless lacZ gene, Tetr (Spaink et al., 1987)

pMP-NB8 NB8 cloned as a 0.3 kb KpnI-XbaI fragment in pMP220. (Fellay et al., 1998)

pMP-NB18 NB18 cloned in pMP220 as a 0.7 kb SacI-XhoI fragment. (Kobayashi et al., 2004)

pMP-NB19 NB19 cloned in pMP220 as a 0.6 kb XbaI-PstI fragment. (Kobayashi et al., 2004)

pMP-TB2 pMP220 derivative containing the TB2 upstream of lacZ. (Marie et al., 2004)

pMP-TB8 pMP220 derivative containing the TB8 upstream of lacZ. (Marie et al., 2004)

pMP-nodD1p Promoter of nodD1 cloned in pMP220 as a 0.7 kb PstI fragment. (Kobayashi et al., 2004)

pRAF115 Promoter of nodD2 cloned in pMP220 as a 1.3 kb PstI-BamHI fragment. (Fellay et al., 1998)

pGT-GFP Vector containing a promoter-less GFP reporter gene. (Miller et al., 2000)

pGT-nopB nopB cloned into pGT-GFP. (Le Quéré et al., 2008)

pGT-nopJ nopJ cloned into pGT-GFP. (Le Quéré et al., 2008)

pGT-fixF fixF cloned into pGT-GFP. (Le Quéré et al., 2008)

pGT-rkpL rkpL cloned into pGT-GFP. (Le Quéré et al., 2008)

pGT-rkpY rkpY cloned into pGT-GFP. (Le Quéré et al., 2008)

pGT-exoK exoK cloned into pGT-GFP. (Le Quéré et al., 2008)

pETBlue-2 E. coli expression vector, Apr Novagen

pETBlue-nodW nodW cloned into pETBlue-2, Apr (Maniatis, 1982)

42

Results and Discussion

Mutation of nodVW

To assess the function of nodV and nodW, a polar mutant was generated. Omega

interposons conferring resistance to spectinomycin or kanamycin were inserted into an

internal EcoRV site, 1520 bp downstream of the nodV start codon (Figure 5). Mutation of

nodVW was also introduced into the nodD1 (Relić et al., 1993) and nodD2 (Fellay et al.,

1995) mutant backgrounds to elucidate any effects of NodVW in the absence of the main

regulator NodD proteins.

EcoRV

1 kbnodVnodWngr156 ngr157 158

nodVnodWngr156 ngr157 158 Ω (spec )r

nodVnodWngr156 ngr157 158 Ω (kan )rNGR 1Ω ΩnodD nodVW

NGRΩnodVW

NGR 2Ω ΩnodD nodVW

NGR234

Figure 5. Mutagenesis of nodV – suicide plasmids containing an omega interposon conferring resistance to spectinomycin or kanamycin inserted into an internal EcoRV site of nodV were created. Triparental matings were used to transfer the resulting plasmid into NGR234, NGRΩnodD1 and NGRΩnodD2.

In the analysis of these mutants, initially genistein and daidzein were used as

flavonoid inducers. These flavonoids are known to induce NGR234 genes (Kobayashi et

al., 2004) and genistein is known to be a strong inducer of nodVW in B. japonicum.

43

Effects the nodVW mutant on Nop secretion

To test the requirement of NodVW for Nop secretion, secreted proteins were

purified from wild type strain and various mutants after 40 hours induction. Western blots

were performed on intracellular and extracellular proteins and probed with antibodies to

specific Nops (Figure 6). T3SS-dependent protein secretion was not abolished in

NGRΩnodVW. Unsurprisingly as Nop secretion is regulated by NodD1 (Marie et al.,

2003), both NGRΩnodD1 and NGRΩnodD1ΩnodVW did not show any Nop secretion.

Nop secretion still occurs in a nodD2 mutant and no significant differences were

observed using NGRΩnodD2ΩnodVW. Thus unlike B. japonicum, where a nodW mutant

blocks expression of ttsI and thus presumably the T3SS (Krause et al., 2002), NGR234

nodVW mutants did not effect secretion of either the T3SS pili subunit NopA or the

effector proteins, NopM, NopL and NopT. Thus NodVW in NGR234 do not have same

effects as their B. japonicum homologues on T3SS function.

NG

R23

4

NG

RΩ

nodV

W

NG

R1

ΩΩ

nodD

nodV

W

NG

R1

Ωno

dD

NG

R2

Ωno

dD

NG

R2

ΩΩ

nodD

nodV

W

D G D G D G D G D G D G

Intracellularprotein

Extracellularprotein

Anti-NopL

Anti-NopM

Anti-NopL

Anti-NopA

Figure 6. Nop secretion by various strains. All strains were grown in the presence of daidzein (D) or genistein (G). Secreted proteins were separated on 15 % SDS-PAGE, transferred to polyvinylideve difluoride (PVDF) membranes, and immuno-blotted using Nop antibodies.

44

Do NodVW of NGR234 affect surface polysaccharides?

In non-inducing conditions NGR234 produces abundant rough-LPS (rLPS,

lacking the O-chain), with only trace amounts of smooth-LPS (sLPS, containing O-chain)

(Gudlavalleti and Forsberg, 2003; Reuhs et al., 1998). Addition of flavonoid to wild-type

NGR234 cultures, however, results in the production of an unique rhamnose-rich O-

antigen containing LPS (Fraysse et al., 2002; Reuhs et al., 2005). Thus NGRΩnodVW

was also tested for the production of rhamnose-rich LPS, as in NGR234 TtsI also controls

rhamnose synthesis (Marie et al., 2004). Figure 7A demonstrates the requirement of TtsI

for the production of rhamnose-rich LPS. NGR234 and derivatives were grown in the

presence of daidzein, polysaccharides were isolated and separated by DOC-PAGE before

staining for LPS. The ttsI mutant does not produce the smeary band indicative of

rhamnose-rich LPS. Complementation of NGR∆ttsI restored synthesis of the rhamnan-

LPS after flavonoid induction. As a further control, it was shown that deletion of rmlB -

wbgA operon, which is under the control of TtsI and encodes enzymes responsible for the

production of rhamnose, also abolished rhamnan synthesis (Broughton et al., 2006).

The nodVW mutants were next tested with strains grown in the presence of

daidzein or genistein. DOC-PAGE revealed that the extracted LPS of the nodVW mutant

has no discernable differences to the wild-type strain LPS (Figure 7B). As expected since

NGRΩnodD1 cannot produce this symbiotically active form of sLPS, neither could

NGRΩnodD1ΩnodVW. NGRΩnodD2 still produces low quantities of rhamnose-rich LPS

(Broughton et al., 2006) but the amount is dramatically reduced compare to the wild-type

strain. In this study, it was difficult to see if NGRΩnodD2 produced the rhamnose-rich

LPS probably due to the amount of sample which were loaded. Regardless of this, there

was no difference observed between NGRΩnodD2 and NGRΩnodD2ΩnodVW. Thus

NodVW do not obviously alter the production of rhamnose-rich LPS in NGR234.

45

NG

R23

4

NG

RΩ

nodV

W

NG

R1

ΩΩ

nodD

nodV

W

NG

R1

Ωno

dD

NG

R2

Ωno

dD

NG

R2

ΩΩ

nodD

nodV

W

D G D G D G D G D G D G

A BA B

NG

R23

4

NG

R∆ t

tsI

NG

R∆ t

tsI

ttsI

p

NG

R∆ r

mlB

-wbg

A

Figure 7. DOC-PAGE of polysaccharides. Polysaccharides synthesized by NGR234 and various mutants after growth in the presence of flavonoid were extracted then separated on DOC-PAGE gel. The gels were specifically stained for LPS. A: Daidzein was used as inducer. The synthesis of the rhamnan components (indicated with black vertical line) were blocked in the ttsI and rmlB-wbgA mutants. Complementation of NGR∆ttsI was obtained by the introduction of pttsI into NGR∆ttsI which restored rhamnan synthesis. B: For inducer, daidzein (D) and genistein (G) were used. NGRΩnodVW did not change the polysaccharides profile of the wild-type strain NGR234. Rhamnan component of lipopolysaccharides is also NodD1-dependent, nodD1 and nodVW double mutant did not show any effect of NodVW compare to NGRΩnodD1. NGRΩnodD2 and NGRΩnodD2ΩnodVW showed similar polysaccharides profile of the wild-type strain NGR234.

Symbiotic phenotype of NGRΩΩΩΩnodVW

In B. japonicum, it was shown that NodVW is required for nodulation of V.

unguiculata, V. radiata and M. atropurpureum, which are all hosts of NGR234 as well.

To evaluate the effect of NodVW from NGR234 on symbiosis, the nodulation abilities of

strains NGR234, NGRΩnodVW, NGRΩnodD2 and NGRΩnodD2ΩnodVW were

inoculated onto differences legume species (Table 3). Besides these three legume plants,

legumes that respond negatively/positively to the NGR234 T3SS were also used; Lablab

purpureus, Pachyrhizus tuberosus and Crotalaria juncea. Leucaena leucocephala was

also tested as a ttsI mutant has an improved symbiotic efficiency (Viprey et al., 1998) and

46

thus if NodVW affects ttsI there might be a phenotype. NGRΩnodD1 and

NGRΩnodD1ΩnodVW were not tested as NGRΩnodD1 is unable to nodulate any plant

(Relić et al., 1993).

On all the plants tested, nodulation by NGR234 and NGRΩnodVW was similar,

suggestive that NodVW in NGR234 does not affect symbiosis with these plants. As

expected, NGRΩnodD2 had dramatic effects on the nodulation of many of the tested

legumes - nodulation was completely abolished on M. atropurpureum and L. purpureus.

On L. leucocephala, V. unguiculata and V. radiata, nodule numbers were increased,

decreased or similar compare to NGR234 respectively. NGRΩnodD2ΩnodVW showed

very similar phenotypes to NGRΩnodD2, however, suggesting that the

NGRΩnodD2ΩnodVW phenotypes were due to the absence of NodD2 and not NodVW.

Table 3. Symbiotic phenotype of NodVW on various legume plants.

NGR234 NGRΩnodVW NGRΩnodD2 NGRΩnodD2ΩnodVW

V. unguiculata 60 (+12.7; 4) 58.2 (+11.1; 6) 23.8 (+4.7; 4) 17.8 (+4.6; 6)

V. radiata 33 (+3.8; 4) 38 (+8.3; 5) 43 (+12; 4) 37.4 (+6.4; 5)

M. atropurpureum 20.1 (+4.8; 4) 17.2 (+5.8; 5) 0 (+0; 4) 0 (+0; 5)

L. purpureus 9.4 (+2; 6) 9.4 (+1.7; 9) 0 (+0; 4) 0 (+0; 6)

L. leucocephala 16.8 (+2.7; 3) 22 (+1.8; 5) 29.1 (+7.3; 4) 30.6 (+6.7; 5)

P. tuberosus 0.5 (+0.7; 4) 0.25 (+0.35; 4) - -

C. juncea Fix- Fix- - -

The mean numbers of nitrogen-fixing nodules per plant are listed. For each test, the standard error of mean and the total number of plants are shown in brackets. As a control, sterile water was used as inoculum. Independent experiments were performed at least two times.

47

Electrophoretic Mobility Shift Assays and DNase I footprinting

NodW contains a characteristic DNA-binding domain, and although NodW of B.

japonicum has not been shown to bind to any promoters, it is required for efficient nod-

gene expression. I investigated whether NodW of NGR234 could bind to promoters

containing nod-boxes and tts-boxes. A hexa-histidine tagged NodW protein was

overexpressed and purified from E. coli, see materials and methods. After IMAC-based

purification NodW eluted as the major polypeptide of approximately 27 kDa, which not

present in the un-induced sample (Figure 8A lane 10 and 11) but there were also some

contaminating proteins. Taking use of its presumed DNA-binding ability a subsequent

purification step using a heparin column was performed. This resulted in homogeneous

NodW protein (Figure 8 B lane 4) at 16 mM which was used in all the DNA binding

assays.

1 14131211109765432 801 14131211109765432 80

A B

Figure 8. Overexpression and purification of NodW. Individual fractions at different stages of the overexpression and IMAC purification were eluted and collected, then separated by 15 % SDS-PAGE gel and stained with Coomassie blue. The red arrow indicates a 27 kDa band whichis the predicted size of NodW. A: Eluted sample with IMAC column. Lane 0 indicates Molecular marker. Lane 1 to 14 samples indicates as bellow: 1, non-induction; 2, crude extract; 3, supernatant; 4, pellet; 5, non-eluted induction; 6 to 14 contained different concentrations of imidazole (0.2 mM to 500mM) in elution buffer. B: Washed and eluted sample with Heparin column. Lane 0 indicates Molecular marker. Lane 1 to 14 samples indicates as bellow: 1, NodW from figure A lane 10 and 11; 2, crude extruct; 3 to 11 contained different concentrations of NaCl (0 mM to 1.2 mM) in heparin buffer.

48

The ability of the purified NodW protein to bind to NB3, NB6, NB8, NB18, TB2

and TB8 was tested in electrophoretic mobility shift assays (EMSA). These regulatory

elements act at key stages of the flavonoid dependent regulatory cascade (Freiberg et al.,

1997; Kobayashi et al., 2004; Marie et al., 2004; Perret et al., 1999). In B. japonicum, it

was shown that NodW phosphorylation is essential for nod gene expression (Loh et al.,

1997). By purifying NodW from E. coli, i.e. in the absence of the NodV sensor, NodW is

probably not phosphorylated when used in these EMSA experiments. Thus carbonyl

phosphate was added in reaction mixture to phosphorylate NodW. Except NB18, NodW

bound only to a fragment of DNA containing NB18 (Figure 9, lanes 1-5) and did not bind

to NB3, NB6, NB8, TB2 or TB8 (data not shown). NB18 is in the promoter region of ttsI.

NodW bound to the NB18 fragment even in presence of an unlabeled nonspecific mixture

of oligonucleotide showing that NodW binds to NB18 specifically (Figure 9, lanes 6-10).

NodW bound to NB18 even in the absence of carbamoyl phosphate (Figure 9, compare

lanes 10 and 11). NodD1 and NodD2 purified proteins were used as positive controls, as

both proteins are known to bind to NB18 (R. Wassem, unpublished data).

DNase I footprinting experiments were performed to determine the binding region

of NodW to the NB18-containing fragment of DNA precisely. Although purified NodW

protein was used at different concentrations no obvious footprint could be seen (Figure

10 lane 3 to 6). This contrasts with the positive controls where; NodD1 (lanes 7 and 8)

and NodD2 (lanes 9 and 10) footprinted the NB18 region. NodD1 bound over the entire

fragment whereas NodD2 bound to NB18 specifically – as shown in the laboratory earlier

(R. Wassem, unpublished data). The results of the EMSA suggest that NodW binding to

the NB18 DNA fragment was relatively weak compared to the positive controls, possibly

it is too weak to see distinct footprinting.

49

NodW- + D1 D2

1 2 3 4 5 6 7 8 9 10

NodW NodW+dIdc

-ca

p

No

dD

1

No

dD2

No

dD

1+N

odD

21 2 3 4 5 6 7 8 9 10 11 12 13 14

Figure 9. Electrophoretic mobility shift assay of a DNA fragment containing NB18 with NodW. Lanes 1 to 11 contained different concentrations of the NodW protein (lane 1 to 5 and 6 to 10; 0, 3.2, 8, 16 and 32 nM: lane 11; 16 nM). NodW proteins were incubated with carbonyl phosphate (lane 1 to 10) and competitor oligonucleotide (lane 6 to 10). NodD1 and NodD2 were tested for positive control.

Figure 10. DNase I footprinting to determine the binding site on the NB18 DNA sequence with NodW. Lane 3 and 6 is contained different concentrations of the NodW protein (16, 32, 80 and 160 nM). NodD1 (lanes 7 and 8) and NodD2 (lanes 9 and 10) were tested for positive control. Lane 1 and 2 contain the free radiolabeled DNA without DNase I or with DNase I, respectively. Red bar indicates NB18 region.

50

Preliminary conclusions

A polar mutant of nodVW was constructed and its effects on protein secretion and

synthesis of the rhamnose-rich LPS assessed, however no effects were seen. Thus unlike

the situation in B. japonicum where NodVW are required for ttsI transcription, nodVW

seem dispensable for this role in NGR234. Furthermore nodulation tests did not show any

obvious effect of NodVW on host plants known to be effected by similar mutations in B.

japonicum.

It has not been shown that NodW of B. japonicum binds to DNA. Surprisingly,

EMSA showed that NodW of NGR234 appeared to specifically bind to the NB18-

containing ttsI promoter - although the exact binding site could not be determined. NodW

bound only to NB18, and not other NBs and TBs.

These two results seem to contradict each other, as although NodW appears to

bind to the ttsI promoter region, there does not appear to be any regulatory effect. It

should be noted, however, that quantification of Nop secretion or rhamnose-rich LPS

production is difficult. Thus I attempted transcriptional assays with the nodVW mutant of

NGR234 using reporter gene fusions to various NBs and TBs, to see if there were subtle

alterations in transcription patterns.

51

Transcriptional effects in the nodVW mutant

The EMSA data suggested that NodW might influence the transcription of the

NB18 containing promoter. Previous data has shown that NB18 is only slightly induced

after flavonoid induction (Kobayashi et al., 2004) and so TB2 and TB8 were also tested

as effects on NB18 might be amplified on these promoters, NB8 which controls nodABC

transcription was also included. For each regulatory element pre-exisiting β-galactosidase

fusions (Kobayashi et al., 2004; Wassem et al., 2008) were mobilized into the nodVW

mutant of NGR234. Using daidzein and genistein for the inducer, β-galactosidase

activities were measured at 1 h and 24 h after induction (Figure 11). Comparing these two

inducers, daidzein activated transcription of all the promoters more strongly than

genistein, but there were no major changes in the patterns of activation. Results of

Student T-tests (P ≤ 0.01) that compared induced activities at 1 h and 24 h in NGR234

and NGRΩnodVW transconjugants, confirmed that optimal activation of two tts-box-lacZ

fusions (TB2 and TB8) had significant differences (P ≤ 0.01, t-test) when using genistein

as inducer. β-galactosidase assays also showed that NB18 had a slightly decreased

promoter activity in nodVW mutant but there were no significant differences when

compared statistically with wild-type. It had been shown that 18 of the 19 nod-boxes

promoter activities were abolished completely in absence of nodD1 (Kobayashi et al.,

2004). In this study, NGRΩnodD1ΩnodVW was also tested the transcriptional activity of

nod-boxes (NB8 and NB18) and tts-boxes (TB2 and TB8). There was no induction of

these promoters in NGRΩnodD1ΩnodVW, nor NGRΩnodD1 (data did not show).

52

TB2 TB8

0

1

2

3

4

5

6

7

NGR234 NGRΩnodVW NGR234 NGRΩ nodVW

1h 24h

NB8

0

1

2

3

4

NGR234 NGRΩ nodVW NGR234 NGRΩ nodVW

1h 24h

pMP220 NB18

0

1

2

3

4

NGR234 NGRΩnodVW NGR234 NGRΩnodVW

1h 24h

0

1

2

3

4

5

6

NGR234 NGRΩ nodVW NGR234 NGRΩ nodVW

1h 24h

0

1

2

3

4

5

6

NGR234 NGRΩ nodVW NGR234 NGRΩnodVW

1h 24h

daidzein

genistein

non-induced

TB2 TB8

Figure 11. Activity of promoters in NGR234 and NGRΩnodVW. β-galactosidase assays were used to assess the effect of NodVW on nod-boxes (NB8 and NB18) and tts-boxes (TB2 and TB8) promoter. Activity of the vector pMP220 devoid of an insert is reported. Assays were performed 1 h and 24 h in presence of inducer daidzein, genistein or absence of inducer. The values (x 10-3 Miller’s units) reported represent the means of three independent experiments and errors bars are shown on each time points.

These β-galactosidase assays showed that NodVW from NGR234 are possibly

required for optimal induction of NB18 and thus tts-boxes. At 24 h of induction, NB18,

TB2 and TB8 had slightly reduced the β-galactosidase activity levels in the nodVW

mutant. This result links with the earlier EMSA experiments indicating NodW might

effect ttsI (via NB18) expression and with the known NodVW function from B.

japonicum. Furthermore, these results also indicate why Nop secretion and rhamnose-rich

LPS synthesis was still observed, as promoter activities are reduced but not abolished.

Surprisingly, NodVW also had a significant effect on NB8 expression, after 24

hours of genistein induction the activity of NB8 was reduced in the nodVW mutant. This

unexpected data suggests that NodVW is not only acting on NB18 but on other promoters,

however NodW did not bind to NB8. Thus NodVW activation of NB8 may not be direct,

and instead is a result of NodVW affecting other parts of the regulatory cascade. EMSA

showed that NodW bound to the promoter region of ttsI, containing NB18, but the exact

site could not be determined. Also in this region, is the promoter region of nodD2,

53

containing a syr-box (Figure 12A). It is possibly that NodW binds elsewhere in this

region, explaining why NodW binding with NB18 was weak, and thus could affect

nodD2 promoter activity (Figure 12B). NodD2 is known to be involved in the repression

of Nod-factor synthesis, which would lead to a decrease in NB8 expression (Fellay et al.,

1998). If NodW was a repressor of nodD2, in the nodVW mutant, nodD2 expression

would be increased causing a stronger repression of nodD1. As a consequence of the

subsequent lower levels of NodD1, the activity nod-boxes (NB8 and NB18) and hence

the tts-boxes (TB2 and TB8) would be decreased, as seen in the previous β-galactosidase

assays (Figure 11).

NodV

NodW

TtsINB18NodD2 SB2

A B

nodD2 ttsI

NB18syr-box

1 kb

NodD1SyrM2

Figure 12. A: Region of ttsI and nodD2. The triangles indicate conserved promoter NB18 and syr-box for ttsI and nodD2, respectively. B: Model of the action of NodVW at the nodD2-ttsI promoter region. NodW has been shown that bind to NB18 using EMSA. If NodW bind to NB18 promoter region, is there any effect on nodD2 promoter activity?

To confirm this hypothesis, the expression of nodD2 should be assayed in the

nodVW mutant. Furthermore activities should be measured at longer time points, as both

NodD2 and TtsI act later time points after induction and thus the exact regulatory effects

may only be clear after 24 hours of induction. β-galactosidase activities of the nodD2

promoter (Fellay et al., 1998) and NB8, NB18, TB2 & TB8 were tested every 24 h up to

96 h using genistein for inducer in NGR234 and the nodVW mutant (Figure 13).

Expression of nodD2 promoter peaked at 48 h, agreeing with a previously study (Fellay

54

et al., 1998). In the nodVW mutant the nodD2 expression pattern was the same as wild-

type, however the expression levels were constantly halved. Thus in contradiction to our

earlier model, lack of NodVW actually led reduction of nodD2 promoter transcription

level and thus NodVW apparently activates NodD2.

NGR234 induced NGR inducedΩnodVW

NGR234 non-induced NGR non-inducedΩnodVW

pMP220

0

400

800

1200

1600

1h 24h 48h 72h 96h

pnodD2

0

400

800

1200

1600

2000

1h 24h 48h 72h 96h

NB18

0

400

800

1200

1600

1h 24h 48h 72h 96h

TB2

0

1000

2000

3000

4000

5000

6000

1h 24h 48h 72h 96h

TB8

0

2000

4000

6000

8000

10000

12000

1h 24h 48h 72h 96h

NB8

0

2000

4000

6000

8000

10000

12000

1h 24h 48h 72h 96h

Figure 13. Activity of the NB, TB and nodD2 promoter in NGR234 and NGRΩnodVW. β-galactosidase assays were used to assess the effects of nodVW on various promoters. Activity of the vector pMP220 devoid of an insert is reported. Assays were performed 1 h to 96 h in presence of inducer genistein or in absence of inducer. The values reported represent the means of three independent experiments and errors bars are shown on each time points.

NB18 promoter activity was repressed after peaking at 24 h in NGR234.

Expression of NB18 was variable in the nodVW mutant, initially lower than wild-type

levels, but after 40 hours (and all subsequent time points) it was higher than wild type.

Both expression patterns show that NB18 is initially induced until 24 h and then

repressed, possibly due to NodD2, as normally NodD2 should start to repress NodD1

(and thus NB18/TtsI) at later time points. In the nodVW mutant, the expression level of

nodD2 is lower than wild type, thus with less NodD2 NB18 would not be suppressed as

much as in the wild type strain – agreeing with the data obtained.

55

NB8, TB2 and (except at 96 hr) TB8 showed similar expression patterns - lower

in the nodVW mutant relative to wild-type at all time points after induction. Based upon

the expression of NB18 (and thus TtsI levels) after 48 h the expression levels of the TBs

should be higher than wild-type. Additionally, a transcriptional effect of NodVW on TBs

could be observed at early time point (24 h) suggesting that NodW may also activate ttsI

directly.

Although NB8 expression was unchanged in the nodVW mutant or wild-type at 24

h, at later time points its expression was consistently lower in the nodVW mutant. NB8 is

thought to be under the direct NodD1 mutant (Kobayashi et al., 2004). The differences of

activity of NB8 between wild type and nodVW mutant were clear and suggested that the

NodW regulation could be at the NodD1 level. Does NodVW effect nodD1 expression?

To answer to this question, the expression levels of the promoter region of NodD1 were

determined in the nodVW mutant. Additionally, the promoter region of syrM2 (NB19)

was also tested as SyrM2 is another regulator in the cascade. Testing NB19 will also

determine whether NodW acts on nodD2 expression directly, or through alterations to

syrM2 expression. Although EMSAs will be necessary to see whether NodW actually

binds at the promoter region of nodD2. A model of the latest NodW sites of action to be

tested is shown in Figure 14.

56

Flavonoids

Tim

e

NodV

NodW

NB3

NodD1

SyrM2

NodD2

NB19

SB2

Figure 14. Model of the hypothesis regulatory cascade of NodVW. nodVW mutants have a reduced activity of the nodD2 promoter. But at what point in the cascade does NodW function to alter expression of the nodD2 promoter?

NB19 and nodD1 promoter activities were tested every 24 h up to 96 h using

genistein for inducer in the nodVW mutant (Figure 15). Formerly, the nodD1 promoter

was shown to be expressed at a very low level, even in absence of flavonoid condition

(Kobayashi et al., 2004). In this study, the promoter region of nodD1 showed that

promoter activity was further repressed after flavonoid induction and this repression

pattern occurred in both, wild-type and NGRΩnodVW. Strikingly however in the nodVW

mutant, the nodD1 promoter at the earliest time point assayed had very high expression

levels. The presence of flavonoids led to a reduction in nodD1 expression in the nodVW

mutant, although in the absence of flavonoid expression remained relatively high. NodD1

is main regulator which controls many symbiosis factors. Therefore its expression is most

likely preserved carefully at a low level in the absence of flavonoids, even in the presence

of flavonoids the activity of nodD1 promoter is quickly repressed by NodD2 by feedback

inhibition (Fellay et al., 1998). The lack of NodVW leads to a higher level of expression

of NodD1 which after flavonoid induction may over-stimulate the regulatory cascade

57

leading to confusing transcriptional data observed. Thus NodVW is mostly likely a

repressor system for NodD1 in the absence of flavonoids.

NGRΩnodVW also had a strong effect on the expression of NB19. Compared to

NGR234, NB19 expression level is much higher, for example 12 times, 6 times at 24h,

48h respectively. Without flavonoid induction, NGRΩnodVW also had a remarkable

effect, in wild-type strain, NB19 strongly is repressed in the absence of flavonoids,

however NB19 in NGRΩnodVW was expressed at levels 20 times higher than wild-type.

It was shown that NodD protein binds to conserved 49 bp motifs (nod-boxes) even in the

absence of flavonoid (Feng et al., 2003) nevertheless flavonoids are required for the

activation of nod-loci (Fisher and Long, 1993). As the effects of NodVW were also

observed non-induced condition, this suggests that NodW is also a suppressor of NB19

in the absence of flavonoids.

pnodD1

0

400

800

1200

1h 24h 48h 72h 96h

NB19

0

400

800

1200

1600

2000

1h 24h 48h 72h 96h

NGR234 induced

NGR inducedΩnodVW

NGR234 non-induced

NGR non-inducedΩnodVW

Figure 15. Activity of the NB19 and nodD1 promoters in NGR234 and NGRΩnodVW. β-galactosidase assays were used to assess the effect of nodVW on various promoter. Assays were performed 1 h to 96 h in presence of inducer genistein or in absence of inducer. The values reported represent the means of three independent experiments and errors bars are shown on each time points.

The NodVW system may thus act to maintain low expression of the main

activators NodD1 and SyrM2 of the flavonoid inducible cascade. NodV is most likely

capable of sensing environmental stimuli, quite possibly as a sensor of the amount or

structure of flavonoids present. Thus at sub-optimal concentrations of flavonoid, NodVW

may repress the main regulators to restrain the symbiosis system. In nature, plant-

produced flavonoids are the first signals for symbiosis between NGR234 and legume host

58

plants. It is known that is NGR234 induced by flavonoids upto 10-8 M and the expression

levels of symbiotic factor increases up to a flavonoid concentration of 10-5 M (Kobayashi,

unpublished). In the laboratory we use the optimal concentration for NGR234 which is at

10-7 M. In the rhizosphere flavonoid concentrations could be low until NGR234 is in

close proximity to the plant root. To prevent premature release of symbiotic signals

(energetically wasteful) NodVW might repress symbiosis regulator cascade until precise

condition to activate. To examine this possibility, the expression levels of various

symbiotic factors will be compared in NGRΩnodVW and wild-type strain with various

concentrations of flavonoids.

Promoter activity level with GFP

For the β-galactosidase transcriptional assays, only daidzein and genistein were

used for inducer, and a few promoters were tested. As the effects of NodVW might be at

the global level of symbiotic signal expression and might depend on the nature of the

flavonoid used as the inducer. The study of promoter activities was expanded, using high-

throughput techniques developed in our laboratory (Le Quéré et al., 2008). Promoters

were fused to GFP and expression levels tested in 96 wells plate by measuring GFP

fluorescence levels (see materials and methods). This technique enables to test many

promoter activity as well as difference inducer relatively quickly with few manipulative

steps.

The effect of the nodVW mutant on seven differences promoters were tested by

comparing the fluorescence level of GFP (Figure 16). Various inducers; apigenin,

daidzein, genistein, kaempfenrol and naringenin were selected for the experiment known

to trigger activation of nod-boxes (Kobayashi et al., 2004). The final fluorescence levels

of GFP were calculated as described in materials and methods to compare each

independent experiment. Promoters of nopB, nopJ and rmlB are part of the TtsI-

dependent regulon (shown in chapter 1: named TB2, TB4 and TB8 respectively), are

inducible with all five flavonoids (Figure 16 A to C). All the flavonoids showed a

statistically significant difference on nopB promoter expression between wild-type strain

and NGRΩnodVW at 42 h to 72 h. The expression of nopB promoter in NGRΩnodVW

59

was decreased about 40 % to NGR234 with four different flavonoids. The reduction of

nopJ promoter expression in NGRΩnodVW was remarkable with all inducers. The

expression level of nopJ promoter in NGRΩnodVW were about 26 % (e.g. with

naringenin at 42 h) to 65 % (e.g. with daidzein at 96 h) of nopJ promoter in NGR234 and

these were showed a statistically significant difference. The nopB promoter (TB8) was

also analyzed its activity with β-galactosidase assay and its expression patterns were in

agreement using both methods with genistein for inducer.

The promoter of fixF (NB6) has been shown to be inducible by daidzein and

highly dependent on NodD2 (Kobayashi et al., 2004). fixF (NB6) promoter is involved in

rhamnan synthesis. The induction of NB6 was delayed and only observed at 24 h after

induction. In this study, promoter of fixF was inducible by all five flavonoids (Figure 16

C). No major effect of NodVW was observed.

The expression of exoK, rkpL and rkpY promoters were not induced by flavonoids,

and were often reduced in comparison to absence of inducer (Figure 16 D to F). Activity

of exoK promoter in NGRΩnodVW was slightly decreased in presence or absence of

inducer compare to NGR234, but there were no significant differences between two

strains. Any effect of NodVW on rkpL and rkpY promoter expressions were not revealed

clearly, only in absence of inducer at 72 h and 96 h, rkpL promoter activity was reduced

in NGRΩnodVW.

Using GFP to measure promoter activity had many advantages; assay time was

brief as GFP is measured directly, using a 96 well plate many samples can be tested at the

same time or in different condition (e.g. inducer, promoter). The major drawback of this

method is that each well is too small for cell growth (the medium is 2 ml maximum), the

cell lysis had probably started at longer time points after induction.

60

Non-induced

0

10

20

30

40

50

60Apigenin

GenisteinDaidzein

0

10

20

30

40

50

60

Kaempferol

0

10

20

30

40

50

60

T0 T24 T42 T48 T72 T96

Naringenin

T0 T24 T42 T48 T72 T96

Non-induced

0

10

20

30

40

50

60

70

80

Genistein

Apigenin

Kaempferol

0

10

20

30

40

50

60

70

80

T0 T24 T42 T48 T72 T96

Daidzein

0

10

20

30

40

50

60

70

80

Naringenin

T0 T24 T42 T48 T72 T96

A. promoternopB B. promoternopJ

Non-induced

0

10

20

30

40

50

60

Genistein

Apigenin

Kaempferol

0

10

20

30

40

50

60

T0 T24 T42 T48 T72 T96

Daidzein

0

10

20

30

40

50

60

Naringenin

T0 T24 T42 T48 T72 T96

C. promoterfixF D. promoter exoK

Non-induced

0

2

4

6

8

Genistein

Apigenin

Kaempferol

0

2

4

6

8

T0 T24 T42 T48 T72 T96

Daidzein

0

2

4

6

8

Naringenin

T0 T24 T42 T48 T72 T96

61

Non-induced

0

2

4

6

8

Genistein

Apigenin

Kaempferol

0

2

4

6

8

T0 T24 T42 T48 T72 T96

Daidzein

0

2

4

6

8

Naringenin

T0 T24 T42 T48 T72 T96

ApigeninNon-induced

0

2

4

6

8

GenisteinDaidzein

0

2

4

6

8

Kaempferol

0

2

4

6

8

T0 T24 T42 T48 T72 T96

Naringenin

T0 T24 T42 T48 T72 T96

E. promoter rkpL F. promoter rkpY

NGR234 NGRΩ nodVW

Figure 16. Activity of the 6 difference promoters in NGR234 and NGRΩnodVW. A to F represent the level of GFP fluorescence (x 10-3) which were measured every 24 h up to 96h. Each promoter was assessed in presence of inducer apigenin, daidzein, genistein, kaempferol or naringenin or in absence of inducer. The values reported represent the means of five independent experiments and errors bars are shown on each time points. The activities of promoters were calculated as described in materials and methods.

RT-PCR

Quantitative RT-PCR was used to measure the mRNA expression levels of nodV

in NGR234 as well as NGRΩnodVW in the presence of genistein. For the relative

expression ratio (R) of targets genes were resulted in comparison to the 16S ribosomal

RNA reference gene. Furthermore, two genes rpsL which encodes house keeping gene

and nopB which encodes secretion protein depend on flavonoid and T3SS were used for

controls. RT-PCR showed that nodV expression was low and not induced by genistein

and as expected were completely missing in the mutant. nodV expression levels were

62

similar after 6 h and 24 h flavonoid induction in NGR234 whereas nopB expression

increased after 24 h (Figure 17). Surprisingly nopB expression levels were not lower in

the nodVW mutant relative to wild-type, which is inconsistent with the β-galactosidase

and GFP transcriptional data described before, which showed significant difference on

nopB promoter activity between NGR234 and NGRΩnodVW at 24 h. This is most likely

due to the different experimental techniques used, i.e. measuring exact RNA levels here

compared to the activity of reporter enzymes earlier. Further RT-PCR will be performed

at different time points to verify these results.

0.10

1.00

10.00

100.00

1000.00

NGR234 NGR234

6h 24h

rpsL nopB nodV

NGRΩnodVW NGRΩnodVW

6h 24h

rpsL nopB nodV

log1

0

log1

0

Figure 17. Quantitative RT-PCR analysis of gene expression at different time points. Expression of rpsL, nopB, and nodV were analyzed at 6 h and 24 h after flavonoid induction. To calculate relative transcript level, 16S ribosomal RNA reference gene was used.

63

Conclusions

To investigate the function of NodV and NodW in NGR234, a nodV polar mutant

was generated, which had no effect on Nop secretion, rhamnose-rich LPS production, or

any phenotype on various host plants. NodW was shown to bind to a DNA fragment

containing NB18, suggesting it might influence ttsI transcription. The transcriptional

activity of several promoters were tested and compared. The nodVW mutants had slightly

or significantly reduced expression levels of NBs (NB8 and NB18) and TBs (TB2 and

TB8) promoter activity after flavonoid induction. This observation coincides with the

profile of secretion protein, polysaccharides and nodulation phenotype. NodVW effects

but does not abolish completely, the expression of these promoters. Further promoter

assays could not reveal the exact regulatory mechanism of NodVW but there was a

distinct effect of NodVW on the promoter regions of nodD1 and syrM2. The strongest

effects of NodVW on these promoters were observed without flavonoid induction. It

suggests that the function of NodVW from NGR234 is most probably as repressors of

nodD1 and syrM2 in absence of flavonoids to maintain low expression level of NodD1

and the symbiosis regulatory cascade. Then once sufficient levels of inducer are found,

repression is lost as nodD1 is activated, leading to symbiotic signal production. As well

as the NodD1 activation of nodD2 leading to NodD2 negative feedback of nodD1

expression, NodVW also appears to enhance activation of nodD2 in the presence of

flavonoids to further repress nodD1, although the mechanism for this is unknown. A

working hypothesis for NodVW functions is summarised in figure 18.

64

Flavonoids

Tim

e

NodV

NodW

NB3

NodD1

SyrM2

NodD2

NB19

SB2

Figure 18. Model of the function of NodVW in the regulatory cascade of NGR234. NodVW is repressor of nodD1 and syrM2 in the absence of flavonoids to maintain low levels of NodD1 and thus the symbiosis regulatory cascade. Then once flavonoids stimulate and promotes symbiosis cascade, NodVW also activates NodD2 which is repressor of nodD1.

Consequently, in the absence of NodVW the timing and induction levels of

symbiosis regulatory cascade was altered. Particularly the high levels of nodD1

expression most likely led to some of the unexplainable β-galactosidase assay data when

trying to determine the mechanism of NodVW function.

Further studies will determine whether NodW functions directly or indirectly on

the promoters of nodD1, syrM2 and nodD2. It will be interesting to test the ability of

NodW to bind to these promoter regions. Despite not having any clear symbiotic

phenotype, the nodVW mutant does have major effects on the induction of various

symbiotic signals. Most probably all the necessary signals can be synthesised but their

temporal regulation could be affected. It is possible that as a consequence the nodVW

mutant is less competitive than the wild-type strains and nodulation ability could be

tested accordingly.

65

For the transcriptional assay, two reporter genes (lacZ and gfp) were used in this

study. β-galactosidase is frequently used as a reporter for studying gene expression. The

enzymatic assays (lacZ) offer more robust analysis of gene activity than GFP. They are

very sensitive but they provide limited temporal or quantitative information due to the

perdurance of beta-galactosidase (Hand and Silhavy, 2000). GFP is particularly useful for

rapid quantification of gene expression in real time and in single cells (Bongaerts et al.,

2002). Quantification of GFP requires more technological investment (e.g. a flow

cytometer, fluorometer, or a fluorescence microscope) and the sensitivity can be a

problem, particularly for weak promoters, the low expression levels of GFP are more

difficult to detect. On the other hand, when the GFP fluorescence signal is detectable, it

can be measured rapidly and accurately in bulk cultures or individual cells in real time.

Initial comparisons on a few NGR234 promoters suggested both reporter genes gave

corresponding expression patterns. The use of GFP in a high-throughput strategy will

offer advantages in the number of promoters that can be assayed in a single experiment.

66

Chapter 3: Characterisation of NopM and the role in symbiosis of NGR234 effector

proteins

Introduction

On pNGR234a, there are three ORFs, nopJ, nopM and nopT (formerly y4lO, y4fR

and y4zC respectively) which have homology to virulence factors secreted in a T3SS-

dependent manner by plant and animal pathogens (Freiberg et al., 1997; Marie et al.,

2001). Thus NopJ, NopM, and NopT are good candidates to be effector proteins secreted

by the T3SS of NGR234 into legumes. As discussed in the previous chapter, each gene

encoding these putative effectors has a tts-box in their promoter (Figure 1). Furthermore

nopJ, nopM and nopT were all shown to be inducible after flavonoid induction, in a TtsI-

dependent manner (Wassem et al., 2008).

y4fR - nopM

TB1

y4fR

y4lO - nopJ

y4lO

fl5

TB4

y4zC - nopT

y4zC

TB112 kb

Figure 1. Genes encoding putative Nops controlled by tts-boxes. Three tts-boxes (TB) TB1, TB4 and TB11 were identified in the promoter regions of genes encoding the putative effector proteins; NopM, NopJ and NopT respectively. tts-boxes are represented by black arrows.

NopJ has homology to the YopJ (Yersinia outer protein J) family of effectors

found in both animal and plant pathogenic bacteria (Table 1). Members of this family

possess a conserved C55 peptidase domain of the CE clan of cysteine proteases (Hotson

et al., 2003; Hotson and Mudgett, 2004) and some have been shown to act on small-

ubiquitin-like modifier (SUMO)-conjugated proteins (Orth, 2002; Roden et al., 2004).

Site-specific mutation of conserved catalytic residues in the protease domain blocks this

activity. Within host cells YopJ interferes with mitogen-activated protein kinase (MAPK)

and nuclear factor kappa B (NF-κB) signalling pathways, and the conserved catalytic

residues are required for this function, although no evidence of any proteolytic activity on

members of these signalling pathways has been obtained (Aepfelbacher et al., 1999; Orth,

2002; Ruckdeschel and Richter, 2002). Recent studies now show that YopJ acts as an

67

acetyltransferase, acetylating serine and threonine residues of MAPKs that are normally

sites of phosphorylation (Mittal et al., 2006; Mukherjee et al., 2006), consequently

inactivating their function. The phytopathogen, Xanthomonas campestris pv. vesicatoria

is known to possess four YopJ-like proteins, AvrXv4, AvrBsT, AvrRxv, and XopJ.

AvrXv4 has been shown to function as a SUMO protease in planta during Xanthomonas-

plant interaction (Roden et al., 2004).

Table 1. Number of amino acids and predicted size of the representative member of YopJ family. Homology to NopJ (similarity and identity) was identified by BLAST-P search. The number of amino acids in a parenthesis indicates over how many amino acids homology existed to NopJ.

Amino acid Size Similarity to NopJ Identity to NopJ

NopJ Rhizobium sp. NGR234

260 aa 29.1 kDa - -

XopJ Xanthomonas campestris pv. vesicatoria

373 aa 40.6 kDa 67 % (215 aa) 47 %

AvrRxv Xanthomonas campestris pv. vesicatoria

373 aa 42 kDa 50 % (205 aa) 37 %

AvrXv4 Xanthomonas campestris pv. vesicatoria

359 aa 39.7 kDa 51 % (253 aa) 36 %

PopP1 Ralstonia solanacearum

368 aa 41 kDa 48 % (252 aa) 35 %

PopP2 Ralstonia solanacearum

488 aa 53 kDa 44 % (170 aa) 28 %

PopP3 Ralstonia solanacearum

378 aa 41.4 kDa 40 % (184 aa) 28 %

YopJ Yersinia pestis biovar Microtus

288 aa 32.5 kDa 45 % (146 aa) 21 %

YopP Yersinia enterocolitica

288 aa 32.3 kDa 45 % (145 aa) 20 %

The YopT family of effectors which include NopT are found in animal and plant

pathogens, as well as two rhizobia (Table 2). Members of the YopT family are also

cysteine (C58) proteases (Shao et al., 2002). They all possess an invariant C/H/D

catalytic core thought to be absolutely required for enzymatic activity. In host cells,

YopT cleaves Rho family GTPases at a specific site in the amino-terminal, releasing it

from the plasma membrane and inactivating it (Shao et al., 2003).

AvrPphB of the phytopathogen Pseudomonas syringae belongs to YopT family,

and AvrPphB is also an auto-protease (Puri et al., 1997; Shao et al., 2002). This cleavage

reveals a myristoylation site at its amino-terminus, leading to this modification within

plant cells causing it to be trafficked to the plant plasma membrane (Nimchuk et al.,

68

2000). The catalytic triad C/H/D in AvrPphB is required for autoproteolytic cleavage and

AvrPphB-induced hypersensitive response (HR) in Arabidopsis plants (Shao et al., 2002;

Warren et al., 1999).

Table 2. Number of amino acids and predicted size of the representative member of YopT family. Homology to NopT (similarity and identity) was identified by BLAST-P search. The number of amino acids in a parenthesis indicates over how many amino acids homology existed to NopT. ns: not significant. At primary amino acids sequence, the homology between NopT and YopT is low as described by Shao et al. (Shao et al., 2002). However NopT and YopT share invariant C/H/D catalytic core as described in chapter 4 Figure 2.

Amino acid size Similarity to

NopT Identity to NopT

NopT Rhizobium sp. NGR234

261aa 28.3 kDa - -

blr2140 Bradyrhizobium japonicum USDA 110

271 aa 29.2 kDa 71 % (270 aa) 58 %

blr2058 Bradyrhizobium japonicum USDA 110

298 aa 32.3 kDa 56 % (205 aa) 39 %

AvrPphB Pseudomonas syringae pv. phaseolicola

267 aa 28.7 kDa 41 % (166 aa) 27 %

YopT Yersinia pestis biovar Microtus

322 aa 36.3 kDa ns ns

Conserved domain searches show that NopM homologues have Leucine-Rich-

Repeats (LRR) (Table 3 and Figure 2). LRRs have been found in the primary structures

of a large number of proteins with diverse functions and cellular locations in a variety of

organisms and are mainly found in cell adhesion factors, hormone receptors, and enzyme

inhibitors (Kobe and Deisenhofer, 1994; Kobe and Kajava, 2001). Most of these proteins

are involved in protein-protein interactions.

Table 3. Number of amino acids and predicted size of the representative member of Leucine-Rich-Repeat family. Homology to NopM (similarity and identity) was identified by BLAST-P search. The number of amino acids in a parenthesis indicates over how many amino acids homology existed to NopM.

Amino acid size Similarity to

NopM Identity to

NopM NopM Rhizobium sp. NGR234

546 aa 60.5 kDa - -

blr1904 Bradyrhizobium japonicum USDA 110

585 aa 64.2 kDa 65 % (484 aa) 53 %

IpaH9.8 Shigella flexneri

545 aa 62 kDa 53 % (510 aa) 36 %

SspH1 Salmonella typhimurium

700 aa 28 kDa 54 % (440 aa) 39 %

SspH2 Salmonella typhimurium

788 aa 87.2 kDa 55 % (434 aa) 41 %

YopM Yersinia pestis biovar Microtus

409 aa 46.2 kDa 50 % (136 aa) 38 %

69

LRR6

LRR6

LRR15

LRR8

LRR12

YopM

NopM

IpaH9.8

SspH1

SspH2

Figure 2. Graphical representation of NopM and its homologues, the gray shaded boxes are the LRR domains (with the number of repeats indicated).

NopM homologues from animal pathogens are often detectable in the host cell

nuclei. SspH1 and SspH2 from S. typhimurium are both translocated into host cells (Miao

et al., 1999) and SspH1 is detectable in the nucleus (Haraga and Miller, 2003). The IpaH

(IpaH9.8, IpaH7.8, and IpaH4.5) proteins can be secreted by Shigella flexneri 2a (YSH6000)

via the T3SS, and immunofluorescence microscopy showed that IpaH9.8 is transported

into the nucleus in the host cells (Toyotome et al., 2001). Finally although YopM

function in host cells is not understood, it is necessary for full virulence of Y. pestis and

Y. enterocolitica as demonstrated by its mutagenesis (Leung et al., 1990; Mulder et al.,

1989). YopM is transported to the nuclei of mammalian cells after T3SS-injection, where

it is thought to modulate host defence responses via a vesicle-associated pathway

(Skrzypek et al., 2003). Thus these proteins all possess LRR, are secreted via T3SS and

are localized into host nucleus although they all lack classical nuclear localization signals

(NLS). Systematic deletions identified that the first three LRRs of YopM and the 32 C-

terminal residues of YopM (YopMC-ter) act as NLSs to target YopM to yeast nuclei

(Benabdillah et al., 2004).

Recently two other studies have shown alternative biochemical functions of two

members of this family of proteins. YopM interacts with mammalian kinases, forming a

complex with protein kinases C-like 2 (PRK2) and a ribosomal protein S6 kinase (RSK1).

The YopM-kinase complexes results in the activation of both PRK2 and RSK1, but the

consequence of their activation by YopM is not clear (McDonald et al., 2003). IpaH9.8

70

acts as an ubiqutin ligase, and a critical residue was identified that is conserved in all

family members, including NopM (Rohde et al., 2007).

Recently a proteomics-based study identified fragments of a NopM-like protein

secreted in a T3SS-dependent fashion by Sinorhizobium fredii HH103 after induction by

plant-derived flavonoids (Rodrigues et al., 2007). Peptides sequenced from this NopM-

like protein show excellent homology to NopM from NGR234. However the role of

NopM in the HH103 symbiotic interaction still remains to be determined. The genomic

sequence of B. japonicum USDA110 shows that it also possesses ORFs encoding

homologues of NopM. It is not known whether they also influence the USDA110

symbiosis. Interestingly no homologues are found in any of the sequenced

phytopathogens, implying that to date the only plant-interacting bacteria with such

putative effector proteins are rhizobia. The work in this chapter describes the

investigation of the T3SS-secretion of NopM, its role in the NGR234 symbiosis and then

the interaction of NopM and other effector Nops in NGR234.

71

Materials and methods

Microbiological techniques

Strains used in his work are listed in table 4. Escherichia coli strains were grown

in Luria-Bertani (LB) media at 37 °C (Sambrook et al., 1989). Rhizobium strains were

grown at 27 °C in either complete (TY) (Beringer, 1974) media or minimal media

containing succinate as the carbon source (RMS) (Broughton et al., 1986). Antibiotics

were added to the media at the following final concentrations; ampicillin (Ap), 50 µg ml-

1; gentamycin (Gm), 10 µg ml-1 (for E. coli), 20 µg ml-1 (for Rhizobium); kanamycin (Kn),

50 µg ml-1; rifampicin (Rif), 50 µg ml-1 and spectinomycin (Sp), 50 µg ml-1.

Mutagenesis of nopM

To obtain NGR∆nopM, cosmid pXBS23 (Perret et al., 1991) was digested with

PstI, and the 3.1 kb fragment containing nopM purified and sub-cloned into pBluescript

KS+. Then, part of insert was excised using BamHI and HindIII and cloned into pUC18

(Maniatis, 1982). This plasmid was then digested with NheI and ClaI to excise a 1-kb

fragment of nopM. The rest of plasmid was treated with the Klenow fragment and self-

ligated. This plasmid was restricted by digestion with PstI and BamHI, and the 1.7 kb

fragment containing deletion nopM were purified and cloned into the rhizobial suicide

vector pJQ-mp18 (Quandt and Hynes, 1993). Triparental mating was used to transfer the

resulting plasmid into NGR234. Double recombination was selected by plating bacteria

onto TY plates containing 5 % sucrose and appropriate antibiotics. Putative mutants were

confirmed by PCR and Southern blots of restricted genomic DNA using standard

procedures (Chen and Kuo, 1993; Sambrook et al., 1989).

The double mutants NGRΩnopJ∆nopM, NGRΩnopL∆nopM, NGR∆nopM∆nopP

and NGR∆nopM∆nopT were obtained by introducing the mutated nopJ (Deakin,

unpublished), nopL (Marie et al., 2003), nopP (Skorpil et al., 2005) and nopT (Saad,

unpublished) genes into NGR∆nopM respectively. The triple mutant

72

NGRΩnopL∆nopM∆nopP was obtained by introducing the mutated nopM gene into

NGRΩnopL∆nopP (Skorpil et al., 2005).

Production of the NopM antibody and over-expression of NopM

Anti-NopM antiserum was conducted using two peptides designed from the

sequence of NopM, N-TAEERPWEGRPQEAV and N-GETMEKVLRGRGLEL.

Immunisation of two rabbits with the coupled peptide mixture was performed to

established protocols (Eurogentec, Herstal, Belgium). To raise anti-NopM antiserum,

NopM was expressed in E. coil with a 6x His-tag at amino-terminal end. nopM DNA

sequence was amplified by PCR, using the primer pair 5’-CGGATATCATGAATGTAC

AACGGCCCGG-3’ and 5’-TCACAGCTCAAGACCGCGACC-3’. The PCR product

was cloned into the SmaI site of pBluescript KS+ and sequenced. Then the fragment of

EcoRV and XbaI site of nopM was subcloned into expression vector pPROEX-1 (BRL,

Bethesda, MD, U.S.A.) to construct pPROEX-nopM. The fusion protein NopM 6x His-

tag was over expressed in E. coil and purified using Ni-NTA resin (QIAGEN). The

presence of the fusion protein was verified by 15 % SDS-polyacryamide gel

electrophoresis.

Analysis of secreted proteins

Secreted proteins were purified and analyzed as described in Marie and associated

(Marie et al., 2003). Rhizobium strains were grown in 100 ml of RMS for 40 h in the

presence of apigenin to a final optical density at 600 nm of approximately 1.0. Secreted

proteins present in the supernatants of cultures were precipitated using ammonium

sulphate (60 %, wt/vol) and subsequently desalted using Sephadex G25-containing

columns (Amersham Biosciences, Uppsala, Sweden). Then proteins were separated by

15 % SDS-polyacryamide gel electrophoresis before staining with silver nitrate. For

immunostaining, proteins were transformed from SDS-PAGE onto Millipore immobilon

PVDF membranes by electroblotting, as described by Ausubel and associates (Ausubel et

al., 1991). Protein-primary NopM antibody complexes and anti-His x4 were visualized

using horseradish peroxidase-labeled anti-rabbit or anti-mouse antibodies respectively.

73

Then these were detected with ECL regent (Amersham). Antisera against NopA, NopB,

NopC, NopL, NopP and NopX has been described previously studies (Ausmees et al.,

2004; Deakin et al., 2005; Marie et al., 2003; Saad et al., 2005).

Plant material and assay

Seed source are listed in Pueppke and Broughton (Pueppke and Broughton, 1999).

Plant seeds were sterilized as described below: Crotalaria juncea; concentrated sulphuric

acid 10 minutes, 0.1 % (v/v) Tween 20 5 minuites and 5 % (v/v) H2O2 5 minutes.

Flemingia congesta; concentrated sulphuric acid 10 minutes, 0.1 % (v/v) Tween 20 10

minuites, 70 % (v/v) ethanol 10 minutes and 5 % (v/v) H2O2 5 minutes. Lablab

purpureus and Tephrosia vogelii; concentrated sulphuric acid 10 minutes, 0.1 % (v/v)

Tween 20 10 minuites and 5 % (v/v) H2O2 5 minutes. Pachyrhizus tuberosus;

concentrated sulphuric acid 25 minutes, 0.1 % (v/v) Tween 20 10 minuites and 5 % (v/v)

H2O2 5 minutes. Vigna unguiculata; concentrated sulphuric acid 10 minutes and 70 %

(v/v) ethanol 5 minutes. Nodulation tests were performed in Magenta jars as described by

Viprey and associates (Viprey et al., 1998). All plants were grown at a day temperature of

28 °C, a night temperature of 18 °C, and a photoperiod of 16 h. Each plant was

inoculated with 107 bacteria and harvested five to seven weeks after inoculation.

74

Table 4. Strains and plasmids used in this study

Strain Relevant characteristics Reference

Escherichia coli

DH5α supE44 ∆lacY169 ( 80lacZ∆M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 BRL, Bethesdda, MD, U.S.A.

Rhizobium strains

NGR234 Broad host-range bacterium isolated from nodules of Lablab purpureus, Rifr (Lewin et al., 1990)

NGRΩrhcN NGR234 derivative containing an Ω insertion in rhcN, Rifr Spr (Viprey et al., 1998)

NGR∆nopA NGR234 derivative with nopA deleted, Rifr (Saad et al., 2005)

NGRnopB::uidA NGR234 derivative containing a uidA insertion in nopB, Rifr (Saad et al., 2005)

NGRΩnopJ NGR234 derivative containing an Ω insertion in nopJ, Rifr Spr W. Deakin, unpublished

NGR∆nopM NGR234 derivative with nopM deleted, Rifr This work

NGRΩnopL NGR234 derivative containing an Ω insertion in nopL, Rifr Kn (Marie et al., 2003)

NGR∆nopPtotal NGR234 derivative in which 0.5 kb of nopP was replaced by an Ω insertion, Rifr Spr

(Skorpil et al., 2005)

NGR∆nopT NGR234 derivative with nopT deleted, Rifr M. Saad, unpublished

NGR∆nopX NGR234 derivative in which the 552-bp BamHI internal fragment of nopX was replaced by an Ω insertion, Rifr Spr

(Marie et al., 2003)

NGR∆ttsI NGR234 derivative with ttsI deleted, Rifr (Wassem et al., 2008)

NGRΩnopL∆nopPtotal NGR∆nopPtotal derivative containing an Ω insertion in nopL, Rifr Knr Spr (Skorpil et al., 2005)

NGRΩnopJ∆nopM NGRΩnopJ derivative with nopM deleted, Rifr Spr This work

NGRΩnopL∆nopM NGRΩnopL derivative with nopM deleted, Rifr Knr This work

NGR∆nopM∆nopPtotal NGR∆nopPtotal derivative with nopM deleted, Rifr Spr This work

NGR∆nopM∆nopT NGR∆nopT derivative with nopM deleted, Rifr This work

NGRΩnopL∆nopM∆nopP NGRΩnopL∆nopPtotal derivative with nopM deleted, Rifr Knr Spr This work

USDA257 Broad host-range strain isolated from Glycine soja, Knr (Keyser et al., 1982)

Plasmids

pBluescript II KS+ High copy number ColE1-based phagemid, Apr Straragene, La Jolla, CA

pBDG98 pBluescript KS+ derivative carrying a nopM in EcoRV and XbaI site, Apr W. Deakin, unpublished

pXBS23 Lorist 2 derivative containing the y4eF to y4gD region of pNGR234a, Knr (Perret et al., 1991)

pUC18 Cloning vector with lacZ reporter gene containing multiple cloning site, Apr (Maniatis, 1982)

pJQ-mp18 Suicide vector used for directed mutagenesis, Gmr (Quandt and Hynes, 1993)

pJQ-nopM pJQ-mp18 derivative construct in which 1.7 kb fragment containing a deletion of nopM was inserted into the PstI and BamHI site, Gmr

This work

pJQ-nopJ pJQ-200SK derivative carrying construct in which an Ω cassette was inserted into the MluI site of nopJ, Gmr Spr

W. Deakin, unpublished

pJQ-nopL pJQ-200SK derivative carrying construct in which an Ω cassette was inserted into the EcoRV site of nopL, Gmr Knr

(Marie et al., 2003)

pJQ-nopP pJQ-200SK derivative carrying a construct in which nopP was replaced by an Ω cassette, Gmr Spr

(Skorpil et al., 2005)

pK18-nopT pK18mobsacB derivative construct containing a deletion of nopT, Gmr Knr M. Saad, unpublished

pRK2013 Tra+ helper plasmid (Figurski and Helinski, 1979)

pPROEX-1 E. coli expression vector with six-His tag, Apr Invitrogen, Carlsbad, CA

pPROEX-nopL pPROEX-1 derivative with EheI and XbaI site of full-length of nopL gene, Apr

(Bartsev et al., 2003)

pPROEX-nopM pPROEX-1 derivative with EcoRV and XbaI site of full-length of nopM gene, Apr

This work

75

Results and Discussion

Identification of NopM and production of antibody

To facilitate detection of NopM from NGR234, polyclonal antibodies were raised

using synthetic peptides designed from NopM. NopM was over-expressed and purified

from E. coli to allow testing of the antisera. A NopM-hexa-histidine tagged fusion protein

was expressed in E. coli and verified using a anti-His monoclonal antibody (Figure 1A).

Anti-His sera detected NopM 6x His-tag fusion protein (carrying a 6x-His tag; apparent

size of ≈63 kDa). For control, pPROEX-1 and pPROEX-nopL (carrying a 6x-His tag;

apparent size of ≈40 kDa) (Bartsev et al., 2004) were also used. The NopM-hexa-

histidine tagged fusion protein was used as a positive control for the polyclonal anti-

NopM antisera in all subsequent experiments.

83

62

47.5

32.5

25

175

kDa

83

62

47.5

32.5

25

175

kDa

A B

pPR

OE

X-n

opL

pPR

OE

X-n

opM

pPR

OE

X- n

opM

pPR

OE

X-0

1

NG

R234

NG

RΩrh

cN

Figure 1. A: Anti-His sera detection of 6x His-tagged Nop fusion proteins. Extracts were used from E. coli containing pPROEX-nopM and as controls, pPROEX-1 and pPROEX-nopL (carrying a 6x His tag; apparent size of ≈40 kDa) (Bartsev et al., 2004). B: Comparison of extracellular proteins from NGR234 and NGRΩrhcN. Secreted proteins from NGR234, NGRΩrhcN and the NopM 6x His-tag fusion protein were separated by SDS-15 % PAGE, transferred to PVDF and probed with the new NopM antibody.

76

NopM was shown to be secreted by the NGR234 T3SS. After 40 h of induction

with 2 x 10−7 M apigenin, proteins found in the supernatants of cultures of NGR234 and

NGRΩrhcN were isolated and separated by SDS-PAGE (Figure 1B). Anti-NopM

detected a 60 kDa band in the NGR234 extracellular proteins (with the NopM his tag

fusion protein also detected at approximately the same size) but not in extracts from

NGRΩrhcN.

Construction of a deletion mutant of nopM

To assess the function of nopM, a deletion mutant was generated. The nopM

region was isolated and a 1 kb fragment of nopM excised which contained its start codon

(Figure 2).

ClaI NheI

nopM y4gA

200 bp

ClaI - NheI

∆nopM y4gA

Figure 2. Mutagenesis nopM.

Proteins found in the supernatants of cultures of NGR234, NGRΩrhcN, and

NGR∆nopM were isolated and separated by SDS-PAGE and then stained with silver

(Figure 3A). Comparison of the distinct profiles of the extracellular proteins showed

clearly that proteins found in induced cultures of NGR234 were missing in induced

cultures of NGRΩrhcN that abolishes secretion of protein via the T3SS. The extracellular

proteins of NGR∆nopM were similar to those of NGR234 and no clear band of 60 kDa

(corresponding to NopM) was obviously missing. Probably NopM which is about 60 kDa

product was covered by NopX (64 kDa). To visualize NopM in NGR234 extracts

compared to NGR∆nopM extracts, two-dimensional gel electrophoresis (2D-gels) could

be used to separate proteins according to their size and isoelectric point, but instead the

77

anti-NopM antisera was used. To confirm that NopM was missing in the nopM deletion

mutant and also determine definitively whether NopM is required for the transit of Nops

into the extracellular medium, secreted proteins from NGR234, NGRΩrhcN and

NGR∆nopM were separated by SDS-15 % PAGE and transferred to PVDF membranes

and then probed with antibodies against NopM, NopX, NopL, NopP, NopB, NopC and

NopA. Western blots using anti-NopM showed that a 60 kDa protein secreted by

NGR234 after the flavonoid induction, cross-reacted with anti-NopM antiserum but not

in extracts from either non-induced NGR234, or induced cultures of NGR∆nopM and

NGRΩrhcN (Figure 3B). Additionally, secretion of Nops occurred in the nopM mutant,

but not the T3SS mutant. Thus NGR∆nopM did not change the pattern of secreted

proteins with the exception of the absence of NopM.

94

67

43

30

20

14

kDa

A

NopA

NopX

NopL

NopP

NopB

NopC

Anti-NopX

Anti-NopM

Anti-NopL

Anti-NopP

Anti-NopB

Anti-NopC

Anti-NopA

NGR234

NGR∆nopM

NGRΩrhcN

+- + +NGR234

NGR∆nopM

NGRΩrhcN

B

Figure 3. Nop secretion by NGR∆nopM. A: Secretion proteins from induced cultures of NGR234, NGRΩrhcN and NGR∆nopM were separated by SDS-15 % PAGE and stained with silver nitrate. B: Immunostaining with antibodies against NopX, NopM, NopL, NopP, NopB, NopC and NopA. Strains were grown in the absence (-) or presence (+) of apigenin.

78

In NGR234 mutations in proteins encoding components of the secretion

machinery block Nop secretion, which was not the case for NGR∆nopM. Considering

this and the homology of NopM to effector proteins from other pathogenic bacteria we

conclude that NopM is not a part of the secretion machinery and is another of the

NGR234 T3SS effector proteins that most likely function within legume cells.

Secretion of NopM by other NGR234 strains

Additionally, immunological detection of NopM was carried out using

intracellular and extracellular proteins of various (induced) mutant derivatives of

NGR234 and USDA257 (Figure 4). As a control, anti-NopL (Marie et al., 2003) was used,

as NopL is known to be detectable in both fractions.

Anti-NopL

Anti-NopMIntracellular

protein

Extracellularprotein

NG

R23

4N

GR

Ωrh

cN

NG

R∆no

pM

USD

A25

7

NG

Rno

pB::

uidA

NG

RΩ

nopL

NG

R∆n

opA

NG

R∆n

opP to

tal

NG

R∆n

opX

NG

R∆t

tsI

Anti-NopL

Anti-NopM

Anti-NopL

Anti-NopMIntracellular

protein

Extracellularprotein

NG

R23

4N

GR

Ωrh

cN

NG

R∆no

pM

USD

A25

7

NG

Rno

pB::

uidA

NG

RΩ

nopL

NG

R∆n

opA

NG

R∆n

opP to

tal

NG

R∆n

opX

NG

R∆t

tsI

Anti-NopL

Anti-NopM

Figure 4. Detection of NopM in various Rhizobium strains. Intracellular and extracellular proteins of induced USDA257 and various derivatives of NGR234 were resolved by SDS-15 % PAGE and transferred to PVDF membranes, followed by immuno-staining with specific anti-Nop antibodies.

As expected NopM and NopL secretion was abolished in T3SS machinery

mutants (NGRΩrhcN, NGR∆nopA and NGRnopB::uidA), but both Nops were detected

intracellularly confirming that it is the absence of the secretion machinery which leads to

their non-secretion. This experiment also demonstrates that under the defined growth

79

conditions used throughout, there is no NGR234 cell lysis and subsequent release of the

Nops. Furthermore, both NopM and NopL were missing in the intracellular and

extracelluar fractions from the transcriptional regulator mutant (NGR∆ttsI), as most

likely the T3SS genes were not induced. NopM and NopL were missing from their

respective mutants, but not from the mutations of NopP or NopX.

Interestingly in the Sinorhizobium fredii USDA257 protein fractions, NopM was

not detectable, NopL could be however, as has been previously shown (Ausmees et al.,

2004). NGR234 and USDA257 have similar legume host ranges (Pueppke and Broughton,

1999) and the genomic organization of their T3SS loci is remarkably similar. In previous

studies, it was shown that at least six Nops were secreted by T3SS of USDA257

(Krishnan, 2002; Krishnan et al., 2003; Lorio et al., 2004). It is possible that either the

peptide sequences used from NopM of NGR234 to generate the anti-NopM antiserum are

not present in a NopM-like protein in USDA257 or USDA257 is missing this Nop. To

answer to this question, Southern blot analysis should be able to confirm whether

USDA257 has sequence homologous to nopM. Recently it has been shown that S. fredii

HH103 probably secretes NopM (Rodrigues et al., 2007). This suggests that there could

be differences in Nop secretion between USDA257 and HH103, which might affect their

different symbioses host plants. The absence/presence of NopM is a good candidate with

which to test this theory.

Symbiotic phenotype of nopM

Nops are known to modulate nodule number and play a role in nodule

development. For some plant species, such as Tephrosia vogelii, Flemingia congesta and

Lablab purpureus, Nops enhance nodule formation, but for the Pachyrhizus tuberosus

and Crotalaria juncea, Nops appear to be deleterious. To evaluate the effect of NopM on

symbiosis, the nodulation abilities of strains NGR234, NGRΩrhcN, and NGR∆nopM

were compared on five different legume species known to have a phenotype with the

NGR234 T3SS (Figure 5A to E).

80

No

dule

num

be

rN

odu

len

umb

er

0

10

20

30

40

50

0

10

20

30

40

50

0

10

20

30

40

50

60

0

10

20

30

40

50

60

0

5

10

15

20

25

0

5

10

15

20

25

0

5

10

15

20

25

0

5

10

15

20

25

No

dule

num

ber

No

dule

num

ber

Nod

ule

num

ber

Nod

ule

num

ber

Nod

ule

num

ber

Nod

ule

num

ber

NGR234 NGRΩrhcN NGR∆nopMNGR234 NGRΩrhcN NGR∆nopM

NGR234 NGRΩrhcN NGR∆nopMNGR234 NGRΩrhcN NGR∆nopM

NGR234 NGRΩrhcN NGR∆nopM H O2

NGR234 NGRΩrhcNNGR∆nopM H O2NGR234 NGRΩ rhcNNGR∆nopM H O2

NGR234 NGRΩrhcNNGR∆ nopM H O2

NGR234 NGRΩrhcN NGR∆ nopM H O2

A: Lablab purpureus B: Pachyrhizus tuberosus

C: Flemingia congesta D: Tephrosia vogelii

E: Crotalaria juncea

NGR : all fix+ΩrhcN

NGR234 NGR∆nopM

Figure 5. Symbiotic phenotype of NopM on

various legume plants. Each bar indicated the

average number of nitrogen-fixing nodules per

plant, and standard errors of the means are

indicated adjacent to the ba

and . For the

control, sterilized water was used. Independent

experiment was performed at least three times.

r.

Ω ∆

Each leguminous

plant was inoculated with NGR234, T3SS null

mutant NGR NGRrhcN nopM

: all fix-

81

In contrast to wild-type NGR234, which produced up to twenty nodules per plant

on L. purpureus, the T3SS null mutant NGRΩrhcN formed few nodules on this plant. On

L. purpureus, in absence of NopM, a significant decrease of nodule number was observed

compared to NGR234. Plants and nodule weights were also decreased. On P. tuberosus, a

functioning T3SS has a globally detrimental effect on nodulation. A slight increase in

nodule number was obtained with NGR∆nopM indicating that NopM is partly

responsible for this block in formation of nodules. On F. congesta, T. vogelii and C.

juncea, NGR∆nopM did not change nodulation.

Interestingly, NGR∆nopM showed different effects depending on the plant

species. On L. purpureus, NGR∆nopM reduced the nodulation ability 50 % compare to

wild-type, and thus NopM acts as a positive effector. On P. tuberosus however,

NGR∆nopM slightly recovered the nodulation ability, suggesting NopM is a negative

effector. In NGR234, NopL and NopP were found and identified as effector proteins,

These Nops were shown only to have positive effects on legumes, for example, NopP and

NopL have positive phenotype on F. congesta and T. vogelii (Skorpil et al., 2005). Thus

in NGR234, NopM is the first effector protein that showed both positive/negative and

effects. This situation mirrors the role of Avr proteins secreted by phytopathogens. For

instance, two Avr proteins (AvrPto and AvrPtoB) from Pseudomonas syringae function

to suppress signalling in the generation of a plant innate defence response in their non-

host Arabidopsis (He et al., 2006) i.e. a positive role in the infection of this plant.

Whereas both AvrPto and AvrPtoB were originally identified as avirulence proteins

recognized by plants triggering a hypersensitive response (HR), a localized programmed

cell death (Dangl and Jones, 2001; Staskawicz et al., 2001) i.e. a negative role in the

infection of this plant. Thus on L. purpureus, NopM may function as suppressor of host

defences facilitating infection by NGR234 and thus improved nodulation, but on P.

tuberosus, NopM could be recognized as an avirulence factor to induce HR.

However as the effect of the NGR∆nopM not as strong as that of NGRΩrhcN on

either plant, suggesting the presence of alternative positive effector(s) for L. purpureus

and negative effector(s) for P. tuberosus. The double mutant, NGRΩnopL∆nopP was

shown to have a more pronounced phenotype than either single mutant, suggesting that

82

the effector Nops may function cooperatively (Skorpil et al., 2005). To test for the

presence of additional positive or negative effector on L. purpureus and P. tuberosus,

multiple Nop mutants could answer this hypothesis. Skorpil and associates (Skorpil et al.,

2005) suggested the presence of a negative effector as well as the positive effector in

NGRΩnopL∆nopP, in the interaction with T. vogelii. NopM is a possible candidate, thus

at the same time a triple mutant NGRΩnopL∆nopM∆nopP was created to test specifically

on T. vogelii.

Construction of multiple mutants

To investigate the interaction of Nops on symbiosis, the double mutants

NGRΩnopJ∆nopM, NGRΩnopL∆nopM, NGR∆nopM∆nopP and NGR∆nopM∆nopT as

well as the triple mutant NGRΩnopL∆nopM∆nopP were generated. To verify the absence

of specific Nops from these mutants extracellular proteins were separated on SDS-PAGE

and Western blot were performed (Figure 6A and B). Each double mutant showed

abolished the secretion of NopM and specifically NopL, NopP or NopT. The triple

mutant abolished secretion of NopL, NopM and NopP. Even though a NopJ antibody has

been constructed it does not detect NopJ in extracellular proteins, however

NGRΩnopJ∆nopM was resistant to spectinomycin as conferred by the omega interposon

inserted into nopJ. All mutants verified by PCR and Southern blots.

83

Anti -NopM

Anti -NopT

Anti -NopL

Anti -NopP

Anti -NopX

A B

Anti -NopM

Anti -NopL

Anti -NopP

Anti -NopM

Anti -NopT

Anti -NopL

Anti -NopP

Anti -NopX

Anti -NopM

Anti -NopL

Anti -NopP

NG

R234

NG

R∆nop

M

NG

R23

4

NG

R

∆nop

M

Ωno

pJ

NG

R

∆nop

P

∆nop

M

NG

R

∆nop

M

Ωno

pL

NG

R

∆nop

T

∆ nop

M

NG

R

∆nop

P

Ωno

pLN

GR

∆nop

M

Ω

∆

nopL

nopP

Figure 6. Profiles of Nops from multiple mutants. Extracellular proteins of multiple Nops mutants were separated by SDS-15 % PAGE and transferred to PVDF membranes and probed with Nop antibodies. A: Double mutants of NGRΩnopJ∆nopM, NGRΩnopL∆nopM, NGR∆nopM∆nopP and NGR∆nopM∆nopT. B: Triple mutant NGRΩnopL∆nopM∆nopP.

To establish whether NopM acts as the unidentified negative effector on T. vogelii,

the triple mutant NGRΩnopL∆nopM∆nopP was tested and compared with each single

mutants and NGRΩnopL∆nopP (Figure 7). As described earlier, the NGRΩnopL∆nopP

double mutant showed a more pronounced phenotype than either single mutants and the

nodulation ability was lower than NGRΩrhcN, indicating the existence of the

unidentified negative effector. The triple mutant NGRΩnopL∆nopM∆nopP generated a

slight increase nodule number but there was no significant difference compared with the

NGRΩnopL∆nopP double mutant. This result suggests that NopM is probably not a

negative effector on T. vogelii and thus the presence of other negative effector(s). As a

first approach to detect negative effector on T. vogelii, mutations of the other known

effector proteins (NopJ and NopT) in NGR234 should be created in NGRΩnopL∆nopP.

84

NG

R2

34N

GR

234

Ωrh

cNΩ

rhcN

ΩL

∆ M∆ P

ΩL

∆ M∆ P

0

5

10

15

20

25

Nod

ule

num

ber

ΩL

∆L

∆PP

∆ nop

∆ nop

∆M

∆M

nop

nop

Ωno

pno

p L

Tephrosia vogelii

ΩrhcN ∆nopM ΩnopL ∆nopP Ω ∆L P H O2NGR234 Ω ∆L P∆M

Figure 7. Symbiotic phenotype of multiple Nop mutants on T. vogelii. A triple mutant NGRΩnopL∆nopM∆nopP was inoculated and compared. Each bar indicated the average number of nitrogen-fixing nodules per plant, and standard errors of the means are indicated adjacent to the bar. For the control, sterilized water was used. Independent experiments were performed at least three times.

The double and triple multiple mutants were tested for their symbiotic ability on P.

tuberosus and L. purpureus - plants affected by NopM (Figure 8A and B). On P.

tuberosus, multiple mutants did not show any significant differences to the single nopM

mutant. This data suggested the presence of another (unidentified) negative effector. To

identify the negative effector(s) on P. tuberosus, a Tn5 transposon (Wilson et al., 1995)

could be used to randomly mutagenesis NGR234 and a mixture of mutants inoculated

onto P. tuberosus. Nodules from plants growing healthily could be harvested and the

rhizobia within extracted. Although it is more likely that Nop secretion is totally blocked

in these mutants, this could be tested, and any transposon mutants still able to secrete

Nops would be good candidates to have an insertion in the gene encoding the negative

effector.

On L. purpureus, the nodulation ability of double mutant NGR∆nopM∆nopP was

lower than NGR∆nopM, and it showed no difference in nodulation ability to T3SS null

85

mutant NGRΩrhcN. This result showed not only identified an additional positive effector,

NopP but also interpreted the difference in nodulation ability between NGR234 and

NGRΩrhcN. Because of missing two positive effectors NopM and NopP, there is a

difference of phenotype between NGR234 and NGRΩrhcN. Furthermore, the nodule

formation ability of NGRΩnopJ∆nopM is approximately equal with wild-type in spite of

the absence of NopM, the nopM single mutant showed 50 % lower nodulation ability

compare to wild-type. This result suggests that NopJ might be a negative effector on L.

purpureus. Furthermore the overall effect of the T3SS might be a result of both positive

and negative Nops. Thus on L. purpureus, the symbiotic phenotype of NGR234 is an

equilibrium result of negative (NopJ) and positive Nops (NopM & NopP). This will be

investigated further in the next section by testing L. purpureus with each single nop

mutants.

H2O

NG

R23

4

Ωrh

cN

∆nop

M

ΩL∆

M

ΩJ∆

M

∆M∆P

ΩL∆

M∆P

B: Pachyrhizus tuberosus

∆M∆T

0

10

20

30

40

50

Nod

ule

num

ber

NG

R23

4

Ωrh

cN

∆nop

M

ΩL

∆M

ΩJ∆

M

∆M∆P

ΩL

∆M∆P

∆M∆T

0

4

8

1 2

1 6

2 0

NG

R23

4

Ωrh

cN

∆nop

M

ΩL

∆M

ΩJ∆

M

∆M∆P

ΩL

∆M∆P

∆M∆T

Nod

ule

num

ber

H2O

NG

R23

4

Ωrh

cN

∆nop

M

ΩL∆

M

ΩJ∆

M

∆M∆P

ΩL∆

M∆P

∆M∆T

A: Lablab pupureus

H2O

NG

R23

4

Ωrh

cN

∆nop

M

ΩL∆

M

ΩJ∆

M

∆M∆P

ΩL∆

M∆P

B: Pachyrhizus tuberosus

∆M∆T

0

10

20

30

40

50

Nod

ule

num

ber

NG

R23

4

Ωrh

cN

∆nop

M

ΩL

∆M

ΩJ∆

M

∆M∆P

ΩL

∆M∆P

∆M∆T

0

4

8

1 2

1 6

2 0

NG

R23

4

Ωrh

cN

∆nop

M

ΩL

∆M

ΩJ∆

M

∆M∆P

ΩL

∆M∆P

∆M∆T

Nod

ule

num

ber

H2O

NG

R23

4

Ωrh

cN

∆nop

M

ΩL∆

M

ΩJ∆

M

∆M∆P

ΩL∆

M∆P

∆M∆T

A: Lablab pupureus

Figure 8. Symbiotic phenotype of multiple Nop mutants. Double and triple effector multiple mutants were inoculated on L. purpureus (A) and P. tuberosus (B). The phenotypes were compared with NGR234, NGRΩrhcN and NGR∆nopM. Each bar indicated the average number of nitrogen-fixing nodules per plant, and standard errors of the means are indicated adjacent to the bar. For the control, sterilized water was used. Independent experiments were performed at least three times.

86

Symbiotic phenotype of nops on Lablab purpureus

In NGR234, so far five effector proteins have been found and identified. On L.

purpureus, these single nop mutants were tested for their nodulation ability (Figure 9).

Plants inoculated with NGR∆nopP showed a dramatic decrease in nodulation ability.

NopP is also a positive effector as NopM, but its effect is stronger than NopM. Thus as

demonstrated earlier with the double mutant NGR∆nopM∆nopP, producing an equal

nodule number as the T3SS null mutant, NopM and NopP are the major positive (T3SS-

derived) determinants for nodulation on L. purpureus. In contrast to these Nops, plants

inoculated by NGRΩnopJ showed an increase in nodule number, although it was not

statistically significant. Thus probably NopJ has negative effect on L. purpureus. The

absence of NopL and NopT had no significant effect on L. purpureus.

Figure 9. Symbiotic phenotype of Nops on Lablab purpureus. Five difference effector Nops single mutants were inoculated on L. purpureus and the phenotype were compared with NGR234 and NGRΩrhcN. Each bar indicated the average number of nitrogen-fixing nodules per plant, and standard errors of the means are indicated adjacent to the bar.

NGR234 was isolated from nodules of L. purpureus and thus should be

considered as its natural host plant (Trinick, 1980). It is thus noteworthy that a functional

T3SS is necessary for efficient nodule formation.

0

5

10

15

20

25

30

35

0

1

2

3

4

5

6

7

8

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NGR234 NGRΩrhcN NGR∆nopM NGRΩnopJ

No

dul

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umbe

r

NGRΩnopL NGR∆nopP NGR∆nopT

Pla

nt w

eig

ht (

g)

0

5

10

15

20

25

30

35

0

1

2

3

4

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NGR234 NGRΩrhcN NGR∆nopM NGRΩnopJ

No

dul

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umbe

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NGRΩnopL NGR∆nopP NGR∆nopT

Pla

nt w

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ht (

g)

87

Chapter 4: Functions of Nops in eukaryotic cells.

Introduction

For many Gram-negative bacterial pathogens, the T3SS is essential for the

initiation of maintenance of host infection (Galan and Collmer, 1999). It is, however, the

cocktail of effector proteins injected by the T3SS into cells of the plant or animal host

that change signal pathways to promote responses beneficial to the pathogen. Presently

the exact molecular functions of only a few effectors have been determined.

Understanding the functions of these effectors has become an urgent goal of both animal

and plant pathology. Determining the mechanism of action of the effectors is difficult,

primary sequence analysis often gives no clues as to their function. Thus a common

approach is to express the effectors in eukaryotic cell culture systems and observe the

consequences. Although this requires the technical ability of developing cell culture

systems from host organisms and methods for transfer of non-host genes and their

subsequent expression.

Some of the Nops are homologous to characterised effector proteins from

pathogens of various animal and plant species (see chapter 3). As reported in chapter 3,

Nops can have negative and/or positive effect depending on the host plant. We suggest

that these responses depend upon recognition (or not) of each Nop by host plants. By

comparison to the use of effectors by pathogens, positive effectors may aid infection (and

thus nodulation) by modulating host signal pathways, perhaps suppressing defence

responses or facilitating endocytosis. In contrast, negative effectors might be recognized

as avirulence factors and lead to strong localised defence reactions (HR-like). It is

difficult to work on many of the host plants of NGR234 at the molecular level. Thus

another eukaryotic model, yeast was used to study Nop functions.

To study microbial virulence factors, as a eukaryotic model, Saccharomyces

cerevisiae can serve as a powerful system (Lesser and Miller, 2001). Yersinia YopM

function was demonstrated in yeast cells and these confirmed the localization patterns

showing YopM to be transported to nuclei (Skrzypek et al., 2003). It was also shown

88

using yeast cells, that the catalytic core of YopT C139, H258, and D274 are essential for

YopT cytotoxicity (Shao et al., 2002). YopJ was also shown to inhibit MAPK signalling

in yeast cells (Yoon et al., 2003).

In this study, all the effector Nops were expressed in yeast cells. For NopJ, NopM

and NopT, based upon their homology to characterised effectors we observed whether the

Nops had similar effects. Thus we tried to visualize the location of NopM in yeast cells,

by expressing it as a GFP fusion. For NopJ and NopT, their toxicity in yeast was assessed,

as both effectors have the conserved catalytic residues found in their pathogenic

homologues. As these residues in the YopT family are also necessary for their proteolytic

activity, we looked for autoproteolytic cleavage. NopL and NopP were also expressed in

yeast, to study potential intracellular locations and their effects on yeast cell growth. Both

NopL and NopP have homology only to rhizobial proteins, thus there is no clue to their

functions from their primary sequences, although both may well interfere in

phosphorylation-based signalling pathways.

As well as using yeast, attempts were made to transform the (non-legume) model

plant Arabidopsis thaliana with the effector nops. Finally it was planned to test any

identified function of the effector Nops in host plants of NGR234. Presently it is not

possible to stably transform these plants thus I studied way to improve experimental

techniques required to transform roots of L. purpureus and V. unguiculata. Several A.

rhizogenes species were tested for their transformation frequency on L. purpureus and V.

unguiculata and attempts made to express Nops in their roots.

89

Materials and methods

Microbiological techniques

Escherichia coli strains were grown in Luria-Bertani (LB) media at 37 °C

(Sambrook et al., 1989). Rhizobium strains were grown at 27 °C in either complete (TY)

(Beringer, 1974) media or minimal media containing succinate as the carbon source

(RMS) (Broughton et al., 1986). Antibiotics were added to the media at the following

final concentrations; ampicillin (Ap), 50 µg ml-1; kanamycin (Kn), 50 µg ml-1; rifampicin

(Rif), 50 µg ml-1; spectinomycin (Sp), 50 µg ml-1; tetracycline (Tet), 15 µg ml-1; and

chloramphenicol (Cm), 15 µg ml-1. Escherichia coli strains DH5α was used as a host for

amplification and storage of plasmids. They were grown in Luria-Bertani (LB) media at

37 °C (Sambrook et al., 1989).

Yeast techniques

Plasmids and Strains

Plasmids and strains are summarized in Table 1. The open reading frame

encoding each of the translocated protein (Nops) was PCR amplified from NGR234

genomic DNA and subcloned in-frame with the C-terminus of GFP in pFUS (Johnson,

1991), to create GFP fusion proteins and without fusion tags in pYES2 (Invitrogen) under

the control of the GAL10 promoter. The PCR-amplified fragments used for cloning were

analyzed by DNA sequencing.

Saccharomyces cerevisiae strain W303 MATa/MATα (leu2-3,112 trp1-1 can1-100

ura3-1 ade2-1 his3-11,15) [phi+] (Fan et al., 1996) was used for the expression of NopM

fused to GFP in the expression vector pFUS and all Nop proteins in the expression vector

pYES2. YPD medium (peptone, yeast extract and glucose) was used for routine growth

of yeast. Whereas synthetic defined (SD) medium was used, with appropriate omissions

of amino acids to select for plasmids carrying metabolic markers, and with either glucose

90

(repression), raffinose (no effect) or galactose (induction) of the GAL10 promter.

Transformation of yeast was performed as described in Gietz and Schiestl (Gietz et al.,

1995).

Construction of pYES-nopM

To obtain pYES-nopM, nopM was amplified by PCR from genomic DNA of

NGR234 using the following primer pairs: A (5’-CAGGATCCATGAATGTACAACGG

CCCGG-3’) and B (5’-TCACAGCTCAAGACCGCGACC-3’). The 1.6 kb fragment

containing of nopM was first cloned into the EcoRV site of pBluescript KS+ and verified

by sequencing. HindIII and BamHI were used to excise a nopM fragment and which was

subcloned into the expression vector pYES2. pYES2-nops (nopJ, nopL, nopP, nopT and

nopT point mutants) were constructed using PCR (W. Deakin, unpublished).

Localization of NopM-GFP in yeast using microscopy

Yeast cultures carrying the plasmid pFUS-NopM were grown overnight in non-

inducing selective synthetic media supplemented with 2 % raffinose. Yeast were diluted

to OD600= 1.0. Expression of the fusion protein was induced by addition of 2 % galactose

to the medium. Yeast was observed at designated time points on a fluorescent microscope.

DNA was visualized by staining with DAPI (Roche) or Hoescht (Sigma). For DAPI

staining, yeast cells were collected by centrifugation and resuspended in H2O then

incubated with DAPI or Hoescht (1 µg/ml).

Preparation of protein extracts and immunoblot analysis

To confirm the presence of Nops protein in the yeast cells, total proteins from

yeast cells were extracted using Y-PER (Yeast Protein Extraction Reagent) (PIERCE).

After induction of Nops by 2 % galactose, yeast cells were collected. Cell pellets were

resuspended with appropriate amount of Y-PER and incubated 20 min at room

temperature. Cell debris were removed by centrifuging, the supernatants were used for

immunoblot analysis. Proteins in the soluble fractions from extraction procedure were

equilibrated to be at the same quantities, and then separated by SDS-PAGE. For

91

immunoblot, proteins were transformed from SDS-PAGE onto Millipore immobilon

PVDF membranes by electroblotting.

Growth assays

To compare the growth rates of strains carrying different Nops, saturated

overnight cultures of the strains were grown in non-inducing selective synthetic media

with 2 % raffinose. Each culture was normalized to OD600= 0.1 and then serially diluted

10-fold four additional times. Aliquots (10 µl) of each of the five dilutions were spotted

onto a selective medium plate supplemented with 2 % glucose or 2 % galactose. The

plates were incubated at 30 °C and photographs of the plates were obtained 2 days (with

glucose) or 2-4 days (with galactose) after plating.

Generation of transgenic roots

Plasmids and Strains

Plasmids and strains are summarized in Table 1. Agrobacterium rhizogenes strain

K599 (Cheon et al., 1993) was kindly supplied by Dr. F. Sanchez, A4RSII (Jouanin et al.,

1986), LBA9402 (Hood et al., 1993), 8196 (Hansen et al., 1992), 2659 (Brevet and

Tempe, 1988) and 15834 (Schiemann and Eisenreich, 1989) were kindly supplied by Dr.

D. Tepfer. These strains were tested the transformation frequency on Lablab purpureus.

A. rhizogenes strain strains were grown at 27 °C in either complete (TY) (Beringer, 1974)

media.

Construction of pCHF3-nopM-GFP

The open reading frame encoding NopM protein was subcloned in-frame with the

C-terminus of GFP in pCHF3 binary vector (Ge et al., 2005), to create GFP fusion

proteins under the control of the 35S promoter. To obtain pCHF3-nopM-GFP, nopM was

amplified by PCR from genomic DNA of NGR234 using the following primer pairs: A

(5’-CGAGTACTATGAATGTACAACGGCCCGG-3’) and B (5’-GTCTCGCCTTGGAT

92

CCTGCG-3’). The 1.6 kb fragment containing of nopM was first cloned into the EcoRV

site of pBluescript KS+ and verified by sequencing. KpnI and BamHI were used to excise

a nopM fragment and which was subcloned into the expression vector pCHF3-GFP.

Triparental matings were used to transfer the resulting plasmid into A. rhizogenes A4RSII,

K599 and LBA9402.

Root transformation using A. rhizogenes

For root transformation, L. purpureus and V. unguiculata were used and

transformation were performed as described in Estrada-Navarrete and associates

(Estrada-Navarrete et al., 2006). Plant seeds were sterilized as described in chapter 3, and

plants were grown 4 to 5 days until germination. Young seedlings with unfolded

cotyledons were infected at the cotyledonary node with A. rhizogenes carrying the gene

construct to be tested and the infection sites were kept in an environment of high

humidity. When the emerged hairy roots could support the plants, the main roots were

removed and the transgenic roots could be tested.

GUS staining

Hairy roots were placed whole in 90 % acetone on ice 20 min then washed with

cold sterile water. After fixation, roots were replaced in X-Gluc staining solution [2 mM

5-bromo-4-chloro-3-indolyl-β-d-glucuronide, 5 mM sodium phosphate, 2 mM potassium

ferrocyanide and 2 mM potassium ferricyanide] and incubated for 10 min at 37 °C.

Tissue was destained with 70 % (w/v) ethanol.

93

Table 1. Strains and plasmids used in this study

Strain Relevant characteristics Reference

Escherichia coli

DH5α supE44 ∆lacY169 ( 80lacZ∆M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1

BRL, Bethesdda, MD, U.S.A.

Saccharomyces cerevisiae

W303 MATa/MATα (leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15) [phi+]

(Fan et al., 1996)

Rhizobium strains

NGR234 Broad host-range bacterium isolated from nodules of Lablab purpureus, Rifr

(Lewin et al., 1990)

Agrobacterium rhizogenes

K599 Cucumopine-type strain (Cheon et al., 1993)

A4RSII Agropine-type strain (Jouanin et al., 1986)

LBA9402 Octopine-type strain (Hood et al., 1993)

8196 Mannopine-type strain Ri plasmid: pAr8196b (Hansen et al., 1992)

2659 Cucumopine-type strain (Brevet and Tempe, 1988)

15834 Agropine-type strain (Schiemann and Eisenreich, 1989)

Plasmids

pBluescript II KS+ High copy number ColE1-based phagemid, Apr Straragene, La Jolla, CA

pBDG98 pBluescript KS+ derivative carrying an nopM in EcoRV and XbaI site, Apr

W. Deakin, unpublished

pXBS23 Lorist 2 derivative containing the y4eF to y4gD region of pNGR234a, Knr

(Perret et al., 1991)

pFUS Yeast expression vector to create GFP fusions, LEU2, GAL10 promoter, high copy number

(Lesser and Miller, 2001)

pYES2 Yeast expression vector, URA3, GAL1 promoter, high copy number

Invitrogen

pFUS-nopM nopM cloned into pFUS as a 1.6 kb SacI-XhoI fragment. This work

pYES2-nopM nopM cloned into pYES2. This work

pYES2-nopJ nopJ cloned into pYES2. W. Deakin, unpublished pYES2-nopL nopL cloned into pYES2. W. Deakin, unpublished pYES2-nopP nopP cloned into pYES2. W. Deakin, unpublished pYES2-nopT nopT cloned into pYES2. W. Deakin, unpublished pYES2-nopT C93S nopT C93S cloned into pYES2. W. Deakin, unpublished pYES2-nopT H205A nopT H205A cloned into pYES2. W. Deakin, unpublished pYES2-nopT D220A nopT D220A cloned into pYES2. W. Deakin, unpublished pLG-GFPOAF1 Inserted GFP upstream of Oaf1, LEU2, GPD promoter M. MacLean,

unpublished. p35SGusInt Binary vector expressing GUS under the control of 35S

promoter, the Gus gene has an intron. (Vancanneyt et al., 1990)

pCHF3-GFP Binary vector to create GFP fusions C. Fankhauser, unpublished.

pCHF3-nopM-GFP Binary vector with nopM fused in frame with GFP This work

94

Results and Discussion

Expression of a NopM-GFP fusion in yeast

To determine where NopM localize in eukaryotic cells, NopM was expressed

tagged with GFP in S. cerevisiae strain W303. Yeast cells expressing the fusion protein

were observed by fluorescence microscopy after induction with galactose (Figure 1A).

Comparing the distribution of NopM-GFP with that of GFP alone (expressed from yeast

containing the vector only) showed that the un-tagged GFP had a diffuse distribution,

being present throughout the cytosol, in the nucleus, and sometimes in the vacuole of the

cell. In contrast, NopM-GFP concentrated in the nucleus and co-localized with Hoechst

staining of DNA (i.e. nuclei) at 1.5 h and 3 h after induction. This finding demonstrated

that NopM of NGR234 does preferentially localize within eukaryotic cell nuclei.

However, after 6 h of induction and all subsequent time points, GFP fluorescence could

be observed in throughout yeast cells containing NopM-GFP (data did not show),

suggesting that the ability of NopM to be trafficked to nuclei was lost. The ability of

NopM to be transiently localised in the nuclei would be an interesting adaptation

compared with YopM, which is stably located in the nucleus (Skrzypek et al., 2003). It

was equally possible that the NopM-GFP fusion could be degrading, however. To check

this, proteins were extracted from yeast cells and western-blots performed using an anti-

NopM antibody (Figure 1B). Although a NopM-GFP fusion protein of approximately 90

kDa could be detected after induction a band of approximately 60 kDa (presumably non-

tagged) NopM single band was also detected. Note that a yeast protein of approximately

60 kDa is detectable with the anti-NopM (or anti-rabbit secondary) antibodies, but with

the NopM-GFP fusions there is clearly an extra detectable band. Thus it appears that the

NopM-GFP fusion protein is unstable, even immediately after induction the fusion started

to degrade and after 6 hours the relative proportion of untagged GFP made microscopic

detection of the NopM-GFP fusion in the nucleus impossible. Thus to confirm the

localization of NopM in yeast cells, use of a yeast protease mutant (e.g. YPL 154c) might

reduce the degradation of the fusion protein. Although the fusion could simply be

inherently unstable and thus alternative techniques might be required.

95

DAPI GFPDAPI/GFP

Control

NopM-GFP fusion

DAPI GFPDAPI/GFP

Positive control

control

Positive control

NopM-GFP fusion

pFUS-NopM-GFP

83

62

kDa0 1.5 3 0 1.5 3

83

62

kDa

pFUS

A

B

Figure 1. A: Immunofluorescence microscopy of yeast expressing NopM-GFP proteins. Yeast (W303) carrying plasmids encoding galactose-inducible nopM-GFP fusion genes were visualized at 0 h, 1.5 h or 3 h after the addition of galactose. Yeast carrying plasmids, pFUS and pLG-GFPOAF1 were used for vector and positive control, respectively. Yeast cells were stained with DAPI to visualize DNA. DNA (blue) is shown in the first panel, GFP (green) is shown in the third panel and the middle panel represents the first two panels merged together such that the turquoise features represent co-localization of NopM-GFP (green) and DNA (blue). B: Immunological detection of NopM in yeast strains. Yeast proteins were extracted at 0 h, 1.5 h or 3 h after the addition of galactose. Then these proteins were separated by SDS-15 % PAGE and transferred to PVDF from SDS-PAGE onto PVDF membranes and probed with NopM antibody. For the control, pFUS vector were expressed in yeast. Note the presence of a non-specific band of approximately the same size as NopM in yeast cells expressing the vector control. As sample concentrations were equilibrated before loading the increased signal at about 60 kDa in samples represents GFP-less NopM.

96

Study of NopT in yeast

As discussed in the previous chapter, NopT is a member of the YopT family of

effectors which have a catalytic triad of amino acids (in YopT - C139, H258, and D274)

essential for function (and for YopT its cytotoxicity in yeast cells) (Shao et al., 2002). In

the homologue AvrPphB of P. syringae these residues are also essential for an auto-

proteolytic event, whereby AvrPphB is cleaved from its 35 kDa precursor form into a

mature peptide of 28 kDa. The cleavage event reveals a myristolation site, and it has been

shown in Arabidopsis that the mature form of AvrPphB is myristolated resulting in its

targeting to the plant plasma membrane (Nimchuk et al., 2000). By alignment NopT and

other rhizobial homologues of YopT/AvrPphB also have the conserved residues (Figure

2).

Various forms of NopT were expressed in yeast cells to study whether NopT

functions similarly to YopT and/or AvrPphB. In particular I examined whether NopT was

capable of autoproteolytic cleavage within yeast cells. NopT after secretion by NGR234

appears to be unprocessed (W. Deakin, unpublished) implying that to function as a

protease NopT might require some feature of the eukaryotic intracellular environment. To

facilitate this study a point mutant in a residue of the catalytic triad of NopT, NopT C93S,

(S. Ardissone, unpublished) was also used. If auto-proteolysis occurs the location of

NopT will be examined.

97

*: conserved catalytic core : other conserved residues

10 20 30 40 50 60 70. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

NopT ----------- MHSPI SGSFTSSTQVHDPI HPANSDGFRETLANVELRTKSPSAECPDKMGCCASKP---blr2140 ----------- MYDRI GGSSTRTSQTDEPSQSVDSGSFTETLADLAPQWSSRSGELPDKMGACCSKPDTLblr2058 ----------- MYNRVDGEYAHTEQAEESSWPADGSECAQTLTEI ARLESLAPGELFDRMGLCFSKPHTSAvrPphB ----------- MKI GTQATSLAVLHNQESHAPQAPI AVRPEPAHAI PEI PLDLAI RPRTRGI HPFLAMTLYopT MNSI HGHYHI QLSNYSAGENLQSATLTEGVI GAHRVKVETALSHSNLQKKLSATI KHNQSGRSMLDRKLT

80 90 100 110 120 130 140. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

NopT ---- QASDPNNPSTSSPAR------------ PSTSLFRYR---------------- TAELAQANADGI CVblr2140 DANVQTSSASEPSTSSPES------------ PATSLFEYR---------------- TADLRDANVDGI CVblr2058 DAI DDSSNTSGLSTSSLSSSSELSVAT--- SPVRPLFDYR---------------- TAELPQANVSGI CVAvrPphB GDKGCASSSGVSLEDDSHT------------- QVSLSDFS---------------- VASR- DVNHNNI CAYopT SDGKANQRSSFTFSMIMYRMI HFVLSTRVPAVRESVANYGGNI NFKFAQTKGAFLHKII KHSDTASGVCE

150 160 170 180 190 200 210. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

NopT GLTAEWLRNLNS- HPSI RMEALVPGSQRHASATVRQKEY- ENLKVHLRRQGAGPSEADFAAQNTMLQKAGblr2140 GLTAEWFRNLSN- SPSTRMSALTPGSQTHASAAERQQQY- QRLKDQLRSRGAGSSQADLQAQNTIL EEAGblr2058 GLAAEWLL DLPS- SASSRMGVLLPGTENHRSAARRQEQS- EKLKTQLKEDKAEGS- HNFQAKSTIL RDAGAvrPphB GLSTEWLVMSSDGDAESRMDHLDYNGEGQSRGSERHQVYNDALRAALSND--- DEAPFFTASTAVI EDAGYopT ALCAHWI RSHAQ-- GQSLFDQLYVGGRKGKFQI DTLYSI KQLQI DGCKADVDQDEVTLDWFKKNGI SERM

220 230 240 250 260 270 280. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

NopT LAPSGK- EKVYKVGEP--- NFPRMLT- KI TADGSNHLL SLYFAEG-- GAHTVATSAMDGN- TTLFDPNFGblr2140 LEPAGE- EKRFAFGKSS-- NVKSMVN- EI NEDGSNHLL SLYFAEG-- GAHTVATSASNGT- TTLFDPNYGblr2058 LEPSAE- ETRYRFGTSS-- CI DKIV N- ELAQDPSVHLVSLKFVQPGAGTHTI ATATSNGT- TIL SDPNYGAvrPphB FSLRREPKTVHASGGSA-- QLGQTVAHDVAQSGRKHLL SLRFANV-- QGHAI ACSCEGSQ- FKLFDPNLGYopT I ERHCLL RPVDVTGTTESEGLDQLL NAIL DTHGI GYGYKKI HLSGQMSAHAI AAYVNEKSGVTFFDPNFG

290 300 310 320 330. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . .

NopT EFTVQSD-- QI DDLFRSLANRYSNPNRQHLTTVTTQKMT-------------------blr2140 EFTVRSDPDQMASLL QSLANRYRNPNGQHLSTI TTQRMQ-------------------blr2058 EFTVPSD-- RVGGLFKSLAERYSTLNKRDI SAVVTQRI RYGHPNATDLALFPRAEPHRAvrPphB EFQSSRS-- AAPQLI KGLI DHYNSLN- YDVACVNEFRVS-------------------YopT EFHFSDK---- EKFRKWFTNSFWGNSMYHYPLGVGQRFRVLTFDSKEV----------

*

* * *** *

**

**

*

: cleavage site and myristoylation sites (glycine residue)

Figure 2. Multiple sequence alignment of representative members of the YopT family of effectors. Members were identified by PSI-BLAST searches and aligned with the Bio Edit program. The following members are shown; NopT from NGR234, blr2140 and blr2058 from B. japonicum, AvrPphB from Pseudomonas syringae pv. phaseolicola and YopT from Y. pestis. The putative catalytic core residues of cysteine, histidine, and aspartic acid are boxed. Other invariant residues are marked with asterisks. The potential cleavage and myristoylation sites (glycine 61) of the rhizobial homologues are indicated with red triangles.

When NopT and NopT C93S were expressed in yeast cell, western-blot analysis

showed an interesting profile (Figure 3). Use of the NopT antibody was complicated by a

relatively high background of non-specific protein detection, but was easily clarified by

comparing the size of expressed proteins to NopT present in secreted proteins isolated

from cultures of NGR234. Thus when wild-type NopT was expressed in yeast after 4

98

hours a band of approximately 28 kDa was visible of the same size as NopT secreted by

NGR234. A second smaller band of approximately 23 kDa was also detected.

Furthermore at 7 hours post induction only this smaller band was detectable. When the

point mutant, NopT-C93S, was expressed and only a band of 28 kDa band was detected

which is probably the unprocessed form of NopT. Thus this result demonstrated that not

only can NopT cleave itself, but when one of the predicted amino acids essential for

protease activity is mutated, the cleavage does not happen. Thus at least this residue of

the predicted catalytic triad is essential for the cleavage of NopT.

NopT

pYES2-nopT

T0 T4 T7 T0 T4 T7 T0 T4 T7

pYES2-nopT C93S pYES2

NG

R2

34

Figure 3. Detection of NopT and NopT-C93S in yeast strains. Yeast proteins were extracted at 0 h, 4 h and 7 h after the addition of galactose. Then these proteins were separated by SDS-15 % PAGE and transferred to PVDF from SDS-PAGE onto PVDF membranes and probed with NopT antibody. For the control, NGR234 supernatant protein and expressing pYES2 vector in yeast were used. Red arrow and triangle indicate full-length of NopT and cleavage NopT bands, respectively.

Unfortunately further experiments to determine the location of NopT after

autoproteolytic cleavage did not reveal a plasma membrane site (data not shown). Most

likely there are differences between the ability of yeast and plant cells to myristolate

99

proteins. The yeast system could however be used to purify the cleaved form of NopT

and to determine whether the cleavage site is where it is predicted to be.

Effects of Nops on yeast growth

To test whether the expression of Nops within yeast affected cell growth and thus

could confer genetically manipulatable growth phenotypes, yeast cells were transformed

with each of the nop genes without any tags (Figure 4A). As YopT is known to be toxic

to yeast growth and based upon the previous results showing NopT is a functional

protease inside yeast cells, all the nopT point mutants in the catalytic triad were assayed.

Expression of the Nops after galactose induction was confirmed by immuno-blotting

(Figure 4B). In yeast expressing NopJ, NopP, NopM and NopT (and the NopT point

mutants) after galactose induction no growth inhibitions were observed. All these strains

grew similarly to a strain expressing vector alone. NopL expression, however conferred a

severe inhibition to growth. When NopL was induced in yeast cell, yeast growth was

completely prevented. Thus NopL must have a function which is a toxic for eukaryotic

cell. It is possible that NopL interferes extensively with signalling pathways within yeast

cells causing this toxicity. NopL is an excellent substrate for plant Ser/Thr kinases and

NopL is phosphorylated by MAP kinases (Bartsev et al., 2003). This phenotype of NopL

within yeast cells, opens up the possibility of functional screens to identify key

domains/residues in NopL important for its function. To identify regions of NopL which

is responsible for growth inhibition, nopL point mutants or deletion mutants will be

generated and tested in yeast cells. Mutated forms of NopL that permit yeast growth after

galactose induction, could reveal an “active site” of NopL. The corresponding nopL

mutants could then be tested for their nodulation ability on F. congesta (Marie et al.,

2003). Screening libraries of yeast mutants with wild-type NopL for strains that are also

capable of growth may indicate the cellular process(es) affected by NopL and thus a

potential enzymatic function.

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pYES2-nopP

Anti-NopP

Anti-NopL

NGR T0 T4 T7

pYES2-nopL

NGR T0 T4 T7

B2% Glucose 2% Galactose

pYES2-nopM

pYES2

pYES2-nopJ

pYES2-nopP

pYES2-nopL

pYES2

pYES2-nopT

pYES2- C93SnopT

pYES2- H205AnopT

pYES2- 220AnopT D

pYES2

A

10 fold dilutions

Figure 4. Effects of Nops on yeast growth. A: Yeast (W303) carrying plasmids encoding galactose-inducible nop genes were grown overnight in non-inducing selective synthetic media containing raffinose as a carbon source. Cultures were then normalized to OD600 = 0.1 and serial 10-fold dilutions were spotted onto selective media plates containing glucose (2 %) and galactose (2 %). For the control, pYES2 vector were used. B: Detection of NopL and NopP in yeast strains. Yeast proteins were extracted at 0 h, 4 h or 7 h after the addition of galactose to confirm the presence of Nops. Then these proteins were separated by SDS-15 % PAGE and transferred to PVDF membranes and probed with NopL or NopP antibody. For the control, proteins were extracted from yeast containing only the pYES vector.

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Expression of Nops within legumes - root transformation using A. rhizogenes

Susceptibility of NGR234 hosts to A. rhizogenes

Ideally the results from the Nop expression in yeast experiments would be

confirmed using legumes and more particularly legumes known to be hosts for NGR234.

Few such legume expression systems exist and thus as a test case, I attempted to

determine whether NopM was also targeted to legume nuclei. As NopM has a positive

role in the nodulation of L. purpureus, this plant was selected. Previous studies had

showed that another NGR234 host V. unguiculata, could be infected by some A.

rhizogenes strains, with A4RSII the most virulent (A. Krause, unpublished). Several A.

rhizogenes species were tested for their transformation ability on L. purpureus. V.

unguiculata was also re-tested for infection by A. rhizogenes A4RSII and K599 strains

and it was reaffirmed that A4RSII had high efficiency (100 %), and formed on average 6

and 18 hairy roots at 2 and 4 weeks after infection respectively. The susceptibility of L.

purpureus was tested with 6 difference A. rhizogenes species after 2 and 4 weeks

injection. Only K599, A4RSII and LBA9402 strains induced hairy root formation at 2

weeks (Figure 5). One to two roots per wound were produced with these A. rhizogenes

strains. The frequencies increased at 4 weeks after injection, with even 2659 and 15834

strains produced hairy roots. Highest frequencies to induce hairy roots were observed

when L. purpureus was infected with A. rhizogenes K599.

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0

1

2

3

4

5

6

K599 A4RSII LBA9402 8296 2659 15834

Ave

rage

nu

mb

erof

har

iry

root

sp

erp

lan

t

2 weeks 4 weeks

Figure 5. Root transformation using various A. rhizogenes on L. purpureus. A: L. purpureus plant was infected with K599 and hairy roots could be seen emerging from globular tumours formed at wounded sites. B: Average number of hairy roots per plant with various A. rhizogenes strains. Hairy roots were counted at 2 or 4 weeks after infection.

Transformation efficiency

Although hairy roots were visible, it was necessary to verify that hairy roots could

be co-transformed with DNA from a binary vector. Thus a reporter system was used to

test this, a binary vector containing a 35S-gusA-intron cassette was integrated into A.

rhizogenes K599, A4RSII and LBA9402 strains by triparental mating. The construct 35S-

gusA-intron contains the β-glucuronidase (GUS) gene under the control of the 35S

promoter and the gusA gene contains a plant intron so it cannot be expressed by

agrobacteria (Vancanneyt et al., 1990). L. purpureus were infected with A. rhizogenes

K599, A4RSII and LBA9402 strains contain p35S-gusA-intron and individual hairy roots

were excised and stained for GUS activity 4 weeks A4RSII infection (Figure 6A). V.

unguiculata was also infected with A. rhizogenes A4RSII strains as a positive control.

The transformed hairy roots were obtained with K599 and A4RSII strains on L.

purpureus, the efficiencies were 33.8 % + 23 % and 38.5 % + 9.5 %, respectively. The

transformed efficiency of V. unguiculata with A4RSII strain (52.8 % + 31.4 %) was

higher however. Compared to V. unguiculata, a high frequency transformed hairy root

103

was not obtained with L. purpureus, however an attempt was made to express a NopM-

GFP fusion in L. purpureus roots (see next section).

0

10

20

30

40

50

60

70

80

90

K599 A4RSII LBA9402 A4RSII

L. purpureus V. unguiculata

Tra

ns

form

edh

air

yro

ots

(%)

0

10

20

30

40

50

60

70

80

90

K599 A4RSII LBA9402 A4RSII

L. purpureus V. unguiculata

Tra

ns

form

edh

air

yro

ots

(%)

Figure 6. Root transformation efficiency on L. purpureus and V. unguiculata. A: GUS staining of hairy roots. Hairy roots were formed by injection with K599 on L. purpureus and stained to see GUS expression. B: Transformed hairy roots per plant (%). Various A. rhizogenes strains were tested the transformation frequency. The hairy roots were collected 4 weeks after infection.

Transformation of pCHF3-nopM-GFP

A. rhizogenes A4RSII and K599 strains containing pCHF3-nopM-GFP were

infected into L. purpureus to see the localization of NopM in plant root cells. NopM is

fused to GFP protein for observation by immunofluorescence microscope with the empty

vector pCHF3-GFP used as a control. In an initial attempt the hairy roots which were

infected with A. rhizogenes A4RSII and K599 strains containing pCHF3-nopM-GFP

showed GFP green fluorescence, however the localization of GFP did not show any

difference compare to vector control (data not shown). Further attempts are required to

refine this technique, and in particular it should be confirmed that NopM-GFP fusion

protein is not degraded after expression in hairy roots.

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Conclusions

Nops were expressed in yeast cells as to five an initial insight into their potential

molecular functions. A NopM-GFP fusion localized to the yeast nucleus 3 h after

induction however the fusion protein was too unstable to be used in further experiments.

For example it would have been ideal to investigate potential nuclear localisation signals

using yeast. Perhaps the use of yeast mutants lacking certain protease would improve the

stability of the NopM-GFP fusion. Immunofluorescence experiments could be performed

to verify the nuclear location of NopM, but are probably too time consuming to screen for

possible NLS. Unlike their Yersinia homologues, neither NopJ nor NopT were toxic to

yeast cells. NopT however was shown to be capable of cleaving itself after expression in

yeast cell, which was verified as the NopT point mutant C93S lost this enzymatic activity.

Perhaps the most useful result to come from the yeast expression assays was that NopL

was extremely toxic for yeast growth. A phenotype that will permit the use of yeast in

functional screens for the domains/residues of NopL responsible for this toxicity and

possibly even the yeast processes effected.

Attempts to express Nops in Arabidopsis were unsuccessful (data not shown), the

reasons for this are unclear but presumably the action of the Nops was detrimental to

plant viability. Preliminary experiments were performed to establish a root

transformation system in legume hosts of NGR234. Several A. rhizogenes strains were

tested for their ability to infect L. purpureus and then for their ability to co-transfer DNA

from a binary vector. For L. purpureus, A. rhizogenes K599 and A4RSII strains showed

high transformation frequency. An initial attempt to express a NopM-GFP fusion in L.

purpureus roots was unsuccessful and significant experimental work remains to establish

such an experimental model.

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Chapter 5: Perspectives

Rhizobium sp. NGR234 establishes a symbiotic interaction with many legume

plants. The primary signal molecules produced by NGR234 have been extensively

studied. A notable feature is the great variety of Nod factor (NF) structures produced and

also the quantity secreted (Broughton et al., 2000). It is apparent though that NFs are not

the only signal molecules and NGR234 also produces a variety of so called secondary

signaling factors, which although not universally essential, are critical for certain

NGR234-host interactions. The work in this thesis details the further characterization of

one of these secondary signals, the T3SS.

Prior to this work it was already known that the T3SS was regulated within the

flavonoid inducible cascade of NGR234 (Kobayashi et al., 2004). My preliminary

projects contributed to the understanding of the TtsI-mediated induction of genes

preceded by tts-boxes, by complementing the NGR∆ttsI mutation (Wassem et al., 2008).

The symbiotic regulatory cascade which is trigged by plant produced flavonoids is

intricate and numerous factors participate including several transcriptional regulators to

control the coordinated synthesis of (at least) Nod-factors, polysaccharides as well as the

T3SS. Approximately half of my studies concerned other potential regulatory influences

on the flavonoid-induced cascade. Particularly the effects of the NodV (sensor) and

NodW (regulator) two-component system that in other rhizobia are key players in the

perception and response to flavonoids. In B. japonicum these regulators are thought to

perceive certain classes of flavonoids and are critical for the nodulation of certain

legumes (Göttfert et al., 1990). Of direct relevance to my work was the essential role of

NodVW in the regulation of the T3SS of B. japonicum potentially by activating ttsI

(Krause et al., 2002). Homologues of nodVW had been identified on the megaplasmid of

NGR234 (Streit et al., 2004) and in this work these were mutated and the effects on the

T3SS and other TB-controlled genes was assessed. There was no obvious effect on either

Nop secretion or the production of rhamnose-rich LPS after mutation of nodVW from

NGR234 and thus they do not have the same function as NodVW from B. japonicum.

Experiments testing NB- and TB-reporter gene fusions in the nodVW mutant, showed that

in some conditions there was a reduction in (flavonoid) induction. Although, as discussed

106

in chapter 2, this was more likely caused by a general effect of NodVW on key symbiosis

regulators, such as NodD1, SyrM2 and NodD2, as it seems there was an early effect of

NodVW on these main regulators. For this reason, attempts to follow the expression of

NB and TB controlled genes was extremely complicated using reporter genes, and other

methods are required to clarify some of these results. In particular to see if NodW binds

to any of the promoters of the other transcriptional activators, expanding on work already

described in chapter 2. Further studies will hopefully determine whether the NodW binds

to the promoters of nodD1, nodD2 and/or syrM1 using EMSA. From these results it

should be possible to see at what point in the regulatory cascade NodVW act.

It was not possible to define the exact biological role of NodVW in NGR234. A

striking observation was the dramatic increase in nodD1 and syrM2 expression in the

absence of flavonoids in the nodVW mutant. As discussed earlier, NodV & NodW are

members of the two-component sensor-regulator family of proteins. NodV shares

homology with sensor proteins. In typical two-component systems, the sensor histidine

protein kinase monitors external stimuli and transmits this information to the response

regulator protein by a phosphorylation step (Pan et al., 1993; Surette et al., 1996). As

flavonoids are the signaling molecules that trigger symbiosis between legumes and

rhizobia, does NodV detect flavonoids, as suspected in B. japonicum? The effects of

NodVW were observed on the nodD1 promoter and NB19 even without the addition of

flavonoids. On the other hand, the expression pattern of pnodD2 did not follow the NB19

expression profile as increased amounts of (NB19-controlled) SyrM2 should up-regulate

nodD2 after flavonoid induction. From these data and other conflicting expression

patterns, I hypothesised that NodV could possibly sense the concentration of flavonoids

in the environment. By monitoring when flavonoid concentrations are sub-optimal it

would activate NodW to bind to key regulatory promoters to suppress their expression

and thus prevent the production of NFs or other signalling molecules when NGR234 is

not in close proximity to the plant root. The NodVW system must have some role in the

repression of the nodD1 and syrM2 promoters, as their expression is increased in the

nodVW mutant background. NGR234 could thus have adapted the NodVW flavonoid

sensing system to prevent wasteful production of symbiotic signaling molecules. Other

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sensing systems could override the NodVW repression system as flavonoid

concentrations increase to symbiotically proficient levels. Experiments should be

performed measuring the expression of various reporter genes at sub-optimal flavonoid

inducing conditions, especially in a nodVW mutant background.

An interesting consequence of my attempts to identify gene regulated specifically

by NodVW led to my involvement in a project to set-up a high-throughput reporter gene

system in NGR234. As detailed in chapter 2, NB- and TB-containing promoters were

fused upstream of the GFP reporter gene allowing gene expression levels to be measured

in vivo without extensive manipulative steps. The direct measurement of GFP expression

in cells, permits reporter assays to be performed in 96-well microplate format and thus

allows a greater number of possible promoters or mutant backgrounds to be assayed at

the same time. It was not clear whether the growth of NGR234 derivatives would be

affected in the smaller volumes present in the 96-well plates, thus as part of my

experiments to measure the expression of key promoters in the nodVW mutant, I

compared the relative expression levels with the β-galactosidase reporter system used

extensively in this laboratory. As described in chapter 2, generally expression levels

monitored using the two reporter systems generally followed the same trends both

temporally and in different mutant backgrounds and thus the high-throughput assay

system seems robust enough to study expression of NGR234 genes (Le Quéré et al.,

2008).

The second part of my thesis was concerned with the study of effector proteins

secreted by the T3SS of NGR234. T3SS effector proteins generally alter cell signalling

and thus host responses induced upon infection. However, their precise biochemical

functions are often difficult to define. In the absence of a suitable legume model to study

effector function, other eukaryotic cells were used to try to identify conserved signalling

pathways targeted by the NGR234 effectors. Finally the roles of individual effector

proteins were assessed on legumes previously shown to be responsive to the functionality

of the NGR234 T3SS.

108

I particularly focused on the effector NopM, initially identified from the

sequencing of pNGR234a as y4fR with homology to yopM of Y. pestis (Freiberg et al.,

1997). Using a specific antibody I demonstrated that NopM was secreted through the

T3SS and constructed a mutant, which was still capable of secreting other Nops, and thus

confirmed that NopM was a candidate NGR234 effector protein. During my study, the

exact function of this family of effectors was not known although many had been shown

to localize in the nuclei of mammalian cells, which was something I also tried to show for

NopM (see below). Recently however, a homologue of NopM has been shown to be an

ubiquitin ligase (Rohde et al., 2007). Furthermore a cysteine residue, conserved in all the

members of this effector family, was shown to be essential for ubiqutin ligase activity in

vitro. As NopM has a correspondingly conserved Cys residue, to confirm whether NopM

also acts as an ubiquitin ligase, nopM should be point mutated at the bases encoding the

Cys residue. The ability of NopM and the point mutant to add ubiquitin in vitro will be

assayed using yeast cells, as described by Rohde and associates (Rohde et al., 2007). The

point mutant will be checked for its secretion by the T3SS and then its nodulation ability

also tested on L. purpureus comparing with wild-type and nopM deletion mutant, to

determine whether its presumed ubiquitin ligase activity is essential for the positive effect

of NopM in the nodulation of this plant.

Initially I wanted to determine whether NopM is also transported into the nucleus

of legume cells. In the absence of a plant cell culture system to test this, I used an

alternative eukaryotic model system, as yeast cells had shown to be capable of modelling

the roles of effector proteins (Lesser and Miller, 2001). As described in chapter 4, I

developed the use of yeast cells to study NopM localisation and also other NGR234

effectors. NopM was shown to localise in yeast cell nuclei, although degradation of the

NopM-GFP fusion protein complicated visualisation, a yeast strain which has lost some

protease activity (e.g. YPL 154c) will be used to avoid the degradation of fusion protein

or in situ hybridization techniques/immunofluoresence will help. Once NopM

localization in the host nucleus is confirmed, in turn the identification of the host

perception mechanism to transit is an exciting future topic. NopM lacks a classical NLS

and thus how is NopM trafficked to nucleus? It was shown that the first three LRRs and

109

carboxy-terminal end (C-ter) of YopM act as NLSs in yeast (Benabdillah et al., 2004).

Constructing multiple NopM deletion mutants will elucidate which domains or residues

are responsible for the migration of NopM to the nucleus of eukaryotic cells. Yeast cells

are of course only a model system, and the results obtained should also be verified in

legume cells. Attempts were made to development a root transformation protocol for L.

purpureus, but relatively low transformation efficiencies made this impractical. Further

studies could be made to improve Lablab transformation, perhaps by trying different A.

rhizogenes strains or growth conditions. The model plant Arabidopsis was also used but it

was not possible to obtain transformed plants, possibly high levels of nop expression

from the 35S promoter were toxic to the plants. Transgenic plants expressing inducible

nopM could be created and thus the location of NopM now checked intracellularly. An

advantage of using Arabidopsis would be to easily identify plant proteins that bind to

NopM (perhaps to traffic it to the nucleus) by established techniques such as yeast 2-

hybrid assays or immunoprecipitation followed by mass spectrometric identification of

interacting plant proteins.

The yeast eukaryotic model system was also used to test the other NGR234

effectors, although expression of NopJ and NopP in yeast cells had no clear phenotypes,

both NopL and NopT provoked changes to yeast cells that could be used as screens to test

for functional domains/residues of these effectors. As described previously, after NopL is

delivered into plant cells, it most likely modulates the activity of signal transduction

pathways that culminate in activation of PR proteins (Bartsev et al., 2004). Expression of

NopL within yeast cells led to a severe growth inhibition. Initially sections of NopL

could be deleted and then these expressed in yeast to see if a particular domain of NopL

is responsible for its apparent toxicity. Assuming that the phosphorylation of NopL is

essential for its ability to disrupt plant kinases, it is possible that this function of NopL

also causes the yeast growth inhibition. Thus by using error-prone PCR, point mutations

in nopL could be generated, then this population could be cloned into a yeast expression

vector, transformed into yeast and then expression induced. Yeast colonies capable of

growth will most likely contain nopL mis-sense mutations or premature stop codons,

which can easily be identified by sequencing. It is also possible that the residues normally

110

phosphorylated could have been mutated preventing phosphorylation, and thus the

disruption to signaling pathways. These candidate mutants could be tested for their

phosphorylation in vitro, and if this is absent then on the legumes F. congesta and T.

vogelii to assess their symbiotic proficiency. In the case of NopT, the presence of key

residues was already suspected from comparison to AvrPphB. The AvrPphB protein from

Pseudomonas syringae was demonstrated to be targeted to the host plasma membrane

after cleaving itself. The cleavage event exposes a myristoylation sites, which after

modification by plant proteins is responsible for the membrane location (Nimchuk et al.,

2000). Expression of a NopT-GFP fusion within yeast cells led to the visualization of

smaller that expected products indicative of a possible auto-proteolytic event (data not

shown). Subsequent experiments expressing only NopT and a NopT point mutation in a

residue critical for its protease activity showed that this autoproteolytic cleavage could

occur (as shown in chapter 4). Further experiments could purify the processed form of

NopT to identify the cleavage site.

Finally in NGR234, the demonstration that NopM is an effector protein brings the

total to five effector proteins which have been identified. Our present hypothesis is that

these are the only effector proteins encoded on pNGR234a, based upon homology

searches and the presence of tts-boxes. Although the T3SS of NGR234 has been shown

to be an important factor in the nodulation of several legumes, the role of individual

effector proteins has not been determined on all these legumes. Thus in chapter 3 when

the symbiotic effects of NopM were being tested, the other four effector mutants were

also included. As described earlier it seems that when the T3SS has a positive effect on

the nodulation of, it actually appears that there is a varied response to the individual

effectors. Some of the effectors appear to be important for nodulation, whilst others are

negative-acting or with little clear role. Thus in this situation the phenotype of the T3SS

appears to be an equilibrium in the action of the individual effector proteins, and is also

indicative that (at least) all five effectors are translocated into the target plant cells. The

situation appears to be different on plants that respond negatively to the T3SS, for

example in the case of C. juncea one of the effectors (NopT) appears to be recognised as

very strongly negative, whereas the other effectors have little phenotype individually,

111

although when double mutants were created in a nopT mutant background, subtle positive

effects of the other effectors (e.g. NopP) were seen (K. Kambara and W. Deakin,

unpublished). It is also tempting to speculate, based upon its strong negative phenotype

and homology to Avr proteins of phytopathogens, that NopT is recognised by a resistance

protein of C. juncea that normally protects against pathogen attack. Although it remains

to be determined whether there is an equivalent hypersensitive type response on the roots

of this legume. The effector responsible for the negative effect of the T3SS on P.

tuberosus was not identified, suggesting that genes encoding other effector proteins are

present in the genome of NGR234. The search for these genes remains an open avenue of

research, one possibility is to use a Tn5 transposon (Wilson et al., 1995) to randomly

mutagenesis NGR234 and then inoculate this mutant population onto P. tuberosus and

select plants that are obviously green and thus nodulated. In this way the gene encoding

the negative effector could have been mutated by the transposon, however in practice this

strategy only identified mutants in the T3SS machinery (Skorpil, unpublished). Thus

alternative strategies need to be developed, bioinformatic approaches are often extremely

useful, based upon the premise that a tts-box should precede any genes encoding potential

effector proteins, upon completion of the NGR234 genome searches for tts-boxes will be

made. Candidate genes will be characterised as described in chapter 3, although the

identification of their exact molecular functions will no doubt continue to be an

extremely difficult subject.

112

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Xiao, H., S.J. Shen, and J.B. Zhu. 1998. Binding of activator SyrM to the site of nodD3 P1 region of Rhizobium meliloti. science in china series c-life scienves. 41:157-162.

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Zhu, H., B.K. Riely, N.J. Burns, and J.M. Ane. 2006. Tracing nonlegume orthologs of legume genes required for nodulation and arbuscular mycorrhizal symbioses. Genetics. 172:2491-9.

128

PUBLICATION LIST

Publication in international journal

Wassem, R., Kobayashi, H., Kambara, K ., Le Quéré, A., Walker, G.C., Broughton, W.J. &

Deakin, W.J. (2008) TtsI regulates symbiotic genes in Rhizobium species NGR234 by binding to

tts boxes. Mol. Microbiol. in press.

Publication in conference proceedings

Boukli, N.M., Deakin, W.J., Kambara, K , Kobayashi, H., Marie, C., Perret, X., Le Quere, A.,

Reuhs, B., Saad, M., Schumpp, O., Skorpil, P., Staehelin, C., Streit, W. & Broughton, W.J.

(2005). Rhizobial control of host-specificity. In ¨Biological Nitrogen Fixation, Sustainable

Agriculture and the Environment¨, p. 217-218, Wang, Y.-P., Lin, M., Tian, Z.X., Elmerich, C.,

and Newton, W.E. (Eds.) Springer, Dordrecht, The Netherlands.

Conference presentations

William J. Deakin, Silvia Ardissone, Kumiko Kambara , Patricia Lariguet, Olivier Schumpp,

and William J. Broughton.

The role of Avirulence-like proteins in the Rhizobium-legume symbiosis

17th Swiss Plant Molecular and Cell Biology Conference, 6-8 February, 2008, Les Diablerets,

Switzerland.

William J. Deakin, Silvia Ardissone, Jeremie Gay-Fraret, Kumiko Kambara , Patricia Lariguet,

Antoine le Quéré, Olivier Schumpp, Roseli Wassem and William J. Broughton.

The role of Avirulence-like proteins in the Rhizobium-legume symbiosis

20th North American Symbiotic Nitrogen Fixation Conference, 10-14 July, 2007, Milwaukee,

USA.

W.J. Deakin, S. Ardissone, J. Gay-Fraret, K. Kambara , P. Lariguet, A. le Quéré, O. Schumpp, R.

Wassem & W.J. Broughton.

129

Symbiotic rhizobia use pathogenic-like protein secretion systems to establish an intracellular life

within plant roots

160th Meeting of the Society for General Microbiology, 26-29 March, 2007, Manchester, UK.

W.J. Deakin, S. Ardissone, J. Gay-Fraret, K. Kambara , P. Lariguet, A. le Quéré, O. Schumpp &

W.J. Broughton

Type III protein secretion systems in the Rhizobium-legume symbiosis

66th Annual Assembly of the Swiss Society for Microbiology, 1-2 March 2007, Interlaken,

Switzerland.

W.J. Deakin, S. Ardissone, J. Gay-Fraret, K. Kambara , P. Lariguet, A. le Quéré, O. Schumpp &

W.J. Broughton

The role of type III protein secretion systems in the Rhizobium-legume symbiosis

7th European Nitrogen Fixation Conference, 22-26 July 2006, Aarhus, Denmark.

W.J. Deakin, S. Ardissone, J. Gay-Fraret, K. Kambara , P. Lariguet, A. le Quéré, M. Saad, O.

Schumpp, P. Skorpil & W.J. Broughton

Type III Secretion Systems Affect the Rhizobium-Legume Symbiosis

ASM-FEMS Conference on Protein Traffic in Prokaryotes, 6-11 May, 2006, Crete, Greece.

K. Kambara , M. MacLean, W.J. Broughton & W.J. Deakin

Characterisation of NopM an effector protein secreted by Rhizobium species NGR234

15th Annual meeting of Japanese Society of Plant Microbe Interactions, 10-12 September, 2005,

Kagawa, Japan.

130

Acknowledgements

I would like to express my sincere appreciation to Dr. William J. Dearkin, my supervisor supporting all my study, and also giving me opportunities, providing guidance,

constant support and invaluable kindness.

I would like to express my sincere appreciation to Prof. William J. Broughton, for supporting my study and also giving me opportunities, providing guidance and advices.

My special thanks are expressed to Dr. Morag MacLean for the supporting and giving advices the yeast work.

My special thanks are expressed to Dr. Hajime Kobayashi and Dr. Roseli Wassem for their invaluable kindness aid and helps for this study.

Thanks to all members of LBMPS for their support and sharing great times. Many thanks to Prof. Xavier Perret, Dr. Michele Crevecoeur, Dr. Olivier Schumpp, Dr. Silvia Ardissone, Dr. Antoine Le Quéré, Dr. Patricia Lariguet, Dr. Maged Saad,

Dr. Sonia Guimil and Ms. Nadia Bakkou for their advice and kindness. Special thanks to Mme. Dora Gerber, Mme. Yin Yin Aung, Mme. Florenea Ares,

and Mme. Rosa Pimenta de Abreu for their kindness and help.

Finally, I am grateful to my family and all my friends for their love, understanding and giving me the encouragement.

TtsI regulates symbiotic genes in Rhizobium species NGR234by binding to tts boxes

Roseli Wassem,1,2 Hajime Kobayashi,1,3

Kumiko Kambara,1 Antoine Le Quéré,1

Graham C. Walker,3 William J. Broughton1 andWilliam J. Deakin1*1Laboratoire de Biologie Moléculaire des PlantesSupérieures (LBMPS), Sciences III, 30 QuaiErnest-Ansermet, Université de Genève, CH-1211Geneva 4, Switzerland.2Departamento de Genética, Universidade Federal doParaná, Caixa Postal 19071, CEP 81531–990, Curitiba,PR, Brazil.3Department of Biology, Massachusetts Institute ofTechnology, 77 Massachusetts Avenue, Cambridge,MA 02139, USA.

Summary

Infection of legumes by Rhizobium sp. NGR234 andsubsequent development of nitrogen-fixing nodulesare dependent on the coordinated actions of Nodfactors, proteins secreted by a type III secretionsystem (T3SS) and modifications to surface poly-saccharides. The production of these signal mol-ecules is dependent on plant flavonoids which triggera regulatory cascade controlled by the transcriptionalactivators NodD1, NodD2, SyrM2 and TtsI. TtsI isknown to control the genes responsible for T3SSfunction and synthesis of a symbiotically importantrhamnose-rich lipo-polysaccharide, most probably bybinding to cis elements termed tts boxes. Eleven ttsboxes were identified in the promoter regions oftarget genes on the symbiotic plasmid of NGR234.Expression profiles of lacZ fusions to these tts boxesshowed that they are part of a TtsI-dependent reguloninduced by plant-derived flavonoids. TtsI was purifiedand demonstrated to bind directly to two of thesetts boxes. DNase I footprinting revealed that TtsIoccupied not only the tts box consensus sequence,but also upstream and downstream regions in aconcentration-dependent manner. Highly conservedbases of the consensus tts box were mutated and,

although TtsI binding was still observed in vitro, gfpfusions were no longer transcribed in vivo. Randommutagenesis of a tts box-containing promoterrevealed more nucleotides critical for transcriptionalactivity outside of the consensus.

Introduction

Symbioses between legumes and rhizobia which result inthe formation of nitrogen-fixing root nodules are the resultof a complex signal exchange between both partners.Initially, flavonoids exuded by the plant trigger synthesis ofNod factors (NF) that are secreted from the bacteria andare critical for rhizobial infection (Broughton et al., 2000;Perret et al., 2000). Nevertheless, establishment of func-tional nitrogen-fixing nodules requires other bacterialsignals such as surface polysaccharides and secretedproteins (Fraysse et al., 2003; Broughton et al., 2006;Soto et al., 2006; Jones et al., 2007). Rhizobium sp.NGR234 (hereafter NGR234) is the most promiscuousknown microsymbiont, capable of establishing symbioseswith more than 112 genera of legumes (Pueppke andBroughton, 1999). Its genome is partitioned into threereplicons: the chromosome and two large plasmids,pNGR234a and pNGR234b (Viprey et al., 2000).pNGR234a is also called the symbiotic plasmid as it con-tains all genes necessary for NF synthesis and nitrogenfixation. Sequencing of this 536-kb plasmid showed that italso contains orthologues of a type III protein secretionsystem (T3SS) (Freiberg et al., 1997). Until this discovery,such secretion systems were thought to be characteristicof pathogenic bacteria, where they play important roles inhost infection. T3SS form an apparatus that injects bac-terial proteins directly into eukaryotic cells to disruptnormal functioning of the cell, facilitating infection (Hueck,1998). The T3SS of NGR234 is capable of secreting Nops(nodulation outer proteins), and is an important determi-nant of host range (Viprey et al., 1998).

Production of NF requires flavonoids and the LysR-type regulator NodD1, which binds to conserved motifs(termed nod boxes) found in the promoter regions ofgenes/operons responsible for NF synthesis. Nop secre-tion also requires flavonoids, NodD1 and another regula-tory protein TtsI (Viprey et al., 1998; Marie et al., 2004). Anod box is located in the promoter region of ttsI, and

Accepted 27 February, 2008. *For correspondence. Email william.deakin@bioveg.unige.ch; Tel. (+41) 22 379 3128; Fax (+41) 22 37913009.

Molecular Microbiology (2008) doi:10.1111/j.1365-2958.2008.06187.x

© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd

NodD1 is thought to activate TtsI which in turn initiatestranscription of T3SS genes (Kobayashi et al., 2004). TtsIshares characteristics with the DNA-binding responseregulators of two-component regulatory systems (Vipreyet al., 1998; Marie et al., 2004). Usually, such regulatorsare activated by their partner sensor, histidine proteinkinases, which auto-phosphorylate at a histidine residueupon sensing an environmental signal. The phosphorylgroup is subsequently transferred to an aspartate residuein the response regulator, inducing a conformationalchange that leads to its activation. Once phosphorylated,response regulators act as transcriptional activators bybinding to cis elements in the promoters of genes requiredto process the initial environmental signal. TtsI, however,has a glutamate residue instead of the conservedaspartate. In other bacterial response regulators, such asubstitution leads to constitutive activation, bypassing therequirement for the sensor kinase partner. It is thus pos-sible that TtsI functions as a transcriptional activator inde-pendent of phosphorylation and a sensor kinase partner.Instead, transcription of ttsI and therefore function(s)regulated by TtsI, are modulated by NodD1 in a flavonoid-dependent manner (Kobayashi et al., 2004; Marie et al.,2004).

Sequencing numerous rhizobial genomes has revealedthat T3SS and TtsI control are relatively common. Acomparison of the promoter regions of T3SS genesfrom several rhizobia identified a putative cis-regulatoryelement termed a tts box (TB). TtsI is thought to bind TBand stimulate transcription of downstream genes (Krauseet al., 2002). Using this consensus sequence, 11 putativeTBs (TB1–TB11) were identified on pNGR234a (Marieet al., 2004). Five of the TBs are located upstream ofgenes/operons involved in the assembly of the type IIIsecretion machine; others precede genes encoding pos-sible secreted proteins. TBs are also found in the promot-ers of genes encoding proteins not directly related toT3SS functions (Fig S1). Thus TtsI potentially regulatesmore than the T3SS, and indeed mutation of ttsI leads todifferent symbiotic phenotypes compared to a T3SSmutant alone (Viprey et al., 1998). As well as beingimpaired in protein secretion, the ttsI mutant failed toproduce a rhamnose-rich lipo-polysaccharide (LPS)known to be important for successful nodulation (Marieet al., 2004; Reuhs et al., 2005; Broughton et al., 2006). Aflavonoid-inducible operon encoding enzymes respon-sible for rhamnose synthesis was shown to require TtsI foractivation and to contain a TB in its promoter region.Evidence that the TB is essential for the activity of thisoperon was obtained by deleting a small region containingthe TB which abolished TtsI-mediated induction (Marieet al., 2004).

In this work, we determined whether the promoterregions containing the 11 predicted TB are inducible in a

TtsI- and flavonoid-dependent manner. We then testedif TtsI could physically bind to TB-containing promotersand mapped the actual binding site in vitro by DNaseIfootprinting. We also mutated key residues in the TBconsensus sequence and randomly mutated aTB-containing promoter to identify further importantresidues. It seems likely that the TtsI/TB regulatorysystem is a basic feature of rhizobial T3SS as Brady-rhizobium japonicum USDA110, Mesorhizobium lotiMAFF303099 and Sinorhizobium fredii USDA257 allpossess T3SS, TtsI homologues, as well as predicted TBsequences (Krause et al., 2002; Krishnan et al., 2003;Hubber et al., 2004). Thus our findings are applicable tomultiple genera of rhizobia.

Results

NGRDttsI, a non-polar deletion mutant of ttsI

Polar mutation of ttsI (NGRWttsI ) demonstrated thatTtsI is required for Nop secretion and synthesis of arhamnose-rich polysaccharide, as well as the transcrip-tional activation of two of the 11 TBs, TB2 and TB8.Although introduction of a plasmid-born copy of ttsI intoNGRWttsI allowed complementation of rhamnan synthe-sis, it failed to restore Nop secretion (Viprey et al., 1998;Marie et al., 2004). As ttsI, rhcC2 and y4xK are predictedto form an operon (Perret et al., 2003), the insertion of anW cassette into ttsI most probably blocked transcriptionof these downstream genes. In other bacteria, rhcC2encodes an essential component of the T3SS: its additionin trans (possibly y4xK as well) would thus be required forcomplementation of Nop secretion by NGRWttsI. Addi-tional regulatory controls of T3SS gene expression havebeen shown in some bacteria: if the machinery fails toassemble correctly (because of mutation of a key gene forexample), then expression of genes encoding secretedproteins is suppressed (Wei et al., 2000). It is thus pos-sible that the block in TB expression in NGRWttsI is due tothe absence of RhcC2, causing the T3SS assemblymachine to fail. In this case, TB expression was probablysuppressed by another regulator. For this reason, weconstructed a non-polar deletion mutant of ttsI (NGRDttsI )to maintain transcription of rhcC2 and y4xK from nod box18 and thus avoid this possibility (Fig. 1). To characterizethe new mutant, secreted proteins and surface polysac-charides were isolated from NGRDttsI and NGR234. Nopsecretion and de novo synthesis of rhamnose-rich LPSwere blocked in NGRDttsI (Fig. S2), consistent with ourprevious observations of the polar mutant NGRWttsI(Marie et al., 2004). To complement NGRDttsI, ttsI with itsown promoter region was subcloned into pRG960 (Vanden Eede et al., 1992), giving rise to pttsI-2. Introductionof pttsI-2 into NGRDttsI restored both Nop secretion and

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© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology

production of rhamnose-rich LPS. Thus NGRDttsI doesnot appear to dramatically affect transcription of rhcC2 (ory4xK) and this mutant was used in all subsequent work.

Promoter activities of TB-containing loci

To examine the function of all TBs, we subcloned the11 predicted TBs into pMP220, a broad-host-rangetranscriptional-lacZ reporter system (Spaink et al., 1987),thus creating pMP-TB1 to pMP-TB11 (see Table 1). Toassess the flavonoid and TtsI dependence on transcrip-tion of the TB, tri-parental matings were used to introduceeach of the constructs into NGR234 and NGRDttsI. Liquidcultures were grown to an OD600 of 0.1 in RMS and theflavonoid daidzein added at 2 ¥ 10-7 M. At 1, 6 and 24 hpost induction (hpi), b-galactosidase activities were moni-tored in transconjugants of NGR234 (red bars in Fig. 2)or at 1 and 24 hpi for NGRDttsI transconjugants (pink barsin Fig. 2). In the absence of an inducer, and with theexception of pMP-TB5, only low promoter activitieswere observed with the different constructs (open bars

in Fig. 2), but addition of daidzein caused significantincreases in b-galactosidase activities 24 hpi (Fig. 2).At 24 hpi, TB8, which controls expression of thenopBrhcJnolUVrhcNy4yJrhcQRSTU operon, is the stron-gest promoter (2500 130 Miller units), whereas thelowest activity was recorded with TB11 (157 5 units).Although expression of TB11 appears to be low, it repre-sents a sixfold induction over that found in the NGRDttsImutant (Fig. 2K). By mobilizing the different TB–lacZfusions into the mutant strain NGRDttsI, the role of TtsI inthe activation of each individual TB was assessed. All TBslost flavonoid inducibility in NGRDttsI (Fig. 2). Introductionof pttsI-2 into four randomly selected NGRDttsI (pMP-TB2, TB4, TB8 and TB10) transconjugants restoredflavonoid-dependent induction (closed bars, Fig. S3).

TtsI binds to TB2 and TB8

TtsI contains an N-terminal conserved receiver domainand a C-terminal helix-turn-helix (HTH) domain, bothtypical of response regulators of two-component systems.The HTH domain usually interacts directly with a DNAbinding site present in the promoters of genes controlledby these regulators. Although the HTH structure is highlyconserved, it has the ability to recognize specific bases,and thus discriminate small modifications in a binding site(Stock et al., 2000). TtsI of NGR234 and other rhizobiahas been shown to control the expression of severalgenes containing a TB in the promoter region, but directcontact had never been shown. To further characterize thebinding of TtsI to the TB-containing promoter regions,we cloned the NGR234 ttsI coding sequence into thepETBlue2 vector, then overexpressed and purified a

ttsI rhcC2 y4xK

N PP S

Δ0.5 kb

nopL

A

nod box NB18 Ω

Fig. 1. Physical map of the ttsI locus of pNGR234a. Restrictionsites are as follows: ApaI (A); NheI (N); PstI (P); SacII (S). Locationof the deleted region in NGRDttsI is shown by D and the site of theomega cassette insertion in NGRWttsI shown by W.

Fig. 2. Expression analyses of TBs inpNGR234a. Flavonoid inducibility of TBs inthe wild-type and NGRDttsI backgrounds. A–Lrepresent the levels of b-galactosidase activity(¥ 103 Miller’s units). Activity of the vectorpMP220 devoid of an insert is reported in L.Assays were performed 1, 6 and 24 hpi with2 ¥ 10-7 M daidzein. In the absence of aninducer, basal levels of b-galactosidaseactivity are shown as open bars. Valuesobtained with induced transconjugants arecoded as: NGR234 (red bars); NGRDttsI (pinkbars). The values reported represent themeans of three independent experiments.Error bars are shown on top of each column.Numbers on top of each bar represent therelative increase in activity of induced- ascompared with non-induced cultures.

A B C D

E F G H

I J K L

TtsI-mediated regulation 3

© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology

Table 1. Bacterial strains and plasmids and primers.

Relevant characteristics Reference

StrainsNGR234 RifR derivative of the isolate NGR234 Lewin et al. (1990)NGRDttsI ttsI deletion mutant of NGR234 RifR This workNGRDrmlB-wbgA Rhamnose synthesis mutant of NGR234, RifR, KmR Marie et al. (2004)

PlasmidspBluescript II KS+ Phage f1, lacZ, ApR StratagenepUC::ttsI pUC18 containing ttsI and its promoter as a 2.4-kb PstI fragment Marie et al. (2004)pJQ200mp18 Suicide vector used in directed mutagenesis, GmR Quandt and Hynes (1993)pJQDttsI As above, but carrying DttsI fragment, subcloned into PstI site, GmR This workpRK2013 Tra+ helper plasmid for mobilisation, KmR Ditta et al. (1980)pRG960 IncQ vector containing promoter-less uidA, SpR Van den Eede et al. (1992)pttsI-2 pRG960 containing ttsI and its promoter as a 2.4 kb PstI fragment This workpMP220 IncP vector containing promoter-less lacZ, TetR Spaink et al. (1987)pMP-TB1 TB1 cloned in pMP220 as a XbaI-PstI fragment This workpMP-TB2 Also called pMP220-rmlB; TB2 cloned as a BamHI fragment in pMP220 Marie et al. (2004)pMP-TB3 TB3 cloned in pMP220 as a XbaI-PstI fragment This workpMP-TB4 TB4 cloned in pMP220 as a XbaI-PstI fragment This workpMP-TB5 TB5 cloned in pMP220 as a XbaI-PstI fragment This workpMP-TB6 TB6 cloned in pMP220 as a XbaI-PstI fragment This workpMP-TB7 TB7 cloned in pMP220 as a XbaI-PstI fragment This workpMP-TB8 Also called pMP220-B; TB8 cloned as a XbaI-PstI fragment in pMP220 Marie et al. (2004)pMP-TB9 TB9 cloned in pMP220 as a XbaI-PstI fragment This workpMP-TB10 TB10 cloned in pMP220 as a XbaI-PstI fragment This workpMP-TB11 TB11 cloned in pMP220 as a XbaI-PstI fragment This workpPROBE-GT pVS1/p15a vector, GmR Miller, 2000pPROBE-GT′ As above but with inverted MCS Miller, 2000pGT-nopB TB8 region in pPROBE-GT′ as a HindIII fragment This workpGT-GAATG Site-directed mutation of TB8 region in pPROBE-GT as an EcoRI fragment This workpGT-A117 Random mutation of TB8 region in pPROBE-GT as an EcoRI fragment This workpGT-A2D Random mutation of TB8 region in pPROBE-GT as an EcoRI fragment This workpGT-A3G Random mutation of TB8 region in pPROBE-GT as an EcoRI fragment This workpGT-A518 Random mutation of TB8 region in pPROBE-GT as an EcoRI fragment This workpGT-A62 Random mutation of TB8 region in pPROBE-GT as an EcoRI fragment This workpGT-A74 Random mutation of TB8 region in pPROBE-GT as an EcoRI fragment This workpGT-A812 Random mutation of TB8 region in pPROBE-GT as an EcoRI fragment This workpGT-A9BG Random mutation of TB8 region in pPROBE-GT as an EcoRI fragment This workpGT-B312 Random mutation of TB8 region in pPROBE-GT as an EcoRI fragment This workpGT-B620 Random mutation of TB8 region in pPROBE-GT as an EcoRI fragment This workpGT-B1127 Random mutation of TB8 region in pPROBE-GT as an EcoRI fragment This work

PrimersTB1-F 5′-tctcatctctagaccaatcggcg-3′TB1-R 5′-cctgcagcgatattgtctcctcg-3′TB3-F 5′-tctagagccgtcagtcatctttcg-3′TB3-R 5′-tgtcctgcagatcgttgatgagg-3′TB4-F 5′-tctagacgatggcgatgttgctc-3′TB4-R 5′-acgactgcaggccttcaagatgg-3′TB5-F 5′-tgctctagagcaccggaagc-3′TB5-R 5′-tcctgcaggggaaagtagcatc-3′TB6-F 5′-cctctagagcctgtcttttctcg-3′TB6-R 5′-gctgcaggcttgcgtttagtgg-3′TB7-F 5′-tcgtctagacgttgaacggtctac-3′TB7-R 5′-aaggctgcaggccgacattgtg-3′TB9-F 5′-tctctagatggcgtcaaatgctgc-3′TB9-R 5′-ttctgcagccatttgcttgctgg-3′TB10-F 5′-catctagactgagagagcttcacg-3′TB10-R 5′-acctgcagctactcctgccttag-3′TB11-F 5′-cgaatctagacgactttcgatcgc-3′TB11-R 5′-gactctgcagggcgttcgtttccc-3′rmlB-F 5′-aagcaccccgaaaactacct-3′rmlB-R 5′-gcattgcgaaatttggatgga-3′nopB-F 5′-ctcgtcttgataaaccaaatctgaa-3′nopB-R 5′-ggactcgattacttaactctttgac-3′TB8-F mut 5′-ggccggtagaatgcgtgtcgtcagctcgcctc-3′TB8-R mut 5′-ggaggcgagctgacgacacgcattctaccggc-3′

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© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology

His-tagged TtsI protein. High levels of expression wereobtained, but more than 50% of the TtsI-His wasinsoluble, which was also observed with other membersof this family (Kumagai et al., 2006). To test the ability ofTtsI-His to bind to TB sequences, we initially performedelectrophoretic mobility shift assays using a 150-bp regionof the nopB promoter that contains TB8. When increasingamounts of TtsI-His were added to the reaction, slowermigrating bands were observed, consistent with DNA–protein interactions (Fig. 3). The slowest band appearedonly at increasing concentrations of TtsI, which could be aresult of oligomerization of TtsI-His upon binding to DNA,or the presence of more than one TB at different loca-tions in the DNA. Inspection of the sequence did notreveal any other candidate region, suggesting that TtsIforms oligomers upon binding TB8. Addition of up to50 mg ml-1 poly(dI-dC) as heterologous competitor DNAdid not disrupt the DNA–protein complexes, suggestingthat binding of TtsI to TB8 is specific (Fig. S4). This speci-ficity was confirmed using a double-stranded oligo-nucleotide specific to TB8 which disrupted TtsI–nopBpromoter complexes (data not shown). A second double-stranded oligo-nucleotide composed of the adjacentsequence upstream of TB8 (originally designed as acontrol) also disrupted the complexes, however. Thisunexpected effect suggested that the TtsI binding regionextends outside of the TB consensus sequence and, forthis reason, we mapped exactly where TtsI bound to thenopB promoter.

The identification of the precise interaction site ofTtsI with the nopB promoter was determined by DNaseI cleavage protection (footprinting) assays in whichevidence of protein binding is seen as modifications ofthe cleavage pattern that result in both decreases and

increases in the intensity of cleaved fragments. When thepolymerase chain reaction (PCR) fragment containing theTB8 site used in the mobility shift assays described abovewas labelled and used as the probe, it clearly interactedwith TtsI-His as revealed by the presence of protectedand hyperreactive bands in the consensus sequence(Fig. 4A). Increasing concentrations of TtsI produced aclearer pattern of interaction, but did not reveal any newinteraction sites. Furthermore, TtsI caused a modificationto the cleavage pattern of bases located upstream anddownstream of the TB, suggesting that it occupies a broadsection of the target DNA (Fig. 4A). This result is consis-tent with oligomerization of TtsI upon binding to DNA, assuggested by the mobility shift assays. The hyperreactivebands observed are an indication that TtsI induces majordistortions in the bound DNA, exposing these sites toDNase I cleavage.

To test whether such a broad footprint is specific to TB8,a TB2-containing portion of the rmlB promoter region wasalso used as a template in identical experiments. As seenwith TB8, modified bases were also observed within andoutside of the TB, and increasing the quantity of TtsI in thereactions sharpened the footprint (Fig. 4B). Thus TtsI hasa broader than predicted binding site on both promoterregions. Bases modified by TtsI binding in both TBs weremapped (Fig. 4C and D), but it was not possible to detectany trend of base cleavage modification which couldpinpoint bases important for binding outside both TBs.Nevertheless, it is evident from the protection and hyper-reactivity of the highly conserved bases of the TB that theyare clearly involved in direct interaction with TtsI. It shouldbe noted that the modified cleavage pattern induced byTtsI towards the 5′ and 3′ ends of the templates may notnecessarily indicate direct contact of these sequenceswith the protein, but could represent distortion of the DNAcaused by the binding of the activator with the TB.

Site-directed mutagenesis of a TB

Specific point mutations were introduced to three highlyconserved bases of the TB8 consensus sequence (spe-cifically GTCAG to GAATG), and the ability of TtsI to bindand activate transcription was tested in vitro and in vivo.Mobility shift assays showed that TtsI binds in vitro toboth wild-type and mutant TBs at similar concentrations(Fig. 5). The ability of the mutated promoter to activatetranscription of a reporter gene was then tested using thegreen fluorescent protein (GFP) to facilitate reporterassays. First, the non-mutated TB8 promoter was sub-cloned upstream of a promoter-less gfp gene in plasmidpPROBE-GT′ (Miller et al., 2000) and then introduced intoNGR234 as well as NGRDttsI by tri-parental matings.Cultures were induced with flavonoids for 40 h and GFPproduction measured in terms of cellular fluorescence. As

0.1

1 3 50TtsI (μM)

Free probe

TtsI-probe

TtsI-probe

Fig. 3. TtsI binding to the nopB (TB8) promoter. Mobility shiftassay using a 32P-labelled 150-bp PCR fragment containing thenopB promoter region. Purified TtsI-His was added at increasingconcentrations, incubated for 20 min and subject to electrophoresisunder native conditions.

TtsI-mediated regulation 5

© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology

expected, a strong GFP signal was observed in NGR234containing the TB8–gfp fusion, whereas no expressionwas observed from NGRDttsI (Fig. 6). The mutated TB8promoter was also subcloned upstream of gfp in plasmidpPROBE-GT (Miller et al., 2000). When the activity of thisplasmid was assayed in NGR234 (under the same con-ditions of induction), no GFP signal was observed (Fig. 6).To answer the question of why the mutated TB8 promoterwas inactive in vivo but apparently unaffected in its

binding in vitro led us to test the ability of TtsI to bind to theGAATG TB8 by the more sensitive assay of DNase Ifootprinting. Using the same experimental conditionsdescribed earlier, differences between TB8 and GAATGTB8 were found, especially near the mutated bases, but

Fig. 4. DNase I footprints of TtsI bound tothe nopB (TB8) and rmlB (TB2) promoterregions. 32P-labelled PCR fragments wereincubated with different concentrations of TtsIand digested using limiting concentrations ofDNase I. After purification, DNA fragmentswere separated in a 6% sequencing gel,dried, exposed to Phosphoimager screensand scanned. The TB8- and TB2-containingPCR fragments are shown in A and Brespectively. Numbers on the right refer to theDNA position compared with ATG of thedownstream gene. Brackets indicate the limitsof the TB. Open and closed circles pinpointDNase I protected and hyperreactive bandsrespectively. Below the panels, the DNAsequences show the organization of the nopB(C) and rmlB (D) promoter regions and theirreactivity to DNase I cleavage. Underlinedbases delineate the TBs with the highlyconserved bases in bold font. Capital lettersrepresent DNase I hyperreactive bases anditalics show protected bases. Numbers referto positions upstream of the ATG of thedownstream gene.

1 2 40 0.25

0.5

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TtsI (μM)

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- 331

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TtsI (μM) 0.25

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BA

aatctCccat gcgGTTcaac tcgACtaaCa TcactctTca AtgggcaAgc gacgctgccgtts box (TB8)

gTagtcagcg tgtcgTcagc tcgcctcgCt agagttccAc gTcaaagagt taagtaatcg

C

-133

tts box (TB2)tacccgTcgC ctAtggatgt cctaaaTagg agagttcgTc agctttTcga aagctcagcC

gaaTagcagT gggaaggcCa agaCcgacCa acctacctga attcagaaaT gg

D-467

0.25

0.5

1 2 40 0.25

0.5

1 2 40TtsI (μμM)

mutantWild type

Free probe

TtsI-probe

TtsI-probe

Fig. 5. TtsI binds to the site-directed TB8 mutant. Mobility shiftassays were performed as before, with 32P-labelled 150-bp PCRfragments containing the nopB promoter regions.

0

200

400

600

800

1000

1200

1400

1600

pPROBE/wt pnopB/wt pnopB/ttsI- GAATG/wt

Fig. 6. In vivo expression of the site-directed TB8 mutant. Themutated nopB promoter region was fused to a promoter-less gfpgene and assayed after 40-h induction with 2 ¥ 10-6 M apigenin.Numbers shown (¥ 103) are an average of at least three replicateexperiments. pPROBE/wt, the vector control in NGR234;pnopB/ttsI-, the wild-type nopB promoter in NGRDttsI; pnopB/wt,the wild-type nopB promoter in NGR234; GAATG/wt, thesite-directed mutant nopB promoter in NGR234.

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also elsewhere in the TB8 consensus (Fig. 7). Thusalthough the GAATG TB8 mutation is clearly insufficient toblock TtsI binding, it alters the nature of the binding whichin turn may well lead to the inability of GAATG TB8 to betranscribed, as observed in vivo.

Random mutagenesis of a TB

As TtsI bound to regions of TB-containing promotersoutside of the TB, we used a random mutagenesisapproach to identify other potential bases important fortranscriptional control by TtsI. Error-prone PCR was per-formed on the nopB promoter, the products cloned into

pGEM-T, and then sequenced. Because of the mutagenicconditions used, several bases were often changed ineach mutant. A number of these PCR products wereselected according to the location of the mutations andsubcloned upstream of the promoter-less gfp gene andmobilized into NGR234. Most of the mutants analysed hadno activity, some had partial activity when induced byapigenin, but none had constitutive activity in the absenceof flavonoid (Fig. 8). Three mutants had no mutationswithin the TB (A62, A117 and A74) and had zero, 6% and18% of the wild-type TB8 activity, suggesting that either theconsensus sequence is longer than predicted (in agree-ment with the large footprint), or the mutated bases alteredbinding of other components of the transcriptional machin-ery. Interestingly, one mutant exhibited higher activitiesthan the wild type upon induction. Attempts to delimit thenopB promoter by footprinting assays using the holoen-zyme Escherichia coli RNA polymerase were not success-ful, possibly as a result of differences in the housekeepingsigma factors of both species. However, the nopB pro-moter region of S. fredii USDA257 is identical to NGR234,and the exact position of the start of transcription wasmapped to a cytidine residue located at the -30 positionfrom the ATG (Kovács et al., 1995). Inspection of theupstream sequence using the proposed consensussequence for rhizobial promoters (MacLellan et al., 2006)allowed the identification of a compatible -10 RNA poly-merase binding site (Fig. 9). Although the -10 sequence isfairly well conserved (four out of six bases), the -35 regiondoes not resemble the consensus, and indeed overlaps thehighly conserved GTCAG bases of the TB. The majority ofinactive mutants had mutations either in the TB or theputative -10 site (Fig. 9). On the other hand, the threemutants that remained active (B1127) or partially active(A2D andA3G) did not have mutations in the TB consensus

TtsI (μM) 0.25

0.5

1 20

TB80.

25

0.5

1 20

***

A B

-113

-8

°

°

°

Fig. 7. DNase I footprint of the site-directed TB8 mutant.32P-labelled PCR fragments (A, wild-type and B, site-directedmutant) were incubated with increasing concentrations of TtsI andthen digested with limiting concentration of DNase I. Afterpurification, DNA fragments were separated in a 6% sequencinggel, dried, exposed to Phosphoimager screens and scanned.Numbers on the right refer to DNA positions compared with ATG ofdownstream genes. Asterisks show the mutated bases; opencircles highlight the differences in DNase I activity observed (ascompared with the wild-type TB8).

0200400600800

100012001400160018002000

pPROBE wt

ttsI-

A1 17

A2 D

A3 G

A5 18

A6 2

A7 4

A8 12

A9BG

B3 12

B6 20

B1127

Fig. 8. In vivo expression levels of the randomly mutated nopBpromoter regions. Mutated nopB promoter regions were fused topromoter-less gfp genes, transferred to NGR234 and NGRDttsI andassayed after 40-h induction with 2 ¥ 10-6 M apigenin. The results(¥ 103) are averages of at least three replicate experiments.pPROBE, vector only in NGR234; wt, the wild-type nopB promoterin NGR234; ttsI-, the wild type nopB promoter in NGRDttsI; thecode names refer to randomly mutated nopB promoters.

TtsI-mediated regulation 7

© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology

sequence or the putative -10 promoter. Interestingly, theA518 mutant possessed four mutations, none of whichmapped to either of binding sites, but it still exhibited verylow transcriptional activity. This indicates that basesoutside of the TB are important for nopB transcription,presumably by modulating binding of TtsI.

Discussion

Phytopathogenic bacteria use T3SS to facilitate infectionor conversely to trigger plant defences (Mudgett, 2005;Grant et al., 2006). Many different effector proteins can besecreted by such T3SS and they have been cataloguedbased upon homology searches or by assessing the poten-tial for co-regulation with other T3SS genes (Cunnac et al.,2004; Lindeberg et al., 2006). Specific plant compoundsthat induce expression of T3SS genes are not known and,as a consequence, the proteins used as sensors by phy-topathogens have not been identified. Nevertheless, manyplayers in the intermediate stages have been identifiedthrough mutagenesis and two broad classes of gene regu-lation are now recognized (Mole et al., 2006; Tang et al.,2006): in group I (represented by Erwinia spp., Pantoeaspp. and Pseudomonas syringae), the activator is a sigmafactor, termed HrpL which is thought to bind directly to a ciselement (hrp boxes) in promoter regions of T3SS genes. Ingroup II, the direct activators are members of the AraCfamily of transcriptional regulators, called HrpX in Xanth-omonas spp. and HrpB in Ralstonia solanacearum, both ofwhich are proposed to bind to cis elements in the promoterregions of T3SS genes. HrpL andAraC-type transcriptionalactivators are encoded by genes in the T3SS loci.

In contrast, flavonoids trigger induction of T3SS (alongwith other symbiotic genes) in rhizobia (Krause et al.,2002; Krishnan et al., 2003; Kobayashi et al., 2004;Viprey et al., 1998). Members of the NodD/LysR family of

transcriptional regulators act at the top of this cascade,and by binding to nod boxes activate both genes encodingthe synthesis of symbiotic signalling molecules along withother regulatory proteins (Schlaman et al., 1998). Thegene encoding one such regulator, TtsI, is found within theT3SS loci of several rhizobia and in all cases is precededby a nod box. ttsI is the only gene encoding a transcrip-tional regulator in all T3SS loci and, when translated, isproposed to recognize a further cis element, the TB, foundin the promoters of T3SS-related genes. The TtsI/TBregulatory system is found in for all rhizobia possessingT3SS (Krause et al., 2002; Krishnan et al., 2003; Marieet al., 2004). Thus the demonstration that the responseregulator transcriptional activator TtsI binds directly to TBsuggests that the regulation of rhizobial T3SS is differentfrom that found in phyto-pathogens.

TtsI binding is not specific to the TB consensussequence, however, and the DNase I footprint extendedup to 100 bp up- and downstream of the TB. Furthermore,a double-stranded oligonucleotide (located from -111 to-79) paired to sequences adjacent to the TB and com-peted with TtsI binding to the nopB promoter region. Itshould be noted that other members of the responseregulator family of transcriptional activators also demon-strate large footprints despite specific consensussequence predictions (Cullen et al., 1996). It is possiblethat other proteins bind TtsI and, in doing so, increasespecificity of the interaction with TB. Mixing the solubleprotein fraction of cell extracts with TtsI and TB-containingpromoter regions had no effect on binding. Furthermore,TtsI did not bind to promoter regions lacking TB, nor did itbind to its own promoter (containing a nod box – data notshown), demonstrating that TtsI recognizes specific DNAsequences and that auto-regulation is absent.

Site-specific mutation of TB8 blocked promoter activitybut did not abolish TtsI binding. A similar result was

-148......-138......-128......-118......-108......-98.......-88.......-78.......-68.......-58.......-48.......-38.......-28.......-18.......-8|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|ATAAACCAAATCTGAAAATCTCCCATGCGGTTCAACTCGACTAACATCACTCTTCAATGGGCAAGCGACGCTGCCGGTAGTCAGCGTGTCGTCAGCTCGCCTCGCTAGAGTTCCACGTCAAAGAGTTAAGTAATCGAGTCC

TB8 ............................................................................GTAGTCAGCGTGTCGTCAGCTCGCCTCGCT...................................

POL .................................................................................Cagcgt.................CTAgAg...............................POL CON .................................................................................CTTGAC.................CTATAT...............................

A117..........................T..G...T....G...T.G.......................G.......C.................................C.....C.............................A2D..............................G........G.........G.......................G...C.T............C..........G..............T............................A3G..........................................................CC...............A..................G..............A.....................................A518.......................C.....................T............................................A........................C..............................A62....................................C...........................................................................G....C.............................A74....................................C........................................G......................................C.....................G........A812................................T...........G.................A.....................A...A...........................C...........G.................A9BG..................................................G..................................................C....C....G..................................B312..................................AC.......A..............C......................................T.C......A...C.A.................................B620.......................C............C................G.......GA...................C.A......G......T...............................................B1127..........................................A.............CC..............G......A........................T........................................GAATG....................................................................................AAT..........................................................

Fig. 9. Positions of the mutated bases in the nopB promoter region. Top line of the DNA sequence shows the wild-type nopB promoter withthe probable transcription start, TB8 underlined. The highly conserved bases of TB8 are also underlined. The putative -35/-10 promoter(labelled as POL) is shown, with bases not matching the consensus sequence (labelled as POL CON) in lower case. Dots representunchanged bases, whereas mutated bases are lettered, code names are indicated at the left. Dotted lines below represent specific mutatednopB promoter regions (labelled with code names), mutated bases are lettered. Numbers refer to positions upstream of the nopB ATG.

8 R. Wassem et al.

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observed with HrpL of Pantoea agglomerans wheredirect binding was unaffected by mutations of the hrpbox consensus sequence that did not permit transcrip-tion (Nissan et al., 2005). Given that TtsI has a largefootprint, it may well bind to or interact with a relativelylarge stretch of DNA, and thus site-directed mutantswould not be expected to prevent binding. However,application of the more sensitive DNase I cleavage pro-tection assay to promoter regions containing the site-specific mutations revealed subtle differences comparedwith the non-mutated promoter. Modulation of cleavageprotection was observed at the sites of mutation, butalso at a second location in the TB, suggesting that theinteraction between TtsI and the TB is altered and tran-scription thus blocked. It should also be noted that thenature of the mutations may have indirectly affected theability of DNase I to digest the target DNA as this abilityis very dependent on the nature of the sequence(Herrera and Chaires, 1994).

Random mutagenesis of the nopB promoter region iden-tified further bases that are important for TtsI-dependenttranscriptional activity. In one case, transcriptional activitywas higher than wild-type levels, although flavonoids andTtsI were always required. No constitutively active muta-tions were generated. The mutants A117, A518 and A74, inwhich activity was blocked, were particularly revealing asnone of the mutated residues were within the TB consen-sus sequence. As the nopB promoter regions of S. frediiUSDA257 and NGR234 have identical DNA sequence, weused the experimentally proven transcription start ofUSDA257 to identify a putative rhizobial RNA polymerasebinding site. Although the nopB promoter is not highlysimilar to the consensus sequences of S. meliloti (MacLel-lan et al., 2006), the -10 box is a good match, whereas the-35 region is poorly conserved. However the sequenceencompassing the -35 region overlaps with numeroushighly conserved residues of the TB. It is possible that thehigh activity of the nopB promoter may be due to thetransition from a normally inactive state to one in which thechanged conformation upon TtsI binding, exposes thepromoter to RNA polymerase. Indeed, other transcriptionalactivators are known to interact with RNA polymerase andbind to sites located very close to the promoter (Browningand Busby, 2004), some by binding to sites which partiallyoverlap the -35 region of the promoter and interact directlywith the s70 subunit of RNA polymerase (Dhiman andSchleif, 2000; Wickstrum and Egan, 2004). Transcriptionalcontrol of genes by the response regulator PhoB, wherethe conserved -35 hexamer in the RNA polymerasebinding site is absent and replaced by a pho box to whichPhoB binds (Makino et al., 1993), might be a good modelfor the TtsI/TB system. In these promoters, RNA poly-merase is only able to bind in the presence of phosphory-lated PhoB.

Following comparison of promoter regions of T3SSgenes from several rhizobial species, Krause et al. (2002)predicted a consensus TB sequence. Using this sequenceto search the symbiotic plasmid of NGR234, we identified11 TBs, the majority of which are involved in regulatingT3SS functions. Although we had previously demon-strated that TB2 and TB8 are active (Marie et al., 2004), itis now clear that 10 of the 11 known TB loci of pNGR234aare functional flavonoid- and TtsI-dependent promoters.TB3, which is upstream of y4gJ, an open reading frame(ORF) without obvious homologues (but part of a clusterof genes involved in LPS modification, some of which arecontrolled by TB2), is also induced to relatively high levelsin a TtsI-dependent manner. Taken together, these datasuggest that genes downstream of TB3 (e.g. y4gJ whichis involved in modifying LPS) may also have a role insymbiosis.

In NGR234, at least 20 genes are thought to be involvedin T3SS, including those that encode NopA, NopB, NopC,NopL, NopP and NopX (Viprey et al., 1998; Marie et al.,2003; Ausmees et al., 2004; Deakin et al., 2005; Saadet al., 2005). NopA, B and X are associated with pilus-likecell surface appendages, and are therefore thought to be apart of extracellular component of the T3SS machine(Krishnan et al., 2003; Deakin et al., 2005; Saad et al.,2005). NopL and NopP are known effectors, which areprobably injected into the cytoplasm of host cells (Bartsevet al., 2003; 2004; Marie et al., 2003; Ausmees et al.,2004; Skorpil et al., 2005). Generally, genes involved in theformation of the membrane-spanning secretion apparatus,such as nopBrhcJnolUVrhcNy4yJrhcQRSTU (TB8), nopX(TB7) and nopCAy4yQrhcVy4yS (TB10), are under thecontrol of moderate to strong promoters. In contrast, TB6and TB9 that control transcription of two effector proteins(NopL and NopP) are only weakly induced (threefold to12-fold induction as compared with non-induced levels)24 hpi (Fig. 2F and I). Furthermore, the ORFs y4fR (TB1),y4lO (TB4) and y4zC (TB11) are all homologous to knownT3SS effector proteins (Marie et al., 2001) and theirexpression profiles are lower than TB controlling themachinery (sixfold, 13-fold and sixfold respectively). Thatthese promoters are active in a TtsI-dependent mannerand after flavonoid induction suggests that they may wellbe upstream of genes encoding functional effector pro-teins. Generally, promoters upstream of potential effectorproteins have variable expression levels. As an example,the expression profile of pMP-TB5 differs significantly fromthose of other constructs. Although a slight induction (1.2-fold) by daidzein was observed 24 hpi, TB5 had basaltranscriptional activities as high as 3440 190 Miller unitseven under non-induced conditions. As a null mutation inttsI had no affect on its basal activity, TB5 is probably underadditional regulatory control. ORF downstream of TB5 arenot obviously related to T3SS or LPS functions, and seem

TtsI-mediated regulation 9

© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology

to be involved in stabilization of pNGR234a (Dombrechtet al., 2001).

Use of the TB consensus sequence in a bio-informaticsscreen to identify TtsI-regulated genes on pNGR234a waslargely successful as 10 of the 11 predicted TBs areactive. Indeed, extending such screens to the other rep-licons of NGR234 as well as other rhizobia should befeasible, although the predictions will need to be verifiedon a case-by-case base as the example of TB5 shows.Differences in the TtsI regulon could be specific to T3SSfunction, as rhizobia possess and may well secrete differ-ent effector proteins, which TB-based searches can helpidentify. Alternatively, production of diverse signallingmolecules could also be controlled by TtsI, such as therhamnose-rich LPS synthesized by NGR234 but not by B.japonicum USDA110 or S. fredii USDA257(Marie et al.,2004). Control of rhamnose synthesis is brought about bygenes recruited into the TtsI regulon out of temporalnecessity, as all are induced relatively late, or as afunctional requirement linked to T3SS activity. Over 30predicted TB sequences are present in the B. japonicumUSDA110 genome (Suss et al., 2006). Completion ofmore rhizobial genomes, particularly those with T3SS andTB, will allow ever more powerful comparative studies intothe roles of flavonoid-inducible genes in symbiosis.

Conclusions

We have extended the inventory of members of theflavonoid-regulatory cascade of NGR234 which bringabout the exceptionally broad host range of thisRhizobium. Methods used here to analyse the TtsIregulon of NGR234 are applicable to other TB-possessingrhizobia, although it is very probable that genes modu-lated by TtsI will vary considerably. What remains is to testTB expression levels in planta, as the potential cocktail ofinducing compounds and environments may well revealmore mechanisms of regulatory control.

Experimental procedures

Microbiological techniques

Escherichia coli recombinants were grown at 37°C on Luria–Bertani medium (Sambrook et al., 1989). NGR234 and itsderivatives were raised at 28°C in Rhizobium minimalmedium containing succinate as the carbon source (RMS)(Broughton et al., 1986) or TY (Beringer, 1974). Ampicillin (forBluescript/pGem), gentamycin, kanamycin (for pRK2013),rifampicin, spectinomycin and tetracycline were added atconcentrations of 50, 20, 50, 100, 50 and 15 mg ml-1

respectively. The flavonoids, apigenein or daidzein were usedto induce ttsI, as both have been shown to induce NB18(Viprey et al., 1998; Kobayashi et al., 2004; Marie et al.,2004).

Construction of NGRDttsI and pttsI-2

To obtain a mutant with a deletion in ttsI, the 2.4-kb PstIfragment from pUC::ttsI (Marie et al., 2004), which carriesttsI as well as its flanking region (Fig. 1), was digested withSacII and NheI, purified by electrophoresis, treated with theKlenow fragment, and self-ligated. Then, the remaining0.8-kb PstI fragment, lacking ttsI, was purified and sub-cloned into the PstI site of pJQ200-mp18 (Quandt andHynes, 1993). The resulting plasmid was mobilized intoNGR234 by tri-parental matings using the pRK2013 helperplasmid (Ditta et al., 1980). Marker exchange in NGRDttsIwas confirmed by Southern hybridization. To complementNGRDttsI, ttsI along with its own nod box-containing pro-moter region, the 2.4-kb PstI fragment of pUC::ttsI (Fig. 1),were subcloned into the PstI site of pRG960 (Van den Eedeet al., 1992), a vector which is compatible with pMP220-based lacZ fusion constructs described below, giving rise topttsI-2.

Extraction and analysis of Nops and LPS

After 40-h induction with 10-6 M apigenin, secreted pro-teins and LPS were recovered from the various NGR234strains as described in Viprey et al. (1998) and Marie et al.(2004) respectively. Aliquots of purified proteins were sepa-rated on SDS-polyacrylamide (PAGE) gels and stainedwith silver (Ausubel et al., 1991) or, for immuno-detection,separated proteins were transferred to PVDF Immobilon-Pmembranes (Millipore Corporation, Bedford, Massachusetts,USA) and probed with antibodies against NopL, NopPand NopX. Horseradish peroxidase-labelled goat anti-rabbit immunoglobulin antibodies of the ECL kit (GE Life-sciences Amersham Pharmacia Biotech, Uppsala, Sweden)were used as secondary antibodies. Reactions were visu-alized by enhanced chemi-luminescence. Extracted LPSsamples were separated on 16% deoxycholic acid (DOC)-PAGE and stained with silver nitrate (Tsai and Frasch,1982).

Cloning of the 11 TBs in pMP220

Predicted promoter regions containing TB1, TB3–TB11 wereamplified by PCR using the primer pairs listed in Table 1. Theamplified products were subcloned into pBluescript KS+

(Stratagene, La Jolla, California) and verified by sequencingthe inserts. Then, the inserts were excised and cloned intopMP220. pMP220 constructs containing TB2 (pMP-TB2) andTB8 (pMP-TB8) correspond to plasmids pMP220-rmlBand pMP220B (Marie et al., 2004) respectively. Promoterconstructs cloned into the broad-host-range reporter vectorpMP220 (Spaink et al., 1987) were mobilized into NGR234and its derivatives by tri-parental matings using pRK2013 asthe helper plasmid (Ditta et al., 1980). Flavonoid inductionwas performed as described previously (Kobayashi et al.,2004): rhizobial cultures grown to a density of 1cmOD600

0.5–0.6 were diluted to 1cmOD600 0.1 in RMS medium andinduced with 2 ¥ 10-7 M daidzein or 1 ¥ 10-6 M apigenin.b-Galactosidase activity was assayed according to Miller(1972).

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DNase I footprinting

DNA fragments were amplified with polynucleotide kinase-end-labelled primers. The rmlB and nopB promoter regionswere amplified with the following primers (respectively):rmlB-F and rmlB-R, and nopB for and nopB rev (Table 1).DNase I footprinting assays were performed in a total volumeof 50 ml of TAP buffer without polyethyleneglycol (50 mMTris-acetate pH 8.0, 100 mM potassium acetate, 8 mM mag-nesium acetate, 27 mM ammonium acetate, 1 mMdithiothreitol). Labelled fragments were added at a final con-centration of approximately 5 nM and incubated with the pro-teins for 30 min at room temperature. After incubation, 0.05 Uof freshly diluted DNase I (Invitrogen, Carlsbad, CA) wasadded, and the reaction was allowed to run for 2 min at roomtemperature. Then, the samples were extracted with phenol,precipitated with ethanol, washed, re-suspended in loadingbuffer and loaded on a 6% DNA sequencing gel. Images wereobtained by a Cyclone imager (Packard Institute, DownersGrove, Illinois) after exposing the dried gel to a Phosphoim-ager screen or by exposing the gels to X-ray film andscanning. Sequence ladders were prepared using the Seque-nase Quick Denature Plasmid DNA Sequencing Kit asdescribed by the manufacturer (USB, Cleveland, Ohio).

Electrophoretic mobility shift assays

32P-labelled PCR fragments were incubated for 30 min inTAP buffer (50 mM Tris-acetate, 100 mM potassium acetate,8 mM magnesium acetate, 3.5% polyethylene glycol 8000,1 mM DTT, pH 7.9) with the indicated concentrations of TtsIand reactions loaded into a 4% native PAGE gel. Runningbuffer contained 50 mM Tris, 400 mM glycine, 2 mM EDTA,8 mM MgSO4 at pH 8.5. Dried gels were exposed to X-rayfilms and images obtained by scanning the film.

Site-directed mutagenesis of TB8

The non-mutated TB8 promoter was excised from pMP-TB8as a HindIII fragment and subcloned upstream of a promoter-less gfp gene in plasmid pPROBE-GT′. Point mutations inTB8 were introduced by PCR using a pair of primers thatreplace the highly conserved triplet TCA for AAT. Two inde-pendent PCR reactions were carried out using primers TB8-Fmut (5′-ggccggtagaatgcgtgtcgtcagctcgcctc-3′) versus nopBrev (5′-ggactcgattacttaactctttgac-3′) and TB8-R mut (5′-ggaggcgagctgacgacacgcattctaccggc-3′) versus nopB for (5′-ctcgtcttgataaaccaaatctgaa-3′). PCR products were pooled,amplified, cloned into pGEM-T (Promega, Madison, Wiscon-sin) and sequenced. The mutated promoters were subse-quently transferred into pPROBE-GT as an EcoRI insert andorientation was checked. The constructs were mobilized intoNGR234 or NGRDttsI as described previously.

Random mutagenesis of TB8

Mutagenesis of the nopB promoter region was accomplishedusing mutagenic PCR as described by Vartanian et al.(1996). PCR reactions contained 1¥ taq buffer, 2.5 mMMgCl2, 0.5 mM MnCl2, 50 mM dATP and dCTP, 1 mM dGTP

and dTTP, 2.5 mM each primer, template DNA and taqpolymerase (Eppendorf). PCR fragments were clonedinto pGEM-T (Promega), sequenced and transferred topPROBE-GT as an EcoRI insert and orientation waschecked.

Assay of GFP intensity

Rhizobial cultures were grown to an OD600 of 0.5, diluted to anOD600 of 0.1 in RMS medium and induced with 1 ¥ 10-6 Mapigenin. Both optical density (OD595) and fluorescence (exci-tation filter at 485 nm and emission filter at 535 nm) weremeasured on 100 ml of cultures 40 hpi using a Plate Chame-leon Multilabel Detection Platform (Hidex Oy, Turku, Finland).Optical densities and fluorescence were corrected to back-ground levels using un-inoculated media, and the resultsrepresent the means of at least three independentexperiments.

Acknowledgements

This work was supported by the Fonds National Suisse dela Recherche Scientifique (Projects 3100AO-104097 and3100A0-116858 to W.J.B. and W.J.D.), the Département del’Instruction Publique du Canton de Genève (to W.J.B. andW.J.D.), the Universitè de Genève (to W.J.B.) and a grantfrom the National Institute of Health (GM31010 to G.C.W.).G.C.W. is also supported by an American Cancer SocietyResearch Professorship and a Howard Hughes Medical Insti-tute Professorship. R.W. was supported by a CAPES post-doctoral fellowship. H.K. is supported by JSPS PostdoctoralFellowships for Research Abroad. We wish to thank Y.-Y.Aung and D. Gerber for their unstinting help, M.M. Saad foruseful discussions on Nop preparation and K.E. Gibson forkind help with LPS preparation.

References

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