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Role of Arabidopsis and Tomato Phytohormones in the Response to Bacterial Pathogens

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Title:
Role of Arabidopsis and Tomato Phytohormones in the Response to Bacterial Pathogens
Creator:
BLOCK, ANNA KATHERINE ( Author, Primary )
Copyright Date:
2008

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Subjects / Keywords:
Diseases ( jstor )
Gene induction ( jstor )
Infections ( jstor )
Inoculation ( jstor )
Pathogens ( jstor )
Plant growth regulators ( jstor )
Plant nutrition ( jstor )
Plants ( jstor )
Symptomatology ( jstor )
Tomatoes ( jstor )

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University of Florida
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Copyright Anna Katherine Block. Permission granted to University of Florida to digitize and display this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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Full Text


















THE ROLE OF


ARABIDOPSIS AND TOMATO PHYTOHORMONES INT THE
RESPONSE TO BACTERIAL PATHOGENS


By

ANNA KATHERINE BLOCK


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA INT PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2004

































Copyright 2004

by

Anna Katherine Block


































This thesis is dedicated to my family who support me without question, no matter how
unusual they think my choices are.
















ACKNOWLEDGMENT S

This work was supported in part by a grant to Harry Klee from the National

Science Foundation (IBN0091064) and the Florida Agricultural Experiment Station. I

thank my advisor Harry Klee and my committee members Jeffrey Jones, John Davis and

Shouguang Jin for their guidance; Eric Schmelz for the measurement of the

phytohormones by GC-MS; the Klee lab for its support and advice; the Jones lab for the

use of its greenhouse and help in tomato plant maintenance and the Settles lab for use of

Arabidopsis growth facilities and other equipment.



















TABLE OF CONTENTS
Page

ACKNOWLEDGMENT S ........._.._ ..... ._._ ..............iv.....

LI ST OF T ABLE S ........._.. ..... ._ ._ ..............vii....

LI ST OF FIGURE S.............. ..............viii

AB S TRAC T .........._.._.._ ..............x...._.... ....

CHAPTER

1 INTRODUCTION ................ ................. 1...............


Plant-Pathogen Interactions ............ .....___ ......__ ... .. ......1
Pathogenesis-Related (PR) Genes ................ ................. 2......... ....
Bacterial Pathogens ................. ........... ........ ... .............. 3.....
The Role of Salicylic Acid in Arabidopsis Defense Responses ................. ..............4
The Role of Ethylene in Arabidopsis Defense Responses. ................ ............... ... 7
The Role of Jasmonates in Arabidopsis ................. ..............7.. .............
The Interaction of Tomato and Xcy ..........._..._ ....... ........._ ..........8
Symptom Development and Systemic Responses in Tomato ..........._..._ .............. 12

2 THE EFFECT OF THE REMOVAL OF SA ON PHYTOHORMONE SIGNALING
NETWORKS IN ARABIDOPSIS INFECTED WITH VIRULENT PST................ 14

Differences in Disease Progression and Symptom Development in Salicyclic Acid-
Deficient Arabidopsis Mutants infected with Pst. ..............._ ........... ......... 14
SA and Jasmonate Induction in Response to Pst Infection ................. ................. 17
The Effect of Coronatine Production by Pst.............. .................. 19
The Role of Ethylene in SA-Deficient Arabidopsis ................ ................ ...._ 22
Discussion ................ ................. 23..............



3 SYSTEMIC ACQUIRED TOLERANCE IN TOMATO ................ ............... ...28

Introduction ................... .. ........... .... .. .......... .. ...... ........2
Virulent and Avirulent Xcy Led to Systemic Acquired Tolerance in Tomato..........3 1
Prior Inoculation with Xcy Led to an Early Production of Ethylene upon Challenge
with Virulent Xcy ..........._.._ ....... .............._ 3 4...












PR Gene Expression Indicated the Necessity of Ethylene and SA in Systemic Signal
Generation. ........._... .... ... .. .._._ .. ....... ..... ... ........3
Avirulent Xcy Induced Greater Local and Systemic PR Gene Expression than
Virulent Xcy, but they Result in Similar PR Gene Induction During Challenge .. 39
Discussion ........._... ......___ .............._ 40...


4 DISCUS SION ........._... ......___ .............._ 46...


Phytohormone Networks in the Responses of Tomato and Arabidopsis to Virulent
Bacterial Pathogens. ........._........... ....._ ......_ ............ 4
The Compatible Tomato-Virulent Xcy Interaction. ..........._..._ ....._._ ............. 48
The Compatible Arabidopsis-Virulent Pst Interaction ................ ..................... 49
Systemic Responses to Infection.............. ............... 51


5 MATERIALS AND METHODS ................ ..............53. ..............


Plant Materials and Treatments.............. ............... 53
Bacterial Culture ................. ................. 54.............
Ion Leakage ................. .......... ................. 54....
Ethylene Measurements ................. .......... ................. 55....
SA, Jasmonate and Coronatine Measurements ................ ......... ................ 55
RNA Extraction.............. ............... 55
RNA Gel Blot Analysis ................ ................. 56.............
Real-time RT-PCR ................ ................. 56..............
MCP Treatment ................ ................. 57..............


LIST OF REFERENCE S.............. ............... 58


BIOGRAPHICAL SKETCH ................. ..............66.......... ......
















LIST OF TABLES

Table pag

4-1 Comparative phenotypes of phytohormone mutants in Arabidopsis and tomato......47

4-2 Comparative phytohormone profiles of Arabidopsis and tomato mutants ...............47


















LIST OF FIGURES


Finure pag

1-1 The role of sid2 in SA biosynthesis and nahG in SA removal ................. .............. .6

1-2 Jasmonate biosynthesis. ................ ..............9.. ......... ....

1-3 The structure of coronatine ................ ................. 10......... ...

1-4 Ethylene biosynthesis ................ ................. 11......... ....

2-1 Bacterial growth in Pst DC3000 infected Arabidopsis ................. ............. ...... 15

2-2 PR1 and PDF l.2 gene expression in Pst infected Arabidopsis .............. ... ........... 16

2-3 SA production in Pst infected Arabidopsis ................. .............. ......... .... 17

2-4 JA and OPDA production in Pst infected Arabidopsis ................ ............. ...... 18

2-5 Coronatine production in Pst infected Arabidopsis ................ ................ ...._ 19

2-6 Bacterial growth at 72 hpi in Pst DC3000 and Pst DC3661infected Arabidopsis .... 20

2-7 Disease symptom development in Pst infected Arabidopsis ................ ................ 21

2-8 Ethylene production in Pst infected Arabidopsis. ................ ... .............. .......23

3-1 Symptom development of Xcy infected tomato ................ ................ ........ 3 1

3-2 Cell death due to a second infection of tomato with virulent Xcy.. ..........._..... ......... 32

3-3 Bacterial growth during the first and second infections of tomato with Xcy. ........... 33

3-4 Local ethylene and SA production during the first and second infections of tomato
with Xcy. ..........._.._ ..............._ 3....._._ 5....

3-5 Local and systemic PR gene induction in ethylene and SA deficient tomato lines in
response to infection with virulent Xcy ..........._..._ ........._._ ...... 38._.__...

3-6 The measurement of local and systemic PR gene expression in tomato during
infections with virulent or avirulent Xcy .............._ ........._._ ...... 41._.__...











3-7 Induction of PR genes during virulent Xcy infection in tomato plants in the presence
or ab sence of S AT ................. ................. 42............

4-1 A model for the signaling network in tomato in response to virulent Xcy........._......49
















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

THE ROLE OF ARABIDOPSIS AND TOMATO PHYTOHORMONES INT THE
RESPONSE TO BACTERIAL PATHOGENS

By

Anna Katherine Block

December 2004

Chair: Harry Klee
Maj or Department: Plant Molecular and Cellular Biology

Phytohormone networks are used to regulate the plant response to virulent bacterial

pathogens. In this work by the use of salicylic acid (SA) deficient lines it is shown that

SA, a major player in the interaction between Arabidopsis and virulent Pseudomonas

syringae py. tomato (Pst), down-regulates ethylene and jasmonates. SA influences

ethylene production in an NPR1-independent manner and jasmonate production in an

NPR1-dependent manner. Pst-produced coronatine, a jasmonate mimic, acts

independently of SA and does not compromise SA-dependent basal resistance. These

three phytohormones are also involved in the response of tomato to Xanthomonas

campestris py. vesicatoria (Xcy). However, the hormone network is dramatically

different to that of Arabidopsis. It is demonstrated, by the use of phytohormone deficient

plants and pathogenesis-related marker gene expression profiling, that a systemic signal

is generated in tomato in response to an infection with virulent Xcy. This signal is SA-

dependent and leads to the development of a systemic acquired tolerance in tomato to










subsequent infections with virulent Xcy. This systemic acquired tolerance is similar to the

systemic response that is generated in response to avirulent Xcy.















CHAPTER 1
INTTRODUCTION

Plant-Pathogen Interactions

The normal growth of a plant can be disrupted due to interactions with pathogenic

organisms such as bacteria, fungi, parasitic higher plants, nematodes, mycoplasmas and

viruses. Pathogens live in or on another organism, from which they derive nutrients.

Substances released by the pathogen, in order to penetrate plant cell walls and make the

nutrients accessible for their use, cause damage to the host tissues. These substances

include enzymes, toxins, growth regulators and polysaccharides, some of which are

induced upon host recognition.

Not all pathogens can cause disease on all hosts as some lack the necessary abilities

to penetrate and survive that host's endogenous structural and chemical defenses. The

host range of a pathogen is defined as the host plants on which a particular pathogen can

recognize and grow successfully (Agrios, 1988).

Resistant hosts can specifically recognize avirulent pathogens and cause an

incompatible interaction. Incompatible interactions often include a hypersensitive

response HR that consists of a rapid induction of host defenses and the death of cells in

contact with the pathogen. The faster this response occurs, the more resistant the plant is

to the pathogen. Compatible interactions occur between susceptible plants and virulent

pathogens, leading to successful invasion of host tissues (Ausubel et al., 1995;

Hammond-Kosack and Jones, 1996).











In both compatible and incompatible interactions the plant attempts to limit damage

and control pathogen growth. This is done by the production of toxic substances around

the site of injury, as well as by the formation of protective layers such as callus and cork.

Some compounds are produced at high enough concentrations to limit pathogen growth.

These compounds include phenolic compounds such as chlorogenic and caffic acids,

oxidation products of phenolic compounds and phytoalexins (Agrios, 1988; Jackson and

Taylor, 1996).

Pathogenesis-Related (PR) Genes

As well as the production of substances and structures, several plant genes are

induced in response to pathogens. PR genes are used as markers of the disease response

in many plant species. They are defined as plant-encoded genes that are only expressed in

the tissue in response to a pathogen or related stress. PR genes are classified by function

and homology; these classes include PR-2 ((p-1,3-glucanase), PR-6 (proteinase-inhibitor) ,

PR-12 (defensin) and PR-1 whose function is unknown (Van Loon and Van Strien,

1999).

Although PR genes are induced during infection, only a few have been shown to

have a direct impact on pathogen growth. The most well characterized of these is the PR-

1 family that has antifungal activities, although the mechanism by which it acts has yet to

be identified (Alexander et al., 1993; Niderman et al., 1995). The combined effect of

many PR gene products may be required to have a significant effect on pathogen growth.

Particular classes of PR genes have been linked to specific phytohormone induction

events. For example the PR gene Thi 2. 1 of Arabidopsis is induced during wounding in a

jasmonate dependent manner (Bohlmann et al., 1998). The phytohormones, however,










affect one another to such an extent that any direct interpretation of the roles of

phytohormones from PR gene expression is inherently risky.

The responses covered so far are induced at a local level by pathogen infection. The

plant, however, also shows a systemic response to infection that includes some of the

same response as those at the local level. For example pathogen infection can lead to the

production of a systemic signal. This signal can induce resistance in uninfected tissues of

the plant 2-3 days after infection. It can also induce the systemic expression of PR genes.

This systemic induced resistance (SAR) can last several weeks and is effective against a

wide range of normally virulent pathogens.

Bacterial Pathogens

The response to pathogens varies depending on the host-pathogen interaction. To

simplify the study of plant pathogen interactions the focus of this work will be limited to

bacterial pathogens, specifically to Xanthomonas and Pseudomonas. These are

necrotizing biotrophic pathogens that live within the apoplast. They can multiply for

some time within host tissues before causing necrosis. They use a type III secretion

system to introduce substances into host cells that cause the release of nutrients and

suppress host defense responses. They also produce toxins and other extracelluar

substances, which affect the plant and cause disease symptom development (Alfano and

Collmer, 1996).

Xanthomonas and Pseudomonas are rod shaped gram negative bacteria of the

family pseudomonadaceaze. They use flagella to move through liquid and can enter host

tissues from water on the surface of the tissue. For example they can enter the leaves

through wounds or stomatal openings. The symptoms these bacteria cause include the









formation of necrotic lesions on infected tissues. These lesions are sometimes surrounded

by a chlorotic halo. The necrotic lesions may coalesce and form large necrotic regions or

even kill the entire tissue.

Bacterial species can be further subdivided into subspecies or pathovars (pv),

which are distinguished by their host range. The two pathogens this work will focus on

are Pseudomonas syringae py. tomato (Pst) that causes bacterial speck both in tomato

and in the model plant Arabidopsis and Xanthomonas campestris pv vesicatoria (Xcy)

that causes bacterial spot in tomato and pepper (Agrios, 1988).

Many of the plant responses to pathogens have been characterized. However, how

the plant forms and coordinates its defense response has yet to be fully elucidated

(Glazebrook, 2001). One of the major tools in such studies is mutant screening for plants

altered in their pathogen responses. The screens focus either on disease phenotype or on

PR gene induction with the plant Arabidopsis thaliana as a favored model (Glazebrook et

al., 1997). These screens have identified mutants that are compromised in their resistance

to avirulent pathogens. Some of these mutants also show enhanced susceptibility to

virulent pathogens and thus demonstrate the existence of a basal resistance, in which the

plant limits the growth of virulent pathogens (Glazebrook et al., 1996). Such mutants

often also have altered phytohormone profiles, indicating important roles for

phytohormones in coordinating the defense responses to both virulent and avirulent

pathogens (Dewdney et al., 2000; Nawrath et al., 2002).

The Role of Salicylic Acid in Arabidopsis Defense Responses

The phytohormone salicylic acid (SA) is involved in maintenance of basal

resistance, the response to avirulent pathogens and development of SAR in Arabidopsis.









Mutants that show enhanced susceptibility to virulent pathogens such as Pst DC3000

often accumulate less SA than their wild type parents following infection. For example

pad4, which has enhanced susceptibility to virulent Pseudomonas syringae pv maculicola

ES4326 (Psm), has reduced and delayed SA synthesis (Zhou et al., 1998).

In several plant species the removal of SA has been accomplished by introduction

of the nahG transgene encoding a Pseudomonas putida salicylate hydroxylase (EC

1.14. 13.1), which converts SA into catechol (Yamamoto et al., 1965). Arabidopsis plants

carrying this transgene have enhanced susceptibility to several pathogens including Pst

(Delaney et al., 1994; Lawton et al., 1995).

An Arabidopsis SA biosynthesis mutant, sid2-2, that fails to accumulate SA in

response to pathogens has also been identified. This line contains a loss of function

mutation in the enzyme isochorismate synthase 1 (EC 5.4.4.2) that converts chorismate to

isochorismate (Figure 1-1) (Wildermuth et al., 2001). sid2-2 has both enhanced

susceptibility to Pst and fails to accumulate SA in response to infection (Nawrath and

Metraux, 1999).

NahG and sid2-2 respond differently to the non-host pathogen Pseudomonas

syringae py. phaseolicola strain 3121, suggesting that the catechol produced by the nahG

transgene may influence the disease process (Van Wees and Glazebrook, 2003). The

conclusion that nahG has an effect beyond elimination of SA is further supported by

global expression phenotyping of Arabidopsis in response to infection by virulent Psm

(Glazebrook et al., 2003). This showed major differences between NahG and sid2-2 in

terms of the expression patterns of genes induced in the two lines by infection with Psm.










H--O O

,H isechorismale H
O' synthase

O w sic ~ 'h 0




ch ri smale i seho~rismale










I H-O O
O-

Q-OaH salicyclateO-
nah G

calchol salic~ic acid
Figure 1-1 .The role of sid2 in SA biosynthesis and nahG in SA removal.

Signal transduction from SA can occur in an NPR1-dependent or independent

fashion (Clarke et al., 1998). NPR1 is a protein with a BTB/BOZ domain and an ankyrin

repeat domain, both of which are involved in protein-protein interactions (Cao et al.,

1997). Upon infection NPR1 is translocated to the nucleus where it interacts with bZIP

transcription factors that are involved in the SA-dependent activation of PR genes

(Despres et al., 2000). NPR1 also has a role in suppressing jasmonate signaling in the

cytoplasm (Spoel et al., 2003). However, nuclear localization of NPR1 is required for PR










gene expression (Kinkema et al., 2000). The loss of function mutant nprl-1 has enhanced

susceptibility to virulent Pseudomonas syringae and has reduced expression of PR genes

after infection (Cao et al., 1994).

The Role of Ethylene in Arabidopsis Defense Responses

The major role of SA in basal resistance is demonstrated by SA-deficient

Arabidopsis that are more susceptible to the virulent pathogen Pst. Acting either

agonistically or antagonistically to SA in these interactions are other phytohormones such

as ethylene and jasmonic acid (JA). For example, the Arabidopsis ethylene insensitive

mutant etrl-1 has faster disease progression than wild type plants in response to virulent

Xanthomonas campestris py. campestris (Xcc). This suggests that ethylene negatively

regulates disease symptom development in this system. However, NahG is deficient in

ethylene production following Xcc infection, suggesting that these two phytohormones

act agonistically in this response (O'Donnell et al., 2003). The loss of ethylene in NahG is

not supported by sid2-2, which was shown to accumulate ethylene in response to

infection with Pst (Heck et al., 2003). Thus, the failure to produce ethylene in pathogen

infected NahG Arabidopsis may be a specific consequence of the nahG transgene and not

due to the failure to accumulate SA.

The Role of Jasmonates in Arabidopsis

Two Arabidopsis mutants that are SA-deficient, eds4 and pad4, have increased

sensitivity to compounds that induce the expression of JA response genes (Gupta et al.,

2000), and NahG and sid2 produce higher levels of JA in response to infection with Pst

than does Columbia (Heck et al., 2003; Spoel et al., 2003). JA is produced from alpha-

linolenic acid via the active precursor 12-oxo-phytodieonic acid (OPDA). It can also be

converted into the volatile compound methyl-jasmonate (Figure 1-2). These two










compounds, in addition to JA and other oxylipins, are commonly referred to as

jasmonates (Seo et al., 2001; Stintzi et al., 2001). Currently, little is known about the

roles of individual jasmonates in pathogen defense.

The evidence for a role for jasmonates in compromising the basal resistance of

Arabidopsis to Pst is further supported by the involvement of Pst-produced coronatine.

Coronatine is a phytotoxin that mimics OPDA (Weiler et al., 1994) (Figure 1-3). A Tn5

transposon insertion into Pst DC3000 produced a coronatine-deficient Pst mutant, Pst

DC3661 (Moore, 1989). This mutant strain was shown to be less virulent than Pst

DC3000 in Arabidopsis in dip infections but not in injection inoculations. However, the

disease symptoms in both forms of infection were reduced when compared to the parental

strain (Mittal and Davis, 1995).

The hypothesis that coronatine is an important virulence factor for Pst is reinforced

by studies with the Arabidopsis coronatine insensitive mutant coil (Feys et al., 1994).

COII is part of an E3 ubiquitin-ligase involved in a jasmonate response pathway (Xie et

al., 1998). The coil mutants are both more resistant to Pst DC3000 and have higher SA

levels (Kloek et al., 2001). These data along with similar studies in tomato (Zhao et al.,

2003) suggest that coronatine acts by stimulating the jasmonate-signaling pathway.

The Interaction of Tomato and Xcy

Not all plants use phytohormones in the same way in their defense responses. In

tomato phytohormones are involved in the response to both virulent and avirulent Xcy. In

the response to virulent Xcy, there is an induction of ethylene followed by accumulation

of SA.










Phospholipid?


Slipolytic enzyme (dadl)



O-

Alpha-linolenic acid



Lipoxygenase (Lox)



0O -1-
O--

13-hydroperoxy-linolenic acid


methyl-jasmonate


aliene oxide synthase


Jasmonic acid
carboxyl
methyltransferase
(JMT)


O--H


allene oxide


Saline oxide cyclase






12-oxo-phytodienoic acid (OPDA)


Jasmonic acid


beta oxidation


OPDA
reductase
(opr3)


O--H


3-oxo-2(2'-pentenyl)-cyclopentane-1l-octanoic acid


Figure 1-2 Jasmonate biosynthesis.






















Figure 1-3 The structure of coronatine.

SA production in this response is dependent on the previous production of ethylene

(O'Donnell et al., 2001), and both are dependent on the ability of the plant to produce

jasmonates.

Antisense plants for the jasmonate biosynthesis enzyme allene oxide synthase (as-

AOS) are resistant to virulent Xcy and show no production of either ethylene or SA

(O'Donnell et al., 2003). Transgenic tomato plants that express 1-aminocyclopropane- 1-

carboxylate (ACC) deaminase (ACD) (EC 3.5.99.7) (Figure 1-4) do not produce ethylene

and are compromised in SA induction during infection. NahG plants, which do not

produce SA, have normal ethylene production. Both ACD and NahG are tolerant to

virulent Xcy and unaffected in their ability to produce jasmonates (O'Donnell et al.,

2003).

The tolerance of a plant to a pathogen can be defined as substantial growth of the

pathogen within the host tissues combined with an absence of full symptom development.

Another plant that is compromised in phytohormone signaling and shows tolerance is the

ethylene insensitive (ein2) mutant of Arabidopsis. It is tolerant to both Pst and Xcc (Bent

et al., 1992).


O O-H






















S- Aden osy-L-rneth ion in e

ACC sqnthase


ACC chlarnin ase


2- Oxotutancate


1 -arninco ycl opro ene-1-car o:= ate (ACC)


ACC oxiciase


H H

eth ~ene


Figure 1-4 Ethylene biosynthesis.


N--H
H


.;;.aO










The response of tomato to avirulent Xcy also involves phytohormone induction.

There is a major early induction of ethylene that is larger than that seen in the susceptible

response. There is also an early induction of SA. Increased ethylene sensitivity in tomato

caused by reduced expression of an ethylene receptor LeETR4 enhances the

hypersensitive response to avirulent Xcy (Ciardi et al., 2001). This demonstrates that

ethylene is also important in the response to avirulent pathogens.

Symptom Development and Systemic Responses in Tomato

Symptom development in tomato infected with Xcy can be categorized into two

stages. The primary response present in resistant, susceptible and tolerant interactions

consists of localized lesion formation. The secondary phenotype, which is only present in

the susceptible response, consists of the development of chlorosis and necrosis that

spreads from the sites of the primary lesions (O'Donnell et al., 2003).

Perhaps there is an advantage for the plant to produce these phytohormones and the

consequent chlorosis and necrosis development in response to virulent infections. In

addition to the necrosis of the tissue effectively removing the pathogen from the plant, it

is possible that this response produces a systemic signal that primes the plant's defenses

in case of repeat infections. It has been demonstrated in several systems, including

tobacco and cucumber that infections with virulent pathogens that produce high levels of

necrosis can lead to the induction of SAR (Cohen and Kuc, 1981; Strobel et al., 1996).

Therefore the phytohormones induced during the interaction with virulent Xcy and

tomato may be involved in the development of SAR and this could be the reason why

they remain even though their absence would lead to tolerance. SAR is induced in tomato










by tobacco necrosis virus (TNV) (Jeun et al., 2000) and Phytophthora infestans (Enkerli

et al., 1993) and leads to resistance to P. infestans.

Here two different aspects of phytohormone signaling in response to infections

with virulent bacterial pathogens are studied. The first, in chapter 2, investigates the

effect of the removal of SA accumulation on the synthesis of ethylene and jasmonates

during the interaction between Pst and Arabidopsis. This chapter focuses on

phytohormone networks in the local response to infection. The second, in chapter 3, is on

the interaction between tomato and Xcy. In this chapter the focus is the systemic response

to virulent bacterial infection and the possible roles of phytohormones in this response.

Although in both cases the focus is on the role of phytohormones in response to

pathogens, it can clearly be seen that the phytohormones play different roles in the two

systems and therefore information gained from studying one plant-pathogen interaction is

not necessarily applicable to another.















CHAPTER 2
THE EFFECT OF THE REMOVAL OF SA ON PHYTOHORMONE SIGNALING
NETWORKS IN ARABIDOPSIS INFECTED WITH VIRULENT PST

Differences in Disease Progression and Symptom Development in Salicyclic Acid-
Deficient Arabidopsis Mutants infected with Pst

Phytohormones do not act in a discrete fashion. The action often attributed to one

phytohormone can be due to the combined action of several. SA plays an important role

in the basal resistance of Arabidopsis to virulent Pst. However, it is not the only

phytohormone induced in response to Pst. Ethylene and jasmonates are also induced in

this response. The aim of this chapter is to determine the effect of removing SA on the

phytohormone-signaling network in Arabidopsis in response to virulent Pst. This will

provide a clearer resolution of the complex relationships between the signaling pathways

in this system.

As a first step into understanding the effect of altered SA production or signaling,

levels of basal resistance and the disease phenotypes in four different lines were

compared. These lines are the SA biosynthesis mutant sid2-2, the SA signaling mutant

nprl-1 and the transgenic line NahG that cannot accumulate SA. The infection of

Columbia (wild type), nprl-1, sid2-2 and NahG plants with virulent Pst led to differential

bacterial growth and disease phenotypes in the four lines.

NahG supported significantly more bacterial growth than the other lines (Figure 2-

1). The mutants sid2-2 and nprl-1 had bacterial growth that was intermediate between

NahG and the wild type. The sid2-2 mutant is a better model for the loss of SA

production than NahG due to the side effects of catechol production (Heck et al., 2003).










Therefore, as sid2-2 supports the same levels of bacterial growth as nprl-1, this

demonstrates that the effect of SA on basal resistance is NPR1-dependent (Figure 2-1).

The development of disease symptoms showed further differences between the four

lines. Columbia exhibited patchy chlorosis at 48 h post infection (hpi). These patches

coalesced at 96 hpi. NahG showed the most severe symptom development, with

widespread chlorosis followed by complete tissue collapse at 96 hpi. The sid2-2 mutant

exhibited less severe symptoms than NahG, with widespread chlorosis and necrosis at 96

hpi although it lacked the complete tissue collapse of NahG. The nprl-1 mutant

developed chlorosis but not necrosis and had an intermediate phenotype between

Columbia and sid2-2. These data demonstrate that although sid2-2 and nprl-1 showed

the same level of basal resistance, sid2-2 exhibited necrosis and tissue collapse that did

not occur in nprl-1 under these conditions. This result indicates that the effect of SA on

repressing necrosis and tissue collapse is NPR1-independent.


-o-Col
-*-nprl-1
8 -1 -0--sid2-2
-m-NahG










0 20 40 60 80 100
hours post infection
Figure 2-1 Bacterial growth in Pst DC3000 infected Arabidopsis. Columbia, nprl-1,
sid2-2 and NahG were dip infected with Pst DC3000. Colony forming units
per square centimeter were determined at the time points indicated.










The expression of the PR genes PR1 and PDF 1.2 was monitored during disease

progression in all four lines. PR1 was induced in Columbia and slightly induced at 96 hpi

in nprl-1 but was not induced in the SA-deficient lines. PDF1.2 was induced to a similar

extent in all lines upon pathogen infection (Figure 2-2). The reduction in PR1 gene

expression in NahG and sid2-2 is consistent with that seen upon infection with avirulent

Pst (Nawrath and Metraux, 1999) and the loss of localized expression of PR genes in

nprl-1 as demonstrated by Cao et al. (1994). Consistent with my findings, PDF1.2

expression was unaltered in NahG and sid2-2, when compared to wildtype, in response to

infection with Alternaria brassicicola (Nawrath and Metraux, 1999).



p~cf 1.2 PR1










Fiur 2- R n D eeepeso nPs netdAaioss oa N a
extrcte fro Couba npl1 si22adNh lnt inetdwt









Figre -2PR DF l.2 o plantsta weresso mockst oculated, or 48 ad 96s hours post


infection.











SA and Jasmonate Induction in Response to Pst Infection

The study of the effect of removal of SA accumulation or perception on the

induction of other hormones was the focus of this chapter. Therefore the pattern of SA

induction in the different lines was confirmed. SA levels increased during infection in the

Columbia and nprl-1 lines, with nprl-1 accumulating significantly more SA than

Columbia. This is consistent with the results of Clarke et al. (2000), who demonstrated

that nprl-1 produces more SA than Columbia. SA remained at a low level in NahG and

sid2-2 consistent with the findings of Nawrath and Metraux (1999) (Figure 2-3).


7000
-o-Col
6000~ ---pri-1
-0- sid2-2
5000 -1 -m-NahG

~4000-

S3000-

2000

1000


0 20 40 60 80 100
hours post infection

Figure 2-3 SA production in Pst infected Arabidopsis. Columbia, nprl-1, sid2-2 and
NahG dip infected with Pst DC3000. SA was measured at the time points
indicated.

The jasmonate response of a plant can be described in terms of an oxylipin

signature consisting of different forms of JA and its precursors (Kramell et al., 2000). To

partially characterize this oxylipin signature in Arabidopsis infected with Pst, the

induction of jasmonic acid (JA) and its active precursor 12-oxo-phytodienoic acid


(OPDA) was measured. Both JA and OPDA were induced in all four lines. The highest

amounts of induction were observed in NahG and sid2-2, followed by nprl-1 with











Columbia producing the lowest amounts of these jasmonates (Figure 2-4). It has been

demonstrated by Heck et al. (2003) that Pst infection leads to higher JA levels in sid2-2

and NahG than in wild type Arabidopsis. Here, it can be seen that OPDA follows the


profile of JA expression and jasmonate production in nprl-1 is intermediate between

those of the SA-deficient lines and wild type.


-m-11NahG

600


400-


200-



0 20 40 60
hours post infection


80 100


2000


cnl500


, 1000


500


0 20 40 60 80 100
hours post infection

Figure 2-4 JA and OPDA production in Pst infected Arabidopsis. Columbia, nprl-1,
sid2-2 and NahG were dip infected with Pst DC3000. The amount of (A) JA
and (B) OPDA was quantified at the time points indicated.










The Effect of Coronatine Production by Pst

Jasmonate accumulation was higher in Arabidopsis that showed enhanced

susceptibility due to SA-deficiency. Therefore, jasmonate signaling may also reduce

defense responses. Pst produces the phytotoxin coronatine that is a mimic of OPDA and

may act by stimulating jasmonate responses (Weiler et al., 1994). The total jasmonate

response consists of jasmonates and coronatine. Therefore, coronatine accumulation

during infection in the different lines was measured. NahG had a significantly higher

level of coronatine accumulation than sid2-2, nprl-1 and Columbia (Figure 2-5).

500
-o-Col
-*-npri-1
40- -- sid2-2
-m-NahG


p" 300


S200-


100



0 20 40 60 80 100
hours post infection

Figure 2-5 Coronatine production in Pst infected Arabidopsis. Columbia, nprl-1, sid2-2
and NahG were dip infected with Pst DC3000. Coronatine was measured at
the time points indicated.

Coronatine's action as a virulence factor may utilize the jasmonate response

pathways to repress SA. To investigate if coronatine's action is SA-dependent, SA-

deficient lines were infected with Pst DC3661. Pst DC3661 is a Tn5 mutant that is

disrupted in its ability to produce coronatine (Mittal and Davis, 1995). Bacterial growth

was reduced by a factor of ten in all lines infected with Pst DC3661 compared to those










infected with Pst DC3000 but the relative differences in susceptibility between the

Arabidopsis lines remained (Figure 2-6).


0 DC3000
mDC3661
10-







S8-








Col nprl-1 sid2-2 NahG

Figure 2-6 Bacterial growth at 72 hpi in Pst DC3000 and Pst DC3661 infected
Arabidopsis. Columbia, nprl-1, sid2-2 and NahG were dip infected with Pst.
The amount of bacteria in the tissues was determined at the time points
indicated.

Disease symptom development was also reduced in all lines infected with Pst

DC3661 when compared to those infected with Pst DC3000 (Figure 2-7). However, the

comparative severity of the disease in the different lines remained the same. These results

were confirmed with an additional coronatine-deficient Pst mutant, Pst DC3118 (Moore,

1989; Ma et al., 1991). These data demonstrate that coronatine's action is independent of

SA, as coronatine-deficiency still reduced virulence on SA-deficient lines. These data

indicate that the JA and SA pathways act in an independent manner in the control of basal

resistance.








DC3000


DC3661


C r


1'-:~

~dP- "
1 ..


Columbia


~uia ,i~ii~
~c~~
:m


/P


npr l- 1







sid2-2


4"8~-5-~81a


Figure 2-7 Disease symptom development in Pst infected Arabidopsis. Columbia, nprl-
1, sid2-2 and NahG plants were dip infected with wild type Pst (DC3000) or
coronatine-deficient Pst (DC3661). Photographs were taken 96 hpi.


;;

~ ;;E"~i~


"(










The Role of Ethylene in SA-Deficient Arabidopsis

Recent work has demonstrated that the catechol produced by NahG has side effects

in the NahG plant that are not due to the loss of SA (Heck et al., 2003). NahG had been

used to demonstrate an agonistic relationship between SA and ethylene in the interaction

between Arabidopsis and Xce (O'Donnell et al., 2003). To verify this relationship in sid2-

2 and to investigate the role of NPR1 in this interaction, ethylene evolution in response to

Pst infection was measured.

Ethylene was induced to similar levels at 48 hpi in Columbia and nprl-1 indicating

that the control of ethylene synthesis by SA is NPR1-independent. A large increase in

ethylene, also at 48 hpi, was observed in sid2-2. NahG showed no induction of ethylene

during disease progression (Figure 2-8). These results indicate that catechol or its further

metabolites suppress ethylene production and that there is an antagonistic relationship

between the synthesis of SA and ethylene, rather than the agonistic relationship suggested

by the NahG phenotype. These results are also consistent with the observation that

ethylene insensitive plants synthesize more SA than wild type plants following pathogen

infection (O'Donnell et al., 2003).

To investigate if it was this suppression of ethylene signaling that caused the

differences in disease symptoms between sid2-2 and NahG, plants were treated with the

ethylene inhibitor 1 -methylcyclopropene (MCP) 24 hours prior to infection and the

resultant effects on disease were assessed. MCP blocks ethylene binding to it receptors,

thus inhibiting its perception (Sisler and Serek, 1997). No differences in disease

development between those plants treated with MCP and untreated plants were observed

(data not shown). These data indicate that although ethylene production is inhibited in










NahG, this absence of ethylene is not responsible for the enhanced disease phenotype

observed in NahG plants relative to sid2-2.


-o-- Col
-*-nprl-1
-0--sid2-2
20 I- -m-NahGI








~ 0



0 20 40 60 80 100
hours post infection

Figure 2-8 Ethylene production in Pst infected Arabidopsis. Columbia, nprl-1, sid2-2
and NahG were dip infected with Pst DC3000. Ethylene production was
measured at the time points indicated.

Discussion

The effect of an inability to accumulate SA on the induction of ethylene and

jasmonates during the interaction between Arabidopsis thaliana and the virulent bacterial

pathogen Pst DC3000 was investigated. Two lines that do not accumulate SA during

infection (NahG and sid2-2) were used to determine these effects. The effects of SA on

ethylene and jasmonates accumulation are believed to be NPR1-dependent (Clarke et al.,

2000). Consequently the levels of ethylene and jasmonates in nprl-1, follow ing infection

were also measured.

In accordance with previous reports (Cao et al., 1994; Delaney et al., 1994;

Naw'rath and Metraux, 1999) an enhanced susceptibility to Pst in NahG, sid2-2 and nprl-

1 was observed, with more severe symptom development in NahG and sid2-2 than in

nprl-1. This result correlates with the partial loss of SA signaling in nprl-1 compared to










the other two lines. NahG displayed a rapid and complete tissue collapse that did not

occur in sid2-2. NahG also supported more bacterial growth than sid2-2, although both

were more susceptible to Pst than Columbia. This result, coupled with the recent data

proposing side effects in NahG associated with catechol (Van Wees and Glazebrook,

2003), led to the comparasion of the NahG and sid2-2 lines for effects on ethylene and

jasmonate accumulation.

As has been reported previously, the SA levels in NahG and sid2-2 are roughly

equivalent following Pst infection (Nawrath and Metraux, 1999). The massive increase of

SA in nprl-1 compared to that of Columbia is probably due to a loss of feedback

inhibition on SA synthesis that relies on an NPR1-dependent SA pathway (Clarke et al.,

2000).

As well as SA, jasmonates have been reported to play a role in disease with both JA

and its precursor OPDA believed to be active signaling compounds (Stintzi et al., 2001).

It was therefore determined how the removal of SA affected the induction of these

jasmonates following infection. In Columbia, both JA and OPDA increase following

infection. In the SA-deficient lines an increase in jasmonates was observed compared to

wild type, with NahG and sid2-2 producing the most jasmonates. nprl-1 produced

intermediate jasmonate levels. This suggests that SA represses jasmonate production in a

partially NPR1 dependent manner. As NahG and sid2-2 produce similar levels of

jasmonates catechol is unlikely to affect jasmonate production. The enhanced

susceptibility of SA-deficient Arabidopsis to Pst may be due to the combined effects of

reduced SA and increased jasmonates.










The use of PR gene expression is often used to investigate the roles of specific

phytohormones in plant pathogen interactions. In order to determine the degree that two

of these markers represented the levels of the phytohormones produced, the expression of

PR1 and PDF1.2 was determined in the different lines during infection. The expression of

PR1 is absent in accordance with the loss of SA accumulation in both sid2-2 and NahG.

It is also much reduced in nprl-1 when compared to Columbia. These results demonstrate

that the loss of SA accumulation in these lines is sufficient to prevent the induction of SA

response pathways and that PR1 is a good marker of SA signaling.

There was also an induction of PDF1.2 to a similar extent in all lines. PDF1.2

requires the activation of both ethylene and JA response pathways to be induced in

response to Alteranria brassicicola (Penninckx et al., 1998). PDF1.2 is not visible in

sid2-2 or NahG at 96hpi due to the poor quality RNA obtained from this highly necrotic

tissue. These data indicate that although the absence of SA affects the levels of ethylene

and jasmonates this is not necessarily reflected in the expression of all downstream genes.

This indicates that marker genes may not always be the best approach to mapping

hormone networks.

The production of coronatine by Pst in the different Arabidopsis lines was

measured to further study the relationship between SA and jasmonates. Coronatine is a

non-host specific phytotoxin whose biological effects include induction of leaf chlorosis

(Mittal and Davis, 1995). It has been suggested to act as a mimic of OPDA (Weiler et al.,

1994). Coronatine production was observed in all of the lines infected with Pst, with the

most production occurring in NahG. The coronatine levels were different to those of

jasmonates. This suggests that coronatine acts via jasmonate signaling rather than by










stimulating jasmonate biosynthesis. The production of coronatine was the same in sid2-2

as Columbia so it is not directly related to bacteria levels. This suggests that host factors

or bacteria thresholds must be present to stimulate coronatine production.

To determine if coronatine affects SA-dependent basal resistance, the lines were

infected with the coronatine-deficient mutants Pst DC3661 and Pst DC3118. All lines

infected with coronatine-deficient Pst had reduced bacterial growth compared with those

infected with Pst DC3000 and consequently developed less severe disease symptoms.

However, the relative differences in susceptibility between the lines remained. This

indicates that role of coronatine as a virulence factor is independent of SA, as SA-

deficient lines still show an enhanced susceptibility to coronatine-deficient Pst despite its

reduced virulence. It is then both the accumulation of coronatine and the loss of SA

signaling that lead to the severe disease phenotype observed in the SA deficient lines

upon infection with Pst.

Ethylene synthesis is transient during the post-infection period, peaking around 48

hpi in Columbia in response to Pst. This induction does not occur in NahG. The faster

development of chlorosis in the ethylene insensitive mutant etrl-1 in response to Xcc

infection (O'Donnell et al., 2003) suggests that ethylene is required to slow the

development of chlorosis. The growth of Xcc in etrl-1 remains unchanged indicating that

ethylene does not play a direct role in limiting pathogen growth in this interaction. As the

Arabidopsis response to Pst and Xcc are similar, it can be concluded that ethylene does

not play a major role in basal resistance to either pathogen.

A major difference between the two SA-deficient lines with respect to this ethylene

induction was observed, with sid2-2 showing a two-fold increase in ethylene induction at










48 hpi relative to Columbia. These data agree with the recent study by Heck et al. (2003).

The completely opposite nature of this response in NahG when compared to sid2-2

suggests that catechol or a further metabolite negatively affects ethylene production.

However, the treatment of sid2-2 and NahG with the ethylene inhibitor MCP before

infection caused no change in their respective phenotypes. This suggests that although

ethylene may play a role in fine-tuning the disease response in Arabidopsis, it is not

responsible for the difference in susceptibility between sid2-2 and NahG. The

suppression of ethylene production by catechol in NahG therefore is independent of the

changes in susceptibility and disease phenotype.

These data indicate that NahG is not always an appropriate model for studying the

role of SA induction in plant-pathogen interactions. They also demonstrate an

antagonistic relationship between SA and ethylene in this system, as the removal of SA

increases ethylene synthesis. As no difference in ethylene induction in nprl-1 compared

with Columbia was observed, it might also be concluded that the effect of SA on ethylene

is NPR1-independent.















CHAPTER 3
SYSTEMIC ACQUIRED TOLERANCE INT TOMATO

Introduction

Pathogens have a high negative impact on agriculture. In 1993, for example, an

estimated 12% of crops were lost to disease (Agrios, 1997). The application of chemicals

such as fungicides help to limit the damage they cause. However, utilizing plant defense

responses remains an important research area for the limiting pathogen damage. For

instance application of Benzothiadiazole stimulates plant defense responses and induces

resistance to several pathogens (Lawton et al., 1996). Infected plants can in many

instances limit the extent of pathogen growth and symptom development. Resistance

occurs via an incompatible interaction and results in rapid activation of defense responses

that limit pathogen growth (Alfano and Collmer, 1996; Hammond-Kosack and Jones,

1996). These responses include: strengthening of cell walls with hydroxy-Proline-rich

glycoproteins (Showalter et al., 1985), callose (Parker et al., 1993) and lignin

(Moerschbacher et al., 1990); rapid expression of PR proteins (Linthorst, 1991); and the

synthesis of antimicrobial compounds (Hain et al., 1993; Epple et al., 1995; Penninckx et

al., 1996).

Tolerance is the repression of symptom development without restricting pathogen

growth. The visible phenotype of the two responses is similar. Resistance is a well-

studied phenomenon yet tolerance remains an enigma. Tolerance has been identified in

several phytohormone compromised plant lines. One of these is the ethylene-insensitive

ein2 mutant of Arabidopsis, which has increased tolerance to several virulent bacterial










pathogens (Bent et al., 1992). Increased tolerance has also been demonstrated to virulent

Xcy in ethylene and SA-deficient tomato lines (Lund et al., 1998; O'Donnell et al., 2001).

Disease development in tomato infected with virulent Xcy can be defined in two

stages: primary and secondary disease development. Primary disease development

consists of localized lesion formation and it is unaltered in the tolerant lines. Secondary

disease development consists of chlorosis and necrosis that spreads from the primary

lesions. These secondary disease symptoms require the cooperative action of ethylene

and SA and are abolished in tolerant lines. It remains a mystery why the plant retains

these responses and thus increases the amount of tissue damage. Organized necrosis may

help the plant withdraw nutrients from the infected tissue. The phytohormones may also

be required for the induction of a systemic response that allows the plant as a whole to

respond to the increased pathogen presence.

While tolerance due to phytohormones-deficiency might be a valuable outcome for

agriculture, it is likely to have costs. Ethylene and SA are involved in many plant-

pathogen interactions. For instance, ethylene is necessary for resistance to certain fungal

pathogens and for the generation of induced systemic resistance (Knoester et al., 1998;

Knoester et al., 1999). SA is necessary for resistance to avirulent pathogens and the

generation of SAR in several species (Gaffney et al., 1993). SAR is the activation of

systemic defense responses that occurs due to the formation of necrotic lesions (Ryals et

al., 1996). The hypersensitive response during an incompatible interaction and tissue

death during a compatible interaction can both induce SAR (Hunt and Ryals, 1996). SAR

results in the development of a broad-spectrum, systemic resistance. It is not effective

against, or induced by, all pathogens. For example, the infection of Arabidopsis with










Botrytis cinerea fails to induce SAR and inoculations with Pseudomonas syringae does

not affect B. cinerea challenge (Govrin and Levine, 2002). In tomato SAR is induced by

pathogens such as tobacco necrosis virus and Phytophthoria infestans (Enkerli et al.,

1993; Jeun et al., 2000).

It is important to understand the roles of phytohormones in systemic responses, as

the loss of these responses could reduce any advantage gained by reduced symptom

development due to engineered ethylene or SA-defieiency, I focus here on tomato, as the

absence of ethylene and SA causes tolerance to virulent Xcy. The systemic response in

tomato to this pathogen has not been previously examined. I investigate if Xcy is capable

of inducing systemic responses in tomato and possible roles of SA and ethylene in its

generation and action. To avoid confusion I will use the term "inoculation" for the first

treatment of a tomato plant and the term "challenge" for the subsequent treatment on

distal leaves. I demonstrate that both virulent and avirulent Xcy generate a systemic

response in tomato. I show that both virulent and avirulent Xcy cause an SA and ethylene-

dependent PR gene induction and sensitize systemic defense responses. However, in

contrast to the well-established SAR, the Xcy-induced response generates tolerance to

subsequent challenge with virulent Xcy. I term this response systemic acquired tolerance

(SAT) and define it as prior pathogen exposure reducing necrosis in response to virulent

pathogen infection in distal tissues without affected pathogen growth. SAT involves a

rapid increase in both PR gene expression and ethylene production in response to

subsequent challenges with virulent Xcy.










Virulent and Avirulent Xcy Led to Systemic Acquired Tolerance in Tomato

To investigate systemic responses to Xcy, wild type tomato plants at the three-leaf

stage were mock inoculated, inoculated with virulent Xcy strain 93-1 or avirulent Xcy

strain 87-7 (Bonas et al., 1993) on their lowest two leaves. The inoculations were then

permitted to run their full course of around 14 days, at which point those leaves

inoculated with virulent Xcy were fully necrotic and those inoculated with avirulent Xcy

had developed lesions associated with the hypersensitive response (Figure 3-1A).
















avirulent virulent


B r






moc-viulet vrulnt-irlen avrulnt-iruen





~~~~kvirulent Xcyand(B)symtom reuletin f vromcaleng-iue wth iuetXy










To determine if this inoculation with Xcy affected responses to subsequent

pathogen exposure, a challenge was performed with virulent Xcy on uninoculated

systemic leaves. Prior inoculations with either virulent or avirulent Xcy, but not mock

inoculations, reduced the necrotic development resulting from the challenge (Figure 3-

1B). The primary symptoms such as lesion formation were unaffected and secondary

symptom development such as chlorosis and some confluent necrosis were still apparent.

The major difference in secondary symptom development during challenge was reduced

necrosis in plants with prior Xcy inoculation.

As the response consists of two independent interactions between two biological

entities a high degree of variation is to be expected. With this in mind the level of

necrosis was determined in large population groups by measuring ion leakage at 16 days

after challenge (Figure 3-2).


35

30-

25 -2





g 10-




mock- vir-vir avr-vir mock- vir- avr-
vir mock mock mock


Figure 3-2 Cell death due to challenge of tomato with virulent Xcy. Cell death was
measured in the form of percent ion leakage in plants with a mock challenge
or challenged with virulent Xcy. The plants were exposed to a mock
inoculation or inoculation with virulent (vir) or avirulent (avr) Xcy prior to
chall enge.
































-o-Vlf















-=-- mock-vir

-*-avr-vir


Percent ion leakage, an indicator of cell death, was two-fold higher upon

challenge in plants previously mock inoculated than those with prior Xcy inoculations. In

contrast to previously mock-inoculated plants there was no significant difference between

symptoms in plants with prior virulent or avirulent inoculations upon challenge with

virulent Xcy. The reduction of symptom development due to previous pathogen exposure

is consistent with SAR generation. Bacterial growth measurements confirmed resistance

to avirulent Xcy but not virulent Xcy, as growth of avirulent Xcy was 10-fold lower than

that of virulent Xcy (Figure 3-3A).


A 12,
10 -



o


2-

]B




'4

2
1
0


9 11


1 35 7
days post infection


Figure 3-3 Bacterial growth during inoculation and challenge of tomato with Xcy. (A)
The growth of virulent (vir) and avirulent (avr) Xcy was measured during
inoculations. (B) A challenge with virulent Xcy was then performed on these
plants and mock-inoculated controls and the bacterial growth was measured.










When bacterial populations in challenge infections were determined there was no

difference in bacterial growth due to prior inoculation with either virulent or avirulent

Xcy compared with plants with prior mock inoculations (Figure 3-3B). This result leads

to the conclusion that avirulent and virulent Xcy induced SAT rather than SAR, as they

reduced symptom development but not bacterial growth due to challenge with virulent

Xcy. As to my knowledge SAT has not been previously described I defined it as the prior

exposure to a pathogen leading to reduced necrosis development in response to

subsequent challenge with a virulent pathogen in distal tissues.

Prior Inoculation with Xcy Led to an Early Production of Ethylene upon Challenge
with Virulent Xcy

Ethylene and SA are involved in the development of systemic responses in several

plants. Ethylene and SA were measured during inoculation and challenge with Xcy.

Inoculation with virulent Xcy led to ethylene production in local tissues at 5 days post

infection (dpi), while avirulent Xcy caused a greater induction of ethylene at 4 dpi (Figure

3-4A). Virulent Xcy induced SA at 10 dpi, while avirulent Xcy induced SA at 4 dpi that

peaked at 10 dpi (Figure 3-4B). The induction of ethylene and SA was later and at

reduced magnitude in response to virulent Xcy than avirulent Xcy. No systemic

production of ethylene was observed in response to primary Xcy inoculations and at 16

dpi no systemic induction of SA was observed in plants inoculated with Xcy (data not

shown).

Loss of secondary disease symptoms occurs in virulent Xcy-infected ethylene or SA

deficient tomato plants (Lund et al., 1998; O'Donnell et al., 2001). Therefore it is possible

that SAT might similarly be the result of reduced phytohormone synthesis during

challenge. To determine if SAT was due to loss of ethylene or SA, their accumulation
































3 5 7 9 11













4 6 8 10 12 14 16


following challenge with virulent Xcy was determined. Results indicated that the


tolerance associated with SAT is not due to reduced ethylene or SA accumulation.


Instead, earlier ethylene induction upon challenge accompanies SAT (Figure 3-4).


B 700
600
500
en400

g 300




0


0 1 23 45 67 891011
D 500
450 -m--rnock-vir
-o-I-VIf-V
400 *avr-vir
350-
,300
~250-
~200-
150-
100


O 2 4 6 8 10 12 14 16
days post infection
Figure 3-4 Local ethylene and SA production during inoculation and challenge of tomato
with Xcy. Tomato plants were mock inoculated or inoculated with virulent

(vir) or avirulent (avr) Xcy and (A) ethylene and (B) SA were measured.
Fourteen days later a challenge with virulent Xcy was performed on
uninfected leaves and (C) ethylene and (D) SA induced by the challenge was
measured.









The ethylene induction in response to a challenge with virulent Xcy, following prior

mock inoculation, reached its maximum at 10 dpi. However, plants with prior Xcy

inoculations produced additional ethylene around 5 dpi when challenged (Figure 3-4C).

Although at a reduced magnitude, this induction resembles a response to avirulent Xcy.

Challenge with virulent Xcy caused ethylene induction at 8 to 10 dpi in all plants, the

magnitude of which was dependent on the prior inoculation. Plants with prior avirulent

Xcy inoculation produced the most ethylene when challenged. This ethylene induction

was lower in plants with prior virulent Xcy inoculations and lowest in the mock-

inoculated controls. However, the significant difference remains the early ethylene

induction that is only present in plants with prior Xcy inoculations.

Plants with prior mock or virulent Xcy inoculations produced similar SA induction

profiles in response to challenge with virulent Xcy. However, a reduction in SA

production was observed in plants with a prior avirulent Xcy inoculation. These plants

also produced an early SA peak (Figure 3-4D) that resembled the response to avirulent

Xcy although reduced in magnitude. The following production of SA was delayed and

resembled that caused by virulent Xcy although at reduced magnitude.

The difference in SA production during a challenge and the increased strength and

speed of phytohormone induction during prior inoculations had no effect on the overall

SAT phenotype. Therefore the early induction of ethylene upon challenge in plants with

prior Xcy infections correlates with the tolerant phenotype, whereas SA induction does

not. Despite the lack of correlation between SA induced during challenge and SAT, SA

and ethylene induction during inoculation may be required for systemic signal formation.










PR Gene Expression Indicated the Necessity of Ethylene and SA in Systemic Signal
Generation

As ethylene or SA-deficient plants develop less secondary disease symptoms due

to virulent Xcy than plants displaying SAT, a direct approach to determining their role in

SAT is difficult. Therefore PR gene expression was assayed instead of ion leakage as a

marker for defense responses in phytohormone-deficient plants. To determine if ethylene

and SA are necessary for systemic signal transduction, two transgenic lines altered in

their ability to accumulate ethylene or SA were used. Loss of ethylene was achieved with

transgenic ACD plants. These plants do not accumulate the ethylene precursor ACC and

thus under-produce ethylene. The ACD line was compared to its isogenic parent UC82B.

The role of SA was determined using transgenic tomato plants expressing the bacterial

salicylate hydroxylase, nahG that cannot accumulate SA. The NahG line was compared

to its isogenic parent Money Maker (MM).

Induction of PR genes is often used as an indicator of early defense responses.

They show both local and systemic induction during SAR (Ward et al., 1991). The

expression of PRla and PRlb was induced in the local and systemic tissues of tomato

during infection with virulent Xcy (Figure 3-5). PRla expression was lower than PRlb.

The local expression of both genes was induced in all lines in response to virulent Xcy.

The timing and level of induction of PRla varied between different cultivars, with an

earlier and a greater induction in MM than UC82B. The expression of PRlb was also

higher in MM than UC82B, although the timing of induction was the same. When

compared to their isogenic parents, infected ACD and NahG plants had reduced local

expression of both genes. However, this reduction did not affect pathogen growth

(O'Donnell et al., 2001).













40 25
local PRla -M-M -*--UC82B I ncal PRla
35 -a-NahG -o-ACD






20



0I 1

E 12








losmca PRlb localM PRlt C2 sseib Pl

0. 01


0.08


012








4 0.08

00.0


00.0
0.1

10246 i I2468
days ~ ~ ~ ~ ~ ~~I potifeto ay otineto

Figur 3- oa n ytmcP gn nuto nehlneadS-eettmt







inlcladsystemi c tissues.16I~~I ytei Pl










Virulent Xcy induced systemic expression of both genes in MM and UC82B. In

MM systemic induction of PRla and PRlb occurred at the same time, whereas in UC82B

the expression of PRlb was later than PRla. ACD and NahG did not show significant

systemic induction of PR gene expression in response to virulent Xcy. Disease symptoms

in ACD and NahG are comparable to their isogenic parents up to 8 dpi (O'Donnell et al.,

2001). As widespread necrosis does not occur before 8 dpi the lack of systemic PR gene

expression is due to loss of ethylene and SA accumulation rather than the loss of

widespread necrosis in these lines. This lack of systemic PR gene induction indicates that

ethylene and SA-deficient lines might be compromised in their systemic signal

transduction. However, the tolerant phenotype of these lines makes direct investigation

difficult.

In UC82B systemic PR gene induction occurred within 6 dpi, whereas the major

local phytohormone induction occurred at 8 to 10 dpi in response to virulent Xcy.

Measurable phytohormone induction, therefore, was not responsible for systemic PR

gene induction. Direct damage to cells infected by the pathogen may cause local ethylene

and SA production limited to cells that are under attack. This phytohormone production

may be involved in the induction of both local and systemic defense responses.

Avirulent Xcy Induced Greater Local and Systemic PR Gene Expression than
Virulent Xcy, but they Result in Similar PR Gene Induction During Challenge

The induction of ethylene and SA is more rapid in response to avirulent than

virulent Xcy, yet the resulting systemic response to these two pathogens is the same. To

determine if the early defense responses follow this trend, induction of PR gene

expression by avirulent and virulent Xcy inoculations was assayed. Avirulent Xcy induced

higher levels and earlier local expression of PR genes than virulent Xcy (Figure 3-6). The










timing of systemic PRlb induction was similar in response to either pathogen, but

systemic PRla induction was faster in response to avirulent Xcy. Faster and stronger local

PR gene response to avirulent than virulent Xcy may reflect the increased defense

responses that limit pathogen growth.

Prior avirulent and virulent Xcy inoculations produced the same induction of

ethylene and changes in symptom development during challenge. The levels of PR gene

expression were assayed to determine if increased systemic PR gene expression

translated into stronger PR gene induction upon challenge. The difference between the

two prior Xcy inoculations in PR gene expression upon challenge was negligible. Both

caused an early peak of PR gene expression at 1 dpi upon challenge that was absent in

plants with prior mock inoculation (Figure 3-7). These results are consistent with the

rapid ethylene induction following challenge. These results lead to the hypothesis that

inoculation with Xcy primed the systemic defenses and caused a faster induction of

defense responses upon challenge. This primed defense response led to the formation of

tolerance upon challenge.

Discussion

In this chapter the systemic responses of tomato to both virulent and avirulent Xcy

were studied. A primary infection with virulent Xcy led to local as well as ethylene and

SA-dependent systemic PR gene induction within 6 dpi. PR gene induction occurred

prior to measurable ethylene or SA induction, indicating that undetectable localized

ethylene and SA production occurred within days of infection with virulent Xcy and led

to the generation of a systemic signal. The systemic signal, as well as inducing PR gene

expression, altered the systemic defense response to challenge with virulent Xcy. This












altered defense response is tolerance and not resistance. It caused rapid PR gene


expression and ethylene induction in response to subsequent challenge, leading to


suppressed symptom development but not resistance.


60


'45








S30

15



o


30




15


O


0.251-ev

S0.2


S0.1 -

S0.05




days post infection

Figure 3-6 The measurement of local and systemic PR gene expression in tomato during
inoculations with virulent or avirulent Xcy. Wild type (UC82B) tomato plants
were mock inoculated or inoculated with virulent (vir) or avirulent (avr) Xcy
and the local and systemic expression of PRla and PRlb was determined by
real time RT-PCR.











12
PRla -mmock-vir
-oVil-Vil
-* avr-vir
a 9












1 .2 -1 PRlb -w-mock-vir
-oi-VirVf
-* avr-vir

< 0.9



.0.6


0.3-


0.0
012345678
days post infection

Figure 3-7 Induction of PR genes in tomato plants during challenge with virulent Xcy in
the presence or absence of SAT. Wild type (UC82B) tomato plants were mock
inoculated or infected with virulent (vir) or avirulent (avr) Xcy. Fourteen days
later a challenge was performed with virulent Xcy. The expression of PRla
and PRlb was determined with real time RT-PCR during this challenge.

The ability of a plant to send a systemic signal in response to a biological stimulus


has long been established. This communication allows separate tissues to act in concert to


the many stimuli they perceive. Systemic signals have been characterized in terms of


responses to pathogens, symbionts and wounding. SA, ethylene, jasmonates and systemin

are all involved in generating systemic signals (Pearce et al., 1991; Gaffney et al., 1993;


Pieterse et al., 1998). SA is a key player in SAR in many species (Ryals et al., 1996).


NahG plants are unable to mount SAR to bacterial, viral or fungal pathogens (Gaffney et


al., 1993; Friedrich et al., 1995; Lawton et al., 1995) and exogenous SA can induce SAR










(Ward et al., 1991; Vernooij et al., 1995). However, grafting experiments with NahG and

wild type tobacco indicate that although SA is required for SAR it is unlikely to be the

transmitted signal (Vernooij et al., 1994).

The role of SA in the systemic responses of tomato may differ somewhat from

that in other plant species. For example, the induction of SAR in tomato against P.

infestans demonstrated neither systemic SA accumulation upon inoculation, nor increased

SA production upon challenge, both of which are common to SAR in other plant species

(Jeun et al., 2000). While local induction of SA is observed in response to virulent and

avirulent pathogens, its accumulation is only observed after the systemic induction of PR

genes (O'Donnell et al., 2001)

In tomato, ethylene and SA are induced in response to both virulent and avirulent

Xcy, although in neither response are they involved in limiting bacterial growth (Lund et

al., 1998; Ciardi et al., 2001; O'Donnell et al., 2001; O'Donnell et al., 2003). Loss of

ethylene causes tolerance to virulent Xcy by reducing cell death and lesion size following

infection with avirulent Xcy. SA loss also leads to tolerance to virulent Xcy. However,

loss of SA increases cell death and lesion size in response to avirulent Xcy. The

development of systemic acquired tolerance to Xcy rather than resistance may be a

specific response to this pathogen and reflect the roles of ethylene and SA in this

interaction.

In agreement with the work of Ciardi et al. (2000), avirulent Xcy induces a rapid

and more intense expression of PR genes than virulent Xcy. Here I show that this

translates into an increased systemic PR gene induction. This increased PR gene

expression coupled with a faster and greater local induction of ethylene and SA may










affect SA production during challenge but it has no impact on SAT. Therefore, changes

in SA production during challenge are not important for the formation of SAT.

Interestingly the work of Jeun et al. (2000) demonstrates that changes in SA induction in

tomato during challenge with P. infestans are also not necessary for SAR.

The PR gene induction and ethylene profiles caused by SAT during challenge

resembled those of an incompatible interaction. However, they failed to develop with the

speed and intensity required for successful resistance to develop. Systemic induction of

defense responses and PR genes that do not lead to SAR have been observed in the

interaction of Arabidopsis and the necrotizing fungal pathogen B. cinerea (Govrin and

Levine, 2002). Virulent Pseudomonas syringae py. tomato also fails to induce SAR in

Arabidopsis, but in this case there is no systemic induction of PR genes (Cameron et al.,

1999). An incompatible-interaction between Arabidopsis and Pseudomonas syringae py.

syringae leads to a reduction of symptom development but no changes in bacterial

growth during a repeat challenge with the same incompatible pathogen (Summermatter et

al., 1995). These data suggest that systemic responses to pathogens exist as a continuum

between no response, tolerance and resistance.

Transgenic tobacco over-expressing PRla show enhanced tolerance to

Peronospora tabacina (Alexander et al., 1993). While tobacco plants deficient in catalase

show that low level activation of defense responses, including PR gene induction, by

HO, can lead to enhanced pathogen tolerance, higher HO, levels cause resistance

(Chamnongpol et al., 1998). These data again suggest that a range of systemic responses

to pathogen invasion occur, with a moderate response repressing symptom development

and a strong response suppressing both symptom development and pathogen growth.










Here I have described the phenomenon of systemic acquired tolerance that adds to the

ranges of known systemic responses induced by pathogens and suggests that plants are

capable of more diversity in their systemic responses than previously suspected.















CHAPTER 4
DISCUSSION

Phytohormone Networks in the Responses of Tomato and Arabidopsis to Virulent
Bacterial Pathogens

Plant signaling networks utilize ethylene, SA and jasmonates to coordinate defense

responses. Different plants use these phytohormones in a variety of ways to achieve the

same result. In this chapter I compare and contrast their roles of in the response of

Arabidopsis and tomato to infection with virulent bacterial pathogens. Plants

compromised in production or signaling of a phytohormone were used to determine its

effects. However, phytohormones do not act in isolation the levels of one can often affect

the levels of another. Many studies do not look at the effects of altering the synthesis or

perception of one phytohormone on others. They therefore cannot address whether the

phenotype observed is strictly due to the phytohormone lost.

Phytohormone measurements demonstrate if the altered phenotype is due to the

compromised phytohormone or if others may be involved. Table 4-1 shows the

phenotypes of Arabidopsis and tomato phytohormone mutants infected with virulent

bacterial pathogens. Table 4-2 shows the effect of these mutations or transgenes on the

production of other phytohormones in response to infection. The Arabidopsis responses

are to virulent Pst and the tomato responses are to virulent Xcy.









Table 4-1 Comparative phenotypes of phytohormone mutants in Arabidopsis and
tomato.
Compatible Salicylic acid Ethylene Jasmonates
interactions
Arabidopsis and Pst
Mutants sid2-2 etrl -1 coi 1 and fad3-2 fad7-
2fa8(a)
Local phenotype enhanced increased symptoms resistant (coil)
susceptibility (1) (2) no effect fad3-2 fad7-2
fad8) (3)
Systemic phenotype normally none (4) ND ND
Tomato and Xcy
Mutants NahG ACD, Nr AOCas
Local phenotype tolerant (5) tolerant (6) resistant (7)
Systemic phenotype no SAT (8) no SAT (8) ND
References are: 1 (Nawrath and Metraux, 1999); 2 (O'Donnell et al., 2003); 3 (Kloek et
al., 2001); 5 (O'Donnell et al., 2001); 6 (Lund et al., 1998); 7 (O'Donnell et al., 2003); 8
(chapter 3). ND = not determined.


Table 4-2 Com 3arative phytohormone profiles of Arabidopsis and tomato mutants.
Arabidopsis and Salicylic acid Ethylene Jasmonates
Pst
Mutants sid2-2 etrl -1 coi 1 and fad3-2
fad7-2 fad8
Salicylic acid none (1) ND increased (coil)(2)
Ethylene increased (3) insensitive (4) ND
Jasmonates increased (5) ND insensitive (coil)(6)
none (fad3-2 fad7-2
fad8)(7)
Tomato and Xcy
Mutants NahG ACD, Nr as-AOC
Salicylic acid none (8) none (9) none (10)
Ethylene normal (11) none (ACD) (9) none (10)
insensitive (Nr) (12)
Jasmonates normal (10) normal (10) none (13)
References are: 1 (Nawrath and Metraux, 1999); 2 (Kloek et al., 2001); 3 (chapter 2); 4
(Bleecker et al., 1988); 5 (Heck et al., 2003); 6 (Feys et al., 1994); 7 (McConn and
Browse, 1996); 18 (Oldroyd and Staskawicz, 1998);9 (O'Donnell et al., 2001); 10 (Lund
et al., 1998); 11 (O'Donnell et al., 2003); 12 (Lanahan et al., 1994); 13 (Stenzel et al.,
2003). ND = not determined.










Arabidopsis has many phytohormone mutants including those for SA (sid2-2),

ethylene (etrl -1) and jasmonates (facd3-2 fad7-2 fad8 and coil). The etrl -1 mutant is a

dominant-insensitive ethylene receptor mutant (Bleecker et al., 1988). The fad3-2 fad7-2

fad8 mutant is a loss of function triple mutant of the three fatty acid desaturases. Due to

its failure to synthesize linolenic acid, it has reduced jasmonate biosynthesis (McConn

and Browse, 1996). The coil mutant is a jasmonate-insensitive ubiquitin E3 ligase mutant

(Xie et al., 1998). Tomato also has mutants and transgenic lines compromised in

phytohormones such as SA (NahG), ethylene (ACD and Nr) and jasmonates (as-AOC).

Nr is a dominant ethylene-insensitive receptor mutant (Rick and Butler, 1956). The

transgenic plant as-AOC is an antisense line of the jasmonate biosynthesis enzyme allene

oxide cyclase (Stenzel et al., 2003).

These data allow the construction of signaling network models for these plant

pathogen interactions. However, not all phenotype and phytohormone measurements

have been addressed in each system. These models are based on only two sets of

interactions and may vary greatly in response to different pathogens or in different plant

systems.

The Compatible Tomato-Virulent Xcy Interaction

A model for the signaling network in tomato in response to virulent Xcy is shown in

Figure 4-1. In the interaction of tomato with Xcy the relationship of these phytohormones

appears to be linear, with SA production dependent on ethylene production that is in turn

dependent on jasmonate signaling (Lund et al., 1998; O'Donnell et al., 2001; O'Donnell et

al., 2003).










JA ethylene SA
Primary Secondary
X~cy 4
symptoms symptoms




Resistance Tolerance
Figure 4-1 A model for the signaling network in tomato in response to virulent Xcy.

As demonstrated in chapter 3, systemic signaling in tomato is dependent upon

ethylene and SA. Their action appears to precede their measurable induction, in a manner

similar to JA. JA-deficient lines show a loss of ethylene despite the fact that measurable

JA production does not occur until after ethylene production (O'Donnell et al., 2003).

This suggests that early, local phytohormone productions can have a profound impact on

disease response.

It is clear in the case of the interaction between virulent Xcy and tomato that

jasmonates are the major players when it comes to the formation of susceptibility to Xcy.

At the local level the induction of ethylene and SA are required for the formation of

secondary disease symptoms (O'Donnell et al., 2003). The interaction between tomato

and virulent Pst also requires a functional jasmonate-signaling pathway for full Pst

virulence. This system, however, is strongly influenced by the phytotoxin coronatine

(Zhao et al., 2003).

The Compatible Arabidopsis-Virulent Pst Interaction

SA-deficient Arabidopsis plants produce increased levels of jasmonates and

ethylene in response to infection with virulent Pst. The prevalent hypothesis for

phytohormones in the response of Arabidopsis to pathogens proposes two pathogen-

response pathways in Arabidopsis (Glazebrook et al., 2003). One pathway utilizes SA










and acts in response to necrotic bacterial pathogens and the other utilizes ethylene and

jasmonates, acting in response to fungal and insect pathogens (Penninckx et al., 1998;

Vijayan et al., 1998).

SA, ethylene and jasmonates are all produced in the interaction between

Arabidopsis and virulent Pst. However, SA limits the production of both ethylene and

jasmonates and is the major phytohormone controlling defense responses in this

interaction. SA represses bacterial growth and thus also the development of chlorosis and

necrosis. Ethylene represses symptom development and jasmonates appear to act by

repressing defense responses.

These data suggest that co-ordinate signaling determines the overall response to the

pathogen. A combination of SA and jasmonates determine the level of bacterial growth

while ethylene influences the level of symptom development. It is likely to be the ratios

and timing of the various phytohormone inductions that control the plant responses. Pst

uses this network to its advantage by producing the jasmonate mimic coronatine.

The reduced virulence of coronatine-deficient Pst on SA-deficient and wild type

Arabidopsis demonstrated in chapter 2, suggests that coronatine's action as a virulence

factor is SA-independent. Further experiments to confirm this include the measurement

of SA in plants infected with coronatine-deficient Pst. Similar SA levels of these plants

and those infected with wild type Pst would support SA-independent coronatine action.

The link between jasmonates and susceptibility could also be investigated using T-

DNA knock-out mutants in jasmonate biosynthesis genes. A good candidate for this is

allene oxide cyclase for which there is a single gene in Arabidopsis (Kubigsteltig et al.,

1999; Park et al., 2002). This mutant would be more appropriate than the fad3-2 fad7-2










fad8 as it is less likely to affect the levels of other metabolites. These experiments require

the coronatine-deficient Pst mutant, as coronatine masks jasmonate-deficiency (Kloek et

al., 2001). Changes in bacterial growth in these lines would demonstrate if jasmonates are

definitively involved in repressing defense responses in Arabidopsis.

Systemic Responses to Infection

Systemic responses to virulent and avirulent pathogens have been previously

described in both tomato and Arabidopsis, with SA playing a similar role in both plants.

In both systems pathogen infections cause a local increase in SA (Nawrath and Metraux,

1999; O'Donnell et al., 2001). SA is necessary for the induction of a systemic response,

however, it is not itself believed to be the transmitted signal (Vernooij et al., 1994;

Lawton et al., 1995). The systemic induction of SA differs between the two systems, as

Arabidopsis has strong systemic SA induction where as tomato does not (Summermatter

et al., 1995). Arabidopsis also shows a strong induction of SA upon challenge that does

not occur in tomato (Jeun et al., 2000).

Ethylene is not required for the induction of SAR in Arabidopsis (Delaney et al.,

1994; Lawton et al., 1995). However, both ethylene and SA appear to be involved the

induction of SAT in tomato (Chapter 3). Virulent pathogens can induce systemic

responses in Arabidopsis, however, SAR is not induced by virulent Pst (Cameron et al.,

1999). Both avirulent and virulent Xcy induce systemic responses in tomato.

Additional experiments are required to confirm the roles of ethylene and SA in the

generation of systemic signaling in tomato. One approach to this could be the use of

grafting to determine if a NahG stock is incapable of systemic signaling to a wild type










scion. The best approach to this would be to monitor PR gene expression in the scion in

response to an inoculation of the stock.

Further study is required in both systems before the complex roles of

phytohormones in these interactions can be fully understood. It is clear from this study

that information gained from studying one system will not necessarily be applicable to

another system. However, certain parallels and common responses exist. By the study of

a wide range of plant-pathogen interactions the common points and differences can be

determined. This will eventually lead to a more complete understanding of general

defense responses, perhaps allowing broad-spectrum engineering or breeding of plants

with increased resistance or reduced symptoms to a wide range of diseases.















CHAPTER 5
MATERIALS AND METHODS

Plant Materials and Treatments

Arabidopsis thaliana Columbia, the NahG line (Delaney et al., 1994), nprl-1 (Cao

et al., 1994) and sid2-2 (courtesy of Julia Dew dney), all in the Columbia background,

were grow n in soil under long night conditions (8-h day; 16-h night) for six weeks to

encourage vegetative grow th. Forty-eight hours prior to infection, plants were transferred

to long day conditions (16-h day; 8-h night). Twelve hours before infection, the plants

were enclosed in a humidity dome to aid bacterial entry. Plants were inoculated with Pst

by submerging the aerial parts of the plant in 1x107 efu mll bacterial suspension,

containing 10mM MgC1, and 0.02% (v/v) Silw'et L-77 (Lehle seeds, Round Rock, TX,

U.S.A.), for 30 seconds. A vacuum w'as then applied to the plants for 2 min to aid

bacterial entry and the plants were returned to the humidity chamber overnight.

Lycopersicon esculentwn cvs Moneymaker and UC82B are the parental lines for

NahG (Oldroyd and Staskaw'icz, 1998) and ACD (Klee et al., 1991) respectively. Wild

type refers to UC82B only in all experiments except PR gene analysis of ethylene and

SA-deficient lines. Plant grow th and treatments were performed under ambient

temperature and lighting in a greenhouse. Plants were inoculated by submersion of the

leaves for 15 seconds in a bacterial suspension of 1x107 efu mll of Xcy strain 93-1

(virulent) or 87-7 (avirulent) containing 10mM MgC1, and 0.02% (v/v) Silw'et L-77.

Mock inoculations were performed by dipping plants in buffered Silwet. For analysis of

the systemic response to infection, inoculations were performed on three week-old plants










by dipping leaves 1 and 2 in infection media. Challenges were then performed on the

same plant 14 days later by dipping leaves 5 and 6 in the infection media containing

virulent Xcy. For PR gene analysis leaves 1 and 2 of six week-old plants were infected

and tissue from the local infection (leaves 1 and 2) and the systemic response (leaves 5

and 6) were removed at the indicated time points and flash frozen in liquid nitrogen.

Bacterial Culture

Pst and Xcy cultures were grown as previously described by O'Donnell et al.

(2001). Leaf colony counts were determined as previously described by Lund et al.

(1998). Briefly, five Icm2 leaf disks were sampled from each line at each time point

indicated. The disks were ground in 10mM MgCle, and serial dilutions were incubated at

room temperature for 2 days on solid media. The average colony-forming unit per square

centimeter (cfu cm 2) for each sample was determined by counting individual colonies.

Ion Leakage

Amount of cell death was estimated by measuring percentage of ion leakage. At 16

dpi the 5th leaf of each plant was placed in 6 ml deionized water and a vacuum of 20 psi

applied for 5 min. The samples were then shaken at room temperature for one hour. 3 ml

of the water was then removed and its conductivity was measured. The samples were

then placed in a boiling water bath for an hour and the conductance of the remaining 3 ml

of water measured. Percent ion leakage was determined by conductivity of first 3 ml

divided by conductivity of second 3 ml multiplied by 100. Measurements of tissue

challenged with virulent Xcy were made from 30 plants per treatment. Measurements on

mock challenges were made for 10 plants per treatment. Each experiment was repeated

on at least two independent occasions.










Ethylene Measurements

Ethylene measurements were performed on a minimum of three independent biological

replicates. Each biological sample was replicated at least three times within an

experiment. Ethylene production was determined by sampling the headspace above either

a whole rosette for Arabidopsis or a single leaf for tomato, enclosed in 5cm3 tubes for 1

hour as described by Lund et al. (1998). Ethylene concentration in a 1 ml sample was

determined by a gas chromatography (Model 5890, Hewlett-Packard, Palo Alto, CA).

SA, Jasmonate and Coronatine Measurements

SA, jasmonates and coronatine were extracted from Arabidopsis tissue and SA

from tomato tissue and derivatized using trimethylsilyl-diazomethane. The volatile

methyl esters were collected from the complex matrix using vapor phase extraction and

quantified by isobutene chemical- ionization gas-chromatography/mass spectrometry as

described in Schmelz et al. (2003). Each biological sample was replicated at least twice

with a minimum of two independent experiments.

RNA Extraction

Total RNA was extracted from 1.0g of tissue from each genotype per time point

with (phenol:chloroform: isoamyl alcohol (PCI) (25:24: 1 (v/v/v))) : extraction buffer (1%

triisopropylnaphthalene-sulfonic acid (w/v); 6% p-amino salicylic acid (w/v); 0. 1M Tris

pH 8; 50 mM EGTA; 0. 1M NaCl; 1% SDS (w/v); 0.039% -Mercaptoethanol (v/v)) (1:1).

The extraction mixture was homogenized with a polytron and incubated at 500C for 20

min. The phases were separated and an equal volume of PCI added to the aqueous phase.

The RNA was precipitated overnight at -800C following the addition of 2.5 volumes of

ethanol and 1/10 volume of sodium acetate. The nucleic acids were pelleted by

centrifugation, (14,000 g for 30 min) resuspended in water and precipitated in 2M LiC1.









The LiCl precipitation was repeated and a subsequent sodium acetate/ethanol

precipitation performed.

RNA Gel Blot Analysis

RNA gel blot analysis was performed using 20 Eog total RNA for each sample as

described by Kneiss1 and Deikman (1996). The RNA was transferred onto a charged

membrane (Hybond-N+; Amersham Life Science inc., Arlington Heights, IL). The PR1

(Genbank accession AY117187) and PDFl .2 (full length cDNA from gene at5g44420)

probes were obtained from The Arabidopsis information resource (Ohio State

University). DNA probes were random primer labeled with 32P with the Prime-It II

labeling kit (Stratagene, La Jolla, CA).

Real-time RT-PCR

PRla (TC117463) and PRlb (TC115947) mRNA levels were quantified by Real-

time quantitative RT-PCR using Taqman@ one-step RT-PCR reagents (Applied

Biosystems, Foster City, CA) and an Applied Biosystems GeneAmp 5700 sequence-

detection system. Each determination was performed using 250ng of Dnase-1 treated

total RNA isolated using an RNeasy@ Plant mini kit (Qiagen, Valencia, CA), in a 25ol1

reaction volume. RT-PCR conditions were: 480C for 30 min, 950C for 10 min followed

by 40 cycles of 950C for 15 sec and 600C for 1 min. Absolute mRNA levels were

quantified using synthesized sense strand RNAs as standards. Primers and probes were

designed using PRIMER EXPRESS software (Applied Biosystems) and were as follows:

PRlb probe 5'-/56-FAM/CAACGGATGGTGGTTCATTTCTTGCA/3B H1-'; PRla

Probe 5' -/56-FAM/TGTGGGTGTCCGGAGAGCCA GA/3BH _1/-3' PR Ib forward

primer 5'-GGTCGGGCACGTTGCA-3 '; PRlb reverse primer 5'-GATCCAGTT-






57


GCCTACAGGACAT-A-3'; PRla forward primer 5'-GAGGGCAGCCGTGCAA-3';

PRla reverse primer 5'-CACATTTTTCCACCAACACATTG-3 (Intergrated DNA

Technologies, Coralville, IA). Each sample was a minimum of two biological replicates

and each experiment was repeated at least twice.

MCP Treatment

MCP treatment was performed 24 hours prior infection as described in Ciardi et al

(2000).
















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BIOGRAPHICAL SKETCH

Anna Block was born on the 13th Of May 1978 in Nottingham, England. She

completed her GCSE's and A levels at John Mason School in Abingdon, Oxfordshire, in

1994 and 1996 respectively. She then went on to receive her master's degree in

biochemistry from the University of Bath in 2000. During her master's degree she

accomplished two 6-month internships. The first internship was in 1998 for Rhone-

Poulenc in Essex and the second in 1999 in the laboratory of Harry Klee at the University

of Florida. She returned to the University of Florida in 2000 for her doctoral studies in

the plant cellular and molecular biology program




Full Text

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THE ROLE OF ARABIDOPSIS AND TOMATO PHYTOHORMONES IN THERESPONSE TO BACTERIAL PATHOGENSByANNA KATHERINE BLOCKA DISSERTATION PRESENTED TO THE GRADUATE SCHOOLOF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENTOF THE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYUNIVERSITY OF FLORIDA2004

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Copyright 2004byAnna Katherine Block

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This thesis is dedicated to my family who support me without question, no matter howunusual they think my choices are.

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ivACKNOWLEDGMENTSThis work was supported in part by a grant to Harry Klee from the NationalScience Foundation (IBN0091064) and the Florida Agricultural Experiment Station. Ithank my advisor Harry Klee and my committee members Jeffrey Jones, John Davis andShouguang Jin for their guidance; Eric Schmelz for the measurement of thephytohormones by GC-MS; the Klee lab for its support and advice; the Jones lab for theuse of its greenhouse and help in tomato plant maintenance and the Settles lab for use ofArabidopsis growth facilities and other equipment.

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vTABLE OF CONTENTSPage ACKNOWLEDGMENTS..............................................................................................ivLIST OF TABLES........................................................................................................viiLIST OF FIGURES......................................................................................................viiiABSTRACT....................................................................................................................xCHAPTER1INTRODUCTION....................................................................................................1Plant-Pathogen Interactions......................................................................................1Pathogenesis-Related (PR) Genes.............................................................................2Bacterial Pathogens..................................................................................................3The Role of Salicylic Acid in Arabidopsis Defense Responses.................................4The Role of Ethylene in Arabidopsis Defense Responses..........................................7The Role of Jasmonates in Arabidopsis....................................................................7The Interaction of Tomato and Xcv...........................................................................8Symptom Development and Systemic Responses in Tomato..................................122THE EFFECT OF THE REMOVAL OF SA ON PHYTOHORMONE SIGNALINGNETWORKS IN ARABIDOPSIS INFECTED WITH VIRULENT PST................14Differences in Disease Progression and Symptom Development in Salicyclic Acid-Deficient Arabidopsis Mutants infected with Pst................................................14SA and Jasmonate Induction in Response to Pst Infection......................................17The Effect of Coronatine Production by Pst............................................................19The Role of Ethylene in SA-Deficient Arabidopsis.................................................22Discussion..............................................................................................................233SYSTEMIC ACQUIRED TOLERANCE IN TOMATO.........................................28Introduction............................................................................................................28Virulent and Avirulent Xcv Led to Systemic Acquired Tolerance in Tomato...........31Prior Inoculation with Xcv Led to an Early Production of Ethylene upon Challengewith Virulent Xcv...............................................................................................34

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viPR Gene Expression Indicated the Necessity of Ethylene and SA in Systemic SignalGeneration.........................................................................................................37Avirulent Xcv Induced Greater Local and Systemic PR Gene Expression thanVirulent Xcv, but they Result in Similar PR Gene Induction During Challenge..39Discussion..............................................................................................................404DISCUSSION........................................................................................................46Phytohormone Networks in the Responses of Tomato and Arabidopsis to VirulentBacterial Pathogens............................................................................................46The Compatible Tomato-Virulent Xcv Interaction...................................................48The Compatible Arabidopsis-Virulent Pst Interaction.............................................49Systemic Responses to Infection.............................................................................515MATERIALS AND METHODS............................................................................53Plant Materials and Treatments...............................................................................53Bacterial Culture....................................................................................................54Ion Leakage............................................................................................................54Ethylene Measurements..........................................................................................55SA, Jasmonate and Coronatine Measurements........................................................55RNA Extraction......................................................................................................55RNA Gel Blot Analysis..........................................................................................56Real-time RT-PCR.................................................................................................56MCP Treatment......................................................................................................57LIST OF REFERENCES...............................................................................................58BIOGRAPHICAL SKETCH.........................................................................................66

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viiLIST OF TABLESTable page 4-1 Comparative phenotypes of phytohormone mutants in Arabidopsis and tomato......474-2 Comparative phytohormone profiles of Arabidopsis and tomato mutants ...............47

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viiiLIST OF FIGURESFigure page 1-1 The role of sid2 in SA biosynthesis and nahG in SA removal...................................61-2 Jasmonate biosynthesis.............................................................................................91-3 The structure of coronatine.....................................................................................101-4 Ethylene biosynthesis.............................................................................................112-1 Bacterial growth in Pst DC3000 infected Arabidopsis............................................152-2 PR1 and PDF1.2 gene expression in Pst infected Arabidopsis................................162-3 SA production in Pst infected Arabidopsis.............................................................172-4 JA and OPDA production in Pst infected Arabidopsis............................................182-5 Coronatine production in Pst infected Arabidopsis.................................................192-6 Bacterial growth at 72 hpi in Pst DC3000 and Pst DC3661infected Arabidopsis....202-7 Disease symptom development in Pst infected Arabidopsis....................................212-8 Ethylene production in Pst infected Arabidopsis.....................................................233-1 Symptom development of Xcv infected tomato.......................................................313-2 Cell death due to a second infection of tomato with virulent Xcv............................323-3 Bacterial growth during the first and second infections of tomato with Xcv............333-4 Local ethylene and SA production during the first and second infections of tomatowith Xcv...............................................................................................................353-5 Local and systemic PR gene induction in ethylene and SA deficient tomato lines inresponse to infection with virulent Xcv.................................................................383-6 The measurement of local and systemic PR gene expression in tomato duringinfections with virulent or avirulent Xcv...............................................................41

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ix3-7 Induction of PR genes during virulent Xcv infection in tomato plants in the presenceor absence of SAT................................................................................................424-1 A model for the signaling network in tomato in response to virulent Xcv................49

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xAbstract of Dissertation Presented to the Graduate Schoolof the University of Florida in Partial Fulfillment of theRequirements for the Degree of Doctor of Philosophy THE ROLE OF ARABIDOPSIS AND TOMATO PHYTOHORMONES IN THE RESPONSE TO BACTERIAL PATHOGENSByAnna Katherine BlockDecember 2004Chair: Harry KleeMajor Department: Plant Molecular and Cellular BiologyPhytohormone networks are used to regulate the plant response to virulent bacterialpathogens. In this work by the use of salicylic acid (SA) deficient lines it is shown thatSA, a major player in the interaction between Arabidopsis and virulent Pseudomonassyringae pv. tomato (Pst), down-regulates ethylene and jasmonates. SA influencesethylene production in an NPR1-independent manner and jasmonate production in anNPR1-dependent manner. Pst-produced coronatine, a jasmonate mimic, actsindependently of SA and does not compromise SA-dependent basal resistance. Thesethree phytohormones are also involved in the response of tomato to Xanthomonascampestris pv. vesicatoria (Xcv). However, the hormone network is dramaticallydifferent to that of Arabidopsis. It is demonstrated, by the use of phytohormone deficientplants and pathogenesis-related marker gene expression profiling, that a systemic signalis generated in tomato in response to an infection with virulent Xcv. This signal is SA-dependent and leads to the development of a systemic acquired tolerance in tomato to

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xisubsequent infections with virulent Xcv. This systemic acquired tolerance is similar to thesystemic response that is generated in response to avirulent Xcv.

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1CHAPTER 1INTRODUCTIONPlant-Pathogen InteractionsThe normal growth of a plant can be disrupted due to interactions with pathogenicorganisms such as bacteria, fungi, parasitic higher plants, nematodes, mycoplasmas andviruses. Pathogens live in or on another organism, from which they derive nutrients.Substances released by the pathogen, in order to penetrate plant cell walls and make thenutrients accessible for their use, cause damage to the host tissues. These substancesinclude enzymes, toxins, growth regulators and polysaccharides, some of which areinduced upon host recognition.Not all pathogens can cause disease on all hosts as some lack the necessary abilitiesto penetrate and survive that hosts endogenous structural and chemical defenses. Thehost range of a pathogen is defined as the host plants on which a particular pathogen canrecognize and grow successfully (Agrios, 1988). Resistant hosts can specifically recognize avirulent pathogens and cause anincompatible interaction. Incompatible interactions often include a hypersensitiveresponse HR that consists of a rapid induction of host defenses and the death of cells incontact with the pathogen. The faster this response occurs, the more resistant the plant isto the pathogen. Compatible interactions occur between susceptible plants and virulentpathogens, leading to successful invasion of host tissues (Ausubel et al., 1995;Hammond-Kosack and Jones, 1996).

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2In both compatible and incompatible interactions the plant attempts to limit damageand control pathogen growth. This is done by the production of toxic substances aroundthe site of injury, as well as by the formation of protective layers such as callus and cork.Some compounds are produced at high enough concentrations to limit pathogen growth.These compounds include phenolic compounds such as chlorogenic and caffic acids,oxidation products of phenolic compounds and phytoalexins (Agrios, 1988; Jackson andTaylor, 1996).Pathogenesis-Related (PR) GenesAs well as the production of substances and structures, several plant genes areinduced in response to pathogens. PR genes are used as markers of the disease responsein many plant species. They are defined as plant-encoded genes that are only expressed inthe tissue in response to a pathogen or related stress. PR genes are classified by functionand homology; these classes include PR-2 (!-1,3-glucanase), PR-6 (proteinase-inhibitor),PR-12 (defensin) and PR-1 whose function is unknown (Van Loon and Van Strien,1999).Although PR genes are induced during infection, only a few have been shown tohave a direct impact on pathogen growth. The most well characterized of these is the PR-1 family that has antifungal activities, although the mechanism by which it acts has yet tobe identified (Alexander et al., 1993; Niderman et al., 1995). The combined effect ofmany PR gene products may be required to have a significant effect on pathogen growth.Particular classes of PR genes have been linked to specific phytohormone inductionevents. For example the PR gene Thi 2.1 of Arabidopsis is induced during wounding in ajasmonate dependent manner (Bohlmann et al., 1998). The phytohormones, however,

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3affect one another to such an extent that any direct interpretation of the roles ofphytohormones from PR gene expression is inherently risky.The responses covered so far are induced at a local level by pathogen infection. Theplant, however, also shows a systemic response to infection that includes some of thesame response as those at the local level. For example pathogen infection can lead to theproduction of a systemic signal. This signal can induce resistance in uninfected tissues ofthe plant 2-3 days after infection. It can also induce the systemic expression of PR genes.This systemic induced resistance (SAR) can last several weeks and is effective against awide range of normally virulent pathogens.Bacterial PathogensThe response to pathogens varies depending on the host-pathogen interaction. Tosimplify the study of plant pathogen interactions the focus of this work will be limited tobacterial pathogens, specifically to Xanthomonas and Pseudomonas. These arenecrotizing biotrophic pathogens that live within the apoplast. They can multiply forsome time within host tissues before causing necrosis. They use a type III secretionsystem to introduce substances into host cells that cause the release of nutrients andsuppress host defense responses. They also produce toxins and other extracelluarsubstances, which affect the plant and cause disease symptom development (Alfano andCollmer, 1996).Xanthomonas and Pseudomonas are rod shaped gram negative bacteria of thefamily pseudomonadaceae. They use flagella to move through liquid and can enter hosttissues from water on the surface of the tissue. For example they can enter the leavesthrough wounds or stomatal openings. The symptoms these bacteria cause include the

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4formation of necrotic lesions on infected tissues. These lesions are sometimes surroundedby a chlorotic halo. The necrotic lesions may coalesce and form large necrotic regions oreven kill the entire tissue.Bacterial species can be further subdivided into subspecies or pathovars (pv),which are distinguished by their host range. The two pathogens this work will focus onare Pseudomonas syringae pv. tomato (Pst) that causes bacterial speck both in tomatoand in the model plant Arabidopsis and Xanthomonas campestris pv vesicatoria (Xcv)that causes bacterial spot in tomato and pepper (Agrios, 1988).Many of the plant responses to pathogens have been characterized. However, howthe plant forms and coordinates its defense response has yet to be fully elucidated(Glazebrook, 2001). One of the major tools in such studies is mutant screening for plantsaltered in their pathogen responses. The screens focus either on disease phenotype or onPR gene induction with the plant Arabidopsis thaliana as a favored model (Glazebrook etal., 1997). These screens have identified mutants that are compromised in their resistanceto avirulent pathogens. Some of these mutants also show enhanced susceptibility tovirulent pathogens and thus demonstrate the existence of a basal resistance, in which theplant limits the growth of virulent pathogens (Glazebrook et al., 1996). Such mutantsoften also have altered phytohormone profiles, indicating important roles forphytohormones in coordinating the defense responses to both virulent and avirulentpathogens (Dewdney et al., 2000; Nawrath et al., 2002).The Role of Salicylic Acid in Arabidopsis Defense ResponsesThe phytohormone salicylic acid (SA) is involved in maintenance of basalresistance, the response to avirulent pathogens and development of SAR in Arabidopsis.

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5Mutants that show enhanced susceptibility to virulent pathogens such as Pst DC3000often accumulate less SA than their wild type parents following infection. For examplepad4, which has enhanced susceptibility to virulent Pseudomonas syringae pv maculicolaES4326 (Psm), has reduced and delayed SA synthesis (Zhou et al., 1998).In several plant species the removal of SA has been accomplished by introductionof the nahG transgene encoding a Pseudomonas putida salicylate hydroxylase (EC1.14.13.1), which converts SA into catechol (Yamamoto et al., 1965). Arabidopsis plantscarrying this transgene have enhanced susceptibility to several pathogens including Pst(Delaney et al., 1994; Lawton et al., 1995).An Arabidopsis SA biosynthesis mutant, sid2-2, that fails to accumulate SA inresponse to pathogens has also been identified. This line contains a loss of functionmutation in the enzyme isochorismate synthase 1 (EC 5.4.4.2) that converts chorismate toisochorismate (Figure 1-1) (Wildermuth et al., 2001). sid2-2 has both enhancedsusceptibility to Pst and fails to accumulate SA in response to infection (Nawrath andMetraux, 1999).NahG and sid2-2 respond differently to the non-host pathogen Pseudomonassyringae pv. phaseolicola strain 3121, suggesting that the catechol produced by the nahGtransgene may influence the disease process (Van Wees and Glazebrook, 2003). Theconclusion that nahG has an effect beyond elimination of SA is further supported byglobal expression phenotyping of Arabidopsis in response to infection by virulent Psm(Glazebrook et al., 2003). This showed major differences between NahG and sid2-2 interms of the expression patterns of genes induced in the two lines by infection with Psm.

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6 Figure 1-1 .The role of sid2 in SA biosynthesis and nahG in SA removal.Signal transduction from SA can occur in an NPR1-dependent or independentfashion (Clarke et al., 1998). NPR1 is a protein with a BTB/BOZ domain and an ankyrinrepeat domain, both of which are involved in protein-protein interactions (Cao et al.,1997). Upon infection NPR1 is translocated to the nucleus where it interacts with bZIPtranscription factors that are involved in the SA-dependent activation of PR genes(Despres et al., 2000). NPR1 also has a role in suppressing jasmonate signaling in thecytoplasm (Spoel et al., 2003). However, nuclear localization of NPR1 is required for PR

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7gene expression (Kinkema et al., 2000). The loss of function mutant npr1-1 has enhancedsusceptibility to virulent Pseudomonas syringae and has reduced expression of PR genesafter infection (Cao et al., 1994).The Role of Ethylene in Arabidopsis Defense Responses The major role of SA in basal resistance is demonstrated by SA-deficientArabidopsis that are more susceptible to the virulent pathogen Pst. Acting eitheragonistically or antagonistically to SA in these interactions are other phytohormones suchas ethylene and jasmonic acid (JA). For example, the Arabidopsis ethylene insensitivemutant etr1-1 has faster disease progression than wild type plants in response to virulentXanthomonas campestris pv. campestris (Xcc). This suggests that ethylene negativelyregulates disease symptom development in this system. However, NahG is deficient inethylene production following Xcc infection, suggesting that these two phytohormonesact agonistically in this response (O'Donnell et al., 2003). The loss of ethylene in NahG isnot supported by sid2-2, which was shown to accumulate ethylene in response toinfection with Pst (Heck et al., 2003). Thus, the failure to produce ethylene in pathogeninfected NahG Arabidopsis may be a specific consequence of the nahG transgene and notdue to the failure to accumulate SA.The Role of Jasmonates in ArabidopsisTwo Arabidopsis mutants that are SA-deficient, eds4 and pad4, have increasedsensitivity to compounds that induce the expression of JA response genes (Gupta et al.,2000), and NahG and sid2 produce higher levels of JA in response to infection with Pstthan does Columbia (Heck et al., 2003; Spoel et al., 2003). JA is produced from alpha-linolenic acid via the active precursor 12-oxo-phytodieonic acid (OPDA). It can also beconverted into the volatile compound methyl-jasmonate (Figure 1-2). These two

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8compounds, in addition to JA and other oxylipins, are commonly referred to asjasmonates (Seo et al., 2001; Stintzi et al., 2001). Currently, little is known about theroles of individual jasmonates in pathogen defense.The evidence for a role for jasmonates in compromising the basal resistance ofArabidopsis to Pst is further supported by the involvement of Pst-produced coronatine.Coronatine is a phytotoxin that mimics OPDA (Weiler et al., 1994) (Figure 1-3). A Tn5transposon insertion into Pst DC3000 produced a coronatine-deficient Pst mutant, PstDC3661 (Moore, 1989). This mutant strain was shown to be less virulent than PstDC3000 in Arabidopsis in dip infections but not in injection inoculations. However, thedisease symptoms in both forms of infection were reduced when compared to the parentalstrain (Mittal and Davis, 1995).The hypothesis that coronatine is an important virulence factor for Pst is reinforcedby studies with the Arabidopsis coronatine insensitive mutant coi1 (Feys et al., 1994).COI1 is part of an E3 ubiquitin-ligase involved in a jasmonate response pathway (Xie etal., 1998). The coi1 mutants are both more resistant to Pst DC3000 and have higher SAlevels (Kloek et al., 2001). These data along with similar studies in tomato (Zhao et al.,2003) suggest that coronatine acts by stimulating the jasmonate-signaling pathway.The Interaction of Tomato and XcvNot all plants use phytohormones in the same way in their defense responses. Intomato phytohormones are involved in the response to both virulent and avirulent Xcv. Inthe response to virulent Xcv, there is an induction of ethylene followed by accumulationof SA.

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9 Figure 1-2 Jasmonate biosynthesis.

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10 Figure 1-3 The structure of coronatine.SA production in this response is dependent on the previous production of ethylene(O'Donnell et al., 2001), and both are dependent on the ability of the plant to producejasmonates.Antisense plants for the jasmonate biosynthesis enzyme allene oxide synthase (as-AOS) are resistant to virulent Xcv and show no production of either ethylene or SA(O'Donnell et al., 2003). Transgenic tomato plants that express 1-aminocyclopropane-1-carboxylate (ACC) deaminase (ACD) (EC 3.5.99.7) (Figure 1-4) do not produce ethyleneand are compromised in SA induction during infection. NahG plants, which do notproduce SA, have normal ethylene production. Both ACD and NahG are tolerant tovirulent Xcv and unaffected in their ability to produce jasmonates (O'Donnell et al.,2003).The tolerance of a plant to a pathogen can be defined as substantial growth of thepathogen within the host tissues combined with an absence of full symptom development.Another plant that is compromised in phytohormone signaling and shows tolerance is theethylene insensitive (ein2) mutant of Arabidopsis. It is tolerant to both Pst and Xcc (Bentet al., 1992).

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11 Figure 1-4 Ethylene biosynthesis.

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12The response of tomato to avirulent Xcv also involves phytohormone induction.There is a major early induction of ethylene that is larger than that seen in the susceptibleresponse. There is also an early induction of SA. Increased ethylene sensitivity in tomatocaused by reduced expression of an ethylene receptor LeETR4 enhances thehypersensitive response to avirulent Xcv (Ciardi et al., 2001). This demonstrates thatethylene is also important in the response to avirulent pathogens.Symptom Development and Systemic Responses in TomatoSymptom development in tomato infected with Xcv can be categorized into twostages. The primary response present in resistant, susceptible and tolerant interactionsconsists of localized lesion formation. The secondary phenotype, which is only present inthe susceptible response, consists of the development of chlorosis and necrosis thatspreads from the sites of the primary lesions (O'Donnell et al., 2003).Perhaps there is an advantage for the plant to produce these phytohormones and theconsequent chlorosis and necrosis development in response to virulent infections. Inaddition to the necrosis of the tissue effectively removing the pathogen from the plant, itis possible that this response produces a systemic signal that primes the plants defensesin case of repeat infections. It has been demonstrated in several systems, includingtobacco and cucumber that infections with virulent pathogens that produce high levels ofnecrosis can lead to the induction of SAR (Cohen and Kuc, 1981; Strobel et al., 1996).Therefore the phytohormones induced during the interaction with virulent Xcv andtomato may be involved in the development of SAR and this could be the reason whythey remain even though their absence would lead to tolerance. SAR is induced in tomato

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13by tobacco necrosis virus (TNV) (Jeun et al., 2000) and Phytophthora infestans (Enkerliet al., 1993) and leads to resistance to P. infestans.Here two different aspects of phytohormone signaling in response to infectionswith virulent bacterial pathogens are studied. The first, in chapter 2, investigates theeffect of the removal of SA accumulation on the synthesis of ethylene and jasmonatesduring the interaction between Pst and Arabidopsis. This chapter focuses onphytohormone networks in the local response to infection. The second, in chapter 3, is onthe interaction between tomato and Xcv. In this chapter the focus is the systemic responseto virulent bacterial infection and the possible roles of phytohormones in this response.Although in both cases the focus is on the role of phytohormones in response topathogens, it can clearly be seen that the phytohormones play different roles in the twosystems and therefore information gained from studying one plant-pathogen interaction isnot necessarily applicable to another.

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14CHAPTER 2THE EFFECT OF THE REMOVAL OF SA ON PHYTOHORMONE SIGNALINGNETWORKS IN ARABIDOPSIS INFECTED WITH VIRULENT PSTDifferences in Disease Progression and Symptom Development in Salicyclic Acid-Deficient Arabidopsis Mutants infected with PstPhytohormones do not act in a discrete fashion. The action often attributed to onephytohormone can be due to the combined action of several. SA plays an important rolein the basal resistance of Arabidopsis to virulent Pst. However, it is not the onlyphytohormone induced in response to Pst. Ethylene and jasmonates are also induced inthis response. The aim of this chapter is to determine the effect of removing SA on thephytohormone-signaling network in Arabidopsis in response to virulent Pst. This willprovide a clearer resolution of the complex relationships between the signaling pathwaysin this system.As a first step into understanding the effect of altered SA production or signaling,levels of basal resistance and the disease phenotypes in four different lines werecompared. These lines are the SA biosynthesis mutant sid2-2, the SA signaling mutantnpr1-1 and the transgenic line NahG that cannot accumulate SA. The infection ofColumbia (wild type), npr1-1, sid2-2 and NahG plants with virulent Pst led to differentialbacterial growth and disease phenotypes in the four lines.NahG supported significantly more bacterial growth than the other lines (Figure 2-1). The mutants sid2-2 and npr1-1 had bacterial growth that was intermediate betweenNahG and the wild type. The sid2-2 mutant is a better model for the loss of SAproduction than NahG due to the side effects of catechol production (Heck et al., 2003).

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15Therefore, as sid2-2 supports the same levels of bacterial growth as npr1-1, thisdemonstrates that the effect of SA on basal resistance is NPR1-dependent (Figure 2-1).The development of disease symptoms showed further differences between the fourlines. Columbia exhibited patchy chlorosis at 48 h post infection (hpi). These patchescoalesced at 96 hpi. NahG showed the most severe symptom development, withwidespread chlorosis followed by complete tissue collapse at 96 hpi. The sid2-2 mutantexhibited less severe symptoms than NahG, with widespread chlorosis and necrosis at 96hpi although it lacked the complete tissue collapse of NahG. The npr1-1 mutantdeveloped chlorosis but not necrosis and had an intermediate phenotype betweenColumbia and sid2-2. These data demonstrate that although sid2-2 and npr1-1 showedthe same level of basal resistance, sid2-2 exhibited necrosis and tissue collapse that didnot occur in npr1-1 under these conditions. This result indicates that the effect of SA onrepressing necrosis and tissue collapse is NPR1-independent. Figure 2-1 Bacterial growth in Pst DC3000 infected Arabidopsis. Columbia, npr1-1,sid2-2 and NahG were dip infected with Pst DC3000. Colony forming unitsper square centimeter were determined at the time points indicated.

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16The expression of the PR genes PR1 and PDF 1.2 was monitored during diseaseprogression in all four lines. PR1 was induced in Columbia and slightly induced at 96 hpiin npr1-1 but was not induced in the SA-deficient lines. PDF1.2 was induced to a similarextent in all lines upon pathogen infection (Figure 2-2). The reduction in PR1 geneexpression in NahG and sid2-2 is consistent with that seen upon infection with avirulentPst (Nawrath and Metraux, 1999) and the loss of localized expression of PR genes innpr1-1 as demonstrated by Cao et al. (1994). Consistent with my findings, PDF1.2expression was unaltered in NahG and sid2-2, when compared to wildtype, in response toinfection with Alternaria brassicicola (Nawrath and Metraux, 1999). Figure 2-2 PR1 and PDF1.2 gene expression in Pst infected Arabidopsis. Total RNA wasextracted from Columbia, npr1-1, sid2-2 and NahG plants dip infected withPst DC3000. Northern blot analysis was performed with 32P labeled PR1 andPDF1.2 on plants that were mock inoculated, or 48 and 96 hours postinfection.

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17SA and Jasmonate Induction in Response to Pst InfectionThe study of the effect of removal of SA accumulation or perception on theinduction of other hormones was the focus of this chapter. Therefore the pattern of SAinduction in the different lines was confirmed. SA levels increased during infection in theColumbia and npr1-1 lines, with npr1-1 accumulating significantly more SA thanColumbia. This is consistent with the results of Clarke et al. (2000), who demonstratedthat npr1-1 produces more SA than Columbia. SA remained at a low level in NahG andsid2-2 consistent with the findings of Nawrath and Metraux (1999) (Figure 2-3). Figure 2-3 SA production in Pst infected Arabidopsis. Columbia, npr1-1, sid2-2 andNahG dip infected with Pst DC3000. SA was measured at the time pointsindicated.The jasmonate response of a plant can be described in terms of an oxylipinsignature consisting of different forms of JA and its precursors (Kramell et al., 2000). Topartially characterize this oxylipin signature in Arabidopsis infected with Pst, theinduction of jasmonic acid (JA) and its active precursor 12-oxo-phytodienoic acid(OPDA) was measured. Both JA and OPDA were induced in all four lines. The highestamounts of induction were observed in NahG and sid2-2, followed by npr1-1 with

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18Columbia producing the lowest amounts of these jasmonates (Figure 2-4). It has beendemonstrated by Heck et al. (2003) that Pst infection leads to higher JA levels in sid2-2and NahG than in wild type Arabidopsis. Here, it can be seen that OPDA follows theprofile of JA expression and jasmonate production in npr1-1 is intermediate betweenthose of the SA-deficient lines and wild type. Figure 2-4 JA and OPDA production in Pst infected Arabidopsis. Columbia, npr1-1,sid2-2 and NahG were dip infected with Pst DC3000. The amount of (A) JAand (B) OPDA was quantified at the time points indicated.

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19The Effect of Coronatine Production by PstJasmonate accumulation was higher in Arabidopsis that showed enhancedsusceptibility due to SA-deficiency. Therefore, jasmonate signaling may also reducedefense responses. Pst produces the phytotoxin coronatine that is a mimic of OPDA andmay act by stimulating jasmonate responses (Weiler et al., 1994). The total jasmonateresponse consists of jasmonates and coronatine. Therefore, coronatine accumulationduring infection in the different lines was measured. NahG had a significantly higherlevel of coronatine accumulation than sid2-2, npr1-1 and Columbia (Figure 2-5). Figure 2-5 Coronatine production in Pst infected Arabidopsis. Columbia, npr1-1, sid2-2and NahG were dip infected with Pst DC3000. Coronatine was measured atthe time points indicated.Coronatines action as a virulence factor may utilize the jasmonate responsepathways to repress SA. To investigate if coronatines action is SA-dependent, SA-deficient lines were infected with Pst DC3661. Pst DC3661 is a Tn5 mutant that isdisrupted in its ability to produce coronatine (Mittal and Davis, 1995). Bacterial growthwas reduced by a factor of ten in all lines infected with Pst DC3661 compared to those

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20infected with Pst DC3000 but the relative differences in susceptibility between theArabidopsis lines remained (Figure 2-6). Figure 2-6 Bacterial growth at 72 hpi in Pst DC3000 and Pst DC3661 infectedArabidopsis. Columbia, npr1-1, sid2-2 and NahG were dip infected with Pst.The amount of bacteria in the tissues was determined at the time pointsindicated.Disease symptom development was also reduced in all lines infected with PstDC3661 when compared to those infected with Pst DC3000 (Figure 2-7). However, thecomparative severity of the disease in the different lines remained the same. These resultswere confirmed with an additional coronatine-deficient Pst mutant, Pst DC3118 (Moore,1989; Ma et al., 1991). These data demonstrate that coronatines action is independent ofSA, as coronatine-deficiency still reduced virulence on SA-deficient lines. These dataindicate that the JA and SA pathways act in an independent manner in the control of basalresistance.

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21 Figure 2-7 Disease symptom development in Pst infected Arabidopsis. Columbia, npr1-1, sid2-2 and NahG plants were dip infected with wild type Pst (DC3000) orcoronatine-deficient Pst (DC3661). Photographs were taken 96 hpi.

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22The Role of Ethylene in SA-Deficient ArabidopsisRecent work has demonstrated that the catechol produced by NahG has side effectsin the NahG plant that are not due to the loss of SA (Heck et al., 2003). NahG had beenused to demonstrate an agonistic relationship between SA and ethylene in the interactionbetween Arabidopsis and Xcc (O'Donnell et al., 2003). To verify this relationship in sid2-2 and to investigate the role of NPR1 in this interaction, ethylene evolution in response toPst infection was measured.Ethylene was induced to similar levels at 48 hpi in Columbia and npr1-1 indicatingthat the control of ethylene synthesis by SA is NPR1-independent. A large increase inethylene, also at 48 hpi, was observed in sid2-2. NahG showed no induction of ethyleneduring disease progression (Figure 2-8). These results indicate that catechol or its furthermetabolites suppress ethylene production and that there is an antagonistic relationshipbetween the synthesis of SA and ethylene, rather than the agonistic relationship suggestedby the NahG phenotype. These results are also consistent with the observation thatethylene insensitive plants synthesize more SA than wild type plants following pathogeninfection (O'Donnell et al., 2003).To investigate if it was this suppression of ethylene signaling that caused thedifferences in disease symptoms between sid2-2 and NahG, plants were treated with theethylene inhibitor 1-methylcyclopropene (MCP) 24 hours prior to infection and theresultant effects on disease were assessed. MCP blocks ethylene binding to it receptors,thus inhibiting its perception (Sisler and Serek, 1997). No differences in diseasedevelopment between those plants treated with MCP and untreated plants were observed(data not shown). These data indicate that although ethylene production is inhibited in

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23NahG, this absence of ethylene is not responsible for the enhanced disease phenotypeobserved in NahG plants relative to sid2-2. Figure 2-8 Ethylene production in Pst infected Arabidopsis. Columbia, npr1-1, sid2-2and NahG were dip infected with Pst DC3000. Ethylene production wasmeasured at the time points indicated.DiscussionThe effect of an inability to accumulate SA on the induction of ethylene andjasmonates during the interaction between Arabidopsis thaliana and the virulent bacterialpathogen Pst DC3000 was investigated. Two lines that do not accumulate SA duringinfection (NahG and sid2-2) were used to determine these effects. The effects of SA onethylene and jasmonates accumulation are believed to be NPR1-dependent (Clarke et al.,2000). Consequently the levels of ethylene and jasmonates in npr1-1, following infectionwere also measured.In accordance with previous reports (Cao et al., 1994; Delaney et al., 1994;Nawrath and Metraux, 1999) an enhanced susceptibility to Pst in NahG, sid2-2 and npr1-1 was observed, with more severe symptom development in NahG and sid2-2 than innpr1-1. This result correlates with the partial loss of SA signaling in npr1-1 compared to

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24the other two lines. NahG displayed a rapid and complete tissue collapse that did notoccur in sid2-2. NahG also supported more bacterial growth than sid2-2, although bothwere more susceptible to Pst than Columbia. This result, coupled with the recent dataproposing side effects in NahG associated with catechol (Van Wees and Glazebrook,2003), led to the comparasion of the NahG and sid2-2 lines for effects on ethylene andjasmonate accumulation.As has been reported previously, the SA levels in NahG and sid2-2 are roughlyequivalent following Pst infection (Nawrath and Metraux, 1999). The massive increase ofSA in npr1-1 compared to that of Columbia is probably due to a loss of feedbackinhibition on SA synthesis that relies on an NPR1-dependent SA pathway (Clarke et al.,2000).As well as SA, jasmonates have been reported to play a role in disease with both JAand its precursor OPDA believed to be active signaling compounds (Stintzi et al., 2001).It was therefore determined how the removal of SA affected the induction of thesejasmonates following infection. In Columbia, both JA and OPDA increase followinginfection. In the SA-deficient lines an increase in jasmonates was observed compared towild type, with NahG and sid2-2 producing the most jasmonates. npr1-1 producedintermediate jasmonate levels. This suggests that SA represses jasmonate production in apartially NPR1 dependent manner. As NahG and sid2-2 produce similar levels ofjasmonates catechol is unlikely to affect jasmonate production. The enhancedsusceptibility of SA-deficient Arabidopsis to Pst may be due to the combined effects ofreduced SA and increased jasmonates.

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25The use of PR gene expression is often used to investigate the roles of specificphytohormones in plant pathogen interactions. In order to determine the degree that twoof these markers represented the levels of the phytohormones produced, the expression ofPR1 and PDF1.2 was determined in the different lines during infection. The expression ofPR1 is absent in accordance with the loss of SA accumulation in both sid2-2 and NahG.It is also much reduced in npr1-1 when compared to Columbia. These results demonstratethat the loss of SA accumulation in these lines is sufficient to prevent the induction of SAresponse pathways and that PR1 is a good marker of SA signaling.There was also an induction of PDF1.2 to a similar extent in all lines. PDF1.2requires the activation of both ethylene and JA response pathways to be induced inresponse to Alteranria brassicicola (Penninckx et al., 1998). PDF1.2 is not visible insid2-2 or NahG at 96hpi due to the poor quality RNA obtained from this highly necrotictissue. These data indicate that although the absence of SA affects the levels of ethyleneand jasmonates this is not necessarily reflected in the expression of all downstream genes.This indicates that marker genes may not always be the best approach to mappinghormone networks.The production of coronatine by Pst in the different Arabidopsis lines wasmeasured to further study the relationship between SA and jasmonates. Coronatine is anon-host specific phytotoxin whose biological effects include induction of leaf chlorosis(Mittal and Davis, 1995). It has been suggested to act as a mimic of OPDA (Weiler et al.,1994). Coronatine production was observed in all of the lines infected with Pst, with themost production occurring in NahG. The coronatine levels were different to those ofjasmonates. This suggests that coronatine acts via jasmonate signaling rather than by

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26stimulating jasmonate biosynthesis. The production of coronatine was the same in sid2-2as Columbia so it is not directly related to bacteria levels. This suggests that host factorsor bacteria thresholds must be present to stimulate coronatine production.To determine if coronatine affects SA-dependent basal resistance, the lines wereinfected with the coronatine-deficient mutants Pst DC3661 and Pst DC3118. All linesinfected with coronatine-deficient Pst had reduced bacterial growth compared with thoseinfected with Pst DC3000 and consequently developed less severe disease symptoms.However, the relative differences in susceptibility between the lines remained. Thisindicates that role of coronatine as a virulence factor is independent of SA, as SA-deficient lines still show an enhanced susceptibility to coronatine-deficient Pst despite itsreduced virulence. It is then both the accumulation of coronatine and the loss of SAsignaling that lead to the severe disease phenotype observed in the SA deficient linesupon infection with Pst.Ethylene synthesis is transient during the post-infection period, peaking around 48hpi in Columbia in response to Pst. This induction does not occur in NahG. The fasterdevelopment of chlorosis in the ethylene insensitive mutant etr1-1 in response to Xccinfection (O'Donnell et al., 2003) suggests that ethylene is required to slow thedevelopment of chlorosis. The growth of Xcc in etr1-1 remains unchanged indicating thatethylene does not play a direct role in limiting pathogen growth in this interaction. As theArabidopsis response to Pst and Xcc are similar, it can be concluded that ethylene doesnot play a major role in basal resistance to either pathogen.A major difference between the two SA-deficient lines with respect to this ethyleneinduction was observed, with sid2-2 showing a two-fold increase in ethylene induction at

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2748 hpi relative to Columbia. These data agree with the recent study by Heck et al. (2003).The completely opposite nature of this response in NahG when compared to sid2-2suggests that catechol or a further metabolite negatively affects ethylene production.However, the treatment of sid2-2 and NahG with the ethylene inhibitor MCP beforeinfection caused no change in their respective phenotypes. This suggests that althoughethylene may play a role in fine-tuning the disease response in Arabidopsis, it is notresponsible for the difference in susceptibility between sid2-2 and NahG. Thesuppression of ethylene production by catechol in NahG therefore is independent of thechanges in susceptibility and disease phenotype.These data indicate that NahG is not always an appropriate model for studying therole of SA induction in plant-pathogen interactions. They also demonstrate anantagonistic relationship between SA and ethylene in this system, as the removal of SAincreases ethylene synthesis. As no difference in ethylene induction in npr1-1 comparedwith Columbia was observed, it might also be concluded that the effect of SA on ethyleneis NPR1-independent.

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28CHAPTER 3SYSTEMIC ACQUIRED TOLERANCE IN TOMATOIntroductionPathogens have a high negative impact on agriculture. In 1993, for example, anestimated 12% of crops were lost to disease (Agrios, 1997). The application of chemicalssuch as fungicides help to limit the damage they cause. However, utilizing plant defenseresponses remains an important research area for the limiting pathogen damage. Forinstance application of Benzothiadiazole stimulates plant defense responses and inducesresistance to several pathogens (Lawton et al., 1996). Infected plants can in manyinstances limit the extent of pathogen growth and symptom development. Resistanceoccurs via an incompatible interaction and results in rapid activation of defense responsesthat limit pathogen growth (Alfano and Collmer, 1996; Hammond-Kosack and Jones,1996). These responses include: strengthening of cell walls with hydroxy-Proline-richglycoproteins (Showalter et al., 1985), callose (Parker et al., 1993) and lignin(Moerschbacher et al., 1990); rapid expression of PR proteins (Linthorst, 1991); and thesynthesis of antimicrobial compounds (Hain et al., 1993; Epple et al., 1995; Penninckx etal., 1996).Tolerance is the repression of symptom development without restricting pathogengrowth. The visible phenotype of the two responses is similar. Resistance is a well-studied phenomenon yet tolerance remains an enigma. Tolerance has been identified inseveral phytohormone compromised plant lines. One of these is the ethylene-insensitiveein2 mutant of Arabidopsis, which has increased tolerance to several virulent bacterial

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29pathogens (Bent et al., 1992). Increased tolerance has also been demonstrated to virulentXcv in ethylene and SA-deficient tomato lines (Lund et al., 1998; O'Donnell et al., 2001).Disease development in tomato infected with virulent Xcv can be defined in twostages: primary and secondary disease development. Primary disease developmentconsists of localized lesion formation and it is unaltered in the tolerant lines. Secondarydisease development consists of chlorosis and necrosis that spreads from the primarylesions. These secondary disease symptoms require the cooperative action of ethyleneand SA and are abolished in tolerant lines. It remains a mystery why the plant retainsthese responses and thus increases the amount of tissue damage. Organized necrosis mayhelp the plant withdraw nutrients from the infected tissue. The phytohormones may alsobe required for the induction of a systemic response that allows the plant as a whole torespond to the increased pathogen presence. While tolerance due to phytohormones-deficiency might be a valuable outcome foragriculture, it is likely to have costs. Ethylene and SA are involved in many plant-pathogen interactions. For instance, ethylene is necessary for resistance to certain fungalpathogens and for the generation of induced systemic resistance (Knoester et al., 1998;Knoester et al., 1999). SA is necessary for resistance to avirulent pathogens and thegeneration of SAR in several species (Gaffney et al., 1993). SAR is the activation ofsystemic defense responses that occurs due to the formation of necrotic lesions (Ryals etal., 1996). The hypersensitive response during an incompatible interaction and tissuedeath during a compatible interaction can both induce SAR (Hunt and Ryals, 1996). SARresults in the development of a broad-spectrum, systemic resistance. It is not effectiveagainst, or induced by, all pathogens. For example, the infection of Arabidopsis with

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30Botrytis cinerea fails to induce SAR and inoculations with Pseudomonas syringae doesnot affect B. cinerea challenge (Govrin and Levine, 2002). In tomato SAR is induced bypathogens such as tobacco necrosis virus and Phytophthoria infestans (Enkerli et al.,1993; Jeun et al., 2000).It is important to understand the roles of phytohormones in systemic responses, asthe loss of these responses could reduce any advantage gained by reduced symptomdevelopment due to engineered ethylene or SA-deficiency, I focus here on tomato, as theabsence of ethylene and SA causes tolerance to virulent Xcv. The systemic response intomato to this pathogen has not been previously examined. I investigate if Xcv is capableof inducing systemic responses in tomato and possible roles of SA and ethylene in itsgeneration and action. To avoid confusion I will use the term inoculation for the firsttreatment of a tomato plant and the term challenge for the subsequent treatment ondistal leaves. I demonstrate that both virulent and avirulent Xcv generate a systemicresponse in tomato. I show that both virulent and avirulent Xcv cause an SA and ethylene-dependent PR gene induction and sensitize systemic defense responses. However, incontrast to the well-established SAR, the Xcv-induced response generates tolerance tosubsequent challenge with virulent Xcv. I term this response systemic acquired tolerance(SAT) and define it as prior pathogen exposure reducing necrosis in response to virulentpathogen infection in distal tissues without affected pathogen growth. SAT involves arapid increase in both PR gene expression and ethylene production in response tosubsequent challenges with virulent Xcv.

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31Virulent and Avirulent Xcv Led to Systemic Acquired Tolerance in TomatoTo investigate systemic responses to Xcv, wild type tomato plants at the three-leafstage were mock inoculated, inoculated with virulent Xcv strain 93-1 or avirulent Xcvstrain 87-7 (Bonas et al., 1993) on their lowest two leaves. The inoculations were thenpermitted to run their full course of around 14 days, at which point those leavesinoculated with virulent Xcv were fully necrotic and those inoculated with avirulent Xcvhad developed lesions associated with the hypersensitive response (Figure 3-1A). Figure 3-1 Symptom development of Xcv infected tomato. (A) Representative diseasesymptoms at 16 dpi of wild type tomato plants inoculated with avirulent andvirulent Xcv and (B) symptoms resulting from challenge with virulent Xcv.

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32To determine if this inoculation with Xcv affected responses to subsequentpathogen exposure, a challenge was performed with virulent Xcv on uninoculatedsystemic leaves. Prior inoculations with either virulent or avirulent Xcv, but not mockinoculations, reduced the necrotic development resulting from the challenge (Figure 3-1B). The primary symptoms such as lesion formation were unaffected and secondarysymptom development such as chlorosis and some confluent necrosis were still apparent.The major difference in secondary symptom development during challenge was reducednecrosis in plants with prior Xcv inoculation.As the response consists of two independent interactions between two biologicalentities a high degree of variation is to be expected. With this in mind the level ofnecrosis was determined in large population groups by measuring ion leakage at 16 daysafter challenge (Figure 3-2). Figure 3-2 Cell death due to challenge of tomato with virulent Xcv. Cell death wasmeasured in the form of percent ion leakage in plants with a mock challengeor challenged with virulent Xcv. The plants were exposed to a mockinoculation or inoculation with virulent (vir) or avirulent (avr) Xcv prior tochallenge.

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33Percent ion leakage, an indicator of cell death, was two-fold higher uponchallenge in plants previously mock inoculated than those with prior Xcv inoculations. Incontrast to previously mock-inoculated plants there was no significant difference betweensymptoms in plants with prior virulent or avirulent inoculations upon challenge withvirulent Xcv. The reduction of symptom development due to previous pathogen exposureis consistent with SAR generation. Bacterial growth measurements confirmed resistanceto avirulent Xcv but not virulent Xcv, as growth of avirulent Xcv was 10-fold lower thanthat of virulent Xcv (Figure 3-3A). Figure 3-3 Bacterial growth during inoculation and challenge of tomato with Xcv. (A)The growth of virulent (vir) and avirulent (avr) Xcv was measured duringinoculations. (B) A challenge with virulent Xcv was then performed on theseplants and mock-inoculated controls and the bacterial growth was measured.

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34When bacterial populations in challenge infections were determined there was nodifference in bacterial growth due to prior inoculation with either virulent or avirulentXcv compared with plants with prior mock inoculations (Figure 3-3B). This result leadsto the conclusion that avirulent and virulent Xcv induced SAT rather than SAR, as theyreduced symptom development but not bacterial growth due to challenge with virulentXcv. As to my knowledge SAT has not been previously described I defined it as the priorexposure to a pathogen leading to reduced necrosis development in response tosubsequent challenge with a virulent pathogen in distal tissues.Prior Inoculation with Xcv Led to an Early Production of Ethylene upon Challengewith Virulent XcvEthylene and SA are involved in the development of systemic responses in severalplants. Ethylene and SA were measured during inoculation and challenge with Xcv.Inoculation with virulent Xcv led to ethylene production in local tissues at 5 days postinfection (dpi), while avirulent Xcv caused a greater induction of ethylene at 4 dpi (Figure3-4A). Virulent Xcv induced SA at 10 dpi, while avirulent Xcv induced SA at 4 dpi thatpeaked at 10 dpi (Figure 3-4B). The induction of ethylene and SA was later and atreduced magnitude in response to virulent Xcv than avirulent Xcv. No systemicproduction of ethylene was observed in response to primary Xcv inoculations and at 16dpi no systemic induction of SA was observed in plants inoculated with Xcv (data notshown).Loss of secondary disease symptoms occurs in virulent Xcv-infected ethylene or SAdeficient tomato plants (Lund et al., 1998; O'Donnell et al., 2001). Therefore it is possiblethat SAT might similarly be the result of reduced phytohormone synthesis duringchallenge. To determine if SAT was due to loss of ethylene or SA, their accumulation

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35following challenge with virulent Xcv was determined. Results indicated that thetolerance associated with SAT is not due to reduced ethylene or SA accumulation.Instead, earlier ethylene induction upon challenge accompanies SAT (Figure 3-4). Figure 3-4 Local ethylene and SA production during inoculation and challenge of tomatowith Xcv. Tomato plants were mock inoculated or inoculated with virulent(vir) or avirulent (avr) Xcv and (A) ethylene and (B) SA were measured.Fourteen days later a challenge with virulent Xcv was performed onuninfected leaves and (C) ethylene and (D) SA induced by the challenge wasmeasured.

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36The ethylene induction in response to a challenge with virulent Xcv, following priormock inoculation, reached its maximum at 10 dpi. However, plants with prior Xcvinoculations produced additional ethylene around 5 dpi when challenged (Figure 3-4C).Although at a reduced magnitude, this induction resembles a response to avirulent Xcv.Challenge with virulent Xcv caused ethylene induction at 8 to 10 dpi in all plants, themagnitude of which was dependent on the prior inoculation. Plants with prior avirulentXcv inoculation produced the most ethylene when challenged. This ethylene inductionwas lower in plants with prior virulent Xcv inoculations and lowest in the mock-inoculated controls. However, the significant difference remains the early ethyleneinduction that is only present in plants with prior Xcv inoculations.Plants with prior mock or virulent Xcv inoculations produced similar SA inductionprofiles in response to challenge with virulent Xcv. However, a reduction in SAproduction was observed in plants with a prior avirulent Xcv inoculation. These plantsalso produced an early SA peak (Figure 3-4D) that resembled the response to avirulentXcv although reduced in magnitude. The following production of SA was delayed andresembled that caused by virulent Xcv although at reduced magnitude.The difference in SA production during a challenge and the increased strength andspeed of phytohormone induction during prior inoculations had no effect on the overallSAT phenotype. Therefore the early induction of ethylene upon challenge in plants withprior Xcv infections correlates with the tolerant phenotype, whereas SA induction doesnot. Despite the lack of correlation between SA induced during challenge and SAT, SAand ethylene induction during inoculation may be required for systemic signal formation.

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37PR Gene Expression Indicated the Necessity of Ethylene and SA in Systemic SignalGenerationAs ethylene or SA-deficient plants develop less secondary disease symptoms dueto virulent Xcv than plants displaying SAT, a direct approach to determining their role inSAT is difficult. Therefore PR gene expression was assayed instead of ion leakage as amarker for defense responses in phytohormone-deficient plants. To determine if ethyleneand SA are necessary for systemic signal transduction, two transgenic lines altered intheir ability to accumulate ethylene or SA were used. Loss of ethylene was achieved withtransgenic ACD plants. These plants do not accumulate the ethylene precursor ACC andthus under-produce ethylene. The ACD line was compared to its isogenic parent UC82B.The role of SA was determined using transgenic tomato plants expressing the bacterialsalicylate hydroxylase, nahG that cannot accumulate SA. The NahG line was comparedto its isogenic parent Money Maker (MM).Induction of PR genes is often used as an indicator of early defense responses.They show both local and systemic induction during SAR (Ward et al., 1991). Theexpression of PR1a and PR1b was induced in the local and systemic tissues of tomatoduring infection with virulent Xcv (Figure 3-5). PR1a expression was lower than PR1b.The local expression of both genes was induced in all lines in response to virulent Xcv.The timing and level of induction of PR1a varied between different cultivars, with anearlier and a greater induction in MM than UC82B. The expression of PR1b was alsohigher in MM than UC82B, although the timing of induction was the same. Whencompared to their isogenic parents, infected ACD and NahG plants had reduced localexpression of both genes. However, this reduction did not affect pathogen growth(O'Donnell et al., 2001).

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38 Figure 3-5 Local and systemic PR gene induction in ethylene and SA-deficient tomatolines in response to inoculation with virulent Xcv. Ethylene-deficient ACD,SA-deficient NahG and their isogenic parents were inoculated with virulentXcv. PR1a and PR1b expression levels were determined by real time RT-PCRin local and systemic tissues.

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39Virulent Xcv induced systemic expression of both genes in MM and UC82B. InMM systemic induction of PR1a and PR1b occurred at the same time, whereas in UC82Bthe expression of PR1b was later than PR1a. ACD and NahG did not show significantsystemic induction of PR gene expression in response to virulent Xcv. Disease symptomsin ACD and NahG are comparable to their isogenic parents up to 8 dpi (O'Donnell et al.,2001). As widespread necrosis does not occur before 8 dpi the lack of systemic PR geneexpression is due to loss of ethylene and SA accumulation rather than the loss ofwidespread necrosis in these lines. This lack of systemic PR gene induction indicates thatethylene and SA-deficient lines might be compromised in their systemic signaltransduction. However, the tolerant phenotype of these lines makes direct investigationdifficult.In UC82B systemic PR gene induction occurred within 6 dpi, whereas the majorlocal phytohormone induction occurred at 8 to 10 dpi in response to virulent Xcv.Measurable phytohormone induction, therefore, was not responsible for systemic PRgene induction. Direct damage to cells infected by the pathogen may cause local ethyleneand SA production limited to cells that are under attack. This phytohormone productionmay be involved in the induction of both local and systemic defense responses.Avirulent Xcv Induced Greater Local and Systemic PR Gene Expression thanVirulent Xcv, but they Result in Similar PR Gene Induction During ChallengeThe induction of ethylene and SA is more rapid in response to avirulent thanvirulent Xcv, yet the resulting systemic response to these two pathogens is the same. Todetermine if the early defense responses follow this trend, induction of PR geneexpression by avirulent and virulent Xcv inoculations was assayed. Avirulent Xcv inducedhigher levels and earlier local expression of PR genes than virulent Xcv (Figure 3-6). The

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40timing of systemic PR1b induction was similar in response to either pathogen, butsystemic PR1a induction was faster in response to avirulent Xcv. Faster and stronger localPR gene response to avirulent than virulent Xcv may reflect the increased defenseresponses that limit pathogen growth.Prior avirulent and virulent Xcv inoculations produced the same induction ofethylene and changes in symptom development during challenge. The levels of PR geneexpression were assayed to determine if increased systemic PR gene expressiontranslated into stronger PR gene induction upon challenge. The difference between thetwo prior Xcv inoculations in PR gene expression upon challenge was negligible. Bothcaused an early peak of PR gene expression at 1 dpi upon challenge that was absent inplants with prior mock inoculation (Figure 3-7). These results are consistent with therapid ethylene induction following challenge. These results lead to the hypothesis thatinoculation with Xcv primed the systemic defenses and caused a faster induction ofdefense responses upon challenge. This primed defense response led to the formation oftolerance upon challenge.DiscussionIn this chapter the systemic responses of tomato to both virulent and avirulent Xcvwere studied. A primary infection with virulent Xcv led to local as well as ethylene andSA-dependent systemic PR gene induction within 6 dpi. PR gene induction occurredprior to measurable ethylene or SA induction, indicating that undetectable localizedethylene and SA production occurred within days of infection with virulent Xcv and ledto the generation of a systemic signal. The systemic signal, as well as inducing PR geneexpression, altered the systemic defense response to challenge with virulent Xcv. This

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41altered defense response is tolerance and not resistance. It caused rapid PR geneexpression and ethylene induction in response to subsequent challenge, leading tosuppressed symptom development but not resistance. Figure 3-6 The measurement of local and systemic PR gene expression in tomato duringinoculations with virulent or avirulent Xcv. Wild type (UC82B) tomato plantswere mock inoculated or inoculated with virulent (vir) or avirulent (avr) Xcvand the local and systemic expression of PR1a and PR1b was determined byreal time RT-PCR.

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42 Figure 3-7 Induction of PR genes in tomato plants during challenge with virulent Xcv inthe presence or absence of SAT. Wild type (UC82B) tomato plants were mockinoculated or infected with virulent (vir) or avirulent (avr) Xcv. Fourteen dayslater a challenge was performed with virulent Xcv. The expression of PR1aand PR1b was determined with real time RT-PCR during this challenge.The ability of a plant to send a systemic signal in response to a biological stimulushas long been established. This communication allows separate tissues to act in concert tothe many stimuli they perceive. Systemic signals have been characterized in terms ofresponses to pathogens, symbionts and wounding. SA, ethylene, jasmonates and systeminare all involved in generating systemic signals (Pearce et al., 1991; Gaffney et al., 1993;Pieterse et al., 1998). SA is a key player in SAR in many species (Ryals et al., 1996).NahG plants are unable to mount SAR to bacterial, viral or fungal pathogens (Gaffney etal., 1993; Friedrich et al., 1995; Lawton et al., 1995) and exogenous SA can induce SAR

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43(Ward et al., 1991; Vernooij et al., 1995). However, grafting experiments with NahG andwild type tobacco indicate that although SA is required for SAR it is unlikely to be thetransmitted signal (Vernooij et al., 1994).The role of SA in the systemic responses of tomato may differ somewhat fromthat in other plant species. For example, the induction of SAR in tomato against P.infestans demonstrated neither systemic SA accumulation upon inoculation, nor increasedSA production upon challenge, both of which are common to SAR in other plant species(Jeun et al., 2000). While local induction of SA is observed in response to virulent andavirulent pathogens, its accumulation is only observed after the systemic induction of PRgenes (O'Donnell et al., 2001)In tomato, ethylene and SA are induced in response to both virulent and avirulentXcv, although in neither response are they involved in limiting bacterial growth (Lund etal., 1998; Ciardi et al., 2001; O'Donnell et al., 2001; O'Donnell et al., 2003). Loss ofethylene causes tolerance to virulent Xcv by reducing cell death and lesion size followinginfection with avirulent Xcv. SA loss also leads to tolerance to virulent Xcv. However,loss of SA increases cell death and lesion size in response to avirulent Xcv. Thedevelopment of systemic acquired tolerance to Xcv rather than resistance may be aspecific response to this pathogen and reflect the roles of ethylene and SA in thisinteraction.In agreement with the work of Ciardi et al. (2000), avirulent Xcv induces a rapidand more intense expression of PR genes than virulent Xcv. Here I show that thistranslates into an increased systemic PR gene induction. This increased PR geneexpression coupled with a faster and greater local induction of ethylene and SA may

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44affect SA production during challenge but it has no impact on SAT. Therefore, changesin SA production during challenge are not important for the formation of SAT.Interestingly the work of Jeun et al. (2000) demonstrates that changes in SA induction intomato during challenge with P. infestans are also not necessary for SAR.The PR gene induction and ethylene profiles caused by SAT during challengeresembled those of an incompatible interaction. However, they failed to develop with thespeed and intensity required for successful resistance to develop. Systemic induction ofdefense responses and PR genes that do not lead to SAR have been observed in theinteraction of Arabidopsis and the necrotizing fungal pathogen B. cinerea (Govrin andLevine, 2002). Virulent Pseudomonas syringae pv. tomato also fails to induce SAR inArabidopsis, but in this case there is no systemic induction of PR genes (Cameron et al.,1999). An incompatible-interaction between Arabidopsis and Pseudomonas syringae pv.syringae leads to a reduction of symptom development but no changes in bacterialgrowth during a repeat challenge with the same incompatible pathogen (Summermatter etal., 1995). These data suggest that systemic responses to pathogens exist as a continuumbetween no response, tolerance and resistance.Transgenic tobacco over-expressing PR1a show enhanced tolerance toPeronospora tabacina (Alexander et al., 1993). While tobacco plants deficient in catalaseshow that low level activation of defense responses, including PR gene induction, byH2O2 can lead to enhanced pathogen tolerance, higher H2O2 levels cause resistance(Chamnongpol et al., 1998). These data again suggest that a range of systemic responsesto pathogen invasion occur, with a moderate response repressing symptom developmentand a strong response suppressing both symptom development and pathogen growth.

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45Here I have described the phenomenon of systemic acquired tolerance that adds to theranges of known systemic responses induced by pathogens and suggests that plants arecapable of more diversity in their systemic responses than previously suspected.

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46CHAPTER 4DISCUSSIONPhytohormone Networks in the Responses of Tomato and Arabidopsis to VirulentBacterial PathogensPlant signaling networks utilize ethylene, SA and jasmonates to coordinate defenseresponses. Different plants use these phytohormones in a variety of ways to achieve thesame result. In this chapter I compare and contrast their roles of in the response ofArabidopsis and tomato to infection with virulent bacterial pathogens. Plantscompromised in production or signaling of a phytohormone were used to determine itseffects. However, phytohormones do not act in isolation the levels of one can often affectthe levels of another. Many studies do not look at the effects of altering the synthesis orperception of one phytohormone on others. They therefore cannot address whether thephenotype observed is strictly due to the phytohormone lost.Phytohormone measurements demonstrate if the altered phenotype is due to thecompromised phytohormone or if others may be involved. Table 4-1 shows thephenotypes of Arabidopsis and tomato phytohormone mutants infected with virulentbacterial pathogens. Table 4-2 shows the effect of these mutations or transgenes on theproduction of other phytohormones in response to infection. The Arabidopsis responsesare to virulent Pst and the tomato responses are to virulent Xcv.

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47Table 4-1 Comparative phenotypes of phytohormone mutants in Arabidopsis andtomato. Compatibleinteractions Salicylic acid Ethylene Jasmonates Arabidopsis and Pst Mutants sid2-2 etr1-1 coi 1 and fad3-2 fad7-2 fad8 (fad3) Local phenotype enhancedsusceptibility (1) increased symptoms(2) resistant (coi1)no effect fad3-2 fad7-2fad8)(3) Systemic phenotype normally none (4) ND ND Tomato and Xcv Mutants NahG ACD, Nr AOCas Local phenotype tolerant (5) tolerant (6) resistant (7) Systemic phenotype no SAT (8) no SAT (8) ND References are: 1 (Nawrath and Metraux, 1999); 2 (O'Donnell et al., 2003); 3 (Kloek etal., 2001); 5 (O'Donnell et al., 2001); 6 (Lund et al., 1998); 7 (O'Donnell et al., 2003); 8(chapter 3). ND = not determined.Table 4-2 Comparative phytohormone profiles of Arabidopsis and tomato mutants. Arabidopsis andPst Salicylic acid Ethylene Jasmonates Mutants sid2-2 etr1-1 coi 1 and fad3-2fad7-2 fad8 Salicylic acid none (1) ND increased (coi1)(2) Ethylene increased (3) insensitive (4) ND Jasmonates increased (5) ND insensitive (coi1)(6)none (fad3-2 fad7-2fad8)(7) Tomato and Xcv Mutants NahG ACD, Nr as-AOC Salicylic acid none (8) none (9) none (10) Ethylene normal (11) none (ACD) (9)insensitive (Nr) (12) none (10) Jasmonates normal (10) normal (10) none (13) References are: 1 (Nawrath and Metraux, 1999); 2 (Kloek et al., 2001); 3 (chapter 2); 4(Bleecker et al., 1988); 5 (Heck et al., 2003); 6 (Feys et al., 1994); 7 (McConn andBrowse, 1996); 18 (Oldroyd and Staskawicz, 1998);9 (O'Donnell et al., 2001); 10 (Lundet al., 1998); 11 (O'Donnell et al., 2003); 12 (Lanahan et al., 1994); 13 (Stenzel et al.,2003). ND = not determined.

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48Arabidopsis has many phytohormone mutants including those for SA (sid2-2),ethylene (etr1-1) and jasmonates (fad3-2 fad7-2 fad8 and coi1). The etr1-1 mutant is adominant-insensitive ethylene receptor mutant (Bleecker et al., 1988). The fad3-2 fad7-2fad8 mutant is a loss of function triple mutant of the three fatty acid desaturases. Due toits failure to synthesize linolenic acid, it has reduced jasmonate biosynthesis (McConnand Browse, 1996). The coi1 mutant is a jasmonate-insensitive ubiquitin E3 ligase mutant(Xie et al., 1998). Tomato also has mutants and transgenic lines compromised inphytohormones such as SA (NahG), ethylene (ACD and Nr) and jasmonates (as-AOC).Nr is a dominant ethylene-insensitive receptor mutant (Rick and Butler, 1956). Thetransgenic plant as-AOC is an antisense line of the jasmonate biosynthesis enzyme alleneoxide cyclase (Stenzel et al., 2003).These data allow the construction of signaling network models for these plantpathogen interactions. However, not all phenotype and phytohormone measurementshave been addressed in each system. These models are based on only two sets ofinteractions and may vary greatly in response to different pathogens or in different plantsystems.The Compatible Tomato-Virulent Xcv InteractionA model for the signaling network in tomato in response to virulent Xcv is shown inFigure 4-1. In the interaction of tomato with Xcv the relationship of these phytohormonesappears to be linear, with SA production dependent on ethylene production that is in turndependent on jasmonate signaling (Lund et al., 1998; O'Donnell et al., 2001; O'Donnell etal., 2003).

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49 Figure 4-1 A model for the signaling network in tomato in response to virulent Xcv.As demonstrated in chapter 3, systemic signaling in tomato is dependent uponethylene and SA. Their action appears to precede their measurable induction, in a mannersimilar to JA. JA-deficient lines show a loss of ethylene despite the fact that measurableJA production does not occur until after ethylene production (O'Donnell et al., 2003).This suggests that early, local phytohormone productions can have a profound impact ondisease response.It is clear in the case of the interaction between virulent Xcv and tomato thatjasmonates are the major players when it comes to the formation of susceptibility to Xcv.At the local level the induction of ethylene and SA are required for the formation ofsecondary disease symptoms (O'Donnell et al., 2003). The interaction between tomatoand virulent Pst also requires a functional jasmonate-signaling pathway for full Pstvirulence. This system, however, is strongly influenced by the phytotoxin coronatine(Zhao et al., 2003).The Compatible Arabidopsis-Virulent Pst InteractionSA-deficient Arabidopsis plants produce increased levels of jasmonates andethylene in response to infection with virulent Pst. The prevalent hypothesis forphytohormones in the response of Arabidopsis to pathogens proposes two pathogen-response pathways in Arabidopsis (Glazebrook et al., 2003). One pathway utilizes SA

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50and acts in response to necrotic bacterial pathogens and the other utilizes ethylene andjasmonates, acting in response to fungal and insect pathogens (Penninckx et al., 1998;Vijayan et al., 1998).SA, ethylene and jasmonates are all produced in the interaction betweenArabidopsis and virulent Pst. However, SA limits the production of both ethylene andjasmonates and is the major phytohormone controlling defense responses in thisinteraction. SA represses bacterial growth and thus also the development of chlorosis andnecrosis. Ethylene represses symptom development and jasmonates appear to act byrepressing defense responses.These data suggest that co-ordinate signaling determines the overall response to thepathogen. A combination of SA and jasmonates determine the level of bacterial growthwhile ethylene influences the level of symptom development. It is likely to be the ratiosand timing of the various phytohormone inductions that control the plant responses. Pstuses this network to its advantage by producing the jasmonate mimic coronatine.The reduced virulence of coronatine-deficient Pst on SA-deficient and wild typeArabidopsis demonstrated in chapter 2, suggests that coronatines action as a virulencefactor is SA-independent. Further experiments to confirm this include the measurementof SA in plants infected with coronatine-deficient Pst. Similar SA levels of these plantsand those infected with wild type Pst would support SA-independent coronatine action.The link between jasmonates and susceptibility could also be investigated using T-DNA knock-out mutants in jasmonate biosynthesis genes. A good candidate for this isallene oxide cyclase for which there is a single gene in Arabidopsis (Kubigsteltig et al.,1999; Park et al., 2002). This mutant would be more appropriate than the fad3-2 fad7-2

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51fad8 as it is less likely to affect the levels of other metabolites. These experiments requirethe coronatine-deficient Pst mutant, as coronatine masks jasmonate-deficiency (Kloek etal., 2001). Changes in bacterial growth in these lines would demonstrate if jasmonates aredefinitively involved in repressing defense responses in Arabidopsis.Systemic Responses to InfectionSystemic responses to virulent and avirulent pathogens have been previouslydescribed in both tomato and Arabidopsis, with SA playing a similar role in both plants.In both systems pathogen infections cause a local increase in SA (Nawrath and Metraux,1999; O'Donnell et al., 2001). SA is necessary for the induction of a systemic response,however, it is not itself believed to be the transmitted signal (Vernooij et al., 1994;Lawton et al., 1995). The systemic induction of SA differs between the two systems, asArabidopsis has strong systemic SA induction where as tomato does not (Summermatteret al., 1995). Arabidopsis also shows a strong induction of SA upon challenge that doesnot occur in tomato (Jeun et al., 2000).Ethylene is not required for the induction of SAR in Arabidopsis (Delaney et al.,1994; Lawton et al., 1995). However, both ethylene and SA appear to be involved theinduction of SAT in tomato (Chapter 3). Virulent pathogens can induce systemicresponses in Arabidopsis, however, SAR is not induced by virulent Pst (Cameron et al.,1999). Both avirulent and virulent Xcv induce systemic responses in tomato.Additional experiments are required to confirm the roles of ethylene and SA in thegeneration of systemic signaling in tomato. One approach to this could be the use ofgrafting to determine if a NahG stock is incapable of systemic signaling to a wild type

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52scion. The best approach to this would be to monitor PR gene expression in the scion inresponse to an inoculation of the stock.Further study is required in both systems before the complex roles ofphytohormones in these interactions can be fully understood. It is clear from this studythat information gained from studying one system will not necessarily be applicable toanother system. However, certain parallels and common responses exist. By the study ofa wide range of plant-pathogen interactions the common points and differences can bedetermined. This will eventually lead to a more complete understanding of generaldefense responses, perhaps allowing broad-spectrum engineering or breeding of plantswith increased resistance or reduced symptoms to a wide range of diseases.

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53CHAPTER 5MATERIALS AND METHODSPlant Materials and TreatmentsArabidopsis thaliana Columbia, the NahG line (Delaney et al., 1994), npr1-1 (Caoet al., 1994) and sid2-2 (courtesy of Julia Dewdney), all in the Columbia background,were grown in soil under long night conditions (8-h day; 16-h night) for six weeks toencourage vegetative growth. Forty-eight hours prior to infection, plants were transferredto long day conditions (16-h day; 8-h night). Twelve hours before infection, the plantswere enclosed in a humidity dome to aid bacterial entry. Plants were inoculated with Pstby submerging the aerial parts of the plant in 1x107 cfu ml-1 bacterial suspension,containing 10mM MgCl2 and 0.02% (v/v) Silwet L-77 (Lehle seeds, Round Rock, TX,U.S.A.), for 30 seconds. A vacuum was then applied to the plants for 2 min to aidbacterial entry and the plants were returned to the humidity chamber overnight.Lycopersicon esculentum cvs Moneymaker and UC82B are the parental lines forNahG (Oldroyd and Staskawicz, 1998) and ACD (Klee et al., 1991) respectively. Wildtype refers to UC82B only in all experiments except PR gene analysis of ethylene andSA-deficient lines. Plant growth and treatments were performed under ambienttemperature and lighting in a greenhouse. Plants were inoculated by submersion of theleaves for 15 seconds in a bacterial suspension of 1x107 cfu ml-1 of Xcv strain 93-1(virulent) or 87-7 (avirulent) containing 10mM MgCl2 and 0.02% (v/v) Silwet L-77.Mock inoculations were performed by dipping plants in buffered Silwet. For analysis ofthe systemic response to infection, inoculations were performed on three week-old plants

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54by dipping leaves 1 and 2 in infection media. Challenges were then performed on thesame plant 14 days later by dipping leaves 5 and 6 in the infection media containingvirulent Xcv. For PR gene analysis leaves 1 and 2 of six week-old plants were infectedand tissue from the local infection (leaves 1 and 2) and the systemic response (leaves 5and 6) were removed at the indicated time points and flash frozen in liquid nitrogen.Bacterial CulturePst and Xcv cultures were grown as previously described by ODonnell et al.(2001). Leaf colony counts were determined as previously described by Lund et al.(1998). Briefly, five 1cm2 leaf disks were sampled from each line at each time pointindicated. The disks were ground in 10mM MgCl2, and serial dilutions were incubated atroom temperature for 2 days on solid media. The average colony-forming unit per squarecentimeter (cfu cm-2) for each sample was determined by counting individual colonies.Ion LeakageAmount of cell death was estimated by measuring percentage of ion leakage. At 16dpi the 5th leaf of each plant was placed in 6 ml deionized water and a vacuum of 20 psiapplied for 5 min. The samples were then shaken at room temperature for one hour. 3 mlof the water was then removed and its conductivity was measured. The samples werethen placed in a boiling water bath for an hour and the conductance of the remaining 3 mlof water measured. Percent ion leakage was determined by conductivity of first 3 mldivided by conductivity of second 3 ml multiplied by 100. Measurements of tissuechallenged with virulent Xcv were made from 30 plants per treatment. Measurements onmock challenges were made for 10 plants per treatment. Each experiment was repeatedon at least two independent occasions.

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55Ethylene MeasurementsEthylene measurements were performed on a minimum of three independent biologicalreplicates. Each biological sample was replicated at least three times within anexperiment. Ethylene production was determined by sampling the headspace above eithera whole rosette for Arabidopsis or a single leaf for tomato, enclosed in 5cm3 tubes for 1hour as described by Lund et al. (1998). Ethylene concentration in a 1 ml sample wasdetermined by a gas chromatography (Model 5890, Hewlett-Packard, Palo Alto, CA).SA, Jasmonate and Coronatine MeasurementsSA, jasmonates and coronatine were extracted from Arabidopsis tissue and SAfrom tomato tissue and derivatized using trimethylsilyl-diazomethane. The volatilemethyl esters were collected from the complex matrix using vapor phase extraction andquantified by isobutene chemicalionization gas-chromatography/mass spectrometry asdescribed in Schmelz et al. (2003). Each biological sample was replicated at least twicewith a minimum of two independent experiments.RNA ExtractionTotal RNA was extracted from 1.0g of tissue from each genotype per time pointwith (phenol:chloroform:isoamyl alcohol (PCI) (25:24:1 (v/v/v))) : extraction buffer (1%triisopropylnaphthalene-sulfonic acid (w/v); 6% p-amino salicylic acid (w/v); 0.1M TrispH 8; 50 mM EGTA; 0.1M NaCl; 1% SDS (w/v); 0.039% -Mercaptoethanol (v/v)) (1:1).The extraction mixture was homogenized with a polytron and incubated at 50C for 20min. The phases were separated and an equal volume of PCI added to the aqueous phase.The RNA was precipitated overnight at -80C following the addition of 2.5 volumes ofethanol and 1/10 volume of sodium acetate. The nucleic acids were pelleted bycentrifugation, (14,000 g for 30 min) resuspended in water and precipitated in 2M LiCl.

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56The LiCl precipitation was repeated and a subsequent sodium acetate/ethanolprecipitation performed.RNA Gel Blot AnalysisRNA gel blot analysis was performed using 20 g total RNA for each sample asdescribed by Kneissl and Deikman (1996). The RNA was transferred onto a chargedmembrane (Hybond-N+; Amersham Life Science inc., Arlington Heights, IL). The PR1(Genbank accession AY117187) and PDF1.2 (full length cDNA from gene at5g44420)probes were obtained from The Arabidopsis information resource (Ohio StateUniversity). DNA probes were random primer labeled with 32P with the Prime-It IIlabeling kit (Stratagene, La Jolla, CA).Real-time RT-PCRPR1a (TC117463) and PR1b (TC115947) mRNA levels were quantified by Real-time quantitative RT-PCR using Taqman" one-step RT-PCR reagents (AppliedBiosystems, Foster City, CA) and an Applied Biosystems GeneAmp 5700 sequence-detection system. Each determination was performed using 250ng of Dnase-1 treatedtotal RNA isolated using an RNeasy" Plant mini kit (Qiagen, Valencia, CA), in a 25lreaction volume. RT-PCR conditions were: 48C for 30 min, 95C for 10 min followedby 40 cycles of 95C for 15 sec and 60C for 1 min. Absolute mRNA levels werequantified using synthesized sense strand RNAs as standards. Primers and probes weredesigned using PRIMER EXPRESS software (Applied Biosystems) and were as follows:PR1b probe 5-/56-FAM/CAACGGATGGTGGTTCATTTCTTGCA/3BQH_1/-3; PR1aprobe 5-/56-FAM/TGTGGGTGTCCGAGAGGCCAGA/3BHQ_1/-3;PR1b forwardprimer 5-GGTCGGGCACGTTGCA-3; PR1b reverse primer 5-GATCCAGTT-

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57GCCTACAGGACAT-A-3; PR1a forward primer 5-GAGGGCAGCCGTGCAA-3;PR1a reverse primer 5-CACATTTTTCCACCAACACATTG-3 (Intergrated DNATechnologies, Coralville, IA). Each sample was a minimum of two biological replicatesand each experiment was repeated at least twice.MCP TreatmentMCP treatment was performed 24 hours prior infection as described in Ciardi et al(2000).

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65Vernooij B, Friedrich L, Goy PA, Staub T, Kessmann H, Ryals J (1995) 2,6-dichloroisonicotinic acid-induced resistance to pathogens without the accumulationof salicylic-acid. Mol Plant Microbe Interact 8: 228-234Vernooij B, Friedrich L, Morse A, Reist R, Kolditz-Jawhar R, Ward E, Uknes S,Kessmann H, Ryals J (1994) Salicylic acid is not the translocated signalresponsible for inducing systemic acquired resistance but is required in signaltransduction. Plant Cell 6: 959-965Vijayan P, Shockey J, Levesque CA, Cook RJ, Browse J (1998) A role for jasmonatein pathogen defense of Arabidopsis. Proc Natl Acad Sci U S A 95: 7209-7214Ward ER, Uknes SJ, Williams SC, Dincher SS, Wiederhold DL, Alexander DC,Ahl-Goy P, Metraux JP, Ryals JA (1991) Coordinate gene activity in response toagents that induce systemic acquired resistance. Plant Cell 3: 1085-1094Weiler EW, Kutchan TM, Gorba T, Brodschelm W, Niesel U, Bublitz F (1994) ThePseudomonas phytotoxin coronatine mimics octadecanoid signalling molecules ofhigher plants. FEBS Lett 345: 9-13Wildermuth MC, Dewdney J, Wu G, Ausubel FM (2001) Isochorismate synthase isrequired to synthesize salicylic acid for plant defence. Nature 414: 562-565Xie DX, Feys BF, James S, Nieto-Rostro M, Turner JG (1998) COI1: An Arabidopsisgene required for jasmonate-regulated defense and fertility. Science 280: 1091-1094Yamamoto S, Katagiri M, Maeno H, Hayaishi O (1965) Salicylate hydroxylase, amonooxygenase requiring flavin adenine dinucleotide. I. Purification and generalproperties. J Biol Chem 240: 3408-3413Zhao Y, Thilmony R, Bender CL, Schaller A, He SY, Howe GA (2003) Virulencesystems of Pseudomonas syringae pv. tomato promote bacterial speck disease intomato by targeting the jasmonate signaling pathway. Plant J 36: 485-499Zhou N, Tootle TL, Tsui F, Klessig DF, Glazebrook J (1998) PAD4 functionsupstream from salicylic acid to control defense responses in Arabidopsis. Plant Cell10: 1021-1030

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66BIOGRAPHICAL SKETCHAnna Block was born on the 13th of May 1978 in Nottingham, England. Shecompleted her GCSEs and A levels at John Mason School in Abingdon, Oxfordshire, in1994 and 1996 respectively. She then went on to receive her masters degree inbiochemistry from the University of Bath in 2000. During her masters degree sheaccomplished two 6-month internships. The first internship was in 1998 for Rhone-Poulenc in Essex and the second in 1999 in the laboratory of Harry Klee at the Universityof Florida. She returned to the University of Florida in 2000 for her doctoral studies inthe plant cellular and molecular biology program