INFORMATION TO USERS · SAR 4.1 The relationship between SA and cell death 28 4.2 The role of cell death in the immunization and 29 establishment stages of SAR 4.3 Summary of the

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ACTIVE OXYGEN SPECIES ACCUMULATION IN THE IMMUNIZATION AND MANIFESTATION STAGES OF SYSTEMIC ACQUIRED RESISTANCE DURING AN

ARABIDUPSIS THALIANA-PSEUDOMONAS SYRINGAE PV TOMA TO INTERACTION

Carolyn Jamie Hutcheon

A thesis submitted in conformity with the requirements for the degree of Master of Science

Graduate Department of the Department of Botany University of Toronto

Q Copyright by Carolyn Jamie Hutcheon 1998

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Active Oxygen Species Accumulation in the Immunization and Manifestation Stages of Systemic Acquired Resistance during an

Arabidopsis thaliana- Pseudomonas syringae pv tomato l n te ract i o n

Degree of Master of Science, 1998

Carolyn Jamie Hutcheon

Department of Botany University of Toronto

Abstract

Active oxygen species (AOS) accumulation was studied in the signalling

and manifestation stages of the systemic acquired resistance (SAR) response in

the Arabidopsis thaliana-Pseudornonas syringae pv tomato pathosystem.

During the manifestation of SAR, AOS do not accumulate to a magnitude

comparable to that observed following inoculation with avirulent Pst.

lmmunization of plants with the glucose/glucose oxidase (G/GO) AOS

generating system resulted in the establishment of SAR, but did not result in cell

death, suggesting an involvement of AOS, but not necessarily of cell death, in

SAR signalling. In response to avirulent bacteria, the SAR mutants nprl and

dirl demonstrated AOS accumulation comparable to that observed in wild type

Arabidopsis, but salicylic acid (SA) deficient NahG Arahiclopsis did not.

Immunization of NahG plants with G/GO or avirulent Pst did not result in the

induction of pathogenesis-related proteins, nor did it result in cell death,

suggesting a dependence in both of these processes on SA.

Acknowledgements

I would like to thank Dr. Robin Cameron for giving me the opportunity to work in her lab. I am also grateful to Dr. Michele Heath and Dr. Peter McCourt for giving me direction and suggestions . My appreciations to Alice, Lu, and Dr. Verna Higgins for sharing their knowledge with me, and to the members of the Cameron lab for interesting, and often unusual conversations. I would like to thank Hannah Parsons, Jacquie Bede, Steve Mezyk, Andrea Gilpin, and Siobhan Brady for help with proof reading. To NSERC for providing me with the funding for this research.

Thanks to Andrea (for her guidance, encouragement, and for knowing the good camping spots), to Camille (for her guidance and support at a difficult time), to Nocha (for advice that I wish I was better at following and because she knows how to make a wicked shepard's pie), to Michelle (because she knows which side of a leaf is abaxial), to the Malloch lab (for evenings of Mari Mac), to Pamy (for keeping me running), and especially (especially) to Jacki (because she knows the seven stages of a time course and because she thinks that a bicycle can fit through a TTC turn style.)

I am very grateful to Hannah and Jeff, for putting up with me and for making me laugh.

Finally, I would like to thank my family, for their love, advice, encouragement, and perspective.

iii

Table of Contents

Section

Abstract Acknowledgments Table of Contents List of Abbreviations List of Figures List of Tables

Introduction The gene-for-gene response The Arabidopsis thaliana(Rps2)-Pseudomonas syringae pv tomato(avrRpt2) gene-for-gene system

The hypersensitive response Defence responses associated with the hypersensitive response

(a) Strengthening of physical barriers (b) PR protein expression

Systemic acquired resistance Defence responses correlated with the establishment and manifestation stages of SAR

(a) Strengthening of physical barriers (b) PR protein expression (c) Salicylic acid

SAR mutants in Arabidopsis (a) nprl (b) dirl (c) NahG

Active oxygen species accurnulation Chemistry of active oxygen species Detection and generation of AOS

(a) Detection of AOS (b) Generation of AOS

Roles of active oxygen species in resistance responses

(a) Antimicrobial effects of AOS (b) Strengthening of physical barriers (c) Involvement of AOS in the SAR signalling

pathway (d) Involvement of AOS in cell death

Page

i i iii iv vii ix xi

1 2 6

7 8

9 10 I I 14

14 15 15 17 17 18 18 18 19 23 23 23 24

24 25 25

27

4. A possible relationship between SA, cell death, and 28 SAR

4.1 The relationship between SA and cell death 28 4.2 The role of cell death in the immunization and 29

establishment stages of SAR 4.3 Summary of the possible relationship between 29

AOS, SA, cell death, and SAR 5. ResearchAims 30 5.1 To characterize the manifestation stage of SAR 30

with respect to AOS accumulation and the expression of proteins thought to be involved to be involved in defence responses

5.2 To characterize the role of AOS in the immunization 30 stage of SAR

Materials and Methods 32 1. Plant growth conditions 33 2. Bacterial growth conditions 33 3. Induction of SAR 34 4. Quantification of bacteria 34 5. Qualitative hydrogen peroxide assay 34 6. Quantification of fluorescence 35 7. Use of the oxygen electrode to measure hydrogen 35

peroxide production 8. Exogenous application of the g lucose/glucose 36

oxidase hydrogen peroxide generating system 9. Determination of cell viability 37

(a) Chlorophyll autofluorescence 37 (b) FDA staining 37

10. Statistical analysis 38 1 1. Preparation of RNA from plant tissue 38 12. Northern analysis 39 13. Preparation of radiolabelled DNA 39

Results 42 1 . Characterization of eli gene expression and 43

AOS accumulation in the manifestation stage of SAR

1 .I Expression of eli I I , eli 18, and ap3 in the 43 establishment and manifestation stages of SAR

1.2 Hydrogen peroxide accumulation in leaves 52 manifesting SAR

1.3 Quantitative approaches to AOS detection 70 1.4 Use of the oxygen electrode for the quantification 70

of H202 2. Characterization of AOS accumulation in the 73

immunization stage of SAR AOS accumulation in mutants unable to establish 73 SAR

(a) dirl 78 (b) nPrl 78

AOS in the immunization stage of SAR 91 AOS induced cell death in the immunization stage 96 of SAR AOS accumulation, PR protein expression, and cell 97 death in NahG Arabidopsis AOS accumulation in NahG Arabidopsis in response 97 to Pst and G/GO

Cell death in NahG Arabidopsis in response to Pst 1 10 GIGO PR gene expression and bacterial growth in NahG 1 10 plants in response to Pst and G/GO

Discussion 115 1. Characterization of the manifestation stage of SAR 116 1 .I Expression of eli I I, eli 18, and ap3 in the 115

manifestation stage of SAR 1.2 AOS in the manifestation stage of SAR 118 2. AOS accumulation and cell death in the 120

immunization stage of SAR

2.1 AOS accumulation in mutants defective in the 121 establishment and manifestation of SAR

3. AOS accumulation and cell death in nahG 122 A ra bio dupsis

3.1 Cell death and SA accumulation 124 4. Different signalling pathways may result in the 125

establishment of SAR

References 127

List of Abbreviations

3-AT 4CL A acd AOS a vr cfu CHS Col-0 DCFH-DA DCFH DCF dir dnd DPI ELI FDA FW G/GO GRP HR HRGP hrp INA Isd M MS MOPS NAD(P)H NahG ndr *Pr OD pad PAL PR Psm psg Pst PV RPM SA SAM SAR

3-amino-l,2,4-triazole 4-coumarate CoA-ligase treatment with avirulent Pst accelera fed cell death active oxygen species avirulence gene colony forming units chalcone synthase Arabidopsis thaliana ecotype Columbia 2',7'-dichlorofluorescin diacetate 2',7'-dichlorofluorescin 2',7'-dichlrorfluorescein defective in induced resistance defence no death diphenylene iodinate elicitor induced Fluorescein diacetate fresh weight glucose/glucose oxidase glycine rich protein hypersensitive response hydroxyproline rich glycoprotein hypersensitive response and pathogenicity 2,6-dichloroisonicotinic acid lesion simulating disease resistance treatment with MgC12 Murishige and Skoog 3-[N-rnorpholino]propanesulphonic acid nicotinamide adenine dinuclotide phosphate salicylate hydroxylase non-specific disease resistance non-expressor of PR genes optical density phytoalexin deficient phenylalanive ammonia-lyase pathogenesis related Pseudomonas syringae pv maculicola Pseudomnas syringae pv glycinea Pseudomonas syringae pv tomato pathovar rotations per minute salicylic acid S-adenosyl- L-methionine systemic acquired resistance

SMS SOD SSC TMV uv v ws WXO

S-adenosyl-L-methionine synthase superoxide dismutase sodium chloride/sodium citrate solution tobacco mosaic virus ultraviolet treatment with virulent Psf Arabidopisis thaliana ecotype Wassilewskija xanthine/xanthine oxidase

viii

List of Figures

Figure

Figure 1 Figure 2 Figure 3

Figure 4

Figure 5

Figure 6

Figure7 Figure 8

Figure 9

Overview of plant defence responses Overview of stages of SAR Proposed deacetylation of DCFH-DA by cellular esterase to DCFH and oxidation of DCFH by Hz02 mediated by cellular peroxidases Northern analysis of total wild type and dirl RNA probed with ELI 18 cDNA Northern analysis of total wild type and dirl RNA probed with ELI 11 cDNA Northern analysis of total wild type and dirl RNA probed with AP3 cDNA Bacterial growth in wild type and dirl Arabisdopsis Ranking scale of relative In planta fluorescence of DCF under UV light AOS accumulation during the manifestation of SAR in ecotype Col-0, as obsetved from 7 to 16 hours after inoculation

Figure 10 ln planta fluorescence of DCF under UV light 57 Figure 11 AOS accumulation during the' 59

manifestation stage of SAR in ecotype Col-0, as observed from 7 to 23 hours after inoculation

Figure 12 Bacterial growth in Col-0 Arabidopsis 61 Figure 13 Bacterial growth in Col-0 Arabidopsis 63 Figure 14 Relative Fluorescence in leaves with 66

exogenously added H202 Figure 15 AOS accumulation as measured by 68

fluorometry during the manifestation stage of SAR in Co 1-0 Ara bidopsis

Figure 16 AOS accumulation during the 71 immunization stage of SAR in dirl Arabidopsis

Figure 17 Bacterial growth in nprl Arabiodopsis 74 Figure 18 AOS accumulation during the 76

immunization stage of SAR in nprl Arabidosis Figure 19 AOS accumulation during the 81

immunization stage of SAR in ecotype Col-0 following exogenous addition of the glucose/glucose oxidase Hz02 generating system

Figure 20 AOS accumulation during the 83 immunization stage in ecotype Col-0 failing to establish SAR following exogenous addition of G/GO Hz02 generating system

Figure

Figure Figure

21 AOS accumulation in ecotype Col-0 following exogenous addition of G/GO

22 Bacterial growth in ecotype Col-0 23 Northern analysis of wild type leaf RNA with

PR-1 cDNA Figure 24 Cell death measured by FDA fluorescence or

chlorophyll autofluorescence Figure 25 Cell death in ecotype Col-0 following infection

with Pst or treatment with G/GO Figure 26 Chlorophyll autofluorescence in ecotype Col-0

leaf mesophyll under blue light Figure 27 Cell death in the immunization stage in ecotype

Col-0 failing to establish SAR following infection with Psi or treatment with WGO

Figure 28 AOS accumulation during the immunization stage of SAR in NahG Arabidopsis

Figure 29 AOS accumulation in the immunization stage of SAR in NahG Arabidopsis following exogenous addition of G/GO

Figure 30 Cell death in NahG Arabiodopsis following infection with Psi or treatment with G/GO

Figure 31 Chlorophyll autofluorrescence in nahG transgenic leaf mesophyll under blue light

Figure 32 Northern analysis of wild type and nahG leaf RNA with PR-1 cDNA

Figure 33 Bacterial growth following infection with Psi

List of Tables

Table Page

Table 1 Probes used for northern analysis 40 Table 2 Summary of experiments involving G/GO 79

induction of AOS, cell death, pr gene expression and inhibition of bacterial growth

Introduction

Plants face an onslaught of environmental stresses, including attack by a large

variety of fungal, bacterial, and viral pathogens. Consequently, plants have evolved

several modes of resistance against these pathogens. Perhaps the most prevalent

form of defence is non-host resistance, which consists of mechanisms that allow a

plant to repel a majority of pathogens. However, for any given plant species or

cultivar, there exists a strain of pathogen which has evolved to overcome that species'

non-host resistance mechanisms. In response to these specialized pathogen strains,

plants have developed a gene-for-gene resistance response: a resistance response

in which a specific cultivar of plant responds to a specific strain of pathogen that is not

repelled by non-host resistance mechanisms. A gene-for-gene interaction will result in

localized responses in the plant. Occasionally, it will initiate a signalling cascade,

ultimately resulting in systemic acquired resistance (SAR), a broad spectrum

resistance response, throughout the plant and lasting for extended periods of time.

Interestingly , despite differences in the mode in which these varied resistance

responses occur, the mechanisms which constitute the response are similar, if not the

same, and include such things as the strengthening of cell wall barriers, and

expression of pathogenesis related (PR) proteins (figure 1).

1. The gene-for-gene response

Faced with a potential pathogen, a plant may defend itself in a number of

ways. Perhaps the most common forms of defence are the mechanisms involved in

non-host resistance. These include physical barriers such as the cuticle, lignin and

suberin in the cell wall, and pectic substances, as well as toxic substances such as

phenolic and alkaloid phytoalexins, small molecular weight antimicrobial compounds

(Heath 1991). The defence tactics of any given plant species or cultivar are invariably

Figure 1

Overview of plant defence responses.

SAR -SA accumulation -PR proteins expressed -strengthening of cell walls

interaction -HR -strengthening of cell walls -PR proteins expressed -AOS accumulate -SA accumulates

-b

Non host resistance -strengthening of cell walls -production of toxic compounds -frequently an HR

with Plant with R gene Non host pathgen avirulence gene

overcome by a small subset of pathogens, which will consequently become

"compatible", causing disease in that species or cultivar. This in turn leads to

adaptations by the plant to develop further defence responses to limit pathogen

growth, thus making the pathogen once again "incompatible" (Flor 1971, Jackson &

Taylor 1 996). These responses, built upon underlying non-host resistance responses,

are often mediated through a gene-for-gene mechanism, in which an avirulence gene

product from the pathogen interacts with a resistance gene product in the plant,

allowing the plant to "recognize" the pathogen and mount a defence response. If

either the avirulence gene or the resistance gene is lacking, the onset of disease will

result (Staskawicz et a1 1995).

Plant resistance genes have been proposed to act as receptors for avirulence

gene products (Keen 1990, de Wit 1992). This idea has been substantiated by in vivo

and in vitro evidence that the Pto resistance gene product of Lycopersicon esculentum

(tomato) interacts directly with the avrPto avirulence gene product of Pseudomonas

syringae pv tomato (Pst) (Tang et a1 1996, Scofield et a1 1996). The Pto gene,

however, does not resemble a number of the resistance genes that have since been

cloned: Pto has been shown to have serine-threonine kinase activity, indicating that it

may initiate a phosphorylation cascade which functions to transduce the signal for the

transcription of genes involved in resistance (Martin et a! 1993), whereas resistance

genes such as Resistant to Pseudomonas syringae (Rps2) of Arabidopsis are

predicted to contain an N-terminus leucine zipper motif, a nucleotide binding site, and

leucine rich repeats, suggesting that protein-protein interactions are involved.

Besides the regions of recognized homology described above, resistance genes

contain small, distinct domains of homology to one another, the function of which is

unknown (Bent 1996).

The finding to date that a majority of resistance gene products are cytoplasmic

appears at first to counter the hypothesis that plants have membrane receptors that

recognize and interact with secreted pathogen avirulence gene products. Studies of

avirulence gene products suggest that the receptor-avirulence gene product

interaction may be occurring cytoplasrnically. Avirulence gene products are generally

hydrophillic and contain no signal sequence, suggesting (1) that the interaction

between avirulence and resistance gene products is not occurring at the plant-

pathogen cellular interface, and (2) that the pathogen avirulence gene product is not

secreted into the apoplast (Alfano and Collmer, 1996). The discovery of the

hypersensitivity response and pathogenicity (hrp) locus in several strains of bacteria

revealed a set of genes important for both pathogenicity and for gene-for-gene

resistance (Huang et a1 1992). These genes share homology to genes responsible for

type I I I secretion systems of such bacteria as Salmonella, Shigella, and Yersinia

(Lamb 1996). This evidence generated the proposal that the avirulence gene product

is secreted directly into the plant cell through a pilus-like pore, followed by a

cytoplasmic interaction between the plant resistance gene and the pathogen

avirulence gene products (Tang et al1996, Scofield et a1 1996).

1 .I The Arabidopsis thaliana (Rps2)-Pseudomonas syringae pv tomato

(avrRpt2) gene-for-gene system

Arabidopsis thaliana has become a model organism for genetic and molecular

research (Estelle and Sornmerville 1986). Use of Arabidopsis has facilitated genetic

dissection of plant-pathogen interactions: its small genome size and well studied

genetics allow for the cloning of resistance genes with far greater ease than is

possible in the majority of crop plants (Whalen et a1 1991). Furthermore, because

Arabidopsis has become a model organism for many aspects of plant growth and

development, use of Arabidopsis in the study of plant pathology should allow for an

increased understanding of how other aspects of development relate to a plant's

ability to defend itself against an onslaught of pathogens. Several gene-for-gene

systems have been developed in Arabidopsis, among them the avrRpt2lRps2 system

developed by Whalen et a1 (1 991). The avrRpt2 avirulence gene was isolated by

determining variation in pathogenicity among several pathovars of the gram negative

bacterium Pseudomonas syringae pv tomato (Pst), a well characterized pathogen

causing bacterial speck disease in tomato (Cuppels 1986). An Arabidopsis mutant

susceptible to Pst containing avrRpt2 was then isolated (Whalen et a1 1991) and the

mutant and wild type alleles of the resistance gene Rps2 were cloned by chromosome

walking (Bent et a1 1994). Upon interaction between the resistance gene product and

the avirulence gene product, stimulation of a signal transduction pathway likely

occurs, resulting in a hypersensitive response approximately 16 hours later

(Hammond-Kosack and Jones 1996).

1.2 The hypersensitive response

A gene-for-gene interaction will result in a series of inducible defence

responses. The most common, and seemingly ubiquitous, is the hypersensitve

response (HR), in which host cells die in a localized region surrounding the site of

pathogen invasion, and which has been correlated with the limitation of pathogen

growth (Jackson and Taylor 1996, He 1996). In biotrophic relationships, the HR is

thought to result in the starvation of the pathogen. In necrotrophic or hemibiotrophic

relationships, it is possible that death of the cell releases toxic substances from the

vacuole, thus killing the pathogen (Hammond-Kosack and Jones 1996). There is

debate about whether the HR is a programmed cell death response, or death resulting

from pathogen infection (Dangl et a1 1996). The discovery of a series of "lesion mimic"

mutants in Zea mays (maize) and Arabidopsis suggest that the HR may be under

genetic control and is therefore programmed. Arabidopsis lesion simulating disease

(Isd) (Dietrich et a1 1 994) and gccelerated cell death (acd) (Greenberg et a1 1 994)

mutant plants appear to mimic an HR defence response in the absence of a pathogen.

Inoculation of lsd or acd mutant plants with virulent Pseudomonas syrhgae pv

maculicola (Psm) results in a reduction in pathogen growth in comparison to that

observed in wild type plants, and in some cases lesions which surpass the boundaries

of pathogen spread, suggesting that the HR-like lesions are not directly resulting from

pathogen infection.

The HR does not appear to be necessary for resistance. It has not been

observed in the potato Rx gene-for-gene interaction (Hammond-Kosack and Jones

1996), nor is it seen in the defense _no death (dnd) Arabidopsis mutants, which show

resistance to avirulent and virulent Pst without detectable signs of an HR (Yu et a1

1998). Conversely, the presence of an HR does not necessarily result in resistance:

The Don race specific disease resistance (ndrl) mutant of Arabidopsis is susceptible

to Pseudomonas syringae despite an apparent HR (Century et a1 1 995). The HR

therefore does not appear to be directly correlated with resistance.

1.3 Defence responses associated with the hypersensitive response

A number of defence responses are associated with the HR: active oxygen

species accumulate (discussed in section 3), salicylic acid (SA) is synthesized

(discussed in section 24, the cell wall is strengthened through lignin deposition and

glycoprotein crosslinking, and pathogenesis-related (PR) proteins accumulate.

(a) Strengthening of physical barriers

The strengthening of cell wall components is thought to play a role in plant

defence by limiting penetration of a pathogen into the plant tissue. A number of

processes believed to be involved in the strengthening of the cell wall have been

studied, including the crosslinking of hydroxyproline rich glycoproteins (HRGP) and

glycine rich proteins (GRP), and lignification.

Hydroxyproline rich glycoproteins (HRGP) are important structural components

of the cell wall. Interacting with polysaccharide and polyphenolic cell wall components

(Hammond-Kosack and Jones 1996), they contribute to strengthening of the cell wall

in both developmental processes and in response to inducible stimuli such as

pathogen infection or wounding in soybean or bean cell cultures (Bradley et a1 1992,

Sticher et a1 1997). The role of HRGP and GRP in plant defence responses have not

been fully characterized. Overexpression of a GRP in tobacco does not increase

resistance to tobacco mosaic virus (Linthorst et a1 1989). A possible explanation for

this finding is that the role of HRGP and GRP in the creation of structural barriers is

dependent, not on the amount of HRGP or GRP present, but on the structure of these

proteins: in response to pathogen elicitation, de novo synthesis of HRGP occurs

relatively slowly, while oxidative crosslinking by isotyrosine linkages within the HRGP

or GRP is rapid (Hammond-Kosack & Jones 1996). The formation of isotyrosine

linkages has been proposed to result in the immobilization of these proteins (Bradley

et a1 1992) which in turn results in a decrease in cell wall digestibility (Brisson et a1

1994). HRGP and GRP have also been proposed to be the initiation site for the

polymerization of lignin (de Oliveira et a1 1990).

Lignin results from the polymerization of phenylpropanoid subunits (Collinge

and Slusrenko 1 987), including coniferyl alcohol, para-coumeryl alcohol, and sinapyl

alcohol (Whetten and Sederoff 1995). These monomers are oxidized to phenoxy

radicals by either phenol oxidase or a cell wall bound peroxidase, and will polymerize

in what is thought to be random fashion (Baker and Orlandi 1998, Whetten and

Sederoff 1995). Lignin is predominantly involved in the strengthening of cell walls;

however, coniferyl alcohol is toxic in vitro and may have an antimicrobial function

(Hammerschmidt et al1982).

(b) PR protein expression

Pathogenesis related (PR) proteins have been defined as proteins that

accumulate in intact plant tissue (or cell culture) following attack by a pathogen or

treatment with an elicitor (Hammond-Kosack & Jones 1996). Acidic PR proteins are

usually secreted, while basic PR proteins are sequestered in the vacuole, with the

exception of PR-1 a, which is secreted (Hammond-Kosack & Jones 1996). Acidic PR

proteins have been divided into 5 classes: classes one and four have an unknown

function, class three are chitinases, class two are 0-1,3-glucanases, and class 5 are

thaumatin-like proteins and have amylase and proteinase inhibiting activities in maize

(Ward et a1 1991). Basic forms of PR-1, PR-2, and PR-3 have also been discovered;

however, they are usually only functionally, and not structurally, homologous to the

proteins in the acidic families (Ward et a/ 1991). Chitinases and 0-1,3- glucanases are

both effective antimicrobial agents, degrading fungal cell walls and lysing bacteria in

vitro; however it is not known whether they also act against bacterial or viral

pathogens, nor how the other three classes of PR proteins might act. A synergistic

increase in antimicrobial activity has been observed in transgenic Nicotiana tabacum

(tobacco) when PR-2 and PR-3 are ectopically expressed together (Zhu et a1 1 994b).

Overexpression of bean chitinase (PR-3) in tobacco has been shown to result in

increased resistance to the fungal pathogen Rhizoctonia solani (Broglie et a1 1 99 1 ). In

contrast, ectopic expression of PR-I in transgenic tobacco does not result in

increased resistance to tobacco mosaic virus (Linthorst et a1 1989). These varied

results might be explained by differing roles of PR-I , PR-2, and PR-3, or by the

different pathogens used: PR proteins may be predominantly effective against fungal

pathogens.

Besides the five classes of PR proteins, expression of other proteins is induced

following elicitation with a pathogen. A series of Elicitorjnduced (ell) genes have been

isolated from both Petroselinum crispum (parsley) and Arabidopsis (Trezzin i et a1

1993). These include HRGP (ell 9), peroxidases (eli 1 1 and ap 3), and tyrosine

decarboxylase (eli5). The phenylpropanoid metabolic pathway is involved in the

synthesis of SA, phytoalexins, and lignin. Enzymes involved in the phenylpropanoid

metabolic pathway, including phenylalanine ammonia lyase (PAL), Ccoumarate Co-A

ligase (4CL), and chalcone synthase (CHS) are upregulated following elicitation

(Collinge and Slusarenko 1987, Treuini et a1 1993, Wanner et a1 1995). Interestingly,

the eli genes 14, 18, 19 are involved in the activated methyl group cycle of amino acid

catabolism, suggesting that primary metabolism is also influenced by plant defence

responses (Somssich and Hahlbrock 1998).

2. Systemic acquired resistance

In addition to non-host and gene-for-gene resistance responses, plants have

the ability to establish systemic acquired resistance (SAR), in which an initial

immunization with certain pathogens, leads to systemic, broad spectrum resistance

throughout the plant (Ryals et a1 1995). Four stages are thought to be involved in the

establishment of SAR: (1) the immunization stage involving an initial infection by

certain pathogens (often necrotizing), (2) the signalling stage in which a phloem

mobile SAR signal travels throughout the plant (Malamy et a1 1990, Metraux et a1

1990, Shulaev eta/ 1995), (3) the establishment stage in which the systemic leaves

perceive and respond to the SAR signal, and (4) the manifestation stage which occurs

Figure 2

Ovewiew of the stages of SAR.

Details in text.

4. Manifestation

3. Establ

1 .Immunization

upon challenge by a normally virulent pathogen (figure 2). SAR has been observed in

a diversity of plants, including Cucumis sativus (cucumber) and Cucumis meio

(muskmelon; Kuc, 1982), tobacco (Ward eta1 1991), and Arabidopsis (Uknes et ai

1992, Cameron et a1 1994). The timing and level of immunity varies with the plant

species and inducer: for instance, cucumber infected with Pseudomonas syringae

will establish SAR after 7 hours, whereas SAR will only be established 2-3 weeks after

inoculation of tobacco with Peronspora parasitica pv tabaci (Sticher et a1 1997). The

duration of SAR also varies, ranging from weeks to months (Ward et al 1991).

2.1 Defence responses correlated with the establishment and manifestation of

SAR

(a) Strengthening of physical barriers

Several defence responses have been correlated with the establishment of

SAR, including the strengthening of cell walls (described in section 1.3(a)). Elevated

levels of GRPI transcription can be seen in Arabiodpsis following treatment with

salicylic acid (SA), an inducer of SAR (de Oliveira et ai 1990). Lignification has also

been detected during the SAR response. An increase in lignin accumulation has been

observed in immunized cucumber leaves challenged with the fungus Colletrichum

lagenarium at levels comparable to those seen in gene-for-gene resistance

(Hammerschmidt and Kuc 1982). An increase in peroxidase activity has also been

observed to correspond with the induction of SAR in cucumber (Hammerschmidt et a1

1 982).

(b) PR proteins

SAR has also been correlated with the transcription of what have been termed

"SAR genes". In tobacco, SAR genes are expressed in systemic leaves 6 days after

immunization, correlating with the initiation of immunity (Ward ef al1991). These

genes have been divided into nine gene families, five of which correspond to the five

families of PR proteins (described in section 1.3(b)). The remaining four correspond

to basic forms of these proteins (Ward et al1991). PR protein expression can

therefore be used as a indicator that SAR has been established. Upon immunization,

PR mRNA expression can be detected in both inoculated leaves and in systemic

leaves. In Arabidopsis the expression of PR-1 in leaves infected with Pst(avrRpt2) is

relatively strong one day after inoculation, and reaches a maximum 4 days after

inoculation. Two days after infection, expression can be observed in systemic leaves

(Alvarez et a1 1998). In contrast, leaves inoculated with virulent Pst begin to express

PR-1 mRNA approximately two to three days after inoculation and usually only in the

inoculated leaves. In leaves manifesting SAR, PR gene expression patterns are

similar to those of leaves inoculated with avirulent pathogens. PR proteins therefore

appear to play a role in both the establishment (stage 3) and manifestation (stage 4)

of SAR (Cameron unpublished).

(c) Salicylic acid

The signal which results in the establishment of SAR is unknown. Perhaps the

best candidate for this signal is salicylic acid (SA). A strong correlation has been

observed between SA accumulation and the immunization stage of SAR. Application

of exogenous SA to tobacco induces SAR gene expression (Malamy et a1 1990).

Tobacco and Arabidopsis plants transformed with the bacterial salicylate hydroxylase

gene (NahG) which hydrolyzes SA, are unable to mount a SAR response (Delaney et

ai 1994). SA accumulates in the phloem of tobacco 20 fold over basal levels after 42

to 48 hours following infection with tobacco mosaic virus (TMV; Malamy et a1 1990)

and in cucumber phloem to up to 10 times upon infection with either TMV or

Colletotichum lagenarium (Metraux et al 1990). SA also appears to accumulate in

systemic leaves five to 10 times basal levels approximately 48 hours after

immunization of tobacco, and these levels can remain elevated for up to one week

(Malamy et a1 1990). Labelling experiments suggest that up to 70% of SA which

accumulates in uninfected leaves after the induction of SAR is translocated from

infected leaves, suggesting that SA is the mobile SAR signal (Metraux et a1 1990,

Shulaev et a1 1995).

There is evidence against the role of SA as the SAR signal. In cucumber, when

an immunized leaf is removed after 6 hours, SA does not accumulate in the phloem,

although SAR is induced (Rasmussen et a1 1991). Grafting experiments in tobacco

support this finding. If a NahG scion is grafted to a wild type rootstock, immunization

of the rootstock with TMV followed by challenge of the scion with either TMV or

Cerospora nicotanea does not result in the manifestation of SAR. However, if a wild

type scion is grafted to a NahG rootstock, SAR is established, suggesting that the

accumulation of SA above basal levels is not required in the immunization stage of

SAR, although it is necessary in the establishment stage (Vernooij et al1994).

Although these experiments do not support the hypothesis that SA is the translocated

SAR signal, neither do they completely disprove it. Low levels of SA are present in

NahG plants. Labelling of SA with 180 suggests that approximately 0.71 pg/g FW is

translocated from immunized leaves to sytemic leaves (Shulaev et a1 1995). NahG

plants retain basal levels of SA at approximately 0.3 pg/g FW (Shulaev et al1995,

Vernooij et a1 1994). It is therefore possible that 0.3 pg/g FW is sufficient for

production of the SAR signal in tobacco. Regulation of signalling may occur by

sequestering of SA in the chloroplast (Vernooij et a1 1994); SA may be involved in

SAR signalling upon release into the cytosol.

2.2 SAR mutants in Arabidopsis

The precise mechanisms involved in the signalling, establishment, and

manifestation of SAR are unknown. In an attempt to elucidate this pathway, a genetic

approach has been taken, resulting in the isolation of several mutants which are

altered in their ability to establish SAR. Among these mutants are Don expressor of

PR genes (npr) and defective in induced ~esistance (dir). Plants defective in the -

ability to establish SAR have also been generated by transforming Arabidopsis and

tobacco with the NahG gene, which hydrolyzes SA.

Arabidopsis mutants in Non expressor of PR genes (Npr) were isolated in a

screen for plants which do not express PR-2 in the presence of a SAR inducer, the SA

analogue 2,6-dichlororisonicotinic acid (INA) (Cao et a1 1994). Several alleles of nprl

have been isolated in different screens, including Don inducible bmuni ty (nim 1;

Delaney et al1994) and the ~alicylicpcidjnsensitive mutant sail (Shah et a1 1997).

Mutant nprl plants demonstrate reduced expression of PR-1 ,and PR-5, as well as PR

-2 in inoculated and systemic leaves. They accumulate SA, but cannot establish SAR

following treatment with INA, indicating that the lesion lies downstream of SA

accumulation. Infection of niml plants with avirulent Perenospora parasitica results in

a disease response (Delaney et a1 1994). Interestingly, a similar infection with

Psm(avrRpt2) results in the formation of a typical HR, although SAR is not

established (Cao et a1 1994).

(b) dirl

The defective in induced_resisfance (dirl) gene was isolated in a screen for

Arabidopsis plants unable to limit virulent Pst growth following immunization. These

mutants retain a functional HR in response to Pst(avrRpt2) and Psm(avrRpml), do not

accumulate SA, and can establish SAR following treatment with INA, suggesting that

the lesion lies upstream of SA accumulation. PR-1 gene expression is absent in

systemic leaves following immunization of dirl plants, yet is present in both the HR

and disease response, suggesting that the dirl lesion is specific to the SAR response

pathway (Cameron et a1 unpublished).

(c) NahG

Both tobacco and Arabidopsis have been transformed with the bacterial

salicylate hydroxylase (NahG) gene, a flavoprotein which catalyzes the conversion of

salicylate to catechol (Gaffney et a1 1993, Delaney et a1 1994). This results in a line of

plants which are unable to accumulate SA, although basal levels remain much the

same as in wild type. NahG transgenic plants are unable to mount an HR, and are

more susceptible to virulent pathogens (Delaney et a1 1 994), suggesting that SA is

required in both disease and resistance responses. NahG transgenic plants are

unable to establish SAR (Delaney et a1 1994).

3. Active oxygen species

Accumulation of active oxygen species (AOS) has been observed in plants in

response to infection by a variety of bacterial and fungal pathogens. It was first

documented in potato tubers, which, when inoculated with incompatible Phytopthera

infestans, demonstrated an increase in superoxide radicals. This increase was not

detectable when the tubers were inoculated with a virulent race, and could be inhibited

by superoxide dismutase enzymes, which convert superoxide to hydrogen peroxide

(Doke 1983). Increases in AOS in response to race specific avirulent or non-host

pathogens has since been observed in planta in Vigna unguiculata (cowpea; Heath

1998), tobacco (Allan and Fluhr 1997), tomato (Lu and Higgins 1998), and

Arabidopsis (Wolfe 1998). Active oxygen species accumulation has also been well

characterized in cell culture. In soybean and tobacco cell culture, a burst of AOS is

observed in response to both virulent and avirulent Pseudomonas syringae strains

within minutes after inoculation, and lasts up to 30 minutes (Baker and Oriandi 1995).

This burst has been termed "Phase I", and has been proposed to be elicited by

flagellin, a protein found on the surface of all Pseudomonas syringae strains (Baker

and Orlandi 1998). The Phase I burst is followed by a Phase II burst in cuitures

inoculated with incompatible races of Pseudomonas syringae. This Phase II burst

occurs between 1.5 to 3 hours after inoculation and can last up to 6 hours (Baker and

Orlandi 1995). The two different phases and their timing have not been well

characterized in planta, although it is assumed that the response in plants is similar to

that observed in plant cell cultures.

3.1 Chemistry of active oxygen species

Active oxygen species (AOS) are produced from the reduction of molecular

oxygen (02). The initial reduction, in which 0 2 is converted to superoxide (Of).

requires a slight input of energy, frequently provided by NAD(P)H (Baker and Orlandi,

1995). Superoxide has been proposed to be generated by a membrane-bound

NADPH oxidase, cell wall peroxidases, lipoxygenase (LOX), or through the aberrant

transfer of electrons in the electron transport chain of the mitochondria and

chloroplasts (Bolwell and Wojtaszek 1997, Bolwell et a1 1998, Baker and Orlandi

1998). In the cell, 02' is found in equilibrium with hydroperoxyl radicals (HOz'),

which, being neutral in charge, are more lipophilic than 02- and therefore more

capable of membrane peroxidation (Adam et a1 1 989). At cellular pHs, superoxides

will decay spontaneously to hydrogen peroxide (H202), as shown in equation (1)

(Lamb and Dixon 1997)

Equation (I) H02- + 02- + H+ -----z H202 + 0 2 .

If catalyzed by superoxide dismutase (SOD), Hz02 will also be formed as shown in

equation (2).

Equation (2) 02- + 02- + 2H+ -----> H202 + 0 2

Hydrogen peroxide is the most stable of the AOS. As such it can diffuse farther

than other AOS, and, due to its non-polar nature, can cross lipid bilayers (Baker and

Orlandi 1998). Hydrogen peroxide will oxidize metals or organic molecules,

especially in the presence of peroxidases (Baker and Orlandi 1995), potentially

resulting in cellular damage.

The hydroxyl radical (OH-) is formed through the Haber-Weiss reaction,

described in equation (3).

Equation (3) 02- + Hz02 ----z OH- + OH- + 0 2

This reaction requires the presence of ~ e 3 + , and so the production of OH- is

determined by the availability of this metal (Baker and Orlandi 1998). Hydroxyl

radicals initiate radical chain reactions with organic molecules, resulting in

peroxidation of lipids, inactivation of enzymes, and degradation of n ucleic acids (Lamb

and Dixon 1997).

Figure 3

Proposed deacetylation of DCFH-DA by cellular esterase to DCFH and oxidation

of DCFH by H202 mediated by cellular peroxidases (adapted from Lu 1998)

2',7'-Dichlorofluorescin Diacetate (DCFH-DA)

(non-fluorescent)

2',7'-Dichlorofluorescin

(DCFH, non-fluorescent)

Cell membrane

Intracellular deacetylation by esterases

~04'v'- \ H

C1 \

I Hz (& + Peroxidase

I

2',7'-Dichlorofluorescein (DCF, Fluorescent)

I H

l c 1

~ O O H

++/

I

3.2 Detection and generation of AOS

(a) Detection of AOS

Several methods of detecting AOS have been developed, many of which detect

H202, since it is relatively stable and has the ability to oxidize molecules in the

presence of peroxidase. The fluorescent probe 2',7'-dichlorofluorescin diacetate

(DCFH-DA) is one example. It was developed in mammalian cell culture systems

(Bass et a1 1983), but has since been used in planta in tobacco (Allan and Fluhr 1997)

and tomato (Lu and Higgins 1998). Due to its non-polar nature, DCFH-DA is believed

to be membrane permeable. Once inside the cell, cytosolic esterases cleave the

diacetate group (DA), rendering the probe polar, trapping it inside the ceil (Bass et a1

1 983). in the presence of peroxidase and either Hz02 or hydroxyl radicals (Zhu et a1

1994a), DCFH will be oxidized, giving the fluorescent compound dichlorofluorescein

(DCF), which can be visulalized under blue light (figure 3; Bass et a1 1983). However,

it has been reported that DCFH and DCF cross cellular membranes and therefore may

detect extracellular as well as intracellular Hz02 (Royall and lschiropoulos 1993).

(b) Generation of AOS

To study the effects of AOS, it is of interest to be able to exogenously add

H202. There are problems inherent with exogenous application of H202, however.

Plants contain anitoxidants such as ascorbic acid, p-carotene, peroxidase, catalase,

and polyubiquitin which will rapidly degrade AOS (Durner et a1 1997), for instance

IOmM exogenously added H202 to soybean cell culture was found to be degraded

within 10 minutes (Levine et al1994). This problem may be overcome in part by the

addition of AOS generating systems which result in the persistant production of AOS . The enzyme xanthine oxidase generates 02- and uric acid from xanthine (Nishino

1994). Similarily, glucose oxidase synthesizes Hz02 and glucanate from a molecule

of P-D-glucose (Gochman and Schmitz 1972). Glucose oxidase has been

overexpressed in potaio (Wu et a1 1997), tobacco, and canola (Kazan et a1 1998),

resulting in the ectopic generation of H202. Alternative approaches have also been

taken: tobacco was transformed with an antisense catalase construct, resulting in a

decrease in catalase (Du and Klessig 1997). Since catalase catalyzes the

degradation of Hz02 to water, catalase antisense transgenic plants will exhibit higher

levels of H202, especially if placed under high light conditions when increases in AOS

are produced as a byproduct of photosynthesis (Chamnongpol et al1998).

3.3 Roles of active oxygen species in resistance responses

Active oxygen species have been proposed to have a number of functions in

plant pathogen interactions, including direct antimicrobial action, oxidative

crosslinking of HRGP and GRP, the lignification process in cell walls, initiating cell

death, and in the SAR signalling pathway.

(a) Antimicrobial effects of AOS

The toxic properties of AOS have been demonstrated both in vitro anc i in vivo:

addition of the glucose oxidase H202 generating system, as well as exogenous

addition of H202, inhibits Verticillium dahliae growth (Kim et a1 1988), and low

concentrations of Hz02 prevent germination of Peronospora tabacina

sporangiospores, and of Cladosporium cucumerinum and Colietotrichum lagenarium

conidia in vitro (Peng and Kuc 1992). In vivo, exogenous addition of H202 appears to

reduce the severity of blue mould infection on tobacco leaf disks (Peng and Kuc

1992). These observations suggest that AOS accumulation in plant-pathogen

interactions may directly inhibit the invading microbe.

The role of AOS as antimicrobial agents has not been emphasized due to the

idea that the levels of AOS necessary to be toxic to the pathogen are never reached in

the leaves (Baker and Orlandi 1998); however, Bestwick et. a/. (1 997) have localized

pathogen-induced Hz02 accumulation to specific regions close to the cell wall. This

implies that the concentration of Hz02 may be much higher in localized areas than

reported in previous calculations, in which the amount of Hz02 measured was

averaged over the whole leaf (Baker and Orlandi, 1998). The direct cytotoxic role of

AOS against microbial pathogens may therefore still be important.

(b) Strengthening of physical barriers

AOS have also been proposed to play a role in structural defence. Hz02 is

involved in the oxidation of coniferyl alcohol, the subunits of lignin polymers. It is also

believed to be involved in peroxidase mediated cross-linking of cell wall proteins: in

elicited soybean cell culture, the timing of insolubilization of HRGP appears to be

correlated with the observation of an oxidative burst (Bradley et at 1992). These

observations suggest that crosslinking of cell wall proteins such as HRGP and GRP is

dependent upon AOS accumulation.

(c) Involvement of AOS in the SAR signalling pathway

The idea that AOS accumulation is correlated with SA accumulation was

proposed when an SA binding protein was identified as catalase (Chen et a/ 1993).

SA was suggested to inhibit catalase by binding to it, thereby resulting in an increase

in H202 AOS could subsequently act as secondary messengers in the signalling

pathways for both gene-for-gene induced defence responses and SAR. Several

criticisms to this hypothesis have been raised: (1) SA has been found to bind, not only

catalase, but other enzymes containing heme groups (Ruffer et al1995). (2) AOS do

not induce PR gene expression in NahG transgenic tobacco, suggesting a

requirement for SA downstream of AOS accumulation in defence gene induction

(Neuenschwander et a/ 1995). (3) Hz02 accumulation is not always observed where

SA is known to increase: in systemic leaves of tobacco, for instance,

Neuenschwander et a/ (1 995) observed an increase in PR-1 expression without a

correlated increase in Hz02 (4) According to the model proposed by Chen et. a/.

(1 993), addition of SA would be expected to result in a decrease in antioxidant activity;

however, exogenous addition of SA to elicited soybean cell culture did not inhibit the

capacity of cells to degrade Hz02 (Tenhaken and Rube1 1997). Finally, (5)

endogenous SA does not increase to levels high enough to inhibit catalase, except

possibly around the region of necrosis resulting from an HR (Neuenschwander et a1

1995). Although SA may bind catalase in these necrotic regions, its involvement in the

SAR signalling pathway is likely through a different mechanism.

An alternative role for AOS in SAR signalling has been proposed. increases in

AOS can be correlated with increases in SA: high concentrations of exogenously

added Hz02 stimulated an increase in SA production (Neuenschwander et a i 1995,

Leon et al1995). Potato plants overexpressing glucose oxidase displayed

constitutively high levels of Hz02 and constitutively levels of high SA (Wu et a1 1997).

Conversly, H202 accumulation can be induced by the addition of SA (Chen et a/ 1993,

Rao et a/. 1993). It has therefore been proposed that SA potentiates the accumulation

of Hz02 and vice versa until a certain threshold has been reached after which SA,

H202, or both may act as signals for the induction of other defence responses

(Shirasu et a/ 1 997).

Recently, Hz02 has been demonstrated to have a direct role in signalling ir: the

SAR response pathway. In vivo intercellular production of Hz02 through the

glucose/glucose oxidase (G/GO) H202 generating system resulted in the

establishment of SAR in the absence of a biotic immunizing agent in Arabidopsis

(Alvarez et a/ 1998). Similarily, when individual leaves of tobacco plants with

suppressed catalase activity were exposed to high light conditions resulting in AOS

accumulation, PR-1 expression was detected in light-protected systemic leaves

(Chamnongpol et a1 1998). The light induced Hz02 accumulation may result in the

production of a mobile signal, which is translocated to light protected leaves and

results in PR-1 expression.

(d) Involvement of AOS in cell death

H202 accumul~tion has been proposed to trigger the HR. In Arabidopsis Isd

mutants, which form spontaneous lesions similar to an HR, addition of 02' induced

lesion formation (Jabs et a1 1996). When an increase in AOS is generated in planta

by placing catalase antisense transgenic tobacco under high light, spontaneous HR-

like lesions are also observed (Du and Klessig 1997). Addition of the GIGO Hz02

generating system to Arabidopsis results in an increase in microscopic cell death in

inoculated leaves (Alvarez et a/ 1998). Similarily, transgenic plants expressing

glucose oxidase demonstrate an increase in cell death (Kazan et al1998). Studies

using AOS inhibitors substantiate these results. Diphenylene iodinate (DPI), an

inhibitor of NADH flavoproteins including the NADPH oxidase thought to be

responsible for the oxidative burst (Lamb and Dixon 1997), blocks cell death in

response to avirulent Pseudomonas syringea pv glycinia (Psg; Levine et a1 1994).

Cell death is enhanced in the presence of the catalase inhibitor 3-aminotiazole (Levine

et a1 1994). Correlations therefore exist between AOS accumulation and cell death,

suggesting that AOS may be a component in the signal transduction pathway leading

to the HR cell death.

However, AOS alone may not be sufficient to induce cell death. Infection of

soybean cell cultures by Pseudomonas fluorescens containing a mutated region of the

hrp locus elicited an oxidative burst, yet plant cell death was not observed (Glazner et

a1 1996). Accumulation of AOS therefore does not necessarily cause cell death,

indicating that the potential link between AOS accumulation and cell death may not be

direct. Recent evidence suggests that another molecule, nitric oxide, may be

involved. Addition of the G/GO H202 generating system, or the X / X O 02- generating

system to soybean cell culture results in a weak cell death response. Addition of a

nitric oxide (NO) donor system also resulted in little cell death, however, addition of a

nitric oxide donor system in conjunction with an AOS generating system resulted in a

large increase in cell death, suggesting that nitric oxide may potentiate AOS induced

cell death (Delledonne et a1 1998). Although AOS may be an important component in

the cell death signalling pathway, alone, they do not appear to be sufficient for the

induction of cell death.

4. A possible relationship between SA, cell death, and SAR

4.1 The relationship between SA and cell death

Although possible correlations have been observed between increases in SA

and increases in AOS, and between increases in AOS and the observation of cell

death, a direct correlation between SA accumulation and cell death has not been

observed. In NahG transgenic plants, gene-for-gene HR cell death is abolished,

suggesting a requirement for SA (Delaney et a1 1994). Genetic crosses between lsd

and NahG Arabidopsis, and between catalase antisense and NahG tobacco suggest

that HR cell death may be independent of SA accumulation (Weymann et a1 1995, Du

and Klessig 1997). Both lsd and catalase suppressed plants form spontaneous HR-

like lesions under long day or high light conditions respectively. The F1 progeny of

these crosses would be expected to be unable to form lesions if SA was required for

lesion formation. This has not proven to be the case, however; F1 progeny form

spontaneous lesions, suggesting that SA is not required for HR-like cell death.

4.2 The role of cell death in the immunization and establishment stages of SAR

Common in the definition of SAR is that it is established upon immunization with

a "necrotizing pathogen" (Dietrich et a1 1994, Greenberg et a1 1994, Shulaev et a1

1995, He 1996), implicating cell death in the SAR signalling pathway. Mutants in isd

and acd show increased resistance following the formation of spontaneous lesions

(Jabs et a1 1996), suggesting that SAR may be established through a signalling

pathway involving cell death. In Arabidopsis, periveinal "micro-HRs" in systemic

leaves have been correlated with the establishment of SAR (Alvarez et a1 1998),

suggesting a role for cell death in the establishment stage of SAR. By constrast, the

dnd mutant, which does not respond with an HR upon infection with avirulent Pst

nevertheless appears to have constitutive SAR-like resistance (Yu et a1 1998). SAR

has also been reported in the absence of HR cell death in rps2 Arabidopsis mutants

(Cameron et a1 1 994).

4.3 Summary of the possible relationship between AOS, SA, cell death, and

SAR

In response to avirulent pathogens, hydrogen peroxide is thought to potentiate

an increase in salicylic acid and vice versa until a certain threshold level has been

reached. Subsequently, AOS may be involved in signalling for cell death, although

recent evidence suggests that involvement of other molecules, such as nitric oxide,

may also be necessary. Cell death in the immunization stage may also be required for

signalling for the establishment of SAR, athough there is only correlative evidence to

support this. Microscopic cell death in the establishment stage of SAR suggests that

cell death may be important in the establishment stage of SAR.

5. Research aims

5.1 To characterize the manifestation stage of SAR with respect to Hz02

accumulation and the expression of proteins thought to be involved in defence

responses.

The role of active oxygen species in the manifestation stage of SAR remains

largely uncharacterized. SAR appears to resemble the gene-for-gene response: PR

proteins are expressed, physical barriers are strengthened, and SA accumulates. We

hypothesized that AOS, which have been shown to accumulate during a gene-for-

gene interaction, also accumulate during the manifestation stage of SAR. Certain

elicitor induced (elf) genes have also been shown to be expressed during a

hypersensitive response in parsley and Arabidopsis. If the manifestation stage of SAR

resembles a gene-for-gene hypersensitive response, these genes would be predicted

to be expressed during SAR manifestation. This thesis will compare the SAR

response to a gene-for-gene response with respect to AOS accumulation and eli gene

expression in the Arabiodopsis-Pst pathosystem.

5.2 To characterize the role of AOS in the immunization stage of SAR

Active oxygen species are potentially involved in the establishment of SAR.

The role of AOS accumulation during immunization is not known; one hypothesis is

that AOS accumulation and/or cell death must occur for the production of the mobile

SAR signal which results in the establishment of SAR. This thesis attempts to test this

prediction by correlating G/GO generated AOS accumulation and microscopic cell

death in the immunization stage with the establishment and manifestation of SAR.

SA may be required for both the accumulation of AOS and for HR cell death.

These hypotheses are tested by determing whether AOS accumulation and

microscopic cell death occur in NahG Arabidopsis in response to avirulent Pst. The

role of AOS during the immunization stage of SAR might also be elucidated by

characterizing the oxidative burst in mutants defective in their ability to establish and

manifest SAR. The SAR mutants nprl and dir I are characterized with respect to

active oxygen species accumulation during the immunization stage of SAR.

Materials and Methods

1. Plant growth conditions

Two wild type ecotypes of Arabidopsis thaliana were used: Columbia (Col-0)

and Wassilewskija (Ws). Mutants are in either of these two backgrounds: nprl and

NahG are in a Col-0 background while dirl is in a Ws background.

Seeds were sterilized in 70% ethanol for two minutes, 18% commercial bleach

and 0.1 % Tween 20 (J.T.Baker) for 10 minutes, washed with sterile water at least four

times, and resuspended in 0.1% (w/v) phyagar (GibcoBRL). Seeds were imbibed

overnight at 4°C to synchronize germination, then plated on Murashige and Skoog

(MS) media containing 2% (w/v) sucrose (GibcoBRL). Seeds were germinated under

continuous 60 pE m-2s-1 light . Seedlings were transplanted to Promix soil (Plant

Products) at approximately the four leaf stage. Plants were grown on soil for

approximately 2 weeks at 22OC, 150 pE m-2 s-2 , with a 9 hour day/ 15 hour night light

cycle.

2. Bacterial growth conditions

Pseudomonas syringae pv tomato (Pst) DC3000 containing either the plasmid

pV288 (containing the avrRpt2 avirulence gene) or pVSP6I (without avrRpt2; both

strains courtesy Dr. Andrew Bent, University of Illinois) were grown overnight at 21 OC,

with shaking at 200 RPM on a VWR Scientific orbital shaker in 5 mL of King's media B,

with 50 pg/mL kanamycin (Sigma). Overnight cultures were centrifuged at l6OOg for

10 minutes in a Beckman GS-15R centrifuge, and resuspended in 10 mM MgC12 (ACP

Chemicals). Optical densities were obtained at )c=600nm using a Novaspec II

spectrophometre (Pharmacia Biotech). Assuming that one OD unit corresponds with

109 cfu/mL, bacteria were diluted in I OmM MgC12 to a final concentration of 106 or

3. Induction of SAR

SAR was induced as described previously (Cameron et a1 1 994). Avirulent Pst

(1 o7 cfu/rnL) or 10mM MgC12 were pressure infiltrated with a 1 cc needleless syringe

into one to two leaves per plant. Two to 4 days later, the plants were challenged by

pressure infiltration of either virulent or avirulent Pst at 106 cfu/mL, or with 10 mM

MgC12 into two to four leaves on each immunized plant.

4. Quantification of bacteria

Three days after inoculation, 4mm diameter leaf disks were obtained from eight

individual leaves for each treatment. Leaf disks were homogenized in 500 pL 10 mM

MgC12 using a Ryobi D18C (540 RPM) drill and sterile bit. The hornogenate was

diluted 1 o4 fold and 106 fold in 10 mM MgC12 and spread on King's media B with

1.5% (w/v) agar (Oxoid Ltd) and 50 pg/ rnL kanamycin (Sigma) and 50 pg1mL

rifampicin (Sigma). Plates were incubated at either 21°C or 28°C for two days.

Bacterial colonies were counted, and bacterial growth (cfdleaf disk) was calculated

using the formula [bacterial growth = (5 x dilution factor x number of colonies) I

number of leaf disks.]

5. Qualitative hydrogen peroxide assay

Dichlorofluorocin diacetate (DCFH-DA; Molecular Probes) was solublized in

anhydrous ethanol (Commercial Alcohols Inc) to a concentration of 20 mM. At each

time point, leaves were harvested and placed into a 1.6 mL microfuge tube containing

1.5 mL 0.02% (v/v) Silwet L-77 (OSI Specialities Inc) in distillled water. An aliquot of

22.5 pL of 20 mM DCFH-DA was added, to a final concentration of 0.3mM. The

microfuge tubes were mixed by manual inversion and the leaves were vacuum

infiltrated three times, for three minutes each time. Leaves were then removed from

the tubes, blotted, and the abaxial side of the leaf observed under UV light (two 15 W

SW tubes, Chromato-Vue Ultra-Violet Products Inc.). The relative fluorescence of

each leaf was scored according to the ranking system described in figure 8.

6. Quantification of fluorescence

At each time point, three replicates of eight leaves for each treatment were

harvested and placed into microtitre plate wells containing 3 mL of 0.02% Silwet L-77

(OSI Specialities Inc). An aliquot of 45 pL of 10 mM DCFH-DA was added to each

well, to a final concentration of 0.3 mM. The leaves were then vacuum infiltrated three

times, for three minutes each time, blotted dry, and place into 3 cc syringes. Wire

mesh was placed at the bottom of the syringe in order to prevent leaf debris from

falling through. The syringes were rested in 30 mL centrifuge tubes, and centrifuged

at 1 OOOg for 10 minutes at 4°C. An aliquot of 50 pL of the intercellular fluid collected

at the bottom of the centrifuge tube was diluted in 3 mL distilled water and fluorometric

readings were obtained at an excitiation wavelength of 488 and an emission

wavelength of 525 in a Hitachi F-4000 fluorometer.

7. Use of the oxygen electrode to measure hydrogen peroxide production

At 12 hours after inoculation, intercellular fluid was collected as described in

section 6. Alternatively, eight 4mm diameter leaf disks were place into a 1.6mL

microfuge tube and immediately frozen in liquid nitrogen. Samples were ground on

dry ice using a Ryobi D l 8C (540 RPM) drill and drill bit, homogenized in 200 yL l0mM

NaHP04 (pH7.2), centrifuged at 1 1500g for 20 min at 4OC in a Beckman GS-15R

centrifuge, and the supernatant collected.

The oxygen elctrode was equilibrated with 4 mL 10mM NaHP04 (pH7.2) or

distilled water, and 2 yL of 3.7 x 106 U/mL catalase (from bovine liver; Sigma).

Samples were than added, and the rate of oxygen production observed. To measure

oxygen production in whole leaves, two leaves were placed in the oxygen electrode

chamber, previously equilibrated with 10 mM NaHP04 (pH7.2). An aliquct of 2 pL of

3.7 x 106 U/mL catalase (from bovine liver; Sigma) was then added and the rate of

oxygen production observed.

8. Exogenous application of the glucose/glucose ox!.lase (GIGO) hydrogen

peroxide generating system

Inoculation of plants with avirulent or virulent Pst, or with 10 mM MgC12 was

designated as time zero. At 6.75 hours, plants reserved for treatment with G/GO or

the glucose alone control were pressure infiltrated using a needleless 1 cc syringe

with 2.5mM glucose (BDH lnc) in 20 mM Nap04 (pH 6.5), with glucose oxidase

(Calbiochem) freshly added to a final concentration of 2.5 U/mL, or with 2.5mM

glucose (BDH lnc) in 20 mM Nap04 (pH 6.5), respectively. For the 7 hour time point,

1.5 mL G/GO or glucose with 0.02% (v/v) Silwet 1-77 (OSI Specialties Inc) was added

with 22.5 pL DCFH-DA (final concentration of 0.3 mM) to a 1.6 mL microfuge tube with

freshly harvested leaves. The contents of the microfuge tube were mixed by manual

inversion and vacuum infiltrated three times, for 3 min each time. Leaves were then

blotted, viewed under UV light, and ranked qualitatively, as described in section 5.

9. Determination of cell viability

(a) Chlorophyll autofluorescence

Three leaves from each treatment were placed in 1.5 mL 0.020h (v/v) Silwet L-

77 (OSI Specialties Inc) in 1.6 mL microfuge tubes and vacuum infiltrated three times

for 3 min each time. A 4mm leaf disk was excised from a region of the leaf

approximately 4mm from the centre of the inoculation site and placed on a microscope

slide in water, with the adaxial side facing upwards. Tissue was viewed using a

Reichert-Jung Polyvar microscope under 18x magnification and blue light (BP 450-

495, DS 510, LP 520). The image was transferred to the Northern Exposure software

system (Empix Imaging lnc). Images were converted to monochrome and integrated

until cells were distinguishable1 . The percentage cell death was determined by

adjusting the threshold range so that cells judged to be alive could be separated from

those deemed dead? Under blue light, chlorophyll in cells that are alive will fluoresce

red; when converted to monochrome, this fluorescence will appear white, while dead

cells will remain dark.

(b) FDA staining

Fluorescein diacetate (FDA; Sigma) was solubilized in acetone to a

concentration of 5 mg/mL. At each time point, two leaves from each treatment were

harvested and placed in a 1.6 mL microfuge tube containing 1.5 mL 0.02% (v/v) Silwet

L-77 (OSI Specialties Inc) in distilled water and 15 pL FDA (final concentration of 0.3

mg/mL). The contents of the tubes were mixed by manual inversion and vacuum

infiltrated three times for 3 min each time. Leaf disks 4mm in diameter were excised,

then viewed and analyzed as described in section 9(a).

values ranged from 30 to 1 00 2values ranged from 0-1 25 to 0-1 90

10. Statistical Analysis

Differences were determined using student t-tests, with p0.05 considered to

significant.

11. Preparation of RNA from plant tissue

Three leaves (corresponding to approximately 0.1 g FW) were harvested and

immediately frozen in liquid nitrogen. Samples were stored at -80°C until ready for

extraction. Tissue was ground on dry ice using a Ryobi D l 8C (540 RPM) drill and

sterile bit. An aliquot of 500 pL of 80°C extraction buffer? phenol ( I :l ; Sigma) was

added to each sample. Samples were vortexed for 5 rnin, 250 pL of chloroform

(ACP): isoamylalcohol (1 :24; ICN Biomedicals Inc) were added, and samples were

vortexed further. Samples were then centrifuged for 5 min at 15500g at 4OC. The

aqueous layer was removed and the chloroform extraction repeated. One volume of

4 M LiCl (J.T. Baker Inc) was added to the aqueous layer, and the RNA was allowed

to precipitate overnight at -20°C. The sample was then centrifuged for 5 min at

15500g at 4°C and the resulting pellet was resuspended in 250 yL water. An aliquot

of 0.1 volumes of 3 M NaOAc (ACP) and two volumes of ethanol was added, and the

RNA was allowed to precipitate at -20°C for at least 30 minutes. The sample was

again centrifuged at 15500g at 4°C for 15 minutes, and the pellet was washed in 70%

ethanol and resuspended in 15 pL water.

- -- --

3 ~ . 1 M LiCl (J.T.Baker lnc), 100 mM Tris (Sigma) pH8.0. 10 mM EDTA (Sangon Ltd), 1 % SDS (EM Science)

12. Northern analysis

The RNA sample (5 pL) was denatured with formaldehyde (ACP Chemicals Inc)

and formamide (EM Science) and interchelated with ethidium bromide. Samples were

separated on a 1.5% (w/v) agarose (Gibco BRL), 18% (vh) formaldehyde (ACP

Chemicals Inc) gel in 1X MOPS. RNA was transferred to Hybond nylon membrane

(Amersham) by capillary transfer in 1 O X SSC (Sambrook et al1989). RNA was

cross-linked to the membrane by UV radiation at 1200 pwatts cm-2 using a UV

Stratalinker 2400. The membrane was prehybridized in Church's buffer (Church and

Gilbert, 1984), then hybridized overnight at 65°C with radiolabelled DNA in a

Hybridiser HB-ED (Techne). The membrane was washed stringently with Church's

wash solution (Church and Gilbert 1984) at 65°C. The membrane was then placed on

autoradiography film (DuPont) and stored at -80°C until ready to be developed.

13. Preparation of radiolabelled DNA

Plasmid DNA was isolated using standard techniques (Sambrook et a1 1989).

Copy DNA (cDNA) fragments were isolated by digestion with the appropriate

restriction enzyme (MBI; Table I ) , separated on a 0.7% (w/v) agarose (Gibco BRL)

gel and isolated using a Qiaex II Gel Extraction Kit (Qiagen). Approximately 20 ng

were labelled with a 3 2 ~ dCTP (Amersham) using the Rediprime labelling kit

(Amersharn).

Table 1

Probes used for northern analysis

Fragment Used

0.75 kB Eco RI and Xho fragment

2 kB Pvu II fragment

I kB Pvu I1 fragment

1.4 Pvu II fragment

Purpose

encodes pathogenesis related protein 1 ; used to determine the establishment of SAR encodes SMS 1 ; the expression pattern was determined during the manifestation and establishment of SAR encodes an anionic peroxidase; the expression pattern was determined during the manifestation and establishment of SAR encodes a peroxidase; the expression pattern was determined during the manifestation and establishment of SAR

Reference

Uknes et a1 1994, obtained courtesy of Dr. Sharon Potter, Ciba- Geigy Corp.

-

Trezzini ef a1 1993

Trezzini et a1 1993

Trezzini et a1 1993

Results

1. Characterization of eli gene expression and AOS accumulation in the

manifestation stage of SAR

The SAR response has been considered to be a manifestation of the same

defence responses observed during a gene-for-gene interaction. Indeed, Kuc

observed that, "symptoms on protected plants following challenge were

microscopically and macroscopically indistinguishable from the normal resistance

reactions produced in response to inoculation with a cultivar non pathogenic race"

(Kuc 1982). It is, however, difficult to make a complete comparison between the SAR

response and an incompatible interaction, since many of the defensive mechanisms

observed during a gene-for-gene interaction have not been characterized during the

manifestation of SAR.

1.1 Expression of eli 11, eli 18, and ap3 in the establishment and manifestation

stages of SAR

The elicitor induced (eli) genes are upregulated in Arabidopsis cell culture

following treatment with the fungal elicitor Pmg (Trezzini et a1 1993). They might

therefore also be expressed during the manifestation, and possibly establishment,

stages of SAR, and, as such could be considered markers for the SAR response.

Determination of the expression patterns of eligenes in SAR defective dirl mutant

plants could lead to further understanding about the involvement of these genes in

SAR. ELI 18 has been shown to have homology to S-adenosyl-L-methionine

synthase 1 (SMS 1) which is involved in the activated methyl cycle (Somssich and

Hahlbrock 1 998). Preliminary studies demonstrated that expression appears to be

induced in leaves of Ws and dirl following inoculation with avirulent Pst (MAin

treatment). The eli 78 gene was expressed in Ws leaves manifesting SAR (AVh

Figure 4

Northern analysis of wild type and dirl leaf total RNA with ELI 18 cDNA.

(MM) refers to immunization and challenge with lOmM MgC12, (MA) refers to

immunization with 1 0mM MgC12 and challenge with avirulent Pst ( I 06 cfu/mL), (MV)

refers to immunization with 10mM MgC12 and challenge with virulent Pst ( I 06 cfu/rnL),

(AV) refers to immunization with avirulent Pst (1 o7 cfu/rnL) and challenge with virulent

Pst ( I 06 cfulml). (In) denotes challenge inoculated leaves and (un) denotes

uninoculated, systemic leaves. (A) demonstrates northern analysis, (B) demonstrates

ethidium bromide staining. Tissue was harvested 24 hours after the challenge

inoculation. This analysis was not repeated.

Figure 5

Northern analysis of wild type and dirl leaf total RNA with ELI 11 cDNA.

(MM) refers to immunization and challenge with I OmM MgC12 (MA) refers to

immunization with 10mM MgC12 and challenge with avirulent Pst (1 o6 cfu/mL), (MV)

refers to immunization with 10mM MgC12 and challenge with virulent Pst (1 06 cfu/mL),

(AV) refers to immunization with avirulent Pst ( I 07 cfu/mL) and challenge with virulent

Pst (1 06 cfu/mL). (In) denotes challenge inoculated leaves and (un) denotes




L~~ dL dirl I

Figure 6

Northern analysis of wild type and dirl leaf total RNA with AP3 cDNA.

(MM) refers to immunization and challenge with 1 OmM MgC12, (MA) refers to

immunization with 10mM MgC12 and challenge with avirulent Pst (1 06 cfulml), (MV)


(AV) refers to immunization with avirulent Pst ( I 07 cfulml) and challenge with virulent

Pst (1 06 cfulml). (In) denotes challenge inoculated leaves and (un) denotes




Figure 7

Bacterial growth in wild type and dirl Arabidopsis.

(Un) refers to uninoculated plant tissue, (MM) refers to immunization and challenge

with 10mM MgC12, (MA) refers to immunization with 1 OmM MgC12 and challenge with

avirulent st (106 cfdml), (MV) refers to immunization with 10mM MgC12 and

challenge with virulent Pst (1 06 cfu/mL), (AV) refers to immunization with avirulent Pst

(1 07 cfu/ml) and challenge with virulent Pst (1 06 cfu/mL). Error bars denote standard

deviations. (*) indicates significant difference from MV treatment at p0.05

treatment), but not in similarly treated dirl leaves (figure 4). In a preliminary northern

analysis of Ws and dirl RNA probed with ELI 1 1, which has homology to an anionic

peroxidase (Trezzini et a1 1993), a slight induction in expression was observed in

response to inoculation with avirulent Pst (MAin treatment) in Ws, but not in dirl plants

(figure 5). It is difficult to make comparisons, however, due to differential loading of

the RNA sample (figure 55). The ap3 gene also encodes a peroxidase enzyme

(Trezzini etal 1993). Like eli 18, it may be induced following inoculation with avirulent

Pst (MAin treatment; figure 6). Expression may increase slightly in leaves

manifesting SAR (AVin) and in response to virulent Pst (MVin), although this

interpretation may be skewed by differential loading of the RNA samples (figure 6B).

The eli I I and 18 genes are not strongly induced in systemic leaves of plants

immunized with avirulent Pst (AVun; figures 4 and 5). Expression of ap3 was

observed in all treatments including leaves inoculated with 10 mM MgC12, an indicator

of wound induced expression, and in uninoculated leaves of plants immunized with 10

mM MgC12, an indicator of constitutive expression.

A complicating factor in these northern analyses is that a full SAR response

was not observed. Bacterial growth was limited five fold (figure 7) compared to 10-50

fold previously described (Cameron et a1 1994), and the characteristic strong systemic

expression of PR-I in plants immunized with avirulent Pst (Uknes etal 1992) was not

observed (Cameron, data not shown).

1.2 Hydrogen peroxide accumulation in leaves manifesting SAR

Active oxygen species have been observed to accumulate during incompatible

interactions in a wide variety of plant species (Allan and Fluhr 1997, Doke 1983, Heath

1998, Lu and Higgins 1998). It is therefore of interest to determine whether a similar

response is observed during the manifestation of SAR.

Figure 8

Ranking scale of relative in planta fluorescence of DCF under UV light.

Leaves fluorescing (0) show either no fluorescence or red chlorophyll

autofluorescence. (+) refers to faint, inconsistent fluorescence. (++) denotes faint,

consistent fluorescence or bright pathchy fluorescence, and (+++) denotes bright,

consistent fluorescence throughout the entire leaf.

Figure 9

AOS accumulation during the manifestation stage of SAR in ecotype Col-0, as

observed from 7 to 16 hours after inoculation.

At each time point, six leaves from each treatment were vacuum infiltrated with DCFH-

DA and scored qualitatively under UV light, according to the ranking system described

in figure 8. (0 ) refers to immunization and challenge with IOmM MgC12, (*) refers to

immunization with 10mM MgC12 and challenge with avirulent Psi (1 o6 cfu/mL), (A)

refers to immunization with 10mM MgC12 and challenge with virulent Pst (1 o6 cfu/ml),

(-T) refers to immunization with avirulent Pst (I 07 cfu/mL) and challenge with virulent

Psi (I 06 cfu/mL). Error bars denote standard error. This was repeated twice with

similar results.

1 I t I I

8 10 12 14 16

Time After Inoculation (Hours)

Figure 10

In pianta fluorescence of DCF under UV light in leaves manifesting SAR.

Leaves were vacuum infiltrated with DCFH-DA and placed under UV light. (MM)

refers to immunization and challenge with lOmM MgC12 (MA) refers to immunization

with 10mM MgC12 and challenge with avirulent Pst (1 o6 cfu/mL), (MV) refers to

immunization with I OmM MgC12 and challenge with virulent Pst (1 06 cfu/mL), (AV)

refers to immunization with avirulent Pst (1 07 cfufml) and challenge with virulent Pst

(1 06 cfu/mL), (UN) refers to uninoculated leaves.

Figure 11

AOS accumulation during the manifestation stage of SAR in ecotype Col-0, as

observed from 7 to 23 hours after inoculation.



in figure 8. (0) refers to immunization and challenge with 10mM MgC12, (U-) refers

to immunization with 1 OmM MgC12 and challenge with avirulent Pst (1 06 cfu/mL), ( I )

refers to immunization with 10mM MgC12 and challenge with virulent Pst (1 06 cfu/ml),

(-v) refers to immunization with avirulent Pst (1 07 cfu/rnL)and challenge with virulent

Pst (I 06 cfulml). Error bars denote standard error.

7 I I 15 19 23


Figure 12

Bacterial growth in ecotype Col-0.

(MM) refers to immunization and challenge with 10mM MgC12, (MA) refers to

immunization with I OmM MgC12 and challenge with avirulent Pst (I 06 cfulml), (MV)

refers to immunization with 10mM MgC12 and chalienge with virulent Pst (1 o6 cfufml),

(AV) refers to immunization with avirulent Pst (107 cfulml) and challenge with virulent

Pst (1 06 cfulrnl). This assay was performed on the same plants as that shown in

figure 9. Error bars denote standard deviation.

Treatment

Figure 13


(MM) refers to immunization and challenge with 10mM MgC12, (MA) refers to

immunization with 1 OmM MgC12 and challenge with avirulent Pst (1 06 cfulml), (MV)

refers to immunization with 10mM MgC12 and challenge with virulent Pst (I o6 cfu/mL),

(AV) refers to immunization with avirulent Pst (I o7 cfu/mL)and challenge with virulent

Pst (1 06 cfu/mL). This assay was performed on the same plants as that shown in

figure 10. Error bars denote standard deviation. (*) indicates significant difference

from MV treatment at p10.05

Treatment

An AOS assay using the fluorescent probe DCFH-DA in planta has been

developed in tomato (Lu and Higgins 1998) and tobacco (Allen and Fluhr 1997), but

has not yet been documented in Arabidopsis. Consequently, an assay for the

detection of AOS using the fluorescent probe DCFH-DA was first established in

Arabidopsis, by amending the protocol described previously (Lu and Higgins 1998).

A qualitative approach was taken. DCFH-DA was infiltrated into infected

leaves, and the relative fluorescence of each leaf ranked according to the ranking

scale illustrated in figure 8.

During a Pst(avrRpt2)-Arabidopsis(Rps2) interaction, fluorescence (++ to +++)

was observed between approximately 8 and 17 hours after inoculation (figure 9, Wolfe

1998). Accumulation of AOS during the manifestation stage of SAR was therefore

measured within this time period. Fluorescence was detected in response to avirulent

Pst but not in response to either virulent Pst or 10 mM MgC12 (figures 9 and 10). In

leaves manifesting SAR, AOS accumulation was low; there was no significant

difference (pr0.05) between fluorescence in leaves manifesting SAR (the AV

treatment) and in leaves inoculated with 10 mM MgC12 (the MM treatment; figure 9).

Since the possibility existed that AOS accumulates in leaves manifesting SAR at a

later time period than in leaves undergoing an HR, AOS accumulation was obsewed

until 23 hours after inoculation (figure 11). A characteristic fluorescence response was

detected in leaves inoculated with avirulent Pst (figure 1 1 ). No fluorescence was

observed in leaves manifesting SAR until approximately 23 hours after inoculation,

closely parallelling the accumulation in response to virulent Pst (figure 11). To

determine whether a SAR response occurred, in planta bacterial growth was

measured. Bacterial growth was limited in leaves manifesting SAR (figures 12 and

13): a 10 to 100 fold reduction in bacterial growth was observed in leaves treated with

avirulent Pst (MA treatment) and leaves manifesting SAR (AV treatment) when

compared to bacterial growth in leaves infected with virulent Pst (MV treatment).

Figure 14

Relative fluorescence of DCF oxidized by exogenously added H202.

Six leaves were treated with the appropriate concentration of H202, vacuum

infiltratred with 0.3 mM DCFH-DA, and scored under UV light according to the

rankings system described in figure 8. Error bars denote standard deviation. This

experiment was repeated three times with similar results.

H,O, Concentration

Figure 15

AOS accumulation as measured by fluorometry during the manifestation of SAR

in Col-0 Arabidopsis.

At each time point, three replicates of 8 leaves from each treatment were vacuum

infiltrated with DCFH-DA. Intercellular fluid was collected, and fluorescence was

determined using a fluorometer. (0) refers to immunization and challenge with 10mM

MgC12. (+) refers to immunization with 10mM MgC12 and challenge with avirulent Pst

(1 06 cfu/mL), ( A-) refers to immunization with 10mM MgC12 and challenge with

virulent Pst (1 06 cfu/rnL), (-v) refers to immunization with avirulent Pst (1 07

cfu/mL)and challenge with virulent Pst (1 06 cfu/mL). Error bars denote standard

deviation.

4 6 8 10 12 14 16 18


Mock treatment with MgC12 (MM treatment) resulted in little bacterial growth.

From these results, the relative levels of AOS accumulation can be determined,

yet the concentration of AOS accumulating in each response is not known.

Fluorescence of leaves inoculated with avirulent Pst can be compared to the

fluorescence resulting from inoculation of leaves with known concentrations of H202

(figure 14). Relative fluorescence increased with increasing concentrations of Hz02

until 225mM exogenous 5202 was added. There is no statistical difference (pS0.05)

between AOS fluorescence observed in response to avirilent Pst (figures 9 and 11)

and fluorescence observed following exogenous addition of H202 concentrations

between five and 20 mM (figure 14).

1.3 Quantitative Approaches to AOS detection

In order to determine the concentration of AOS accumulating, a quantitative

assay was developed using DCFH-DA and fluorometry. Erratic fluorescence was

observed in intercellular fluid obtained from leaves responding to virulent Pst, while

little fluorescence was observed in response to avirulent Pst (figure1 5), a result which

is contrary to results obtained using the qualitative assay (figures 9 and 11). Leaves

manifesting SAR demonstrated significantly higher fluorescence; however, in light of

the inconsistent fluorescence observed in the controls, these results were not deemed

reliable and this method was not used in further experiments.

1.4 Use of the Oxygen electrode for the Quantification of H202

Other assays for the quantification of Hz02 have been explored, including use

of the oxygen electrode. Catalase degrades hydrogen peroxide to water and oxygen;

Figure 16

AOS accumulation during the immunization stage of SAR in dirl Arabidopsis.



in figure 8. (0) refers to inoculation with 10mM MgC12, (*) refers to inoculation

with avirulent Pst (I 06 cfu/mL), ( A-) refers to inoculation with virulent Pst (I 06

cfu/mL). Error bars denote standard error. This experiment was repeated twice with

similar results.


therefore the amount of H202 present in a system can theoretically be quantified

using an oxygen electrode by adding catalase and measuring the amount of oxygen

released. Catalase was added to intercellular fluid collected from leaves 12 hours

after inoculation with virulent or avirulent Pst, or 10 mM MgC12. No detectable

differences in oxygen production were obsewed between treatments. Since it is

possible that the protocol for collecting intercellular fluid resulted in the release of

antioxidants, the assay was repeated with tissue ground in liquid nitrogen. Again, no

detectable differences were observed (data not shown).

One microlitre of 10M H202 injected into the tissue before grinding produced a

large decrease in oxygen production compared to the same amount of Hz02 added

directly to the catalase in the electrode chamber. It is probable that antioxidant activity

remained high during the procedure, so that any Hz02 present in the tissue was

degraded before it could be measured. In an attempt to circumvent this problem,

whole leaves were placed in the oxygen electrode, and catalase added;

however,once again, no differences were observed in response to the different

treatments. It is likely that the amount of H202 diffusing out of the leaves was low:

the amount of Hz02 substrate available for catalase activity was low, and differences

between treatments were negligable.

2. Characterization of AOS accumulation in the immunization stage of SAR

2.1 AOS accumulation in mutants unable to establish SAR

Recent evidence has substantiated the suggestion that AOS is involved in the

SAR signalling pathway (Alvarez et al1998, Chamnongpol et al1998). It is therefore

of interest to determine whether certain mutants defective in the SAR response are

also unable to produce an oxidative burst. Furthermore, characterization of AOS in

these mutants may allow for additional dissection of the SAR pathway: it may be

Figure 1 7

Bacterial growth in nprl Arabidopsis.

(M) refers to inoculation with 10mM MgC12 (A) refers to inoculation with avirulent Pst

(1 o7 cfu/mL), (V) refers to inoculation with virulent Pst (I o7 cfu/mL). Error bars

denote standard deviation.

Figure 18

AOS accumulation during the immunization stage of SAR in nprl Arabidopsis.



in figure 8. (0) refers inoculation with 10mM MgCI2, (+) refers to inoculation with

avirulent Pst (1 07 cfu/mL), (-I) refers to inoculation with virulent Pst (I 07 cfu/mL).

Error bars denote standard error. This experiment was repeated once with similar

results.


possible to determine where AOS accumulation lies relative to NPRl and DlRl protein

function in SAR signalling.

(a) dirl

Mutants of dirl can establish an HR in response to avirulent bacteria (Cameron

et a1 unpublished). AOS accumulation in dirl plants was therefore predicted to mirror

that of wild type Ws in the immunization stage of SAR. Mutants in dirl were assayed

for AOS accumulation in response to virulent and avirulent Pst and to 10 mM MgC12

control using the qualitative approach. Fluorescence was observed in response to

avirulent Pst between 7 and 16 hours at a magnitude comparable to that observed in

wi!d type plants (figures 16 and 9). Little fluorescence was observed in response to

either virulent bacteria or MgC12, also comparable to levels observed in wild type

plants.

(b) nprl

Mutants in nprl demonstrate a typical HR in response to Psm (avrRpt2)

including necrotic lesions, autofluorescence of phenolic compounds, and a reduction

in bacterial numbers (Cao et a1 1994). It was therefore predicted that AOS

accumulation associated with an HR in response to Pst (avrRpt2) would be similar to

that seen in wild type plants. A reduction in bacterial growth was seen in nprl plants

comparable to that observed in Col-0 (figure 17), indicating that nprl responds to Pst

(avrRpt2) as it does to Psm (avrRpt2). Assaying nprl plants for AOS accumulation

during the immunization stage of SAR revealed that AOS accumulated in response to

avirulent Pst in a manner comparable to the Col-0 response (figure 18). In response

to virulent Pst or MgC12, AOS accumulation was relatively low, also paralleling the wild

type response (figure 18).

Table 2

Summary of experiments involving G/GO induction of AOS, cell death, pr gene

expression, and inhibition of bacterial growth.

to ecotype Col- 0; SAR was established G/GO addition to ecotype Col- 0; SAR was not established G/GO addition to NahG transgenic ~ lan ts

AOS accumulation figure 19

figure 20

figures 21 and 29

Cell death

figures 25 and 26

figure 27

figures 30 and 31

Bacterial arowth figure 22

data not shown

figure 33

-- - -

PR gene expression figure 23

data not shown

Figure 19

AOS accumulation during the immunization stage of SAR in ecotype Col-0

following exogenous addition of the glucose1 glucose oxidase H202 generating

system.



in figure 8. (0) refers inoculation with IOmM MgC12, (+) refers to inocuiation with

avirulent st (106 cfu/mL), (-A) refers to inoculation with virulent Pst (1 06 cfu/mL),

(-@-) refers to treatment with glucose (2.5 mM) and glucose oxidase (2.5 WmL) at the

7 hour time point, and (+ ) refers to treatment with glucose (2.5 mM) at the 7 hour

time point. Time 0 refers to the time of inoculation with Pst and 10 mM MgC12 Error

bars denote standard error. This experiment was repeated twice with similar results.

i I I 1

7 9 11 13


Figure 20

AOS accumulation during the immunization stage in ecotype Cob0 failing to

establish SAR following exogenous addition of the GIGO H202 generating

system,



in figure 8. (0) refers inoculation with 1 OrnM MgC12, (%) refers to inoculation with

avirulent Pst (1 06 cfulrnl), (A-) refers to inoculation with virulent Pst (1 06 cfulml),

(-@I-) refers to treatment with glucose (2.5 mM) and glucose oxidase (2.5 U/mL) at the

7 hour time point, and (+) refers to treatment with glucose (2.5 mM) at the 7 hour

time point. Time 0 refers to the time of inoculation with 10 mM MgC12 and Pst Error

bars denote standard error.

1 I I I 1 1

6 8 10 12 14 I6


Figure 21

AOS accumulation in ecotype Col-0 following exogenous addition of the GIGO

H202 generating system.



in figure 8. (0) refers inoculation with I OmM MgC12, (9) refers to inoculation with

avirulent Pst (1 06 cfu/mL), ( I) refers to inoculation with virulent Pst (1 06 cfu/mL),

(-@- ) refers to treatment with glucose (2.5 mM) and glucose oxidase (2.5 U/mL) at the

7 hour time point, and (+) refers to treatment with glucose (2.5 mM) at the 7 hour

time point. Time 0 refers to the time of inoculation with 10 mM MgC12 and Pst. Error

bars denote standard error. Repeated once with similar results.

1 I I I

7 9 11 13

Time After lnoculation (Hours)

Figure 22


(MM) refers to immunization and challenge with 10mM MgC12 (MA) refers to

immunization with 1 0mM MgC12 and challenge with avirulent Pst (1 06 cfu/mL), (MV)


(AV) refers to immunization with avirulent Pst (1 07 cfu/mL) and challenge with virulent

Pst (I 06 cfu/ml), (GV) refers to immunization with glucose (2.5 mM) and glucose

oxidase (2.5 U/mL), and challenge with virulent Pst ( lo6 cfu/mL), and (PV) refers to

immunization with glucose (2.5 mM), and challenge with virulent Pst (1 06 cfu/mL).

Error bars denote stnadard deviation. (*) indicates significant difference from MV

treatment at p10.05. This analysis was repeated once with similar results.

Figure 23

Northern analysis of wild type leaf total RNA with PR-1 cDNA

(MM) refers to immunization and challenge with I OmM MgC12, (MA) refers to

immunization with 10mM MgC12 and challenge with avirulent Pst ( I 06 cfu/mL), (MV)


(AV) refers to immunization with avirulent Pst ( I 07 cfu/mL) and challenge with virulent

Pst (1 06 cfu/mL), (GV) refers to immuniztion with glucose (2.5mM) and glucose

oxidase (2.5 U/rnL) and challenge with virulent Pst (1 o6 cfuImL), (PV) refers to

immunization with glucose (2.5 mM) and challenge with virulent Pst (I 06 cfdrnl). (In)

denotes challenge inoculated leaves and (un) denotes uninoculated, systemic leaves.

(A) demonstrates northern analysis, (B) demonstrates ethidium bromide staining.

Tissue was harvested 24 hours after the challenge inoculation. This experiment was

repeated once with similar results.

2.2 AOS in the immunization stage of SAR

Hz02 has been shown to signal for the establishment and manifestation of

SAR (Alvarez et a1 1998, Chamnongpol et a1 1998). The mechanisms by which

thissignalling occurs remain open to question. The possibility that signalling may

occur through a cell death pathway was explored by correlating the Hz02 levels

produced by addition of the G/GO H202 generating system with levels of microscopic

cell death. In each set of experiments, AOS levels, microscopic cell death, PR gene

expression, and bacterial growth were determined. These assays will be discussed

individually; the figures corresponding with each experimental set are outlined in table

2.

Hydrogen peroxide levels following addition of G/GO were not as high as

those seen upon inoculation with avirulent Pst approximately (+) compared with (++;

figures 19, 20, and 21). By comparing these values with the standard curve of

fluorescence obtained from exogenously added H202 (figure 14), it can be estimated

that the G/GO generated Hz02 levels correspond with 4 5 mM exogenously added

H202 (~50.05). G/GO generates Hz02 in planta for less than two hours (figures 19

and 21).

Immunization with G/GO results in a reduction in bacterial growth comparable

to that observed in leaves manifesting SAR (figure 22). This was not observed in

plants immunized with glucose alone (PV): bacterial growth in the PV treatment group

is not significantly different (p0.05) from that in leaves from the MV treatment group

(figure 22). However, only a five-fold reduction in bacterial growth was observed

following challenge of plants immunized with either G/GO (GV) or avirulent Pst (AV)

rather than the 10 to 50 fold reduction previously documented (Cameron et a1 1994).

Although PR-1 gene expression was detected in leaves challenged with avirulent or

virulent Psf (the MA, MV, and AV treatments), no PR-I gene expression was observed

in systemic leaves of plants immunized with avirulent Pst, suggesting that a complete

Figure 24

Cell death measured by FDA fluorescence or chlorophyll autofluorescence.

At each time point, two leaves from each treatment were vacuum infiltrated with FDA

and two leaves were infiltrated with water. Leaves were then viewed under the

microscope under blue light. , (0) refers inoculation with 10mM MgC!2, !+) refers to

inoculation with avirulent Pst (1 06 cfu/mL), ( I ) refers to inoculation with virulent Pst

(1 06 cfu/mL), (4) refers to uninoculated leaves. Crosses in the middle of the symbol

indicate measurement using FDA, symbols without crosses indicate measurement

using chlorophyil autofluorescence. Error bars denote standard error.

I 1 1

8 12 16


Figure 25

Cell death in ecotype Col-0 following infection with Pst or treatment with GIGO.

At each time point, three leaves per time point were observed microscopically under

blue light. Chlorophyll autofluorescence was used as an indicator of cell vitality. (0)

refers inoculation with 10mM MgC12, (+) refers to inoculation with avirulent Pst (I o6

cfulml), (-A) refers to inoculation with virulent Pst (106 cfu/mL), (-8- ) refers to

inoculation with glucose (2.5 rnM) and glucose oxidase (2.5 UlmL) at the 7 hour time

point, and (+) refers to inoculation with glucose (2.5 mM) at the 7 hour time point.

Time 0 refers to the time of inoculation with I OmM MgC12 and Pst. Error bars denote

standard error. This experiment was repeated once with similar results.

SAR response was not established (figure 23). This phenomenon in which bacterial

growth is limited in leaves manifesting SAR (AVin), but PR gene expression in

systemic (AVun) leaves is not induced has been termed "partial SAR".

SAR is frequently not established, as determined both by a lack of inhibition of

bacterial growth in the AVin treatment and by a lack of PR gene induction in the AVun

treatment. In this situation, AOS nevertheless accumulated following inoculation with

avirulent Pst (figure 20).

2.3 AOS induced cell death in the immunization stage of S A R

It has been proposed that cell death is a component in the signal transduction

pathway resulting in the mobile SAR signal and the establishment of SAR (Dietrich et

a1 1994, Cao et al1994, Ryals et a/ 1994). The finding that generation of H202 using

G/GO results in the establishment of SAR (Alvarez et al1998), provides an

opportunity to determine if cell death is involved in the SAR signalling pathway. Cell

death was measured using an assay for chlorophyll autofluorescence: when the cell

dies, chlorophyll will no longer fluoresce red under blue light. It has been reported as

a measure of cell viability (Ryerson and Heath 1996), and levels of cell death

observed using this assay are comparable to those seen using the cell viability stain

FDA (figure 24). Chlorophyll autofluorescence was therefore used as an assay for cell

viability in leaves immunized with G/GO. Levels of cell death induced by addition of

GIGO were compared microscopically to cell death levels following inoculation with

avirulent and virulent Pst (figure 25). Deaths resulting from inoculation with MgC12

and glucose alone were also observed as controls for wound induced cell death. Cell

death in leaves undergoing an HR occurs rapidly: at 10 hours after inoculation, cell

death in tissue inoculated with avirulent Pst was comparable to that observed in

MgC12 controls (approximate 0 to 6%), while two hours later, 60 to 100% of the tissue

inoculated with avirulent Pst observed was dead (figures 25 and 26). Lack of

chlorophyll autofluorescence was frequently correlated with green or orange

fluorescence under blue light, possibly from the depostion of phenolics. This

autofluorescence has been previously described (Yu e t a1 1993) and has been used

as a marker for the HR in Arabidopsis (Cao et a1 1994, Yu et a1 1998). In contrast,

within the first 14 hours, cell death levels in leaves inoculated with G/GO were

comparable to those seen in leaves inoculated with virulent Pst, 10mM MgC12, or

20mM phosphate buffer with 2.5 mM glucose (figure 25).

When SAR is not established, as determined by PR gene expression patterns

and bacterial growth, cell death is nevertheless observed following immunization with

avirulent Pst (figure 27).

3. AOS accumulation, PR gene expression, and cell death in NahG

A rabidopsis

SA has been proposed to potentiate the increase in AOS accumulation and vice

versa (Shirasu et a1 1997). It is therefore difficult to distinguish the individual roles of

these compounds in each stage of the SAR signalling pathway. Use of NahG

transgenic plants, in which SA does not accumulate, and the GIGO AOS generating

system should prove to be useful in the dissection of the signalling pathway in the

immunization stage of SAR.

3.1 AOS accumulation in NahG Arabidopsis in response to Pst and G/GO

AOS do not accumulate in NahG transgenic plants following inoculation with

avirulent Pst fluorescence levels detected in leaves treated with avirulent bacteria

were similar to those detected in leaves treated with MgC12 or virulent Pst (figure 28).

Figure 26

Chlorophyll autofluorescence in ecotype Col-0 leaf mesophyll under blue light.

Leaves were vacuum infilatrated with water and viewed under blue light. (A)

mesophyll cells 8 hours after inoculation with avirulent Pst, (B) mesophyll cells 12

hours after inoculation with avirulent Pst, (C) mesophyll cells 5 hours after inoculation

with G/GO, (D) rnesophyll cells 12 hours after inoculation with 10 mM MgC12. The bar

represents 50 pm.

Figure 27

Cell death in the immunization stage in ecotype Col-0 failing to establish SAR

following infection with Pst or treatment with the GIGO.

At each time point, three leaves per time point were obselved microscopically under


refers inoculation with lOmM MgCI*,(A-) refers to inoculation with avirulent Pst (1 06

cfu/mL), (t) refers to inoculation with virulent Pst (1 06 cfu/mL), (-@--) refers to

treatment with glucose (2.5 mM) and glucose oxidase (2.5 U/mL) at the 7 hour time

point, and (+) refers to treatment with glucose (2.5 mM) at the 7 hour time point.

Time 0 refers to the time of inoculation with 10 mM MgC12 and Pst Error bars denote

standard error.


Figure 28

AOS accumulation during the immunization stage of SAR in NahG trangenic

Arabidopsis.



in figure 8. (0) refers to inoculation with lOmM MgC12, (-)) refers to inoculation

with avirulent Pst (1 06 cfu/mL), (A-) refers to inoculation with virulent Pst (1 06

cfu/mL). Error bars denote standard error. This experiment was repeated three times

with similar results.

8 9 10 11 12


Figure 29

AOS accumulation in the immunization stage of SAR in NahG transgenic

Arabidapsis following exogenous addition of GIGO.



in figure 8. (0) refers to inoculation with lOmM MgC12, (+) refers to inoculation

with avirulent Pst (1 06 cfu/ml), (I) refers to inoculation with virulent Pst ( I 06

cfu/mL), (-v) refers to treatment with glucose (2.5 mM) and glucose oxidase (2.5

U/mL) at the 7 hour time point, and (+) refers to treatment with glucose (2.5 mM) at

the 7 hour time point. Time 0 refers to the time of inoculation with 10 mM MgC12 and

Pst. Error bars denote standard error. This experiment was repeated once with

similar results.

I I 1 I

7 9 11 13


Figure 30

Cell death in NahG transgenic Arabidopsis following infection with Pst or

treatment with GIGO.

At each time point, three leaves per time point were observed microscopically under


refers inoculation with I OmM MgClz, (* ) refers to inoculation with avirulent Pst

(1 06 cfu/rnL), (A- ) refers to inoculation with virulent Pst (1 06 cfu/mL), (-

inoculation with glucose (2.5 mM) and glucose oxidase (2.5 U/mL) at the 7 hour time

point, and (+) refers to inoculation with glucose (2.5 mM) at the 7 hour time point. A

cross in the center of the symbol indicates a response in Col-0 wild type Arabidopsis.

Time 0 refers to the time of inoculation with 10 mM MgC12 and Pst. Error bars denote

standard error. This experiment was repeated once with similar results.


Figure 31

Chlorophyll autofluorescence in Nahg transgenic leaf mesophyll under blue

light.

Leaves were vacuum infiltrated with water and viewed under blue light. (A) mesophyll

cells 12 hours after inoculation with avirulent Pst (6) mesophyll cells 5 hours after

inoculation with G/GO (C) mesophyll cells 12 hours after inoculation with 10 mM

MgCI2 (D) mesophyll cells 12 hours after inoculation with virulent Pst The bar

represents 50 pm.

Inoculation of NahG transgenic plants with GIGO results in (+) fluorescence (figure

29), comparable to the (+) fluorescence obsewed in wild type plants (figure 21), G/GO

generated Hz02 is detectable for less than two hours (figure 29).

3.2 Cell death in NahG Arabidopsis in response to Pst and GIGO

HR cell death is not observed macroscopically in NahG transgenic plants

(Delaney et a1 1994). When NahG plants were inoculated with avirulent Pst, no

microscopic cell death was observed: cell death remained at approximately 0-6%,

comparable to levels observed in tissue treated with MgC12 or virulent Pst (figure 30).

Inoculation of NahG transgenic plants with G/GO did not result in an increase in cell

death beyond these background levels (figure 30 and 31).

3.3 PR gene expression and bacterial growth in NahG plants in response to Pst

and GIGO

The PR-1 gene was not expressed in NahG plants in response to avirulent or

virulent Pst, or in response GIGO (figure 32). PR-1 was expressed in the wild type

controls; however, only in leaves inoculated with Pst (the MAin, MVin, and AVin

treatments) and not in the uninoculated leaves of plants immunized with avirulent Pst

(AV,,; figure 32). This suggests that SAR was not established. A five fold inhibition

of bacterial growth was observed in the wild type plants (figure 33) indicating that

"partial SAR" was manifested in this experiment. Bacterial growth was not limited in

NahG Arabidopsis following inoculation with avirulent Pst (MA) or in leaves immunized

with avirulent Pst (AV): no statistically significant difference ( ~ ~ 0 . 0 5 ) was observed

between avirulent or virulent bacterial growth, or in virulent bacterial growth in plants

previously immunized with avirulent Pst (figure 30).

Figure 32

Northern analysis of wild type and NahG transgenic leaf total RNA probed with

PR-1 cDNA

(MM) refers to immunization and challenge with 10mM MgC12 (MA) refers to

immunization with 10mM MgC12 and challenge with avirulent Pst (1 06 cfu/rnL), (MV)

refers to immunization with I OmM MgCIz and challenge with virulent Pst (1 06 cfu/mL),

(AV) refers to immunization with avirulent st (1 07 cfu/mL) and challenge with virulent

Pst (1 o6 cfu/mL). (In) denotes challenge inoculated leaves and (un) denotes

uninoculated, systemic leaves. Tissue was harvested 24 hours after the challenge

inoculation. (A) Col-0 leaf RNA, (B) NahG leaf tissue.

Figure 33

Bacterial growth in ecotype Col-0 and NahG transgenic Arabidopsis.

(MM) refers to immunization and challenge with lOmM MgC12 (MA) refers to

immunization with 10mM MgC12 and challenge with avirulent Pst (I 06 cfu/mL), (MV)

refers to immunization with 10mM MgC12 and challenge with virulent Pst (106 cfu/mL),

(AV) refers to immunization with avirulent Pst (1 07 cfu/mL) and challenge with virulent

Pst (106 cfu/mL), (GV) refers to immunization with glucose (2.5 rnM) and glucose

oxidase (2.5 U/mL), and challenge with virulent Pst (106 c f u h l ) , and (PV) refers to

immunization with glucose (2.5 mM), and challenge with virulent Pst (1 06 cfu/mL).

Error bars denote standard deviation. (*) indicates significant difference from MV

treatment at p10.05.

Discussion

The phenomenon of systemic acquired resistance (SAR) has been studied for

the past sixty years, yet despite this, the signalling pathway and responses involved in

the establishment and manifestation of SAR remain relatively unknown. Salicylic acid

(SA) has been proposed to be involved in the signalling (Malamy et a1 1990, Metraux

et a1 1990, Shulaev et a1 1995), establishment (Vernooij et a1 1994), and manifestation

stages of SAR in tobacco, active oxygen species (AOS) have been implicated in the

signalling pathway in tobacco and Arabidopsis (Alvarez et a1 1998, Champagnopol et

a1 1998), pathogenesis related (PR) protein expression can be correlated with the

establishment of SAR in tobacco and Arabidopsis (Ward et a1 1991, Uknes et a1 1992),

and lignification and peroxidase activity have been observed in the manifestation

stage in cucumber (Harnmerschmidt and Kuc 1982, Harnmerschmidt et a1 1982).

However, an understanding of the mechanisms involved in the signalling,

establishment and manifestation of SAR is by no means complete.

1. Characterization of the manifestation stage of SAR

The manifestation of SAR in Arabidopsis is not well characterized. PR proteins

accumulate (Alvarez et al1998), bacterial growth is limited, and HR-like cell death is

occasionally observed (Cameron et a1 1994). To further characterize this final stage of

SAR, the expression pattern of several genes induced during a gene-for-gene

response (eli 11, 18, and ap3) and the accumulation of AOS have been determined.

1.1 Expression of eli 1 I , eli 18, and ap3 in the manifestation of SAR

A number of genes determined to be upregulated during a gene-for-gene

interaction have been isolated from parsley and Arabidopsis (Trezzini et a1 1993).

Determining the expression pattern of these genes during the manifestation and

establishment stages of SAR allows for the further characterization of the SAR

response. Observation of the expression patterns in the SAR defective mutant dirl

might also provide insight into the mechanisms involved in SAR. Preliminary

expression patterns were obtained for three elicitor activated (eli) genes: eli I I, eli 18,

and ap3.

Eli I I and ap3 encode two peroxidases (Trezzini et a1 1993). During the

manifestation of SAR, peroxidase activity increases in cucumber (Hammerschmidt et

a1 1982) suggesting that upregulation of peroxidase expression could be involved in

this stage. Preliminary evidence demonstrated that ap3 was constitutively expressed,

although a two fold induction in transcription was observed following inoculation with

avirulent or virulent Pst and in leaves manifesting SAR. AP3 may be involved in the

manifestation of SAR; however the observation of its increased expression in a

disease response indicates that its upregulation may not be specific to the SAR

response. Transcription of the eli I I peroxidase was induced in Ws plants in

response to avirulent Psi, although unequal loading of the RNA sample and the lack of

repetition of this analysis make conclusions impossible. The in planta induction of ap3

and eli 7 I expression in Ws following inoculation with avirulent Pst was slight

compared to that observed in cell culture after 1.5 hours in response to elicitation with

Pmg (Trezzini et a1 1993). While plant responses to incubation with preformed

elicitors such as Pmg are generally more rapid than to bacteria such as Pst, it is

nevertheless possible that ap3 expression was elevated to a greater extent prior to 24

hours after inoculation, the time at which these samples were collected.

The eli 18 gene encodes S-adenosyl-L-methionine synthase, an enzyme

involved in the activated methyl cycle (Trezzini et a1 1993) which converts methionine

to S-adenosyl-rnethionine (SAM; Somssich and Hahlbrock 1998). SAM may be

involved in the biosynthesis of ethylene, a potential signal during plant defence

responses (Dangl et a1 1996), and in the biosynthesis of potentially antimicrobial

furanocoumarins (Somssich and Hahlbrock 1998, Sticher et a1 1997). Expression of

eli 18 transcripts were increased following inoculation with avirulent Pst and in the

manifestation stage of SAR in wild type plants. In dirl mutant plants, upregulation of

transcription was observed following infection with avirulent Pst but not in the

manifestation stage of SAR. This preliminary evidence supports the hypothesis that

dirl is deficient in the ability to produce or perceive the SAR signal. Increased

expression of eli 18 may prove to be important during SAR, however, further analysis

is necessary before conclusions can be made.

Eli 11 and eli 18 gene expression did not occur in systemic leaves of plants

immunized with avirulent Pst, and ap3 is constitutively expressed; therefore these

genes should not be considered as markers for the establishment stage of SAR. It

should be noted, however, that a partial SAR response was observed in this

experiment. Replication of these results is necessary in order to determine if these

genes are involved in the SAR response and whether they may be useful as molecular

markers for SAR.

1.2 AOS in the manifestation stage of SAR

The oxidative burst has been demonstrated in a variety of plant species during

a gene-for-gene defence response. This burst has been detected in pianta by a

number of methods, including use of the probe dichlorofluorescin diacetate (DCFH-

DA; Allan and Fluhr 1997, Lu and Higgins 1998, Heath 1998). Using the probe

DCFH-DA, two assays, one qualitative and one quantitative, have been developed for

detection of AOS in the Arabidopsis-Pst pathosystem.

The quantitative method developed for measurement of DCFH-DA

fluorescence in response to Pst infection was not successful. Results obtained were

inconsistent, both between replications of the experiment and with results obtained

using the qualitative approach. lnconsistencies could be explained by the lengthiness

of the procedure: approximately 1 hour passed between the infiltration of the leaves

with DCFH-DA, and the fluorometric readings. It is therefore likely that a certain

degree of autooxidation of the probe occurred (Heath 1998, Wofe 1998), obscuring

the results. Additionally, centrifugation of the leaves to obtain intercellular fluids likely

resulted in cellular damage, causing the release of antioxidants such as catalase,

superoxide dismutase, or glutathione-S-transferase. AOS may also be released. As

cells die, AOS are thought to be released from the peroxisome (Bestwick et a1 1997).

AOS also increase with the decline of photosynthesis: pigments release singlet

oxygen species, superoxide is transferred from electron carriers, and lipoxy radicals

are produced in the thylakoid membrane (Baker and Orlandi 1998).

The qualitative approach developed is not ideal. Small, localized regions of

fluorescence would not be detected, since the technique was not adapted for

microscopic use. The simplicity and speed of the qualitative protocol generated

consistent results, however, and was therefore used for a majority of the experiments

involving detection of AOS accumulation.

An oxidative burst was observed in Arabidopsis by 7 hours after inoculation with

avirulent Pst. However, no hydrogen peroxide accumulation during the manifestation

of SAR between 7 and 23 hours after a challenge inoculation was detected at a

macroscopic level. The difference between levels of AOS accumulation during a

gene-for-gene interaction and in the manifestation of SAR indicates that the

manifestation stage of SAR is not identical to the HR; although certain responses may

occur in both processes, it appears that macroscopic AOS accumulation occurs only in

the gene-for-gene, and perhaps in non host (Mellersh and Heath, unpublished)

resistance responses. Considering that the SAR response differs from the gene-for-

gene response in at least one respect, it will be interesting to see if SAR-specific

responses will be discovered.

2. AOS accumulation and cell death in the immunization stage of SAR

Direct application of H202 does not result in the establishment of SAR in

tobacco (Neuenschwander et al1995). However, due to the efficiency of antioxidant

enzymes, direct application of H202 may be ineffectual. When AOS are allowed to

accumulate in planta, whether through exogenous addition of an H202 generating

system or through a suppression of catalase expression, an inhibition of bacterial

growth or accumulation of PR-1 results, suggesting that AOS are involved in the SAR

signalling pathway (Alvarez et a1 1998, Champangopol et al1998).

Hz02 induces microscopic cell death in planta in Arabidopsis (Alvarez et a1

1998). However, in Arabidopsis plants inoculated with the GIGO Hz02 generating

system, cell death of the magnitude observed during the HR was not obsewed (this

thesis). While contradicting previous results, this finding does not disprove them. The

levels of H202 detected following addition of G/GO are less than those observed

following inoculation with avirulent Pst. AOS have been proposed to initiate cell death

when a certain threshold level has been reached (Levine et al1994): i t is therefore

possible that the level of Hz02 required to trigger cell death was not attained in these

experiments.

it is also possible that a prolonged period of AOS accumulation is necessary for

the initiation of HR cell death. AOS accumulation can be seen up to 6 hours before

cell death is observed in leaves treated with avirulent Pst. By contrast, Hz02

production following G/GO addition frequently does not persist beyond 2 hours.

Future experiments to determine whether higher or more prolonged levels of G/GO

generated Hz02 induce HR-like cell death could answer this question.

Recent evidence suggests that AOS alone are insufficient to induce cell death.

In soybean cell culture, addition of G/GO does not result in cell death, nor does

addition of the NO donor system sodium nitroprusside (SNP). When SNP is added in

conjunction with G/GO, however, cell death is induced to levels comparable to those

observed during a Psginduced HR (Delladone et al1998). In tobacco plants, NO was

found to induce PR gene expression in inoculated leaves (Durner et a1 1998). NO

might therefore be necessary for AOS induced cell death in planta, and may have a

role in SAR signalling.

Cell death does not appear to be necessary for the production of a mobile SAR

signal. Despite the inability of 2.5 units/mL G/GO to induce cell death, it nevertheless

produced a SAR response as measured by bacterial limitation following challenge with

virulent Pst. This implies that an HR-like cell death is not necessary for the

establishment of SAR. Substantiating these results, the dnd mutant in Arabidopsis.

which does not mount an HR in response to avirulent Pst, has been found to inhibit

virulent pathogen growth to levels similar to those observed during the SAR response

(Yu et a1 1998).

2.1 AOS accumulation in mutants defective in the establishment and

manifestation of SAR

The Arabidopsis mutant dirl is unable to establish SAR. Macroscopical[y, it

has retained the ability to mount an HR, and SA levels appear to be unaffected,

indicating that the mutation may be downstream of the production of the mobile SAR

signal (Cameron et a1 unpublished). It can be predicted that AOS accumulate in the

immunization stage in dirl mutant plants as it does in wild type. When infected with

avirulent Pst, dirl plants accumulate AOS to levels comparable to similarly treated

wild type plants. The lesion resulting in the inability of dirl to establish SAR is

therefore likely downstream of AOS accumulation during the HR, and DlRl does not

affect a plant's ability to accumulate AOS during a gene-for-gene response.

Mutants in nprl retain the ability to mount a functional HR in response to

Psm(avrRptZ), as measured by autofluorescence of phenolics, necrotic lesions, and a

reduction in bacterial growth (Cao et a1 1993). A reduction in in planta bacterial

growth was observed in response to Pst(avrRpt2). It is therefore not surprising that

nprl retains the ability to accumulate AOS in response to infection with avirulent Pst.

Mutants in nprl cannot establish and manifest SAR. Consequently, the lesion in nprl

plants is thought to lie within the SAR signalling pathway. Since AOS accumulation

results in the induction of SARI it is likely that the nprl lesion lies downstream of AOS

accumulation in the immunization stage of SAR.

3. AOS accumulation and cell death in NahG Arabidopsis

SA is required for the establishment of SAR. NahG transgenic plants contain

the bacterial SA degrading enzyme salicylate hydroxylase (NahG; Delaney et a1

1994), and therefore do not accumulate SA or establish SAR. SA has been proposed

to be the mobile SAR signal (Shulaev et a1 1995) and is thought to have a role in the

establishment stage of SAR (Vernooij et a1 1994), as well as in the HR and in disease

interactions (Delaney et al1994, Gaffney et a1 1993). The precise SA signalling

pathway has not been elucidated, however. Following immunization, SA and H202

have been proposed to potentiate their accumuiation until a certain concentration

threshold has been reached, after which either SA or Hz02 or both will act in the SAR

signalling pathway (Shirasu et a1 1997). This model is supported by the finding that

NahG transgenic plants, which do not accumulate SA, also do not accumluate AOS as

measured macroscopically using DCFH-DA. This is not due to an increase in

antioxidant activity, since addition of G/GO to NahG plants results in H202 levels

similar to those seen in wild type plants. Hz02 accumulation during a gene-for-gene

response therefore appears to be an SA-dependent process.

If AOS have a role downstream of SA accumulation in the immunization stage

of SARI NahG plants may not establish SAR due to a failure to accumulate AOS. The

observation that exogenous addition of the G/GO Hz02 generating system results in

the establishment of SAR in wild type plants supports this idea, but does not prove it:

the addition of H202 may result in an increase in SA (Leon et a1 1995, WU et a1 1997),

which will then act as a SAR signal (Shulaev et a1 1995). Studies to determine

whether H202 is downstream of SA in the SAR signalling pathway resulting in the

production of the mobile SAR signal might be examined by supplementing SA

deficient plants with exogenous H202: if Hz02 is downstream of SA in the signalling

pathway, SAR should be established. However, this potentially simple experiment is

obscured by the fact that SA is also required in the establishment and manifestation

stages of SAR (Vemooij et al1994). Even if the role of SA in the immunization stage

of SAR is only to potentiate the increase of H202, a H202-mediated signal would not

be perceived in NahG plants where SA does not sccumulate in distal leaves.

Extrapolation of results from grafting experiments involving NahG and wild type

tobacco contradict the idea that accumulation of Hz02 can overcome a deficiency in

SA in the signalling stage of SAR. Results from this thesis demonstrate that NahG

Arabidopsis do not accumulate AOS in response to avirulent Pst. The assumption that

a NahG tobacco rootstock will also likely not accumulate AOS can be made. Were

AOS required for producing the mobile SAR signal, SAR would not be established in a

wild type scion grafted onto a NahG stock. This is not the case; the wild type scion

establishes SAR as measured by PR gene expression and limitation of pathogen

growth (Vernooij et a1 1994). This does not irrevocably negate a necessity of AOS in

the immunization stage of SAR, however. AOS accumulation was observed

macroscopically. It is possible that "microbursts" similar to those detected in systemic

leaves (Alvarez et a1 1998) are present in NahG transgenic plants and play a role in

the production of a mobile SAR signal.

Indeed, the levels of GIGO generated Hz02 able to induce SAR were lower

than those observed during a gene-for-gene interaction. The threshold level

necessary for SAR signalling was not determined: it is conceivable that Hz02 will

signal for SAR at levels much lower than those used in this thesis. Future studies to

determine the level at which AOS induce the establishment of SAR, and at which they

may be necessary for localized defence responses such as the strengthening of cell

wall barriers, in antimicrobial action, or in a potential induction of HR cell death, may

prove to be interesting.

Two possible responses may be occurring in the immunization stage. In the

first, a potentiation cycle could result in a large accumulation of SA and AOS, which is

necessary for the initiation of localized defence responses such as gene-for-gene

responses and the manifestation stage of SAR. These high levels of SA and/or AOS

in the immunization stage may result in the production of a mobile SAR signal: G/GO

generated H202 accumulation could potentiate an increase in SA which acts as the

mobile SAR signal. In the second, SA and AOS levels could remain low. Regulation

of SA may occur through the sequestering of SA in the chloroplast, thereby

inactivating it. Upon elicitation, low levels of SA may be released into the cytosol

(Vernooij et a1 1994) where they are involved in the production of a mobile SAR signal.

This process may involve AOS accumulation at levels too low to be detected using

DCFH-DA, or may involve "microbursts" of AOS requiring a microscopic detection

assay.

3.1 Cell death and SA accumulation

Whether SA accumulation is required for HR cell death remains open to

question. The gene-for-gene HR is abolished in NahG plants both macroscopically

(Delaney et a1 1994) and microscopically (this thesis), since no significant cell death

was detected using chlorophyll autofluorescence as a cell death assay. This suggests

that accumulation of SA is required for HR cell death. In contrast, crosses between

spontaneous lesion forming mutants (Isd in Arabidopsis and catalase antisense

transgenic tobacco) and NahG plants result in F1 progeny that retain the ability to form

lesions (Weymann et a1 1995, Du and Klessig 1997). It is possible, however, that the

lesions in the lsd Arabidopsis lines that trigger cell death lie downstream of a

requirement for SA in the initiation of cell death, or that LSD proteins are not involved

the HR. Variation between results obtained in NahG catalase antisense tobacco (Du

and Klessig 1997) and observation of the effects of G/GO addition to NahG

Arabidopsis could be due to differences in the responses of Arabidopsis and tobacco,

or could be due to differences in the AOS levels involved: tobacco with suppressed

catalase activity may accumulate AOS to cytotoxic levels: the cell death induced may

not occur through similar mechanisms as those involved in the HR.

The finding that NahG plants do not mount an HR can be correlated with their

inability to accumulate AOS, a correlation which implicates AOS as a signal for HR cell

death. However, addition of Hz02 did not result in cell death in either wild type or

NahG plants, suggesting that AOS accumulation alone is not sufficient to signal for the

HR. Recent evidence suggests that nitric oxide is necessary for AOS signalling of cell

death (Delledone et al1998). It will be interesting to determine whether the addition of

an NO donor system and an AOS generating system to NahG plants will result in cell

death. These experiments may differentiate whether SA signalling for cell death

merely induces the accumulation of AOS and potentially NO (Durner et al1998), or

whether SA is involved further downstream in the SAR pathway.

4. Different signalling pathways may result in the establishment of

SAR

In gene-for-gene responses, the observation of AOS accumulation has been

correlated with a limitation of pathogen growth. During the manifestation of SAR,

however, bacterial limitation is observed in the absence of AOS levels comparable to

those observed during a gene-for-gene interaction, suggesting that a macroscopic

oxidative burst is not required for pathogen limitation. Characterization of mutants

defective in other defence responses indicate that AOS accumulation is not unique in

this respect. Arabidopsis mutants in nprl are deficient in their ability to accumulate

PR proteins, yet can mount an HR and limit pathogen growth in response to

Psm(avrRpf2) (Cao et a1 1994) and Pst(avrRpt2). The dnd mutant is defective in HR

cell death in response to avirulent pathogens, yet pathogen growth is limited (Yu et a1

1998). The phytoalexin deficient (pad) mutants are deficient in the ability to produce

phytoalexins, yet inhibition of pathogen growth is observed (Hain et al 1 993). PR

proteins, cell death, phytoalexins, and AOS each are not sufficient for resistance.

It has been suggested that the disruption of one response, such as cell death,

can be compensated by an increase in other responses (Yu et a1 1998). The

possibility remains that, even if AOS and SA accumulation is involved in signalling for

SAR, when their accumulation is prevented another pathway may compensate. The

role of cell death in the signalling pathway of SAR may be equally obscured. It is

possible that the establishment of SAR is initiated by any one of a number of events in

the immunization stage. For instance, in a disease interaction or in lsd plants, SAR

might be established through a cell death pathway. By contrast, addition of Hz02

could result in the establishment of SAR through a cell death-independent pathway.

The roles of AOS and cell death in the production of the SAR signal have yet to be

elucidated; future advances in this field should prove to be interesting.

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INFORMATION TO USERS · SAR 4.1 The relationship between SA and cell death 28 4.2 The role of cell death in the immunization and 29 establishment stages of SAR 4.3 Summary of the