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The Pennsylvania State University

The Graduate School

Intercollege Graduate Program in Plant Physiology

MOLECULAR GENETIC ANALYSIS OF CELL DEATH AND DEFENSE

SIGNALING IN ARABIDOPSIS hrl1 MUTANT

A Thesis in

Plant Physiology

Sendil Devadas

Submitted in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy

May 2002 We approve the thesis of Sendil Devadas.

Date of Signature

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Ramesh Raina Assistant Professor of Biology Thesis Advisor Chair of Committee

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Richard J. Cyr Professor of Biology

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Hong Ma Associate Professor of Biology

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Timothy W. McNellis Assistant Professor of Plant Pathology

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Teh-hui Kao Professor of Biochemistry and Molecular Biology Chair of the Intercollege Graduate Program in Plant Physiology

ii ABSTRACT

Defense against pathogens in Arabidopsis is orchestrated by at least three signaling molecules: salicylic acid (SA), jasmonic acid (JA), and ethylene (ET). The hrl1 (for hypersensitive response-like lesions 1) mutant of Arabidopsis is characterized by spontaneous necrotic lesions, accumulation of reactive oxygen species (ROS), constitutive expression of SA- and ET/JA-responsive defense genes, and enhanced resistance to virulent bacterial and oomycete pathogens. Epistasis analyses of hrl1 with npr1, etr1, coi1, and SA-depleted nahG plants revealed novel interactions between SA and ET/JA signaling pathways in regulating PR gene expression and cell death. Northern analysis of the RNA isolated from the lesion+ (local) and the lesion− (systemic) leaves of double mutants of hrl1 uncovered different signaling requirements for the expression of defense genes in these tissues. Expression of ET/JA-responsive PDF1.2 gene was markedly reduced in hrl1 npr1 and in the SA-depleted hrl1 nahG plants. In hrl1 nahG plants, expression of PDF1.2 was regulated by benzathiadiazole (BTH) in a concentration dependent manner: induced at low concentration and suppressed at high concentration. The hrl1 etr1 plants lacked systemic PR-1 expression, and exhibited compromised resistance to virulent Pseudomonas syringae. Inhibiting JA responses in hrl1 coi1 plants lead to exaggerated cell death and severe stunting of plants. Finally, the hrl1 mutation led to elevated expression of AtrbohD, which encodes a major sub-unit of the NADPH oxidase complex. These results indicate that cell death and defense induction in hrl1 is regulated synergistically by SA and ET/JA defense pathways.

The preexisting defense responses enable hrl1 plants to resist virulent pathogen infection. However, the same preexisting defense responses in hrl1 suppress HR cell death against virulent pathogens. Furthermore, the high PR-1 expression observed in hrl1 remains unaltered following avirulent and virulent pathogen infections. The suppressed HR phenotype in hrl1 is observed even when an elicitor is expressed endogenously from an inducible promoter, suggesting that an impaired transfer of avirulent factors is not the reason. Interestingly, the lack of HR phenotype in hrl1 is reversed if the constitutive defense responses are compromised either by a mutation in NPR1 or by depleting

iii salicylic acid (SA) due to the expression of the nahG gene. The rescue of HR cell death in hrl1 npr1 and in hrl1 nahG depends on the extent to which the constitutive systemic acquired response (SAR) is compromised. Pre-treating Arabidopsis wild-type plants with SAR-inducers, prior to pathogen infection resulted in a significant decrease in HR cell death. The suppressed HR cell death and the failure to enhance PR gene expression following avirulent pathogen infection in hrl1 suggest that the preexisting defense responses serve as negative feedback loops to regulate extensive cell death and defense- related gene expression. In hrl1, the chronic stress response due to PR gene expression may alter the cellular physiology at various nodes that lead to insensitivity to subsequent pathogen attack. Down-regulation of HR cell death in the presence of an already existing systemic resistance response may be a way for plants to prevent excessive cell death and further defense induction

Microarray analyses of plant-pathogen interactions have revealed a complex interplay of multiple signaling pathways (Schenk et al., 2000; Maleck et al 2000). The changes in the transcript levels of approximately 1200 Arabidopsis ESTs were examined in hrl1 and the various double mutants of hrl1. Consistent with the earlier Northern results, genes like PR-1, PR-2, GST1 and PDF1.2 were induced both in the lesion+ and in the lesion- tissue of hrl1. However several GST1-like and PR-1-like genes were not induced in the lesion- tissue suggesting that they are regulated differently in the lesion+ and lesion- tissue. The lesion+ tissue had a stronger overall response than the lesion− tissue suggesting that the lesion+ tissue mimic local HR cell death and the lesion− tissue resemble the distant SAR response. Approximately 50 genes (22%) that have altered expression pattern in hrl1 encode proteins that are involved in metabolism. This suggests that cell death and defense signaling in hrl1 may interact with components involved in cellular homeostasis. These studies further highlight the complexity involved in the regulation of the defense response in plants against pathogens.

iv TABLE OF CONTENTS

LIST OF FIGURES……………………………………………………………… vii

LIST OF TABLES………………………………………………………………. ix

ACKNOWLEDGMENTS….……………………………………………………. x

CHAPTER 1 Introduction to Plant Defense and Cell Death During Pathogen Attack…………………………………………………… 1

CHAPTER 2 Identification and Analysis of a Spontaneous Cell Death Mutant in Arabidopsis………………………………………………………… 8

Introduction……………………………………………….. 8 Materials and Methods……………………………………. 10 Results…………………………………………………….. 17 Discussion………………………………………………… 29

CHAPTER 3 Role of Salicylic Acid and NPR1 in Cell Death, Gene expression, and Disease Resistance in hrl1……………………………………. 33

Introduction………………………………………………. 33 Materials and Methods…………………………………… 35 Results……………………………………………………. 38 Discussion………………………………………………... 50

CHAPTER 4 Effects of Ethylene and Jasmonic Acid Signaling in hrl1………… 54

Introduction……………………………………………….. 54 Materials and Methods……………………………………. 56 Results…………………………………………………….. 58 Discussion………………………………………………… 68

CHAPTER 5 Analysis of the Suppressed HR Phenotype of hrl1……………….. 74

Introduction……………………………………………….. 74 Materials and Methods……………………………………. 77 Results…………………………………………………….. 79 Discussion………………………………………………… 91

CHAPTER 6 Global Analysis of Gene Expression in hrl1………………………. 97

Introduction……………………………………………….. 97 Materials and Methods……………………………………. 100 Results…………………………………………………….. 106

v Discussion…………………………………………………. 115

LITERATURE CITED……………………………………………………………. 122

APPENDIX A: Preexisting systemic acquired resistance suppresses hypersensitive response-associated cell death in Arabidopsis hrl1 mutant …….... 142

APPENDIX B: The Arabidopsis gain-of-function mutant dll1 spontaneously develops lesions mimicking cell death associated with disease……………. 144

vi LIST OF FIGURES

Figure 2-1: Phenotype of hrl1 mutant at various stages of growth…………… 18

Figure 2-2: Accumulation of defense-related biochemical markers in hrl1...... 20

Figure 2-3: ROI accumulation in hrl1 mutant………………………………… 21

Figure 2-4: Analysis of defense-related gene expression in hrl1……………... 23

Figure 2-5: Growth of bacterial and oomycete pathogens in hrl1 Mutant……. 25

Figure 3-1: Phenotype of hrl1 double mutants………………………………… 39

Figure 3-2: Comparison of SA and SAG Levels in hrl1, Col-0, nrp1, nahG, hrl1 npr1, and hrl1 nahG Plants…………………………… 41

Figure 3-3: Effects of npr1 on defense-related gene expression in hrl1………. 43

Figure 3-4: Effects of nahG on defense-related gene expression in hrl1……… 44

Figure 3-5: Expression of PR-1 and PDF1.2 genes in hrl1 and hrl1 nahG . . Plants in response to BTH treatments…………………………….. 47

Figure 3-6: Effects of npr1, nahG and on Growth of Pst DC3000 in hrl1……. 48

Figure 3-7: Effects of npr1, and nahG on Growth of P. parasitica Ahco2 in hrl1…………………………………………………………….. 49

Figure 4-1: Double mutants of hrl1 with etr1, coi1 and transgenic hrl1 coi1 nahG…………………………………………………… 59

Figure 4-2: Ethylene levels in the lesion+ and lesion- tissue of hrl1………….. 60

Figure 4-3: Expression of PR-1 and PDF1.2 genes in hrl1 and Col-0 Plants . in response to SA and MJ……………………………………….. 62

Figure 4-4: Effects of etr1 and coi1 on the expression of defense-related . genes in hrl1 mutant……………………………………………… 63

Figure 4-5: Effects of etr1 on Growth of Pst DC3000 in hrl…………………. 65

Figure 4-6: Effects of etr1 on Growth of P. parasitica Ahco2 in hrl1……….. 66

vii Figure 5-1: Suppressed HR of hrl1 in response to avirulent pathogen……. 80

Figure 5-2: Electrolyte leakage and pathogen growth in hrl1 after . avirulent pathogen infiltration……………………………….. 81

Figure 5-3: Analysis of PR-1 expression in hrl1 and Col-0 after pathogen . infection and BTH treatment…………………………………. 84

Figure 5-4: Defense-related gene expression analysis in hrl1 and other double . mutants of hrl1. ……………………………………………… 87

Figure 5-5: Quantitative representation of the HR in hrl1, hrl1 npr1 and . hrl1 nahG plants following avirulent pathogen infection…… 89

Figure 5-6: Electrolyte leakage in SAR-induced Col-0, npr1 and nahG . plants following avirulent pathogen infection……………….. 90

Figure 5-7: A model for the possible suppression of HR cell death by . pre-existing SAR pathways…………………………………. 94

Figure 6-1: Graphical representation of K-means clustering of . differentially expressed genes in hrl1 and the double mutants...... 114

Figure 6-2: Functional classification of differentially expressed genes in hrl1...... 119

viii LIST OF TABLES

Table 2-1: Primer sequences for CAPS markers…………………………… 26

Table 2-2: Restriction patterns for the CAPS markers…………………….. 27

Table 2-3: Recombination analyses for the hrl1 mutation based on the . known marker positions……………………………………….. 28

Table 6-1: Global gene expression pattern in hrl1 and the double mutants... 106

Table 6-2: Categorization of genes based on their induction pattern………. 117

ix ACKNOWLEDGMENTS

I am greatly indebted to Dr. Ramesh Raina, my thesis advisor – who gave me an opportunity to learn and practice Science in his lab. His endless enthusiasm for experiments and to teaching undergraduates has always been inspirational. There was never a time in the lab when he did not have time for his students nor was any of us left short on resources. Besides all these, the fact that he believed in me more than I did gave me the strength I needed during difficult times.

Dr. Richard Cyr, I sincerely thank you for your valuable suggestions not just on my research but also for your helpful advice on my career and future. Dr. Hong Ma, I sincerely thank you for your continuous assistance in directing my research and also for your exceptional advice on several occasions. Dr. Tim McNellis, I thank you for all the valuable advice and also for providing me with some important clones and constructs.

I am extremely grateful to Dr. Nina Fedoroff, for her valuable insights and for letting me use her lab premises through out my research. I am also thankful for her lab personnel for various practical suggestions and discussions.

I sincerely thank the Plant Physiology program and its chair Dr. Teh-Hui Kao for the support and encouragement through out my graduate study. I also thank the Biology Department for granting me teaching assistantships and several travel awards, which enabled me to successfully complete this research.

One of the greatest experiences of any graduate study is enjoying the lab “atmosphere”. I was blessed with incredible lab-mates who made my stay a thoroughly enjoyable one. Anamaria Gomez, thank you for all the great things – from demonstrating RNA isolation to teaching Spanish. Rachel Pilloff, thanks for being a great friend and for your several thoughtful deeds. Guru Jagadeeswaran, thank you very much for always encouraging me to be positive and also for all those sumptuous dinners. I will never forget the lively discussions I had and all the help I received from a lot of undergraduates in the lab, especially August Denicco, Rohit Soans, Derek Dreschel, Preeti Shah, Suzanne

x Sayles, Nathan Majenski, and Michael Brincat. I thank you all for encouraging me to recover from the inevitable rejects and also to rejoice when my very first paper got accepted.

I extend my warmest gratitude to Dr. John Bylander for all those scientific discussions on the golf course. Thank you for introducing me to the most fascinating sport I ever played and also for reinforcing in me the belief “Remember only your good shots”. I would like to thank Drs. Bob Dietrich and Manuel Sainz at Syngenta Biotechnology Inc., who provided me with an opportunity to experience the other side of research and also for their words of support.

Finally, I would like to thank those who mean a lot to me personally. My wonderful parents Dr. Ambalavanan Devadas and Mrs. Chandralekha Devadas whose sacrifice and unconditional love through all these years have helped me achieve one of my dreams – a Ph.D. My sister, Dr. Aneetha Bharathi has always been a symbol of strength and perseverance for me to look up to. Thank you for all the support and excitement. My best friend from 3rd grade, Natarajan, was always there when I needed and often performing all the duties of a son to my parents. With you being there, I was able to better concentrate on my research.

Dedicated to my wife Binu

Whose impeccable love and Whole hearted support will reverberate in Every word in this thesis for ever…..

xii

Chapter 1 Introduction to plant defense and cell death during pathogen attack

When a pathogen invades a host plant there are two possible outcomes. The plants may show resistance, thus preventing any further pathogen growth, or they may succumb and develop disease. Interestingly, cell death is often associated with both resistance response (incompatible interaction) and disease susceptibility (compatible interaction) to the invading pathogens (Baker et al., 1997). Successful host resistance against pathogen invasion requires expeditious recognition and activation of necessary defense repertoire. One such robust response in plants involves the recognition of pathogen-derived elicitors and initiation of localized cell necrosis at the site of pathogen infection. Undoubtedly, the most noticeable feature of this type of resistance response is the rapid cell death that is well defined within the attempted infection site, a process known as hypersensitive response (HR) (Agrios, 1988). Ion channel fluxes, production of reactive oxygen intermediates (ROI), fortification of cell walls, synthesis of anti-microbial compounds and induction of several defense related genes are presumed to contain the pathogen spread during HR (Dixon et al., 1994). Several individual plant genes have been identified that control the gene-for-gene resistance, and these genes are known as resistance (R) genes (Bent, 1996). The corresponding pathogen gene is called the avr gene if its expression produces a signal that can trigger a strong defense response in a plant with the cognate R gene (Alfano and Collmer, 1996; Keen, 1990). A distinguishing hallmark of most gene-for-gene interactions is the hypersensitive response (HR). HR is characterized as an incompatible interaction involving elicitor recognition by the host R gene product. Following HR, a broad-spectrum resistance called systemic acquired resistance (SAR) is established in the uninfected distal tissues (Ryals et al., 1996). Once SAR is induced, the entire plant is resistant towards any further pathogen attack. Thus HR and SAR constitute the two key aspects of active resistance responses.

1.1 Early events during pathogen perception

Once the plant perceives the invading pathogen, the effectiveness of resistance mechanisms depends on the rapidity and magnitude of the various defense responses. The early events after the recognition of the pathogen include pH changes in the extracellular matrix, generation of reactive oxygen intermediates (ROI) and ion fluxes across the cellular membrane. In parsley suspension cells, a transient influx of Ca2+ and H+ and efflux of K+ and Cl- are initiated within two to five minutes after the addition of a fungal elicitor (Hahlbrock et al., 1995; Yang et al., 1997). Elicitor induced ion fluxes are likely to be mediated by protein phosphorylation and dephosphorylation events (Levine et al., 1994; Ligterink et al., 1997). Increase in intracellular calcium is also presumed to play a role in defense (Levine et al., 1996). This capability is also supported by a recent report of two calmodulin isoforms that are induced in response to pathogen infection in a SA independent fashion (Heo et al., 1999).

The oxidative burst during plant-pathogen interaction follows a biphasic pattern. The accumulation of ROI in phase I occurs within 10-20 minutes after elicitation and the response is both transient as well as non-specific. The phase II accumulation occurs 2-6 h after elicitation and is long-lived and a specific response. The ROI-mediated resistance .- network involves superoxide radical (O2 ), hydrogen peroxide (H2O2), hydroxy peroxyl

(HO2) and nitric oxide (NO) (Alvarez et al., 1998; Delledonne et al., 1998; Desikan et al., 1996; Dixon et al., 1994). Generation of superoxide radical is thought to be dependent on the activation of a plasma membrane bound NADPH oxidase similar to that present in mammalian phagocytes (Desikan et al., 1996). Superoxide is not very reactive at physiological pH and often dismutates to H2O2 and O2 either spontaneously or by the

enzymatic action of superoxide dismutase (SOD). H2O2 is a relatively stable, electrically

neutral molecule. Being not very reactive, H2O2 can easily diffuse across cell membranes

and reach locations remote from the site of its formation. H2O2 can also be generated by peroxidases depending upon the cellular pH and the avaialability of the reductants. In contrast, various peroxidases can use H2O2 as a substrate for the generation of phenoxyl

radicals during lignin synthesis for cell wall fortification. In addition, H2O2 can

disproportionate to form H2O and molecular oxygen either spontaneously or through

catalase activity. However, the mechanisms by which these early events induce and amplify the downstream responses remain relatively unknown.

1.2 Role of cell death in plant pathogen interaction

Cell death takes place in a variety of contexts during plant development, as it does in animals. In fact, the first cells ever to be observed under a microscope by R. Hooke, 334 years ago, were pith cells of cork that had undergone programmed cell death (PCD) (Hooke, 1665). Unlike animal cells, plant cells are fixed in place by their cell walls. Since they have no circulating scavenger cells to dispose of cellular corpses, a tightly regulated mechanism must exist to prevent the untoward spread of toxic waste products to the neighboring cells. This tightly regulated mechanism seems to have allowed plants to effectively utilize cell suicide in response to pathogen ingress (Martienssen, 1997; Williams, 1994). As mentioned earlier, cell death is an essential feature of disease symptoms during compatible interactions also. In many instances during disease progression, host cells are killed by the action of pathogen-derived toxins (Hammond- Kosack and Jones, 1996). It is unknown whether the mechanism of cell death during HR is the same as that invoked during cell death leading to disease symptoms. Furthermore, no systematic study has addressed disease development at a molecular level. Many key questions regarding cell death during pathogenesis remain unanswered. Is the cell death in HR and disease, programmed genetically by the host plant cells, or is it simply killing of plant cells by pathogen-derived toxins? Is HR-associated cell death absolutely required for successful display of defense (Century et al., 1995; Yu et al., 1998)? Are there different cell death pathways controlling HR and disease? What are the executioners of HR mediated cell death (Dangl et al., 1996; Shirasu and Schulze-Lefert, 2000)?

1.3 Defense-related gene expression

In various plant species, numerous genes have been identified whose mRNA levels increase significantly upon pathogen infection (Delaney et al., 1994; Dixon and Harrison, 1990; Hammond-Kosack and Jones, 1996; Ryals et al., 1996; Vernooij et al.,

1994). While both HR (resistance) and disease (susceptibility) induce defense gene expression, the rapidity and the high magnitude of induction during HR seems to play an important role in ensuring resistance to pathogens. Several families of pathogenesis- related (PR) proteins have been characterized from a wide variety of species such as tobacco, tomato, Arabidopsis, parsley, and radish (Van Loon and Van Strien, 1999). Among them, the PR-1 family of genes is one of the tightly regulated genes in plant- pathogen interaction. Ironically, the function of PR-1 protein, the first PR protein to be isolated is still unknown. However, functions of other PR proteins are better understood. For example, PR-2 encodes a b-1,3-glucanase, PR-3 and PR-4 encode chitinases, PR-5 encodes a thaumatin-like protein and PR-6 encodes a proteinase-inhibitor. The rest of the PR protein members encode peroxidases, endoproteinases and thionins (reviewed in Van Loon and Van Strien, 1999). Moreover the induction of several of these genes appears to have a synergistic effect in controlling pathogen growth. Systemic acquired response (SAR) is a result of the concerted expression of a battery of defense genes and hence this form of broad-spectrum resistance is preferred for rendering protection towards pathogens.

1.4 Chemical regulators of defense signal transduction

In plants, the early signals derived from the pathogen are further transduced and amplified by salicylic acid (SA), or jasmonic acid (JA) or ethylene or a combination of these three regulators. The reason why plants regulate most of their PR expression using just three signal molecules is unknown. It is also not known when during evolution these pathways appeared to resist what type of pathogens (Dong, 1998; Reymond and Farmer, 1998). Several lines of evidence have clearly established that SA plays a critical role in the activation of defense responses. First, increases in the levels of SA and its conjugates precede or parallel the expression of defense genes (Dempsey et al., 1997; Uknes et al., 1992). Second, exogenous application of SA induces almost all of the SAR associated defense genes. Third, when SA accumulation is prevented by the expression of the transgene nahG from Pseudomonas putida, which encodes salicylate hydroxylase, both SAR gene induction and resistance to pathogens are compromised (Delaney et al., 1994;

Gaffney et al., 1993). However, at present it is unclear whether SA is the long distance signal that activates SAR in the distant uninfected tissues. Evidence both for and against this notion strongly argue for additional experiments to clarify SA's role as the transported signal (Enyedi et al., 1992; Vernooij et al., 1994).

Ethylene levels have been shown to increase during HR (Enyedi et al., 1992). Ethylene treatment induces certain defense genes and enhances the SA-induced expression of PR-1 in Arabidopsis (Vogeli et al., 1988). In fact, tobacco genes for vacuolar PR-2, PR-3 and PR-5 are induced more efficiently by ethylene than by SA (Xu et al., 1994). Ethylene also plays an important role in disease. In susceptible tomato plants, cell death induced by a fungal elicitor AAL- toxin was partially blocked by the use of ethylene action inhibitors (Moussatos et al., 1994). In support of this finding, Arabidopsis ethylene insensitive mutant, ein2, develops very little or no disease symptoms when infected with a virulent bacterial pathogen (Bent et al., 1992). Besides HR and disease, ethylene also plays a role in the timing of the onset of senescence in plants, a form of programmed cell death (Guarente et al., 1998; Wang et al., 1996). In contrast, induction of resistance response to pathogens seems to be ethylene independent in ein1, ein2 and etr1 mutants of Arabidopsis (Bent et al., 1992; Lawton et al., 1994). From these observations it is clear that ethylene by itself does not induce cell death or resistance to pathogens, but may serve as a modulator of cell death and resistance processes. Jasmonates are involved in various aspects of plant development and pathogen defense (Creelman and Mullet, 1997). Methyl jasmonate along with systemin mediate the defense responses after wounding and insect attack (Ryan and Pearce, 1998; McConn et al., 1997). Recently, it was shown that the induction of a defensin gene PDF1.2 in Arabidopsis needs functional ethylene and jasmonate response pathways but not SA (Penninckx et al., 1998). Treatment of tobacco seedlings with SA and MeJa leads to superinduction of PR-1 gene compared to that observed with SA or MeJa alone (Xu et al., 1994). These inferences strengthen the models that describe inter-digitating networks for SA, ethylene and jasmonate mediated defense responses against pathogen attack as well as in the cell death process.

1.5 Genes controlling expression of defense responses in Arabidopsis

One approach to understanding the signal transduction networks that control defense responses is to use genetic tools to identify signaling components and elucidate their functions within the network. During the last decade, considerable progress has been made using this approach in identifying Arabidopsis genes that regulate defense-related gene expression (Boyes et al., 1996; Dangl et al., 2001; Nimchuck et al., 2001). The non-specific disease resistance 1 (NDR1) and enhanced disease susceptibility 1 (EDS1) genes are required for gene-for-gene mediated resistance to avirulent strains of the bacterial pathogen Pseudomonas syringae and the oomycete pathogen Peronospora parasitica (Century et al., 1995; Century et al., 1995; Parker et al., 1996; Falk et al., 1999). Interestingly, ndr1 mutants are susceptible to one set of avirulent pathogens, whereas eds1 is susceptible to a non-overlapping set of avirulent pathogens (Aarts et al., 1998). NDR1 encodes a protein with two predicted transmembrane domains and EDS1 encodes a protein with similarity to triacyl glycerol lipases. Expression of several PR genes such as PR-1, PR-2 and PR-5 in response to SA requires a gene called non-expressor of PR 1 (NPR1) or non-inducible immunity 1 (NIM1) (Cao et al., 1994; Cao et al., 1997; Shah et al., 1997; Ryals et al 1997). The npr1 mutant plants cannot activate SAR and are highly susceptible to virulent pathogen infections. NPR1 encodes a novel protein with ankyrin repeats (protein-protein interaction domains) and the protein product is localized to the nucleus upon pathogen attack or SA treatment. In the nucleus NPR1 may interact with TGA transcription factors that in turn induce PR gene expression. The coronatine insensitive 1 (coi1) and jasmonate resistant 1 (jar1) mutants of Arabidopsis fail to respond to JA treatment and are susceptible to pathogens like Pythium and Alternaria brassisicola that induce JA-responsive genes (Staswick et al 1998; Xie et al., 1998). COI1 has been cloned and found to encode a protein with a degenrate F-box motif and LRR repeats. Both coi1 and jar1 mutants do not induce PDF1.2, an anti-fungal defensin in response to JA. The ethylene insensitive 2 (EIN2) and the ethylene resistance 1 (ETR1) mutants show increased disease symptoms upon virulent pathogen infection (Bent et al 1992). It has been shown that the concomitant activation of both JA and ethylene signaling is

required for PDF1.2 induction and resistance to A. brassisicola infection in Arabidopsis (Penninckx et al., 1998) Through the identification of the genes discussed above and others, it is now possible to construct at least three independent defense pathways in Arabidopsis that differ significantly in their requirements of signal regulators.

Avirulent pathogens A. brassisicola, P. fluorescens (gene-for-gene resistance) Pythium sp. (colonization)

TIR-NBS-LRR LZ-NBS-LRR R genes R genes JA/COI1

Ethylene JA EDS1 NDR1 Ethylene/ETR1 ETR1 COI1 SA EIN2 JAR1

NPR1 NPR1

PR genes & other PDF1.2 & other unidentified factors resistance factors ?

Resistance to P. syringae, Resistance to Resistance to P.syringae P. parasitica A.brassisicola, F. oxysporum Pythium sp.

SA-dependent JA/ethylene- Induced systemic SAR dependent SAR resistance (ISR)

Chapter 2

Identification and analysis of a spontaneous cell death mutant in Arabidopsis

2.1 Introduction

In recent years, identification and analysis of several Arabidopsis mutants with altered response to pathogens and SAR-inducing chemicals have helped unravel the molecular basis of defense activation in plants. This screening approach has been useful in finding several new pathways that are under both negative and positive control. By screening for mutants that sponatanesously show resistance and cell death, the molecular genetic elements that control defense regulation can be elucidated. Several mutants constitutively express resistance to virulent bacterial and oomycete pathogens and accumulate high levels of SA. They include accelerated cell death (acd) (Greenberg et al., 1994; Rate et al., 1999), lesions simulating disease resistance (lsd) (Dietrich et al., 1994; Weymann et al., 1995), constitutive PR (cpr) (Bowling et al., 1994; Bowling et al., 1997; Clarke et al., 1998), constitutive immunity (cim) (Lawton et al., 1993), suppressor of SA insensitive (ssi) (Shah et al., 1999; Shah et al., 2001), suppressor of NPR1 insensitivity (sni1) (Li et al., 1999), map kinase 4 (mpk4) (Petersen et al., 2000), and defense no death (dnd) (Yu et al., 1998; Yu et al., 2000). In addition to the constitutive SAR expression, some of these mutants spontaneously develop lesions and are referred as lesion-mimic mutants. Another mutant in this class, enhanced disease resistance (edr), exhibits enhanced resistance to pathogens without constitutive SAR (Frye and Innes, 1998). In addition there are several transgenic plants that show lesion formation due to the expression of transgenes such as a bacterial proton pump, an invertase, a transcription factor AmMYB308 and certain other avirulence factors (Herbers et al., 1996; Mittler et al., 1995; Tamagnone et al., 1998). In several plant species other than Arabidopsis, mutants have been observed with a lesion phenotype that resembles the lesions caused by pathogen attack. They have been

identified in maize (disease lesion mimics; Walbot, 1991), tomato (autogenous necrosis; Langford, 1948), barley (mlo; Wolter et al., 1993), and rice (Sekiguchi lesion; Marchetti et al., 1983). The phenotypes associated with these mutants, especially the high frequency of non-allelic mutations in maize suggest that host factors alone are sufficient to initiate cell death that may be analogous to pathogen-induced cell death. However, disease response-specific markers were not tested in these mutants.

Biochemical and molecular markers are often associated with the necrosis induced by virulent or avirulent pathogens (Dixon and Lamb, 1990). Pathogen-induced cell death, in the form of HR during resistance or disease during susceptibility, result in a systemic long-lasting broad-spectrum resistance called SAR. Biochemical markers of pathogen-triggered cell death include irreversible membrane leakage, callose deposition, and accumulation of autofluorescent compounds in and around dead cell foci (Lamb, 1989). Several genes are induced locally as well as systemically following pathogen infection. Among these genes, certain pathogenesis-related genes PR-1, PR-2, PR-5 and GST1 are frequently used as molecular markers of plant defense response.

Here I describe the identification and characterization of a spontaneous cell death mutant of Arabidopsis that shows constitutive resistance against virulent bacterial and oomycete pathogens. Molecular and genetic analysis of this mutant will help to better understand the signaling networks that orchestrate the defense responses in plants against pathogens.

2.2 Methods

2.2.1 Plant growth conditions

Plants (Arabidopsis thaliana Col-0) were grown in soil (Metro-Mix 360; The Scotts Company, Marysville, OH) or on plates containing Murashige and Skoog (MS) media (Life Technologies, Grand Island, New York) supplemented with 1% sucrose and 0.8% agar (Gibco-BRL). The plant growth chambers were set at 25/23oC (day/night), 60-70% relative humidity, and a photosynthetic photon flux density (PPFD) of 100-150 µmolm- 2sec-1 with a 10-hr photoperiod unless otherwise specified. Soil-grown plants were supplemented with Miracle Grow fertilizer once in two-weeks (25g/gallon of water). For MS media, 4.64 g of MS salts were used for 1L and supplemented with 10 g of sucrose and 8 g of bacto-agar.

2.2.2 Pathogen inoculations and growth curves

Bacterial pathogens Pst DC3000 with an empty vector and Pst DC3000 carrying a plasmid borne avrRpm1 gene [Pst DC3000 (avrRpm1)] or avrRpt2 gene [Pst DC3000 (avrRpt2)] (Whalen et al., 1991) were grown at 28oC on King’s B agar plates or in liquid medium (King et al., 1954) supplemented with 100 µg/ml rifampicin and 25 µg/ml kanamycin. Bacterial culture was prepared by resuspending the overnight grown cells in 9 10 mM MgCl2 to a final optical density (OD600) of 0.0001 (1 OD600= 10 cfu/ml). The bacterial suspension was pressure-infiltrated on the abaxial side of the leaves using a 1 ml syringe to permiate the entire leaf surface. For growth curves, eight leaf discs (0.5 cm in diameter) from eight different plants were collected with a no. 2 cork-borer for each time

point at indicated times and ground in 10 mM MgCl2 using a pestle. To maintain uniformity, every tissue sample was ground for 1 min and vortexed for 15 seconds before plating. Serial dilutions were plated on King’s B agar plates supplemented with appropriate antibiotics. Plates were incubated at 28oC for 2 days to determine the number of colony-forming units. Peronospora parasitica Ahco2 infection assay was performed by spraying a water suspension of conidospores (5 x 104 spores/ml) on 3-week-old soil grown plants. The

spores were counted using a hemocytometer. Inoculated plants were kept covered to maintain high humidity throughout the course of experiment and were grown in a growth chamber at 17oC with a 12-hr photoperiod. After seven days, conidiophores on individual leaves of the plants were counted under a light microscope.

2.2.3 Histochemistry and Microscopy

The protocols for autofluorescence and callose staining were adapted from Dietrich et al., (1994).

(I) Autofluorescence Leaves for autofluorescence were boiled for 2 min in alcoholic lactophenol (95% ethanol:lactophenol, 2:1), rinsed in 50% ethanol, and then rinsed in water. Fresh alcoholic lactophenol was used to destain multiple leaves. Cleared leaves were mounted in 70% glycerol and autofluorescence was observed using UV epifluorescence (excitation filter, 365 nm; and barrier filter 420 nm).

(II) Aniline blue staining callose deposition For callose detection, leaves were cleared following the method described in autufluorescence. The cleared leaves were stained for 1 hr at room temperature in a

0.01% (w/v) solution of aniline blue in 0.15 M K2HPO4. Stained leaves were viewed under UV epifluorescence as described for autofluorescence.

(III) Trypan blue staining of dead cells Staining for dead cells was performed by the trypan blue uptake method and adapted from Vogel and Somerville, (2000) and Bowling et al., (1997). 0.25 mg/ml of trypan blue (EM Biosciences) was dissolved in lactophenol (25% v/v lactic acid, 25% v/v water saturated phenol, 25% v/v glycerol, and 25% v/v water). The solution was heated to 70oC and the leaves were vacuum infiltrated for 5 min with slow-release of pressure. The infiltration was repeated to ensure complete coverage. The leaves in the trypan blue-lactic acid solution was boiled for 2 min and cooled for 1 h at room temperature. The leaves were rinsed in water 3-4 times and destained in 10 g/ml chloral hydrate solution (Sigma,

class IV restricted compound) with multiple exchanges for more than 24 h. If the destaining was not complete the process was extended at 37oC. After the destaining, the leaves were equilibrated in 70% glycerol for several hours before viewing under the microscope. Destaining in chloral hydrate makes the leaf very brittle and equilibration in glycerin helps to avoid accidental breakage of the leaf tissue. If the destaining was not complete even after multiple exchanges at 37oC, lower concentrations of trypan blue (upto 10 µg/ml) were used. Mature, fully grown leaves were harder to destain than young seedlings. Hence different concentrations of trypan blue and longer destaining were tried. The two-minute boiling step was eliminated if destaining was a recurring problem. Sometimes, partial destaining was be carried out in lactophenol without chloral hydrate.

(IV) Staining for peroxidase activity assoaciated with H2O2 accumulation I adapted the protocol followed by Thordal-Christensein et al. (1997). I vacuum- infiltrated (25 p.s.i; 5 min) the leaves with freshly prepared diamino benzidine-HCl (DAB; Sigma) 1 mg/ml in water whose pH was adjusted to 3.8. If the leaves did not get infiltrated properly, the vacuum-infiltration was repeated. After infiltration, the leaves were incubated at 28oC with gentle shaking (100 rpm) for 3 hours. The leaves were then cleared in boiling ethanol for 2 min or until all the chlorophyll is de-pigmented. Complete clearing of chlorophyll pigments was essential for proper viewing under the microscope. The leaves were rinsed in water briefly and mounted in 70% glycerol and viewed at 10- 20 X magnification under bright-field illumination. The reddish brown precipitates

indicated H2O2 accumulation and peroxidase activity.

(V) Staining for the presence of superoxide through NBT This protocol was adapted from Jabs et al. (1996). The leaves were vacuum-infiltrated

with freshly prepared 10 mM sodium nitro-prusside (NaN3; made in 10 mM potassium phosphate buffer [pH 7.8]) and then immersed for 15-30 min in the same buffer containing 0.1% nitroblue tetrazolium (NBT; Sigma). The leaves were cleared in lactophenol overnight with several changes. Mounting and viewing was done as described above.

2.2.4 Chemical treatment of plants

Mutant and wild-type plants were sprayed with an aqueous solution of SA (1 mM), rose bengal (RB; 20 µM) or methyl jasmonate (50 µM in 0.01% ethanol) until run off. Control plants were sprayed with water or 0.01% ethanol. The plants were covered overnight to maintain high humidity. Leaves were harvested at the indicated times after treatment, flash frozen in liquid nitrogen and stored at -80oC until further analysis.

2.2.5 RNA isolation and Northern analysis

Tissue samples were collected from plants grown on soil at indicated time points. Samples were flash frozen in liquid nitrogen and total RNA was isolated using TRIzol reagent according to manufacturer’s instructions (Gibco BRL, Gaithersburg, MD) with the following modifications: a) TRIzol solution was heated to 75oC before addition; b) after TRIzol addition, the samples were spun at 10,000 g and the clear supernatant was

used for CHCl3 extraction; c) the isopropanol incubation was carried out for 1 h; d) the RNA pellet after the 70% ethanol wash was never allowed to over-dry. The RNA concentration was determined by UV absorbance at 260 nm using water and the quality (260/280 ratio) was determined in 10 mM Tris-Cl pH 7.5. For Northern analysis, ten µg of total RNA was fractionated by electrophoresis through denaturing formaldehyde-agarose gels and transferred to the Hybond N+ hybridization membrane (Amersham-Pharmacia, IL) according to Ausubel et. al. (1994). Gene-specific probes were synthesized by random primed 32P-labelling of gel-purified DNA fragments using RediPrime kit according to the manufacturer’s instructions (Amersham-Pharmacia, IL). cDNAs for GST1, and PDF1.2 genes were obtained from Dr. Fredrick Ausubel (Harvard Medical School, MA), and 18s rRNA from Dr. Jill Deikman (Monsanto, MO) . PR-1, and PR-2 gene-specific probes were obtained by PCR from Col-0 genomic DNA. Primers used for PR-1 were 5’-CCACAAGATTATCTAAGGGTTC-3’ (sense) and 5’- GGCTTCTCGTTCACATAATTCC-3’ (antisense), and for PR-2 were 5’- GATCTTGAACGTCTCGCCTCCAGTC-3’ (sense), 5’- GGCCTTCTCGGTGATCCATTCTTC-3’ (antisense). Hybridizations and washes were

performed following the methods described in Ausubel et. al. (1994). The hybridized membranes were exposed onto Kadak BioMax MS films with intensifier screens.

2.2.6 Arabidopsis genomic DNA extraction for mapping

For mapping, 50-100 mg of leaf tissue was ground to a fine powder using liquid nitrogen n an Eppendorf tube using a motor-driven plastic pestle. The powder was resuspended in 250 µl extraction buffer that was supplemented with β-mercaptoethanol (BME). The samples were gently mixed by inversion. After incubating the samples at 65oC for 10

min, an equal volume of CHCl3 was added and shaken gently. The clear upper aqueous phase was transferred to a fresh tube and 0.6 volumes of isopropanol was added. After a 20-min incubation at room temperature, the samples were centrifuged at 12,000 g for 15 min at 4oC. The pellets were washed twice with 70% ethanol, air dried briefly and resuspended in 100 µl of DNAse free water. To remove the contaminating RNA, 2 µl of 1 mg/ml RNAse was added and the samples were incubated at 37oC for 30 min. Usually 1 µl was enough for PCR analysis.

2.2.7 PCR for CAPS and SSLP analysis

PCR for CAPS and SSLP marker analysis was usually carried out in a 10-µl reaction volume with the following constituents: 20-50 ng of DNA; 1 pmole of forward and reverse primers; 0.2 mM dNTPs; 1X PCR buffer with no Mg2+; and 0.2U Taq. Mg2+ concentration used in the different reactions varied depending on the primer pair as well as the quality of the DNA prep. Usually the Mg2+ that was precipitated during the DNA extraction protocol (part of the chlorophyll structure) was enough for the PCR. For restriction analysis, 2 µl of the PCR products were run on a 1.2 % agarose gel and 8 µl was used for restriction digestion with the appropriate enzyme. The restriction digestion was done in a 30-µl reaction volume with 0.5U of the enzyme.

2.2.8 Sub-cloning of BAC fragments into a binary vector

The vector used for sub-cloning bacterial artificial chromosome (BAC) inserts was a binary cosmid pCLD04541 (kind gift from Dr. Tim McNellis, Penn State University). The plasmid DNA was isolated using a Qiagen large plasmid DNA extraction kit (Qiagen, Valencia, CA). For sub-cloning, 20 mg of the vector DNA was digested with 30U of BamH1 for an hour. Longer digestions with BamHI resulted in a drastic reduction in cloning efficiency. The digested vector was split into half and one half was treated with 1U of calf-intestine alkaline phosphatase (CIP) for 1 h at 37oC. The CIP-treated and the untreated samples were gel-purified for further analysis. The BAC plasmid DNA was isolated using the large DNA extraction kit from Qiagen. 40 mg of BAC DNA was used for a partial digestion with Sau3AI (1U, 0.25U, 0.06U, and 0.01U) for 1 h in a 40-µl reaction volume and size selected for 15-20 kb fragments. The gel purified 15-20 kb fragments were resuspended in water and then ligated to CIP-treated pCLD04541 vector as follows. For ligation, 20-40 ng of the gel-purified inserts, 100 ng of CIP-treated vector, 1 mM ATP, 1 mg/ml BSA, and 10U of T4-DNA ligase were used in a 20-µl reaction. The samples were incubated at 16oC for 16 h. The ligated samples were drop-dialysed against water using a 0.45-µm porous nitrocellulose filter disc (Millepore) for 1 hr. This steps removes excess salts that may affect the electroporation. Electrocompetent E.coli DH10b cells were electroporated with the ligated samples following the manufacturer’s suggestions (Gibco-BRL, Gaithersburg, MD). The colonies were screened on LB agar plates supplemented with 10 µg/ml of tetracycline. The extent of coverage was determined by amplifying a 1-kb region that was specific to each BAC. For BAC F20B18 primers5’-CTCAGAGTCTTGGACTTGTCG-3’; 5’- TTCGACGGATGGACTCTCGTG- 3’, for BAC T25K17 primers 5’- GGTGATCTCTTTGGTGACTGG-3’; 5’- CGAGCTGTTCCTAGCAATGCG-3’, for BAC F14M19 primers 5’- CTTCGTCGGTCAGGATCTGCT-3’; 5’- GGCGTATCACATCATTCAAAGC-3’ were used to amplify a 1-kb fragment.

2.2.9 Transformation of Agrobacterium and Arabidopsis plants

Plasmid DNA was isolated from the positive clones and transformed into an electrocompetent Agrobacterium strain GV3101 following the same procedure described above. The transformants were screened on LB agar supplemented with 3 µg/ml of tetracycline, 50 µg/ml of kanamycin, and 30 µg/ml of gentamycin. The colonies appear within two days of electroporation. The colonies were also tested for the presence of NPTII (encodes neomycin phospho transferase for kanamycin resistance) gene by PCR amplifying a 500-bp fragment using primers 5’- TGCTCGACGTTGTCACTGAAG-3’ and 5’- GTCAAGAAGGCGATAGAAGGC-3’.

A pre-inoculum of 2-ml of Agrobacterium culture was used to inoculate 250 ml of LB supplemented with appropriate antibiotics. The Agrobacterium culture was grown for 16 h and the cells were centrifuged at 5000 g at 4oC for 15 min. The cell pellet was washed twice with 5% sucrose solution and then resuspended in 500 ml of 5% sucrose solution supplemented with 0.015% Silwet L-77, a surfactant used as a wetting agent. The final OD of the Agrobacterium culture was at least 0.8. The Arabidopsis plants that have bolted with a good mixture of opened and unopened buds were preferred for Agrobacterium-mediated transformation through a floral-dip method (Clough et al., 1998). Approximately 4 plants were used per construct. The floral-dip method was repeated 2 more times with a 5-day interval. Once the siliques turned brown, the watering was stopped and the plants were allowed to dry. The seeds were collected, sterilized, and screened on MS media supplemented with 50 µg/ml kanamycin (marker linked to the T- DNA) and 200 µg/ml ampicillin to arrest Agrobacterium contamination.

2.3 Results

2.3.1 Isolation of hrl1 mutant

We screened EMS-mutagenized populations of Arabidopsis ecotype Col-0 for mutants that displayed resistance (less water-soaked chlorotic lesions) in response to infection by a virulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) (see Methods). From one of these screens, we identified a mutant designated hrl1 (for hypersensitive response-like lesions 1) that spontaneously developed HR-like lesions (Figure 2-1) and displayed reduced disease symptoms. Lesions in hrl1 start as random necrotic patches of dead cells on the leaf blades of 2-week-old plants that are confined to the rosette and cauline leaves; no visible lesions develop on the stem or on the inflorescence. Once formed, the lesions do not enlarge significantly. Lesions develop on aseptically grown plants, indicating that exposure to pathogens is not necessary. The rosettes of the mature hrl1 plants are significantly smaller than those of the wild-type parent (Figure 2-1). The flowering time of hrl1 is normal with no apparent defect in fertility. All the experiments were performed with a mutant line that was backcrossed three times to the wild-type parent.

2.3.2 Genetic analyses of hrl1 Genetic crosses were performed to determine the segregation of hrl1 locus

Cross (recipient x Type Total Lesionb χ2 a pollen donor ) Positive No hrl1 / hrl X F1 37 0 37 HRL1/ HRL1 hrl1 / HRL1 X F2 334 82 252 0.036 hrl1 / HRL1 (P>0.95) a The recipients for the crosses were all in Col-0 background. b The phenotype was scored on plants grown in short day (10 h light) growth conditions when the plants were 6 weeks old. Spontaneous lesions represent the hrl1

17 A B

Col-0 hrl1 Col-0 hrl1 C D

hrl1 Col-0 E

(i) (ii)

Figure 2-1: Phenotype of hrl1 mutant at various stages of growth A: 3-week old Col-0 and hrl1 plants, B: 4-week old Col-0 and hrl1 plants, C: 6-week old hrl1 plant, and D:6-week old Col-0 plant, E: lesion minus (i) and lesion plus (ii) leaves of 6-week-old hrl1 plant The pictures in C and D were photographed from the same distance. Scale bars in C and D equal 2.5 cm.

phenotype. This closely approximates a 3:1 ratio (χ2= 0.036; P>0.95) indicating that the phenotype is caused by a recessive nuclear mutation at a single locus. The reduced size of the plants always cosegregated with the lesion phenotype.

2.3.3 Mutation in the HRL1 locus induces lesions with extensive similarity to pathogen-induced cell death

Pathogen-induced cell death is often accompanied by the presence of several biochemical markers that distinguish this type of cell death from other cell death processes like senescence and wounding. To analyze the nature of hrl1 lesions and to test if they phenocopy pathogen-induced HR, we analyzed the presence of the markers associated with hypersensitive response (HR). (Goodman and Novacky, 1996; Hammond-Kosack and Jones, 1996). Trypan blue staining strongly supports the hypothesis that the lesions seen in hrl1 is the result of cell collapse and the staining pattern mirrors the visible necrotic patches. Comparison of whole mounted leaves of hrl1 with controls revealed substantial deposition of autofluorescent material (Figure 2-2),

callose and accumulation of H2O2 in and around the lesions in hrl1, and in the control tissue displaying HR. None of stains used above tested positive in the leaves with no

visible lesions. Often, the vein endings in hrl1 was characterized by strong H2O2 staining. In addition, hrl1 plants stained positive for superoxide through NBT precipitation. In contrast to the H2O2 staining pattern, accumulation of superoxide was evident even in the lesion minus tissue also. These results suggest that hrl1 mutant constitutively expresses cellular and biochemical markers associated with plant's hypersensitive response to avirulent pathogens (Baker and Orlandi, 1995).

2.3.4 hrl1 activates the expression of several pathogenesis-related (PR) genes If the lesions in hrl1 resemble pathogen-induced cell death, we hypothesized that the lesion formation in the hrl1 mutant will be accompanied by transcriptional activation of PR genes. Transcriptional activation of these genes during pathogen infection is a

19 hrl1 Col-0/avr Col-0/MgCl2 A B C

D E F

Callose

G H I Trypan blue

Figure 2-2: Accumulation of defense-related biochemical markers in hrl1. Vertical columns represent genotype of the plant/ treatment used and the horizontal rows represent the biochemical marker tested. (A-C) Auto-fluorescent materials (AF) visualized by UV microscopy, (D-F) Callose deposition revealed by aniline blue staining, (G-I) Dead cells stained by trypan blue. Col-0/avr, wild-type Col-0 leaves infiltrated with 107 cfu/ml of avirulent bacterial pathogen Pst DC3000/avrRpm1 (positive control for HR-mediated cell death); Col-

0/MgCl2, Col-0 leaves infiltrated with 10 mM MgCl2 (mock inoculated control). Leaves were harvested for analysis 24 hr after infiltration.

20 hrl1 Col-0/avr Col-0/MgCl2 A B C

H2O2 (DAB)

D E F Superoxide (NBT)

Figure 2-3: ROI accumulation in hrl1 mutant

Diamino benzidine (DAB) staining for peroxidase activity and H2O2 accumulation in hrl1 (A), Col-0 infiltrated with an avirulent pathogen (B) and buffer infiltrated Col-0 (C). Nitroblue tetrazolium staining for the presence of superoxide in hrl1 mutant (D), Col-0 infiltrated with an avirulent pathogen (E) and buffer infiltrated Col-0 (F). The bar shown in (F) equals 1 mm and represents the magnification in A through F. The inset in (A) represents the frequently observed staining pattern near the vein endings in hrl1.The inset in (D) and (E) represent whole leaves stained with NBT.

characteristic defense response and many of the gene products may have direct antimicrobial effects. The total RNA was isolated from hrl1 leaf tissue and Northern blots were probed with cDNAs encoding PR-1, PR-2, PR-5, and GST1 (markers of SA- responsive defense pathway); and PDF1.2 (marker of ethylene/jasmonic acid-responsive defense pathway) (Uknes et al., 1992; Greenberg et al., 1994; Penninckx et al., 1996; Manners et al., 1998). Since visible cell necrosis in hrl1 appears at 2 weeks and intensifies throughout development, we analyzed the transcript accumulation of these genes at various stages of growth. Furthermore, to specifically distinguish the lesion- associated HR-like response from the SAR-like response, we analyzed the expression of these genes in both the lesion+ leaves and in the lesion- leaves of 6-week old hrl1 plants. Typically, hrl1 leaves shown in (Fig 2-Ei) represent lesion+ and leaves- shown in (Fig 2- Eii) represent lesion- samples. No dead cells were found in lesion- leaves even after staining with trypan blue. Results in (Fig 2-4) demonstrate that the accumulation of defense-related gene transcripts positively correlates with the age as well as the lesion status of the plants. In addition, both lesion+ and lesion- leaves of 6-week old hrl1 plants show heightened defense gene activation. These results suggest that the lesion+ leaves mimic at least some aspects of HR and the lesion- leaves mimic some of the SAR responses of the plants to pathogen infection.

2.3.5 Growth of virulent bacterial and oomycete pathogens in hrl1

Since hrl1 plants constitutively express cellular and molecular markers associated with HR and SAR, we sought to determine whether they were resistant to virulent pathogens. The growth of the virulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) was tested in the leaves of hrl1 plants in which lesions had just initiated. Results presented in figure 2-5A demonstrate that hrl1 is more resistant to Pst DC3000. Compared to wild type Col-0, bacterial levels were 200 fold lower in hrl1 plants 4-days post infiltration. Similar results were obtained with the leaves that had no lesions at the beginning of the experiment. However, since these leaves developed lesions during the course of the experiment, they cannot be considered true lesion− leaves and therefore were not included in the subsequent experiments. Growth of virulent oomycete pathogen Peronospora parasitica Ahco2 was also tested on lesion+ leaves of hrl1 plants. Virulent

22 Col-0 hrl1 SA RB Treatment - avr --- - Age (weeks) 6 6 6 6 2 4 6 6 Lesion - + + - Lane 1 2 3 4 5 6 7 8

PR-1

PR-2

PR-5

GST1

PDF1.2

rRNA

Figure 2-4. Analysis of defense-related gene expression in hrl1

Expression of PR-1, PR-2, PR-5, GST1, and PDF1.2 genes was determined at various stages of growth in hrl1 mutant. Age of the plants and the lesion status of the hrl1 leaves used for RNA isolation are indicated. RNA in lanes 7 and 8 was isolated from lesion+ and lesion− (as in Figure 1E) leaves of the same 6-week-old hrl1 plants. Lesion− leaves had no macroscopic or microscopic lesions. Six-week-old wild-type Col-0 plants were infiltrated with 107 cfu/ml of Pst DC3000/avrRpm1 (avr) or sprayed with 1mM salicylic acid (SA) (control for SA-responsive defense genes) or sprayed with 20 µM Rose Bengal (RB) (control for PDF1.2 gene). Leaves treated with Pst DC3000/avrRpm1 and SA were harvested 24 hr after treatment, and leaves treated with RB were harvested 48 hr after treatment. Blot containing 10 µg total RNA of each sample was serially probed with the indicated gene-specific probes. The 18S ribosomal subunit gene- specific probe (rRNA) was used as a loading control. The vertical line seprating the blot is shown to indicate that some unwanted lanes were removed during the scanning of the autoradiographs.

P. parasitica require live host cells to survive and are therefore considered biotrophs. As shown in figure 2-5B, growth of P. parasitica Ahco2 is strongly suppressed in hrl1 plants. These results demonstrate that hrl1 plants are more resistant to virulent bacterial and oomycete pathogens compared to the wild-type control plants.

2.3.6 Mapping the hrl1 locus using PCR-based CAPS and SSLP markers

The hrl1 mutant in the Col-0 background was isolated as an ethylmethane sulfonate (EMS) mutant. EMS causes point mutations that may result in a frameshift and/or a premature stop codon. EMS has been widely used as a mutagenizing agent for Arabidopsis (Koornneef et al, 1982). Mutants that are generated by EMS are subjected to marker based positional cloning strategy (Arondel et al., 1992; Miquel, 2000). The mutant locus is identified using a set of markers that are ecotype specific. The mutant line in one particular background is crossed to another ecotype that can complement the mutation. The resulting F2 progeny are first screened for mutant phenotype and then analyzed with a set of markers. Due to the available genetic information for Col-0 and Ler ecotypes, they are the preferred choices to initiate map-based cloning approach. Several PCR based markers that span all 5 chromosomes have been identified and available for pubic use (www.arabidopsis.org). PCR based codominant amplified polymorphic sequences (CAPS; Konieczny and Ausubel, 1993) and simple sequence length polymorphism (SSLP; Bell and Ecker, 1994) markers accelerate the map-based cloning approach. These markers differentiate the different ecotypes based on the differences in the restricted PCR product (CAPS) or a difference in the length of the PCR products (SSLP). To map the hrl1 locus, a set of 12 different CAPS and SSLP markers that cover all 5 chromosomes of Col-0 and Ler ecotypes were used.

24 A 108

107 hrl1/vir 106 Col-0/vir hrl1/avr 105 Col-0/avr sc i d f 104

/lea 103 cfu 102

101 024 B Days after infection

40 hrl1 30 Col-0

10 Number of plants 0 0123

Disease rating

Figure 2-5. Growth of bacterial and oomycete pathogens in hrl1 mutant. (A) Leaves of 6-week-old wild-type Col-0 and lesion+ leaves of hrl1 mutant were infiltrated with a suspension of Pst DC3000 (vir) or Pst DC3000 expressing avrRpm1 (avr) in 10 mM MgCl2 at a 5 dose of 10 cfu/ml (OD600= 0.0001). The bacterial counts + SD are presented as colony-forming units (cfu) per leaf disc and represent averages from three independent experiments. (B) Three-week-old wild-type Col-0 and hrl1 mutant seedlings were sprayed with a spore suspension of P. parasitica Ahco2 in water (2 X 104 spores/ml) and the number of conidiophores were counted on each plant 7 days after infection. Disease ratings are as follows: 0, no conidiophore on the plant; 1, less than 5 conidiophores per infected leaf; 2, 6 to 20 conidiophores on a few infected leaves; 3, six or more conidiophores on most of the infected leaves. The data are represented as average values + SD from three independent experiments.

Chromosme Marker Primer sequence I m235 Forward 5’-GAATCTGTTTCGCCTAACGC-3’ Reverse 5’-AGTCCACAACAATTGCAGCC-3’ I ADH Forward 5’-GCGTGACCATCAAGACTAAT-3’ Reverse 5’-AAAAATGGCAACACTTTGAC-3’ II GPA Forward 5’-GGGATTTGATGAAGGAGAAC-3’ Reverse 5’-ATTCCTTGGTCTCCATCATC-3’ II ER Forward 5’-CTAATGTAGTGATCTGCGAGGTAATC-3’ Reverse 5’-GAGTTTATTCTGTGCCAAGTCCCTG-3’ II m429 Forward 5’-TGGTAACATGTTGGCTCTATAATTG-3’ III GAPA Reverse 5’-GGCAGTTATTATGAATGTCTGCATG-3’ III TSA Forward 5’-TCTTGGTAGCATGATTCTCAGTC-3’ Reverse 5’-CCTTTCCGCTTACAGATGATC-3’ III ArLIM15 Forward 5’-GCCAGTTTTTTCCTGCACATCAATC-3’ Reversr 5’-TGCTGCTTTATTTTGTCGCGATGTT-3’ IV RPS2 Forward 5’-CTCAGAGTCTTGGACTTGTCG-3’ Reverse 5’-TTCGACGGATGGACTCTCGTG-3’ IV g8300 Forward 5’-TAAAAGCTTGGACTGGCGTGATTGA-3’ Reverse 5’-GAATTCCCGACGGCATTGCCAG-3’ IV CBF Forward 5’-CCTTATCCAGTTTCTTGAAACAGAG-3’ Reverse 5’-CGAATATTAGTAACTCCAAAGCGAC-3’ IV nga1139a Forward 5’-TAGCCGGATGAGTTGGTACC-3’ Reverse 5’-TTTTTCCTTGTGTTGCATTCC-3’ IV g4539 Forward 5’-GGTCATCCGTTCCCAGGTAAAG-3’ Reverse 5’-GGACGTAGAATCTGAGAGCTC-3’ V PAI2 Forward 5’-CAGTTAATGAAACAAGCTTTGTTC-3’ Reverse 5’-GTTGAGAAAATCACTTTGGTG-3’ V EG7F2 Forward 5’-GATCTGTGTAGGACTACGAGAC-3’ Reverse 5’-GCATAGAATTTGACGATAACGAGC-3’ a: nga1139 is an SSLP marker

Table 2-1: Primer sequences for CAPS markers

The markers were selected from all five chromosomes of Arabidopsis based on the information given in the Arabidopsis community web page at http://www.arabidopsis.org

Marker PCR Restriction Restriction pattern product enzyme Col-0 Ler m235 530 bp HindIII 330 bp, 200 bp 530 bp ADH 1290 bp XbaI 1290 bp 1090 bp, 262 bp GPA 1590 bp AflIII 705 bp, 680 bp, 209 bp 1385 bp, 209 bp ER 500 bp DdeI 500 bp 360 bp, 140 bp m429 316 bp ScrF1 316 bp 216 bp, 100 bp GAPA 771 bp DdeI 420 bp, 178 bp, 100 240 bp, 190 bp, bp, 33 bp, 19 bp, 10 bp 178 bp, 100 bp TSA 380 bp AluI 240 bp, 130 bp 380 bp ArLIM15 500 bp EcoRI 450 bp, 50 bp 500 bp RPS2 785 bp Sau3AI 605 bp, 180 bp 350 bp, 250 bp, 180 bp g8300 700 bp HindIII 700 bp 500 bp, 200 bp CBF 680 bp MboI 360 bp, 300 bp 300 bp, 220 bp, 140 bp nga1139 114/118 N/A 114 bp 118 bp g4539 600 bp HindIII 600 bp 480 bp, 120 bp PAI2 644 bp AflIII 590 bp, 50 bp 644 bp EG7F2 1200 bp XbaI 1200 bp 700 bp, 500 bp

Table 2-2: Restriction patterns for the CAPS markers

The table summarizes the PCR product size and the restriction enzymes used to generate the characteristic patterns for Col-0 and Ler ecotype.

Marker Chr Col-0a Lerb Col-0 Total Total Rec. Distance /Lerc Col-0d Lere Freq (cM)g (%)f m235 I 10 10 38 58 58 50 N/A ADH I 8 16 38 54 70 56 N/A GPA/ER II 13 5 24 50 34 40 56 m429 II 18 2 48 84 56 39 52 GAPA/ III 18 10 14 50 34 40 56 ArLIM15 TSA III 8 1 23 39 25 39 52 g4539 IV 183 6 52 418 64 13 13 CBF IV 83 0 3 169 3 1.7 1.74 RPS2 IV 234 0 3 471 3 0.6 0.63 g8300 IV 157 2 18 332 22 6.21 6.25 nga1139 IV 117 5 25 259 35 11.9 12.14 PAI2 V 10 4 4 24 14 31 36 EG7F2 V 20 8 30 70 46 39 54

a: no. of samples displaying Col-0 pattern b: no. of samples displaying Ler pattern c: no. of samples displaying both Col-0 and Ler patterns d: total no. of samples displaying Col-0 pattern (2X Col-0 + Col-0/Ler samples) e: total no. of samples displaying Ler pattern (2X Ler + Col-0/Ler samples) f: recombination frequency = total no. of samples displaying Ler phenotype X 100 over total no. of samples displaying Col-0 and Ler phenotypes g: Map distance calculated according to the Kosambi relation (Koornneef and Stam, 1992) 25 ln [(100+2*Rec. Freq)/(100-2*Rec. Freq)]

Table 2-3: Recombination analyses for the hrl1 mutation based on the known marker positions

Total number of CAPS and SSLP marker analyses on F2 progenies generated from a

cross between hrl1 (Col-0 back ground) and Ler. Genomic DNA was isolated from the F2 progenies that showed hrl1 symptoms and subsequent mapping was done.

2.4 Discussion

Based on our analysis with biochemical and molecular markers, the lesions in hrl1 resemble many characteristics of pathogen-induced cell death. In addition, hrl1 mutant shows enhanced resistance to virulent bacterial and oomycete pathogens. Several of the PR genes that are induced in hrl1 encode antimicrobial proteins. These antimicrobials, along with high ROI in hrl1 may be responsible for the high level of resistance to virulent pathogens. From these observations and due to the recessive nature of hrl1 mutation, it is tempting to speculate that HRL1 may encode a negative regulator of cell death and defense in plants. Alternatively, HRL1 may encode a component that activates an anti-death pathway. Programmed cell death in plants is involved in various developmental processes such as anther, megagametophyte, and vascular tissue development (Jones and Dangl, 1996). However, no clear biochemical or molecular markers of apoptosis have been characterized in plants. It is not known if any of these cell death processes and the cell death during pathogen attack share any similarities. Despite the presence of various defense-related responses, it is quite possible that hrl1 mutant is perturbed in a metabolic or a photosynthetic process that results in a stressed cellular state. Since such metabolic lesions have not been previously tested for defense- related responses, the possibility remains open. Among the spontaneous cell death mutants with disease resistance phenotype in plants, only a few have been cloned so far. LSD1 encodes a novel protein with three zinc finger domains and is believed to negatively regulate a superoxide-dependent cell death process (Dietrich et al., 1997). Interestingly, ACD2 gene product shows significant and extensive similarity to red chlorophyll catabolite reductase, which catalyzes one step in the breakdown of the porphyrin component of chlorophyll (Mach et al., 2000). It is hypothesized that cell death in acd2 plants is caused by the accumulation of chlorophyll breakdown products. It should be noted that acd2 plants undergo cell death in response to bacterial infection and also to nonspecific stress response like wounding. Unlike acd2 plants, the lesions in hrl1 plants do not initiate due to mechanical stress or pathogen inoculation. The appearance of lesions in hrl1 follows a strict developmental pattern that seems to be unaffected by nonspecific external stimuli. When the expression levels of a

protoporphyrinogen IX oxidase (PPO), the last common enzyme of the biosynthesis of the haem group and chlorophyll was reduced by antisense, the leaves developed necrotic lesions that resembled pathogen induced cell death (Molina et al., 1999). Overexpression of a myb transcription factor that represses phenolic acid metabolism lead to precocious cell death and abnormal leaf development in tobacco (Tamagnone et al., 1998). These results demonstrate that genetic disruption of a metabolic pathway can lead to the induction of a set of defense responses including activation of SAR and HR-like cell death. The hrl1 mutant accumulates high levels of both superoxide and hydrogen peroxide. This phenomenon can be explained by two simple possibilities: uncontrolled generation of ROI or failure to detoxify the normal levels of ROI. Increased ROI can activate a multitude of downstream signaling events (Grant et al., 2000). From the Northern analysis, it is evident that hrl1 mutant accumulates high levels of GST1, one of the main players of antioxidant responses. Hence it is unlikely that hrl1 mutant fails to activate the antioxidant responses to normal cellular ROI generation. However, it is still possible that other forms of ROI detoxification are not recruited in hrl1. But, in favor of the first possibility we have observed increased accumulation of AtrbohD, an Arabidopsis homolog of mammalian gp91phox gene. AtrbohD encodes a major subunit of NADPH oxidase multienzyme complex and is thought to be the predominant source of superoxide during the pathogen-induced oxidative burst. This scenario will be discussed in the subsequent chapters. In summary, it is possible that HRL1 encodes a signaling component whose functional presence is essential to prevent premature cell death. The cell death process in hrl1 may be triggered due to the excessive accumulation of ROI, which normally would occur during pathogen attack. Although the mode of ROI generation is not precisely known, the molecular genetic analysis of hrl1 mutant will help us to better understand the mechanisms that are required to initiate and enhance the cell death phenotype. Signal molecules like SA, JA and ethylene have been implicated in modulating defense and cell death during pathogen attack. But a definitive role for each of these molecules in the mutants that show constitutive defense has not been analyzed thoroughly. The next two chapters will focus on the roles of SA, JA, ethylene and their

cognate transducers NPR1, COI1 and ETR1 respectively in regulating defense and cell death in hrl1.

2.3.1 Mapping of hrl1 locus

Using a set of 12 PCR based markers and 300 F2 progeny, I mapped the hrl1 locus to a 6-cM interval in the bottom arm of chromosome IV. Other lesion-mimic mutations that map to chromosome IV include ssi1, acd6, lsd1, acd2, and cpr20 (Dietrich et al., 1994; Greenberg et al., 1994; Rate et al., 1999; Shah et al., 1999). Among these, lsd1 and acd2 map closest to hrl1 (Dietrich et al., 1997; Mach et al., 2001). Therefore, to test if hrl1 is allelic to lsd1 or acd2, complementation tests were performed. All these

mutations are recessive and none of the F1 progeny from the hrl1 x lsd1 (n=25) or hrl1 x acd2 (n=20) crosses developed lesions. Thus, hrl1 is not allelic to these lesion-mimic mutants. Since the hrl1 locus maps very close to RPS2 resistance gene (0.63 cM), it is possible that hrl1 has a mutation in the RPS2 gene that results in a novel lesion-mimic phenotype. To test this possibility, a complementation test was performed with rps2-

201C mutant line and none of the F1 progeny (n= 45) from this cross had lesions and all of them elicited normal HR in response to P. syringae expressing AvrRpt2, an elicitor corresponding to RPS2 gene (Kunkel et al., 1993). From these results we conclude that hrl1 defines a novel locus with a lesion-mimic phenotype. Since hrl1 maps very close to RPS2 marker, I attempted a shot-gun complementing approach. Three overlapping BACs that span a 200-kb region on chromosome IV were selected and complementation was attempted with entire BAC insert (Mozo et al., 1999). Due to the inherent instability of large DNA fragments in Agrobacterium, this approach was abandoned. A cosmid library was constructed from each of these 3 BACs that contained 15-20 kb fragments. Twenty-five clones per BAC were randomly picked from the cosmid library and complementation of hrl1 mutant was attempted through Agrobacterium-mediated transformation. None of the clones from BAC F20B18 and BAC F14M19 complemented hrl1 mutation. Even though, 2 Agrobacterium clones were selected per construct for transformation, it is still possible that some rearrangements could have occurred during the transformation step. This might have resulted in the loss of functional HRL1 gene. Alternatively, these BACs may not

contain the wild-type copy of hrl1 gene. For a better map position an estimated 1200 F2 progeny need to be screened with additional markers. The success rate of PCR with the DNA samples isolated from hrl1 mutant is rather low (45%) due to the constitutive accumulation of phenolics and other degraded cell wall components that affect PCR.

Hence, the DNA samples have to be purified with double phenol-CHCl3 extraction and a double 70% ethanol wash. Owing to this tedious approach, further fine mapping of hrl1 locus is a lengthy process. I decided to look for candidate genes in the overlapping BACs. However, this chromosomal region is extremely gene rich (~ 4kb/gene) and careful analysis failed to yield any potential candidates. In addition, only a few genes have been cloned from the lesion-mimic mutants that share some similarity to hrl1 and so the information available to search for candidates is rather limited. LSD1 encodes a novel zinc finger transcription factor, ACD2 is a red chlorophyll catabolite reductase, CPR5 is a novel protein with a transmembrane domain and a nuclear targeting sequence, and SSI2 encodes a fatty acid desaturase (Dietrich et al., 1997; Mach et al., 2000; Kachroo et al., 2001; Kirik et al., 2001). For example, in the 3 overlapping BACs of interest, there are 8 putative proteins, 6 hypothetical proteins, 4 transcription factors, a mitochondrial carrier protein, and 2 nucleic acid binding proteins. Hence finding potential candidates is difficult with limited prior information.

Chapter 3

Role of salicylic acid and NPR1 in cell death, gene expression, and disease resistance in hrl1

3.1 Introduction

Over the past decade or so, SA has emerged to be a key signaling molecule in regulating disease resistance in a wide variety of plant species (Ryals et al., 1996; Klessig and Malamy, 1994). SA accumulation is critical in the establishment of SAR, a broad- spectrum resistance response. An essential tool to understand role of SA has been the identification of a salicylate hydroxylase encoded by the nahG gene of Psuedomonas putida (Gaffney et al., 1993; Delaney et al., 1994). This salicylate hydroxylase converts SA into an inactive catechol and therefore prevents accumulation of active SA or its derivatives. Expression of the nahG transgene in Arabidopsis, tobacco, and rice resulted in susceptibility to a variety of bacterial and oomycete pathogens due to the reduced accumulation of SA (reviewd in Ryals et al., 1996). In addition, SA treatment prior to pathogen attack enhanced resistance to several virulent pathogens. The role of SA in regulating cell death is not clear. The Arabidopsis transgenic plants expressing the nahG gene show normal HR cell death against P. syringae expressing avrRPM1 but not to the one expressing avrRpt2 (Rate et al., 1999). In the case of lesion-mimic mutants, SA depletion suppressed all of the cell death phenotype in some of the mutants but had no effect on the others. Together, these results suggest that SA is required for the induced defense response against many virulent pathogens but does not have a defined role in regulating cell death in response to pathogen ingress.

The Arabidopsis NPR1 gene controls the onset of systemic acquired resistance to a broad spectrum of pathogens that is normally established after a primary exposure to avirulent pathogens (Cao et al., 1994; Cao et al., 1997). The npr1 mutants fail to respond to various SAR- inducing treatments, displaying very low expression of pathogenesis-

related (PR) genes and exhibiting increased susceptibility to infections. NIM1 shows sequence homology to the mammalian signal transduction factor I κ β subclass α. Necrosis factor NF-κ β/I κ β signaling pathways are implicated in disease resistance responses in a range of organisms from Drosophila to mammals, suggesting that the SAR signaling pathway in plants may have some signaling counterparts in animals (Ryals et al., 1997).

Overexpression of NPR1 in Arabidopsis leads to broad-spectrum resistance with no obvious detrimental effect on the plants (Cao et al., 1998). Upon induction by SA or pathogen infection, the NPR1 protein is localized to the nucleus where it is thought to interact with a subclass of transcription factors in the basic leucine zipper protein family (Kinkema et al., 2000). These b-zip transcription factors interact with the promoter elements of PR genes (Zhou et al., 2000)

Since hrl1 mutant shows activation of several PR genes and displays enhanced resistance to virulent pathogens, we sought to determine if the defense signaling in hrl1 follows a SA/NPR1-dependent pathway.

3.2 Methods

3.2.1 Construction of hrl1 npr1 double mutant

The hrl1 npr1 double mutant was generated using pollen from npr1-1 mutant (Cao et al., 1994) to fertilize flowers of hrl1. Success of the cross was judged by the loss

of hrl1 phenotype in F1 plants. Several F1 plants were allowed to set seeds and screened

for homozygosity in the F2 generation. The F2 seeds were plated in MS media containing 100 µM SA. The npr1 mutants bleach when grown in high SA, We made use of this phenotype and rescued the bleaching seedlings by transferring to plain MS. After 1 week the rescued seedlings were transferred to soil and were maintained under high humidity for 2 days. Two weeks after transfer to soil, the plants were selected for hrl1-like symptoms. To further confirm the presence of npr1 mutant allele, PCR analysis for npr1 was performed with the DNA isolated from the plants that displayed hrl1-like phenotype. After confirmation by PCR, the plants were allowed to set seeds and used for further th analysis. About 1/16 of the total F2 plants (136) screened were homozygous for hrl1 and npr1.

3.2.2 Construction of hrl1 nahG transgenic line

To construct the hrl1 nahG line, the nahG gene was introduced into hrl1 plants by a genetic cross, using pollen of nahG to fertilize hrl1 flowers. Transgenic nahG line in the Col-0 ecotype (line B15) was obtained from Syngenta Biotechnology Inc. (formerly

Novartis, Inc.). Success of the cross was judged by the loss of hrl1 phenotype in F1 plants and resistance to the antibiotic kanamycin. F2 seeds were plated on MS media supplemented with 50 µg/mL kanamycin (marker linked to nahG gene). Since the nahG gene is expressed from a 35-s promoter, it is dominant when linked to the kanR backbone. th R Therefore roughly 3/16 of the total F2 seedlings (112) screened was kan and showed hrl1-like symptoms. Kanamycin-resistant seedlings were transferred to soil and scored for hrl1-like phenotype. Homozygous hrl1 lines expressing nahG gene were identified as kanamycin-resistant plants showing hrl1-like phenotype. Lines homozygous for hrl1 and

nahG loci were identified by screening F3 populations of individual F2 lines. The F2 lines that showed 100% resistance to kanamycin in the F3 population and displayed hrl1-like phenotype were considered homozygous for hrl1 and nahG loci. The presence of the nahG transgene was also confirmed by PCR using nahG gene-specific primers.

3.2.3 Sterilization of Arabidopsis seeds for plating on MS media

Approximately 1000 seeds were sterilized in 1 mL of 20% bleach and 0.1% SDS by shaking in an Eppendorf shaker twice for 15 min. The bleach solution was removed and sterile water was used to wash off the remaining bleach and SDS. This washing step was repeated 5 times in the laminar airflow hood to ensure complete removal of bleach and SDS. The seeds were then stored at 4oC for 2 days for further analysis.

3.2.4 Preparation of MS media (solid)

To prepare 1L of MS media, 4.64 g of MS salts (Gibco, MD), 8 g of bacto-agar, and 10 g of sucrose were mixed in double-distilled water and autoclaved for 15 min using standard autoclaving settings. Antibiotic or other chemical supplements were usually added after autoclaving.

3.2.5 PCR analysis of npr1 allele and nahG transgene

Plant genomic DNA was isolated using Qiagen DNAeasy method following manufacturer’s protocol. For PCR, the following conditions were used. Genomic DNA template: 20-50 ng Final dNTP concentration: 0.2 mM Final primer concentration: 2.0 pmoles (2.0 µM) Taq polymerase: 1.0 unit Buffer (supplied with Taq): 1X o Tm= 57 C

Since the quality of the DNA varies with the method used, PCR conditions have to be slightly adjusted. Sometimes using the buffer with no Mg2+ was effective. Primer sequence for the npr1-1 allele: Forward 5’-CGAATGTACATAAGGCACTTG-3’ Reverse 5’-AGTTGCTTCTGATGCACTTGC-3’ Primer sequences for the nahG transgene: Forward 5’- TTGCCGACCGTACTTCGGAG-3’ Reverse 5’- TAGGCGTCCTCAAGCCCTTG-3’

3.2.6 Salicylic acid and ethylene measurements

Salicylic acid (SA) was extracted from leaf tissue (500 mg) and quantified by spectrofluorescence HPLC using the method of Enyedi and Raskin (1993). To determine the concentration of salicylic acid-glucoside (ß-glucosylsalicylic acid; SAG), the methanolic leaf extract was dried and resuspended in 1.25 mL of hydrolysis buffer (100 mmol.L-1 sodium acetate buffer, pH 5.5) containing 20 units of ß-glucosidase (EC 3.2.1.21; almond). After 1.5 hr incubation at 37 °C, extracts were acidified to pH 1.0 with 10% (w/v) trichloroacetic acid and subjected to SA extraction and quantitation.

3.3 Results

3.3.1 npr1 and SA attenuate lesion formation in hrl1

NPR1 functions downstream of SA and is required for some aspects of SA signal transduction in response to pathogen infection (Cao et al., 1994; Glazebrook et al., 1996; Cao et al., 1997). To understand the role of NPR1 in regulating cell death and defense activation in hrl1, we constructed hrl1 npr1 double mutants (see Methods). Although the initial appearance of lesions on the first true leaves of hrl1 npr1 double mutant was very similar to the lesions on hrl1 plants, development of lesions on the secondary leaves was delayed by at least 10 days (compare Fig 3-1A and Fig 2-1E). Furthermore, the rosettes of hrl1 npr1 plants are larger in size than those of hrl1 plants but smaller than the wild- type parent Col-0, indicating that npr1 partially suppressed cell death and the reduced stature of hrl1 plants. The DAB staining for the presence of H2O2 associated with peroxidase activity and the trpan blue staining for dead cells of hrl1 npr1 leaves were very similar to that of hrl1 (Fig 3-1 E, G). To determine the role of SA in regulating the cell death and defense expression in hrl1, we constructed hrl1 nahG plants. Plants expressing the nahG gene do not accumulate SA and are more susceptible to virulent pathogens (Gaffney et al., 1993; Delaney et al., 1994; Vernooij et al., 1994; Friedrich et al., 1995). To construct hrl1 nahG line, the nahG gene was introduced into hrl1 plants by a genetic cross using a well- characterized Arabidopsis line harboring the nahG gene (see Methods). The onset and the nature of lesions on the first true leaves of hrl1 nahG plants were very similar to that of the hrl1 plants, but their formation on the subsequent leaves was delayed by more than 2 weeks (Fig 3-1B). The rosettes of hrl1 nahG plants were significantly larger than hrl1 plants but somewhat smaller than that of the wild type parent. Moreover, these plants reveal a novel lesion phenotype. In contrast to the punctate and non-spreading lesions on hrl1 plants, lesions on hrl1 nahG plants always commence as chlorotic regions at the tip and the margins of the leaves and spread towards the base (Fig 3-1B). This spreading of lesions resembles the lesions that develop on the "propagation class" of lesion mimic mutants such as lsd1 and acd2 (Dietrich et al., 1994; Greenberg et al., 1994). This

38 A B

hrl1 npr1 hrl1 nahG C D

100µM BTH water Col-0 nahG hrl1 nahG 100µM BTH

hrl1 npr1 hrl1 nahG E F

H2O2 (DAB)

G H Trypan blue

Figure 3-1: Phenotype of hrl1 double mutants (A): 6-week old hrl1 npr1 plant, (B): 6-week old hrl1 nahG plant, (C): BTH-induced lesions in hrl1 nahG after 6 days (arrows), and (D):6-week old Col-0 and nahG plants treated with BTH, H2O2 accumulation in hrl1 npr1 (E) and hrl1 nahG (F), trypan blue staining for dead cells in hrl1 npr1 (G) and hrl1 nahG (H) Pictures in A and B were photographed from the same distance. Scale bars in A and B equal 2.5 cm.

spreading type of lesions was also evident through DAB and trypan blue staining (Fig 3- 1F, H)

3.3.2 hrl1 accumulates elevated levels of SA

SA is a key endogenous signal required for the expression of SA-dependent defense signaling pathway (Gaffney et al., 1993; Delaney et al., 1994). Furthermore, several lesion-mimic mutants accumulate elevated levels of SA (Ryals et al., 1996). We analyzed the endogenous levels of free SA and salicylate glucoside (SAG) in the rosette leaves of 6-week-old soil-grown hrl1 plants (see Methods). The levels of free SA and SAG in hrl1 plants are 2- and 5-fold higher, respectively, than in the parental Col-0 plants (Fig 3-2). However, the increase in the levels of SA and SAG in hrl1 is significantly lower compared to the increase in levels (up to 30-fold) observed in several other SAR mutants such as cpr1, cpr5, cpr6, lsd6, lsd7, and ssi1 (Bowling et al., 1994; Weymann et al., 1995; Bowling et al., 1997; Clarke et al., 1998; Shah et al., 1999). The levels of SA and SAG were reduced to the background amounts in hrl1 nahG plants (Fig 3-2). This result indicates that the delayed cell death observed in hrl1 nahG plants is most likely due to the lack of SA accumulation. Nonetheless, these results indicate that elevated levels of SA in part, may activate SA-mediated defense pathways leading to constitutive defense gene expression and enhanced resistance to virulent pathogens in hrl1 plants.

3.3.3 NPR1 regulates expression of both SA- and ET/JA-responsive genes in hrl1

Expression of SA- and ET/JA-responsive defense genes was analyzed in the lesion+ and lesion− leaves of hrl1 npr1 plants by Northern blot analysis. Lesion+ leaves of hrl1 npr1 and hrl1 plants used in these experiments had similar levels of cell death. The defense-related genes were differentially expressed in the lesion+ and lesion− leaves of hrl1 npr1 plants (Fig 3-3). In the lesion+ leaves, the npr1 mutation moderately suppressed constitutive expression of the PR-1, but had little effect on the expression of PR-2 and GST1 genes. In the lesion− leaves, however, expression of PR-1 was markedly reduced

40 per gramfreshweight, andareaverages analyz (B) Sugar-conjugatedSA(SAG). (A) Freesalicylicacid(SA). hrl1 nahG Figure 3-2:Compari SA andSAGwereextractedfromtherosette ed byHPLCa

Plants. µg/g fresh wt. 0.2 0.4 0.6 0.8 10 2 4 6 8 0 0 1 s des s on of

c hr ribed l h 1 SAandSAGLe rl 1 np in Methods.Thevalues r1 h rl 1 na hG from 4setsofsam leaves of6-week-oldsoil-grownplantsa vels in

41 n pr 1 hrl1

arepresentedasmicrogram ofSA na , Col-0, hG p les pe C o l-0 SAG SA nr r genotype. p1 , nahG

, hrl1 npr 1 nd , and + SD

but GST1 expression was minimally affected. These results suggest that NPR1-dependent and NPR1-independent pathways regulate the expression of PR genes in hrl1.

NPR1, a key regulator of SA-mediated defense signaling pathway, is not required for the expression of the ET/JA-mediated defense pathway (Penninckx et al., 1996). Therefore, to determine if NPR1 has any role in regulating the expression of ET/JA- mediated defense pathways in hrl1, expression of PDF1.2 was analyzed in hrl1 npr1 plants. Northern blot analysis revealed that the expression of PDF1.2 is reduced 3-4-fold both in lesion+ and lesion− leaves of hrl1 npr1 plants (Fig 3-3). The npr1 mutants have been shown to accumulate elevated levels of SA compared to the wild-type parent (Delaney et al., 1995; Clarke et al., 2000). Based on these results, NPR1 has been suggested to regulate feedback accumulation of SA (Clarke et al., 2000). Hence, one possible explanation for reduced PDF1.2 expression in hrl1 npr1 plants is that the npr1 allele increases SA levels, which in turn suppress PDF1.2 expression. To test this possibility, we determined the levels of SA and SAG in hrl1 npr1 and control plants. The levels of SA and SAG in hrl1 npr1 plants are slightly elevated compared to hrl1 plants (Fig 3-2). However, this slight increase in the levels of SA in hrl1 npr1 plants is unlikely to suppress the levels of PDF1.2 by 3-4-fold. These results suggest that the reduced PDF1.2 expression in hrl1 npr1 is probably not the result of high SA levels.

3.3.4 Effects of SA on SA- and ET/JA-responsive defense genes in hrl1

We analyzed the expression of SA- and ET/JA-responsive defense genes in lesion+ and lesion− leaves of hrl1 nahG plants by Northern blot analysis. The extent of cell death in the lesion+ leaves of hrl1 nahG and in hrl1 plants used in these experiments was similar. Results in Figure 3-4 show that defense-related genes are differentially expressed in lesion+ and lesion− leaves of hrl1 nahG plants. Expression of nahG gene in hrl1 nahG plants suppressed the expression of all tested defense-related genes in the lesion− leaves. Preventing SA accumulation in hrl1 nahG plants lead to significant reduction of PDF1.2 expression (2-fold) in the lesion+ leaves and to undetectable levels

r p

hrl1

n Co Lesions + - + -

PDF1.2

11 25 4 6 1 1

PR-1

48 27 10 1 1 1

GST1

11 7 8 4 1 1

AtrbohD

3 7 27 1 1 rRNA

Figure 3-3: Effects of npr1 on defense-related gene expression in hrl1. Transcript levels of PR-1, GST1, PDF1.2, and AtrbohD in the leaves of 6-week-old plants of the indicated genotypes were determined by northern blot analysis. Gene expression was determined separately in lesion+ (+) and lesion− (-) leaves that were collected from the same set of plants. Blot containing 10 µg total RNA of each sample was serially probed with the indicated gene-specific probes. The 18S ribosomal subunit gene-specific probe (rRNA) was used as a loading control. Signals were quantified using the PhosphorImager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA) and were normalized relative to the loading control. The values under each row represent the fold induction of gene expression for each sample compared to that of the untreated control Col-0.

r o

hrl1

h C nahG Lesions + - + -

PR-1

48 27 1 1 1 1

PDF1.2

11 25 6 1 1 1

GST1

11 7 9 1 -5 1 AtrbohD

32711 1

rRNA

Figure 3-4: Effects of nahG on defense-related gene expression in hrl1. Transcript levels of PR-1, GST1, PDF1.2, and AtrbohD in the leaves of 6-week-old plants of the indicated genotypes were determined by northern blot analysis. Blot containing 10 µg total RNA of each sample was serially probed with the indicated gene-specific probes. The 18S ribosomal subunit gene-specific probe (rRNA) was used as a loading control. Signals were quantified using the PhosphorImager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA) and were normalized relative to the loading control. The values under each row represent the fold induction of gene expression for each sample compared to that of the untreated control Col-0.

in the lesion− leaves (Fig 3-4). Thus, SA appears to positively regulate the expression of both SA- and ET/JA-responsive genes in hrl1.

3.3.5 Expression of PDF1.2 in hrl1 nahG plants in response to BTH treatment

Results described above and by others (Shah et al., 1999) demonstrate that SA is required for constitutive expression of PDF1.2 in some Arabidopsis mutants. However, several studies indicate that SA treatment suppresses the expression of JA signaling pathway (Doherty et al., 1988; Doares et al., 1995). To explain this paradoxical result we hypothesized that PDF1.2 induction in hrl1 requires an optimal concentration of SA and deviation from this concentration suppresses PDF1.2 expression. To test this hypothesis we used hrl1 nahG plants that are unable to accumulate SA, and analyzed the expression of PDF1.2 in response to increasing concentrations of BTH. BTH, a biologically active analog of SA induces PR gene expression in the wild-type and SA-depleted nahG transgenic plants (Gorlach et al., 1996; Lawton et al., 1996). Since removal of SA in hrl1 nahG plants blocked punctate lesions, abolished PR-1 expression and partially affected PDF1.2 expression, we tested if BTH treatment would restore these SA-dependent phenotypes. BTH was sprayed at 3 different concentrations (1, 10, 100 µM) on 6-week old hrl1 nahG and the control plants. We found that BTH treatment at 10 and 100 µM concentrations, restored spontaneous punctate lesions within 6 days in the old and in the newly emerging leaves of hrl1 nahG plants (Fig 3-1C) with no effect on Col-0 or nahG plants (Fig 3-1D). Similar to SA treatment, constitutive expression of PR-1 and PDF1.2 in hrl1 plants was induced and suppressed respectively in response to BTH. Furthermore, increasing amounts of BTH restored PR-1 expression in hrl1 nahG plants with significant induction occurring at 100 µM (Fig 3-5A). However, PDF1.2 expression was the highest at 1 µM BTH and then declined with increasing concentrations of BTH. Similar results were obtained with the RNA samples isolated from the lesion free tissue samples harvested 24 hr after BTH treatment, demonstrating that BTH-dependent PDF1.2 induction in hrl1 nahG plants is not due to cell necrosis (Fig 3-5A). As expected, BTH did not induce PDF1.2 expression in Col-0 or nahG plants (Fig 3-5B). These results demonstrate that depending on the

relative concentration, BTH/SA can stimulate as well as suppress PDF1.2 expression in hrl1.

3.3.6 hrl1 constitutively activates AtrbohD gene

AtrbohD, an Arabidopsis homolog of mammalian gp91phox gene, encodes a putative major subunit of NADPH oxidase multienzyme complex and is induced during HR (Keller et al., 1998; Torres et al., 1998), (Gomez and Raina, unpublished results). As shown in Figure 6, hrl1 plants express elevated levels of AtrbohD compared to the wild type Col-0. The expression of AtrbohD is two-fold higher in the lesion- leaves compared to the lesion+ leaves. While the constitutive expression of this gene remains unaffected in hrl1 npr1 plants, it is reduced to background levels in hrl1 nahG plants. These results suggest that the induction of AtrbohD in hrl1 is independent of NPR1 but requires SA accumulation.

3.3.7 Resistance to virulent bacterial pathogens in hrl1 requires both NPR1 and SA

Lesion+ leaves of hrl1 nahG, hrl1 npr1, and control plants were inoculated with Pst DC3000 at a dose of 105 cfu/ml. Bacterial titer was determined 4-days after infection (see Methods). Consistent with the previous reports, npr1 and nahG expressing plants were more susceptible to Pst DC3000 compared to Col-0. Resistance in hrl1 was compromised in hrl1 npr1 and hrl1 nahG plants, although these double mutants were less susceptible than npr1 and nahG plants respectively (Fig 3-6). These results suggest that full resistance displayed by hrl1 requires functional NPR1 and SA accumulation. However, the super-susceptibility exhibited by npr1 and nahG plants was abated due to hrl1 mutation.

3.3.8 Oomycete resistance in hrl1 is NPR1-independent The growth of the virulent oomycete pathogen P. parasitica Ahco2 was also tested on the double mutants mentioned above. We found that the resistance displayed by hrl1 to P. parasitica Ahco2 was abolished in hrl1 nahG plants but was only

46 A hrl1 hrl1 nahG 24 hr 24 hr 6 days

BTH (µM) 0 1 10 100 0 1 10 100 0 1 10 100

PR-1

rRNA

B Col-0 (24 hr) nahG (24 hr)

BTH (µM) 0 1 10 100 0 1 10 100

PR-1

PDF1.2 rRNA

Figure 3-5: Expression of PR-1 and PDF1.2 genes in hrl1 and hrl1 nahG Plants in response to BTH treatments. Six-week-old hrl1 and hrl1 nahG plants (A), and Col-0 and nahG plants (B) were sprayed with the indicated concentrations of BTH and leaves were harvested at the indicated times after treatment. BTH treatment induced hrl1-like lesions on hrl1 nahG leaves 6-days after treatment (Figure 3-1 E) but not within 24 hr. Blot containing 10 µg total RNA of each sample was serially probed with the indicated gene-specific probes. The 18S ribosomal subunit gene- specific probe (rRNA) was used as a loading control. This experiment was replicated twice with different sets of plants and similar results were obtained.

47 109 Day 0 Day 4

1000103 108

107 disc

106 cfu/leaf

105

102 100 104

1 1 r1 G 0 1 G l1 r G -0 r1 G l h l- r r p h l h r p a o p h h n a o p a h n n n a 1 n C n n l1 C n rl 1 r l1 h rl h r h h

Figure 3-6: Effects of npr1, nahG and on growth of Pst DC3000 in hrl1. Leaves of 6-week-old plants of the indicated genotypes were infiltrated with a

suspension of virulent strain of bacterial pathogen Pst DC3000 in 10 mM MgCl2 at a 5 dose of 10 cfu/ml (1OD600= 0.0001). Eight leaf discs from each genotype were collected 4 days after infiltration and bacterial count was determined as described in Methods. The bacterial counts + SD are presented as colony-forming units (cfu) per leaf disc and are averages of three independent experiments.

48 des on ea Figure 3-7: of Three-week-old seedlingsof inde P. par c pe ribed inFigure2-5.Thedata ch pla nde as nt experim itica n Effects of t wa Number of plants Ahco2 inwater(2 s counted7da 10 20 30 40 50 0 e nts. npr 1 01 , and indic ys afterinfe are representedasaveragevalues nahG Col-0 a nahG X10 npr1 hrl1 nahG hrl1 hrl1 npr1 ted ge Disease rat 4 on Grow notypes weres s p ction. Di ores/m 49 l) andthenum th of sea 2 i ng s e rating P. par p ra yed withasporesus asitica wa b er ofconidi 3 + s determ Ahco2 in SD fromthree ined as ophores p hrl1 ension .

3.4 Discussion

Impairments in SA signaling either by depleting SA accumulation or by a mutation in NPR1 resulted in delayed lesions, suppressed PR gene expression, and loss of resistance to virulent bacterial pathogens in hrl1. SA is not required for HR cell death triggered by an avirulent bacterial pathogen Pst DC3000 expressing avrRpm1. However, Pst DC3000 expressing avrRpt2 requires SA for HR cell death. Thus, SA is required for HR cell death depending on which signaling pathway is activated. In addition, lesion- mimic mutants lsd2 and lsd4 develop spontaneous lesions in the absence of SA. In both of these mutants SA depletion did not delay the lesion formation. In hrl1, the nahG expression delayed the lesion development by two weeks. Interestingly the initial appearance of lesions was not affected in hrl1 nahG. Whereas, the nahG expression suppressed all of the mutant phenotypes in acd6, ssi1, and dnd1. Hence, lesion formation is determined both prior to and after SA accumulation.

Expression analyses of defense-related genes in the leaves of hrl1, hrl1 npr1 and hrl1 nahG plants revealed that multiple defense pathways are induced in hrl1. Particularly, we found different signaling pathways regulate the subset of defense-related genes in the lesion+ leaves (tissue mimicking HR) and in the lesion− leaves (tissue mimicking SAR) of hrl1. For example, expression of PR-1 is partially suppressed in the lesion+ and abolished in the lesion− leaves of hrl1 npr1. Reduced level of PR-1 transcript in lesion+ leaves is not the result of leaky expression of npr1 because in the lesion− leaves constitutive expression of PR-1 is completely blocked. However, removal of SA in hrl1 nahG plants blocked the expression of PR-1 in both lesion+ and lesion− tissue. These results indicate the participation of an additional signal generated only in the cells undergoing necrosis, which together with SA can activate PR-1 gene expression independent of NPR1. Apparent from these results, at least three defense pathways; NPR1-dependent, NPR1-independent but SA-dependent, and SA-independent pathways regulate the expression of PR genes in hrl1. Detailed expression analyses in the lesion+ and lesion- leaves separately provided us with new insights on the requirements for local and systemic signaling in a lesion mimic mutant. Previous analyses with lesion mimic-

mutants overlooked this important difference and hence were inconsistent in their PR gene expression profiles.

In the absence of a functional NPR1, hrl1 npr1 plants retain partial and full resistance against bacterial and oomycete pathogens respectively. However, resistance to both these pathogens is severely compromised in the SA depleted hrl1 nahG plants. These results suggest that while resistance to a bacterial pathogen Pst DC3000 is partially regulated through NPR1, resistance to at least one isolate of the oomycete pathogen P. parasitica is independent of NPR1. Existence of a SA-dependent but NPR1-independent pathway for regulation of PR-1 expression and resistance to bacterial pathogens has been previously suggested (Bowling et al., 1997; Clarke et al., 1998; Rate et al., 1999; Kachroo et al., 2000).

Induction of PDF1.2 expression in response to A. brassicicola infection in Arabidopsis has been shown to be independent of both NPR1 and SA (Penninckx et al., 1996). However, Bowling et al. (1997) observed elevated levels of PDF1.2 expression in npr1 plants grown on agar plates compared to the wild-type plants (Bowling et al., 1997). In ssi1 mutant, constitutive expression of PDF1.2 was found to be higher in the npr1 background (Shah et al., 1999). Based on these studies, it has been suggested that NPR1 negatively regulates PDF1.2 expression (Shah et al., 1999). In contrast to these reports, constitutive expression of PDF1.2 in hrl1 plants is reduced 3-4-fold in the absence of NPR1 function. Since the presence of npr1 in hrl1 npr1 plants leads to only a slight increase in the levels of SA, this increase is unlikely to be the reason for the observed reduction in PDF1.2 expression. In fact, the presence of npr1 in other Arabidopsis mutants leads to a significant increase in SA levels, yet constitutive PDF1.2 expression is not suppressed (Shah et al., 1999; Clarke et al., 2000). Furthermore, if the increase in SA levels in hrl1 npr1 was the cause for the suppressed PDF1.2 expression, then the removal of SA from hrl1 plants should alleviate the repression. However, constitutive PDF1.2 expression is suppressed in the SA-depleted hrl1 nahG plants. Although we can not exclude the possibility that elevated levels of SA in hrl1 npr1 plants is responsible for the lowered PDF1.2 expression in these plants, a more plausible explanation is that in hrl1

plants, PDF1.2 expression is regulated through an additional pathway involving NPR1 or an NPR1-derived signal. Involvement of NPR1 in regulating SA-independent defense pathways is not without precedent. For example, SA-independent but JA/ethylene- dependent induced systemic resistance (ISR) activated by P. fluorescens requires NPR1 function (Pieterse et al., 1996; Pieterse et al., 1998). Together, these results demonstrate that NPR1 can respond to multiple signals from various pathogen defense pathways in Arabidopsis. Since NPR1 contains ankyrin repeats that can function in a wide range of protein-protein interactions, it has been suggested to interact with 'adapter' molecules from different defense pathways to mediate these signals (Glazebrook, 1999). One possible mechanism by which NPR1 integrates JA/ethylene-mediated expression of PDF1.2 may involve these ‘adapter’ molecules. Although, the hrl1 npr1 leaves used in the Northern analysis had similar levels of lesion compared to that of hrl1 leaves, it is still possible that npr1 mutation leads to an uncharacterized qualitative difference in the lesions and this difference results in a reduced PDF1.2 expression. However, this possibility does not explain the observed reduction in PDF1.2 in the lesion- leaves of hrl1 npr1 as well.

Studies in several plant species have shown that SA and JA signaling are mutually inhibitory (Doherty et al., 1988; Pena-Cortes et al., 1993; Doares et al., 1995). At the same time, instances of synergism between SA and JA signaling have also been reported (Xu et al., 1994; Schweizer et al., 1997). We speculate that synergism or antagonism between SA and JA signaling probably depends on the relative concentration of the signaling molecules. How does SA function both as an inducer and as a suppressor of ET/JA signaling in hrl1 nahG plants? One possible explanation is that in hrl1 nahG plants, an inert signal is present that needs activation by SA. This SA-activated signal either transduces or activates the components of ET/JA pathway to induce PDF1.2 expression. In the wild type plants this signal is absent and hence SA or BTH alone cannot induce PDF1.2 expression. Also, pathogens may overcome this hrl1-derived signal to induce PDF1.2 independent of SA. A similar conjecture has been postulated to explain the SA-dependent induction of PDF1.2 in ssi1, but the role of cell death was never ruled out (Shah et al., 1999). However, when endogenous SA concentration

exceeds a certain critical threshold, it blocks JA/ET biosynthesis or their downstream signals, and thus suppresses the constitutive expression of PDF1.2.

It is interesting that spontaneous cell death formation, most likely due to an oxidative stress in hrl1 requires classical SA signaling components for defense-related gene expression and defense. Though, some bacterial elicitors require SA for cell death and PR expression, most of the elicitors do not depend on SA or NPR1 for local defense- related gene expression. Recently, it was shown that EDS1 and PAD4 are required for superoxide driven cell death in a lesion-mimic mutant lsd1 (Rusterucci et al., 2001). EDS1 and PAD4 that share homology to lipases are likely to encode lipid-based derivatives involved in SA signal transduction (Feys et al., 2001).

Chapter 4

Effects of ethylene and jasmonic acid signaling in hrl1

4.1 Introduction

Although SA is an important signal mediating defense against a variety of pathogens, resistance response to some pathogens such as Alternaria brassicicola and Botrytis cinerea is independent of SA and NPR1 (Penninckx et al., 1996; Penninckx et al., 1998). This SA/NPR1-independent pathway is characterized by the induction of PDF1.2 and thionin genes that encode antimicrobial peptides and requires functional ethylene (ET) and jasmonic acid (JA) signaling pathways (Penninckx et al., 1996; Penninckx et al., 1998). Another JA/ET signaling-dependent defense pathway, known as induced systemic resistance (ISR) is activated by root colonizing bacteria P. fluorescens. Intriguingly, although ISR is independent of SA, it requires NPR1 (Pieterse et al., 1996; Pieterse et al., 1998).

SA- and JA/ET-mediated signaling, although, appear to regulate distinct defense pathways, several studies indicate cross-talk between these pathways (Dong, 1998; Maleck and Dietrich, 1999). For example, ethylene has been shown to potentiate SA- mediated induction of PR-1 in Arabidopsis (Lawton et al., 1994a). Simultaneous application of JA and SA superinduces PR-1 in tobacco (Xu et al., 1994). Cell death of Arabidopsis protoplasts induced by fumonisin, a toxin produced by the fungus Fusarium moniliforme, requires functional SA, JA and ET signaling pathways (Asai et al., 2000). Experiments with cDNA microarrays revealed that relatively large numbers of Arabidopsis genes are coordinately regulated by SA and methyl jasmonate (MJ) (Schenk et al., 2000). Recently, analysis of Arabidopsis cpr mutants that constitutively express SA- and JA-responsive genes, revealed that components of JA/ET-mediated resistance pathway are required for SA-mediated, NPR1-independent resistance (Clarke et al., 2000). NPR1, a key regulator of SA-mediated SAR response, is also required for mediating the JA/ET-mediated ISR response (Pieterse et al., 1998). Simultaneous

activation of SA-dependent SAR and JA/ET-dependent ISR in Arabidopsis has an additive effect on induced resistance against Pseudomonas syringae (van Wees et al., 2000). While these studies demonstrate the synergistic effects of various defense- signaling pathways, several studies have reported antagonistic effects between these pathways. For example, SA and its derivative acetyl SA suppress JA biosynthesis and downstream signaling in tomato (Pena-Cortes et al., 1993; Doares et al., 1995). SA also inhibits ethylene biosynthesis in apple (Leslie and Romani, 1988). NPR1 has been suggested to negatively regulate the expression of JA/ET-responsive defense gene PDF1.2 (Shah et al., 1999). While SA promotes HR-related cell death, JA represses superoxide-driven cell death and thus lesion containment (Overmyer et al., 2000; Rao et al., 2000). These studies suggest that ethylene, JA and SA play an antagonistic as well as a synergistic role in regulating cell death and defense. In this chapter I will analyze the relationships of ethylene and JA signaling in modulating cell death and disease resistance in hrl1.

4.2 Methods

4.2.1 Construction of hrl1 etr1 double mutant

To construct hrl1 etr1 double mutant, pollen from etr1-1 (Chang et al., 1993) was used to fertilize the hrl1 flowers. Success of the cross was judged by loss of the hrl1 phenotype

in F1 progeny. To identify the hrl1 etr1 double mutants, F2 seeds were plated on 0.8% agar plates containing 50 µΜ 1−amino-cyclopropoane-1-carboxylic acid (ACC), incubated in the dark for 5 days and screened for ET-mediated triple response. Seedlings that failed to display the triple response were transferred to soil and scored for hrl1

symptoms. Homozygous lines for hrl1 and etr1 loci were identified by screening F3 seeds from the individual F2 plants for the lack of triple response. F2 lines that displayed 100%

hrl1 phenotype and lacked the triple response in the F3 progeny were considered homozygous for hrl1 and etr1.

4.2.2 Construction of hrl1 coi1 double mutant and hrl1 coi1 nahG triple mutant

To construct hrl1 coi1 double mutant, pollen from hrl1 was used to fertilize the male

sterile coi1-1 flowers (Xie et al., 1998). F2 seeds were plated on MS media containing 50 µM methyl jasmonate (Me-JA) and 3% sucrose. Seedlings that lacked JA-induced responses (inhibition of root growth and accumulation of excessive anthocyanin) were transplanted to soil, and were scored for hrl1-like phenotype. The presence of coi1 mutation was further confirmed by CAPS analysis using the primers: forward 5’- GGTTCTCTTTAGTCTTTAC-3’ and reverse 5’-CAGACAACTATTTCGTTACC-3’

(Xie et al., 1998). Since coi1 mutant is male sterile, for every experiment, F2 seeds from a line homozygous for hrl1 and heterozygous for coi1 were screened for insensitivity to methyl jasmonate to identify hrl1 coi1 homozygotes.

A line homozygous for hrl1 and coi1 was then crossed with a homozygous transgenic hrl1 line expressing nahG. Since hrl1 coi1 was male sterile, pollen from the hrl1 nahG line was used to fertilize the flowers of hrl1 coi1. The silique formation

confirmed the success of the cross. All of the F1 seedlings displayed hrl1-like phenotype

when germinated and the seeds were collected. The F2 seedlings were later screened on MS Kan containing 50 µM MJ to identify a hrl1 coi1 line expressing at least one copy of the nahG transgene.

4.2.3 Ethylene measurements

Leaves for ethylene measurements were collected and placed immediately on MS media in airtight vials sealed with silicone septum. After 12 hr, 1 ml of gas sample was withdrawn with a syringe and analyzed by gas chromatography (GC) on a Hewlett Packard 6890 instrument equipped with an alumina column and a flame ionization detector.

4.2.4 SA and MJ treatments

Six-week old hrl1 and Col-0 plants were sprayed with 1mM SA or 50-µM MJ solutions. 1 mM SA was prepared by dissolving appropriate amount of the sodium salt of SA in water. 50 µM MJ solution was prepared by making a stock solution of 5 mM in 0.1% ethanol from a 4.37 M 99.9% pure MJ solution. The treated plants were kept covered for 3-4 h to ensure a longer retention of the sprayed solution.

4.3 Results

4.3.1 hrl1 generates 5-fold more ethylene than the wild-type Col-0

We quantified the ethylene produced by hrl1 plants and found that they evolved significantly more ethylene compared to the wild-type parent. Interestingly, the lesion- leaves of hrl1 evolved 5-fold more ethylene than the wild-type Col-0 compared to less than 2-fold by the lesion+ leaves (Fig 4-2). It is possible that the lesion+ leaves do not have enough living cells to evolve higher amounts of ethylene. The lesion+ leaves used to measure ethylene had roughly 32.8+2.8 % living cells (measured by visual comparison using scanned images of the leaf green area with the aid of NIH image analysis software version II). This 2/3 reduction in the number of living cells may account for the reduced ethylene emission in the lesion+ leaves compared to the lesion− leaves. Hence it is very likely that the lesion+ leaves do not have enough living cells to generate the amount of ethylene observed in the lesion− leaves.

4.3.2 Ethylene signaling regulates cell death in hrl1

The results described in Chapter 3 demonstrate that the signaling components of the SA-mediated defense pathway (SA and NPR1) positively regulate the expression of both SA- and ET/JA-response genes. Next, we tested the effects of etr1 mutation on the expression of SA- and ET/JA-response genes in hrl1. ETR1 encodes an ethylene receptor and etr1-1 mutants are defective in ethylene perception (Chang et al., 1993). The appearance of lesions on the first true leaves of hrl1 etr1 was similar to that of the hrl1, but lesion development in the subsequent leaves was delayed by 10 days. Similar to hrl1 nahG and hrl1 npr1 plants, the rosette of hrl1 etr1 was significantly larger than that of hrl1 plants but smaller than the parent Col-0, indicating that etr1 partially suppressed the cell death and the reduced stature of hrl1 plants (Fig 4-1A).

58 A B

hrl1 etr1 hrl1 coi1 C D

hrl1 coi1 nahG hrl1

Figure 4-1: Double mutants of hrl1 with etr1, coi1 and transgenic hrl1 coi1 nahG. (A): 6-week old hrl1 etr1 plant, (B): 6-week old hrl1 coi1 plant, (C): 6-week old hrl1 coi1 nahG plants. Notice that the severe stunting of hrl1 coi1 plant is reversed when the transgene nahG is expressed. (D): 6-week old hrl1 plant. Pictures in A through D were photographed from the same distance and the scale bars equal 2.5 cm.

t 7

s 6

r f

g /

r 4

/ e

n 3

l y

h 2

t e

m p

p 0 ) -) + n l- n io o io s s le C le ( 0 ( l1 l1 r r h h

Figure 4-2: Ethylene levels in the lesion+ and lesion- tissue of hrl1 Ethylene levels were measured from 6-week-old hrl1 and Col-0 plants as described in methods. On an average, 26 leaves per genotype were included for each experiment. The values indicate averages from 3 independent experiments and the error bars represent standard error.

4.3.3 Analysis of defense-related gene expression in hrl1 etr1

Expression of ET/JA- and SA-responsive genes was analyzed in the lesion+ and lesion− leaves of hrl1 etr1 plants by Northern blot analysis. As expected, constitutive expression of PDF1.2 was markedly suppressed in both the lesion+ and lesion− leaves of hrl1 etr1 plants (Fig 4-4). Although the constitutive expression of SA-response genes was unaffected in the lesion+ leaves of hrl1 etr1 plants, expression of PR-1 was markedly suppressed and that of PR-2 and GST1 were moderately reduced in the lesion− leaves. These results imply that in addition to regulating the expression of ET/JA-responsive genes, ethylene signaling positively regulates the SAR expression of a SA-responsive gene in hrl1 plants.

4.3.4 Antagonistic effects of JA and SA on PR-1 and PDF1.2 expression

Several studies indicate that SA treatment suppresses the expression of JA biosynthesis and the expression of a proteinase-inhibitor gene in tomato (Doherty et al., 1988; Doares et al., 1995). Therefore, to understand how SA- and JA-signaling pathways might interact in hrl1, we analyzed the effects of SA and MJ treatments on the constitutive expression of PR-1 and PDF1.2 genes. Consistent with the idea of antagonistic effects, SA treatment suppressed the expression of PDF1.2 and MJ treatment suppressed the expression of PR-1 in hrl1 plants (Fig 4-3). These results suggest that the high PR-1 and PDF1.2 expression hrl1 indicates a delicate balance in the endogenous levels SA and JA. A significant shift in this equilibrium may favor one pathway or the other. It is also important to mention that among all the lesion-mimic mutants analyzed for PR-1 and PDF1.2 expression so far, only hrl1 spontaneously expresses both of these genes to an equally high level.

4.3.5 Mutation in COI1 gene aggravates lesion formation in hrl1

COI1 is required for sensitivity to coronatine and jasmonates in Arabidopsis. The coi1 mutant is defective in jasmonate signaling and does not induce PDF1.2 expression

61 Ethanol SA MJ

0 0 0 - l- - l l1 l1 l l1 o r o r o r C h C h C h Lesions + - + - + -

PR-1

PDF1.2

rRNA

Figure 4-3: Expression of PR-1 and PDF1.2 genes in hrl1 and Col-0 plants in response to SA and MJ. Six-week-old Col-0 and hrl1 plants were sprayed with 1 mM SA, 50 µM MJ, and 0.01% ethanol (solvent for MJ). Leaf samples were harvested 24 hr after treatment. Expression in lesion+ (+) and lesion− (-) leaves of hrl1 was analyzed separately. Blot containing 10 µg total RNA of each sample was serially probed with the indicated gene-specific probes. The 18S ribosomal subunit gene-specific probe (rRNA) was used as a loading control. This experiment was replicated twice with different sets of plants and similar results were obtained.

62 1 i1 r t o 0 e c 1 - 1 1 1 i l l l1 l r o r r r t o h h h e c C Lesions + - + - + - - -

PDF1.2

PR-1

PR-2

GST1

rRNA

Figure 4-4. Effects of etr1 and coi1 on the expression of defense-related genes in hrl1 mutant. Expression of PR-1, PR-2, GST1, and PDF1.2 in the leaves of 6-week-Old plants of the indicated genotype was determined by the northern blot analysis. Gene expression in hrl1 and hrl1 etr1 plants was determined separately in the lesion+ (+) and lesion- (-) leaves that were collected from the same set of plants. No lesion- leaves could be obtained from hrl1 coi1 plants. Blots containing 10 µg total RNA of each sample was serially probed with the indicated gene-specific probes. The 18S ribosomal subunit gene- specific probe (rRNA) was used as the loading control. This experiment was repeated once with different sets of plants and similar results were obtained. The vertical line indicates the separation of autoradiograph during scanning.

in response to chemical or biological inducers (Xie et al., 1998). To assess the role of COI1 in regulating the expression of PDF1.2 and SA-responsive genes in hrl1, we constructed hrl1 coi1 double mutant, by a genetic cross with coi1-1 mutant (see Methods). Interestingly, unlike hrl1 npr1, hrl1 nahG or hrl1 etr1 plants, in which lesion formation was attenuated, the lesions in hrl1 coi1 plants were exaggerated and the plant rosette was severely stunted (Fig 4-1B). Lesions in hrl1 coi1 plants had severe bleaching and the entire leaf collapsed within a week following lesion initiation.

4.3.6 COI1 is necessary for PDF1.2 induction in hrl1

We analyzed the expression of PDF1.2 and SA-response defense genes in the lesion+ leaves of hrl1 coi1 plants by Northern blot analysis. Consistent with its signaling requirements, PDF1.2 gene expression was significantly reduced in these plants (Fig 4- 4). Similar to hrl1 etr1 plants, expression of PR-1, PR-2 and GST1 remained unaffected in the lesion+ leaves. Since hrl1 coi1 plants were extremely dwarfed with severe lesions, we could not obtain lesion-free tissue samples from these plants for Northern analysis. These results show that in addition to transducing JA-dependent defense signals, COI1 is necessary to limit the severity of cell death in hrl1.

4.3.7 Role of ET/ETR1 in regulating resistance against pathogens in hrl1

Due to the very small stature and severe lesions on hrl1 coi1 plants, we could not reliably infect them to study their response to pathogens. Lesion+ leaves of hrl1, hrl1 etr1, and control plants were inoculated with Pst DC3000 at a dose of 105 cfu/ml. Bacterial titer was determined 4-days after infection (see Methods). Consistent with the previous reports Col-0 and etr1 plants had similar levels of Pst DC3000 (Fig 4-5). Interestingly, hrl1-mediated resistance was compromised in hrl1 etr1 plants. These results suggest that resistance to Pst DC3000 in hrl1 is mediated by the simultaneous expression of SA- and ET-signaling pathways. The growth of the virulent oomycete pathogen P. parasitica Ahco2 was also tested on the double mutants mentioned above. We found that the resistance displayed by hrl1 to P. parasitica Ahco2 was abolished in hrl1 etr1. The double mutant had more

64 1000103 Day 0 disc cfu/leaf

102 100 1000000000109 Day 4

100000010008

10000001007 disc

1000100006 cfu/leaf 100010005

10001004 0 1 1 l- r1 l tr t r e o e h 1 C rl h

Figure 4-5: Effects of etr1 on growth of Pst DC3000 in hrl1. Leaves of 6-week-old plants of the indicated genotypes were infiltrated with a suspension of virulent strain of bacterial pathogen Pst DC3000 in 10 mM MgCl2 at a 5 dose of 10 cfu/ml (1OD600= 0.0001). Eight leaf discs from each genotype were collected 4 days after infiltration and bacterial count was determined as described in Methods. The bacterial counts + SD are presented as colony-forming units (cfu) per leaf disc and are averages of three independent experiments.

65 40 hrl1 hrl1etr1 Col-0 etr1 30 ants l

20 No. p

0 0123 Disease rating

Figure 4-6: Effects of etr1 on growth of P. parasitica Ahco2 in hrl1. Three-week-old seedlings of indicated genotypes were sprayed with a spore suspension of P. parasitica Ahco2 in water (2 X 104 spores/ml) and the number of conidiophores on each plant was counted 7 days after infection. Disease rating was determined as described in Figure 2-5. The data are represented as average values + SD from three independent experiments. Double mutant hrl1 coi1 was not included in this experiment due to its extreme dwarf and severe lesions.

conidiophores compared to hrl1, indicating that ethylene signaling is required for resistance to P. parasitica in hrl1. These results suggest that resistance to virulent oomycete in hrl1 also requires the concurrent expression of SA- and ET-signaling pathways.

4.4 Discussion

The role of ethylene in the transduction of SA-dependent defense responses against pathogens is not well understood. Analysis of Arabidopsis mutants impaired in ethylene signaling indicate that although sensitivity to ethylene is required for development of disease symptoms in response to virulent bacterial pathogens, it is not essential for elicitation of the hypersensitive response against avirulent pathogens (Bent et al., 1992; Lawton et al., 1994a). Ethylene-insensitive tomato plants infected with Xanthomonas campestris pv. vesicatoria do not accumulate SA and show reduced necrosis (O'Donnell et al., 2001). However, ethylene insensitive soybean mutants are altered in their resistance response to certain avirulent pathogens (Hoffman et al., 1999). Similarly, transgenic tobacco plants expressing an Arabidopsis etr1 gene show normal hypersensitive response to tobacco mosaic virus infection, but lack non-host resistance against the soil borne fungi Pythium sylvaticum (Knoester et al., 1998). These results suggest that although ethylene may not be a global signal for establishing plant immunity, it can supplement some of the dominant resistance responses in a subset of host-pathogen interactions. The role of ethylene signaling in regulating the SAR response is also not clear. For example, although PR-1 expression in response to chemical inducers of SAR is not affected in the ethylene insensitive mutants, upon SA treatment wild-type Col-0 plants showed enhanced PR-1 induction following ethylene exposure (Lawton et al., 1994b). Presence of ein2 in Arabidopsis mutants cpr5 and cpr6 does not significantly alter the constitutive PR-1 gene expression or resistance to P.s. maculicola ES4326 (Clarke, 2000). The results from these genetic analyses support a model in which SA-dependent SAR gene expression and resistance do not depend on ethylene signaling. However, in the hrl1 etr1 double mutant, constitutive expression of PR-1 is not affected in the tissue mimicking HR (lesion+ leaves) but is blocked in the tissue mimicking SAR (lesion− leaves) (Fig 4-4). Based on these results, we believe that ethylene plays an important role in relaying or amplifying the signal(s) that emanate from the local necrotic tissue to potentiate SA-dependent SAR gene expression in the distal healthy tissue. By analyzing the expression of defense genes in lesion+ and in lesion− leaves, we are able to assign

distinct spatial dependence for ethylene-mediated transcriptional regulation of PR gene expression. The down-regulation of PR gene expression in the systemic tissue of hrl1 etr1 also correlates with the suppressed resistance against virulent bacterial and oomycete pathogens in these plants (Fig 4-5). Thus, ethylene seems to enhance SA-mediated PR gene expression in the lesion− leaves of hrl1 and also aids in strengthening the SAR response against normally virulent pathogens. COI1 defines a crucial control element in transmitting JA-regulated responses against pests and pathogens in Arabidopsis and other plant species. Based on its sequence homology to Arabidopsis TIR1, human Skp2 and yeast Grr1, COI1 has been hypothesized to play a role in targeting regulators of defense and pollen development for modification by ubiquitination (Xie et al., 1998). The hrl1 coi1 double mutant has exacerbated lesions and is extremely dwarfed compared to hrl1. This phenotype is in sharp contrast to what we observed with the other double mutants of hrl1. The nahG expressing hrl1, hrl1 npr1, and hrl1 etr1, all had attenuated lesions and larger rosette compared to hrl1, presumably due to the reduced accumulation of toxic defense-related compounds and a reduced metabolic burden associated with their synthesis. Although the expression of PDF1.2 and possibly other COI1-regulated defense genes is blocked, hr1 coi1 double mutant has severe lesions and stunted rosettes compared to hrl1. We speculate that the absence of COI1-mediated signaling may lead to further accumulation of toxic compounds constitutively produced in hrl1. Alternatively, unidentified signaling pathways that are repressed by JA/COI1 may be turned on in hrl1 coi1 plants leading to severe lesions. The fact that MJ pretreatment of ozone-sensitive Arabidopsis ecotype Cvi- 0 and rcd1 mutant mitigated the propagation of cell death, and MJ-insensitive jar1 and MJ-deficient fad3/7/8 developed spreading lesions in response to ozone suggest a protective role for jasmonates in containing cell death (Overmyer et al., 2000; Rao et al., 2000). The phenotype of hrl1 coi1 leads us to believe that COI1-assisted JA signaling may also serve to protect the cells against ROI-driven cell death. Interestingly, transgenic tobacco plants expressing a variant ubiquitin showed necrotic cell death and altered response to TMV infection, indicating a causal link between ubiquitination and pathogen induced cell death (Bachmair et al., 1990; Becker et al., 1993).

4.4.1 Possible roles of HRL1 in regulating cell death and defense

How does HRL1 regulate cell death and multiple defense pathways? Although it is difficult to predict the position of HRL1 gene in defense signaling pathway, we hypothesize that wild type HRL1 protein might be involved in regulating an early step leading to ROI production during plant-pathogen interactions. In support of this hypothesis we show that hrl1 mutant accumulates elevated levels of AtrbohD transcript. AtrbohD encodes a major subunit of NADPH oxidase multienzyme complex and may

participate in superoxide generation. Superoxide can be rapidly dismutated to H2O2 and high levels of H2O2 can result in cell necrosis. Formation of hrl1-associated lesions are attenuated but not blocked in hrl1 npr1, hrl1 etr1 and hrl1 nahG plants suggesting that cell death in hrl1 is modulated by, but not dependent on, signals downstream of SA, NPR1, and ETR1.

Accumulation of H2O2 can lead to synthesis of SA and SA in turn can stabilize

H2O2 by inhibiting catalase activity (Chen et al., 1993; Leon et al., 1995). Elevated levels of SA along with H2O2 can activate PR and GST1 gene expression locally and can serve as a systemic signal to activate SAR in the distal (lesion−) leaves. In addition, superoxide generated due to the overexpression of AtrbohD can induce PDF1.2 gene. Chemicals such as paraquat or rose bengal that generate superoxide and singlet oxygen respectively, also induce PDF1.2 gene expression (Bowler et al., 1992; Green and Fluhr, 1995). Although SA alone cannot induce cell death or accumulation of ROI, it can potentiate an elicitor-mediated generation of ROI and HR-associated cell death (Shirasu et al., 1997). In our experiments, removal of SA in hrl1 nahG plants significantly reduces AtrbohD expression (Fig 3-4). This raises the possibility that SA or a SA-regulated signal may enhance the accumulation of ROI by positively regulating the transcription of AtrbohD and possibly other related genes.

SA alone cannot induce cell death or accumulation of ROI, but it can potentiate an elicitor-mediated generation of ROI and HR-associated cell death (Shirasu et al., 1997). We find that the depletion of SA in hrl1 nahG plants significantly reduces

AtrbohD expression (Figure 3-5). This raises the possibility that SA or a SA-regulated signal may enhance the accumulation of ROI by positively regulating the transcription of AtrbohD and possibly other related genes. Recent evidence suggests that EDS1 and PAD4 may enhance resistance by processing ROI- and SA-activated molecules (Rusterucci et al., 2001).

4.4.2 Conclusion and perspectives on studies using lesion-mimic mutants

Signaling during pathogen-induced cell death and disease resistance is emerging to be an intricate web, where perturbation of one or more signals leads to collateral effects. Genetic screens for pathogen resistance in Arabidopsis have resulted in the identification of a number of constitutive SAR mutants and some of them have lesion- mimic phenotype. Except a few, it has been difficult to define the exact roles of these genes despite the fact that most of them have high SA levels and increased pathogen resistance. There is a growing concern that many of the observed defense signaling in either the constitutive SAR or the lesion-mimic mutants do not necessarily follow the expected patterns. I feel it is important to emphasize here that these mutants, including hrl1, are subject to multiple chronic stress conditions and are under constant pressure to adapt to these permanent changes. Whereas, in the experiments that involve pathogen infections, the stimulus and responses are usually specific, acute as well as transient. Hence it is not illogical to expect some of the known regulators of defense to deviate from their characteristic signaling requirements. For example no pathogen induces both PR-1 and PDF1.2 to high levels. However simultaneous activation of PR-1 and PDF1.2 has been observed in a good number of lesion-mimic mutants including hrl1. Hence, these mutants provide an interesting platform to understand how these two seemingly independent signaling pathways interact and provide resistance to diverse pathogen species. These constitutive defense mutants serve as alternate experimental systems to evaluate the various stress responses resulting from signals such as ROI, SA, JA and ethylene.

Considering the genetic complexity involved in maintaining the cellular homeostasis, it is not surprising that many genetic and environmental insults are likely to induce cell death. Indeed, many genes known to be involved in regulating basic metabolism are differentially expressed in response to pathogen infection (Schenk et al., 2000) Gomez and Raina, unpublished results). Many of these genetic components are likely to be the part of overlapping signaling pathways and might act as ‘funnels’ to guide different signals to activate cell death in response to various stimuli. Such genetic components have been reported in animal systems (Anderson, 2000; Hatada et al., 2000). In spite of the significant overlap among different signaling pathways, it is important to note that while cell necrosis can be induced by many stresses or genetic alteration, not all activate downstream SAR responses. For example, mechanical injury, application of inorganic chemicals and catalase inhibitor 3-aminotriazole can induce necrotic lesions that are cytological similar to pathogen-induced lesions, they do not trigger SAR (Dekayos, 1972; Tighe and Heath, 1982; Neuenschwander et al., 1995). Furthermore, while a large number of lesion-mimic mutants or transgenes whose overexpression induces lesions, have been reported, only some trigger increased SA levels, PR-1 expression and pathogen resistance, hallmarks of SAR (reviewed in (Mittler and Rizhsky, 2000; Shirasu and Schulze-Lefert, 2000). Finally, recent epistasis analysis of constitutive SAR mutants (cpr1, cpr6, dnd1 and dnd2) and lesion-mimic mutants (crp5 and lsd1,) with known defense regulators (eds1, pad4 and ndr1) demonstrate that defense expression in these mutants requires known defense regulators (Clarke et al., 2001; Jirage et al., 2001; Rusterucci et al., 2001).

In summary, we provide new evidence that the signaling components of SA- and ET/JA-regulated defense pathways may function synergistically to regulate the expression of both the SA- and ET/JA-responsive genes and resistance against virulent strains of bacterial and oomycete pathogens. Furthermore, we provide support for an emerging paradigm that both the presence and relative concentrations of various endogenous signals enable plants to fine tune their transcriptional read out against a wide variety of stress responses through synergistic or antagonistic regulation (Reymond and

Farmer, 1998; Feys and Parker, 2000). These studies further highlight the complexity involved in the regulation of the defense response in plants against pathogens.

Chapter 5

Analysis of the suppressed HR phenotype of hrl1

5.1 Introduction

The HR cell death is often preceded by changes in ion fluxes, oxidative burst and cross-linking of cell wall proteins. Most of the HR cell death processes are accompanied by an increase in salicylic acid (SA) biosynthesis, transcriptional activation of various pathogenesis-related (PR) genes and the establishment of a long lasting systemic response known as systemic acquired resistance (SAR) (Hammond-Kosack and Jones 1996; Ryals et al. 1996).

Several lines of evidence indicate that HR cell death is a form of programmed cell death that resembles apoptotic cell death in other organisms (Mittler and Lam 1996; Morel and Dangl 1997). Identification and analysis of several Arabidopsis mutants with spontaneous cell death that mimic pathogen-induced cell death support the idea that HR cell death may be controlled by plant’s own genetic mechanisms (Dangl et al. 1996; Greenberg 1997; Glazebrook 1999). Although, HR cell death is intrinsically controlled by the plant, the relative importance of cell death in conferring resistance to pathogens is not well understood. For example, the Arabidopsis nonspecific disease resistance1 (ndr1) mutant exhibits normal HR cell death but is susceptible to certain avirulent Pseudomonas syringae (Century et al. 1995; Century et al. 1997). Conversely, in barley, Mlo-conferred resistance against an obligate biotroph Erysiphe graminis f. sp. hordei was abolished when HR cell death was inhibited (Schiffer et al. 1997). These and other studies suggest that HR cell death is not always required for resistance and may vary depending on the pathogen species involved (Bendahmane et al. 1999). Although, the role of HR cell death is not clear, the role of SAR induction that immediately follows HR is well established.

Signal molecules like SA and H2O2 that emanate from the local infected tissue spread systemically to induce a broad-spectrum resistance response in many plant species (Lamb and Dixon 1997; Alvarez et al. 1998). Despite the lack of a clear role for HR cell death in disease resistance, its prevalence in most of the gene-for-gene mediated resistance

signaling is intriguing. This well conserved nature of HR cell death suggests that the ability to undergo rapid cell death may offer other benefits to plants that are not yet fully understood.

Genetic screens aimed at identifying the loss of resistance to avirulent pathogens have resulted in the cloning of several R genes. These R genes, when mutated, fail to develop HR against one or more avirulence factors and they represent genetic components that are required rather early in the signal transduction leading to HR cell death (Bent 1996; Dangl and Jones 2001). However, further attempts to identify additional components failed to yield any new mutations other than the targeted R genes (Innes 1998). The relative scarcity in identifying new mutants that lack HR cell death may be due to the presence of functionally redundant genes, embryo lethality and weaker HR− phenotypes that were overlooked. In recent years, some of the Arabidopsis mutants with elevated resistance response were shown to possess partial loss of HR phenotype against avirulent pathogens (Yu et al. 1998; Rate et al. 1999; Yu et al. 2000; Rate and Greenberg 2001). Among them, the Arabidopsis defense no death1 (DND1) gene has been cloned and found to encode a cyclic nucleotide-gated ion channel (Clough et al. 2000). Many of these mutants have high SA levels, increased PR-gene expression, and enhanced resistance to virulent pathogens. In aberrant growth and death2 (agd2) mutant, the loss of HR phenotype was reversed in the nonexpressor of PR-1 (npr1) and in the SA- depleted (nahG) genetic backgrounds (Rate and Greenberg 2001). In accelerated cell death6 (acd6) mutant, upon delivery of the AvrRpt2 elicitor inside the plant cell, HR cell death was rescued (Rate et al. 1999). Apart from these genetic studies, there are instances in a wide range of plant species, where HR cell death was suppressed by treatments with a transcriptional inhibitor, an actin polymerization inhibitor, and incubation in low oxygen (Tomiyama, Sato et al. 1982; Mittler, Shulaev et al. 1996; Schiffer, Görg et al. 1997).

The fact that a significant number of the constitutive SAR mutants of Arabidopsis are compromised in eliciting HR against avirulent pathogens indicated a feedback role for defense responses to suppress further HR induction. Here we report the detailed

characterization of the suppressed HR phenotype of hypersensitive response like lesions1 (hrl1) mutant against avirulent bacterial pathogens and in response to an endogenously expressed elicitor. The rescue of normal HR cell death in hrl1 npr1 and hrl1 nahG plants strongly suggests that the elevated SAR response in hrl1 plays an active role in suppressing HR cell death. This is further supported by our observation that pre-inducing SAR in wild-type plants also restricts HR cell death in response to avirulent pathogens.

5.2 Methods

5.2.1 Bacterial inoculations

Bacterial pathogens Pst DC3000 with an empty vector and Pst DC3000 carrying a plasmid borne avrRpm1 gene [Pst DC3000 (avrRpm1)] or avrRpt2 gene [Pst DC3000 (avrRpt2)] (Whalen et al., 1991) were grown at 28oC on King’s B agar plates or in liquid medium (King et al., 1954) supplemented with 100 µg/ml rifampicin and 25 µg/ml kanamycin. Bacterial culture was prepared by resuspending the overnight grown cells in 9 10 mM MgCl2 to a final optical density (OD600) of 0.01 or 0.1 (1 OD600= 10 cfu/ml). The bacterial suspension was pressure-infiltrated on the abaxial side of the leaves using a 1 ml syringe to cover the entire leaf surface. For growth curves, eight leaf discs (0.5 cm in diameter) from eight different plants were collected for each time point at indicated times and ground in 10 mM MgCl2 using a pestle. Serial dilutions were plated on King’s B agar plates supplemented with appropriate antibiotics. Plates were incubated at 28oC for 2 days to determine the number of colony-forming units.

5.2.2 Dexamethasone treatments

A 30 mM Dexamethasone (Sigma, St. Louis, MO) stock solution was made in 100% ethanol and was stored at –20oC in a dark vial. For treatments, the 30 mM stock solution was diluted to a final concentration of 30 µM in water and was pressure-infiltrated on the abaxial side of the leaves using a 1 ml syringe. Control infiltrations were performed with 0.1% ethanol solution. Plant responses were recorded 24 hr post inoculation.

5.2.3 Electrolyte leakage measurements

For electrolyte leakage measurements, four leaf punches (0.5 cm diameter) were taken at indicated time points and were shaken for 10 min at 28oC in 2 ml water. The solution was transferred to a portable VWR brand conductivity meter (VWR Scientific Products, Pittsburgh, PA) for conductivity measurements.

5.2.4 Construction of a transgenic hrl1 line expressing inducible AvrRpt2

To construct a transgenic hrl1 line expressing inducible avrRpt2, pollen from a transgenic Col-0 line transformed with a glucocorticoid-inducible avrRpt2 cassette was used to fertilize hrl1 flowers (McNellis, Mudgett et al. 1998). The resulting F1 seedlings were selected on MS media containing 20 mgL-1 hygromycin B (Sigma, St. Louis, MO) and -1 were allowed to set seeds. The F2 seedlings were again selected on 20 mgL hygromycin B and the resistant ones were scored for hrl1 phenotype. Genomic DNA was isolated from the hygromycin-resistant hrl1 plants using a DNeasy isolation kit following the manufacturer’s protocol (Qiagen, Valencia, CA). A 400-bp avrRpt2 fragment was PCR- amplified using primers 5’-GCTCCAGTTGCCATAAATCACA-3’ (forward) and 5’- CAGGCATACCAACATCCCATT-3’ (reverse) to confirm the presence of the transgene.

5.3 Results

5.3.1 Delayed and attenuated HR in hrl1 mutant

The hypersensitive response-like lesions1 (hrl1) mutant shows constitutive SAR gene expression and spontaneous cell death. In addition, the hrl1 mutant is resistant to virulent bacterial and oomycete pathogens (described elsewhere). To characterize the hypersensitive response of hrl1 to avirulent bacterial pathogens, we infiltrated the leaves of 6-week-old hrl1 plants with P. syringae pv. tomato DC3000 expressing avrRpm1 (Pst DC3000:avrRpm1) at a dosage of 108 cfu/ml. Usually, within 4-5 hours, Col-0 plants show collapse of tissue at the site of pathogen infiltration , a characteristic feature of HR- associated cell death (Fig 5-1A). However, hrl1 plants did not show any visible HR even after 8 hours and a small percentage developed a very weak HR after 24 hours (Fig 5-1 A, B). The weak HR in the mutant plants was restricted to a small area surrounding the point of infiltration and was not confluent. The hrl1 leaves are smaller then the Col-0 leaves because hrl1 plants are significantly smaller than the corresponding Col-0 plants.

We also measured the electrolyte leakage in hrl1 and in the wild-type Col-0 following Pst DC3000 (avrRpm1) infection (Fig 5-2A). Electrolyte leakage due to membrane damage is a characteristic feature of HR cell death collapse (Goodman and Novacky 1996). The Col-0 plants infiltrated with 107 cfu/ml Pst DC3000 (avrRpm1) showed maximal conductivity within 8 hours. The hrl1 mutant did not show a substantial increase in ion leakage within 8 hours and the levels were stagnant even after 24 hours (Fig. 5-2A). These results indicate that unlike the wild-type parent Col-0, hrl1 mutant is significantly impaired in its ability to elicit HR cell death against avirulent bacterial pathogens.

79 A 8 hr B 24 hr

hrl1

+ - lesion lesion Col-0 lesion+ lesion- Col-0 hrl1 hrl1

C D

lesion- lesion+ npr1 hrl1 nahG nahG hrl1 npr1

E F

hrl1: Col-0: Col-0 Col-0 Col-0 avrRpt2 avrRpt2 + Dex (SAR pre-induced) + Dex + Dex

Figure 5-1: Suppressed HR of hrl1 in response to avirulent pathogen. A: Leaves of six-week old hrl1 and Col-0 plants were infiltrated with 108 cfu/ml of P. syringae (avrRpm1) and photographed after 8-hr. B: The pathogen-infiltrated hrl1 and Col-0 leaves after 24 hours as described in (A). C: Leaves of six-week old hrlnpr1 and npr1 plants, 12 hours after pathogen infiltration as described in (A). D: Leaves of six-week old hrl1nahG and nahG plants, 12 hours after pathogen infiltration as described in (A). E: Leaves of six-week old transgenic hrl1 plants expressing avrRpt2, transgenic Col-0 expressing avrRpt2, and wild-type Col-0 infiltrated with 30 µM dexamethasone. The picture was taken 24-hr after treatment. All the photographs shown above are representative samples from a large experimental pool.

80 A

120 Col-0 hrl1 100 S/cm) µ

( 80 y t 60

ductivi 40 Con 20

0 246824 hr post infiltration B

12,000 hrl1 Col-0

10,000 sc i

d 8,000 f 6,000 /lea 4,000 cfu 2,000

0 03924 hr post infiltration

Figure 5-2. Electrolyte leakage and pathogen growth in hrl1 after avirulent pathogen infiltration A: Electrolyte leakage in hrl1 and Col-0 following pathogen infiltration (P. syringae DC3000 (avrRpm1; 108 cfu/ml)) . The error bars represent + SD from 3 independent trials. µS., micro Siemens. B: Growth of P. syringae (avrRpm1) in hrl1 and in Col-0. The graph displays the reduction in the growth of P. syringae DC3000 (avrRpm1) in hrl1 and in Col-0. Each time point represents the average from three independent growth curve experiments. A minimum set of eight plants per genotype was included in each experiment. The pathogen inoculum was 107 cfu/ml.

5.3.2 Suppressed HR in hrl1 is not due to an immediate decrease in viable pathogen concentration

The timing and the magnitude of the visible HR (macroscopic HR) in plants are often pathogen-dosage dependent. If the pathogen concentration falls below a certain threshold, then there is no visible HR (Goodman and Novacky 1996). Since hrl1 mutant has high defense-related gene expression and perhaps accumulates antimicrobial compounds, it is possible that, upon infiltration, the viability of pathogens is affected leading to a suppressed HR. Another possibility is that, not enough pathogen is infiltrated in the mutant leaves compared to the wild-type control. Therefore, we tested the growth of an avirulent pathogen, Pst DC3000 (avrRpm1), in Col-0 and in hrl1 plants at a dose of 107 cfu/ml (see Methods). The zero hour time point (Fig 5-2B) demonstrates that similar amount of pathogen was infiltrated in both the mutant as well as the wild-type. The extent of pathogen growth in hrl1 and in Col-0 is very similar through 24 hours indicating that the avirulent bacterial population is reduced to similar levels in both the genotypes. These results suggest that the delayed HR in hrl1 is not due to a preferential decrease in viable bacterial population because of the pre-existing defense responses. Furthermore, these results indicate that despite the lack of a visible HR, hrl1 plants mount similar level of resistance to avirulent bacterial pathogen compared to Col-0 plants.

5.3.3 Direct expression of a bacterial elicitor protein in hrl1 plants fails to induce HR

Since P. syringae pathogens depend on type III secretion apparatus for efficient delivery of avirulence factors into the plant cell, the suppressed HR in hrl1 plants could be due to an impaired transfer of Avr proteins into the plant cell (Bonas and Ackervaken, 1997; Alfano and Collmer, 1997). Therefore, to circumvent the pathogen-based delivery of Avr protein into the plant cells, we constructed a transgenic hrl1 line expressing avrRpt2 from a glucocorticoid-inducible promoter (see Methods). Plants harboring this construct express AvrRpt2 protein in response to dexamethasone (Dex) treatment and induce HR-associated cell death through the cognate RPS2-dependent signaling (McNellis et al., 1998). If the lack of HR in hrl1 is primarily due to a block in the

delivery of the Avr proteins, then the direct expression of an Avr protein within the plant cells should trigger normal HR. Such endogenous expression of bacterial elicitor proteins in plant cells has been shown to trigger HR cell death in a variety of experimental systems (Gopalan et al., 1996; Leister et al., 1996; McNellis et al., 1998). Dex treatment of transgenic Col-0 plants expressing the Dex-inducible avrRpt2 induced HR within 24 hours (Fig 5-1E). The Dex-infiltrated region of the leaf showed HR within 8 hours and the entire leaf collapsed within 24 hours. However, the hrl1 transgenic lines harboring avrRpt2 gene did not develop HR even after 24 hours following Dex treatment (Fig 5- 1E). These results strongly suggest that the suppressed HR in hrl1 plants is not due to defect in the transfer of AvrRpt2 protein into the cells of hrl1.

5.3.4 PR-1 Expression in hrl1 Remains Unaltered After Pathogen Infection

Active host resistance in plants is often accompanied by the induction of several pathogenesis-related (PR) genes ( Uknes et al., 1991). Proper recognition of the Avr factors by the cognate resistant gene products in plants leads to rapid induction of these defense-related genes during an incompatible interaction. The PR gene induction is slower and weaker during a compatible interaction. In hrl1 plants, many of these PR genes are expressed to high levels in the lesion+ as well as in the lesion− tissue (Chapter 3 and Chapter 4). We tested the expression levels of PR-1 in hrl1 and in Col-0 plants following avirulent and virulent bacterial pathogen infections. Prior to infection, PR-1 transcript levels were elevated in hrl1 plants (Fig 5-3A). Within 24 hours after infection with an avirulent P. syringae pathogen, Col-0 plants showed a significant increase in PR- 1 expression. However, hrl1 plants did not show any further enhancement of PR-1 expression even 3 days post infection. This unaltered PR-1 induction pattern was also observed in the virulent pathogen-infected hrl1 plants. Although the hrl1 plants failed to show an increase in PR-1 induction after pathogen infection, they displayed similar levels of resistance to an avirulent pathogen as observed in the control plants (Fig 5-2B).

83 A hrl1 Col-0

avr vir avr vir d.p.i 0 1 2 3 1 2 3 0 1 2 3 1 2 3

PR-1

rRNA

B hrl1 Col-0 (24 hr) BTH -+ -+ PR-1 rRNA

Figure 5-3. Analysis of PR-1 expression in hrl1 and Col-0 after pathogen infection and BTH treatment. The RNA blot represents 10 µg of total RNA isolated from pathogen-infected (A) and BTH-treated (B) tissues of hrl1 and Col-0. Avr denotes avirulent P. syringae DC3000 (avrRpm1) and vir denotes virulent P. syringae DC3000 at 107 cfu/ml. d.p.i refers to days post infection. The100 µM BTH was sprayed as an aqueous solution and the samples were collected 24 hours later. The 18S ribosomal subunit gene-specific probe (rRNA) was used as a loading control.

Treatment of hrl1 plants with 100 µM benzothiadazole (BTH), an active analog of SA, lead to a two-fold increase in PR-1 expression 24-hours after treatment (Fig 5-3B). This suggests that the ability of hrl1 to induce PR gene expression is not saturated, at least to a SAR-inducing chemical, but the defense machinery is selectively unresponsive to pathogen stimulus. These results lead us to infer that hrl1 mutation restrains any further PR-1 induction and possibly other defense-related responses including HR in response to avirulent pathogens.

5.3.5 The reversal of HR− phenotype in hrl1 depends on the extent of SAR induction

Since hrl1 plants exhibited several defense responses associated with pathogen attack we analyzed if the pre-existing SAR has any role in desensitizing the HR induction in hrl1 plants. The npr1 mutant was isolated based on its inability to transduce some of the SA-mediated responses and the nahG gene encodes a salicylate hydroxylase that converts SA into an inactive catechol (Gaffney, Friedrich et al. 1993; Cao, Bowling et al. 1994; Cao, Glazebrook et al. 1997). The hrl1 npr1 double mutant and the transgenic hrl1 nahG plants have reduced PR-1 gene expression (Fig 5-4) and compromised resistance response (described elsewhere). The PR-1 expression in the lesion  leaves of hrl1 npr1 plants is reduced compared to the lesion+ leaves of hrl1 and is undetectable in the lesion− leaves of hrl1 npr1 plants (Fig 5-4). Northern analysis demonstrates that PR-1 expression in both the lesion+ and lesion− leaves of hrl1 nahG was reduced to background levels (Fig 5-4). In addition, expression levels of other SAR responsive genes that were induced in hrl1 were also reduced to undetectable levels in hrl1 nahG plants.

We monitored the HR of lesion+ and lesion− leaves of hrl1 plants in response to an avirulent bacterial pathogen over a 24-hr period (Fig 5-5). Within 6 hours, more than 95 % of the infiltrated wild-type Col-0 plants showed HR, while less than 10% of the lesion− leaves of hrl1 showed weak HR (Fig 5-5). The suppressed effect was slightly pronounced in the lesion+ leaves of hrl1. Even after 24 hours, less than 20 % of the infiltrated leaves of hrl1 developed a visible HR. The hrl1 plants exhibited delayed-HR

when the pathogen concentration was increased by 10-fold to 109 cfu/ml or in response to another avirulent strain of Pst DC3000 expressing avrRpt2 elicitor (data not shown). We analyzed HR induction in lesion+ and lesion− leaves of hrl1 npr1 and hrl1 nahG plants. Similar to hrl1, lesion+ leaves of hrl1 npr1 elicited delayed and attenuated HR in response to Pst DC3000 (avrRpm1) (Fig 5-1C-D, Fig 5-5B). However, the lesion  leaves of hrl1 npr1 exhibited HR similar to wild-type Col-0, in terms of both timing and magnitude (Fig. 1C, 5-5B). In addition, the suppressed HR phenotype was completely reversed in the lesion+ as well as in the lesion− leaves of hrl1 nahG (Fig. 1D, 5-5C). The positive HR in the lesion− leaves of hrl1 npr1 and in the leaves of hrl1 nahG plants correlated well with the loss of PR-1 expression in these tissues. These results suggest that the pre-existing induced defense responses in hrl1 plants may down regulate further induction of HR against pathogen attack.

5.3.6 Strong SAR inducing treatments before pathogen inoculation suppress HR cell death

Results described above demonstrate that constitutive SAR expression suppresses HR-associated cell death in hrl1 plants. This prompted us to evaluate the effects of SAR- inducing treatments in wild-type Col-0 in suppressing HR cell death. SAR was induced in Col-0 plants either by application of 1.5 mM SA or by infiltration with a low dose (105 cfu/ml) of an avirulent pathogen, Psm E4326 (avrRpm1). At this low dose, Psm E4326 (avrRpm1) does not elicit macroscopic HR, but induces several SAR-associated genes (Gomez and Raina, unpublished). The npr1 and nahG plants, incapable of activating SAR in response to SA, were included as controls. Twenty-four hours after SA or pathogen treatment, these plants were infiltrated with an avirulent pathogen Psm E4326 (avrRpm1) at a dose of 107 cfu/ml to assess the effects of the preexisting SAR on HR elicitation. SA- or pathogen-treated Col-0 plants experienced significantly reduced HR- associated cell death compared to the water-treated Col-0 plants, as judged by the reduced electrolyte leakage (Fig 5-6). SA treated npr1 mutant and the nahG transgenic line did not show any reduction in electrolyte leakage levels. These results clearly

86 G r1 h p a n n 1 1 1 r1 G 0 rl rl p h l- h rl h n a o h n C Lesions + - + - + - - --

PR-1

GST1

rRNA

Figure 5-4. Defense-related gene expression analysis in hrl1 and other double mutants of hrl1. Northern blot containing 10 µg of total RNA, isolated from the indicated genotypes was hybridized with PR-1, GST1 gene-specific probes. The 18S ribosomal subunit gene-specific probe (rRNA) was used as a loading control. The lesion+ and lesion− represent leaves from the same set of six-week-old plants that had necrotic cell death. In this figure, all the wild- type plants were considered lesion−.

demonstrate that pre-inducing SAR in wild-type plants leads to a substantial decrease in HR cell death.

88 hrl1 (lesion+) A hrl1 (lesion-) Col-0 120

100 es

v 80

lea Figure 5-5. Quantitative

+ 60

R representation of the HR in 40 hrl1, hrl1 npr1 and hrl1 nahG

% H plants following avirulent 20 pathogen infection. 0 One half of the leaf of six-week 36hr 91224 old plants were infiltrated with 108 cfu/ml of P. syringae (avrRpm1) and scored for the B hrl1 npr1 (lesion+) characteristic HR cell death at hrl1 npr1 (lesion-) the indicated time points. About npr1 120 30 leaves from six plants per genotype were infiltrated. The 100 graphs represent the percentage 80 of leaves that developed HR in

leaves hrl1 and Col-0 (A), in hrl1npr1 + 60 and npr1 (B), and in hrl1 nahG R 40 and nahG (C) plants. The mean

% H values + standard deviation (SD) 20 from three independent 0 experiments are plotted. The + − 3 6 hr 9 12 24 lesion and lesion denote the leaves in which the lesions had just initiated and the leaves C hrl1 nahG (lesion+) hrl1 nahG (lesion-) without any visible lesions nahG respectively. 120

100

80 leaves

+ 60 R 40 % H 20

0 3691224 hr

89 acid C following avirulentpathogeninfection. prior toinoculationwith10 with 10mM Figure 5-6. o nductivity m (SA) , 10 Electrolyte leak MgCl

5 Conductivity (µS/cm) cfu/m 100 120 easure 20 40 60 80 0 2 . Errorbarsindicate+ l of m ents insix-weekoldplan 246 P . npr1 Col-0 (SAR) Col-0 7 syringae cfu/ml age inSA (S hr postinfiltration A Psm ) m a R-induced C c u E4326 ( licola SD fromthreeinde 90 ES4326 ( ts thatweretreatedwith1.5mM avrRpm1 8 o l-0, Col-0 (mock) nahG Col-0 (SA) avrRpm npr ). 2 4 Mo 1 pe (SA) an ck ndent e 1 d represents infiltration ) nahG (SAR) x perim plants or water,24-hr e nts. salicylic

5.4 Discussion

In an attempt to understand the regulation of HR response against pathogens in plants, we employed an Arabidopsis constitutive SAR mutant hrl1, and showed that the preexisting defense responses antagonize HR cell death. The HR was compromised even when the AvrRpt2 elicitor was expressed within the hrl1 plants suggesting that the repressed HR cell death was not due to a defective transfer of Avr proteins into the plant cells. Our results suggest that constitutive SAR expression suppresses the HR-associated cell death in hrl1 plants. In support of this finding, we showed that the HR− phenotype could be reversed in the double mutants of hrl1 in which the elevated defense-responses were compromised. In addition, we demonstrated that pre-treating wild-type Col-0 plants with SAR-inducing agents suppressed HR-associated cell death.

The induction of HR cell death in resistant plants upon pathogen attack is probably the most well recognized active resistance response. Although, the exact role of cell death during HR is unclear, the controlled initiation and execution of HR cell death are thought to limit the spread of pathogens and other unwanted toxic products into healthy cells (Morel and Dangl 1997). Hence, it is necessary for the host cellular machinery to precisely control the untoward spread of HR cell death. The fact that the resistance gene product RPM1 undergoes rapid degradation soon after HR initiation strongly suggests the existence of a negative feedback loop modulating the extent of cell death at the site of infection (Boyes et al. 1998). The best line of evidence for the genetic control of HR-like cell death stems from the analyses of several lesion-mimic mutants that may be perturbed in regulating certain aspects of pathogen-induced cell death. Despite the isolation and characterization of several constitutive cell death mutants, genetic screens have failed to identify clear loss of HR mutants. However, mutations in the R genes that deprive the plants of their ability to recognize the avirulence factors are HR  with the caveat that they all represent genes very early in the signal transduction cascade (Dangl, 1995). The Arabidopsis dnd1 mutant, originally identified in a screen for reduced HR against Pseudomonas syringae pv. glycinea (avrRpt2), was later found to be a rare/conditional lesion mimic mutant (Clough et al., 2000). The DND1 gene encodes a cyclic nucleotide-gated ion channel and its ion-channel activity may be needed for strong

HR induction. However, why such a mutation that affects HR leads to systemic resistance is not clear. Alternatively, the systemic resistance itself may be responsible for the reduced HR in dnd1 mutant.

The constitutive defense responses in hrl1 may lead to desensitization of the HR activating machinery following pathogen attack. The suppression of HR in hrl1 may not be a direct effect of the hrl1 mutation but may be consequence of a sustained resistance response exhibited by hrl1 plants. This resistance-induced suppression of HR is supported by our results with the hrl1 npr1 double mutant and transgenic hrl1 nahG plants. In the presence of npr1 allele, PR-1 expression is partially reduced in the lesion+ leaves but is completely absent in the lesion− leaves of hrl1 npr1 plants. Accordingly, in hrl1 npr1 plants, the lesion+ leaves showed suppressed HR but the lesion− leaves developed normal HR in response to avirulent pathogens. In addition, both the lesion+ and the lesion− leaves of hrl1 nahG plants exhibited normal HR to Pst DC3000 (avrRpm1). The full HR recovery in hrl1 nahG plants correlates well with the loss of PR- 1 expression in the lesion+ as well as in the lesion− leaves. Expression of several other PR genes (PR-2, PR-5) was also significantly reduced in hrl1 nahG (data not shown) suggesting that SAR induction was severely compromised in hrl1 nahG. The role of systemic resistance in suppressing HR cell death is further supported by the reduced electrolyte leakage in Col-0 plants that were pre-treated with inducers of SAR. However, pre-treating npr1 and nahG plants with SA did not alter the electrolyte leakage levels, suggesting that SAR signaling is critical in suppressing HR cell death.

It is not known clearly if the elevated defense responses present in agd2, acd6 or dnd class of mutants played any role in altering HR-associated cell death (Rate et al. 1999; Clough et al. 2000; Yu et al. 2000; Rate and Greenberg 2001). Epistasis analysis in agd2 mutant indicated that NPR1 and SA might negatively regulate HR in response to pathogens (Rate and Greenberg 2001). Our results with hrl1 npr1 and hrl1 nahG clearly suggest that the suppressed constitutive SAR in these plants make them more responsive to eliciting HR during pathogen attack. Such elicitation competency might have been suppressed in hrl1 plants due to the sustained high level of SAR expression. The lack of

enhancement of PR-1 expression in hrl1 plants following pathogen inoculation can also be explained by the reduced elicitation competency in response to pathogen infection. It should be noted however that the magnitude and the nature of SAR induction in hrl1 might not reflect the physiology of other constitutive defense mutants and hence other mutants might respond differently to HR-inducing pathogens. For example, prelesion lsd1 mutant was hyper-responsive and exhibited faster HR-like symptoms to both virulent and avirulent pathogens at a very low dosage (105cfu/ml) (Dietrich et al. 1994). However, lsd1 mutant did not have preexisting SAR prior to pathogen inoculation when grown in the permissive environment (Dietrich, Delaney et al. 1994). In contrast, acd6 mutant is impaired in its ability to perceive the elicitor and therefore does not develop HR against avirulent pathogens (Rate et al. 1999). However, acd6 plant tissue exhibited normal HR cell death when the elicitor was delivered inside the plant cell through biolistic transformation. Interestingly, acd6 nahG plants developed normal HR in response to Pst DC3000 (avrRpm1) (Rate et al. 1999). Tobacco plants that were pretreated with resistance-inducing heat killed Ralstonia solanacearum cells developed reduced HR, presumably due to the activation some of the defense responses (Lozano and Sequeira 1970).

Pre-treatment of tobacco plants with high oxygen pressure before pathogen infection resulted in a delayed HR (Mittler et al. 1999). It was suggested that the antioxidant mechanisms that were activated during the oxidative stress might scavenge the ROI generated during pathogen infection leading to the suppression of HR. However, the expression of SAR genes was not analyzed in those plants. In hrl1 plants, we found the GST1 expression to be high and its expression pattern was not affected in the hrl1 npr1 plants. Nevertheless, the lesion− leaves of hrl1 npr1 with elevated GST1 expression developed normal HR suggesting that the induction of anti-oxidant mechanisms alone is not sufficient to suppress HR. Although the anti-oxidant mechanisms in hrl1 as a consequence of cell death might play a role in suppressing the HR cell death, the constitutive SAR induction might have a greater effect in the compromised HR response.

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SAR inducers (SA) HR cell death

SAR

Figure 5-7. A model for the possible suppression of HR cell death by pre-existing SAR pathways. Various methods of SAR induction by pathogen infection or SA treatment are shown. Similar constitutive SAR response in hrl1 may be responsible for the suppressed HR cell death against avirulent pathogens. Pre-existing SAR either due to SA treatment or a low dose of avirulent pathogen can suppress HR cell death against further pathogen attack.

What are the possible mechanisms that suppress HR in hrl1 plants? There are numerous signaling steps in the HR cascade, which, when affected, can influence HR. For example, down regulation of K+ and Cl- efflux channel activities in hrl1 plants can lead to a severe reduction in HR. Ca2+ channel blockers have been shown to inhibit HR in tobacco and soybean cells (Atkinson et al. 1996). Continuous generation of ROI and other antimicrobial compounds such as phytoalexins may render hrl1 mutant refractory to changes in membrane permeability that is crucial for HR cell death (Hahlbrock et al. 1995). As mentioned earlier, in the case of RPM1 degradation upon elicitation, rapid turnover of various R gene products could prevent HR induction. Although we cannot accurately predict which step is perturbed in hrl1 that leads to suppression of HR, our results indicate a systemic desensitization response that down regulates further HR induction (Fig 5-7).

In addition to the defense-related processes discussed above, there are instances where interfering with normal cellular homeostasis lead to the suppression of HR. For example, treatments of potato cells with inhibitors of actin polymerization such as cytochalasin B and colchicine blocked HR cell death triggered by Phytophthora infestans (Tomiyama et al. 1982). The inhibition of cytoplasmic aggregation in elicitor treated potato cells delayed some of the resistance reactions that are involved in HR cell death (Furuse et al. 1999). These studies provide biochemical evidence for the existence of functional proteins and components of HR signaling that are associated with the cytoskeleton. However, at present, it is not known if perturbation of any of these normal cellular functions in hrl1 affects HR induction.

Desensitization provides a way for cells to adapt to permanent differences in levels of certain signaling compounds. Receptor down-regulation as a tool to achieve desensitization and tolerance is a common cellular adaptation in many hormonal and neuronal responses in animal systems (Pawson 1995). In plants, suspension-cultured tomato cells undergo desensitization to alkalanization of the growth medium in response to repeated stimuli with chitin elicitors (Felix et al. 1998). In addition, elicitor induced oxidative burst in cultured soybean cells render the cells insensitive to further induction

of ROI generation by the same or a different stimuli (Chandra et al. 2000). These observations demonstrate that desensitization can serve as a general mechanism to tightly regulate cellular processes that have significant overlap. In hrl1, the chronic stress response due to PR gene expression may alter the cellular physiology at various nodes that lead to insensitivity to subsequent pathogen attack. Down-regulation of HR cell death in the presence of an already existing systemic resistance response may be a way for plants to prevent excessive cell death and further defense induction (Fig 5-7). Identification of host factors and the mechanisms that lead to desensitization without compromising the resistance response will be valuable in developing plants with enhanced defense in the absence of unwanted cell death.

Chapter 6

Global Analysis of Gene Expression in hrl1

6.1 Introduction

Meaningful success from the genome sequencing efforts can be achieved if only we are able to elucidate the functions of all the genes. The advent of microarray technology represents a significant step in our understanding of the gene regulation at the transcriptional level (Schena et al., 1995; Brown and Botstein, 1999). Various methods are available for detecting and quantitating gene expression levels, including Northern blots, differential display and serial analysis of gene expression (SAGE; Duggan et al., 1999). To augment this coterie are two array-based technologies__cDNA and oligonucleotide arrays. These allow us to study static expression levels of several thousands of genes in parallel. The high degree of digital data extraction and processing requires a good understanding of statistical and mathematical tools and thus provides a new platform, where biologists, mathematicians and statisticians interact better than ever before.

6.1.1 Principle of method

Hybridization between nucleic acids is the essential property that drives any immobilized substrate-probe binding. Traditionally, most applications of this method have employed a single, pure, labeled oligonucleotide or polynucleotide species in the liquid phase and complex mixtures of polynucleotide species attached to a solid support. Transcript abundance is usually assayed by the amount of radioactively or fluorescently labeled probe immobilized to the target. cDNA microarrays adopt a different strategy (Cheung et al., 1999). In an array experiment, several gene-specific fragments of cDNAs are spotted individually on a single matrix, usually a processed glass slide. This matrix is then simultaneously probed with fluorescently tagged cDNA representations of total RNA pools from test and control samples. The differential labeling of test and control

RNA species using wavelength-specific dyes allows us to quantify the relative abundance of transcripts that correspond to the spotted gene.

6.1.2 Fabrication of DNA microarrays

Production of arrays begins with the selection of the cDNA fragments to be printed on the array. In many cases, ESTs from a general public database, cDNA clones from the libraries that are derived from a specific cell type or after special treatments, and PCR amplified products from the annotated regions are chosen for a global analysis of gene expression. However, more specialized arrays are also common where the array contains genes that are selected based on their expected functions. cDNA arrays are generated by spotting PCR products representing specific genes onto a matrix which is usually a specially treated glass slide. The PCR products are usually generated from purified templates to avoid cellular contaminants and also any ambiguous sequences. Typically 200-500 ng/ml of purified PCR product that is resuspended in SSC is printed onto the glass slide. The printing is carried out by a robot that spots several genes at a time onto multiple arrays. There are several methods of printing available that include peizo electric or ink-jet modes are used (Bowtell et al., 1999). The arrays are usually coated with poly-lysine, amino-silanes or amino-reactive silanes. The DNA is cross- linked to the matrix by UV radiation followed by a series of post-print processes.

6.1.3 Target labeling and hybridization

Typically, reverse transcription of total or mRNA pools from an oligo-dT primer is used. The fluorescent Cy3/Cy5 dyes can be incorporated during the cDNA synthesis or can be chemically coupled to the cDNA after synthesis. The labeled product is the single stranded cDNA pool that is complementary to the immobilized targets on the array. The purity of RNA and the shelf life of the dyes are probably the two most important factors in determining the success of microarray hybridizations. Proteins, lipids and carbohydrates that contaminate the RNA samples often affect the fluorescence while

scanning. The fluorescent Cy3 and Cy5 dyes are light labile and their spectral properties are altered upon long storage.

6.1.4 Global analysis of gene expression in hrl1 and in the double mutants of hrl1

Initial gene expression analysis in hrl1 mutant indicated that multiple signaling pathways mediated by SA, JA and ethylene were activated. In addition, through epistasis analysis with npr1, nahG, etr1 and coi1, different signaling requirements were uncovered in the lesion+ and in the lesion- leaves of hrl1. However, these analyses have been focused on a few genes that have been previously associated with their respective defense-signaling pathways. From such studies it is rather difficult to comprehend the global signaling patterns that may involve genes that are not part of the defense repertoire. Quantitative and simultaneous analysis of expression profiles using cDNA microarrays that contain genes enriched for pathogen response and genes that are involved in normal cellular homeostasis will provide useful information regarding cell death and defense regulation in hrl1. The cDNA microarrays used in this study has genes that have been selected from several specialized cDNA libraries. These cDNAs were derived from Arabidopsis plants that have been treated with SA, avirulent and virulent bacterial pathogens, avirulent and virulent oomycete pathogens, ozone, and methyl jasmonate. The cDNA population was further enriched by adopting a PCR-based suppression subtractive hybridization method that increased the likelihood of low- abundant genes. The cDNA microarray also contains ESTs from the public domains that were isolated from untreated Col-0 plants. Thus the combination of selective stress- specific genes and genes that are expressed during standard growth conditions provides a powerful tool to analyze global gene expression patterns in hrl1.

6.2 Methods

6.2.1 Total RNA extraction for microarray hybridization

Total RNA was isolated using Qiagen RNAeasy Plant Mini kit with the following modifications from the protocol that was provided with the kit: a) 200 mg of tissue was ground in liquid nitrogen until it was a fine powder; b) 900 µl of RLT buffer was added and the sample was vortexed vigorously for 15 s; c) Samples were incubated at 65oC for 5 min; d) For elution 50 µl of RNAse-free water (Promega, WI) was heated to 65oC and added to the center of the column. The samples were incubated for 5 min instead of 1 min. The procedure was repeated twice to increase the yield (approx. 140 µg/100 µl); e) For quantification, RNAse-free water was used. To measure the quality (260/280 ratio), 10 mM Tris-Cl pH 8.0 was used

6.2.2 Concentrating RNA

RNA extracted by the Qiagen method needs to be concentrated for chip hybridizations. One-tenth the volume of 3M sodium acetate pH 5.2 (DEPC treated) was added and 300 µl of 100% ethanol was mixed. The samples were incubated at –80oC overnight and spun at 13,000 g for 20 min at 4oC. The pellets were washed with 70% ethanol twice to reduce, briefly air-dry and resuspended in 20 µl of RNAse-free water. OD 260/280 was measured again in water and Tris-Cl as mentioned above. All the samples were measured using the same RNAse-free water (Promega, WI).

6.2.3 Reverse transcription using amino-allyl dUTP

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The reverse transcription reaction for microarrays is similar to the routinely practiced cDNA synthesis protocols. However, a modified amino-allyly dUTP is used which will be necessary for coupling Cy3 and Cy5 dyes. a) 25-30 µg RNA was taken in a final volume of 10 µl RNAse-free water. 3.6 µl of spiking RNA (positive control), 2.5 µl of oligo dT (18-mer from Sigma, PAGE purified), and 0.5 µl of RNAse inhibitor RNAsin (Promega) were mixed to the RNA sample in a PCR tube and the reaction mixture was incubated at 65oC for 10 min in a thermal cycler. Using a thermal cycler gave a better temperature control and consistency. The samples were then allowed to cool at room temperature for 10 min. b) To set up the reverse transcription, 6 µl of 50X first-strand buffer, 3 µl of 0.1M DTT, 3 µl of 10X aadNTP mix (5mM each of dA, dC, dG, 3 mM dTTP, 2 mM aadUTP), and 2 µl of Superscript II 200U/ml (Gibco-BRL) were mixed and then added to the cooled RNA mix. Since the oligo dT is loosely bound to the mRNA, pipetting was avoided and only gentle swirls were performed. The samples were incubated at 42oC for 2 h in a thermal cycler. Note: aadUTP is unstable over long periods of time and hence it should be prepared once every 2 months following this protocol. 1 mg aadUTP (Sigma) was mixed with 189 µl of PCR grade water and the pH was adjusted to 7 with 0.7 µl of 1N NaOH. 47.5 ml of 100 mM dATP, dGTP, dCTP and 28.5 ml dTTP were added. The final volume was made to 950 µl with 589 µl of PCR grade water. The sample was aliquoted into 50 µl and was frozen at –80oC. c) Once the reverse transcription was done, 10 µl of 0.5 M EDTA (pH8.0) was added to the samples to stop the reaction. Then 2.0 µl of freshly prepared 5N NaOH (final concentration is 0.25 N) was added to denature the RNA hybrid. The samples were incubated at 65oC for 15 min. d) 20 µl of 1M HEPES (pH7.5) was added to neutralize the sample. HEPES-free acid was used and the pH was adjusted to 7.5 if needed.

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6.2.4 Post reverse transcription clean-up

To remove the HEPES and other salts left over from the reverse transcription, a 30-kDa cut off membrane filter Nanosep (PALL) was used. Pre-rinsing of the column was done

by adding 500 µl of ddH20 and centrifuging at 5000 g for 5 min. The neutralized reaction

sample was mixed with 450 µl of ddH20 and centrifuged for 5-6 min. The left over liquid in the reservoir was less than 100 µl. The process was repeated for 4 more times and the final concentrated volume was usually 50-75 µl. The remaining liquid was removed and concentrated in a speed-vac with medium heat (45-60 min). The dry sample was stored at –20oC for more than a week until further coupling.

6.2.5 Coupling of Cy3 and Cy5 dyes

The steps that involve the fluorescent dyes have to be done in darkness (no direct lighting). Fresh tubes of mono reactive Cy3/Cy5 NHS-esters were resuspended in 75 µl of DMSO and 4.5 µl aliquots were stored in amber colored tubes at 4oC in a light proof box under dessication. The dyes are usually stable for two weeks after resuspension. The

dried cDNA was resuspended in 4.5 µl of 0.1 M NaHCO3 pH 9.0 (pH is critical). In the dark, 4.5 µl of the cDNA was added to the appropriately labeled dye tubes. Usually the experimental sample was coupled to Cy3 and the control was coupled to Cy5. But the dye needs to be swapped to ensure dye efficiency in the replicate experiments. The samples were incubated at room temperature for 1 h in the dark. 4.5 µl of 4M hydroxylamine was added to prevent cross coupling of dyes at later stages. 70 µl of water was added to one of the sample tubes (for e.g., Cy3) and then transferred to its corresponding Cy5 counterpart. The samples were then purified using QiaQuick (Qiagen) following the manufacturer’s protocol. The eluant (50 µl) was dried in a speed-vac without heat in the dark. This step was completed in approximately 2.5 h.

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6.2.6 Processing of silanized slides

The DNA printed side on the slide was identified by the presence of a layer of spots with ridges. The sides and the underside of the printed area were marked with a diamond scribe. The slide was usually labeled at the bottom side opposite to the printed end. The slide was moistened by placing the slide (face down) over a hot water reservoir in the humidity chamber. Usually this process takes less than a minute or until the dots become shiny and well defined. The moistened slides were snap dried by pressing them against a heating block set at 80oC for 3-5 sec. The snap dried slides are UV-cross linked at 1900 (X100) µjoules. The slides were then immersed in 0.1% SDS for 30 sec and then transferred to water for 30 sec and then in boiling water for 3 min followed by a 1-2 min dip in 70% ethanol. They were spin dried at 500 rpm for 5 min at 18oC.

6.2.7 Pre-hybridization

Pre-hybridization buffer was prepared fresh every time by mixing 30 ml of autoclaved ddH20, 12.5 ml of 20X SSC, 0.5 ml of 10% SDS and 0.5 g of BSA. The components were heat dissolved and filtered if necessary. The processed slides were immersed in a koshland jar containing 50 ml of the pre-hybridization buffer. The slides were incubated at 42oC for 45 min to 1 h.

6.2.8 Hybridization

Coverslips with lifter pads were rinsed in 0.1% SDS, water and 95% ethanol. They were spread over clean paper towels and dried. The slides were rinsed in autoclaved water 10- 12 times and in 95% ethanol for 5-6 times and spin dried as described above. The hybridization buffer is prepared by mixing the following components per sample: 7.5 µl of de-ionized formamide (Sigma), 3.75 µl of 20X SSC, 1.5 µl of 1 mg/ml Herring sperm DNA (Sigma), 1.5 µl of 50X Denhardts solution (Sigma), and 0.75 µl of 2% SDS. To each dried labeled cDNA sample, 15 µl of the hybridization buffer was added. This probe was denatured in boiling water for 2 min followed by cooling at room temperature for 5

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min. In the mean time, the hybridization cassettes were prepared by adding 10 µl water into the tiny slots in the cassette cover. The probe was spun down briefly. Cover slips were placed over the marked area on the slide and the probe is gently under the cover slips. If the probe did not spread properly, slight pressure was applied to ensure thorough spread of the probe under the cover slip. Hybridization was carried out for 18 h at 42oC in a water bath or in air incubator.

6.2.8 Post-hybridization washes

The slides were plunged first in a beaker containing 2X SSC/0.1% SDS for 3 min with gentle shaking followed by a 2 min immersion in 1X SSC. The slides were then transferred to a 0.2X SSC solution for 1 min and then to 0.05X SSC for 15 sec. After briefly spin drying at 500 rpm for 5 min at 18oC, the slides were scanned in the GenePix scanner.

6.2.9 Scanning and data analysis

The slides were scanned using GenePix Pro version 3.0 software interfaced with a confocal scanner. The spots in the array were aligned with the appropriate grids manually and the results were analyzed using the same software. The final data files were retrieved with the feature and background intensities for each dye channel with other statistical analysis. The normalization was done on a chip per chip basis and involves the use of spiking controls. For the hybridizations performed here, the following methods were adopted (i) Spots with intensity less than 500 were not included (ii) Non-uniform distribution of signal on the spots were flagged (iii) If the values from two experiments differed by more than 3-fold, then those were flagged for further analysis (iv) 2.5-fold induced or repressed were included for clustering for the lesion- tissue sample and 3.5-fold for the lesion+ tissue sample of hrl1

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(v) For normalization the following spots were included : 10 spots for each a) Myosin heavy chain (MHC), b) bacillus subtilis lysA (LYS), c) insulin like growth factor (IGF). They consist of 5 different concentrations spotted in duplicates. These spiking controls adjust for any difference in RT. To ensure same amount of RNA was taken, homeotic genes like ubiquitin, fatty acid desaturase (FAD2), actin subunits, and elongation factor 1 alpha were included. The ratio of the sum of these intensities for Cy3 and Cy5 channels provides the normalization factor. (vi) The data from the two replicates were not averaged. Instead they were just used to measure the reproducibility of the pattern of induction. The data set with the minimum number of flagged spots was chosen for further analysis

6.2.10 Cluster analysis

The data files from GenePix were further polished by removing the flagged spots and by adjusting with the normalization factor. K-means clustering was performed by using the Cluster 2.0 software (available at http://rana.stanford.edu/software) and the clusters were visualized using TreeView program (available at http://rana.stanford.edu/software) following the instructions given on the accompanying manuals.

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6.3 Results Table 6-1: Global gene expression pattern in hrl1 and the double mutants

Microarray analysis were performed with RNA isolated from 6-week old hrl1, hrl1npr1 (hnpr) and hrl1nahG (hnahG) plants. RNA was isolated separately from lesion positive (+) and lesion negative (-) leaves. The hybridizations were performed twice. The values in red are induced, green are repressed and black are no change in expression. The cut off was 2.5 fold. The empty cells represent the values that were not reproducible.

ID Description hrl1 Hnpr hnahG hrl1 hnpr hnahG Le+ Le+ Le+ Le- Le- Le- NPR1- and SA-dependent rpm4-a3 eukaryotic protein synthesis 8.944 1.495 1.2602 1.387 2.34 0.69565 initiation factor 4A mjf-eg08 RNA-binding protein RNP-T 9.722 1.55 1.6989 1.556 2.634 2.41203 precursor rpm2-f4 disease resistance protein w/ 9.89 0.706 5.4614 1.867 1.689 1.04878 ATP/GTP-binding site mjf-hc10 hypothetical protein 4.754 0.818 1.6501 1.0710.87405 app-dc10 hypothetical protein 4.793 1.357 1.4517 1.604 1.38889 est 98d6t7 cytochrome P450 like protein 6.613 1.933 1.9151 1.248 2.054 0.84099 vppbd6 Erd1 protein precursor 5.516 1.62 1.0601 1.779 2.429 2.10494 aozb68 predicted protein of unknown 5.059 1.572 1.9428 1.444 1.33 1.72917 function similar to b 137j19t7 unknown protein 5.002 2.064 1.6986 1.861 1.236 0.5928 vppgc1 unknown protein 7.431 2.158 1.078 1.71 1.886 0.7227 134e2t7 DNA binding protein-like with 8.148 1.349 1.4343 1.676 1.453 0.82161 RING domain aozc18 hypothetical protein 4.085 1.307 1.9796 1.861 1.188 1.63324 vppfe5 nodulin-like protein 5.808 1.083 1.4258 1.167 1.702 1.22927 rpm2-e2 putative protein 4.218 0.719 2.3825 1.172 0.936 1.87522 sa2-a6 unknown protein, similar to 4.572 0.975 0.7514 2.345 0.638 0.91197 elongation factor 1-ga sa3-a3 putative subtilase 4.157 2.301 1.0934 1.832 1.531 0.92038 avr2e5 auxin-responsive - like-protein 5.831 1.926 1.1403 1.175 1.38 0.6676 moz65 heat shock protein 90 11.52 3.305 1.7382 1.526 1.711 0.78157 rpm4-b10 putative cytochrome P450 11.51 1.876 0.6938 4.035 1.106 0.59139 PR-1 PR-1 22.4 4.56 1.85 12.35 1.054 0.9527

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aozb22 AR781, similar to yeast pheromone 5.221 2.002 1.5962 2.312 2.732 2.01394 receptor moz23 luminal binding protein 7.282 1.744 1.9975 1.539 1 0.90116 dc1-b1 unknown protein 4.221 1.163 2.1785 7.843 2.809 3.42687 mjf-ab02 AP2 domain containing protein 4.421 0.642 3.4437 2.544 0.409 1.40494 RAP2.3 ID Description hrl1+ hnpr+ hnahG+ hrl- hnpr- hnahG- aozg14 tryptophan synthase beta chain 1 4.851 1.068 1.3652 1.671 1.106 0.93043 precursor moz44 putative protein, similarity to crp1 7.824 0.594 0.8614 1.196 1.21 1.52174 protein aozc43 Strong similarity to ser/thr protein 4.868 1.086 1.0056 0.958 1.25 0.75441 kinases from mjf-bc07 chlorophyll a/b-binding protein-like 13.86 0.934 2.4957 1.767 0.945 1.13953 rpm4c6 tryptophan synthase 5.641 1.202 1.3041 1.269 1.478 0.87134 e10d11t7 glutamine dependent asparagine 6.494 1.415 1.5291 1.125 1.901 1.16066 synthase cozib5 similar to nitrate/chlorate transporter 6.267 0.967 1.1555 1.035 1.133 0.77754 aozd15 putative translation initiation factor 5.669 1.353 1.0735 1.539 1.356 1.06627 cozic5 unknown protein 5.522 1.856 1.4697 1.72 1.832 0.86387 mjf-gd06 glycolate oxidase, putative 6.183 1.397 2.0865 1.186 1.366 0.94048 moz26 reticuline oxidase-like protein 6.104 1.254 1.7902 1.564 0.899 0.75291 (alkyloid biosynth aozd67 cytochrome P450-like protein 5.722 1.343 1.8583 1.741 1.328 0.86697 flavonoid rpm5-g4 hexose transporter-like-protein 5.583 1.126 2.1138 1.232 0.894 0.80769 179h9t7 unknown protein with hydrolase 5.414 1.156 1.0229 1.199 1.307 0.85078 fold aozrhb07 L-ascorbate peroxidase 7.584 1.171 1.5934 2.435 1.618 0.70504 vppfe4 drought-induced protein 7.267 0.211 1.3609 3.21 1.404 0.68898 dc5-e1 putative peroxidase ATP2a 5.02 1.231 0.7204 est g6c7t7 pectinesterase, putative 5.12 1.174 1.7693 1.984 1.482 0.94021 moz92 luminal binding protein 5.349 1.173 0.7909 1.141 1.691 1.40742 cozjb3 unknown protein 6.63 1.143 1.9968 2.602 1.492 1.57681 moz50 putative mitochondrial 6.768 1.258 1.5553 2.255 1.877 1.1039 dicarboxylate carrier prote aozc66 putative myosin heavy chain 6.692 1.207 1.6667 2.137 1.972 1 dc7-h8 ankyrin-like protein 21.21 0.9 2.2143 dc2-b6 Ca2+-transporting ATPase-like 48.8 1.268 1.337 1.544 1.093 0.90847 protein vppbe10 putative RNA-binding 24.88 0.79 1.8094 1.597 1.51034

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sa1-e7 glutamate-dependent asparagine 5.146 2.419 1.0442 1.329 1.304 0.65019 synthetase 203c22t7 hypothetical protein similar to 5.221 2.053 1.2531 1.343 1.758 0.97772 glutelins rpm5-g1 putative 60S ribosomal protein L1 5.353 1.782 1.1058 1.299 1.435 0.78481 ID Description hrl1+ hnpr+ hnahG+ hrl- hnpr- hnahG- rpm6-e1 hypothetical protein 5.108 2.142 1.331 1.873 1.3 0.625 moz87 putative ABC transporter 6.987 1.779 1.2127 0.909 1.924 1.12678 aozc40 hypothetical protein 6.939 1.718 1.5772 1.857 2.505 1.87566 aozrhe01 oxoglutarate/malate translocator- 4.347 1.853 1.458 1.852 1.48 0.77355 like protein 110h21t7 germin-like protein 4.545 1.351 1.0906 0.973 1.471 1.05622 aozc64 putative glutamate decarboxylase 4.299 1.483 1.0439 0.93 1.286 0.76839 aozd38 hypothetical protein 4.284 1.891 1.5896 1.558 1.556 1.29759 rpm3-a6 similar to disease resistance protein 4.509 1.99 1.0117 2.038 2.033 1.47516 rps4 vfrbe1 light regulated protein 5.398 1.669 0.958 2.45 1.01167 NPR1-and SA-independent 117p5t7 hypothetical protein with AP2 domain 7.797 4.174 3.1144 2.724 2.456 2.50459 vppce1 unknown protein 13.89 8.787 4.3786 3.829 2.812 0.85747 cozic8 unknown protein w/ prokaryotic 36.44 20.78 7.8618 5.043 3.262 1.39033 membrane liporotein sa1-a3 similar to MAP kinase kinase 4.897 3.387 2.2689 2.347 3.057 2.94681 moz112 putative protein 8.124 6.865 3.0951 2.162 2.603 0.93257 124p16t7 unknown protein with muT domain 12.75 3.893 3.1202 6.119 6.295 4.56234 vir1a1 putative protein 16.56 4.376 2.832 2.829 8.008 2.89045 sa2-a1 hypothetical protein 14.61 9.454 10.541 2.623 1.59 1.68 sa1-e6 serine O-acetyltransferase 14.3 9.054 3.0794 2.682 2.705 0.86314 est 91d3t7 similar to homeotic protein boi1AP1 4.676 3.59 2.5618 2.087 1.72 1.60992 moz36 putative nematode-resistance protein 13.19 5.599 5.4224 3.278 3.904 2.53105 sa2-b9 multicatalytic endopeptidase 4.833 3.098 1.8487 1.519 0.987 0.90508 rpm7-f1 small proteins (~100 aa) that are 8.41 4.97 2.6773 1.841 2.41 1.14202 induced by heat, moz107 photosystem II core complex 22.33 6.568 3.4658 9.721 3.751 1.23288 protein, putative aozc9 12-oxophytodienoate reductase 7.207 4.418 2.9067 2.391 2.023 1.45762 (OPR1) avr2a2 similar to harpin-induced protein 40.34 16.85 4.3597 9.31 2.534 0.90461 hin1 from tobacco

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mjf-cc12 hypothetical protein contains 54.07 31.16 4.697 4.053 0.93023 similarity to TATA vppbe12 putative nematode-resistance protein 6.2 4.545 1.7762 4.64 2.562 1.61111 ID Description hrl1+ hnpr+ hnahG+ hrl- hnpr- hnahG- rpm4-e5 uracil phosphoribosyltransferase- 27.88 12.52 7.3073 3.304 2.854 1.40566 like protein vppff5 ATP-sulfurylase 10.73 4.029 5.9499 3.936 9.088 4.80537 avr2a5 nuclear receptor binding factor-like 8.787 7.099 1.306 1.689 0.85965 protein vppdc9 thioredoxin 8.657 5.244 3.1627 1.832 1.762 1.32813 123k13t7 12-oxophytodienoate reductase 11.44 5.615 1.855 3.996 2.158 0.75165 dc8-a7 nodulin-like protein 4.055 2.562 1.5543 aozla4 GTPase activator protein 4.208 2.136 1.8699 0.97 0.737 0.52449 vppgg9 protein kinase - like KI domain 4.192 3.045 1.499 1.578 1.843 0.79024 interacting kinase aoz lea-m13 late embryogenesis abundant gene 4.086 2.171 1.9269 1.652 1.538 1.21694 sa2-g10 putative glutathione S-transferase 14.26 11.85 4.5 2.215 2.194 1.61168 rpm4-b5 hypothetical protein 14.14 5.283 3.6804 1.722 1.723 0.87456 moz48 germin-like protein 10.68 3.891 2.3595 4.37 2.641 1.11775 app-ha12 unknown protein 14.12 6.283 3.4314 3.791 5 1.99425 PDF1.2 Defensin 16.85 4.58 9.45 22.45 6.213 1.856 moz24 putative endochitinase 3.749 4.234 1.2438 1.7 moz20 hypothetical protein 13.8 6.036 2.0284 6.006 2.958 0.63726 moz115 glutathione S-transferase, putative 4.361 2.208 1.7126 1.747 1.09589 sa1-f11 glutathione S-transferase (GST1) 9.956 7.543 2.674 3.008 14.79 1.33333 vppde10 thaumatin-like protein 4.68 3.6 1.5263 1.544 2.012 1.584 vppeb7 calreticulin, putative 16.92 25 1.9735 19.6 8.955 1.522 Repressed 209o24t7 delta tonoplast integral protein 0.095 0.197 0.3353 0.535 0.67 0.82062 156i16t7 wound induced protein 0.555 0.25 0.3999 0.415 0.36 0.52423 mjf-db05 cyclic nucleotide and calmodulin- 0.098 0.09 0.1735 0.796 0.812 0.68668 regulated ion channel moz25 putative histidyl tRNA synthetase 0.314 0.184 0.2581 0.676 1.075 0.74714 dc4-c9 auxin-responsive-like protein Nt- 0.156 0.257 0.5692 gh3 e10d3t7 putative non-specific lipid transfer 0.089 0.138 0.1984 0.479 0.636 0.68866 protein vppgb10 fatty acid hydroxylase 0.068 0.615 0.7239 0.794 1.084 1.09504 dc7-g3 unknown protein 0.34 0.211 0.5643 154i3t7 unknown protein 0.046 0.119 0.169 0.397 0.672 0.72693

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ID Description hrl1+ hnpr+ hnahG+ hrl- hnpr- hnahG- est 63g9t7 stromal ascorbate peroxidase 0.489 0.199 0.4745 0.392 0.702 0.7277 vppgc9 long-chain-fatty-acid--CoA ligase 0.131 1.1389 232c20t7 unknown protein 0.423 0.176 0.2952 0.328 0.294 0.44148 vppfe6 putative protein (catalase) 0.992 0.013 1.6113 0.767 0.9314 vpped5 phosphoprotein phosphatase 1 0.671 0.235 0.3565 0.361 0.287 0.49313 cozjc8 sugar transport, putative 0.225 0.667 0.8125 114j9t7 senescence associated proein sen-1 0.24 0.144 0.5333 0.284 0.396 0.89512 e5f4t7 unknown protein w/ ATP/GTP- 0.18 0.406 0.4438 0.824 1.231 0.84854 binding site motif A (P-loop) dc7-c9 putative MAP kinase, similar to 0.222 0.921 1.021 1.153 0.99 1.43995 blast and wounding kin1 protein kinase1 0.212 0.11 0.5182 0.246 0.34 0.9905 moz58 unknown protein 0.393 0.225 0.421 0.244 0.347 0.69745 sa1-c12 putative UDP-glucose 4.994 7.251 2.3788 1.81 2.226 0.96184 glucosyltransferse app-bb01 putative AVR9 elicitor response 0.436 6 0.8889 0.878 1.204 1.19595 protein sa1-a11 putative protein contains similarity 1.54 5.231 2.9652 2.128 2.934 1.09805 to NAC-domain aozd27 putative protein 2.16 5 1.0286 1.426 1.128 0.98113 aozd11 putative LOX 2.608 10 1.1606 1.191 1.16 0.6603 cozic7 hypothetical protein 1.109 7.5 0.8599 1.083 1.247 0.91824 sa3-h2 zinc finger protein ZAT7 3.047 20 1.4429 0.929 0.977 0.69281 cozic9 multicatalytic endopeptidsase 0.99 4 0.9087 0.971 1.107 1.06407 complex 97a6t7 unknown protein with C2 domain 20 10.58 15.855 3.882 3.789 0.8705 and proline rich region 93c1t7 putative protein with proline rich 13.06 11.35 10.81 3.55 2.829 0.77645 region 229o3t7(114b putative glutathione S-transferase 4.899 3.389 3.7307 1.877 1.45 1.15022 12) sa3-f3 putative calmodulin-binding protein 7.785 2.503 2.4627 2.281 1.493 1.18486

Induced in the necrotic tissue rpm5-f2 mitochondrial carrier protein-like 8.921 2.611 2.8081 1.194 1.621 0.98971

64c3t7 putative protein 4.731 1.773 4.2387 1.215 1.228 1.04748 cozig1 unknown protein 13.56 2.367 4.168 1.571 1.175 1.12742 rpm1-g2 glutathione transferase, putative 8.7 2.574 2.5041 2.369 1.627 1.31727 dc7-c3 putative protein, similarity to 4.805 5.3 6.0182 1.711 2.043 1.57245 SPINDLY protein

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rpm3-c1 unknown protein 9.571 3.304 3.8916 2.367 2.499 1.9813 ID Description hrl1+ hnpr+ hnahG+ hrl- hnpr- hnahG- cozib2 Ran-binding protein AtRanBP1 13.84 5.169 8.5152 2.859 2.463 1.11387 155b1t7 unknown protein 4.813 3.208 3.0962 1.503 2.554 1.11888 sa1-d4 Ser/Thr kinase 4.65 0.568 11.167 1.432 1.471 1.12889 193n8t7 unknown protein with IQ 4.135 4.674 6.4001 1.425 1.529 0.74177 calmodulin binding motif 204c16t7 unknown protein 5.363 3.069 3.873 1.824 1.181 0.73063 vir1b1 putative protein 5.37 5.119 5.9635 2.063 1.76 1.00488 121g21t7 DNA-binding protien 50.8 3.761 7.6837 1.988 2.011 0.98877 mjf-aa11 unknown protein w/ ATP/GTP- 4.235 1.466 2.0784 1.074 0.846 0.72229 binding site motif A rpm6-g12 hypothetical protein 11.54 3.64 3.6061 1.525 2.799 0.89202 aoztd3 unknown protein 12.02 6.732 5.3473 4.234 3 1.4918 moz32 putative protein NBS/LRR disease 4.064 3.051 2.7591 1.368 1.51 0.70737 resistance protei sa1-g3 putative protein 10.15 8.352 6.9116 2.488 1.958 1.71711 219m16t7 hevein-l-protein percursor (PR-4) 10.45 6.144 10.564 2.54 1.573 0.79704 sa2-g3 putative endoxyloglucan 4.607 4.942 6.514 1.366 1.507 0.96875 glycosyltransferase est g12g1t7 ethylene response sensor (ERS) 4.013 1.726 1.818 2.381 1.336 1.29147 sa3-e7 putative ubiquitin activating enzyme 4.59 3.832 3.8749 1.064 1.383 0.93134 dc2-c12 putative mutT domain protein 4.543 1.121 5.2751 3.304 2.349 4.76656 moz69 vacuolar processing 5.93 3.258 4.0145 2.166 1.372 1.16273 enzyme/asparaginyl endopeptida cozid7 blue copper binding protein 12.06 4.5876 1.992 1.9 1.08692 128a16t7 hypothetical protein with HMG 15.13 9.607 11.905 3.401 3.347 0.80119 domain rpm3-e12 putative endochitinase 4.552 4.642 5.3541 1.758 1.501 1.05612 212b17t7 senescence-associated protein sen1 6.529 4.055 5.2945 1.855 1.581 1.24724 148b7t7 putative lectin/receptor kinase 5.582 1.565 2.3811 0.899 0.591 0.43111 rpm4-b1 unknown protein 5.36 1.768 2.2095 1.9 1.352 1.4497 119p17t7 putative protein kinase 25.39 3.007 7.7254 3.037 7.662 3.30556 aozc14 putative WRKY-type DNA binding 5.018 1.788 2.3056 1.699 1.409 1.17819 protein 245h19t7 putative protein with c3hc4 type 20.68 3.828 3.7643 3.798 4.48 1.65936 RING finger motif 105p15t7 unknown protein 5.415 1.516 2.1368 1.755 1.445 sa2-b3 putative protein, similar to 5.492 2.922 3.1597 1.513 1.492 1.61992 transcription factor

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moz10 hexose transporter-like protein 31.71 6.955 18.985 4.899 4.24 6.85

ID Description hrl1+ hnpr+ hnahG+ hrl- hnpr- hnahG- vpphb1 unknown protein 5.526 2.529 2.3828 1.65 0.877 0.99703 sa3-f1 putative flavonol 17.93 3.547 3.8672 2.609 2.038 1.26687 glucosyltransferase 90l23t7 hypothetical protein with kelch 7.279 6.46 5.0057 1.946 2.162 1.38728 repeat aozb28 receptor serine/threonine kinase-like 5.8 5.518 4.0585 2.307 2.075 1.61099 protein vppcg3 60S ribosomal protein L10, putative 16.08 6.244 5.7415 3.596 2.206 1.02041 aozc56 heat-shock protein 5.733 3.842 3.4729 2.466 2.006 1.4 aozrd1 hypothetical protein 11.8 5.897 1.0811 0.765 1.012 0.62441 vppcg2 beta-1,3-glucanase 2 (BG2) (PR-2) 28 5.9 4.5 24.9 7.6 0.89412 162e13t7 unknown protein 11.59 2.238 2.1515 1.87 1.284 1.05435 sa3-b12 pathogenesis-related PR-1 like 16 3 0.794 16.89 1.013 1.2906 protein vir1b6 probable fibrillarin (Sb21) mRNA 12.23 4.231 0.9367 1.603 1.026 0.72088 Uncorrelated Induction vfrbe6 putative chlorophyll a/b binding 4.021 1.38 1.5654 1.741 1.386 2.11511 protein rpm1-b5 unknown protein 13.87 5.458 1.6218 1.977 2.459 0.85533 vppga12 putative protein 4.169 1.442 1.2166 1.417 1.595 2.51974 rpm6-g9 dehydroascorbate reductase 16.35 4.932 1.9959 4.52 2.544 1.15423 aozlb11 putative phosphoribosylanthranilate 14 3.198 2.6807 1.913 2.115 3.05556 transferase rpm3h12 hypothetical protein with ubiquitin2 16.41 1.992 1.012 1.426 4.915 0.78165 doamin app-ec10 membrane related protein CP5, 17.57 5.568 1.6875 5.86 2.855 1.566 putative sa4-a4 glutathione S-transferase, putative 18.75 4.25 1.5846 3.341 4.62 0.71262 rpm1-e2 putative protein 27.6 3.797 1.234 2.996 2.72 0.97907 rpm3-c10 PR-1-like protein 19 4.5 0.7362 1.079 0.938 0.65875 sa3-f6 Atpm24.1 glutathione S-transferase 19 11.21 1.0798 4.625 0.95294 rpm4-d4 unknown protein contains 22.01 4.894 1.8103 14.58 1.235 1.13341 dienelactone hydrolase f 178m1t7 unknown protein 25.27 3.416 2.9113 1.995 2.448 0.89802 rpm4-f10 GTP-binding protein RAB11D 7.152 2.668 1.5434 1.624 1.536 0.75656 aoze41 phytochelatin synthase 5.275 2.03 1.8291 3.764 5.494 5.52929 ID Description hrl1+ hnpr+ hnahG+ hrl- hnpr- hnahG-

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119m11t7 ubiquitin dependent proteolytic 7.298 1.983 1.2114 protein rpm4-e10 transporter-like protein high-affinity 5.609 1.646 1.7055 1.382 1.608 0.83629 nitrate tra dc3-g9 aspartate aminotransferase (Asp3) 89.86 5.984 3.1376 4.187 1.568 0.87193 rpm5-a4 bZIP transcriptional activator RSG 5.626 3.484 0.9771 3.882 1.23 0.59831 sa2-b12 putative glucosyltransferase 6.5 1.578 0.9888 1.808 0.9345 vppda10 similar to casein kinase 6.614 3.45 1.0686 2.514 4.108 2.04124 rpm5-a12 putative tropinone reductase 11.24 3.265 2.1399 2.069 1.089 0.70388 sa1-f1 putative indole-3-acetate beta- 10.72 2.082 1.8575 0.857 0.64251 glucosyltransferase rpm3-c3 hypothetical protein 10.05 4.335 0.9579 2.704 3.133 1.37198 rpm4-e1 reticuline oxidase-like protein 10.15 3.149 2.0639 4.007 1.489 1.09192 (alkyloid biosynthesis) aozrd4 disulfide isomerase-related protein-p 7.579 2.56 1.1667 1.096 1.124 1.02 rpm4-b3 ABC transporter, putative 8.25 3.342 1.445 2.402 2.741 0.66962 vppgg6 protein kinase - like KI domain 8 4.303 0.9091 2.1 0.9280.67262 interacting kinase vfrbc8 unknown protein 8.301 2.055 1.544 2.188 7.1 1.80454 aozrg6 hypothetical protein with DUF52 4.845 2.367 1.3615 2.699 1.577 0.82336 domain aozb23 ankyrin like protein 4.686 2.16 1.3791 1.361 0.992 0.90556 mjf-ce05 putative small heat shock protein 8.75 4.235 0.8621 2.932 0.985 0.81538 rpm4-f6 cysteine protease 4.692 1.626 1.6784 1.289 1.946 1 105d3t7 unknown protein with dual 6.258 1.742 2.3409 1.263 1.53 0.79963 specificity phosphatase motif

113 + + 1 G r h p a n n + 1 1 1 l l l r r r h h h

- - G 1 r h p a n n

1- 1 1 l l l r r r h h h

Lesion+ Lesion-

Figure 6-1: Graphical representation of K-means clustering of differentially expressed genes in hrl1 and the double mutants K-means clustering was performed with 210 genes that were either 2.5-fold induced or repressed in the lesion+ tissue of hrl1, hrl1 npr1, and hrl1 nahG. For the lesion- tissue, only 113 genes passed the cut-off. All the plants were 6-week-old and grown under identical conditions. The number of clusters was determined by trial and error. Color code is as follows: Red__induction, green__repression, black__no change, gray__data not available. This experiment was replicated twice (see methods).

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6.3 Discussion

Microarray analyses of plant-pathogen interactions have revealed a complex interplay of multiple signaling pathways (Schenk et al., 2000; Maleck et al 2000). In this study I examined the changes in the transcript levels of approximately 1200 Arabidopsis ESTs. These ESTs represent an enriched population of genes that may be involved in pathogen and oxidative stress response. Six-week-old hrl1, hrl1 npr1, hrl1 nahG, npr1, nahG, and Col-0 plants were used in this set of experiments. Total RNA was isolated separately from the lesion positive and lesion negative leaves of the double mutants to analyze cell death and SAR signal transduction respectively. The strongest overall response was observed in the lesion+ tissue of hrl1 mutant and the weakest response was observed in the lesion- leaves of hrl1 nahG. Out of the possible 1200 ESTs representing approximately 5% of the entire Arabidopsis genome, 210 ESTs showed a differential response in the lesion+ tissue of hrl1 and 35 ESTs had differential induction pattern in the lesion- tissue of hrl1 nahG. Interestingly, 30% of the genes that showed either 2.5- fold up-regulation or down-regulation had no known function. However, only 10% of the genes were associated with defense or cell death. The cut-off was kept at 3.5 fold for the lesion+ samples and 2.5 fold for the lesion- samples due to two reasons: first, inconsistencies between replicate experiments were significantly higher between 2 and 2.5 fold. Second, in a constitutive lesion-mimic mutant like hrl1, the likelihood of secondary effects, not directly responsible for the observed phenotype is relatively high in the lesion+ tissue. Hence, to avoid the high background associated with such a pleotrophic effect, I maintained a slightly higher cut-off than the 2-fold cut-off, which is routinely used. When the cut-off was lowered to 2.0-fold, the number of genes that showed differential expression jumped by more than 30%. However, having a stringent cut-off value may result in losing some valuable information about the genes whose small change in expression levels may be biologically significant. For example, most of the genes that encode transcription factors and other regulatory kinases are not induced to high levels. Thus, a global cut-off may be necessary to eliminate a large number of genes that are not important; a more prudent approach should allow room for genes that are expressed to low levels. Although impractical, signal intensities and fold changes should

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be looked at on a gene-per-gene basis, at least for the genes whose expression levels are expected to be low.

6.3.1 Gene expression pattern in the lesion+ (local) and in the lesion- (systemic) tissue of hrl1

Previous Northern analysis with hrl1 indicated that the known defense-related genes are regulated differentially in the tissue undergoing cell death compared to the lesion minus distal tissue. So, I sought to determine the global gene expression profiles in the tissue undergoing cell death and in the lesion minus tissue of hrl1. Overall, lesion+ tissue had twice the number of genes that were differentially expressed. From figure 6-1 and table 6-1, 210 genes were differentially expressed in the lesion+ tissue compared to 114 genes in the lesion- tissue of hrl1. Although, the number of differentially expressed genes in the lesion- tissue was reduced by half, the basic structure of the clusters remained the same (Figure 6-1). Consistent with the earlier Northern results, genes like PR-1, PR-2, GST1 and PDF1.2 were induced both in the lesion+ and in the lesion- tissue of hrl1. However several GST1-like and PR-1-like genes were not induced in the lesion- tissue suggesting that they are regulated differently in the lesion+ and lesion- tissue. For many of the genes that are induced in the lesion- tissue, the induction levels are significantly lower compared to the lesion+ tissue. It is possible that the induction of these genes peak during cell death. Alternatively, the increase in expression levels could be the consequence of cell death. Most of the repressed genes are down regulated to an equal extent in the lesion- and lesion+ tissue (Fig 6-1, Table 6-1).

A small number of genes (a putative hexose transporter, an ATP-sulfurylase, a putative protein kinase, and a putative nematode resistance protein) are induced in the lesion+ and lesion- tissue in a SA-independent manner. This represents the first set of genes that are induced in the lesion- tissue of hrl1 nahG. So far, all of the tested marker genes by Northern analysis required SA in the lesion- tissue of hrl1. In contrast, a significant number of genes that are induced in the lesion- tissue of hrl1 do not seem to require NPR1. Although, the induction levels are low, the pattern of expression remains the same. Despite the similarity in induction of these genes, hrl1 npr1 plants are

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susceptible to virulent bacterial pathogens. It is possible that the magnitude of induction is crucial for resistance against pathogens. In addition, genes that require NPR1 for their induction may be critical for resistance in hrl1. A number of proteins with unknown function are not induced in the hrl1 npr1 mutant. It is possible that some of these unknown proteins may play an important role in conferring resistance to bacterial pathogens in an NPR1-dependent fashion. Constitutive SAR mutants like cpr6, showed unaltered PR gene induction in the npr1 background, yet they were susceptible to virulent bacterial pathogens. It was postulated that unidentified factors regulated through NPR1 might be responsible for the observed susceptibility.

Pattern No. of genes Differentially expressed

SA-dependent 132 63% SA-independent 35 17% SA-partially dependent 42 20%

NPR1-dependent 88 42% NPR1-independent 65 31% NPR1-partially dependent 57 27%

Cell death-dependent 140 67% Cell death-independent 20 10% Cell death-partially dependent 50 24%

Table 6-2: Categorization of genes based on their induction pattern

Genes selected under “independent” category exhibit less than 3-fold change with

respect to hrl1+. Genes with more than 3-fold change with respect to hrl1+ were

grouped under “partially dependent”. The “dependent” category consists of genes

that show no induction when compared to hrl1+.

From the microarray analysis it is clear that majority of the genes (63%-67%) require SA and are also dependent on cell death (Table 6-2). Interestingly, 31 out of the 35 genes that

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are SA-independent are also NPR1-independent. This suggests that signaling pathways that are independent of SA do not require NPR1 also. NPR1- and SA-independent activation of some PR genes during HR cell death has been observed earlier. Most of the genes that do not require either SA or NPR1 need cell death dependent signals. Because, 90% of the SA/NPR1-independent genes are induced only in the lesion+ tissue of hrl1, cell death specific signals may activate these genes.

6.3.2 Functional classification of differentially expressed genes in hrl1

As mentioned earlier, approximately 30 % of the genes with altered expression levels had no known function. Only 10 % of the genes have been previously associated with defense and cell death. A large number of genes (22%) are involved in metabolism. In addition, 7 % of the genes participate in protein synthesis and targeted protein degradation (Fig 6-2). For example, a luminal binding protein is induced to 7-fold in the lesion+ tissue of hrl1. It has been shown that this luminal binding protein is induced in anticipation of increased protein synthesis to alleviate any potential endoplasmic reticulum stress (Leborgne-Castel et al., 1999). Since hrl1 accumulates transcripts of several genes and presumably their protein products, proper targeting and secretion of the proteins is crucial. Transcripts of translation initiation factor and a protein synthesis initiation factor are also up regulated, suggesting that there is an increased demand for translational machinery. Increased transcriptional and translational activities in hrl1 may pose a severe metabolic burden on growth and development. It is evident from the short stature of hrl1 compared to the wild-type. When the metabolic pressure is reduced to some extent in hrl1 npr1, double mutant size is bigger than hrl1. From figure 6-1, hrl1 nahG has the least number of induced genes compared to hrl1 npr1 and hrl1. Hence, the metabolic burden should be significantly less. In support of that, hrl1 nahG is almost similar in size when compared to the wild-type. As expected, the expression of luminal binding protein BiP is not altered in hrl1 nahG (Table 6-1).

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Miscellaneous Unknown function

15% 31%

Metabolism 22%

Defense/ cell death 10%

Signaling Protein synthesis/ degradation 15% 7%

Figure 6-2: Functional classification of genes with altered expression levels in hrl1 The classification is based on MIPS method of assigning categories and includes a total of 210 genes. Miscellaneous category includes sub categories such as energy, secondary metabolism and unclassified proteins. The signaling category has transcription factors, kinases and phosphatases. Protein degradation comprises of proteins such as ubiquitin activating enzyme, proteins with RING finger domains, and ubiquitin-dependent proteolytic protein.

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Since the proteins that accumulate in hrl1 need to be degraded effectively, several components of the ubiquitin-dependent proteolytic pathways are activated. For example a putative ubiquitin activating enzyme and an ubiquitin-dependent proteolytic enzyme are induced in hrl1. Although premature, it is very likely that an ubiquitin-dependent protein degradation pathway is critical to maintain the cellular homeostasis in the already stressed hrl1 mutant. The fact that hrl1 coi1 double mutant is severely stunted with exacerbated cell death supports the earlier contention.

Transcriptional activation genes require appropriate transcription factors. In hrl1 several transcription factors are up regulated. Since the cut-off for differential expression was set between 2.5-3.5-fold, it is possible that some of the transcription factors may have been missed. A bZIP transcriptional activator and a putative WRKY-DNA binding protein are induced in hrl1. The TGA family of transcription factors that are part of the bZIP class are known to bind the promoters of PR genes and activate the transcription (Zhang et al., 1999; Despres et al., 2000). Recently WRKY-type DNA binding proteins are shown to regulate the transcription of genes involved during pathogen attack (Eulgem et al., 2000; Yu et al., 2001; Robatzek and Somssich, 2001). The activation of PR genes and possibly other unidentified defense-related genes in hrl1 could be induced by these transcription factors. Promoter analysis for cis-regulatory elements among the gene clusters generated in this study will help to characterize some of the unknown proteins.

6.3.4 Summary

The microarray analysis of hrl1 and the double mutants of hrl1 with npr1 and the nahG transgene have provided a broader view of gene regulation in hrl1. 1. A significant number of genes are induced in the lesion- tissue of hrl1 suggesting that the systemic lesion- leaves mimic the characteristic SAR response. 2. Approximately 50 genes (22%) that have altered expression pattern in hrl1 encode proteins that are involved in metabolism. This suggests that cell death

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and defense signaling in hrl1 may interact with components involved in cellular homeostasis. 3. SA is required for the induction of nearly 75% of the genes in hrl1 mutant. The effect is more pronounced in the lesion- leaves of hrl1 nahG where only 14% of the genes are induced. 4. Only 10% of the genes that are induced have been previously associated with defense and cell death 5. Roughly 30% of the differentially expressed genes had no known function

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APPENDIX : A

Preexisting Systemic Acquired Resistance Suppresses Hypersensitive Response-Associated Cell Death in Arabidopsis hrl1 Mutant

142 Art: 972239 Input-ams 10:29 02/11/2 4؍balt2/pp-plant/pp-plant/pp0402/pp7157-02a smithl S

Preexisting Systemic Acquired Resistance Suppresses Hypersensitive Response-Associated Cell Death in Arabidopsis hrl1 Mutant1

Sendil K. Devadas and Ramesh Raina* Biology Department, Biotechnology Institute, and Intercollege Graduate Program in Plant Physiology, The Pennsylvania State University, University Park, Pennsylvania 16802

The hypersensitive response (HR) displayed by resistant plants against invading pathogens is a prominent feature of plant-pathogen interactions. The Arabidopsis hypersensitive response like lesions1 (hrl1) mutant is characterized by heightened defense responses that make it more resistant to virulent pathogens. However, hrl1 suppresses avirulent pathogen-induced HR cell death. Furthermore, the high PR-1 expression observed in hrl1 remains unaltered after avirulent and virulent pathogen infections. The suppressed HR phenotype in hrl1 is observed even when an elicitor is expressed endogenously from an inducible promoter, suggesting that an impaired transfer of avirulent factors is not the reason. Interestingly, the lack of HR phenotype in hrl1 is reversed if the constitutive defense responses are compromised either by a mutation in NON EXPRESSOR OF PR-1 (NPR1) or by depleting salicylic acid due to the expression of the nahG gene. The rescue of HR cell death in hrl1 npr1 and in hrl1 nahG depends on the extent to which the constitutive systemic acquired response (SAR) is compromised. Pretreating Arabidopsis wild-type plants with SAR-inducers, before pathogen infection resulted in a significant decrease in HR cell death. Together, these results demonstrate that the preexisting SAR may serve as one form of negative feedback loop to regulate HR-associated cell death in hrl1 mutant and in the wild-type plants.

Fn1 Successful host resistance against pathogen inva- tion and analysis of several Arabidopsis mutants sion requires expeditious recognition and activation with spontaneous cell death that mimic pathogen- of the necessary defense repertoire. One such robust induced cell death support the idea that HR cell response in plants involves resistance (R) gene- death may be controlled by plant’s own genetic dependent recognition of pathogen-derived elicitors mechanisms (Dangl et al., 1996; Greenberg, 1997; and initiation of localized cell necrosis at the site of Glazebrook, 1999). Genetic screens aimed at identi- pathogen infection (Goodman and Novacky, 1996). fying the loss of HR to avirulent pathogens have Undoubtedly, the most noticeable feature of this R resulted in the cloning of several R genes. These R gene-dependent resistance response is the rapid cell genes, when mutated, fail to develop HR against one death that is well defined within the attempted in- or more avirulence factors, and they represent ge- fection site, a process known as hypersensitive re- netic components that are required rather early in the sponse (HR). The HR cell death is often preceded by signal transduction leading to HR cell death (Bent, changes in ion fluxes, oxidative burst, and cross- 1996; Dangl and Jones, 2001). The relative scarcity in linking of cell wall proteins. Most of the HR cell identifying new mutants that lack HR cell death may death processes are accompanied by an increase in be due to the presence of functionally redundant Ϫ salicylic acid (SA) biosynthesis, transcriptional acti- genes, embryo lethality, or weaker HR phenotypes vation of various pathogenesis-related (PR) genes, that were overlooked (Innes, 1998). Although HR cell and the establishment of a long-lasting systemic re- death is intrinsically controlled by the plant, the rel- sponse known as systemic acquired resistance (SAR; ative importance of cell death in conferring resistance Hammond-Kosack and Jones, 1996; Ryals et al., to pathogens is not well understood. For example, 1996). the Arabidopsis non-race-specific disease resistance1 Several lines of evidence indicate that HR cell (ndr1) mutant is susceptible to several strains of death is a form of programmed cell death that resem- Pseudomonas spp., although it elicits HR against some bles apoptotic cell death in other organisms (Mittler of these pathogens (Century et al., 1995, 1997). Con- and Lam, 1996; Morel and Dangl, 1997). Identifica- versely, in barley (Hordeum vulgare), MLA-conferred resistance against an obligate biotroph Erysiphe gra- 1 This work was supported by the Department of Biology and minis f. sp. hordei is abolished when HR cell death is the Intercollege Graduate Program in Plant Physiology (to S.K.D.) inhibited (Schiffer et al., 1997). at Pennsylvania State University. * Corresponding author; e-mail [email protected]; fax 814–863– In recent years, some of the Arabidopsis mutants 1357. with elevated resistance response were shown to pos- Article, publication date, and citation information can be found sess partial loss of HR phenotype against avirulent at www.plantphysiol.org/cgi/doi/10.1104/pp.010941. pathogens (Yu et al., 1998, 2000; Rate et al., 1999; Rate

APPENDIX : B

The Arabidopsis gain-of-function mutant dll1 spontaneously develops lesions mimicking cell death associated with disease

144 The Plant Journal (2002) 30(1), 1±11 TheArabidopsisgain-of-functionmutantdLl1 spontaneously develops lesions mimicking cell death associated with disease

Rachel K. Pilloff1,², Sendil K. Devadas1,², Alexander Enyedi2, and Ramesh Raina1,* 1Biology Department, Biotechnology Institute, and Intercollege Graduate Program in Plant Physiology, The Pennsylvania State University, University Park, PA 16802, USA 2Department of Biological Sciences, Western Michigan University, Kalamazoo, MI 49008, USA

Received 5 November 2001; revised 31 December 2001; accepted 31 December 2001. *For correspondence: (fax 814-863-3157, e-mail [email protected] ²These authors contributed equally to this work.

Summary

We describe the characterization of a novel gain-of-function Arabidopsis mutant, dLl1 (disease-like lesions1), which spontaneously develops lesions mimicking bacterial speck disease and constitutively expresses biochemical and molecular markers associated with pathogen infection. Despite the constitutive expression of defense-related responses, dLl1 is unable to suppress the growth of virulent pathogens. However, dLl1 elicits normal hypersensitive response in response to avirulent pathogens, thus indicating that dLl1 is not defective in the induction of normal resistance responses. The lesion+ leaves of dLl1 support the growth of hrcC mutant of Pseudomonas syringae, which is defective in the transfer of virulence factors into the plant cells, and therefore non-pathogenic to wild-type Col-0 plants. This suggests that dLl1 intrinsically expresses many of the cellular processes that are required for pathogen growth during disease. Epistasis analyses reveal that salicylic acid and NPR1 are required for lesion formation, while ethylene modulates lesion development in dLl1, suggesting that signi®cant overlap exist between the signalling pathways leading to resistance- and disease-associated cell death. Our results suggest that host cell death during compatible interactions, at least in part, is genetically controlled by the plant and DLL1 may positively regulate this process.

Keywords: compatible interactions, PR genes, disease, cell death, dLl1, Arabidopsis.

Introduction Since plants are sessile organisms, they have evolved variety of pathogens (Dong, 2001; Ryals et al., 1996). SAR elaborate defense mechanisms to resist pathogen infec- establishment is preceded by the activation of patho- tion. Most interactions between plant and pathogens can genesis-related (PR) genes and salicylic acid (SA) accumu- be classi®ed as either compatible or incompatible. During lation (Enyedi et al., 1992; Uknes et al., 1992). SA induces an incompatible interaction, the plant recognises the the expression of PR genes and disease resistance in pathogen and rapidly activates an extensive array of several plant species, while SA depletion leads to defense responses at the site of infection that limit enhanced susceptibility to multiple pathogens (Delaney pathogen ingress into neighbouring cells. In contrast, a et al., 1994; Yang et al., 1997). NPR1 (also known as NIM1 compatible interaction is often characterised by a much and SAI1) functions downstream of SA and is a critical delayed and attenuated defense response that fails to component of the SA signalling pathway (Cao et al., 1994; retard pathogen colonisation.UncorrectedRyals et al., 1997; Shah et al proof., 1997). The npr1 mutant Both incompatible and compatible interactions are cannot activate SAR in response to SA application and usually followed by the onset of systemic acquired resist- displays enhanced susceptibility to virulent pathogens. ance (SAR), a distinct plant defense response that results A nearly ubiquitous result of plant±pathogen inter- in a non-speci®c and long-lasting systemic resistance to a actions is host cell death. During incompatible inter-

ã 2002 Blackwell Science Ltd 1 VITA Sendil Kumaran Devadas

EDUCATION: Ph.D. in Plant Physiology. The Pennsylvania State University, University Park, PA B.S. with Distinction in Industrial Biotechnology, May 1996. Anna University, Madras, India.

EXPERIENCE: Spring 2001: Internship at Syngenta Biotechnology Incorporation, R.T.P., NC (formerly Novartis), in the rice functional genomics department

Fall 1996- Fall 2000: Teaching Assistant at the department of Biology, Penn State University, PA

Summer 1995: Summer Internship at TIFR, Bombay, India

Summer 1994: Summer Internship at Karnataka Antibiotic and Pharmaceuticals Ltd., Bangalore, India

PUBLICATIONS: Devadas, S.K. and Raina, R. (2002). Preexisting SAR suppresses hypersensitive response- associated cell death in Arabidopsis hrl1 mutant. Plant Physiology (in press).

Devadas, S.K., Enyedi, A., Raina, R. The Arabidopsis hrl1 mutation reveals novel overlapping roles for salicylic acid, jasmonic acid, and ethylene signaling in cell death and defense against pathogens. Plant J (in press)

Pilloff, R.K., Devadas, S.K., Enyedi, A., Raina, R. (2002). The Arabidopsis gain-of- function mutant dll1 spontaneously develops lesions mimicking cell death associated with disease. Plant J (in press).

Mullin, E., Devadas, S.K., Geiser, D., Raina, R., Kang, S. Arabidopsis as a model system for fusarium infection. Manuscript in preparation.

PROFESSIONAL MEMBERSHIPS:

American Association for the Advancement of Science, American society of Plant Biologists