Flg22- Triggered Immunity and the Effect of Azelaic Acid on the Defense Response in Citrus

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Flg22- Triggered Immunity and the Effect of Azelaic Acid on the Defense Response in Citrus
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Shi,Qingchun
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Master's ( M.S.)
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University of Florida
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Plant Molecular and Cellular Biology
Committee Chair:
Moore, Gloria A
Committee Members:
Jones, Jeffrey B
Febres, Vicente

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Subjects / Keywords:
azelaic -- azi1 -- candidatus -- canker -- citri -- citrus -- defense -- eti -- expression -- flg22 -- grapefruit -- huanglongbing -- immunity -- kumquat -- liberibacter -- npr1 -- pamp -- pathogen -- plant -- pti -- resistance -- sar -- susceptibility -- xanthomonas
Plant Molecular and Cellular Biology -- Dissertations, Academic -- UF
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Plant Molecular and Cellular Biology thesis, M.S.
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theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
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Abstract:
The citrus industry plays an important role in the economy of Florida. However, bacterial diseases such as citrus canker (caused by Xanthomonas citri subsp. citri, Xcc) and huanglongbing (caused by Candidatus Liberibacter asiaticus, Las) have caused considerable losses to citrus production. To have a better understanding of the defense response of citrus against Xcc and Las and to seek alternative ways to control canker and huanglongbing, we studied: 1) The role of Pathogen-Associated Molecular Pattern (PAMP)-triggered immunity (PTI) in the response of resistant kumquat and susceptible grapefruit, to Xcc; 2) The effect of PAMPs from Xcc or Las on PTI; and 3) Evaluated the effect of azelaic acid, a molecule recently found to be important in the priming and signaling of pathogen defense, on the PTI response in grapefruit. The first experiment was performed by treating kumquat and grapefruit with a 22 amino acid peptide from the Xcc bacterial flagellin conserved domain (Xflg22), a PAMP. The results showed that, compared to the mock inoculation, Xflg22 (10 ?M) triggered the expression of a set of defense-associated genes including EDS1, RAR1, SGT1, EDR1, PBS1, NDR1, EDS5, PAL, NPR2, NPR3, RdRP and AZI1 in kumquat, but the corresponding genes in grapefruit were either not induced or showed slight downregulation. In kumquat, the significantly Xflg22 induced genes EDS1, RAR1, SGT1 and NDR1 are thought to be PTI-associated genes, suggesting that Xflg22 initiated PTI in this resistant species. However, no obvious induction of those genes was observed in grapefruit suggesting a weaker PTI and perhaps part of the reason this species is susceptible to canker. In the second experiment we compared the effect of Xflg22 with the flg22 derived from Las (Lflg22) as well as the archetype flg22 on grapefruit. Additionally, azelaic acid was used as a pretreatment before any flg22 infiltration in order to evaluate its defense-eliciting capability in citrus. Comparison between the effects of the three different flg22s showed that most of the defense-associated genes tested were not differentially expressed between the treatments compared with the mock inoculation, except for salicylic acid signaling gene AZI1 and salicylic acid biosynthesis gene PAL. However, when plants were pretreated with azelaic acid (1 mM) two days prior to the PAMP treatments, mock inoculation, flg22 and Xflg22 treatments all showed higher expression levels of EDS1, RAR1, EDR1, EDS5, NPR1, NPR2, NPR3, RdRP and AZI1 than that of in control buffer pretreated plants, except the Lflg22 treatment which significantly reduced the expression of EDS1, RAR1, SGT1, EDR1, NPR1, NPR2 and NPR3 compared with the mock inoculation. Azelaic acid alone induced a set of defense-associated genes including EDS1, RAR1, EDR1, EDS5, NPR1, NPR2, NPR3, RdRP and AZI1, which was comparable with the gene inductions by Xflg22 in kumquat, suggesting that azelaic acid could be used as a potential chemical to control bacterial disease in citrus.
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In the series University of Florida Digital Collections.
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Includes vita.
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Statement of Responsibility:
by Qingchun Shi.
Thesis:
Thesis (M.S.)--University of Florida, 2011.
Local:
Adviser: Moore, Gloria A.
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RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-08-31

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FLG22-TRIGGERED IMMUNITY AND THE E FFECT OF AZELAIC ACID ON THE DEFENSE RESPONSE IN CITRUS By QINGCHUN SHI A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORID A IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2011 1

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2011Qingchun Shi 2

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To my parents who always understand and support me in chasing my dream 3

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ACKNOWLEDGMENTS I would like to express my heartfelt t hanks to all the people who helped and inspired me during my masters study. I want to thank the chair of my academic committee and advisor Dr. Gloria Moor e for her financial support and dedicated advisory during my entire study. Specially th ank her for always being supportive even at the most difficult time studying in this fore ign country, without her trust I would not had been able to achieve this milestone. Dr. Moores kindness and easygoing personality will remain in me and guide me to treat other people like she does. My gratitude also goes to my committ ee members Dr. Vicent e Febres and Dr. Jeffrey Jones. I am very delighted to in teract with them and hav e learned tremendous knowledge from them. Their insi ghts to plant pathology explai n to me what world-class scientists would be like. Repetitive thanks given to Dr. Febres not only because his tireless scientific guidance to me, worki ng with him on a daily basis, he spent large amount of precious time teaching me laborat ory skills and taking care of me in my everyday life. I feel happy and proud to have such a friend and spiritual father. I would also like to thank Dr. Abeer K halaf for her patient explanations and encouragement, and thanks to Kimberly, Latany a, Terry and Fabiana for their help and working with them was really a pleasure Last but not least, deep thanks go to my parents far back in China for their underst anding and support, and thanks to my wife for being with me and taking care of my life. I love you all. 4

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TABLE OF CONTENTS page ACKNOWLEDG MENTS .................................................................................................. 4LIST OF TABLES ............................................................................................................ 7LIST OF FI GURES .......................................................................................................... 8ABSTRACT ................................................................................................................... 10 CHAPTER 1 INTRODUC TION .................................................................................................... 122 LITERATURE REVIEW .......................................................................................... 15The Plant Immune System ...................................................................................... 15PAMPs and PAMPs-Triggered Immunity (PTI) ....................................................... 16Defense Si gnals ...................................................................................................... 17Genes Involved in Pl ant Immuni ty .......................................................................... 19Enhanced Disease Susceptibility 1 (EDS 1) and Nonrace-Specific Disease Resistance 1 (NDR1) .................................................................................... 19Requires for Mla12 Resistance (RAR1) and Suppressor of GTwo Allele of Skp1 (SGT 1) ................................................................................................. 20Enhanced Disease Resi stance (E DR1) ............................................................ 20Enhanced Disease Susceptib ility 5 (E DS5) ...................................................... 21Pathogensis-Related Gene 1 (PR1) and Nonexpressor of PR Genes (NPR1) .......................................................................................................... 21Azelaic Acid Induced 1 (AZI 1) .......................................................................... 23RNA-Dependent RNA Polymerase (RdR P) ...................................................... 24Isochorismate Synthase 1 (ICS1) and P henylalanine Ammonia Lyase (PAL) .. 24Avrpphb Susceptible 1 (PBS1 ) ......................................................................... 25Rationale and Ob jectives ........................................................................................ 253 MATERIALS A ND METHOD S ................................................................................ 28Plant Mate rial .......................................................................................................... 28Flagellin 22-Amino Acids Cons erved Domain Peptides .......................................... 28Challenge Kumquat and Grapefru it with Xflg22 Peptide ......................................... 29Azelaic Acid Pretreatment and Chal lenges with flg22, Lflg22 and Xflg22 Peptides ............................................................................................................... 29RNA Extraction and Pu rificati on .............................................................................. 30cDNA Synt hesis ...................................................................................................... 30Gene Expression Analysis ...................................................................................... 31 5

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4 COMPARISON OF THE RESPON SE TO XFLG22 BETWEEN canker SUSCEPTIBLE AND RESIST ANT CITRUS TYPES .............................................. 33Results .................................................................................................................... 33Discussio n .............................................................................................................. 415 EFFECT OF AZELAIC ACID AND DIFFERENT flg22 PEPTIDES ON THE DEFENSE RESPONSE OF GRAPEFRU IT ............................................................ 46Results .................................................................................................................... 46Discussio n .............................................................................................................. 576 CONCLUS IONS ..................................................................................................... 61LIST OF RE FERENCES ............................................................................................... 62BIOGRAPHICAL SKETCH ............................................................................................ 72 6

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LIST OF TABLES Table page 5-1 Summary of the effect of Lfl g22 on gene expression after azelaic acid pretreatment in grapefru it. .................................................................................. 545-2 Summary of the effect of azelaic acid in grapefruit and the e ffects of Xflg22 in kumquat on gene in duction. ............................................................................... 555-3 Sequence comparisons bet ween the 22 amino acid pe ptides for flg22, Xflg22 and Lflg22. ......................................................................................................... 58 7

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LIST OF FIGURES Figure page 2-1 Simplified model of bacte ria flagellin perception in Arabidopsis .. ....................... 172-2 Nucleotide sequence homology analysis of the Arabidopsis NPR1 protein family ................................................................................................................. 232-3 A simplified salicylic acid ( SA) synthetic pathway [ 99]. ....................................... 254-1 Effect of Xflg22 on expression of EDR1, EDS1, NDR1, PBS1, RAR1 and SGT1 in Nagami kumquat (A) an d Duncan grapefru it (B). .............................. 364-2 Effect of Xflg22 on expression of ED S5, ICS1 and PAL in Nagami kumquat (A) and Duncan grap efruit (B ). .......................................................................... 374-3 Effect of Xflg22 on expression of NPR1, NPR2 and NPR3 in Nagami kumquat (A) and Duncan grapefruit (B). ........................................................... 384-4 Effect of Xflg22 on expression of PR1 and RdRP in Nagami kumquat (A) and Duncan grapefru it (B). ................................................................................ 394-5 Effect of Xflg22 on expression of AZ I1 in Nagami kumquat (A) and Duncan grapefruit (B). ...................................................................................................... 404-6 Phylogenetic tree of NPR1-like protei n sequences from different species, including citrus using the Mi nimum Evolutio n method. ....................................... 445-1 The effect of differ ent flg22 on the expression of EDR1, EDS1, NDR1, PBS1, RAR1 and SGT1 in grapefruit pret reated with MES or azelaic acid. ................... 495-2 The effect of different flg22 on the expression of EDS5, ICS1 and PAL in grapefruit pretreated with MES or azelai c acid. .................................................. 505-3 The effect of different flg22 on the expression of NPR1, NPR2 and NPR3 in grapefruit pretreated with MES or azelai c acid. .................................................. 515-4 The effect of different flg22 on the expression of PR1 and RdRP in grapefruit pretreated with MES or azelaic acid. .................................................................. 525-5 The effect of different flg22 on the expression of AZ I1 in grapefruit pretreated with MES or azel aic acid. ................................................................................... 535-6 Effect of azelaic acid on the expr ession of RAR1, NPR2, NPR3 and EDS1 in grapefruit ........................................................................................................... 565-7 Effect of azelaic acid on the expr ession of AZI1 in grapefruit. Data was selected from Fi gure 5-5. .................................................................................... 56 8

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LIST OF ABBREVIATIONS AzA Azelaic Acid ETI Effector-Triggered Immunity HLB Huanglongbing MES 2-(N-morpholino) ethanesulfonic acid PAMP Pathogen-Associated Molecular Pattern PCR Polymerase Chain Reaction PTI PAMP-Triggered Immunity SA Salicylic Acid SAR Systemic Acquired Resistance Xcc Xanthomonas citri subsp. citri 9

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Abstract of Thesis Pres ented to the Graduate School of the University of Florida in Partial Fulf illment of the Requirements for t he Degree of Master of Science FLG22-TRIGGERED IMMUNITY AND THE E FFECT OF AZELAIC ACID ON THE DEFENSE RESPONSE IN CITRUS By Qingchun Shi August 2011 Chair: Gloria Moore Major: Plant Molecular and Cellular Biology The citrus industry plays an important role in the econom y of Florida. However, bacterial diseases such as citrus canker (caused by Xanthomonas citri subsp. citri Xcc ) and huanglongbing (caused by Candidatus Liberibacter asiaticus, Las) have caused considerable losses to citrus production. To have a better understanding of the defense response of citrus against Xcc and Las and to seek alternative ways to control canker and huanglongbing, we studied: 1) The role of Pathogen-Associated Molecular Pattern (PAMP)-triggered immunity (PTI) in the res ponse of resistant kumquat and susceptible grapefruit, to Xcc ; 2) The effect of PAMPs from X cc or Las on PTI; and 3) Evaluated the effect of azelaic acid, a molecule recent ly found to be important in the priming and signaling of pathogen defense, on the PTI response in grapefruit. The first experiment was performed by treating kumquat and grapefruit with a 22 amino acid peptide from the Xcc bacterial flagellin conser ved domain (Xflg22), a PAMP. The results showed that, compared to the mo ck inoculation, Xflg22 (10 M) triggered the expression of a set of defense-asso ciated genes including EDS1, RAR1, SGT1, EDR1, PBS1, NDR1, EDS5, PAL, NPR2, NPR3 RdRP and AZI1 in kumquat, but the corresponding genes in grapefruit were either not induced or showed slight 10

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11 downregulation. In kumquat, t he significantly Xflg22 induced genes EDS1, RAR1, SGT1 and NDR1 are thought to be PTI-associated genes, suggesting that Xflg22 initiated PTI in this resistant species. However, no obv ious induction of those genes was observed in grapefruit suggesting a weaker PTI and perhaps part of the reason this species is susceptible to canker. In the second experiment we compared the effect of Xflg22 with the flg22 derived from Las (Lflg22) as well as the archetype flg22 on grapefruit. Additionally, azelaic acid was used as a pretreatment before any flg22 in filtration in order to evaluate its defenseeliciting capability in citrus. Comparison between the effects of the three different flg22s showed that most of the defense-associ ated genes tested were not differentially expressed between the treatm ents compared with the mock inoculation, except for salicylic acid signaling gene AZI1 and salicylic acid biosynthesis gene PAL. However, when plants were pretreated with azelaic ac id (1 mM) two days prior to the PAMP treatments, mock inoculation, flg22 and Xflg22 treatments all showed higher expression levels of EDS1, RAR1, EDR1, EDS5, NPR1, NPR2, NPR3, RdRP and AZI1 than that of in control buffer pretreated plants, except the Lflg22 treatment which significantly reduced the expression of EDS1, RAR1 SGT1, EDR1, NPR1, NPR2 and NPR3 compared with the mock inoculation. Azelaic acid alone induced a set of defenseassociated genes including EDS1, RAR1, EDR1, EDS5, NPR1, NPR2, NPR3, RdRP and AZI1, which was comparable with the gene inductions by Xflg22 in kumquat, suggesting that azelaic acid could be used as a potential chemical to control bacterial disease in citrus.

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CHAPTER 1 INTRODUCTION With hundreds of years of growing history, citrus production in Florida has become a multibillion dollar industry contributing most of the value to Floridas agriculture ( http://www.florida-agriculture.com/agfacts.htm ). Citrus production in Florida accounted for 65 percent of the total citrus mark et in the United St ates during the 2009-2010 season, far more than that of Californi a (31%), Texas and Arizona (4% combined) ( http://www.nass.usda.gov/Statistics_by_Sta te/Florida/Publications/Citrus/fcs/200910/fcs0910.pdf ). Multiple citrus types are grown in Florida, among which grapefruit ( Citrus paradise Macf.) is one of the most popular. It is consumed as fresh fruit and juice not only because it is flavorful but it is al so a good sources of heal th promoting nutrients and phytochemicals such as vitamin C, antioxidants and fiber pectin [ 1 2 ]. As the largest grapefruit pr oducing state in the United St ates, Florida is suffering from a serious disease, citrus canker, caused by the bacterium Xanthomonas citri subsp. citri ( Xcc ). Grapefruit is highly susceptible to citrus canker and the disease causes lesions on leaves, stems and fruits. Fruit lesions make them cosmetically deficient and devalue the commodity. When favorable conditions such as high winds and rainfall are present, severe infection can lead to attenuated tree vigor characterized by defoliation, death of y oung shoots, and fruit drop [ 3 ]. In an effort to eliminate canker in Florida, the U.S. Department of Agri culture (USDA) in c ooperation with Florida Department of Agriculture and Consumer Service (FDACS) declared an eradication program in 1994, mandati ng the removal of diseased and surrounding trees, including in backyards. However, the hurricanes in 2004 ex acerbated the spread of canker making the eradication unrealistic. As a result, the USDA withdrew the eradication program and 12

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imposed a statewide quarantine, restricting the out of st ate shipment of citrus ( http://www.freshfromflorida.com/pi/canker/pdf/cankerflorida.pdf ). Several management methods can be used to control canker with various degrees of effectiveness. Since wind-blown rain facilitates canker bacteria dissemination, windbreaks in the infected orchard can re strict disease progress by reducing wind speed [ 4 ]. The Asian leafminer ( Phyllocnistiscitrella ) larvae produce wound galleries in the citrus leaves and make them more susceptible to canker, so either chemical or biological control of the leafminer is an indi rect way to manage citrus canker spreading [ 3 ]. In terms of bactericide use, copper-based sprays so far are more effective than any other antibacterial chemicals tested [ 5 6 ]. However, the antibacterial activity is limited to the surfaces of leaves or fruit and multiple spray times are needed to reach the optimal effect [ 3 6 ]. In Florida, huanglongbing (or citrus greening), caused by Candidatus Liberibacter asiaticus, is another bacterial disease affect ing the citrus industry. Transmitted by the vector psyllid ( Diaphorina citri Kuwayama), bacterial infection can result in small and misshapen fruits and largely reduced yi eld and lifespan of citrus trees. ( http://www.aphis.usda.gov/plant_health/plant_pest_info/citrus_greening/background.sh tml ). Because the disease has long lat ency, nonspecific and environment dependant symptoms, the efficient det ection and the management of huangl ongbing is difficult [ 7 ]. Almost all the cultivated citrus species and citrus relatives can be infected by huanglongbing. The current methods for controlling huanglongbing are through chemical control of psyllid vector, remo val of infected trees and using disease-free nursery materials [8 9 ]. 13

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14 Facing the threat of new diseases in Fl orida, it is necessary to study these diseases. Studying the citrus defense response at the molecular level can be helpful to understand the reasons for the vulnerability to their bacte rial pathogens. One of the supportive evidences for that is th e genomic sequencing of model plants and pathogenic bacteria has provided a better understanding of the plant immune system and plant-microbe interactions at the molecular, genetic and evolutionary levels [ 10, 11]. With little understanding of the citrus-pat hogen system, taking advantage of the latest information on plant immunity and plant-p athogen interactions and combining with the recent availability of a citrus genome database, we intend to explore mechanisms of citrus susceptibility and resistance to it s bacterial diseases and seek alternative effective management methods.

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CHAPTER 2 LITERATURE REVIEW The Plant Immune System Plants use two different pathways of acti ve defense or immunity against potential pathogens to prevent the est ablishment of disease [ 12]. In one pathway plants respond to certain molecules that originate from phytopathogenic microorganisms. These are named Pathogen-Associated Molecular Patterns (PAMPs). Recognition of PAMPs in the plant is mediated by pattern recognition receptors (PRRs),which triggers signaling cascades that can lead to a defense response [ 13, 14]. The PAMP triggered immunity (PTI) in plants is also termed the basal def ense and it deters colonization by a broad range of microbes [ 15]. Pathogens that successfully over come the plant PTI may further induce a stronger defense response, effector -triggered immunity (ETI), which brings about disease resistance in the plant. To be specific, plant resistance (R) genes encode proteins mostly with nucleotid e binding (NB) and leucine rich repeat (LRR) domains that recognize pathogen virulence factor s (effectors), resulting in t he initiation of R-mediated resistance.ETI is typically accompanied by the hypersensitive response (HR) [ 12, 16], a programmed cell death process effectively rest raining pathogen growth at the infection site where lesions serve as visual markers. Additionally, when infected by pathogens plants are not only able to generate defense mechanisms locally but also to tran sduce signals to distal plant tissues triggering systemic resistance to protec t against subsequent pathogen attacks. This process is termed Systemic Acquired Resistance (SAR) [ 17, 18]. One of the examples is that Arabidopsis thaliana R-mediated resistance at the infect ion site leads to elevated systemic protection against virulent bacteria [19]. 15

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PAMPs and PAMPs-Triggered Immunity (PTI) Plant basal defense triggered by PAMPs is essential, without it plants would be vulnerable to even minor microbial challenges. As conserved molecules among microbial species, PAMPs such as flagelli n and the amino-terminus of the elongation factor Tu (EF-Tu) are required for t he perception of bacteria in plants [ 20, 21]. For plant perception of fungi and oomycetes, two known PAMPs are chitin and ergosterol [ 14, 22, 23]. Flagellin is a component of the bacterial flagellum filaments and it contains a conserved 22 amino acids (flg22) domain that is sufficient to be recognized by a LRR receptor-like kinase (FLAGE LLIN SENSING 2, FLS2) in plants, which activates a mitogen-activated protein kinase (MAPK) cascade that results in the regulation of defense-related genes [ 24, 25, 26] (Figure 2-1). In Arabidopsis flagellin treatment can result in cell death and plant developmental impediment, and this effect depends on the amino acid residue at position 43 (as partic acid) on the flagellin protein [ 27]. Arabidopsis plants treated with flg22 produce a microRNA that degrades mRNAs of several auxin signaling components, halting the gr owth of phytopathogenic bacteria [ 28]. Antagonistic to auxin signaling, SA accumulates during flg22 infection and mutation of certain SA signaling genes leads to dampened PTI, i ndicating that an SA -mediated defense response is one consequence of flg22 perception [ 29, 30]. Furthermore, ion fluxes change, such as calcium ions increase [ 31], synthesis of callose [ 32] and stomatal closure [ 33] also contribute to PTI. 16

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Figure 2-1. Simplified model of bacteria flagellin perception in Arabidopsis A conserved 22 amino acid sequence in the bacterial flagellin is recognized and bound by a LRR containing receptor (FLS2), which also has kinase activity that triggers a MAPK signaling cascade [ 26, 34]. Multiple MAP kinases, MAP kinase kinases and MAP kinase kinase kinases are involved in the regulation of WRKY transcription factors, resulting in the modulation of th e transcription of defense genes and PTI [ 24]. Defense Signals The correlation between induction of SAR and accumulation of SA in plants upon pathogen infection [ 35], along with the evidence that exogenous treatment with SA appears to initiate expression of SAR-re lated genes implies that SA may be the chemical involved in SAR signal transduction [ 36, 37]. However, pathogen infected tobacco rootstocks engineered with a bacte rial SA-degrading enzyme NahG are still capable of transmitting SAR signals to the wild type scions and only accumulation of SA in distant tissues is necessary for SAR induction [ 38]. 17

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A subsequent study in the tobacco-Tobacco Mosaic Virus (TMV) system indicates that methyl salicylate (MeSA) was actually a key SAR signal component since MeSA could move from local infection sites to distal healthy leaves and was then hydrolyzed into SA by salicylic acid-binding prot ein 2 (SABP2), which triggered downstream defense signaling that established resistance systemically [ 39, 40]. Nevertheless, it is still too early to conclude that MeSA is the universal SAR inducing signal because similar research in Arabidopsis demonstrates that MeSA is not as effective in triggering SAR as was shown in the tobacco-TMV system [ 41]. A study of vascular sap extracted from Arabidopsis infected with a systemic resistance inducible pathogen revealed that azelaic acid, a nine-carbon dicarboxylic acid, had higher accumulation in SAR-induced exudates than in mock-induced exudates [ 42]. The increase of azelaic acid was found to be involved in the onset of SA accumulation and exogenous application of this chemical induced an earlier and stronger defense response both locally and systemically. Meanwhile, the AZI1 (AZELAIC ACID INDUCED 1) gene, encoding a putative secreted protease inhibitor/seed storage/lipid trans fer protein family protein, was induced by azelaic acid and mutation of this gene resulted in a ttenuated SAR. Taken t ogether, mediated through AZI1, azelaic acid was an important signal involved in priming the plant defense [ 42]. The elevation of jasmonic acid (JA) c ontent and heightened expr ession levels of JA biosynthetic genes observed when Arabidopsis leaves were inoculated by SAR inducing avirulent Pseudomonas syringae plus the evidence that JA treated plants are more competent in systemic resistance, gave the cue that JA was also a good 18

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candidate signal for initiating SAR [ 43]. However, instead of using low inoculum concentration of pathogen, a par allel experiment challenged plants with an HR-eliciting concentration of bacteria suggested that JA signaling components were not always required, and JA accumulation did not positively correlated with SAR activity [ 41, 44], implying that the involvement of JA and its signaling in SAR depends on pathogen pressure [ 45]. Genes Involved in Plant Immunity Enhanced Disease Susceptibility 1 (E DS1) and Nonrace-Specific Disease Resistance 1 (NDR1) Mutant eds1Arabidopsis plants were found to have both attenuated basal resistance against a non-host pathogen and R proteins mediated resistance [ 46, 47]. Biochemical analysis demonstrated that the R proteins involved were characterized by their Toll-Interleukin-1 receptor (TIR) and nucleotide binding-leucine rich repeat (NBLRR) domains [ 48]. EDS1 functions mainly by association with proteins encoded by phytoalexin deficient4 (PAD4) or senescence-associated gene101 (SAG101), forming a protein complex coordinating signali ng events delivered by ETI and PTI [ 49, 50, 51]. It is believed that regulation by the EDS1 complex occurs immediately downstream of Rprotein activation but upstream of both SA accumulation and the oxidative burst that leads to HR [ 51, 52]. In parallel with EDS1 NONRACE-SPECIFIC DISEASE RESISTANCE 1 (NDR1) was found to be a regul ator gene whose protein is associated with a different class of R proteins (coiled-coil motif containing) responding to specific avirulent proteins. Like EDS1, NDR1 is pr oposed to be involved in basal resistance to both compatible and non-host pathogens [53]. The Arabidopsisndr1 mutant exhibits decreased SA production and SAR and NDR1 regul ation was shown to be upstream of 19

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SA synthesis but downstream of reactive oxygen species (ROS) production, which potentially leads to programmed cell death [ 48, 54, 55]. Requires for Mla12 Resistance (RAR1) an d Suppressor of G-TwoAlleleofSkp1 (SGT1) With the concept that R pr otein-effector interaction is mediated by third party protein complexes [ 56], RAR1 and SGT1 were characterized as important components coordinating R-mediated resistance [ 57, 58]. RAR1 mutated barley and Arabidopsis both showed compromised resistance spec ifically triggered by R genes [ 59, 60], and similar weakened R gene-mediated resistance was also observed in SGT1 silenced barley, tobacco and Arabidopsis [ 61, 62, 63]. The binding between RAR1 and SGT1 proteins was confirmed in vitro [ 61] and another molecular chaperone Heat Shock Protein 90 (HSP90) was also found to be part of the RAR1-SGT1 complex [64]. However, Rmediated resistance does not always require the physical interaction between these three proteins [ 57]. Noticeably, SGT1 silenced Nicotiana benthamiana plants inoculated with a non-pathogen microbe accumula ted a higher bacterial popul ation than that of wild type inoculated plants and an Arabidopsis RAR1 mutation lead to subdued basal defense against both compat ible and non-host pathogens [ 62]. Similarly, in soybean RAR1 and SGT1 were found to be conver ging signaling component s for basal defense [ 65]. These findings indicate that RAR1 and SGT1 are not only essential regulators of R gene-mediated defense but are necessary for plant basal defense against a wide range of microbes. Enhanced Disease Resistance (EDR1) EDR1 is a negative regulator of the plant defense response and it has been shown that Arabidopsis edr1 mutants have enhanced resistance to the powdery mildew fungus 20

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Erysiphe cichoracearum [ 66]. EDR1 mediated defense is dependent on SA signaling pathways and do not require JA or ethylene signaling directly [ 67, 68, 69]. However, exogenous application of ethylene to edr1 mutants can cause faster senescence followed by increased expression of PR genes, the marker of SAR, implying that EDR1 may regulate defense response through the senescence process [ 70, 71]. Biochemical analysis revealed that EDR1 encodes a putative Raf family MAP kinase kinase kinase (MAPKKK) having an autophosphorylation acti vity, which negative regulates the defense response [ 70, 72]. Enhanced Disease Susceptibility 5 (EDS5) When inoculated with pathogenic bacteria, susceptible Arabidopsis mutant eds5shows failure to accumulate SA and a reduced transcriptional level of the Pathogenesis Related 1 gene (PR1), which is one of the markers of SAR. However, the susceptibility observed in the eds5mutant was not as strong as in plants expressing SA degrading enzymes (NahG), which had attenuat ed transcription of multiple PR genes (PR-1, PR-2, PR-5) [ 73, 74]. When a cloned EDS5 was used to complement the eds5 mutant the resulting plant showed accumulation of SA and expression of PR-1, implying that EDS5 regulated the defense response upstream of SA synthesis and SAR onset [ 75]. What is more, eds1, pad4 and ndr1 mutants exhibit si gnificantly decreased EDS5 transcription levels upon pathogen inoculat ion, indicating that EDS5 functions downstream of thes e signaling components Pathogenesis-Related Gene 1 (PR1) and Nonexpressor of PR Genes (NPR1) PR1 gene expression is considered a marker of SAR given the evidence that its expression correlates with the initiation of SA R and that PR1 is the most highly induced 21

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PR gene after a plant has been inoculated wit h SAR-inducing pathoge ns or treated with SA. However, the function of t he PR1 protein is still unclear [ 76, 77]. NPR1 plays a key role as a transcr iptional activator for the PR1 gene [ 78]. The Arabidopsis npr1 mutant is not able to respond to the SAR elicitin g chemical SA and fails to induce multiple PR genes including PR1 [ 79, 80, 81]. Cytoplasmic NPR1 exists in anoligomeric form and, upon infection, SA-m ediated cellular redox changes cause the NPR1 oligomers to break into monomers, wh ich are then translocated into the nucleus [ 82]. Nuclear localization of NPR1 is nec essary to trigger PR1 expression [ 83] and a bipartite nuclear localization signal (NLS) in the NPR1 protein is necessary for its translocation to the nucleus [ 84]. Yeast two-hybrid assays s uggest that functional NPR1 binds to the TGA family of transcription factor s, which are regulators of the transcription of PR1 by binding to its SA-responsiv e element in the promoter region [ 78, 83 85]. Moreover, NPR1s transactivation activity is in turn regulated by a proteasome-mediated mechanism: in uninfected plants, nuclear NPR1 is subjected to a cullin3-based ubiquitin ligase-mediated degradation process to avoid consti tutive expression of the PR gene; When the plant is challenged with a pathogen, phosphorylation of NPR1 at the Ser11/Ser15 residues also led to pr oteolysis of NPR1, probably to stimulate an influx of fresh NPR1 [ 86]. In the Arabidopsis genome, there are five NPR1 paralogs that share sequence similarity with NPR1 (NPR2, NPR3, NPR4, BOP1 and BOP2) and the phylogenetic relationships between t hese paralogs are shown in figure 2-2 [ 87, 88]. Functional analysis indicates that NPR3 and NPR4 also interact with the TGA family of transcription factors and negatively regulate expression of the PR1 gene [ 87]. The sequences of BOP1 and BOP2 have the least si milarity with NPR1 and these proteins 22

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also bind to a TGA of subfamily transcripti on factor. However, t he expression of BOP1 and BOP2 is tissue specific and the gene produc ts regulate plant growth asymmetry in Arabidopsis [ 88, 89]. Figure 2-2. Nucleotide sequenc e homology analysis of the Arabidopsis NPR1 protein family. NPR2 is the most homologous to NPR1, forming a NPR1-NPR2 subgroup. PR1 negative regulators NPR3 and NPR4 form another group. BOP1 and BOP2 are in a group that has least homology to NPR1 [ 87, 89]. Azelaic Acid Induced 1 (AZI1) Arabidopsis AZI1 (At4g12470) is one of the genes induced in cutinase transformed plants that are confirmed to be fully resistant to the fungal pathogen Botrytis cinerea and overexpression of AZI1 pr ovides disease resistance in Arabidopsis [ 90]. The protein sequence of AZI1 is homologous to a lipid transfer protein (LTP) family that has the putative functions of fatty acid binding and phospho lipid transfer during plant defense against pathogens [ 91]. The nomenclature of At4g12470 as AZELAIC ACID INDUCED 1 (AZI1) originated because expression of the gene is initiated in azelaic acid triggered resistance and AZI1 can be induced by azelaic acid applications. In addition AZI1 seems to be involved in the regulation or direct translocation of the 23

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SAR signaling to systemic tiss ues, given the evidence that azi1 mutants showed normal local resistance but had a compromised systemic resistance [ 42]. RNA-Dependent RNA Polymerase (RdRP) In the defense against RNA viruses, pl ants use an RNA silencing mechanism to inhibit viral replication in the tissues, during which a surveillance system of the plant detects exogenous RNA and initiates doubl e-strand RNA (dsRNA) mediated RNA degradation [ 92]. It has been shown that RNA-dependent RNA polymerases (RdRPs) are a family of proteins necessary for RNA silencing in terms of dsRNA synthesis and silencing signal amplification [ 93, 94, 95]. Some RdRPs have been shown to be inducible by either SA or infection with an RNA virus in Arabidopsis and Nicotiana tabacum [ 96, 97], whereas a mutation in the SA-inducible RdRP gene in Nicotiana benthamiana caused the plant susceptible to the viruses [ 98], which indicates that this particular RdRp is essential for the plant defense against virus and the antivirus response is also associated with the SA-mediated defense response. Isochorismate Synthase 1 (ICS1) and Phenylalanine Ammonia Lyase (PAL) The synthesis of SA is believed to occur via two parallel pathways [ 99, 100 ] (Figure 2-3).The enzymatic reactions from chor ismate have been shown to be the most important one during pathogeninduced SA biosynthesis in multiple plant-pathogen systems [ 101 102 103 104 ]. Silencing the ics1 gene leads to reduced SA accumulation in tobacco and tomato during pathogen infection and results in a compromised defense response, suggesting ICS1 is a key enzym e for defense-associated SA synthesis [ 101 102 ]. On the other hand, a biochemical st udy using isotope feeding showed that PAL is involved in SA synthesis via cinnamat e intermediates and that inhibiting PAL activity decreases SA accumulation induced by a pathogen [ 105 106 ]. Moreover, PAL 24

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has been shown to be an important protein cata lyzing the first enzymatic reaction in the phenylpropanoid biosynthetic pathway (lignin biosynthesis) [ 107]. Lignin is considered a necessary physical barrier agains t non-host bacterial invasion in Arabidopsis [ 108]. Figure 2-3. A simplified salicylic acid (SA) synthetic pathway [ 99]. The key enzymes are indicated above the arrows: PAL is pheny lalanine ammonia lyase, ICS1 is isochorismate synthase 1 and IPL is isochorismate pyruvate lyase. Avrpphb Susceptible 1 (PBS1) PBS1 is an important component fo r the R-mediated resistance in Arabidopsis against plant pathogenic bacteria(i.e. the resistance induced by the R protein PRS5 recognition of the avrPphB effector secreted from Pseudomonas syringae [109] ). Functional analysis indicated that the PBS1 gene encodes a protein kinase which normally interacts with the Coiled Coil (CC)-NBS-LRR domain of PRS5, inhibiting the initiation of ETI [ 110]. Upon being challenged with Pseudomonas syringae strains, Arabidopsis PBS1 is cleaved from the PRS5-PBS1 complex by the protease activity of avrPphB, in which PRS5 is released and triggers a stronger R-mediated defense response [ 111, 112]. Rationale and Objectives Previous research showed that the citrus relative kumquat exhibited resistance characteristics such as cell death, leaf abscission and restricted bacterial growth in response to canker infection [ 113 ]. By subtractive cDNA li brary analysis and microarray 25

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expression profiling, groups of genes were identified that were putatively involved in the canker-kumquat interaction and some of these genes were homologous to known defense-associated genes, such as transcrip tion factors and receptors, suggesting that canker challenge could trigger an active def ense response and certain signaling events in kumquat [ 113 ]. Preliminary comparisons between canker inoculated grapefruit and kumquat indicated that defense-associ ated genes EDS1, NDR1, NPR2, NPR3, PBS1 and RAR1 were induced both earlier and higher in kumquat than in the susceptible grapefruit, which may correlate with resistance/susceptibility to canker (Febres and Khalaf unpublished results). PTI, previously termed basal defense, provi des one of the first levels of protection against harmful microbial invasion and is typically triggered by the recognition of slowly evolving molecules from microbes know as PAMPs [ 13]. Experimentally PTI can be initiated by challenging plants with the synthetic 22 amino acids peptide (flg22), which is a the conserved domain of the bacterial flagellin, a PAMP [ 25, 27, 34]. In our experiments we used a synthetic peptide derived from Xcc (Xflg22), the causal agent of citrus canker. The first objective of this research was to investigate w hether Xflg22 triggered PTI in citrus, and to compare defense-a ssociated gene expression changes in response to Xflg22 over time between susceptible gr apefruit and resistant kumquat. This may provide a better understanding of the role PT I has in the resistance/ susceptibility response to canker. As previously mentioned, huanglongbing (caused by Candidatus Liberibacter asiaticus is another severe bacterial disease. Candidatus Liberibacter asiaticus is believed not to possess flagella in citrus hosts based on the evidence from electron 26

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microcopy observation and genome sequence information [ 9 114 ]. However, flagellin domain-containing protei ns (GI: 254040208 and GI: 25478051 3) have been identified in the Candidatus Liberibacter asiaticus genome and they also contain the conserved 22 amino acid domain (Lflg22) near the N te rminus with some variations. Our second objective was to compare the response of gr apefruit to Xflg22, Lf lg22 and the archetype flg22. This would provide insights into the basal defense response of grapefruit to different PTI elicitors, which could be helpful in understanding the grapefruit susceptibility. A recent study of the small molecular compounds present in the vascular sap of pathogen inoculated Arabidopsis revealed that a 9 carbon molecule named azelaic acid was important for the resistance to virul ent bacteria. In additi on, the gene AZI1 was identified to be specifically inducible by azelaic acid and mutation of AZI1 in Arabidopsis led to compromised SAR [ 42]. We identified a homologous AZI1 gene in citrus and found that its expression was induced by Xfl g22 in grapefruit, suggesting that it may have a role in the defense response and azelaic acid may also be involved in signaling. Based on the SAR priming characteristic of azelaic acid, our third objective was to evaluate whether exogenous application of this co mpound could enhance the defense response of susceptible grapefruit. Moreov er, comparing the response of defenseassociated genes to different flg22 peptides and investigating how and whether azelaic acid affects this response could help to understand the role of these proteins in the susceptibility of grapefruit to canker and HLB. 27

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CHAPTER 3 MATERIALS AND METHODS Plant Material The plants used were greenhouse maintained Nagami kumquat ( Fortunella margarita (Lour.) Swing. ) (Floyd and Associ ates, Dade city, FL.) and Duncan grapefruit ( Citrus paradise Macf.). All of the plants were watered daily and fertilized once a week (Peters fertilizer 20-20-20, 2.0 g/L). Horticultural oil spray was applied as needed to control mites and scales. Kumquat plants were grown in individual pots and their age were about 2 years old. Kumquat plants were pruned to the height of 70-90 cm and mature leaves were used for the experim ents. Grapefruit plants were grown either individually or in groups of 2-4 plants per pot and their age were about 2-3 years. Approximately a month before the experiments, grapefruit plants were pruned to about 30-40 cm high and mature leaves from the new shoots were used for the experiments. Flagellin22-Amino Acids Conserved Domain Peptides The peptides used for treating the plants we re obtained from GenScript USA Inc. The archetype 22-amino acid peptide (f lg22, QRLSTGSRINSAKDDAAGLQIA) was in stock from the Genscript U SA Inc. and the sequence was refe renced from the study by [ 115 ]. Using BLAST analysis against the NCBI protein database with the flg22 sequence as query identified a flagellin protein from Xcc (GI: 21242719) and two flagellin domain-containing proteins from Candidatus Liberibacte rasiaticus (GI: 254040208 and GI: 254780513). The c onserved domains from Xcc (Xflg22: QRLSSGLRINSAKDDAAGLAIS), and Candidatus Liberibacter asiaticus(LFgl22: DRVSSGLRVSDAADNAAYWSIA) were then synthesized. 28

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Challenge Kumquat and Grapef ruit with Xflg22 Peptide Nagami kumquat and Duncan grapefruit plants were used for flg22 from Xcc (Xflg22) and control (water) treatments. The Xflg22 solution was prepared by dissolving the lyophilized peptide in dist illed water to a final concentration of 10 M. This concentration was chosen based on previous work by Zipfel et al. [ 25]. The Xflg22 challenge was performed by infiltrating the peptide solution in to the abaxial surface of fully expanded mature leaves using a 1cc insulin syringe with a needle. Infiltration with distilled water was the control. The solution was infiltrated until half of the leaf was saturated. Subsequently, leaf samples from each treatment were collected at 0, 6, 24, 72 and 120h after the infiltration. Zero hour samples were collected right before infiltration. Three biological replicates fr om different plants were collected for each treatment, genotype and time point. The samp les (two leaves from each plant) were immediately frozen in liquid nitrogen and stored in the freezer at -80 ; subsequently the infiltrated leaf halves were used for RNA extraction and gene expression analysis. Azelaic Acid Pretreatment and Challenges with flg22, Lflg22 and Xflg22 Peptides The plant material used was Duncan grapefruit. Individual plant s (a total of 24) were divided into two populations of 12 plant s for each of two chemical pretreatments (azelaic acid or MES buffer). Azelaic acid (Acros Organics, NJ) was dissolved in 5mM MES (Acros Organics, NJ) buffer pH 5.6 to r each maximum solubility. The azelaic acid solution final concentration used was 1mM [ 42]. For the pretreat ments, the 1 mM azelaic acid solution was infiltrated into the abaxial surface of leaves using a 1cc insulin syringe with a needle two days prior to the flg22 challenges and 5 mM MES buffer pH 5.6 infiltration was used as control. The 12 plants from each pretreatment were further divided into four groups of three plants as biological replicates and subsequently 29

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challenged with a10 M solution of flg22, Lflg22, Xflg22or distilled water (control). Leaf samples from each plant were collected at 0, 6, 24, 72 and 120h after the peptide or water infiltration, where 0h samples were co llected immediately before any infiltration. The tissue was immediately frozen in liqui d nitrogen and stored in the freezer at 80Cprior to RNA extraction and gene expression analysis. RNA Extraction and Purification For each sample, RNA was extracted using TriZol reagent following the manufacturers instructions (Invitrogen, CA). Each sample consisted of two different leaves from the same plant. The RNA was subsequently treated wit h DNase followed by a cleanup protocol using the RNeasy Plant Mini Kit (QIAGEN Sciences, MD) to eliminate any DNA and prot ein contamination. The c oncentration of RNA was determined with a NanoDrop 2000c spectrophot ometer (Thermo Scientific, PA) and the RNA purity was measured as the OD260/OD280value in which RNA with values 1.80 were considered as clean and were subjected to cDNA synthesis. cDNA Synthesis The purified RNA (1 g) was used for eac h 20 L cDNA synthesis reaction. The 20 L reaction mixture included 2 L of 50 M Random Decamers (Ambion Inc, Applied Biosystem, CA), 2 L of 10 M dNTPs, 2 L of 10X First Strand Buffer (Ambion Inc, Applied Biosystem, CA), 1Lof M-MLV Revers e Transcriptase (100U/L, Ambion Inc, Applied Biosystem, CA) and 1 L of RNase I nhibitor (40U/L, Ambion Inc, Applied Biosystem, CA). The remainder of the volu me was completed with RNase-free water. The RNA, Random Decamers, dNTPs and wate r mixture was incubated at 80C in a water bath for 2-3 minutes and placed on ic e for 3 minutes before adding the First Strand Buffer, RNase Inhibitor and Reverse Tr anscriptase. The 20 L reaction mixture 30

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was then subjected to reverse transcription using the thermal cycle at 42C for 1h and 92Cfor 10 minutes (PTC-100 Programmable T hermal Controller, MJ Research Inc. Canada). The cDNA samples were stored at -20C prior to real-time PCR. Gene Expression Analysis Gene expression levels were determined by quantitative real-t ime PCR using the StepOnePlus Real-Time PCR system (Applied Biosystems, CA). A Fast 96-well Reaction Plate (0.1 mL) (MicroAmp, Applie d Biosystems) was used for the reactions and each well was used to perform the 20 L expression assay of one gene from each sample. The cDNA was diluted to a final concentration of 50 ng/L and gene amplification was performed fr om 2 L of the diluted cDNA Each reaction mixture was composed of 2 L of 50ng/L cDNA, 10L of TaqMan Fast Universal PCR Master Mix (2X) (Applied Biosystems, CA), 1 L of TaqMan probe and primer Assay Mix (20X) (Applied Biosystems, CA) and 7 L of RNase-free water. The Assay Mix was a combination of specific probes and forwar d and reverse primers for each gene. For all the assayed citrus defense genes, the final primer concentrations were 900 nM each and the final probe concentration was 250 nM For the reference gene, 18S, the final primer concentrations were 250 nM and the final probe concentration was 150 nM per reaction. The quantitative real-time PCR experim ent was designed using the StepOne software v2.1 (Applied Biosystems, CA), in which the experiment type was set as comparative CT (CT), using TaqMan Reagent and the amplification speed was set to fast. The fluorescent reporter for the tested genes was FAM and VIC for the 18S.The Quencher was NFQ-MGB for all the probes. The real-time PCR thermal cycle was 95 C 20 for sec, followed by 95C for 1 sec and 60C for 20 sec. Fo r each 96-well plate, 31

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the amplification plots of every gene were manually checked for th e correct threshold levels and proper base line starting and ending points. For the comparative CT analysis, the 0 h samples were selected as refer ence samples and the calculated relative quantitation (RQ) values were exported to Mi crosoft Office Excel for further analysis. The RQ data were subjected to a statistical Q-test [ 116 ] to eliminate any outlier values among the replicates, and subsequently RQ means and stand errors were calculated. The statistical analysis was performed using JMP Genomic 5.0 Model fitting of standard least square means (LS Means) and Students t test (SAS Instit ute Inc. NC). All treatments for each ex periment (time and type of flg22 or time, flg22 and pretreatment) were compared to each other. Mean RQ values that were statistically significant at a specific time point are indicated in the figures. 32

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CHAPTER 4 COMPARISON OF THE RESPONSE TO XFLG22 BETWEEN CANKER SUSCEPTIBLE AND RESI STANT CITRUS TYPES Results Canker resistant kumquat and susceptible grapefruit were challenged withXflg22 (water infiltration was used as the control) and samples were collected at 0, 6, 24, 72 and 120h after the treatments. For each genotype and time point the gene expression level of the following genes was determined using quantitative real-time PCR: EDS1, RAR1, SGT1, EDR1, PBS1, NDR1, EDS5, ICS1, PAL, NPR1, NPR2, NPR3, PR1, RdRP and AZI1. These genes were chosen bec ause they are part of PTI/ETI induction (EDR1, EDS1, NDR1, PBS1, RAR1 and SGT1) (Figure 4-1), SA biosynthesis and signaling (EDS5, ICS1, PAL and AZI1) (Fig ure 4-2 and 4-5), PTI/ETI transcriptional regulation (NPR1, NPR2 and NPR3) (Fi gure 4-3) and PR genes (PR1 and RdRp) (Figure 4-4). Among the PTI/ETI-associated genes in vestigated, EDS1, NDR1, RAR1 and SGT1 displayed significant elevated expressi on levels at 6h after inoculation compared with the water inoculated controls in kumquat (Figure 4-1A). This effect lasted for 24h (RAR1 and SGT1) or for 72 h (EDS1, NDR1) after the treatment (Figure 4-1A).In contrast, the effect of the Xflg22 in grapefruit for those same genes was different, with their expression levels not significant ly different between the Xflg22 and water treatments at all the time point s except 6 h (F igure 4-1B). SA biosynthesis may be accomplished via two distinct pathways in which ICS1 and PAL catalyze the first enzymatic steps respectively. Remarkably, PAL expression was induced by Xflg22 at 6 and 24h after the treatment in kumquat but not in grapefruit 33

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compared with the mock inoculated controls at the same time points, even though in grapefruit PAL showed higher ex pression levels at 6 (water), 72 (water and XFlg22) and 120h (water and XFlg22) after the treatments compared with its expression at 0h (Figure 4-2). In contrast, ICS1 expression appeared not to be affected by XFlg22 treatment in kumquat but significantly induced in grapefruit at 24 and 72h after treatment (Figure 4-2). The SA signaling gene EDS5 did not show obvious induction by Xflg22 in either of citrus genotypes (Figure 4-2). AZI1 is a gene regulated specifically by the defense priming signal azelaic acid, which is believed to be an essential signal for SA accumulation during the defense response in Arabidopsis [ 42]. In our research, AZI1 appeared to be induced by Xflg22 both in kumquat and grapefruit (Figure 4-5) although the induction happened earlier in kumquat (6h after inoculation) than in grapefruit where AZI1 was significantly induced at 72h after inoculation. The significant AZI1 upregulation in grapefruit correlated with the induction of the SA synthesis gene ICS1 but not that of PAL (Figures 4-2B and 4-5B); whereas the AZI1 upregulation in kumquat appeared to be a ssociated with the induction of PAL (Figures 4-2A and 4-5A). The PTI/ETI transcriptional regulation genes are shown in the Figure 4-3. Compared to the controls, expression of NPR2 was significant ly higher at 6 h and 24 h in kumquat plants treated with Xflg22, and similar induction was found in NPR3 at 6, 24 and 72h after the treatment in kumquat (Figure 4-3A). NPR1 was not induced by Xflg22 compared with control, but its expression level at 6h (both water and Xflg22 treatments) was higher than 0h (Figure 4-3A). However, Xf lg22 treatment in grapefruit did not trigger higher expressions of NPR1, NPR2 or NP R3 than the control treatment, even though 34

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the induction of these genes was found in cont rol plants at 6h in respect to 0h, which may be the result of infiltra tion procedure (Figure 4-3B). PR1 is considered as the marker gene fo r SAR because its expression correlates with the resistance response against pat hogens and SA accumulation in systemic tissues [76, 77]. Even though investigating SAR was not our purpose, since the SAmediated defense response is also an essent ial local defense mechanism we measured the expression of PR1 (Fi gure 4-4). In Xflg22 treated kumquat, PR1 expression was significantly lower than the water treated controls at 6, 24 and 72h (Figure 4-4A), which was in contrast with the PR1 expression patte rn in the Xflg22 treated grapefruit where PR1was highly upregulated at 24 and 72h after inoculation (Figure 4-4B). In addition, another PR family protein RdRP encoding gene showed significantly higher expression level than the control at 6, 24 and 72h in kumquat, whereas such induction was not found in grapefruit (Figure 4-4B). 35

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0 1 2 3 4 5 6 7 8 0h6h C6h X24h C24h X72h C72h X120h C120h X RQ EDR1 EDS1 NDR1 PBS1 RAR1 SGT1 A* * * * * 0 1 2 3 4 5 6 7 8 0h6h C6h X24h C24h X72h C72h X120h C120h X RQ EDR1 EDS1 NDR1 PBS1 RAR1 SGT1 B Figure 4-1. Effect of Xflg22 on expre ssion of EDR1, EDS1, NDR1, PBS1, RAR1 and SGT1 in Nagami kumquat (A) and D uncan grapefruit (B). Leaves were infiltrated with 10M Xflg22 or distilled water. Samples were collected at 0, 6, 24, 72 and 120h after the infiltration. Gene expression was quantified by real time PCR followed by comparative CT analysis. The vertical axis indicates the relative quantitation (RQ), where the gene expression level in each sample is compared to the reference sample (0h). The lateral axis shows the names of the biological groups including hours a fter inoculation and treatment, in which C stands for control (water) and X for Xflg22 treatment. Leaves for 0h were collected right before infiltration. T he means and standard errors of three replicates are shown. An asterisk indica tes a statistically significant difference between the control and treatment at the same time point. 36

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0 1 2 3 4 5 6 7 8 0h6h C6h X24h C24h X72h C72h X120h C120h X RQ EDS5 ICS1 PAL A* 0 1 2 3 4 5 6 7 8 0h6h C6h X24h C24h X72h C72h X120h C120h X RQ EDS5 ICS1 PAL B *Figure 4-2. Effect of Xflg22 on expression of EDS5, ICS1 and PAL in Nagami kumquat (A) and Duncan grapefruit (B). Leaves were infiltrated with 10M Xflg22 or distilled water. Samples were collected at 0, 6, 24, 72 and 120h after the infiltration. Gene expression was quantified by real time PCR followed by comparative CT analysis. The vertical axis indicates the relative quantitation (RQ), where the gene expression level in each sample is compared to the reference sample (0h). The lateral axis shows the names of the biological groups including hours after inoculation a nd treatment, in which C stands for control (water) and X for Xflg22 treatment Leaves for 0h were collected right before infiltration. The m eans and standard errors of three replicates are shown. An asterisk indicates a statisti cally significant difference between the control and treatment at the same time point. 37

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0 1 2 3 4 5 6 0h6h C6h X24h C24h X72h C72h X120h C120h X RQ NPR1 NPR2 NPR3 A* * * 0 1 2 3 4 5 6 0h6h C6h X24h C24h X72h C72h X120h C120h X RQ NPR1 NPR2 NPR3 B Figure 4-3. Effect of Xflg22 on expressi on of NPR1, NPR2 and NPR3 in Nagami kumquat (A) and Duncan grapefruit (B). Leaves were infiltrated with 10M Xflg22 or distilled water. Samples were collected at 0, 6, 24, 72 and 120h after the infiltration. Gene expressi on was quantified by real time PCR followed by comparative CT analysis. The vertical axis indicates the relative quantitation (RQ), where the gene expression level in each sample is compared to the reference sample (0h). The lateral axis shows the names of the biological groups including hours a fter inoculation and treatment, in which C stands for control (water) and X for Xf lg22 treatment. Leaves for 0h were collected right before infiltration. T he means and standard errors of three replicates are shown. An asterisk indica tes a statistically significant difference between the control and treatment at the same time point. 38

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0 1 2 3 4 5 6 7 8 9 0h6h C6h X24h C24h X72h C72h X120h C120h X RQ PR1 RdRP A* * * 0 1 2 3 4 5 6 7 8 9 0h6h C6h X24h C24h X72h C72h X120h C120h X RQ PR1 RdRP* *B Figure 4-4. Effect of Xflg22 on expression of PR1 and RdRP in Nagami kumquat (A) and Duncan grapefruit (B). Leaves were infiltrated with 10M Xflg22 or distilled water. Samples were collected at 0, 6, 24, 72 and 120h after the infiltration. Gene expression was quantified by real time PCR followed by comparative CT analysis. The vertical axis indicates the relative quantitation (RQ), where the gene expression level in each sample is compared to the reference sample (0h). The lateral ax is shows the names of the biological groups including hours after inoculation a nd treatment, in which C stands for control (water) and X for Xflg22 treatment Leaves for 0h were collected right before infiltration. The m eans and standard errors of three replicates are shown. An asterisk indicates a statisti cally significant difference between the control and treatment at the same time point. 39

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0 2 4 6 8 10 12 0h6h C6h X24h C24h X72h C72h X120h C120h X RQ AZI1 A Figure 4-5. Effect of Xflg22 on expression of AZI1 in Nagami kumquat (A) and Duncan grapefruit (B). Leaves were infiltrat ed with 10M Xflg22 or distilled water. Samples were collected at 0, 6, 24 72 and 120h after the infiltration. Gene expression was quantified by real ti me PCR followed by comparative CT analysis. The vertical axis indicates t he relative quantitation (RQ), where the gene expression level in each sample is compared to the reference sample (0h). The lateral axis shows the names of the biological groups including hours after inoculation and treatment, in which C stands for control (water) and X for Xflg22 treatment. Leaves fo r 0h were collected right before infiltration. The means and standard errors of three replicates are shown. An asterisk indicates a statistically significant difference between the control and treatment at the same time point. 0 2 4 6 8 10 12 0h6h C6h X24h C24h X72h C72h X120h C120h X RQ AZI1*B 40

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Discussion The results in this study suggest that t he Xflg22 challenge tri ggers the expression of a set of defense-associat ed genes in canker resistant kumquat but not in the susceptible grapefruit. The defense-associat ed genes significantly induced in kumquat as a result of Xflg22 treatment were ED S1, SGT1 NPR2, NPR3, NDR1, RAR1 and PAL (Figure 4-1, 4-2 and 4-3). Among these genes, signaling regulators EDS1 and NDR1 are well known for their roles in R-medi ated resistance, and there have also been reports indicating that EDS1 and NDR1 are necessary for Arabidopsis PTI during pathogen attack and non-host interactions with wheat powdery mildew and Pseudomonas syringae pv. tabaci [ 46, 48, 53, 117 ]. Similarly, Nicotiana benthamiana SGT1 was found to be involved in the non-host resistance to Pseudomonas syringae pv. phaseolicola and Xanthomonas axonopodis pv. vesicatoria [ 62] and both RAR1 and SGT1 in soybean were necessary for PTI signaling [ 65 ]. The transcriptional alterations in flg22 treated Arabidopsis cell cultures and seedlings were screened using microarrays and the majority of the genes that were rapidly elicited were homologous to defense signal perception genes includ ing NDR1, EDS1 like gene and NPR1 [ 118 ]. In our research, the induction of PTI-associ ated genes by Xflg22 in kumquat was in agreement with previous re search results from Arabidopsis and other plant species, suggesting that this PAMP upregulated a similar set of PTI genes in kumquat In contrast, grapefruit PTI signaling genes were not induced by Xflg22 treatment or the effect was too weak to be detected. Alternativ ely, it is also possi ble that there was PTI gene response but it occurred at time points beyond the 6-120h range studied here. These genes would have been either transi ently induced prior to 6h after Xflg22 infiltration or late induced after 120h. However, since grapefruit is highly susceptible to 41

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canker, the most likely scenario is t hat the attenuated imm une response observed during the 6-120h period was indication of its inability to respond to Xflg22 and accounts for its susceptibility. SA accumulation is correlated with pathogentriggered defense responses in both local infection sites and systemic tissues [ 35, 119 120 ]. Upon pathogen recognition, plants can initiate SA biosynthesis in order to activate defense mechanisms and the pathogen inducible SA synthesis is believ ed to occur via two pathways: the PAL pathway and the ICS1 pathway (Figure 2-3). There are reports from different plantpathogen systems implying that either one of the pathways can be responsible for an increase in the SA levels during pathogen invasion [ 102 103 106 ]. In our results, Xflg22 induced significantly higher PAL expression in infiltrated kumquat but ICS1 expression did not appear to be affected, at least beyond the effect of water infiltration (Figure 42A). For canker resistant ku mquat, the PAL catalyzed SA bi osynthetic pathway might be the dominant pathway in response to Xflg22 challenge. However, it is notable that PAL is also the key enzyme in lignin biosynthesis and the lignifications of plant cells is a layer of defense during non-host in compatible interactions [ 106 108 ]. Hence the induction of kumquats PAL could indicate the initiation of lignin synthesis after the flg22 challenge, or perhaps PAL is involved in both SA and lignin biosynthesis during the defense response. On the other hand, ICS1 expression was the significantly induced by Xflg22 in grapefruit (Figure 4-2B). This implies that in grapefruit the ICS1 pathway might be the active SA synthetic pathway upon Xflg22 treatment. In addition, the expression of the SAR marker gene PR1 and the SA synt hesis priming gene AZI1 were well coordinated with ICS1 induction (Figures 4-2B, 4-3B and 4-4B), with all three genes 42

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upregulated at 24 and 72h after Xflg22 infiltrati on, suggesting that the SA regulating PR1 expression was synthesized through the ICS1 pathway. However, RdRP as another SA inducible PR gene, was significantly induced by Xflg22 in kumquat but not in grapefruit, implying that its regulating SA may be synthesized through PAL pathway. In Arabidopsis NPR1 is a positive regulator of the expression of PR1 [ 78]. PR1 gene transcription can be activated by the binding of nuclear localized monomer NPR1 protein to the TGA transcription factor and translocation of NPR1 from its cytosol oligomer form to the nucleus is necessary for the NPR1 transactivation activity [ 82, 84]. In the nucleus, NPR1 is also regulated by proteasome-mediated protein hydrolysis process to ensure continuous influx of active NPR1 from cytoplasm [ 86]. In our results, NPR1 expression was not obviously induced by Xflg22 challenge neither in kumquat nor in grapefruit (Figure 4-3), although PR1 wa s observed to be downregulated in kumquat and upregulated in grapefruit (Figure 4-4). The reason for the unnoticeable NPR1 induction in citrus could be the regulation of NPR1 was at the protein level instead of the mRNA level, where PR1 expression was regula ted by the translocation of NPR1 to the nucleus. Alternatively, regulator(s) other than NPR1 in citrus may have a dominant role in the transcriptional regulation of PR1. Arabidopsis NPR1 has a total of five paralogs in its genome (NPR2, NPR3, NPR4, BOP1 and BOP2, Figure2-2), among which NPR3 and NPR4 form a distinctive group that have been proven to be negative regulators of PR1 expression [ 87]. A previous study using BLAST analysis of AtNPR1 against the citrus EST database revealed that multiple NPR1 homologous sequences exist in citrus. The evolutionary distances between these sequences and NPR1 proteins from other plant species are shown in Figure 4-6 [ 121 ].The citrus genes CpNPR2 and CpNPR3 43

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(from Citrus paradisi ), which share high sequence simila rity at the protein level with AtNPR3 and AtNPR4, were significantly i nduced by Xflg22 in kumquat whereas the expression of the genes appeared to be reduced in grapefruit (Figure 4-3). Meanwhile, the PR1 gene was significantly downregulat ed in kumquat treated with Xflg22 however in grapefruit this gene was induced by the sa me treatment (Figure 4-4). These results suggest that CpNPR2 and CpNPR3 could be in volved in the negative regulation of PR1 in citrus, but the regulating roles of these two genes should be confirmed by functional analysis. Figure 4-6.Phylogenetic tree of NPR1-like pr otein sequences from different species, including citrus using the Minimum Evolution method. The percentage of replicate trees in which the associ ated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The evolutionary distances were computed using the Poisson correction method and are in the units of t he number of amino acid substitutions per site. All positions containing gaps and missing dat a were eliminated from the dataset (Complete deletion option). There were a total of 268 positions in the final dataset. Phylogenetic analyses were conducted in MEGA4. At= A. thaliana ; Cp= C. paradisi ; Cr= Carrizo citrange ( C. sinensis X P. trifoliata ); Pp= Physcomitrella patens; Pt= Populus trichocarpa; Vv= Vitis vinifera [ 121 ] 44

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45 Previous research on the defense responses of resistant kumquat and susceptible grapefruit challenged with Xcc demonstrated that kumquat was capable of inducing the expression of defense genes much earlier and at higher levels upon canker inoculation than was the case with grapefru it and these induced genes in kumquat were believed to be associated with the observed resistanc e (V. Febres and A. Khalaf unpublished results). In this research, Xflg22 also triggered the response of many PTI-related genes and NPR1-like transcriptional coactivators in kumquat but not in grapefruit (Figure 4-1, 4-2, 4-3 and 4-4), suggesting t hat the recognition and response to Xcc flagellin was necessary for citrus plants to successfully defend themselves against the pathogenic bacterium. By comparing kumquat genes whos e induction was triggered by Xflg22 with those induced by canker inoculation, it wa s found that most of the defense-associated genes upregulated in response to Xflg22 (EDS1, NDR1, NPR2, NPR3 and RAR1) were also even more highly induced by canker inoculation (V. Febres and A. Khalaf unpublished), suggesting that t he Xflg22 initiated PTI shar ed similar defense signaling pathways with canker resistance response a nd that the flagellin triggered PTI probably contributes to the total resistance to canker observed in kumquat.

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CHAPTER 5 EFFECT OF AZELAIC ACID AND DIFFERE NT FLG22 PEPTIDES ON THE DEFENSE RESPONSE OF GRAPEFRUIT Results Grapefruit plants were pretr eated with 1mMazelaic acid or 5 mM MES buffer (as a negative control) two days prior to being in filtrated with flg22 fr om three different organisms or water as a control. Samples we re collected at 0, 6, 24, 72 and 120h after treatment. Expression levels of the following genes were determined by real-time PCR: EDS1, RAR1, SGT1, EDR1, PBS1, NDR1, EDS5, ICS1, PAL, NPR1, NPR2, NPR3, PR1, RdRP and AZI1. The results are shown from Figures 5-1to 5-5, where the genes were divided into the groups indicated in each figure. Figures 5-1A, 5-2A, 5-3A, 5-4A and 5-5A show the gene expression in MES bu ffer pretreated plants, from which we could compare the effect of the different peptide treatments. Figures 5-1B, 5-2B, 5-3B, 5-4B and 5-5B show the expression of the same genes in grapefruit pretreated with azelaic acid followed by treatment with va rious peptides, where we could analyze the effect of azelaic acid and possibly the overlapping effects of azelaic acid and flg22 peptides. In MES pretreated grapefruit Xflg22didnot significantly altered the expression of the majority of the defenseassociated genes, given that at most time pointsXflg22 treated and mock inoculated plants showed similar expression levels of EDR1, EDS1, NDR1, PBS1, RAR1 and SGT1 (Figure5-1A), EDS5 and ICS1 (Figure 5-2A), NPR1, NPR2 and NPR3 (Figure 5-3A),PR1 and Rd RP (Figure 5-4A), even though the SA biosynthesis gene PAL was induced at 120h a fter Xflg22 treatment (Figure 5-2A). PR1 expression was increased at 24h in all treatments, including the water control, which intimates that the increase was probably due to the infiltration pr ocedure or some other 46

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environment effect (Figure 5-4A). Noticeably, Xflg22 treatment elevated the expression of AZI1 in MES pretreated gr apefruit at all time points and expression was significantly higher than in the control at 72h (Figure 5-5A), which was in agreement with AZI induction by Xflg22 found from the previous experiment. In addition to investigating the effect of Xf lg22, we compared the effect of the other two flagellin peptides: flg22 and Lflg22. The three types of peptides showed similar gene expression patterns as the mock inoculat ion did (Figures 5-1A, 5-2A, 5-3A and 54A), except that PAL was induced by Lfl g22 and Xflg22 but not by flg22 at 120h after the treatment (Figure 5-2A), and AZI1 ex pression was higher in Xflg22 treated plants than the other two peptide tr eatments (Figure 5-5A). In contrast to what was observed in MES pretreated grapefruit plants, azelaic acid pretreatment had an effect on the expressi on of some defense-associated genes. Regardless of the different fl g22 treatments, the azelaic acid pretreated plants showed induction of a set of genes including ED S1, RAR1, EDR1, EDS5, NPR1, NPR2, NPR3, PR1, RdRP and AZI1 (Figures 5-1B, 5-2B 5-3B, 5-4B and 5-5B).On the other hand, expression of PAL was reduced by azelaic acid (Figure 5-2B). Howe ver, the effect of azelaic acid appeared to be diminished by the Lflg22 challenge, as the expression levels of EDS1, RAR1, EDR1, SGT1, NPR1 NPR2, NPR3, NDR1 and AZI1 were lower than that of the control treat ment at 6, 24 and 72h (Figure 5-1B, 5-2B, 5-3B, 5-4B and 55B) and reduced expression of SGT1, RAR1 NPR1, NPR2, NPR3, EDS1 and EDR1 caused by Lflg22treatment was st atistically significant at the time points shown in Table 5-1. 47

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Since azelaic acid pretreatment was able to induce defense genes in all the treatments (water, flg22, Xflg22 and Lflg22), to investigat e only the effect of azelaic acid, gene expression data from all the mock inoculated plants (both MES and azelaic acid pretreated) was chosen for analysis. Co mpared to MES pretre atment, the set of genes induced by azelaic acid infiltration alone included EDS1, RAR1, EDR1, EDS5, PAL, NPR1, NPR2, NPR3, PR1, RdRP and AZ I1, it was notable that most of these genes were similarly induced in Xflg22 tr eated kumquat (Table 5-2). EDS1, RAR1, NPR2 and NPR3 showed significant induction at 6h after the mock inoculation and the induction of these genes lasted until 72h (Figur e 5-6). As was expected, azelaic acid treatment also elevated the expression of AZI1 and the gene induction appears to last as long as 120h (Figure 5-7). 48

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0 1 2 3 4 5 6 7 80h 6h C 6h F 6h L 6h X 24h C 24h F 24h L 24h X 72h C 72h F 72h L 72h X 120h C 120h F 120h L 120h XRQ EDR1 EDS1 NDR1 PBS1 RAR1 SGT1 MES A 0 1 2 3 4 5 6 7 80h 6h C 6h F 6h L 6h X 24h C 24h F 24h L 24h X 72h C 72h F 72h L 72h X 120h C 120h F 120h L 120h XRQ EDR1 EDS1 NDR1 PBS1 RAR1 SGT1 AzA B Figure 5-1. The effect of different flg22 on the expressi on of EDR1, EDS1, NDR1, PBS1, RAR1 and SGT1in grapefruit pret reated with MES or azelaic acid. Duncan grapefruit leaves were infiltra ted with 5mM MES buffer (A) as control or 1mM azelaic acid (B) two days prio r to flg22 challenges. Using the same infiltration method, 10uM flg22 (F), Lflg22 (L), Xflg22 (X) or water as control (C) were used to challenge the pretreated plants. Leaf samples were collected at 0, 6, 24, 72 and 120h after the flg22 inoculation. Gene expression was quantified by real time PCR and com parative CT analysis. The vertical axis indicates the relative quantitation (RQ), in which gene expression level in each sample is compared to the refe rence sample (0h) within the same pretreatment (azelaic acid or MES) 0h samples were collected right before flg22 infiltration. The mean and standard errors of three replicates is shown. 49

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0 2 4 6 8 10 12 14 160h 6h C 6h F 6h L 6h X 24h C 24h F 24h L 24h X 72h C 72h F 72h L 72h X 120h C 120h F 120h L 120h XRQ EDS5 ICS1 PAL MES A 0 2 4 6 8 10 12 14 160h 6h C 6h F 6h L 6h X 24h C 24h F 24h L 24h X 72h C 72h F 72h L 72h X 120h C 120h F 120h L 120h XRQ EDS5 ICS1 PAL AzA B Figure 5-2. The effect of di fferent flg22 on the expression of EDS5, ICS1 and PAL in grapefruit pretreated with M ES or azelaic acid. Duncan grapefruit leaves were infiltrated with 5mM MES buffer (A) as control or 1mM azelaic acid (B) two days prior to flg22 challenges. Using the same infiltration method, 10uM flg22 (F), Lflg22 (L), Xflg22 (X) or water as control (C) were used to challenge the pretreated plants. Leaf sample s were collected at 0, 6, 24, 72 and 120h after the flg22 inoculation. Gene expression was quantified by real time PCR and comparative CT analysis. The vertical ax is indicates the relative quantitation (RQ), in which gene expression level in each sample is compared to the reference sample (0h) within the same pr etreatment (azelaic acid or MES). 0h samples were collected right before flg22 infiltration. The mean and standard errors of three replicates is shown. 50

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0 1 2 3 4 5 6 7 8 9 100h 6h C 6h F 6h L 6h X 24h C 24h F 24h L 24h X 72h C 72h F 72h L 72h X 120h C 120h F 120h L 120h XRQ NPR1 NPR2 NPR3 MES A 0 1 2 3 4 5 6 7 8 9 100h 6h C 6h F 6h L 6h X 24h C 24h F 24h L 24h X 72h C 72h F 72h L 72h X 120h C 120h F 120h L 120h XRQ NPR1 NPR2 NPR3 AzA B Figure 5-3. The effect of di fferent flg22 on the expression of NPR1, NPR2 and NPR3 in grapefruit pretreated with MES or azelaic acid. Dunc an grapefruit leaves were infiltrated with 5mM MES buffer (A) as control or 1mM azelaic acid (B) two days prior to flg22 challenges. Using the same infiltration method, 10uM flg22 (F), Lflg22 (L), Xflg22 (X) or water as control (C) were used to challenge the pretreated plants. Leaf sample s were collected at 0, 6, 24, 72 and 120h after the flg22 inoculation. Gene expression was quantified by real time PCR and comparative CT analysis. The vertical ax is indicates the relative quantitation (RQ), in which gene expression level in each sample is compared to the reference sample (0h) within the same pr etreatment (azelaic acid or MES). 0h samples were collected right before flg22 infiltration. The mean and standard errors of three replicates is shown. 51

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0 10 20 30 40 50 600h 6h C 6h F 6h L 6h X 24h C 24h F 24h L 24h X 72h C 72h F 72h L 72h X 120h C 120h F 120h L 120h XRQ PR1 RdRP MES A 0 10 20 30 40 50 600h 6h C 6h F 6h L 6h X 24h C 24h F 24h L 24h X 72h C 72h F 72h L 72h X 120h C 120h F 120h L 120h XRQ PR1 RdRP AzA B Figure 5-4. The effect of di fferent flg22 on the expression of PR1 and RdRP in grapefruit pretreated with MES or azelaic acid. Duncan grapefruit leaves were infiltrated with 5mM MES buffer (A) as control or 1mM azelaic acid (B) two days prior to flg22 challenges. Using the same infilt ration method, 10uM flg22 (F), Lflg22 (L), Xflg22 (X) or water as control (C) were used to challenge the pretreated plants. Leaf samples were collected at 0, 6, 24, 72 and 120h after the flg22 inoculation. Gene expression was quantified by real time PCR and comparative CT analysis. The vertical ax is indicates the relative quantitation (RQ), in which gene expression level in each sample is compared to the reference sample (0h) within the same pr etreatment (azelaic acid or MES). 0h samples were collected right before flg22 infiltration. The mean and standard errors of three replicates is shown. 52

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0 10 20 30 40 50 600h 6h C 6h F 6h L 6h X 24h C 24h F 24h L 24h X 72h C 72h F 72h L 72h X 120h C 120h F 120h L 120h XRQ AZI1 MES*A Figure 5-5. The effect of di fferent flg22 on the expression of AZI1 in grapefruit pretreated with MES or azelaic acid. Duncan grapefruit leaves were infiltrated with 5mM MES buffer (A) as control or 1mM azelaic acid (B) two days prior to flg22 challenges. Using the same infilt ration method, 10uM flg22 (F), Lflg22 (L), Xflg22 (X) or water as control (C) were used to challenge the pretreated plants. Leaf samples were collected at 0, 6, 24, 72 and 120h after the flg22 inoculation. Gene expression was quantified by real time PCR and comparative CT analysis. The vertical ax is indicates the relative quantitation (RQ), in which gene expression level in each sample is compared to the reference sample (0h) within the same pr etreatment (azelaic acid or MES). 0h samples were collected right before flg22 infiltration. The mean and standard errors of three replicates is shown. 0 10 20 30 40 50 600h 6h C 6h F 6h L 6h X 24h C 24h F 24h L 24h X 72h C 72h F 72h L 72h X 120h C 120h F 120h L 120h XRQ AZI1 AzA* *B 53

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Grapefuit-AzA-Lflg22 Time Points EDS1* 6h, 24h, 72h RAR1* 6h SGT1* 6h, 24h EDR1* 24h PBS1 NDR1 EDS5 ICS1 PAL NPR1* 24h NPR2* 6h, 24h NPR3* 6h, 72h PR1 RdRP AZI1 Table 5-1. Summary of the effect of Lflg22 on gene expression after azelaic acid pretreatment in grapefruit. Genes i ndicated in blue displayed expression levels that were suppressed by Lflg22 treatment as compared with control. An asterisk means the alteration in expression was statistically significant compared with those of the control at the time points indicated in the right column. 54

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grapefruit-AzA kumquat-Xflg22 EDS1* EDS1* RAR1* RAR1* SGT1 SGT1* EDR1 EDR1 PBS1 PBS1 NDR1 NDR1* EDS5 EDS5 ICS1 ICS1 PAL PAL* NPR1 NPR1 NPR2* NPR2* NPR3* NPR3* PR1 PR1* RdRP RdRP* AZI1* AZI1 Table 5-2. Summary of the effect of azelaic acid in grapefruit and the effects of Xflg22 in kumquat on gene induction. Genes indica ted in red were upregulated by the treatment and genes in blue were suppressed. An asterisk indicates the expression alteration was statistically si gnificant compared to the control at one or more time points. AzA stands for azelaic acid. 55

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0 1 2 3 4 5 6 7 8 9 10 RQ RAR1 NPR2 NPR3 EDS1* **Figure 5-6. Effect of azelaic acid on the expression of RAR1, NPR2, NPR3 and EDS1 in grapefruit. Data was selected from Figur e 5-1and 5-3. RQ values from water infiltration treatment in gr apefruit pretreated 5mM MES buffer (C) as control or 1mM azelaic acid (A) were chosen fo r the comparisons. An asterisk means that the expression alteration was stat istically significant compared with the MES buffer pretreatment at the same time point. 0 5 10 15 20 25 0h6h24h72h120h RQ MES AzA*Figure 5-7. Effect of azelaic acid on the expression of AZI1 in grapefruit. Data was selected from Figure 5-5. RQ values from water infiltration treatment in grapefruit pretreated 5mM M ES buffer (MES) as control or 1mM azelaic acid (AzA) were chosen for the comparis ons. An asterisk means that the expression alternation is statistically significant compared with the MES buffer pretreatment at t he same time point. 56

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Discussion The results shown here indicate that neither flg22nor Xflg22induced the expression of defense-associated genes includ ing PTI-associated genes (EDR1, EDS1, NDR1, RAR1 and SGT1), SA-synthesis rela ted genes (EDS5 and ICS1) or SA induced PR genes (PR1 and RdRP) and their transcr iptional regulators (NPR1, NPR2 and NPR3) in the MES pretreated grapefruit plan ts (Figures5-1A, 5-2A, 5-3A and 5-4A). However, AZI1 was the except ion in that it was induced by Xflg22 but not flg22in grapefruit pretreated with MES (Figure 55A). A sequence comparison between flg22 and Xflg22 revealed that the tw o peptides had four amino acid differences (Table 5-3), suggesting that the alternations in residues could be responsible for the difference in PAMP recognition and response in grapefruit. Moreover, when the plants were pretreated with azelaic acid it appeared that flg22 was capable of inducing AZI1 expression (Figure 5-5B), im plying that the exog enous azelaic acid could complement the low AZI1 induction by flg22 and proper rec ognition of the residue alternations in flg22 may be key for the plants to produce endoge nous azelaic acid. However, even if the Xflg22 challenge could induce AZI1 and potent ially promote azelaic acid level as a defense response in grapefruit, it seemed that the induction of AZI1/azelaic acid was not enough to trigger the induction of expr ession of genes associated with plant defense and SA biosynthesis. We considered the concent ration of 1mMazelaic acid applied in our experiments to be high enough to induce a response [ 42], however it is possible that higher levels are necessary in citrus. 57

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Peptides Amino acid sequence flg22 Q R L S T G S R I N S A K D D A A G L Q I A Xflg22 Q R L S S G L R I N S A K D D A A G L A I S Lflg22 D R V S S G L R V S D A A D N A A Y W S I A Table 5-3. Sequence comparison s between the 22 amino acid peptides from flagellins of archetype (flg22), Xcc (Xflg22) and Candidatus Liberibacter asiaticus (Lflg22). The amino acid differences among the three peptides are shown in red. Amino acid differences in Lfgl22 from the other two sequences are indicated in green. For the huanglongbing causal agent Candidatus Liberibacter asiaticus, it is believed that this intracellular bacterial pathogen does not have a flagellar structure [ 9 114 ], even though we obtained a flagellin domai n containing proteins from its genomic database.TheLflg22 pept ide derived from these prot eins had more amino acid differences from the sequences from the ot her two species (Table 5-3). Compared with the mock and the other two peptide inoculat ions,Lflg22 treatment showed a suppressive effect on the expressions of the majority of the defenseassociated genes in grapefruit that were pretreated with azelaic acid but not in the plants pretreated with MES buffer (Figures5-1, 5-2, 5-3, 5-4, Table 1). G enome sequencing of the bac teria reveals that Candidatus Liberibacter asiaticus has a compar atively smaller genome than other Liberibacter species and does not have type II and type III secretion systems that are necessary for pathogenicity in other bacterial species, as a result of which Candidatus Liberibacter asiaticus is considered to in fect plants using an avoidance strategy [ 114 ]. The sequence variation of Lflg22 in the fl agellin domain containing protein may be a result of faster evolution of Ca. L. asiaticus and may contribute to avoiding the PAMP 58

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recognition [ 114 ], which renders the plants more vulnerable to this pathogenic bacterium. When comparing the effects of different flagellin peptide treatments in MES buffer pretreated plants, it was noticed that PAL was induced by Xflg22 and Lflg22 but not by flg22 and the induction event happened as late as 120h after treatment (Figure 5-2A). As discussed in the previous chapter, PAL is a key enzyme shared by the SA biosynthetic pathway and ligni n biosynthetic pathway [106 108 ] and the induction of PAL could be an indication of initiation of ei ther or both pathways in the response to a PAMP. Increased expression level of PAL by Xflg22 or Lflg22 suggested that this gene may be important for grapefru it recognition of pathogenic bacteria, even though the late induction could lead to insufficient defense level against the pat hogens. On the other hand, in azelaic acid pretreated grapefrui t, the effect of Xflg22 and Lflg22 on PAL expression was not obvious, and the average PAL expression level was lower than that of MES pretreated gra pefruit (Figure 5-2B, Table 5-2), suggesting that azelaic acid may have an antagonistic role to PAL involved pathways. Although demonstrated as an important signal for SAR priming, azelaic acid can confer both local and systemic resistance to virulent bacterial PmaDG3 in Arabidopsis when infiltrated into local leaves, and gene mutants in the SA-mediated defense pathways show compromised resistance after azelaic acid treatment [ 42]. Our results showed that local azelaic acid leaf infilt ration alone induced a set of defense-associated genes including EDS1, RAR1, EDR1, EDS5 PAL, NPR1, NPR2, NPR3, PR1, RdRP and AZI1in grapefruit (Table 5-2).Many of these genes such as EDS1, RAR1, EDS5, PAL, NPR1, PR1, RdRP and AZI1are a ssociated with the SA-mediated defense 59

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signaling, suggesting that azelaic acid app lication induced a similar local defense response in citrus as it did in Arabidopsis Additionally, comparison between the azelaic acid alone infiltrated grapef ruit and Xflg22 challenged kumquat indicated that azelaic acid was capable of triggering the expressions of a set of grapefruit genes that seemed necessary for the basal defense in kum quat (Table 5-2), and these genes had also been shown to be highly induced by Xcc in kumquat (V. Febres and A. Khalaf unpublished results), which was fu rther evidence for the mediat ion of defense by azelaic acid in grapefruit. However, whether the azelaic acid mediated defense response would be sufficient to protect grapefruit plants against pathogen invasion still needs to be confirmed by pathogen challenge assays. In Arabidopsis AZI1 induction was confirmed from 3h-48h after leaf infiltration with 1mM azelaic acid [ 42]. In our research, due to the diffe rent experimental design in which all the samples were collected after the azelaic acid/MES pretreatment (two days before), the time points when the samples were used for gene expression assays were actually 48 hours later. Hence the AZI1 induc tion by the azelaic acid treatment ranges from 0-120h in grapefruit is equival ent to 48h-168h (Figure 5-2) in the Arabidopsis experiment, indicating that azel aic acid can have an effect on the AZI1 expression for at least more than five days. Homologous to a lipid transfer protein family, AZI1 is believed to be the regulator or transporter of SAR priming signal in Arabidopsis [ 42]. In grapefruit, the induction of AZI1 by azelaic ac id implies that AZI1 can have a similar role during the SAR as in Arabidopsis in mediating azelaic acid primed resistance. 60

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CHAPTER 6 CONCLUSIONS The results presented here show tha t: 1) Xflg22 induced a number of PTIassociated genes (EDS1, SGT1, RAR1 and NDR1) and transcriptional activators (NPR2 and NPR3) in canker resistant kumquat but not in susceptible grapefruit, suggesting that PTI has a role in canker resistance; 2) Gr apefruit did not respond differently to flg22, Xflg22 or Lflg22, suggesting that is not capable of sensing this peptide, contributing to its susceptibility; 3) The app lication of Azelaic acid (1 mM) alone induced a defense response in susceptible grapefruit that wa s comparable to Xflg22 induced immunity in resistant kumquat. However, when grapefruit plants were pretreated with azelaic acid, Lflg22 showed a suppressive effect on the expression of defens e-associated genes whereas flg22 and Xflg22 did not; 4) AZI1 wa s induced by Xflg22 and azelaic acid in grapefruit, suggesting it is involved in the defens e response of citrus as is the case in model systems; and 5) It remains to be deter mined whether azelaic acid can be used effectively and practically in t he control of citrus canker. 61

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BIOGRAPHICAL SKETCH Qingchun Shi was born in Baoding, Hebei province, China. He graduated from Agricultural University of Hebei with a Bachelor of Agronomy, majoring in horticulture. During his undergraduate study, he worked in the project Research on Ya Pear and its Eco-geological and Chemical En vironment and finished his thesis titled in Dynamics of Soil Nutrients in Pear Orchar d in Spring. Due to his ex cellent performance in study and research, he was selected as graduate st udent candidate without an entrance exam in Agricultural University of Hebei, majori ng in pomology. During his graduate study, he was involved in the projects Technology and Extension of Improvem ent of Pear fruit quality, concentrating on tissue culture and Agrobacterium -mediated transformation of insect-resistant gene in pear cultivars. In par allel, he completed his masters thesis research Effects of Salicylic Acid on Respiratory Pathway of Postharvest Huang-guan Pear and obtained the Masters degree in 2009. In the fall of 2009, he was admitted to the plant molecular and cellular biology (PMCB) program in University of Florida. During the two years graduate study in PMCB, he did the masters research on Flg22Triggered Immunity and the Effe ct of Azelaic Acid on the Defense Response in Citrus. 72