to my parents who always understand and support me in
TRANSCRIPT
FLG22-TRIGGERED IMMUNITY AND THE EFFECT OF AZELAIC ACID ON THE DEFENSE RESPONSE IN CITRUS
By
QINGCHUN SHI
A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE
UNIVERSITY OF FLORIDA
2011
1
© 2011Qingchun Shi
2
To my parents who always understand and support me in chasing my dream
3
ACKNOWLEDGMENTS
I would like to express my heartfelt thanks to all the people who helped and
inspired me during my master’s study. I want to thank the chair of my academic
committee and advisor Dr. Gloria Moore for her financial support and dedicated
advisory during my entire study. Specially thank her for always being supportive even at
the most difficult time studying in this foreign country, without her trust I would not had
been able to achieve this milestone. Dr. Moore’s kindness and easygoing personality
will remain in me and guide me to treat other people like she does.
My gratitude also goes to my committee members Dr. Vicente Febres and Dr.
Jeffrey Jones. I am very delighted to interact with them and have learned tremendous
knowledge from them. Their insights to plant pathology explain to me what world-class
scientists would be like. Repetitive thanks given to Dr. Febres not only because his
tireless scientific guidance to me, working with him on a daily basis, he spent large
amount of precious time teaching me laboratory 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 Khalaf for her patient explanations and
encouragement, and thanks to Kimberly, Latanya, 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 understanding and support, and thanks to my wife for
being with me and taking care of my life. I love you all.
4
TABLE OF CONTENTS page
ACKNOWLEDGMENTS .................................................................................................. 4
LIST OF TABLES ............................................................................................................ 7
LIST OF FIGURES .......................................................................................................... 8
ABSTRACT ................................................................................................................... 10
CHAPTER
1 INTRODUCTION .................................................................................................... 12
2 LITERATURE REVIEW .......................................................................................... 15
The Plant Immune System ...................................................................................... 15 PAMPs and PAMPs-Triggered Immunity (PTI) ....................................................... 16 Defense Signals ...................................................................................................... 17 Genes Involved in Plant Immunity .......................................................................... 19
Enhanced Disease Susceptibility 1 (EDS1) and Nonrace-Specific Disease Resistance 1 (NDR1) .................................................................................... 19
Requires for Mla12 Resistance (RAR1) and Suppressor of G-Two Allele of Skp1 (SGT1) ................................................................................................. 20
Enhanced Disease Resistance (EDR1) ............................................................ 20 Enhanced Disease Susceptibility 5 (EDS5) ...................................................... 21 Pathogensis-Related Gene 1 (PR1) and Nonexpressor of PR Genes
(NPR1) .......................................................................................................... 21 Azelaic Acid Induced 1 (AZI1) .......................................................................... 23 RNA-Dependent RNA Polymerase (RdRP) ...................................................... 24 Isochorismate Synthase 1 (ICS1) and Phenylalanine Ammonia Lyase (PAL) .. 24 Avrpphb Susceptible 1 (PBS1) ......................................................................... 25
Rationale and Objectives ........................................................................................ 25
3 MATERIALS AND METHODS ................................................................................ 28
Plant Material .......................................................................................................... 28 Flagellin 22-Amino Acids Conserved Domain Peptides .......................................... 28 Challenge Kumquat and Grapefruit with Xflg22 Peptide ......................................... 29 Azelaic Acid Pretreatment and Challenges with flg22, Lflg22 and Xflg22
Peptides ............................................................................................................... 29 RNA Extraction and Purification .............................................................................. 30 cDNA Synthesis ...................................................................................................... 30 Gene Expression Analysis ...................................................................................... 31
5
4 COMPARISON OF THE RESPONSE TO XFLG22 BETWEEN canker SUSCEPTIBLE AND RESISTANT CITRUS TYPES .............................................. 33
Results .................................................................................................................... 33 Discussion .............................................................................................................. 41
5 EFFECT OF AZELAIC ACID AND DIFFERENT flg22 PEPTIDES ON THE DEFENSE RESPONSE OF GRAPEFRUIT ............................................................ 46
Results .................................................................................................................... 46 Discussion .............................................................................................................. 57
6 CONCLUSIONS ..................................................................................................... 61
LIST OF REFERENCES ............................................................................................... 62
BIOGRAPHICAL SKETCH ............................................................................................ 72
6
LIST OF TABLES
Table page 5-1 Summary of the effect of Lflg22 on gene expression after azelaic acid
pretreatment in grapefruit. .................................................................................. 54
5-2 Summary of the effect of azelaic acid in grapefruit and the effects of Xflg22 in kumquat on gene induction. ............................................................................... 55
5-3 Sequence comparisons between the 22 amino acid peptides for flg22, Xflg22 and Lflg22.. ......................................................................................................... 58
7
LIST OF FIGURES
Figure page 2-1 Simplified model of bacteria flagellin perception in Arabidopsis.. ....................... 17
2-2 Nucleotide sequence homology analysis of the Arabidopsis NPR1 protein family. ................................................................................................................. 23
2-3 A simplified salicylic acid (SA) synthetic pathway [99]. ....................................... 25
4-1 Effect of Xflg22 on expression of EDR1, EDS1, NDR1, PBS1, RAR1 and SGT1 in ‘Nagami’ kumquat (A) and ‘Duncan’ grapefruit (B). .............................. 36
4-2 Effect of Xflg22 on expression of EDS5, ICS1 and PAL in ‘Nagami’ kumquat (A) and ‘Duncan’ grapefruit (B). .......................................................................... 37
4-3 Effect of Xflg22 on expression of NPR1, NPR2 and NPR3 in ‘Nagami’ kumquat (A) and ‘Duncan’ grapefruit (B). ........................................................... 38
4-4 Effect of Xflg22 on expression of PR1 and RdRP in ‘Nagami’ kumquat (A) and ‘Duncan’ grapefruit (B). ................................................................................ 39
4-5 Effect of Xflg22 on expression of AZI1 in ‘Nagami’ kumquat (A) and ‘Duncan’ grapefruit (B). ...................................................................................................... 40
4-6 Phylogenetic tree of NPR1-like protein sequences from different species, including citrus using the Minimum Evolution method. ....................................... 44
5-1 The effect of different flg22 on the expression of EDR1, EDS1, NDR1, PBS1, RAR1 and SGT1 in grapefruit pretreated with MES or azelaic acid. ................... 49
5-2 The effect of different flg22 on the expression of EDS5, ICS1 and PAL in grapefruit pretreated with MES or azelaic acid. .................................................. 50
5-3 The effect of different flg22 on the expression of NPR1, NPR2 and NPR3 in grapefruit pretreated with MES or azelaic acid. .................................................. 51
5-4 The effect of different flg22 on the expression of PR1 and RdRP in grapefruit pretreated with MES or azelaic acid. .................................................................. 52
5-5 The effect of different flg22 on the expression of AZI1 in grapefruit pretreated with MES or azelaic acid. ................................................................................... 53
5-6 Effect of azelaic acid on the expression of RAR1, NPR2, NPR3 and EDS1 in grapefruit. ........................................................................................................... 56
5-7 Effect of azelaic acid on the expression of AZI1 in grapefruit. Data was selected from Figure 5-5. .................................................................................... 56
8
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
Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science
FLG22-TRIGGERED IMMUNITY AND THE EFFECT 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 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 µM) 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
10
11
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.
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 Florida’s agriculture
(http://www.florida-agriculture.com/agfacts.htm). Citrus production in Florida accounted
for 65 percent of the total citrus market in the United States during the 2009-2010
season, far more than that of California (31%), Texas and Arizona (4% combined)
(http://www.nass.usda.gov/Statistics_by_State/Florida/Publications/Citrus/fcs/2009-
10/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 also a good sources of health promoting nutrients
and phytochemicals such as vitamin C, antioxidants and fiber pectin [1,2].
As the largest grapefruit producing state in the United States, 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 young shoots, and fruit drop [3]. In an effort to eliminate canker
in Florida, the U.S. Department of Agriculture (USDA) in cooperation with Florida
Department of Agriculture and Consumer Service (FDACS) declared an eradication
program in 1994, mandating the removal of diseased and surrounding trees, including in
backyards. However, the hurricanes in 2004 exacerbated the spread of canker making
the eradication unrealistic. As a result, the USDA withdrew the eradication program and
12
imposed a statewide quarantine, restricting the out of state 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 restrict 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 indirect 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 affecting the citrus industry. Transmitted by the
vector psyllid (Diaphorina citri Kuwayama), bacterial infection can result in small and
misshapen fruits and largely reduced yield 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 latency, nonspecific and environment dependant
symptoms, the efficient detection and the management of huanglongbing 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, removal of infected trees and using disease-free
nursery materials [8,9].
13
14
Facing the threat of new diseases in Florida, 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 bacterial pathogens. One of the
supportive evidences for that is the 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-pathogen system, taking advantage of the latest
information on plant immunity and plant-pathogen interactions and combining with the
recent availability of a citrus genome database, we intend to explore mechanisms of
citrus susceptibility and resistance to its bacterial diseases and seek alternative
effective management methods.
CHAPTER 2 LITERATURE REVIEW
The Plant Immune System
Plants use two different pathways of active defense or immunity against potential
pathogens to prevent the establishment 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 defense and it deters colonization by a broad
range of microbes [15]. Pathogens that successfully overcome 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 nucleotide binding (NB) and leucine rich repeat (LRR) domains that
recognize pathogen virulence factors (effectors), resulting in the initiation of R-mediated
resistance.ETI is typically accompanied by the hypersensitive response (HR) [12,16], a
programmed cell death process effectively restraining 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 transduce signals to distal plant tissues
triggering systemic resistance to protect 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 infection site leads to elevated
systemic protection against virulent bacteria [19].
15
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 flagellin and the amino-terminus of the elongation
factor Tu (EF-Tu) are required for the 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 (FLAGELLIN 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 (aspartic acid) on the flagellin protein [27]. Arabidopsis
plants treated with flg22 produce a microRNA that degrades mRNAs of several auxin
signaling components, halting the growth of phytopathogenic bacteria [28]. Antagonistic
to auxin signaling, SA accumulates during flg22 infection and mutation of certain SA
signaling genes leads to dampened PTI, indicating 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
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 the 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-related genes implies that SA may be the
chemical involved in SAR signal transduction [36,37]. However, pathogen infected
tobacco rootstocks engineered with a bacterial 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
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 protein 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 transfer protein family protein, was induced by azelaic acid
and mutation of this gene resulted in attenuated SAR. Taken together, mediated
through AZI1, azelaic acid was an important signal involved in priming the plant defense
[42].
The elevation of jasmonic acid (JA) content and heightened expression 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
candidate signal for initiating SAR [43]. However, instead of using low inoculum
concentration of pathogen, a parallel 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 (EDS1) and Nonrace-Specific Disease Resistance 1 (NDR1)
Mutant eds1Arabidopsisplants 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 (NB-
LRR) domains [48]. EDS1 functions mainly by association with proteins encoded by
phytoalexin deficient4 (PAD4) or senescence-associated gene101 (SAG101), forming a
protein complex coordinating signaling events delivered by ETI and PTI [49,50,51]. It is
believed that regulation by the EDS1 complex occurs immediately downstream of R-
protein 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 regulator 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 proposed 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 regulation was shown to be upstream of
19
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) and Suppressor of G-TwoAlleleofSkp1 (SGT1)
With the concept that R protein-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 specifically 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, R-
mediated 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 accumulated a higher bacterial population than that of wild
type inoculated plants and an Arabidopsis RAR1 mutation lead to subdued basal
defense against both compatible and non-host pathogens [62]. Similarly, in soybean
RAR1 and SGT1 were found to be converging signaling components 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
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 activity, 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 attenuated 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 EDS5regulated the defense response upstream of SA synthesis and SAR
onset [75]. What is more, eds1, pad4 and ndr1 mutants exhibit significantly decreased
EDS5 transcription levels upon pathogen inoculation, indicating that EDS5 functions
downstream of these 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 SAR and that PR1 is the most highly induced
21
PR gene after a plant has been inoculated with SAR-inducing pathogens or treated with
SA. However, the function of the PR1 protein is still unclear [76,77].
NPR1 plays a key role as a transcriptional activator for the PR1 gene [78]. The
Arabidopsis npr1 mutant is not able to respond to the SAR eliciting chemical SA and
fails to induce multiple PR genes including PR1 [79,80,81]. Cytoplasmic NPR1 exists in
anoligomeric form and, upon infection, SA-mediated cellular redox changes cause the
NPR1 oligomers to break into monomers, which are then translocated into the nucleus
[82]. Nuclear localization of NPR1 is necessary 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 suggest that functional NPR1
binds to the TGA family of transcription factors, which are regulators of the transcription
of PR1 by binding to its SA-responsive element in the promoter region [78,83,85].
Moreover, NPR1’s 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 constitutive expression of the
PR gene; When the plant is challenged with a pathogen, phosphorylation of NPR1 at
the Ser11/Ser15 residues also led to proteolysis 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 these 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 similarity with NPR1 and these proteins
22
also bind to a TGA of subfamily transcription factor. However, the expression of BOP1
and BOP2 is tissue specific and the gene products regulate plant growth asymmetry in
Arabidopsis [88,89].
Figure 2-2. Nucleotide sequence 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 provides 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 phospholipid 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
SAR signaling to systemic tissues, given the evidence that azi1mutants showed normal
local resistance but had a compromised systemic resistance [42].
RNA-Dependent RNA Polymerase (RdRP)
In the defense against RNA viruses, plants 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 double-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 chorismate have been shown to be the most
important one during pathogen-induced 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 enzyme for defense-associated SA synthesis
[101,102]. On the other hand, a biochemical study using isotope feeding showed that
PAL is involved in SA synthesis via cinnamate intermediates and that inhibiting PAL
activity decreases SA accumulation induced by a pathogen [105,106]. Moreover, PAL
24
has been shown to be an important protein catalyzing the first enzymatic reaction in the
phenylpropanoid biosynthetic pathway (lignin biosynthesis) [107]. Lignin is considered a
necessary physical barrier against 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 phenylalanine ammonia lyase, ICS1 is isochorismate synthase 1 and IPL is isochorismate pyruvate lyase.
Avrpphb Susceptible 1 (PBS1)
PBS1 is an important component for 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 library analysis and microarray
25
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 transcription factors and receptors, suggesting that
canker challenge could trigger an active defense response and certain signaling events
in kumquat [113]. Preliminary comparisons between canker inoculated grapefruit and
kumquat indicated that defense-associated 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, provides 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 whether Xflg22 triggered
PTI in citrus, and to compare defense-associated gene expression changes in response
to Xflg22 over time between susceptible grapefruit and resistant kumquat. This may
provide a better understanding of the role PTI 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
microcopy observation and genome sequence information [9,114]. However, flagellin
domain-containing proteins (GI: 254040208 and GI: 254780513) have been identified in
the Candidatus Liberibacter asiaticus genome and they also contain the conserved 22
amino acid domain (Lflg22) near the N terminus with some variations. Our second
objective was to compare the response of grapefruit to Xflg22, Lflg22 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 virulent bacteria. In addition, 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 Xflg22 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 compound could enhance the defense
response of susceptible grapefruit. Moreover, comparing the response of defense-
associated 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
CHAPTER 3 MATERIALS AND METHODS
Plant Material
The plants used were greenhouse maintained ‘Nagami’ kumquat (Fortunella
margarita (Lour.) Swing. ) (Floyd and Associates, 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 experiments. 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 were obtained from GenScript USA Inc.
The archetype 22-amino acid peptide (flg22, QRLSTGSRINSAKDDAAGLQIA) was in
stock from the Genscript USA Inc. and the sequence was referenced 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 conserved domains from Xcc (Xflg22:
QRLSSGLRINSAKDDAAGLAIS), and Candidatus Liberibacter asiaticus(LFgl22:
DRVSSGLRVSDAADNAAYWSIA) were then synthesized.
28
Challenge Kumquat and Grapefruit 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 distilled water to a final concentration of 10 µM. This
concentration was chosen based on previous work by Zipfel et al. [25]. The Xflg22
challenge was performed by infiltrating the peptide solution into 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 from different plants were collected for each
treatment, genotype and time point. The samples (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 plants (a total of 24)
were divided into two populations of 12 plants 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 reach maximum solubility. The azelaic acid
solution final concentration used was 1mM [42]. For the pretreatments, 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
challenged with a10 µM 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 collected immediately before any infiltration.
The tissue was immediately frozen in liquid nitrogen and stored in the freezer at -
80°Cprior to RNA extraction and gene expression analysis.
RNA Extraction and Purification
For each sample, RNA was extracted using TriZol reagent following the
manufacturer’s instructions (Invitrogen, CA). Each sample consisted of two different
leaves from the same plant. The RNA was subsequently treated with DNase followed by
a cleanup protocol using the RNeasy Plant Mini Kit (QIAGEN Sciences, MD) to
eliminate any DNA and protein contamination. The concentration of RNA was
determined with a NanoDrop 2000c spectrophotometer (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 µg) was used for each 20 µL cDNA synthesis reaction. The
20 µL reaction mixture included 2 µL of 50 µM Random Decamers (Ambion Inc, Applied
Biosystem, CA), 2 µL of 10 µM dNTPs, 2 µL of 10X First Strand Buffer (Ambion Inc,
Applied Biosystem, CA), 1µLof M-MLV Reverse Transcriptase (100U/µL, Ambion Inc,
Applied Biosystem, CA) and 1 µL of RNase Inhibitor (40U/µL, Ambion Inc, Applied
Biosystem, CA). The remainder of the volume was completed with RNase-free water.
The RNA, Random Decamers, dNTPs and water mixture was incubated at 80°C in a
water bath for 2-3 minutes and placed on ice for 3 minutes before adding the First
Strand Buffer, RNase Inhibitor and Reverse Transcriptase. The 20 µL reaction mixture
30
was then subjected to reverse transcription using the thermal cycle at 42°C for 1h and
92°Cfor 10 minutes (PTC-100 Programmable Thermal Controller, MJ Research Inc.
Canada). The cDNA samples were stored at -20°C prior to real-time PCR.
Gene Expression Analysis
Gene expression levels were determined by quantitative real-time PCR using the
StepOnePlus Real-Time PCR system (Applied Biosystems, CA). A Fast 96-well
Reaction Plate (0.1 mL) (MicroAmp®, Applied Biosystems) was used for the reactions
and each well was used to perform the 20 µL expression assay of one gene from each
sample. The cDNA was diluted to a final concentration of 50 ng/µL and gene
amplification was performed from 2 µL of the diluted cDNA. Each reaction mixture was
composed of 2 µL of 50ng/µL cDNA, 10µL of TaqMan Fast Universal PCR Master Mix
(2X) (Applied Biosystems, CA), 1 µL of TaqMan probe and primer Assay Mix (20X)
(Applied Biosystems, CA) and 7 µL of RNase-free water. The Assay Mix was a
combination of specific probes and forward 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 experiment was designed using the StepOne
software v2.1 (Applied Biosystems, CA), in which the experiment type was set as
comparative CT (ΔΔCT), 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 95°C for 1 sec and 60°C for 20 sec. For each 96-well plate,
31
the amplification plots of every gene were manually checked for the correct threshold
levels and proper base line starting and ending points. For the comparative CT analysis,
the 0 h samples were selected as reference samples and the calculated relative
quantitation (RQ) values were exported to Microsoft 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 Student’s t test (SAS Institute Inc. NC). All
treatments for each experiment (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
CHAPTER 4
COMPARISON OF THE RESPONSE TO XFLG22 BETWEEN CANKER SUSCEPTIBLE AND RESISTANT 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 because 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) (Figure 4-2 and 4-5), PTI/ETI transcriptional
regulation (NPR1, NPR2 and NPR3) (Figure 4-3) and PR genes (PR1 and RdRp)
(Figure 4-4).
Among the PTI/ETI-associated genes investigated, EDS1, NDR1, RAR1 and
SGT1 displayed significant elevated expression 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 significantly different between the Xflg22 and water
treatments at all the time points except 6 h (Figure 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
compared with the mock inoculated controls at the same time points, even though in
grapefruit PAL showed higher expression 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 associated 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 significantly 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, Xflg22 treatment in grapefruit did not trigger
higher expressions of NPR1, NPR2 or NPR3 than the control treatment, even though
34
the induction of these genes was found in control plants at 6h in respect to 0h, which
may be the result of infiltration procedure (Figure 4-3B).
PR1 is considered as the marker gene for SAR because its expression correlates
with the resistance response against pathogens and SA accumulation in systemic
tissues [76,77]. Even though investigating SAR was not our purpose, since the SA-
mediated defense response is also an essential local defense mechanism we measured
the expression of PR1 (Figure 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 pattern 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
0
1
2
3
4
5
6
7
8
0h 6h C 6h X 24h C 24h X 72h C 72h X 120h C120h X
RQ
EDR1EDS1NDR1PBS1RAR1SGT1
A
* **
**
*
**
**
0
1
2
3
4
5
6
7
8
0h 6h C 6h X 24h C 24h X 72h C 72h X 120h C120h X
RQ
EDR1EDS1NDR1PBS1RAR1SGT1
B
Figure 4-1. Effect of Xflg22 on expression of EDR1, EDS1, NDR1, PBS1, RAR1 and SGT1 in ‘Nagami’ kumquat (A) and ‘Duncan’ grapefruit (B). Leaves were infiltrated with 10µM 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 and treatment, in which ‘C’ stands for control (water) and ‘X’ for Xflg22 treatment. Leaves for 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.
36
0
1
2
3
4
5
6
7
8
0h 6h C 6h X 24h C 24h X 72h C 72h X 120h C120h X
RQ EDS5ICS1PAL
A
**
0
1
2
3
4
5
6
7
8
0h 6h C 6h X 24h C 24h X 72h C 72h X 120h C120h X
RQ EDS5ICS1PAL
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 10µM 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 and treatment, in which ‘C’ stands for control (water) and ‘X’ for Xflg22 treatment. Leaves for 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.
37
0
1
2
3
4
5
6
0h 6h C 6h X 24h C 24h X 72h C 72h X 120h C120h X
RQ NPR1NPR2NPR3
A*
*
*
**
0
1
2
3
4
5
6
0h 6h C 6h X 24h C 24h X 72h C 72h X 120h C120h X
RQ NPR1NPR2NPR3
B
Figure 4-3. Effect of Xflg22 on expression of NPR1, NPR2 and NPR3 in ‘Nagami’ kumquat (A) and ‘Duncan’ grapefruit (B). Leaves were infiltrated with 10µM 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 and treatment, in which ‘C’ stands for control (water) and ‘X’ for Xflg22 treatment. Leaves for 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.
38
0123456789
0h 6h C 6h X 24h C 24h X 72h C 72h X 120h C120h X
RQ PR1RdRP
A
** *
*
**
0123456789
0h 6h C 6h X 24h C 24h X 72h C 72h X 120h C120h X
RQ PR1RdRP
*
*
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 10µM 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 and treatment, in which ‘C’ stands for control (water) and ‘X’ for Xflg22 treatment. Leaves for 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.
39
0
2
4
6
8
10
12
0h 6h C 6h X 24h C 24h X 72h C 72h X 120h 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 infiltrated with 10µM 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 and treatment, in which ‘C’ stands for control (water) and ‘X’ for Xflg22 treatment. Leaves for 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
0h 6h C 6h X 24h C 24h X 72h C 72h X120h C120h X
RQ AZI1
*
B
40
Discussion
The results in this study suggest that the Xflg22 challenge triggers the expression
of a set of defense-associated genes in canker resistant kumquat but not in the
susceptible grapefruit. The defense-associated genes significantly induced in kumquat
as a result of Xflg22 treatment were EDS1, 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-mediated 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 including NDR1, EDS1 like gene and NPR1 [118]. In
our research, the induction of PTI-associated genes by Xflg22 in kumquat was in
agreement with previous research 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. Alternatively, it is also possible 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 transiently induced prior to 6h after Xflg22
infiltration or late induced after 120h. However, since grapefruit is highly susceptible to
41
canker, the most likely scenario is that the attenuated immune 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 pathogen-triggered 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 believed to occur via two pathways: the PAL
pathway and the ICS1 pathway (Figure 2-3). There are reports from different plant-
pathogen 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 4-
2A). For canker resistant kumquat, the PAL catalyzed SA biosynthetic 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 incompatible interactions [106,108]. Hence the
induction of kumquat’s 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 synthesis priming gene AZI1 were well
coordinated with ICS1 induction (Figures 4-2B, 4-3B and 4-4B), with all three genes
42
upregulated at 24 and 72h after Xflg22 infiltration, suggesting that the SA regulating PR-
1 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 was 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 regulated 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
(from Citrus paradisi), which share high sequence similarity at the protein level with
AtNPR3 and AtNPR4, were significantly induced 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 downregulated in kumquat treated with Xflg22 however
in grapefruit this gene was induced by the same treatment (Figure 4-4). These results
suggest that CpNPR2 and CpNPR3 could be involved 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 protein sequences from different species, including citrus using the Minimum Evolution method. The percentage of replicate trees in which the associated 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 the number of amino acid substitutions per site. All positions containing gaps and missing data 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
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 grapefruit and these induced genes in kumquat were believed to
be associated with the observed resistance (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 that the recognition and response to Xcc flagellin was
necessary for citrus plants to successfully defend themselves against the pathogenic
bacterium. By comparing kumquat genes whose induction was triggered by Xflg22 with
those induced by canker inoculation, it was 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 the Xflg22 initiated PTI shared similar defense signaling
pathways with canker resistance response and that the flagellin triggered PTI probably
contributes to the total resistance to canker observed in kumquat.
CHAPTER 5 EFFECT OF AZELAIC ACID AND DIFFERENT FLG22 PEPTIDES ON THE DEFENSE
RESPONSE OF GRAPEFRUIT
Results
Grapefruit plants were pretreated with 1mMazelaic acid or 5 mM MES buffer (as a
negative control) two days prior to being infiltrated with flg22 from three different
organisms or water as a control. Samples were 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 buffer 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 various 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 defense-associated 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 RdRP (Figure 5-4A), even though the SA
biosynthesis gene PAL was induced at 120h after 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 procedure or some other
46
environment effect (Figure 5-4A). Noticeably, Xflg22 treatment elevated the expression
of AZI1 in MES pretreated grapefruit 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 Xflg22, 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 inoculation did (Figures 5-1A, 5-2A, 5-3A and 5-
4A), except that PAL was induced by Lflg22 and Xflg22 but not by flg22 at 120h after
the treatment (Figure 5-2A), and AZI1 expression was higher in Xflg22 treated plants
than the other two peptide treatments (Figure 5-5A).
In contrast to what was observed in MES pretreated grapefruit plants, azelaic acid
pretreatment had an effect on the expression of some defense-associated genes.
Regardless of the different flg22 treatments, the azelaic acid pretreated plants showed
induction of a set of genes including EDS1, 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). However, 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 treatment at 6, 24 and 72h (Figure 5-1B, 5-2B, 5-3B, 5-4B and 5-
5B) and reduced expression of SGT1, RAR1, NPR1, NPR2, NPR3, EDS1 and EDR1
caused by Lflg22treatment was statistically significant at the time points shown in Table
5-1.
47
Since azelaic acid pretreatment was able to induce defense genes in all the
treatments (water, flg22, Xflg22 and Lflg22), to investigate 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. Compared to MES pretreatment, the set of
genes induced by azelaic acid infiltration alone included EDS1, RAR1, EDR1, EDS5,
PAL, NPR1, NPR2, NPR3, PR1, RdRP and AZI1, it was notable that most of these
genes were similarly induced in Xflg22 treated 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 (Figure 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
012345678
0h6h
C6h
F6h
L6h
X24
h C
24h
F24
h L
24h
X72
h C
72h
F72
h L
72h
X12
0h C
120h
F12
0h L
120h
X
RQEDR1EDS1NDR1PBS1RAR1SGT1
MESA
012345678
0h6h
C6h
F6h
L6h
X24
h C
24h
F24
h L
24h
X72
h C
72h
F72
h L
72h
X12
0h C
120h
F12
0h L
120h
X
RQ
EDR1EDS1NDR1PBS1RAR1SGT1
AzAB
Figure 5-1. The effect of different flg22 on the expression of EDR1, EDS1, NDR1, PBS1, RAR1 and SGT1in 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 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 comparative CT analysis. The vertical axis indicates the relative quantitation (RQ), in which gene expression level in each sample is compared to the reference 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
02468
10121416
0h6h
C6h
F6h
L6h
X24
h C
24h
F24
h L
24h
X72
h C
72h
F72
h L
72h
X12
0h C
120h
F12
0h L
120h
X
RQ EDS5ICS1PAL
MESA
02468
10121416
0h6h
C6h
F6h
L6h
X24
h C
24h
F24
h L
24h
X72
h C
72h
F72
h L
72h
X12
0h C
120h
F12
0h L
120h
X
RQ EDS5ICS1PAL
AzAB
Figure 5-2. The effect of different flg22 on the expression of EDS5, ICS1 and PAL 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 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 comparative CT analysis. The vertical axis indicates the relative quantitation (RQ), in which gene expression level in each sample is compared to the reference 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.
50
0123456789
10
0h6h
C6h
F6h
L6h
X24
h C
24h
F24
h L
24h
X72
h C
72h
F72
h L
72h
X12
0h C
120h
F12
0h L
120h
X
RQ NPR1NPR2NPR3
MESA
0123456789
10
0h6h
C6h
F6h
L6h
X24
h C
24h
F24
h L
24h
X72
h C
72h
F72
h L
72h
X12
0h C
120h
F12
0h L
120h
X
RQ NPR1NPR2NPR3
AzAB
Figure 5-3. The effect of different flg22 on the expression of NPR1, NPR2 and NPR3 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 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 comparative CT analysis. The vertical axis indicates the relative quantitation (RQ), in which gene expression level in each sample is compared to the reference 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.
51
0
10
20
30
40
50
60
0h6h
C6h
F6h
L6h
X24
h C
24h
F24
h L
24h
X72
h C
72h
F72
h L
72h
X12
0h C
120h
F12
0h L
120h
X
RQ PR1RdRP
MESA
0
10
20
30
40
50
60
0h6h
C6h
F6h
L6h
X24
h C
24h
F24
h L
24h
X72
h C
72h
F72
h L
72h
X12
0h C
120h
F12
0h L
120h
X
RQPR1RdRP
AzAB
Figure 5-4. The effect of different 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 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 comparative CT analysis. The vertical axis indicates the relative quantitation (RQ), in which gene expression level in each sample is compared to the reference 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.
52
0
10
20
30
40
50
60
0h6h
C6h
F6h
L6h
X24
h C
24h
F24
h L
24h
X72
h C
72h
F72
h L
72h
X12
0h C
120h
F12
0h L
120h
X
RQ AZI1
MES
*
A
Figure 5-5. The effect of different 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 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 comparative CT analysis. The vertical axis indicates the relative quantitation (RQ), in which gene expression level in each sample is compared to the reference 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.
0
10
20
30
40
50
60
0h6h
C6h
F6h
L6h
X24
h C
24h
F24
h L
24h
X72
h C
72h
F72
h L
72h
X12
0h C
120h
F12
0h L
120h
X
RQ AZI1
AzA*
*
B
53
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 indicated 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
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 indicated in red were upregulated by the treatment and genes in blue were suppressed. An asterisk indicates the expression alteration was statistically significant compared to the control at one or more time points. AzA stands for azelaic acid.
55
0123456789
10
RQRAR1NPR2NPR3EDS1
*
*
*
*
Figure 5-6. Effect of azelaic acid on the expression of RAR1, NPR2, NPR3 and EDS1 in grapefruit. Data was selected from Figure 5-1and 5-3. RQ values from water infiltration treatment in grapefruit pretreated 5mM MES buffer (C) as control or 1mM azelaic acid (A) were chosen for the comparisons. An asterisk means that the expression alteration was statistically significant compared with the MES buffer pretreatment at the same time point.
0
5
10
15
20
25
0h 6h 24h 72h 120h
RQ MESAzA
*
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 MES buffer (MES) as control or 1mM azelaic acid (AzA) were chosen for the comparisons. An asterisk means that the expression alternation is statistically significant compared with the MES buffer pretreatment at the same time point.
56
Discussion
The results shown here indicate that neither flg22nor Xflg22induced the
expression of defense-associated genes including PTI-associated genes (EDR1, EDS1,
NDR1, RAR1 and SGT1), SA-synthesis related genes (EDS5 and ICS1) or SA induced
PR genes (PR1 and RdRP) and their transcriptional regulators (NPR1, NPR2 and
NPR3) in the MES pretreated grapefruit plants (Figures5-1A, 5-2A, 5-3A and 5-4A).
However, AZI1 was the exception in that it was induced by Xflg22 but not flg22in
grapefruit pretreated with MES (Figure 5-5A). A sequence comparison between flg22
and Xflg22 revealed that the two 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), implying that the exogenous azelaic acid could complement
the low AZI1 induction by flg22 and proper recognition of the residue alternations in
flg22 may be key for the plants to produce endogenous azelaic acid. However, even if
the Xflg22 challenge could induce AZI1 and potentially 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 expression of genes associated with plant defense
and SA biosynthesis. We considered the concentration 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
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 comparisons 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 domain containing proteins from its
genomic database.TheLflg22 peptide derived from these proteins had more amino acid
differences from the sequences from the other two species (Table 5-3). Compared with
the mock and the other two peptide inoculations,Lflg22 treatment showed a suppressive
effect on the expressions of the majority of the defense-associated 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). Genome sequencing of the bacteria reveals that
Candidatus Liberibacter asiaticus has a comparatively 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 infect plants using an ‘avoidance strategy’ [114].
The sequence variation of Lflg22 in the flagellin domain containing protein may be a
result of faster evolution of Ca. L. asiaticus and may contribute to avoiding the PAMP
58
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 lignin biosynthetic pathway [106,108] and the induction of
PAL could be an indication of initiation of either 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 grapefruit recognition of pathogenic bacteria, even though the late
induction could lead to insufficient defense level against the pathogens. On the other
hand, in azelaic acid pretreated grapefruit, 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 grapefruit (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 infiltration 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 associated with the SA-mediated defense
59
signaling, suggesting that azelaic acid application induced a similar local defense
response in citrus as it did in Arabidopsis. Additionally, comparison between the azelaic
acid alone infiltrated grapefruit 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 kumquat (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 further evidence for the mediation 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 different 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 induction by the azelaic acid treatment ranges
from 0-120h in grapefruit is equivalent to 48h-168h (Figure 5-2) in the Arabidopsis
experiment, indicating that azelaic 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 acid implies that AZI1 can have a similar role
during the SAR as in Arabidopsis in mediating azelaic acid primed resistance.
60
CHAPTER 6 CONCLUSIONS
The results presented here show that: 1) Xflg22 induced a number of PTI-
associated 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) Grapefruit did not respond differently to flg22,
Xflg22 or Lflg22, suggesting that is not capable of sensing this peptide, contributing to
its susceptibility; 3) The application of Azelaic acid (1 mM) alone induced a defense
response in susceptible grapefruit that was 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 defense-associated genes
whereas flg22 and Xflg22 did not; 4) AZI1 was induced by Xflg22 and azelaic acid in
grapefruit, suggesting it is involved in the defense response of citrus as is the case in
model systems; and 5) It remains to be determined whether azelaic acid can be used
effectively and practically in the control of citrus canker.
61
LIST OF REFERENCES
1. Lee HS (2000) Objective Measurement of Red Grapefruit Juice Color. Journal of Agricultural and Food Chemistry 48: 1507-1511.
2. Cerda JJ, Robbins FL, Burgin CW, Baumgartner TG, Rice RW (1988) The effects of grapefruit pectin on patients at risk for coronary heart disease without altering diet or lifestyle. Clinical Cardiology 11: 589-594.
3. Dewdney MM, Graham JH (2011) 2011 Florida Citrus Pest Management Guide: Citrus Canker. Gainesville: EDIS. pp. 6.
4. Gottwald TR, Timmer LW (1995) The efficacy of windbreaks in reducing the spread of citrus canker caused by Xanthomonas campestris pv citri. Tropical Agriculture 72: 194-201.
5. Graham JH, Leite RP (2004) Lack of Control of Citrus Canker by Induced Systemic Resistance Compounds. Plant Disease 88: 745-750.
6. Leite RP, Mohan SK (1990) Integrated management of the citrus bacterial canker disease caused by Xanthomonas campestris pv. citri in the State of Paraná, Brazil. Crop Protection 9: 3-7.
7. Manjunath KL, Halbert SE, Ramadugu C, Webb S, Lee RF (2008) Detection of ‘Candidatus Liberibacter asiaticus’ in Diaphorina citri and Its Importance in the Management of Citrus Huanglongbing in Florida. Phytopathology 98: 387-396.
8. Graça JV, Korsten L (2004) Citrus Huanglongbing: Review, Present status and Future Strategies. In: Naqvi SAMH, editor. Diseases of Fruits and Vegetables Volume I: Springer Netherlands. pp. 229-245.
9. Bove JM (2006) Huanglongbing: A destructive, newly-emerging, century-old disease of citrus. Journal of Plant Pathology 88: 7-37.
10. Nishimura MT, Dangl JL (2010) Arabidopsis and the plant immune system. The Plant Journal 61: 1053-1066.
11. Chisholm ST, Coaker G, Day B, Staskawicz BJ (2006) Host-Microbe Interactions: Shaping the Evolution of the Plant Immune Response. Cell 124: 803-814.
12. Jones JD, Dangl JL (2006) The plant immune system. Nature 444: 323-329.
13. Zipfel C, Felix G (2005) Plants and animals: a different taste for microbes? Curr Opin Plant Biol 8: 353-360.
14. Nurnberger T, Brunner F, Kemmerling B, Piater L (2004) Innate immunity in plants and animals: striking similarities and obvious differences. Immunol Rev 198: 249-266.
62
15. Boller T (1995) Chemoperception of microbial signals in plant-cells. Annual Review of Plant Physiology and Plant Molecular Biology 46: 189-214.
16. Dangl JL, Jones JD (2001) Plant pathogens and integrated defence responses to infection. Nature 411: 826-833.
17. Ryals JA, Neuenschwander UH, Willits MG, Molina A, Steiner HY, et al. (1996) Systemic acquired resistance. Plant Cell 8: 1809-1819.
18. Hunt MD, Ryals JA (1996) Systemic acquired resistance signal transduction. Critical Reviews in Plant Sciences 15: 583-606.
19. Cameron RK, Dixon RA, Lamb CJ (1994) Biologically Induced Systemic Acquired-Resistance in Arabidopsis-Thaliana. Plant Journal 5: 715-725.
20. Felix G, Duran JD, Volko S, Boller T (1999) Plants have a sensitive perception system for the most conserved domain of bacterial flagellin. Plant Journal 18: 265-276.
21. Kunze G, Zipfel C, Robatzek S, Niehaus K, Boller T, et al. (2004) The N terminus of bacterial elongation factor Tu elicits innate immunity in Arabidopsis plants. Plant Cell 16: 3496-3507.
22. Felix G, Regenass M, Boller T (1993) Specific Perception of Subnanomolar Concentrations of Chitin Fragments by Tomato Cells - Induction of Extracellular Alkalinization, Changes in Protein-Phosphorylation, and Establishment of a Refractory State. Plant Journal 4: 307-316.
23. Granado J, Felix G, Boller T (1995) Perception of Fungal Sterols in Plants - Subnanomolar Concentrations of Ergosterol Elicit Extracellular Alkalinization in Tomato Cells. Plant Physiology 107: 485-490.
24. Asai T, Tena G, Plotnikova J, Willmann MR, Chiu WL, et al. (2002) MAP kinase signalling cascade in Arabidopsis innate immunity. Nature 415: 977-983.
25. Zipfel C, Robatzek S, Navarro L, Oakeley EJ, Jones JDG, et al. (2004) Bacterial disease resistance in Arabidopsis through flagellin perception. Nature 428: 764-767.
26. Gomez-Gomez L, Boller T (2000) FLS2: An LRR receptor-like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis. Molecular Cell 5: 1003-1011.
27. Naito K, Taguchi F, Suzuki T, Inagaki Y, Toyoda K, et al. (2008) Amino Acid Sequence of Bacterial Microbe-Associated Molecular Pattern flg22 Is Required for Virulence. Molecular Plant-Microbe Interactions 21: 1165-1174.
63
28. Navarro L, Dunoyer P, Jay F, Arnold B, Dharmasiri N, et al. (2006) A plant miRNA contributes to antibacterial resistance by repressing auxin signaling. Science 312: 436-439.
29. Tsuda K, Sato M, Glazebrook J, Cohen JD, Katagiri F (2008) Interplay between MAMP-triggered and SA-mediated defense responses. Plant Journal 53: 763-775.
30. Wang D, Pajerowska-Mukhtar K, Culler AH, Dong XN (2007) Salicylic acid inhibits pathogen growth in plants through repression of the auxin signaling pathway. Current Biology 17: 1784-1790.
31. Ma W, Berkowitz GA (2007) The grateful dead: calcium and cell death in plant innate immunity. Cellular Microbiology 9: 2571-2585.
32. Bestwick CS, Bennett MH, Mansfield JW (1995) Hrp Mutant of Pseudomonas syringae pv phaseolicola Induces Cell Wall Alterations but Not Membrane Damage Leading to the Hypersensitive Reaction in Lettuce. Plant Physiology 108: 503-516.
33. Lee S, Choi H, Suh S, Doo IS, Oh KY, et al. (1999) Oligogalacturonic acid and chitosan reduce stomatal aperture by inducing the evolution of reactive oxygen species from guard cells of tomato and Commelina communis. Plant Physiology 121: 147-152.
34. Chinchilla D, Bauer Z, Regenass M, Boller T, Felix G (2006) The Arabidopsis receptor kinase FLS2 binds flg22 and determines the specificity of flagellin perception. Plant Cell 18: 465-476.
35. Jocelyn Malamy, John P. Carr, Daniel F. Klessig, Raskin aI (1990) Salicylic Acid: A Likely Endogenous Signal in the Resistance Response of Tobacco to Viral Infection. Science 250: 1002-1004.
36. Gaffney T, Friedrich L, Vernooij B, Negrotto D, Nye G, et al. (1993) Requirement of Salicylic-Acid for the Induction of Systemic Acquired-Resistance. Science 261: 754-756.
37. Ward ER, Uknes SJ, Williams SC, Dincher SS, Wiederhold DL, et al. (1991) Coordinate Gene Activity in Response to Agents That Induce Systemic Acquired Resistance. The Plant Cell Online 3: 1085-1094.
38. Vernooij B, Friedrich L, Morse A, Reist R, Kolditz-Jawhar R, et al. (1994) Salicylic Acid Is Not the Translocated Signal Responsible for Inducing Systemic Acquired Resistance but Is Required in Signal Transduction. The Plant Cell Online 6: 959-965.
39. Park SW (2008) Methyl salicylate is a critical mobile signal for plant systemic acquired resistance (5 October, pg 113, 2007). Science 321: 342-342.
64
40. Klessig DF, Park SW, Liu PP, Forouhar F, Vlot AC, et al. (2009) Use of a Synthetic Salicylic Acid Analog to Investigate the Roles of Methyl Salicylate and Its Esterases in Plant Disease Resistance. Journal of Biological Chemistry 284: 7307-7317.
41. Attaran E, Zeier TE, Griebel T, Zeier J (2009) Methyl Salicylate Production and Jasmonate Signaling Are Not Essential for Systemic Acquired Resistance in Arabidopsis. The Plant Cell Online 21: 954-971.
42. Greenberg JT, Jung HW, Tschaplinski TJ, Wang L, Glazebrook J (2009) Priming in Systemic Plant Immunity. Science 324: 89-91.
43. Grant M, Truman W, Bennettt MH, Kubigsteltig I, Turnbull C (2007) Arabidopsis systemic immunity uses conserved defense signaling pathways and is mediated by jasmonates. Proceedings of the National Academy of Sciences of the United States of America 104: 1075-1080.
44. Chaturvedi R, Krothapalli K, Makandar R, Nandi A, Sparks AA, et al. (2008) Plastid ω3-fatty acid desaturase-dependent accumulation of a systemic acquired resistance inducing activity in petiole exudates of Arabidopsis thaliana is independent of jasmonic acid. The Plant Journal 54: 106-117.
45. Shah J (2009) Plants under attack: systemic signals in defence. Current Opinion in Plant Biology 12: 459-464.
46. Parker JE, Holub EB, Frost LN, Falk A, Gunn ND, et al. (1996) Characterization of eds1, a mutation in Arabidopsis suppressing resistance to Peronospora parasitica specified by several different RPP genes. Plant Cell 8: 2033-2046.
47. Rietz S, Stamm A, Malonek S, Wagner S, Becker D, et al. (2011) Different roles of Enhanced Disease Susceptibility1 (EDS1) bound to and dissociated from Phytoalexin Deficient4 (PAD4) in Arabidopsis immunity. New Phytologist: no-no.
48. Aarts N, Metz M, Holub E, Staskawicz BJ, Daniels MJ, et al. (1998) Different requirements for EDS1 and NDR1 by disease resistance genes define at least two R gene-mediated signaling pathways in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America 95: 10306-10311.
49. Wagner S, Rietz S, Parker JE, Niefind K (2011) Crystallization and preliminary crystallographic analysis of Arabidopsis thaliana EDS1, a key component of plant immunity, in complex with its signalling partner SAG101. Acta Crystallographica Section F-Structural Biology and Crystallization Communications 67: 245-248.
50. Feys BJ, Moisan LJ, Newman MA, Parker JE (2001) Direct interaction between the Arabidopsis disease resistance signaling proteins, EDS1 and PAD4. Embo Journal 20: 5400-5411.
65
51. Wiermer M, Feys BJ, Parker JE (2005) Plant immunity: the EDS1 regulatory node. Current Opinion in Plant Biology 8: 383-389.
52. Shirano Y, Kachroo P, Shah J, Klessig DF (2002) A Gain-of-Function Mutation in an Arabidopsis Toll Interleukin1 Receptor–Nucleotide Binding Site–Leucine-Rich Repeat Type R Gene Triggers Defense Responses and Results in Enhanced Disease Resistance. The Plant Cell Online 14: 3149-3162.
53. Zhang J, Lu H, Li X, Li Y, Cui H, et al. (2010) Effector-Triggered and Pathogen-Associated Molecular Pattern–Triggered Immunity Differentially Contribute to Basal Resistance to Pseudomonas syringae. Molecular Plant-Microbe Interactions 23: 940-948.
54. Century KS, Shapiro AD, Repetti PP, Dahlbeck D, Holub E, et al. (1997) NDR1, a pathogen-induced component required for Arabidopsis disease resistance. Science 278: 1963-1965.
55. Shapiro AD, Zhang C (2001) The role of NDR1 in avirulence gene-directed signaling and control of programmed cell death in arabidopsis. Plant Physiology 127: 1089-1101.
56. Mackey D, Holt BF, Wiig A, Dangl JL (2002) RIN4 interacts with Pseudomonas syringae type III effector molecules and is required for RPM1-mediated resistance in Arabidopsis. Cell 108: 743-754.
57. Shirasu K, Schulze-Lefert P (2003) Complex formation, promiscuity and multi-functionality: protein interactions in disease-resistance pathways. Trends in Plant Science 8: 252-258.
58. Schulze-Lefert P (2004) Plant immunity: The origami of receptor activation. Current Biology 14: R22-R24.
59. Torp J, Jorgensen JH (1986) Modification of Barley Powdery Mildew Resistance Gene Ml-A12 by Induced Mutation. Canadian Journal of Genetics and Cytology 28: 725-731.
60. Muskett PR, Kahn K, Austin MJ, Moisan LJ, Sadanandom A, et al. (2002) Arabidopsis RAR1 Exerts Rate-Limiting Control of R Gene–Mediated Defenses against Multiple Pathogens. The Plant Cell Online 14: 979-992.
61. Azevedo C, Sadanandom A, Kitagawa K, Freialdenhoven A, Shirasu K, et al. (2002) The RAR1 interactor SGT1, an essential component of R gene-triggered disease resistance. Science 295: 2073-2076.
62. Peart JR, Lu R, Sadanandom A, Malcuit I, Moffett P, et al. (2002) Ubiquitin ligase-associated protein SGT1 is required for host and nonhost disease resistance in plants. Proceedings of the National Academy of Sciences of the United States of America 99: 10865-10869.
66
63. Tör M, Gordon P, Cuzick A, Eulgem T, Sinapidou E, et al. (2002) Arabidopsis SGT1b Is Required for Defense Signaling Conferred by Several Downy Mildew Resistance Genes. The Plant Cell Online 14: 993-1003.
64. Boter M, Amigues B, Peart J, Breuer C, Kadota Y, et al. (2007) Structural and functional analysis of SGT1 reveals that its interaction with HSP90 is required for the accumulation of Rx, an R protein involved in plant immunity. Plant Cell 19: 3791-3804.
65. Fu D-Q, Ghabrial S, Kachroo A (2009) GmRAR1 and GmSGT1 Are Required for Basal, R Gene–Mediated and Systemic Acquired Resistance in Soybean. Molecular Plant-Microbe Interactions 22: 86-95.
66. Frye CA, Innes RW (1998) An Arabidopsis mutant with enhanced resistance to powdery mildew. Plant Cell 10: 947-956.
67. Falk A, Feys BJ, Frost LN, Jones JDG, Daniels MJ, et al. (1999) EDS1, an essential component of R gene-mediated disease resistance in Arabidopsis has homology to eukaryotic lipases. Proceedings of the National Academy of Sciences of the United States of America 96: 3292-3297.
68. Alonso JM, Hirayama T, Roman G, Nourizadeh S, Ecker JR (1999) EIN2, a bifunctional transducer of ethylene and stress responses in Arabidopsis. Science 284: 2148-2152.
69. Penninckx IAMA, Thomma BPHJ, Buchala A, Metraux JP, Broekaert WF (1998) Concomitant activation of jasmonate and ethylene response pathways is required for induction of a plant defensin gene in Arabidopsis. Plant Cell 10: 2103-2113.
70. Frye CA, Tang DZ, Innes RW (2001) Negative regulation of defense responses in plants by a conserved MAPKK kinase. Proceedings of the National Academy of Sciences of the United States of America 98: 373-378.
71. Morris K, Mackerness SAH, Page T, John CF, Murphy AM, et al. (2000) Salicylic acid has a role in regulating gene expression during leaf senescence. Plant Journal 23: 677-685.
72. Tang DZ, Innes RW (2002) Overexpression of a kinase-deficient form of the EDR1 gene enhances powdery mildew resistance and ethylene-induced senescence in Arabidopsis. Plant Journal 32: 975-983.
73. Nawrath C, Métraux J-P (1999) Salicylic Acid Induction–Deficient Mutants of Arabidopsis Express PR-2 and PR-5 and Accumulate High Levels of Camalexin after Pathogen Inoculation. The Plant Cell Online 11: 1393-1404.
74. Rogers EE, Ausubel FM (1997) Arabidopsis Enhanced Disease Susceptibility Mutants Exhibit Enhanced Susceptibility to Several Bacterial Pathogens and Alterations in PR-1 Gene Expression. The Plant Cell Online 9: 305-316.
67
75. Nawrath C, Heck S, Parinthawong N, Métraux J-P (2002) EDS5, an Essential Component of Salicylic Acid–Dependent Signaling for Disease Resistance in Arabidopsis, Is a Member of the MATE Transporter Family. The Plant Cell Online 14: 275-286.
76. Alexander D, Goodman RM, Gut-Rella M, Glascock C, Weymann K, et al. (1993) Increased tolerance to two oomycete pathogens in transgenic tobacco expressing pathogenesis-related protein 1a. Proceedings of the National Academy of Sciences of the United States of America 90: 7327-7331.
77. Tornero P, Gadea J, Conejero V, Vera P (1997) Two PR-1 Genes from Tomato Are Differentially Regulated and Reveal a Novel Mode of Expression for a Pathogenesis-Related Gene During the Hypersensitive Response and Development. Molecular Plant-Microbe Interactions 10: 624-634.
78. Zhang Y, Fan W, Kinkema M, Li X, Dong X (1999) Interaction of NPR1 with basic leucine zipper protein transcription factors that bind sequences required for salicylic acid induction of the PR-1 gene. Proceedings of the National Academy of Sciences 96: 6523-6528.
79. Cao H, Bowling SA, Gordon AS, Dong X (1994) Characterization of an Arabidopsis Mutant That Is Nonresponsive to Inducers of Systemic Acquired Resistance. The Plant Cell Online 6: 1583-1592.
80. Delaney TP, Friedrich L, Ryals JA (1995) Arabidopsis signal transduction mutant defective in chemically and biologically induced disease resistance. Proceedings of the National Academy of Sciences 92: 6602-6606.
81. Glazebrook J, Rogers EE, Ausubel FM (1996) Isolation of Arabidopsis Mutants With Enhanced Disease Susceptibility by Direct Screening. Genetics 143: 973-982.
82. Tada Y, Spoel SH, Pajerowska-Mukhtar K, Mou Z, Song J, et al. (2008) Plant Immunity Requires Conformational Charges of NPR1 via S-Nitrosylation and Thioredoxins. Science 321: 952-956.
83. Kinkema M, Fan W, Dong X (2000) Nuclear Localization of NPR1 Is Required for Activation of PR Gene Expression. The Plant Cell Online 12: 2339-2350.
84. Zhang X, Chen S, Mou Z (2010) Nuclear localization of NPR1 is required for regulation of salicylate tolerance, isochorismate synthase 1 expression and salicylate accumulation in Arabidopsis. Journal of Plant Physiology 167: 144-148.
85. Lebel E, Heifetz P, Thorne L, Uknes S, Ryals J, et al. (1998) Functional analysis of regulatory sequences controllingPR-1 gene expression in Arabidopsis. The Plant Journal 16: 223-233.
68
86. Spoel SH, Mou Z, Tada Y, Spivey NW, Genschik P, et al. (2009) Proteasome-Mediated Turnover of the Transcription Coactivator NPR1 Plays Dual Roles in Regulating Plant Immunity. Cell 137: 860-872.
87. Zhang Y, Cheng YT, Qu N, Zhao Q, Bi D, et al. (2006) Negative regulation of defense responses in Arabidopsis by two NPR1 paralogs. The Plant Journal 48: 647-656.
88. Hepworth SR, Zhang Y, McKim S, Li X, Haughn GW (2005) BLADE-ON-PETIOLE-Dependent Signaling Controls Leaf and Floral Patterning in Arabidopsis. The Plant Cell Online.
89. Norberg M, Holmlund M, Nilsson O (2005) The BLADE ON PETIOLE genes act redundantly to control the growth and development of lateral organs. Development 132: 2203-2213.
90. Chassot C, Nawrath C, Métraux J-P (2007) Cuticular defects lead to full immunity to a major plant pathogen. The Plant Journal 49: 972-980.
91. Arondel V, Vergnolle C, Cantrel C, Kader JC (2000) Lipid transfer proteins are encoded by a small multigene family in Arabidopsis thaliana. Plant Science 157: 1-12.
92. Baulcombe D (2002) RNA silencing. Current Biology 12: R82-R84.
93. Nishikura K (2001) A Short Primer on RNAi: RNA-Directed RNA Polymerase Acts as a Key Catalyst. Cell 107: 415-418.
94. Waterhouse PM, Wang M-B, Lough T (2001) Gene silencing as an adaptive defence against viruses. Nature 411: 834-842.
95. Mourrain P, Béclin C, Elmayan T, Feuerbach F, Godon C, et al. (2000) Arabidopsis SGS2 and SGS3 Genes Are Required for Posttranscriptional Gene Silencing and Natural Virus Resistance. Cell 101: 533-542.
96. Yu DQ, Fan BF, MacFarlane SA, Chen ZX (2003) Analysis of the involvement of an inducible Arabidopsis RNA-dependent RNA polymerase in antiviral defense. Molecular Plant-Microbe Interactions 16: 206-216.
97. Xie Z, Fan B, Chen C, Chen Z (2001) An important role of an inducible RNA-dependent RNA polymerase in plant antiviral defense. Proceedings of the National Academy of Sciences 98: 6516-6521.
98. Yang S-J, Carter SA, Cole AB, Cheng N-H, Nelson RS (2004) A natural variant of a host RNA-dependent RNA polymerase is associated with increased susceptibility to viruses by Nicotiana benthamiana. Proceedings of the National Academy of Sciences of the United States of America 101: 6297-6302.
69
99. Vlot AC, Dempsey DA, Klessig DF (2009) Salicylic Acid, a Multifaceted Hormone to Combat Disease. Annual Review of Phytopathology 47: 177-206.
100. Wildermuth MC (2006) Variations on a theme: synthesis and modification of plant benzoic acids. Current Opinion in Plant Biology 9: 288-296.
101. Catinot J, Buchala A, Abou-Mansour E, Metraux JP (2008) Salicylic acid production in response to biotic and abiotic stress depends on isochorismate in Nicotiana benthamiana. Febs Letters 582: 473-478.
102. Uppalapati SR, Ishiga Y, Wangdi T, Kunkel BN, Anand A, et al. (2007) The phytotoxin coronatine contributes to pathogen fitness and is required for suppression of salicylic acid accumulation in tomato inoculated with Pseudomonas syringae pv. tomato DC3000. Molecular Plant-Microbe Interactions 20: 955-965.
103. Wildermuth MC, Dewdney J, Wu G, Ausubel FM (2001) Isochorismate synthase is required to synthesize salicylic acid for plant defence. Nature 414: 562-565.
104. Chen Z, Zheng Z, Huang J, Lai Z, Fan B (2009) Biosynthesis of salicylic acid in plants. Plant signaling & behavior 4: 493-496.
105. Meuwly P, Molders W, Buchala A, Metraux JP (1995) Local and Systemic Biosynthesis of Salicylic Acid in Infected Cucumber Plants. Plant Physiology 109: 1107-1114.
106. Mauch-Mani B, Slusarenko AJ (1996) Production of Salicylic Acid Precursors Is a Major Function of Phenylalanine Ammonia-Lyase in the Resistance of Arabidopsis to Peronospora parasitica. The Plant Cell Online 8: 203-212.
107. Rohde A, Morreel K, Ralph J, Goeminne G, Hostyn V, et al. (2004) Molecular Phenotyping of the pal1 and pal2 Mutants of Arabidopsis thaliana Reveals Far-Reaching Consequences on Phenylpropanoid, Amino Acid, and Carbohydrate Metabolism. The Plant Cell Online 16: 2749-2771.
108. Mishina TE, Zeier J (2007) Bacterial non-host resistance: interactions of Arabidopsis with non-adapted Pseudomonas syringae strains. Physiologia Plantarum 131: 448-461.
109. Warren RF, Merritt PM, Holub E, Innes RW (1999) Identification of Three Putative Signal Transduction Genes Involved in R Gene-Specified Disease Resistance in Arabidopsis. Genetics 152: 401-412.
110. Swiderski MR, Innes RW (2001) The Arabidopsis PBS1 resistance gene encodes a member of a novel protein kinase subfamily. Plant Journal 26: 101-112.
70
71
111. Ade J, DeYoung BJ, Golstein C, Innes RW (2007) Indirect activation of a plant nucleotide binding site-leucine-rich repeat protein by a bacterial protease. Proceedings of the National Academy of Sciences of the United States of America 104: 2531-2536.
112. Zhang J, Li W, Xiang TT, Liu ZX, Laluk K, et al. (2010) Receptor-like Cytoplasmic Kinases Integrate Signaling from Multiple Plant Immune Receptors and Are Targeted by a Pseudomonas syringae Effector. Cell Host & Microbe 7: 290-301.
113. Khalaf A, Moore GA, Jones JB, Gmitter Jr FG (2007) New insights into the resistance of Nagami kumquat to canker disease. Physiological and Molecular Plant Pathology 71: 240-250.
114. Duan Y, Zhou L, Hall DG, Li W, Doddapaneni H, et al. (2009) Complete Genome Sequence of Citrus Huanglongbing Bacterium, ‘Candidatus Liberibacter asiaticus’ Obtained Through Metagenomics. Molecular Plant-Microbe Interactions 22: 1011-1020.
115. Felix G, Duran JD, Volko S, Boller T (1999) Plants have a sensitive perception system for the most conserved domain of bacterial flagellin. The Plant Journal 18: 265-276.
116. Rorabacher DB (1991) Statistical Treatment for Rejection of Deviant Values - Critical-Values of Dixon Q Parameter and Related Subrange Ratios at the 95-Percent Confidence Level. Analytical Chemistry 63: 139-146.
117. Yun B-W, Atkinson HA, Gaborit C, Greenland A, Read ND, et al. (2003) Loss of actin cytoskeletal function and EDS1 activity, in combination, severely compromises non-host resistance in Arabidopsis against wheat powdery mildew. The Plant Journal 34: 768-777.
118. Navarro L, Zipfel C, Rowland O, Keller I, Robatzek S, et al. (2004) The transcriptional innate immune response to flg22. interplay and overlap with Avr gene-dependent defense responses and bacterial pathogenesis. Plant Physiology 135: 1113-1128.
119. Yalpani N, Shulaev V, Raskin I (1993) Endogenous Salicylic-Acid Levels Correlate with Accumulation of Pathogenesis-Related Proteins and Virus-Resistance in Tobacco. Phytopathology 83: 702-708.
120. Metraux JP, Signer H, Ryals J, Ward E, Wyssbenz M, et al. (1990) Increase in Salicylic-Acid at the Onset of Systemic Acquired-Resistance in Cucumber. Science 250: 1004-1006.
121. Febres V, Khalaf A, Gloria M (2010) Phylogenetic Relationships of NPR1-like Proteins from Citrus. American Society of Plant Biologists
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 Environment’ and finished his thesis titled in ‘Dynamics of
Soil Nutrients in Pear Orchard in Spring’. Due to his excellent performance in study and
research, he was selected as graduate student candidate without an entrance exam in
Agricultural University of Hebei, majoring in pomology. During his graduate study, he
was involved in the projects ‘Technology and Extension of Improvement of Pear fruit
quality’, concentrating on tissue culture and Agrobacterium-mediated transformation of
insect-resistant gene in pear cultivars. In parallel, he completed his master’s thesis
research ‘Effects of Salicylic Acid on Respiratory Pathway of Postharvest Huang-guan
Pear’ and obtained the Master’s 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 year’s graduate study in PMCB, he did the master’s research on ‘Flg22-
Triggered Immunity and the Effect of Azelaic Acid on the Defense Response in Citrus’.
72