1 GENETIC AND MOLECULAR ANALYSIS OF PATHOGENICITY GENES IN Xanthomonas citri subsp. citri By JOSÉ FRANCISCO LISSONI FIGUEIREDO A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009
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1
GENETIC AND MOLECULAR ANALYSIS OF PATHOGENICITY GENES IN Xanthomonas citri subsp. citri
By
JOSÉ FRANCISCO LISSONI FIGUEIREDO
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
Type I secretion system ............................................................................................... 14 Type II secretion system .............................................................................................. 15 Type III secretion system............................................................................................. 16 Type IV secretion system ............................................................................................ 18
Hypersensitive Response and Pathogenicity (hrp) Genes ................................................ 19 Harpins a HR elicitor protein ...................................................................................... 22
Type Three Secretion System-Dependent Effectors ......................................................... 23 Project Goals and Objectives...................................................................................................... 25
2 MUTATIONAL ANALYSIS OF TYPE III EFFECTORS GENES FROM Xanthomonas citri subsp. citri.................................................................................................... 27
Introduction ................................................................................................................................. 27 Materials and Methods ................................................................................................................ 28
Bacterial Strains and Plasmids ............................................................................................ 28 Media and Growth Conditions ............................................................................................ 28 Recombinant DNA Techniques .......................................................................................... 29 DNA Amplification ............................................................................................................. 30 Mutation of Candidate Genes by Homologous Recombination ....................................... 30 Complementation of ΩhrpG and ΩhrpX Deficient Mutants of Xcc Strain 306 .............. 31 Plant Material and Inoculations .......................................................................................... 32
Results .......................................................................................................................................... 33 Analysis of HrpX-Regulon Candidate Genes .................................................................... 33 Mutations of the regulatory and structural genes of Xcc 306 hrp pathway cause loss
of pathogenicity ................................................................................................................ 33 Phenotypic Analysis of Nineteen Candidate Effector and Hrp-Regulon Associated
3 IDENTIFICATION OF A HRP-INDEPENDENT HR ELICITOR FROM Xanthomonas citri subsp. citri............................................................................................................................ 50
Introduction ................................................................................................................................. 50 Materials and Methods ................................................................................................................ 52
Bacterial Strains and Plasmids ............................................................................................ 52 Media and Growth Conditions ............................................................................................ 53 Recombinant DNA Techniques .......................................................................................... 53 DNA Amplification ............................................................................................................. 54 Mutagenesis of hrpG and hrpX in X. citri subsp. citri 306 ............................................... 54 Subcloning of HR-Elicitor Candidate from Xaa Library .................................................. 55 Plant Material and Plant Inoculations................................................................................. 57 Measurement of Electrolyte Leakage ................................................................................. 58 Protein Thermal Stability .................................................................................................... 58
Results .......................................................................................................................................... 58 Unusual Incompatible Responses of Mutants 306::ΩhrpG and 306::ΩhrpX in
Tomato .............................................................................................................................. 58 Subclone of X. f. pv. aurantifolli Confers the Ablilty to Trigger an HR in Tomato ....... 59 Sequencing Analysis Revealed Novel Genes Controlling Nonhost HR Elicitor ............ 60
4 GENETIC CHARACTERIZATION OF HRPW OF Xanthomonas citri subsp. citri ............ 76
Introduction ................................................................................................................................. 76 Material and Methods ................................................................................................................. 78
Bacterial Strains and Plasmids ............................................................................................ 78 Media and Growth Conditions ............................................................................................ 78 Recombinant DNA Techniques .......................................................................................... 78 DNA Sequence Alignment and Phylogenetic Analysis of hrpW Gene............................ 79 Generation of hrpW Deletion Mutants ............................................................................... 80 Construction of hrpW-Harpin Domain ............................................................................... 81 Complementation Tests ....................................................................................................... 82 Plant Material ....................................................................................................................... 83 Plant Inoculations ................................................................................................................ 83 Bacterial Populations ........................................................................................................... 84 Translocation Activity in 306::∆harpin Mutant ................................................................. 84 Construction of hrpW::avBs2 Fusion ................................................................................. 85
Results .......................................................................................................................................... 85 Xanthomonas citri subsp. citri hrpW Phylogeny ............................................................... 85 Total Deletion of hrpW Gene Is Irrelevant for Xcc Pathogenicity and HR in Non-
Host Plants ........................................................................................................................ 86 Harpin Domain in hrpW Gene is Required for Pathogenicity .......................................... 87 Complementation of HrpW∆harpin Mutant In-Cis ........................................................... 88 Translocon Machinery, T3SS, Is Not Affected by ∆harpin Mutant in Xcc ..................... 88 HprW is not Translocated into the Mesophyll of Plant Cell ............................................. 89
5 CHARACTERIZATION OF avrGf1 FROM Xanthomonas citri subsp. citri STRAIN AW .............................................................................................................................................. 117
Introduction ............................................................................................................................... 117 Materials and Methods .............................................................................................................. 118
Bacterial Strains and Plasmids .......................................................................................... 118 Media and Growth Conditions .......................................................................................... 118 Recombinant DNA Techniques ........................................................................................ 118 Plant Material and Plant Inoculations............................................................................... 119 Agrobacterium-mediated expression of avrGf1 . ............................................................ 120 Bacterial Expression of avrGf1......................................................................................... 122
Results ........................................................................................................................................ 123 Induction of Hypersensitive Response by Transient Expression of avrGf1 Within
Citrus ............................................................................................................................... 123 The N-terminal and C-terminal Are Required for HR Elicitation .................................. 124 Xanthomonas citri subsp. citri Harboring avrGf1 Induces a Hypersensitive
Reaction in Grapefruit ................................................................................................... 124 AvrGf1 Is Translocated Inside the Host Plant Cell ......................................................... 125
Table page 2-1 Xanthomonas citri pv. citri genes used in the study. ........................................................... 38
2-2 Bacterial strains and plasmids used in the study. ................................................................. 39
2-3 Oligonucleotides sequence used in this study. ..................................................................... 42
2-4 Candidate PIP box (-like) and -10 box-like sequences of the proposed hrpX regulons and putative effector/avirulence genes of Xcc. .................................................................... 44
2-5 Xanthomonas citri pv. citri mutants pathogenicity phenotype in grapefruit leaveas and HR elicitation in tomato leaflets..................................................................................... 45
3-1 Bacterial strains and plasmids used in the study. ................................................................. 64
4-1 Bacterial strains and plasmids used in the study. ................................................................. 93
4-2 Oligonucleotides sequence used in this study. ..................................................................... 95
5-1 Bacterial strains and plasmids used in the study. ............................................................... 128
5-2 Oligonucleotides sequence used in this study. ................................................................... 130
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LIST OF FIGURES
Figure page 2-1 Genetic organization of the hrp genes of X. citri subsp. citri.............................................. 46
2-2 Proposed model for hrp gene regulation cascade in X. citri subsp. citri ............................ 47
2-3 Comparison of the bacterial pathogenicity and HR elicitor in grapefruit leaves and tomato leaflets, respectively .................................................................................................. 48
2-4 Integration by site-specific recombination using one single target sited ............................ 49
3-1 Phenotype of grapefruit leaves were used to distinguish among the strains ...................... 66
3-2 Phenotype of 306 wild type, 306::ΩhrpG and 306:: ΩhrpX in leaflet of tomato cv Bonny Best.............................................................................................................................. 67
3-3 Hypersensitive response assay in tomato cv. Bonny Best and pepper cv. ECW-20R ....... 68
3-4 Electrolyte leakage in tomato plant is changed in WT::pLAFR3, compatible interaction, and WT:: 450, incompatible interaction ........................................................... 69
3-5 Subclone, B38, triggers hypersensitive response (HR) independent of T3SS in X. perforans in non-host plants, tomato cv. Bonny Best and pepper cv. ECW20R ............... 70
3-6 Protein alignment of XAC3857 gene and Xanthomonas spp .............................................. 71
3-7 Protein alignment of XAC3858 gene and Xanthomonas spp .............................................. 72
3-8 Protein alignment of XAC3859 gene and Xanthomonas spp .............................................. 73
3-9 Transcriptional organization of the ORFs XAC3857 to XAC3859 region in Xanthomonas citri subsp. citri............................................................................................... 75
4-1 Neighbor joining analyses using full nucleotide sequence of hrpW gene from X. c. subsp. citri strain 306 ............................................................................................................. 96
4-2 Sequence alignment of Xcc strains collected world-wide ................................................... 97
4-3 Sequence alignment of Xcc strain 306 – wild type (W1); W::∆Harpin mutant (W2); and 306::∆hrpW HrpW (W3) .............................................................................................. 112
4-4 Phenotype assay in grapefruit leaves .................................................................................. 113
4-5 Populations of Xcc 306 (Xcc306) ....................................................................................... 114
4-6 Canker symptoms on abaxial leaf surface of grapefruit .................................................... 115
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4-7 PCR amplification of genomic DNA of Xcc 306 .............................................................. 116
5-1 Diagram of AvrGf1 and truncated versions used to test regions of the protein for elicitor activity ...................................................................................................................... 131
5-2 Agrobacterium-mediated expression of avrGf1 in grapefruit leaf tissue ......................... 132
A-2 Nucleotide sequence of AvrGf11-106::AvrBs262-574 fused protein ..................................... 140
A-3 Nucleotide sequence of HrpW1-109::AvrBs262-574 fused protein ........................................ 141
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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
GENETIC AND MOLECULAR ANALYSIS OF PATHOGENICITY GENES IN Xanthomonas
citri subsp. citri
By
José Francisco Lissoni Figueiredo
May 2009 Chair: James H. Graham Cochair: Jeffrey B. Jones Major: Plant Pathology
Xanthomonas citri subsp. citri (Xcc), which causes citrus canker, relies on a type III
secretion system (T3SS) to successfully develop disease. Various plant pathogenic bacteria
carrying a T3SS inject more than 40 different effector proteins (T3-effectors) into the plant cells
via this apparatus. In Xanthomonas, the genes for the T3SS are regulated by HrpX, which is an
AraC-type transcriptional regulator. The possible roles that nineteen candidate T3-effector genes
play in the ability of Xcc to cause citrus canker were investigated through the use of site-directed
mutagenesis. The candidate genes were selected on the basis of having promoter features similar
to gene regulated by HrpX or by sequence similarity to known T3-effectors in other plant
pathogenic bacteria. Inoculation in grapefruit revealed that none of these mutants were visually
impaired for disease development. Unlike the hrpW null mutant, deletion in the harpin domain
from hrpW resulted in the loss of Xcc pathogenicity symptoms, while not affecting the ability of
of the bacteria to multiply in the plant. The mutants were also assessed for the ability to elicit
hypersensitive response (HR) in non-host plant tomato. The Xcc hrp- mutants, in contrast to
many pathogenic xanthomonads, retained the ability to trigger HR in the nonhost. Using
subcloning procedures and homology search, three candidates open reading frames, XAC3857,
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XAC3858 and XAC3859, were identified in Xcc that might be responsible for this T3SS-
independent HR elicitation. Experiments were also performed to further characterize a new
avirulence gene, avrGf1, isolated from Xcc strain Aw, which induces HR in grapefruit. The
avrGf1 gene was demonstrated to encode a protein that is translocated into plant cells via the
T3SS. Additionally, a transient expression on grapefruit leaves was devised using an
Agrobacterium-mediated delivery system. Different avrGf1 deletion mutants in the N- and C-
terminal coding regions were tested using the system, and the results showed that the first 116
amino acids in the N-terminal and the last 83 amino acids in the C-terminal were crucial for HR
elicitation in grapefruit. In summary, this study reported the presence of candidate T3-effector
genes that did not affect the disease progress under the experimental conditions and a harpin
domain of hrpW gene is essential for symptom development. Additionally, the development of
an efficient transient expression in grapefruit leaves revealed specific regions of avrGf1 are
required for defense activation.
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CHAPTER 1 THE WEAPONRY INVOLVED ON PLANT-PATHOGEN INTERACTION
Introduction
Many complex steps are involved in the interaction of plants and their pathogens.
Although plants are exposed to attack by many bacteria, disease development is a relatively rare
event. Usually, plant defenses retard or stop bacterial ingress without the development of disease
symptoms. The term innate immunity, which is observed in both plants and animals, is often
used to refer to the defenses after the physical barriers are broached by microbes (Menezes et al.,
2000). Microbe invasion commonly induces a series of biochemical-changes that may lead to
resistance to disease development, such as production of the salicylic acid (SA), deposition of
lignin and callose in the cell wall, transcription of pathogenesis-related (PR) genes, and
production of antimicrobial compounds (Loake and Grant, 2007). The plant may also recognize
specific pathogen effectors that interact, directly or indirectly, with resistance (R) proteins,
leading to the activation of a rapid defense response and accompanying hypersensitive reaction
(HR), which is also called effector-triggered immunity (ETI) (Jones and Dangl, 2006). Basal
plant defenses are also triggered by the recognition of common microbial molecules called
pathogen associated molecular patterns (PAMPs). This recognition, which is called PAMP-
triggered immunity (PTI), involves recognition of PAMPs by pattern recognition receptors
(PRRs), which are receptor-like kinases (Jones and Dangl, 2006; Block et al., 2008). The major
known elicitors of PTI include flagellin peptide (flg22) (Zipfel et al., 2004), lipopolysacharides
(LPS) derived from pathogen cell walls (Keshavarzi et al., 2004), peptidoglycans, microbial cell
wall fragments, phospholipids, proteins, double stranded RNA and methylated DNA (Ingle et al.,
2006; Iriti and Faoro, 2007). Activation of basal defense responses by PTI results in rapid
increases of the Ca2+ flux, nitric oxide and reactive oxygen species (Nurnberger et al., 2004).
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Phytopathogenic proteobacteria (Gram-negative bacteria) rely on different weapons to
neutralize the host defenses and promote the colonization of the host tissue. The weapons include
as several protein secretions systems, T3- effector proteins, toxins, small molecule suppressors,
cell wall degradation enzymes, and genes involved in flagellar and biofilm biosynthesis
(Shiraishi et al., 1997; Gross et al., 1999; Alfano et al., 2002; Wolpert et al., 2002; Moissiard
and Voinnet, 2004; Hommais et al., 2008). Pathogens use this diversity of weapons to undermine
core components of plant defenses, such as the HR, cell wall-based defenses, jasmonic acid
signaling, systemic acquired and induced resistance and expression of defense genes
(Abromovitch and Martin, 2004). Most phytopahogenic bacteria carry four secretion systems,
although new studies have demonstrated the presence of up to six secretion systems (Yahr,
2006). Four of the secretion systems have been well characterized in plant pathogenic bacteria
and are fundamental for the ingress, colonization, suppression of host defenses and disease
development. Approximately 20% of all polypeptides synthesized by bacteria are located
partially or completely outside of cytoplasm, and this arsenal of proteins makes use of these
systems for secretion and translocation into the apoplast and into plant cells (Pugsley, 1993;
Salmond and Reeves, 1993). In general, pathogenicity in proteobacteria is dependent upon
secretion machinery that mediates the transport and injection of toxic molecules into target
tissues or cells. These secretion systems are classified into six types (I to VI) and a summary of
the four major types are presented here.
Plant Pathogen Translocon Pathways
Type I secretion system
The route that each protein will be translocated through bacterial membranes is dependent
on the substrate. Translocation of most proteins, in general, is catalyzed by the secretory (Sec)
apparatus although some exported proteins are translocated independently of the core Sec-
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translocon, named sec-independent (Danese and Silhavy, 1998; Cristobal et al., 1998). This
system, which is sec-independent and ABC-transporter dependent, is specific for a particular
protein or a family of closely related proteins (Higgins, 1995). The proteins are exported by ABC
(ATP-binding cassette) transporters, which are involved in the import or export of a great variety
of substrates such as different small peptide signaling molecules, bacteriocins and amino acids,
through the outer membrane (Gray et al., 1989; Wandersman, 1992; Higgins, 1995; Young and
Holland, 1999). Basically, the type I translocator is assembled on two dedicated inner membrane
proteins, first, the ABC transporter (provide energy and it is the initial channel across the inner
membrane), second, the membrane fusion protein (MFP), which starts in the periplasm and form
a channel to the surface with an third protein, TolC, in the outer membrane protein (Holland et
al., 2005). The proteins that are translocated by this system, usually, carry the secretion signal at
the C-terminal and is not removed during transport (Holland et al., 2005). The type I translocon
system plays an important role in both animal and plant bacteria pathogens. A type I mutant in
Actinobacillus pleuropneumoniae, which has hemolytic activity, is defective in protein secretion
and the strain is non-hemolytic (Kuhnert et al., 2005). The importance of the type I system is
demonstrated in plants as well. Rice plants that carry the Xa21 resistance gene, which mediates
recognition of bacterial strains expressing AvrXa21 activity, are resistant to Xanthomonas oryzae
pv oryzae although plants lacking Xa21 are susceptible (Gomez-Gomez and Boller, 2002).
Mutational analysis of X. oryzae pv oryzae genes involved in type I secretion revealed that the
strain is no longer recognized by Xa21 (da Silva et al., 2003).
Type II secretion system
Of the six secretion systems reported in phytopathogenic bacteria, the most prolific
pathway is the type II secretion system from which most proteins are secreted, including elastase,
lipase, phospholipases and exotoxins (Filloux et al., 1998; Ball et al., 2002). For these proteins to
16
reach their final destination, they may be delivered through two routes that are via sec machinery
or Tat export pathways (Douglas et al., 1987; Voulhoux et al., 2001). The proteins translocated
through the type II-dependent system are first translocated across the cytoplasmic membrane via
the sec machinery. The next step in the secretion process involves the mature periplasmic protein
being transported across outer membrane, which requires the function of at least 12 gene
products of a specialized apparatus called the secreton (Pugsley, 1993; Sandkvist, 2001).
Depending on the species, between 12 and 15 genes (named by letters A through O and S) are
essential for type II secretion and minor differences in genetic organization of this cluster in
closely related species have been observed (Pugsley, 1997; Sandkvist, 2001). Studies on
secretion genes in Erwinia chrysanthemi and Erwinia carotovora, which cause soft rot diseases
on various plants, showed that one of the genes, outN, is missing in E. chrysanthemi whereas its
presence in E. carotovora is required for functioning of the system (Lindeberg et al., 1996). da
Silva et al. (2002) describe the presence of two type II secretion systems in Xanthomonas citri
subsp citri (Xcc) and Xanthomonas campestris pv campestris, , that are involved in the secretion
of degradative enzymes and toxins. Although HrpX is known as type III secretion system
regulator in Ralstonia solanacearum (Genin et al., 1992) and Xanthomonas spp (Wengelnik and
Bonas, 1996), it also regulates some type II secretory proteins in Xanthomonas oryzae pv oryzae,
causal agent of bacterial leaf blight (Furutani et al., 2004). This broad regulation is also observed
in X. citri subsp citri where HrpG, which regulates HrpA and HrpX (Brito et al., 1999;
Wengelnik et al., 1996b), also regulates 11 type II secretion system-related proteins (Yamazaki
et al., 2008).
Type III secretion system
A major breakthrough in the plant pathology field was the elucidation of T3SS, which is
used for most Gram-negative pathogenic bacteria and, it is conserved among animal and plant
17
bacteria (Cornelis and Van Gijsegem, 2000). Species in the genera Yersinia, Shigella flexneri,
Salmonella typhimurium, Salmonella typhimurium, enteropathogenic E. coli, Erwinia amylovora,
Pseudomonas syringae, Pseudomonas solanacearum and Xanthomonas spp rely on the T3SS for
successful colonization of their respective hosts (Hueck, 1998; Cornelis and Van Gijsegem,
2000). Phytopathogenic bacteria such as Pseudmonas, Rasltonia, Xanthomonas, Erwinia, and
Pantoea invade their hosts by using natural openings, such as stomata, or wounds. When bacteria
ingress the host they promote the secretion and translocation a diverse group of bacterial
virulence factors through T3SS, refered to here as T3-effectors (Cornelis and Van Gijsegem,
2000). The first demonstration of the translocation of T3-effectors was observed with Yersinia
effector proteins (Rosqvist et al., 1994). Once these T3-effectors are inside the host cell, it is
expected to modulate plant physiology providing an environment beneficial to sustain the growth
of the pathogen outside the cell and suppress host defenses (Mudgett, 2005). The identification
of the P. syringae hypersensitive response and pathogenicity (hrp) gene cluster suggest that the
expression of these genes results in the formation of a T3SS structure or hrp pilus, which creates
a bridge between pathogen and host cell for protein delivery (Aizawa et al., 1998; Kimbrough
and Miller, 2000). All proteins secreted and/or translocated from T3S system are delivered from
the bacterial cytoplasm to the host cell interior passing between the bacterial and host
membranes/cell wall through this channel (Whittam et al., 2004). The remarkable achievement
related with T3S system in mammalian and plant bacterial pathogenesis is the finding that a
defect in this system often leads to complete loss of bacterial pathogenicity (Lindgren et al.,
1986; Zischeck et al., 1987). Despite the obvious importance of the T3SS, which functions in
delivering the type three effectors (T3-effectors) into plant cells (Galan & Collmer, 1999;
Kjemtrup et al., 2000), not all plant pathogens bacteria rely on this particular system, including
TRCGB, TTCGB-N15-TTCHB (B: C/T; H: A/C/T; R: A/G; V: A/C/G) it can have a negative
effect on gene activities (Furutani et al., 2006).
Pathogenicity experiments to assess the influence of each gene for disease development
were carried out. Surprisingly, none of the 19 mutants generated in this study was observed to
affect the ability to incite disease symptoms in susceptible grapefruit leaf. Neither mutated
homologous genes, which have been reported to be required for pathogenicity, such as XAC0661
36
(endopolygalacturonase) and XAC2374 (polygalacturonase) that are similar to E. carotovora
subsp. carotovora PehA- polygalacturonase, a cell wall-degrading enzyme and HR elicitor in
Arabidopsis (Kariola et al., 2003). However, X. o. pv. oryzae T3-effectors suppress a plant basal
defense response elicited by T2-secreted plant cell-wall-degrading enzymes (Jha et al., 2007). In
addition, Yamazaki et al. (2008) suggested that Xcc may suppress the polygalacturonase-
mediated plant defense by coregulating polygalacturonase isozymes.
The pathogenicity assay with 306::Ωhpa1 and 306::ΩhpaF mutants did not show any
macroscopic change in the symptoms compared with the Xcc wild type strain contrasting with
the results reported in X. o. pv. oryzae and X. a. pv. glycines, where hpa1 and hpaF are required
for full virulence in (Zhu et al., 2000; Kim et al., 2003; Cho et al., 2007).. In the class of
Xanthomonas outer proteins, two mutants were assessed 306::ΩxopX (carrying an insertion in
xopX gene) and 306::ΩxopQ (carrying an insertion in xopQ gene) for pathogenicity, neither one
demonstrated canker symptoms. However, xopQ and xopX have been reported to be highly
conserved among Xanthomonas spp. (Roden et al., 2004; Furutani et al., 2009). In Xcv, XopX,
contributes to the virulence on host pepper and tomato plants and is T3SS-dependent (Metz et
al., 2005).
When the avirulence genes, 306avrXacE1, avrXacE2, avrXacE3 and avBs2 were mutated
in Xcc strain they produced typical canker symptoms on grapefruit leaves. Similar results were
reported previously with avrXacE1 and avrXacE3 homologs in Xcv ∆xopE1 and ∆xopE2,
respectively, mutants when tested in susceptible pepper plants – ECW (Thieme et al., 2007).
However, avrBs2 phenotype observed here contrasts with the phenotype demonstrated with
avrBs2 mutant from X. c. pv. vesicatoria, which is required for full virulence (Swords et al.,
37
1996). In conclusion these avirulence genes do not appear to have a pronounced effect on Xcc
fitness in disease development in grapefruit.
We demonstrated above that a putative pathogenicity Xcc genes tested are not required for
disease development in greenhouse conditions. These genes were identified, based on sequence
analysis, by da Silva et al. (2002) and they are considered genes that are regulated by HrpX. Our
results indicate that the putative hrpX-regulon and effector/avirulence genes do not have any
direct role in disease development (Table 2-5). However, we can not conclude that they are not
important for bacterial survival.
In xanthomonads hrp activation cascade, the HrpG up-regulates expression of hrpA and
hrpX genes, and then HrpX activates the transcription the hrpB to hrpF operons (Wengelnik and
Bonas, 1996; Wengelnik et al., 1996a; Wengelnik et al., 1996b). In our experiments, the
disruption of either hrpG or hrpX severely disrupts the induction of any symptoms in grapefruit
leaves but, unexpectedly, both mutants retained the ability to trigger HR in non-host tomato
plants. Similar results were reported by Marutani et al. (2005), where a P. syringae pv. tabaci
hrp-regulatory ∆hrcC mutant also was able to trigger HR in non-host tomato plants in the hrp-
independent manner (Chapter 3). Additionally, a hrp X. perforans mutant harboring a X. fuscans
pv. aurantifolli clone was able to trigger HR in similar manner. Based on these results, we
speculate that an unknown T3SS-independent gene in Xcc is a HR-elicitor. To deepen our
understanding the role of the HrpX-regulon genes for pathogenicity and the model of action of
T3SS-independent HR-elicitor proteins, more detailed mutagenesis and protein secretion analysis
are required.
38
Table 2-1. Xanthomonas citri pv. citri genes used in the study ORF Number Relevant characteristics* Hrp Gene Cluster XAC0416 hpa1 XAC0415 hrpA (hrcC) XAC1265 hrpG XAC1266 hrpX Extended Hrp Conserved Regulon XAC0277 Conserved hypothetical protein XAC0661 endopolygalacturonase XAC1706 Alkanal monooxygenase XAC1886 β-K-adipate enol-lactone hydrolase XAC2374 Polygalacturonase XAC2534 Conserved hypothetical protein XAC3309 aminopeptidase Additional Hrp Regulon Candidate XAC2370 Endopeptidase XAC2922 hprW XAC3230 Actin-ADP-ribosylating toxin domain Putative Effector/Avirulence XAC0286 avrPphE1 XAC3224 avrPphE2 XACb0011 avrPphE3 XAC0393 hpaF XAC3090 Leucine rich protein XAC0076 avrBs2 Xanthomonas outer protein XAC0543 xopX XAC4330 xopQ
* All the genes shown in this table were identified by da Silva et al. (2002)
39
Table 2-2. Bacterial strains and plasmids used in the study Strain or plasmid Relevant characteristics Source Strains Xanthomonas citri subsp. citri 306 Wild type, Asiatic strain, isolated in Brazil,
RifR DPIa
306::ΩavrBs2 avrBs2-, single recombinant of pCRavrBs2, KnR, AmpR
This study
306:: Ω2277 Conserved hypothetical protein, single recombinant of pCR2277, KnR, AmpR
This study
306::ΩavrPphE1 avrPphE1-, single recombinant of pCRavrPphE1, KnR, AmpR
This study
306::ΩhpaF hpaF -, single recombinant of pCRhpaF, KnR, AmpR
This study
306::ΩhrcC hrcC -, single recombinant of pCRhrcC, KnR, AmpR
This study
306::Ωhpa1 hpa1-, single recombinant of pCRhpa1, KnR, AmpR
This study
306::ΩxopX xopX -, single recombinant of pCRxopX, KnR, AmpR
This study
306::Ω0661 Xac0661-, single recombinant of pCR0661, KnR, AmpR
This study
306::ΩhrpG hrpG -, single recombinant of pCRhrpG, KnR, AmpR
This study
306::ΩhrpX hrpX -, single recombinant of pCRhrpX, KnR, AmpR
This study
306::Ω1706 Xac1706-, single recombinant of pCR1706, KnR, AmpR
This study
306::Ω1886 Xac1886-, single recombinant of pCR1886, KnR, AmpR
This study
306::Ω2370 Xac2370-, single recombinant of pCR2370, KnR, AmpR
This study
306::Ω2374 Xac2374-, single recombinant of pCR2374, KnR, AmpR
This study
306::Ω2534 Xac2534-, single recombinant of pCR2534, KnR, AmpR
This study
306::ΩhrpW hrpW -, single recombinant of pCRhrpW, KnR, AmpR
This study
306::Ω3090 Xac3090-, single recombinant of pCR3090, KnR, AmpR
This study
306::ΩavrPphE2 avrPphE2-, single recombinant of pCRavrPphE2, KnR, AmpR
This study
306::Ω3230 Xac3230-, single recombinant of pCR3230, KnR, AmpR
This study
306::Ω3309 Xac3309-, single recombinant of pCR3309, KnR, AmpR
This study
40
Table 2-2. Continued Strain or plasmid Relevant characteristics Source 306::ΩxopQ xopQ -, single recombinant of pCRxopQ, KnR,
AmpR This study
306::ΩavrPphE3 avrPphE3-, single recombinant of pCRavrPphE3, KnR, AmpR
al. (1998) pUFR034 KnR, Tn903, IncW, Mob+ De Feyter et
al. (1990) pCRavrBs2 avrBs2 partial fragment in pCR2.1-TOPO This study pCR0277 Conserved hypothetical protein partial
fragment in pCR2.1-TOPO This study
pCRavrPphE1 avrPphE1 partial fragment in pCR2.1-TOPO This study pCRhpaF hpaF partial fragment in pCR2.1-TOPO This study pCRhrcC hrcC (hrpA) partial fragment in pCR2.1-TOPO This study pCRhpa1 hpa1 partial fragment in pCR2.1-TOPO This study pCRxopX xopX partial fragment in pCR2.1-TOPO This study pCR0661 Endopolygalacturonase partial fragment in
pCR2.1-TOPO This study
pCRhrpG hrpG partial fragment in pCR2.1-TOPO This study pCRhrpX hrpX partial fragment in pCR2.1-TOPO This study pCR1706 Alkanal monooxygenase partial fragment in
pCR2.1-TOPO This study
pCR1886 β-K-adipate enol-lactone hydrolase partial fragment in pCR2.1-TOPO
This study
pCR2370 Endopeptidase partial fragment in pCR2.1-TOPO
This study
pCR2374 Polygalacturonase partial fragment in pCR2.1-TOPO
This study
pCR2534 Conserved hypothetical protein partial fragment in pCR2.1-TOPO
This study
pCRhrpW hrpW partial fragment in pCR2.1-TOPO This study pCR3090 Leucine rich protein partial fragment in
pCR2.1-TOPO This study
pCRavrPphE2 avrPphE2 partial fragment in pCR2.1-TOPO This study
41
Table 2-2. Continued Strain or plasmid Relevant characteristics Source pCR3230 Actin-ADP-ribosylating toxin domain partial
fragment in pCR2.1-TOPO This study
pCR3309 Aminopeptidase partial fragment in pCR2.1-TOPO
This study
pCR4074 Ribonucleotide-disphosphate reductase partial fragment in pCR2.1-TOPO
This study
pCR4090 3-oxoacyl-[ACP] reductase partial fragment in pCR2.1-TOPO
This study
pCRxopQ xopQ partial fragment in pCR2.1-TOPO This study pCRb0011 avrPphE3 partial fragment in pCR2.1-TOPO This study pL22 Xcc strain Aw pLAFR3 cosmid that contains
hrpX, hrpG and hsp90Xo homologues Rybak et al. (2009)
pO∆Harpin HrpW, RifR This study pO∆HrpW Harpin, RifR This study pCR3309 Aminopeptidase partial fragment in pCR2.1-
TOPO This study
pCR4074 Ribonucleotide-disphosphate reductase partial fragment in pCR2.1-TOPO
This study
a BRL, Bethesda Research Laboratories, Gaithersburg, MD. b UB, U. Bonas, Martin Luther-Universität, Halle, Germany.
42
Table 2-3. Oligonucleotide sequences used in this study Gene Primer name Primer sequence length avrBs2 avrBs2F1 5’-CGCATCATCTTCAATCTGCAGC-3’ 22
a PIP box sequence and the distance in base pairs between the two conserved motif. Nucleotides deviating from the consensus are underline. b Distance in base pairs between the end of PIP box and the – 10 promoter motif c – 10 promoter motif sequence and – 10 conserved base pairs are shown in bold. Nucleotides deviating from the consensus are underline.
45
Table 2-5. Xanthomonas citri pv. citri mutants pathogenicity phenotype in grapefruit leaveas and HR elicitation in tomato leaflets
a Bacterial Pathogenicity: (+) positive for symptoms development, (-) no symptoms. b HR induction: (+) positive for HR, (-) negative for HR.
46
Figure 2-1. Genetic organization of the hrp genes of X. citri subsp. citri. The arrows indicate the
orientation of the six hrp operons, hrpA to hrpF. The boxes correspond to open reading frames (ORFs). hrc genes encode proteins conserved among type III secretion system; hrp and hpa genes encode non-conserved proteins (da Silva et al., 2002; Dunger et al., 2005).
47
Figure 2-2. Proposed model for hrp gene regulation cascade in X. citri subsp. citri. The model
was formulated based on the X. axonopodis pv. citri srain 306 genome sequence (da Silva et al., 2002) and the model presented by Büttner and Bonas 2002 for hrp gene regulation in X. campestris pv. vesicatoria. The cascade is initiated with an uncharacterized signal in the bacterial envelope (indicate by a question mark) that senses the outside stimuli and activates hrpG. Consequently, HrpG activates the expression of hrpX and hrpA, and HrpX activates the expression of hrpB-hrpF, xop genes, hrpW (which is part of the hrp cluster although is located downstream) and other hrpX-regulon genes.
48
Figure 2-3. Comparison of the bacterial pathogenicity and HR elicitor in grapefruit leaves and tomato leaflets, respectively. The strains were infiltrated into the leaves at concentrations of 5 x 108 CFU/mL. Labeling: (1) 306::ΩhrpG-comlemented; (2) 306:: ΩhrpG; (3) Xcc strain 306 – wild type; (4) 306:: ΩhrpX; (5) 306:: ΩhrpX-comlemented; (6) 306:: ΩhrpA; (7) 306:: ΩhrpG. The grapefruit leaves were photographed 7 days after infiltration and the tomato leaf (right) was photographed 24 h after infiltration.
49
Figure 2-4. Integration by site-specific recombination using one single target sited. Integration of the complete linearized plasmid is mediated by single crossing-over event. Amp = Ampicillin resistance cassette, Km = kanamycin resistance cassette.
50
CHAPTER 3 IDENTIFICATION OF A HRP-INDEPENDENT HR ELICITOR FROM Xanthomonas citri
subsp. citri
Introduction
Plants are frequently under attack by pathogens. Plants are resistant to most pathogens, and
the responses to pathogen attack involve different innate immunity responses. Innate immunity
responses can be initiated by a local infection where the defense response extends to unaffected
parts, a response called induced systemic resistance (ISR) or systemic acquired resistance (SAR)
(Hunt et al., 1996; Hammerschimidt, 1999). The elicitation of plant innate immunity by bacteria
is sometimes divided, conceptually, into two type of elicitation. PAMP triggered immunity
(PTI) involves the induction of defense response by a variety of common molecular components
of bacteria known collectively as pathogen associate molecular patterns. Effector triggered
immunity (ETI) involves the triggering of defense by effector proteins of the type III secretion
pathway, which are present in many plant and animal pathogenic proteobacteria. Innate
immunity involves the activation of mitogen-activated protein kinase (MAPK) cascades and
synthesis of hormones such as salicylic acid (SA) or jasmonic acid (JA) (Block et al., 2008). The
bacterial pathogens also trigger the production of an oxidative burst, which, ultimately, limits
pathogen spread by induction of host cell death (Baker and Orlandi, 1995; Gozzo, 2003; Block et
al., 2008) and leads to changes in the cell wall composition and the de novo synthesis of
antimicrobial compounds such as phytoalexins and pathogen-related (PR) proteins (Bestwick et
al., 1998; Hammerschimidt, 1999b; Innes, 2001). Other responses include closure of stomata in
response to bacterial attack (Melotto et al., 2006); and the hypersensitive reaction (HR) around
the initial infection sites isolating the pathogen in dead cells, consequently, preventing the spread
throughout the plant (Alfano and Collmer, 2004).
51
Type III secretion systems (T3SS) function to translocate a collection of effectors (T3-
effectors) into the plant cells, where the T3-effectors modulate the cell environment and enhance
the conditions for bacterial multiplication (Cornelis and van Gijsegem, 2000). The genes
encoding most of the components for the T3SS are clustered into the hypersensitive reaction and
pathogenicity (hrp) gene regions (Büttner and Bonas, 2003). Basically, hrp gene clusters in plant
pathogenic bacteria, including Xanthomonas species, are regulated and dependent on the host
interaction. In Xanthomonas species, two key regulatory proteins are HrpG and HrpX
(Wengelnik and Bonas, 1996). In R. solanacearum, the hrp system is regulated through a HrpG
homolog and, downstream, a HrpX homolog originally named HrpB. HrpB and HrpX belong to
to the AraC family of transcriptional regulators (Wengelnik and Bonas, 1996; Brito et al., 1999).
P. syringae possesses three regulatory genes hrpR, hrpS and hrpL. The products of two genes,
HprR and HrpS, positively regulate the expression of hrpL, which encodes an alternative sigma
factor responsible for transcriptional activation of other hrp genes (Xiao et al., 1994). In
addition, homologs of hrpS and hrpL also occur in Erwinia amylovora (Sneath et al., 1990; Wei
et al., 1992).
In 1971, Flor demonstrated that a component from the bacteria, avirulence (avr) gene,
when recognized by the host triggered a defense response. Later, studies showed that T3SS was
the system used by the bacteria to inject the avr genes into the host cells (T3-effectors) and the
recognition was performed by host resistance protein. A malfunction of the T3SS generally
results in the complete loss of disease development and the loss of ETI or Hrp-dependent
elicitation of resistance responses due to the inability to transport the avr protein into the host
(Jones and Dangl, 2006; Block et al., 2008). The first avirulence gene, avrA, was isolated from
P. s. pv. phaseolicola in 1984 (Staskawicz et al., 1984). More than 40 Avr proteins have since
52
been identified. The first direct interaction between an Avr protein and the cognate R protein was
demonstrated with the tomato bacterial speck bacterium P. s. pv. tomato harboring the gene
avrPto, whose product is recognized by the tomato resistance protein Pto (Tang et al., 1996;
Mucyn et al., 2006). Additionally, Pto also recognizes another distinct Avr protein from P. s. pv.
tomato – known as AvrPtoB (Abramovitch et al., 2003,Wu et al., 2004). AvrXa27 in X. oryzae
pv. oryzae also induces resistance in rice plants carrying the cognate R gene Xa27 but the
resistance is induced only in the presence of the pathogen (Gu et al., 2005). PTI, on the other
hand, is the recognition of PAMPs by receptor-like kinases, which are localized in the
extracellular matrix (Jones and Dangl, 2006). PTI response is not generally considered to be a
race-specific defense. Therefore, ETI requires the T3-effectors and is essentially T3SS-
dependent, and PTI is T3SS-independent and is generally not accompanied by an HR.
In chapter 2 was demonstrated that hrpG and hrpX mutants of Xanthomonas citri subsp.
citri strain 306, the causal agent of citrus canker (Stall and Civerolo, 1991), retained the
capability to trigger an HR in the non-host plant tomato, indicating that the HR was T3SS-
XAC3859 exihibited high homology to D-alanyl-D-alanine dipeptidase precursor, including to
D-alanyl-D-alanine dipeptidase precursor (92% identity and 95% similarity) from Xcv strain 85-
10; D-alanyl-D-alanine dipeptidase (82% identity and 89% similarity) from Xoo strain POX99A;
D-alanyl-D-alanine dipeptidase (84% identity and 91% similarity) from X. o. pv. oryzicola strain
BLS256; and vanX (72% identity and 79% similarity) from X. c. pv. campestris strain B100.
Additionally, this gene is part of VanY superfamily, which is related to vancomycin resistance
(Evers and Courvalin, 1996; Bussiere et al,. 1998; Lee et al., 2000). We also search for the
presence plant-inducible promoter box (PIP box) sequence upstream of the XAC3859, which
was not found. Moreover, the translated protein sequence showed certain level of homology with
other Xanthomonas spp. (Figure 3-8). Based on the DNA sequence of B38-3 subclone, where the
tree genes described above are presented, and the transcriptional organization (Figure 3-9), we
can speculate that these genes could be part of an operon due the fact that all the genes are the
same direction.
Discussion
Besides the fact that the hrp-regulators share a degree of homology among some important
plant pathogenic bacteria, most hrp-regulatory mutants are totally impaired with disease
symptoms, bacterial growth and HR elicitation (Wei et al., 1992; Xiao et al., 1994; Wengelnik et
al., 1995; Wengelnik and Bonas, 1996; Brito et al., 1999; Sneath et al., 1990). In this study, the
ability of Xcc to induce symptoms on highly susceptible grapefruit was dependent on a
functional T3SS, which corroborates with the previous studies mentioned above. However, the
ability of 306::ΩhrpG and 306::ΩhrpX mutants to induce an HR on non-host plants is unusual
with respect to hrp mutants. The infiltration of 306::ΩhrpG and 306::ΩhrpX mutants into non-
host tomato plants induced strong hypersensitive responses. This phenotype was reported with a
63
hrcC mutant on P. syringae pv. tabaci, which completely lost pathogenicity on tobacco leaves,
but still induce a strong HR in non-host tomato plants. The HR-elicitor for the hrcC mutant was
determined to be flagellin protein (Marutani et al., 2004).
A parallel study in this laboratory demonstrated that a clone, pXfa450, from X. fuscans pv.
aurantifolli (Xfa) genomic library elicited an HR in tomato plants in the T3SS-independent
manner. Speculating that the genome sequence between X. citri subsp. citri and X. f. pv.
aurantifolli are similar, we used the 450 clone to identify the unknown T3SS-independent HR-
elicitor from X. f. pv. aurantolli. In the search for the Xcc T3SS-independent HR clone, a series
of subclones from the 450 cosmid were isolated into X. perforans strain 91-118 hrp mutant
(91Ωhrp). A 3.0 Kb subclone was identified and called B38-3, which was able to elicit the
nonhost HR phenotype. The sequence analysis of the B38-3 demonstrated that two ORFs,
XAC3857 and XAC3858, are homologous to conserved hypothetical proteins in other
Xanthomonas spp. but have no association with known HR-elicitor was observed. The third
gene, XAC3859 contains a conserved domain, which is part of the VanY superfamily and
involved in resistance to antibiotic, vancomycin (Evers and Courvalin, 1996; Bussiere et al,.
1998; Lee et al., 2000); and it is highly conserved in other Xanthomonas spp. This implies that
this phenotype, probably, is not connected with flagella, or flagella-related proteins. Moreover,
the transcriptional region where the genes are located showed that all 10 genes possess the same
orientation. This may indicate that these genes are part of an operon and the phenotype is linked
to two or more genes, instead of a single gene. However, in order to confirm this hypothesis
mutational analysis, creating a polar affect, must be performed.
64
Table 3-1. Bacterial strains and plasmids used in the study Strain or plasmid Relevant characteristics Source Strains Xanthomonas citri subsp. citri 306 Wild type, Asiatic strain, isolated in
Brazil, RifR DPIa
306::ΩhrpG hrpG , single recombinant of pCRhrpG, KnR, AmpR
This study
306::ΩhrpX hrpX , single recombinant of pCRhrpX, KnR, AmpR
This study
ΩhrpG::XV9 hrpG , complemented with pXV9, KnR, AmpR, TcR
This study
ΩhrpX::XV9 hrpX , complemented with pLHrpG/X, KnR, AmpR, TcR
This study
Xanthomonas perforans 91-118 Wild type, pathogenic to tomato, RifR Jones et al.
(2004) 91::450 91-118 carrying the 450 cosmid This study 91Ωhrp::450 91-118 defective T3SS carrying the 450
cosmid This study
91Ωhrp::Bs3 91-118 defective T3SS carrying a AvrBs3 homolog
This study
91::B38 91-118 carrying the B38 plasmid This study 91:: Ωhrp 91-118 defective T3SS This study 91Ωhrp::B38 91-118 defective T3SS carrying the B38
plasmid This study
91::B38-3 91-118 carrying the B38-3 plasmid This study X. fuscans pv. aurantifolli Xfa-C 5979 Xc 70, isolated in Brazil DPI Opportunistic xanthomononad INA42 Isolated in orange R. E. Stallb
INA42::B38 4.5 Kb Xaa fragment cloned into BamH I pUF034
pUFR034 KnR, Tn903, IncW, Mob+ De Feyter et al. (1990)
pCRhrpG hrpG partial fragment in pCR2.1-TOPO This study pCRhrpX hrpX partial fragment in pCR2.1-TOPO This study pCR3857 3857 ORF and endogenous promoter in This study
65
Table 3-1. Continued pCR2.1-TOPO pCR3859 3859 ORF and endogenous promoter in
pCR2.1-TOPO This study
pXV9 ~25kb of hrp cluster of X. c. pv. vesicatoria 75-3 cloned in pLAFR3
Bonas et al. (1991)
pXfa450 32kb Xaa fragment cloned into pLAFR3 G. Minsavaged
B38 4.5 Kb Xaa fragment cloned into BamH I pUF034
This study
B38.3 3.0 Kb Xaa fragment cloned into BamH I and EcoR I pUF034
This study
B38.1 1.5 Kb Xaa fragment cloned into BamH I and EcoR I pUF034
This study
a DPI, Division of Plant Industry of the Florida Department of Agriculture and Consumer Services, Gainesville, FL, USA. b R.E. Stall, Robert E. Stall, University of Florida, Gainesville, FL. c BJS, B. J. Staskawicz, University of California, Berkley, CA. d G. Minsavage, Gerald Minsavage, University of Florida, Gainesville, FL.
66
Figure 3-1. Phenotype of grapefruit leaves were used to distinguish among the strains. Strains were infiltrated into the leaves at concentrations of 5 x 108 CFU/mL. Labeling: (1) ΩhrpG::XV9; (2) 306::ΩhrpG; (3) Xcc strain 306 – wild type; (4) 306::ΩhrpX; (5) ΩhrpX::XV9. The grapefruit leaves were photographed 7 days after infiltration.
67
Figure 3-2. Phenotype of 306 wild type, 306::ΩhrpG and 306:: ΩhrpX in leaflet of tomato cv Bonny Best. The strains were hand-infiltrated into the leaflets at concentrations of 5 x 108 CFU/mL. The tomato was photographed 24 h after infiltration.
68
Figure 3-3. Hypersensitive response assay in tomato cv. Bonny Best and pepper cv. ECW-20R (A) left: 91Ωhrp::450; right: 91::450. (B) left: 91Ωhrp::450; right-down: 91-118 – wild-type; right-up: 91Ωhrp. The strains were infiltrated into the leaves at concentrations of 5 x 108 CFU/mL. The tomato and pepper leaves were photographed 24 h after infiltration.
69
A
B Figure 3-4. Electrolyte leakage in tomato plant is changed in WT::pLAFR3, compatible
interaction, and WT:: 450, incompatible interaction. (A) Comparisson of ion leakage among compatible interaction WT::pLAFR3; WT::pAvrBs3 (carrying a AvrBs3 homolog) and WT:: 450. (B) Comparisson of ion leakage among incompatible interaction hrp-Mut::pLAFR3; hrp-Mut::pAvrBs3and hrp-Mut::450. The bacterial suspension was infiltrated at concentration of 3 x 108 CFU/ml.
050
100150200250300350400450
0 24 48
Mic
rom
hos
Hours after infiltration
Electrolyte Leakage - Wild-Type
WT + pLAFR3
WT + pAvrBs3
WT + pL450
0
50
100
150
200
250
0 24 48
Mic
rom
hos
Hours after infiltration
Electrolyte Leakage - Hrp Mutant
hrp-Mut + pLAFR3
hrp-Mut + pAvrBs3
hrp-Mut + pL450
70
Figure 3-5. Subclone, B38, triggers hypersensitive response (HR) independent of T3SS in X. perforans in non-host plants, tomato cv. Bonny Best and pepper cv. ECW20R. (A) left: 91::38 (open arrow); right: 91-118 wild-type (open arrow); left: 91Ωhrp::B38 (close arrow); right: 91::Ωhrp (close arrow). (B) left: 91Ωhrp::B38 (open arrow); right: 91::Ωhrp (open arrow); left: OPP::B38 (close arrow) ; right: OPP-INA42 (close arrow). (C) 1. 91-118 wild-type; 2. 91::B38; 3. 91::Ωhrp; 4. 91Ωhrp::B38; 5. OPP-INA42; 6. OPP::B38. The strains were infiltrated into the leaves at concentrations of 5 x 108 CFU/mL. The tomato and pepper leaves were photographed 24 h after infiltration.
Figure 3-6. Protein alignment of XAC3857 gene and Xanthomonas spp. (A) Original protein sequence of the ORF XAC3857 from X. citri subsp. citri strain 306; (B) Protein alignment among X. citri subsp. citri strain 306 (XCC306), XAC3857 clone (3857), X. campestris pv. vesicatoria strain 85-10 (XCV85-10), X. oryzae subsp. oryzicola strain BLS256 (XOOBLS), X. oryzae subsp. oryzae strain POX99A (XOO99), and X. campestris subsp. campestris strain 33913 (XCC333913). Asterisks indicate that the amino acids in that column are identical in all sequences. Colon shows that conserved amino acids substitutions have been observed. Dot means that semi-conserved substitutions are observed.
sequence of the ORF XAC3858 from X. citri subsp. citri strain 306; (B) Protein alignment among X. citri subsp. citri strain 306 (XCC306), X. campestris pv. vesicatoria strain 85-10 (XCV85-10), X. oryzae subsp. oryzae strain POX99A (XOO99), X. oryzae subsp. oryzicola strain BLS256 (XOOBLS), X. campestris subsp. campestris strain 8004 (XCC8004), and X. campestris subsp. campestris strain 33913 (XCC333913). Asterisks indicate that the amino acids in that column are identical in all sequences. Colon shows that conserved amino acids substitutions have been observed. Dot means that semi-conserved substitutions are observed.
sequence of the ORF XAC3859 from X. citri subsp. citri strain 306; (B) Protein alignment among X. citri subsp. citri strain 306 (XCC306), X. campestris pv.
74
Figure 3-8.Continued vesicatoria strain 85-10 (XCV85-10), X. oryzae subsp. oryzae strain POX99A (XOO99), X. oryzae subsp. oryzicola strain BLS256 (XOOBLS), X. campestris subsp. campestris strain B100 (XCCB100), and X. campestris subsp. campestris strain 33913 (XCC333913). Asterisks indicate that the amino acids in that column are identical in all sequences. Colon shows that conserved amino acids substitutions have been observed. Dot means that semi-conserved substitutions are observed.
75
Figure 3-9. Transcriptional organization of the ORFs XAC3857 to XAC3859 region in
Xanthomonas citri subsp. citri. The region contains11 genes. The arrowheads indicate direction of transcription.
76
CHAPTER 4 GENETIC CHARACTERIZATION OF HRPW OF Xanthomonas citri subsp. citri
Introduction
The Gram-negative bacterium, Xanthomonas citri pv. citri (Xcc), is a plant pathogen that
causes citrus canker diseases in most citrus cultivars (Stall and Civerolo, 1991; Schaad et al.,
2006). Many pathogenic proteobacteria rely on the type III secretion system (T3SS), encoded by
hypersensitive reaction and pathogenicity (hrp) genes, to secrete bacterial effector proteins into
the extracellular milieu and translocate them into the eukaryotic host cell cytosol and
consequently modulate host physiology (Chang et al., 2004; Tang et al., 2006). To deliver these
T3-effector proteins into host cells three biological barriers must be crossed, the membranes in
the host and the inner and outer bacterial. This process involves the T3SS pore-forming
translocon complex (Cornelis, 2006). In Xanthomonas spp. the hrpF is a typical translocon
component involved in pore-formation in the planar lipid bilayers but it is not required for
secretion (Rossier et al., 2000; Buttner et al., 2002). Moreover, pathogens carrying a defective
translocon are impaired in T3-effectors translocation, thus, inhibiting disease progress (Rossier et
al., 2000; Sugio et al., 2005; Cornelis, 2006)
Lorenz et al. (2008) classified the T3S substrates in two groups: (I) extracellular
components of the secretion apparatus, and (II) effector proteins that are translocated into the
host cell modifying cells metabolism (He et al., 2004; Mudgett, 2005). One class of proteins that
crosses the Hrp type III secretion apparatus is composed of the harpin proteins, which are T3SS-
dependent and secreted in the extracellular milieu during interaction (Perino et al., 1999;
Tampakaki and Panopoulos, 2000). Harpin proteins are unique to phytopathogens and do not
show homology to any other known protein, heat stable, glycine rich and lack of cysteine
residues. Importantly, harpins trigger hypersensitive response (HR) cell death in non-host plants
77
and may contribute to effector translocation (Wei et al., 1992; He et al., 1993; Arlat et al., 1994;
Charkowski et al., 1998; Kim et al., 2004; Kvitko et al., 2007). Additionally, in recent studies
the harpin from Erwinia amylovora (HrpN) is weakly translocated into tobacco cells (Bocsanczy
et al., 2008); purified harpin modulates the accumulation of salicylic acid (Clarke et al., 2005);
induces activation of mitogen-activated protein kinases (MAPKs) (Desikan et al., 2001),
elevation of cytosolic Ca2+ (Cessna et al., 2001), activation of active oxygen species (AOS) and
ion flux modulation (Reboutier et al., 2007).
Since the first harpin, HrpN, was isolated from E. amylovora (Wei et al., 1992), many
harpin-like proteins or genes have been identified from diverse plant pathogenic bacteria,
including Hpa1 from X. oryzae pv. oryzae (Zhu et al., 2000), HpaG from X. a. pv. glycines and
X. c. pv. vesicatoria (Kim et al., 2003; Thieme et al., 2005), and PopA1 from R. solanacearum
(Arlat et al., 1994), One of the most widely distributed members of the harpin-like protein group
is HrpW. HrpW was first described in E. amylovora, and subsequently identified in P. s. pv.
tomato and Xcc (Charkowski et al., 1998; Kim and Beer 1998; da Silva et al., 2002). HrpW
contains a C-terminal domain homologous to class III pectate lyase (PEL) and an N-terminal
harpin-like domain. While located within the respective hrp gene clusters of E. amylovora and P.
syringae, the hrpW gene of Xcc strain 306 is located downstream of the hrp cluster (da Silva et
al., 2002). The role of harpin-like proteins, including HrpW, play in bacterial pathogenisis or
whether or not HrpW is even required for pathogenesis in Xcc is unknown. Therefore, a genetic
and biochemical analysis of the hrpW gene of Xcc was undertaken. Additionally, because it was
thought that hrpW was important, was hypothesized that hrpW is conserved among diverse X.
citri strains. To address this hypothesis hrpW sequences of X. citri subsp. citri were produced
and compared along with similar sequences derived from different X. citri strains.
78
Material and Methods
Bacterial Strains and Plasmids
Bacterial strains and plasmids used in this study are listed in Table 4-1.
Media and Growth Conditions
Escherichia coli cells were grown in at 37oC in Luria-Bertani (LB) medium (1% (w/v)
tryptone, 0.5% (w/v) yeast extract, 1% (w/v) sodium chloride, pH 7.5). For solid media BD
Bacto agar was added at 15 g l-1. Xanthomonas strains were cultivated at 28oC on nutrient agar
(NA) medium and on tryptone sucrose agar (Zhu et al., 2000). All bacterial strains used in this
study were stored in 20% glycerol in sterile tap water and maintained at -80oC. Triparental
matings were performed on nutritent-yeast extract-glycerol (NYG) agar (Daniels et al., 1984).
Antibiotics were used in the following concentrations: ampicillin (Amp) 100 µg ml-1; kanamycin
Grapefruit cv. Duncan (Citrus paradisi) plants were grown from seed in 15 cm plastic pots
in Terra-Lite® agricultural mix (Scott Sierra Horticultural Products Co. Marysville, OH). The
plants were kept in the glasshouse of the University of Florida in Gainesville, Florida at
temperature ranging from 25-30oC.
Pepper plants (Capsicum annuum) cv. ECW and the near isogenic line ECW20R
containing the Bs2 resistance gene were planted from seeds in Plugmix (W. R. Grace & Co.,
Cambridge, MA, USA). After two weeks, the seedlings were transferred to Metromix 300 (W. R.
Grace & Co) in 10 cm plastics pots. The plants were kept in the glasshouse of the University of
Florida in Gainesville, Florida at temperature ranging from 25-30oC.
Plant Inoculations
All the Xanthomonas spp. and mutants were cultured on nutrient agar plates for 18 h at
28oC. For preparation of bacterial suspensions, 18 h cultures were harvested from the NA plates
and suspended in sterile tap water, and standardized to an optical density at 600 nm (OD600) =
0.3 (5 x 108 CFU/ml) with a Spectronic 20 Genesys spectrophotometer (Spectronic-UNICAM,
Rochester, NY, USA).
For pathogenicity tests, bacterial suspensions of the wild-type, mutant and complemented
Xcc were infiltrated at 5 x 108 CFU/ml into abaxial surface of citrus leaves by using hypodermic
syringe and needle. Plant responses were evaluated after 3-4 days for water-soaking and 6-7 days
for citrus canker symptoms. The inoculated plants were kept in a growth room at 28-30oC up to
10 days after inoculation.
The sensitivity of the Xcc wild-type and its ∆hrpW mutants was evaluated with pin-prick
inoculation in grapefruit leaves. Bacteria were suspended in sterile tap water at a concentration
of 5 x 108 CFU/ml. A drop of the suspension was placed on the adaxial surface of young
84
grapefruit leaf. Immediately after a needle was pierced through the bacterial suspension and
through the leaves, the inoculum drops were wiped off with sterile cotton. The inoculated plants
were then grown in a glasshouse up to 30 days. The diameter of circular lesions, including cork
tissue and water-soaked margin, was measured after inoculation.
Elicitation of HR was tested by infiltrating bacterial suspensions, which were prepared
with sterile tap water at concentration of 5 x 108 CFU/ml, into leaflets of tomato cv. Bonny Best
and pepper plants cv. ECW and ECW20R with a hypodermic syringe and needle. The inoculated
plants were incubated in a growth room at 24-28oC and assessed 24 h after infiltration for HR
induction. Score are the means of those for three leaves. All the experiments were repeated at
least three times.
Bacterial Populations
Bacterial suspensions with 5 x 108 CFU/mL were infiltrated in grapefruit leaves with a
syringe and 27-gauge needle (Klement, 1963). A 0.5 cm2 of inoculated area from each leaf, each
sample was infiltrated in three leaves, was excised and macerated in 1 mL of sterile tap water, 50
µL were spread onto NA plates. The plates were incubated at 28oC for 2 – 3 days, thus, the
colonies were counted.
Translocation Activity in 306::∆harpin Mutant
A pL799 clone, carrying avrGf1 gene from Xcc strain Aw that triggers HR in grapefruit
(Rybak et al., 2009), was conjugated by triparental mating into Xcc wild-type and HrpW∆harpin
mutant to investigate if the mutation on the harpin domain has any affect in the T3SS
functioning. The transconjugant 306::799 and ∆harpin::799 were inoculated at concentration of 5
x 108 CFU/mL in grapefruit and kept at growth room at 28oC. The plants were assessed for HR
induction up to 5 days after infiltration.
85
Construction of hrpW::avBs2 Fusion
For translocation studies, pepper plants cv. ECW and ECW-20R were infiltrated with X.
campestris pv. vesicatoria strain XV1922 (wild type), 1922::WBs (pUWBs) leading to the
expression of the first 109 amino-acids from hrpW gene fused with avrBs2 C-terminal with 62 –
574 amino acids. The strains were resuspended to an OD600 of 0.3 and infiltrated into the
mesophyll of pepper leaves. 24 hr after infiltration, the inoculated pepper plants were assessed
for HR elicitation.
The plasmid was constructed as follows. First, 327 bp fragment was amplified using the
primers NTW-F1 and NTW-R1 introducing a Bgl II site in both ends. The PCR fragment was
excised from the agarose gel 1% and purified with QIAquick Gel Purification Kit system
(QIAGEN). The digested PCR fragment was cloned into pBS1 (BglII::avrBs262-574::HA) plasmid
resulting in pBWBs. For construction of pUWBs plasmid, pBWBs was digested using BamH I
and Kpn I and ligated into the expression vector pUFR034 digested with the same restriction
enzymes. The resulting plasmid pUWBs was used to transform X. c. pv. vesicatoria strain
XV1922 by triparental matings. Furthermore, the selected transconjugants were analyzed for
induction of HR in pepper leaves.
Results
Xanthomonas citri subsp. citri hrpW Phylogeny
Comparison of phylogenies from sequences of conserved chromosomal genes is an
effective method to evaluate the evolution among different pathovars in the same genus. In this
study, we analyzed the full sequence of hrpW gene from Xcc strain 306 with strains collected
world-wide and Florida State, and plant pathogenic bacteria that carry hrpW homologous
sequences. The neighbor joining (NJ) tree obtained via analysis of the hrpW sequence (Figure 4-
1) shows that the genomospecies of X. citri, in general, cluster together very well, with the
86
exception of two X. citri strains XS2003-0004 and XS1997-00018 (represented in the tree as
XC04 and XC018, respectively). However, sequence alignment analysis revealed that these two
strains, XS2003-0004 and XS1997-00018, show a certain level of nucleotide similarity (Figure
4-2). The close similarity between hrpW from Xcc strain 306 and X. c. pv. campestris strain
33913 and E. amylovora has already been demonstrated by da Silva et al. (2002). Meanwhile,
the phylogenies of R. solanacearum strain UW551 (UW551), Acidovorax avenae subsp. citrulli
strain AAC00-1 (AAC00), Pseudomonas viridiflava (PV), and Erwinia tasmaniensis strain
Et1/99 indicate that hrpW sequence is also conserved in these strains (Figure 4-1).
The maximum parsimony (MP) analysis of the hrpW sequence set yielded one
parsimonious tree, which was virtually identical to NJ tree, except for the positions of taxa
within two strains, R. solanacearum strain UW551/ Acidovorax avenae subsp. citrulli strain
AAC00-1 (AAC00). The maximum likelihood (ML) tree was identical to the NJ tree, with the
exception that the positions of Acidovorax avenae subsp. citrulli strain AAC00-1 (AAC00) and
Pseudomonas viridiflava (PV) to E. amylovora and Erwinia tasmaniensis strain Et1/99. The
hrpW phylogeny data in the NJ tree were supported by the bootstrap values of 100 in both the
ML analysis and the MP analysis.
Total Deletion of hrpW Gene Is Irrelevant for Xcc Pathogenicity and HR in Non-Host Plants
To determine the relevance of hrpW gene for the pathogenicity for Xcc strain 306 in a
compatible and incompatible reaction, deletion in the 0.9 kb coding sequence of hrpW was
generated using Aat II restriction enzyme. One clone, 306::∆hrpW, representing a hrpW full
length deletion was selected for further characterization. Using sequence alignment we showed
the precise location of the mutations in the HrpW (Figure 4-3). Grapefruit leaves were infiltrated
with bacterial suspensions of 5 x 108 CFU/ml and the generation of symptoms was assessed 7
87
days after inoculation. 306::∆hrpW mutant induced typical canker symptoms on grapefruit at 7
days postinoculation, additionally, the symptoms incited by the mutant are macroscopically
identical with the Xcc wild type (Figure 4-4). Xcc strain 306 induced hypersensitive response
(HR) in tomato plants. In order determine if the mutation in hrpW affect Xcc HR activity, we
inoculated tomato (Bonny Best) and pepper (ECW and ECW-20R) leaves with bacterial
suspensions of 5 x 108 CFU/ml and observations of macroscopic tissue collapse typical of HR
were made 24 h postinoculation. At this concentration, both strains Xcc wild type and
306::∆hrpW induced an HR. Moreover, no difference was noted in the bacterial population
among the strains tested in this experiment (Figure 4-5).
Harpin Domain in hrpW Gene is Required for Pathogenicity
In order to explore the effect of a deletion in the harpin domain of hrpW gene may have on
pathogenicity and HR elicitor activity of Xcc, we used deletion mutagenesis to generate harpin
mutant, W∆harpin, which 87 bp were deleted (Figure 4-3). The HR activity of the W∆harpin
mutant was the same as that of wild type hrpW. Surprisingly, the W::∆harpin mutant showed a
severe reduction on the ability to induce symptoms compared with the Xcc-306 (Figure 4-4). The
W::∆harpin mutant phenotype was restored when we replaced the deleted harpin portion, using
pOHrpW-EP, by the harpin wild type region in cis. Considering the fact that 306::∆hrpW mutant
did not affect the pathogenicity and W::∆harpin blocked the progress of symptom development
in grapefruit, we may propose that this phenotype is given by the expression of a defective HrpW
protein, which may destabilize some protein interaction necessary for the disease progress.
Although the W::∆harpin mutant is affecting the phenotype in a compatible reaction, no change
in the W::∆harpin mutation population was observed compared with the Xcc wild type or the
306::∆hrpW mutant (Figure 4-5).
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Complementation of HrpW::∆harpin Mutant In-Cis
The first attempts in recovery the W::∆harpin mutant in trans using pUHrpW-EP, which
carry the hrpW gene drive by it native promoter, were unsuccessful. In order to demonstrate a
non-polar mutation effect in W∆harpin mutant, we complemented the W∆harpin mutant using a
0.9 kb PCR fragment from Xcc containing the full length hrpW gene, and cloned into the pOK1
suicide vector, pOHrpW-EP. Inoculations performed in grapefruit leaf showed very similar
symptoms with ∆harpin::harpin complemented and Xcc wild type (Figure 4-4). The results
reported lead us to speculate that this incapability to restore the pathogenicity in trans is due to a
dominant-negative effect of the defective protein, in cis, exerted in the wild type protein
expressed in trans. To determine if our theory is correct, we conjugated the pU∆harpin plasmid
in Xcc strain 306, creating 306::∆harpin(T) transconjugant, that expresses the defective harpin
domain in trans. Therefore, if the expressed defective ∆harpin protein is negatively dominant in
cis, this dominance should be maintained when the defective protein is expressed in trans.
Grapefruit leaves were inoculated by pin-prick method with Xcc wild type, W::∆harpin,
306::∆harpin(T), ∆harpin::harpin and 306::∆hrpW. All strains tested showed typical canker
lesion formation 28 days postinoculation (Figure 4-6). The mutations and complemented mutant
were confirmed by size PCR-fragment (Figure 4-7). The results demonstrated that the loss of
pathogenicity by the W∆harpin mutant it is not caused by negative dominance effect. Therefore,
more experiments need to be performed to explain the role of harpin domain for Xcc virulence.
Translocon Machinery, T3SS, Is Not Affected by ∆harpin Mutant in Xcc
The effect delete of W::∆harpin mutant may have on the ability of Xcc strain 306 to deliver
protein effectors through the T3SS was investigated using pL799 clone, which contain the
avirulence gene, avrGf1, that triggers HR in grapefruit and it is T3SS-dependent (Rybak et al.,
89
2009). The W::∆harpin transconjugant, W∆harpin::799, was infiltrated in grapefruit at 5 x 108
CFU/mL and compared with Xcc wild type strain with avrGf1 clone (pL799). The HR elicitation
was observed in both strains with the same intensity five days after inoculation.
HprW is not Translocated into the Mesophyll of Plant Cell
To determine the targeting of HrpW protein to host cells a fused protein was constructed
with HrpW N-terminal and C-terminal from AvrBs2 of X. c. pv. vesicatoria. The fused protein
was expressed in X. c. pv. vesicatoria strain XV1922, a avrBs2 defective strain, to test HrpW
translocation into pepper leaves. As a positive control we used the XV1922-1 clone, which
contains the avrGf11-106::avrBs262-574 and gives HR in pepper cv ECW-20R. The inoculation
assay shows no HR induction after 24 hours, which means that HrpW protein is not translocated
into the plant cells.
Discussion
HrpW was first identified in E. amylovora (Kim and Beer, 1998). Thereafter, hrpW was
reported in many phytopathogenic bacteria (Gaudriault et al., 1998; Charkowski et al., 1998; da
Silva et al., 2002; Shrestha et al., 2004) including xanthomonads. Therefore, we used the hrpW
sequence from Xcc strain 306 (da Silva et al., 2002), to establish the diversity among several
Xcc strains collected world-wide (Table 4-1). The phylogram of the Xcc strains indicates very
high level of sequence conservation among all Xcc strains analyzed in this study and that it is,
somewhat, less conserved among the other phytopathogenic bacteria (Figure 4-6). With the
exception of Xcc strains XS2003-0004 and XS1997-00018, which diverge from the group
showing high similarity to hrpW sequence from Xcc strain 306. Although the dendrogram
demonstrated that these two strains branched differently from the other strains, the sequence
alignment showed a considerable level of similarity between these two specific strains (Figure 4-
2). The slight diversity reported in the XS2003-0004 and XS1997-00018 strains may be
90
explained due to a flexible element that could affect DNA similarity among pathovars in the
same species, e. g. transposable elements, which are often variable within strains of species
(Parkinson et al., 2007).
Taken together that hrpW is highly conserved among Xanthomonas citri strains and it
downstream location outside of hrp cluster reported by da Silva et al. (2002), we hypothesized
that hrpW might be involved in the Xcc strain 306 pathogenicity. Here, we show that the full
sequence of HrpW is not required for the Xcc pathogenicity and virulence, and neither, for HR
induction in non-host tomato plants. Similar results were reported with hrpW mutants from E.
amylovora and P. s. pv. tomato (Kim and Beer, 1998; Charkowski et al., 1998). Moreover,
previous reports demonstrated that HrpW of E. amylovora inhibits HR induced by the HrpN
(Reboutier et al., 2007), and the expression of hrpW in trans also blocked the HR induction by P.
fluorescens (Charkowski et al., 1998). Based on these reports, we can speculate that the lack of
HrpW in Xcc eliminates an unknown antagonistic interaction with other pathogenic factors.
Studies comparing HrpW protein from Xcc and X. c. pv. campestris with HrpW harpins of
Pseudomonas and Erwinia species, showed that the HrpW protein from both Xanthomonas did
not elicit HR in non-host plants. Surprisingly, the deletion of a few amino acids in the N-
terminal, which encodes the putative harpin domain, totally shutdown the Xcc virulence in
grapefruit plants but does not affect the bacterial population in planta and the ability to trigger
HR in non-host tomato plants. Although it is well known that harpins are HR-elicitors, their role
in bacteria pathogenicity is still unclear. Some of the harpins identified are directly linked with
pathogenicity such as, HrpN in E. amylovora and E. chrysanthemi (Wei et al., 1992), HrpN also
is involved in the translocation of T3-effector DspA/E (Bocsanczy et al., 2008) while HrpZ from
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P. s. pv. phaseolicola is associated with pore-forming activity that might be involved in nutrient
release and/or entry for bacterial effectors (Lee et al., 2001).
Intriguingly, the absence of the entire hrpW gene did not compromise the Xcc
pathogenicity in a compatible reaction in contrast what was observed with the deletion on the
harpin domain. We therefore suggest that the protein-protein interaction complex that HrpW
form with other hrp genes is disrupted due to change in the protein conformation expressed by
the ∆harpin mutant. Alegria et al. (2004) demonstrated that HrpW forms a protein complex with
three other Xcc proteins, HrpD6 and HrpB1, which are cytoplasmic proteins that shown
homology with proteins in X. c. pv. vesicatoria that are essential for pathogenicity and conserved
hypothetical proteins, respectively (Rossier et al., 2000) and HrpB4, although the interaction of
this third proteins is not physically demonstrated. Xcv HrpB4 protein is essential for
pathogenicity and T3-effectors translocation and not secreted or translocated, and shows
association with signal sensors (Rossier et al., 2002; Alegria et al., 2004). These findings support
our suggestion that a defective hrpW protein encoded by harpin mutant destabilizes this complex,
consequently, reducing the HrpB4 interaction or the sense of a specific signal. However, the
translocation of T3-effector, AvrGf1, into the host cell was not terminated in the harpin mutant.
Considering this fact, we may speculate that this phenotype was caused by the interruption or
reduction of the translocation of a specific crucial factor for pathogenicity. Our hypothesis is also
supported by recently studies showing that phytopathogenic bacteria carrying a defective harpin
protein impaired the translocation of several type III secretion system effectors (Kvitko et al.,
2007; Bocsanczy et al., 2008).
In addition, we have characterized the stability of the T3SS in the ∆harpin mutant and
translocation of HrpW from Xcv strain XV1922 to pepper plants. We show that the deletion on
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the harpin domain does not affect the T3-effector translocation inside of plant cells. Furthermore,
the use of AvrBs2 C-terminal, a Xcv T3-effector protein, as a reporter demonstrated that the
HrpW is not translocated into the mesophyll of host cells. However, using CyaA reporter system
demonstrated that HrpN, a harpin protein from E. amylovora, is slightly translocated into tobacco
plant cells (Bocsanczy et al., 2008). The lack of translocation reported here may be due the fact
that harpin domain in Xcc is smaller and has highly homology with the Pel domains of the
Pseudomonas and Erwinia than with harpin domains (Kim et al., 2004).
93
Table 4-1. Bacterial strains and plasmids used in the study Strain or plasmid Relevant characteristics Source Strains X. citri subsp. citri 290 Isolated from Saudi Arabia R. E. Stalla 112 Isolated from China R. E. Stall 131 Isolated from Maldive Islands R. E. Stall 106 Isolated from Australia R. E. Stall 46 Isolated from India R. E. Stall 257-2 Isolated from Thailans R. E. Stall 62 Isolated from Japan R. E. Stall 126 Isolated from Korea R. E. Stall 101 Isolated from Guam R. E. Stall XI2000-00194 Isolated from Florida DPIb
XS1999-0038 Isolated from Florida DPI X1999-12815 Isolated from Florida DPI XN03-2912 Isolated from Florida DPI XI3001-00098 Isolated from Florida DPI X2000-12878 Isolated from Florida DPI XI1999-00112 Isolated from Florida DPI XN03-11#3 Isolated from Florida DPI XS2003-0004 Isolated from Florida DPI XS1997-00018 Isolated from Florida DPI 306 Wild type, Asiatic strain, isolated in Brazil,
RifR DPI
306::∆ hrpW hrpW -, 855 bp deleted, RifR This study W::∆harpin Harpin -, 87 bp deleted, RifR This study 306::∆hrpG hrpG -, single recombinant of pCRhrpG, KnR,
AmpR, RifR This study
∆harpin::Harpin Harpin – complemented with pOHrpW-EP, RifR
This study
306::∆harpin(T) 306 carrying pU∆Harpin, KnR, RifR This study 306::799 306 carrying pL799, TcR, RifR This study X. c. pv. campestris XV1922 Pepper race 6, RifR Jones, J. B.c 1922::WBs strain XV1922 carrying pUWBs, KnR, RifR This study ∆harpin::799 Harpin – carrying pL799, TcR, RifR This study Escherichia coli DH5α FrecAϕ80dlacZ∆M15 Invitrogen
λPIR Host for pOK1; SpR oriR6K replicon UBd Plasmids
Table 4-1. Continued pLAFR3 TcR, rlx+, RK2 replicon BJSe
pUFR034 Tn903, IncW, Mob+, KnR, De Feyter et al. (1990) pCRhrpW hrpW partial fragment in pCR2.1-TOPO This study pCR∆harpin Harpin -, 87 bp deleted with Aat II, KnR,
AmpR
pCRHrpW-EP pCR2.1 with1.8-kb hrpW plus native promoter, KnR, AmpR
This study
pOHrpW-EP pOK1 clone containing hpW plus native promoter, SmR
This study
pO∆Harpin/PEL pOK1 with HrpW-, SmR This study pO∆Harpin pOK1 with Harpin-, SmR This study pL22 Xcc strain Aw pLAFR3 cosmid that contains
hrpX, hrpG and hsp90Xo homologues, TcR Rybak et al. (2009)
pL799 pLAFR3 with DNA fragment from Xcc-Aw that contains avrGf1, TcR
Rybak et al. (2009)
pBS1 BglII::avrBs262-574::HA of pBluescript-KS+/-, AmpR Mudgett, M. B.f
pBWBs hrpW1-109 cloned in pBS1 This study
pUWBs hrpW1-109 fused to avrBs262-574 cloned pUFR034 This study
pUHrpW-EP Xcc pUFR034 clone containing hpW plus native promoter, KnR
a R.E. Stall, Robert E. Stall, University of Florida, Gainesville, FL. b DPI, Division of Plant Industry of the Florida Department of Agriculture and Consumer Services, Gainesville, FL, USA. c Jones, J. B., University of Florida, Gainesville, FL, USA. d UB, Ulla Bonas, Martin-Luther-Universität, Halle, Germany. e BJS, B. J. Staskawicz, University of California, Berkley, CA f Mudgett, M. B., Stanford University, Stanford, CA.
95
Table 4-2. Oligonucleotides sequence used in this study Gene Primer name Primer sequence length hrpW POWF1 5’-ACGCGTCGACGTCAGCCGAAAGGAATATACAG-3’ 32
Figure 4-1. Neighbor joining analyses using full nucleotide sequence of hrpW gene from X. c. subsp. citri strain 306. The numbers represent the parsimony tree bootstrap and maximum likelihood values.
Figure 4-3. Sequence alignment of Xcc strain 306 – wild type (W1); W::∆Harpin mutant (W2); and 306::∆hrpW HrpW (W3). Dots show parts of hrpW gene that were deleted.
113
Figure 4-4. Phenotype assay in grapefruit leaves. A. Xcc strain 306 wild type (1), W::∆harpin
mutant (circle) and ∆harpin::Harpin complemented (2); B. Xcc strain 306 wild type (1) and 306::∆hrpW mutant. The bacterial suspension at 5 x 108 CFU/mL were syringe and needle infiltrated and photographed 7 days later.
mutant (harpin Mut), ∆harpin::harpin mutant complement (harp-Comp), Xcc 306 wild type carrying the pU::∆harpin plasmid (306.1) and 306::∆hrpG mutant (hrpG Mut) in grapefruit leaves times after infiltration of 5 x 108 CFU/mL of each strain into mesophyll.
5
6
7
8
9
0 2 4 6 8 10
Log
10 cf
u/cm
2
Days after Inoculation
Xcc 306
hrpW Mut
harpin Mut
harp-Comp
306.1
hrpG Mut
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Figure 4-6. Canker symptoms on abaxial leaf surface of grapefruit. Symptoms developed 28 days after pin-prick inoculation with Xcc 306 (all circled), 306::∆hrpW mutant mutant (A), W::∆harpin mutant (B), ∆harpin::harpin mutant complement (C), Xcc 306 wild type carrying the pU::∆harpin plasmid (D), and 306::∆hrpG mutant (E).
116
Figure 4-7. PCR amplification of genomic DNA of Xcc 306 (1/2), 306::∆hrpW mutant mutant (3), W::∆harpin mutant (4), ∆harpin::harpin mutant complement (5), Xcc 306 wild type carrying the pU::∆harpin plasmid (6/7). λ: Lambda DNA/EcoR I and Hind III markers (Promega).
117
CHAPTER 5 CHARACTERIZATION OF AVRGF1 FROM XANTHOMONAS CITRI SUBSP. CITRI
STRAIN AW
Introduction
Host resistance is often linked to the recognition of specific elicitors encoded by the
pathogen avirulence (avr) genes by a single resistance (R) gene in the host (Hammond-Kosack
and Jones, 1997). This recognition is now commonly referred to as effector-triggered immunity
(ETI) (Flor, 1971; Jones and Dangl, 2006). Phytopathogenic proteobacteria, such as
Xanthomonas, employ a type III secretion system (T3SS) in the initial infection phase to inject
T3-effector proteins through this apparatus (Bonas et al., 1991; Fenselau et al., 1992). The
activity of T3-effectors is required collectively to modulate the plant cell environment to provide
optimum conditions for bacterial colonization (Cornelis and van Gijsegem, 2000). While the
general function of T3-effectors is to suppress plant defenses, some T3-effectors trigger a rapid
and localized programmed cell death known as the hypersensitive reaction (HR) (Lindgren et al.,
1986; Staskawicz et al., 1995;.Block et al., 2008). The list of T3-effectors with dual activity
include avrRpm1 from Pseudomonas syringae pv. maculicola (Ritter and Dangl, 1995), avrXa7
in X. oryzae pv. oryzae (Yang et al., 2004); avrPto of P. s. pv. tomato (Chang et al., 2000) and
avrPtoB in P. s. pv. tomato DC300 (Scofield et al., 1996; Tang et al., 1999; Kim et al., 2002).
106::AvrBs262-574 was generated for translocation assay in pepper cvs. ECW and ECW20R. For
making pBSNGf1 plasmid, a PCR fragment encoding the N-term for AvrGf1 was amplified from
Xcc 306 genomic DNA using NGf1-F1 and NGf1-R1 primers, and cloned into the Bgl II
restriction site in the pBS(BglII::avrBs262-574::HA) plasmid. The BamH I – Kpn I fragment
containing AvrGf11-106::AvrBs262-574 region was excised from the resulting plasmid and ligated
into the pUFR034 vector previously digested by the same enzymes, creating pUF12. The
resulting plasmid was introduced into X. campestris pv. vesicatoria strain XC1922 by triparental
mating. Xcv carrying the construct was then tested for the ability to elicit an AvrBs2-HR in the
two pepper genotypes.
Results
Induction of Hypersensitive Response by Transient Expression of avrGf1 Within Citrus
To investigate whether the induced HR in grapefruit leaves is triggered by AvrGf1 alone or
in combination with other proteins from Xanthomonas, we expressed the full length of avrGf1
under control of the constitutive cauliflower mosaic virus 35S promoter in grapefruit leaves
using Agrobacterium tumefaciens-mediated gene delivery. The amplified avrGf1 fragment was
cloned into pGWB5 and pGWB2 binary vector (Figure 5-1) using Gateway cloning system and
Agrobacterium transformed with pGGf1B5 and pGGf1B5 (both carrying the avrGf1), and pGWB5 and
pGWB2 (empty vectors) were inoculated in grapefruit leaves. Transient expression of avrGf1 under
the control of the 35S Cauliflower mosaic virus promoter in young grapefruit leaves resulted in a
typical HR 4 – 5 days after inoculation (Figure 5-2). The A. tumefaciens strain GV3101 by itself
or carrying pGWB2 or pGWB5, empty vectors, did not show any visible sign of necrosis (Figure
5-2), implying that the HR is induced by the avrGf1 and not due to an Agrobacterium protein, or
the green fluorescent protein (GFP) or other protein expressed by T-DNA genes delivered by the
empty vectors..
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The N-terminal and C-terminal Are Required for HR Elicitation
Deletion mutants were constructed affecting both ends of avrGf1 (Figure 5-1a). Two
different deletions were performed in each terminal. The plasmids carrying the N- and C-
terminal coding sequence deletions, p5∆N13 and p5∆N116, p5∆C7 and p5∆C83, respectively,
were electroporated into the A. tumefaciens strain GV3101. The resulting strains were tested for
their HR elicitation in grapefruit leaf tissue. The transient expression of the deletion mutants
Gf1::∆N13 and Gf1::∆C7, with 13 deleted amino acids after the start codon and 7 deleted amino
acids before the stop codon, produced an HR comparable to that of A. tumefaciens carrying the
full length avrGf1, 31::Gf1B5 and 31::Gf1B2 (Figure 5-1). However, the HR induction of the
Gf1::∆N116 and Gf1::∆C83, which carry 116 deleted amino acids after the start codon and 83
deleted amino acids before the stop codon, respectively, showed complete absence of a
macroscopical HR induction (Figure 5-1).Thus, both N- and C-terminal are required for HR
elicitation.
Xanthomonas citri subsp. citri Harboring avrGf1 Induces a Hypersensitive Reaction in Grapefruit
To determine if the Xcc, which induces a compatible reaction, was able to delivery AvrGf1
into the plant host cell of grapefruit leaf tissue, and then, trigger an incompatible reaction we
transformed Xcc with pHArGf1, which carries the avrGf1 driver by its own native promoter. The
transconjugants Xcc 306 expressing in-trans the avrGf1 gene (306::avrGf1) were infiltrated into
grapefruit and Valencia leaf tissue at 5 x 108 CFU/ml, and leaves were observed after 5 days for
the development of visible HR (Figure 5-3). The Xcc 306 by itself showed water-soaking in the
edges of the infiltrated area, which is a classical symptom of disease development. The HR in
planta of grapefruit elicited by 306::avrGf1 did not differ among the three repeats.
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AvrGf1 is Translocated Inside the Host Plant Cell
Rybak et al. (2009) reported that avrGf1 requires a functional T3SS and triggers a HR in
grapefruit plants. To further test if the AvrGf1 is translocated into host plant cells or only
secreted into the apoplast, the N-terminal coding region of AvrGf1 (codons 1 – 106) was fused to
the C-terminal coding region of AvrBs2 (codons 62 – 574), a X. campestris pv. vesicatoria type
III effector (Kearney and Staskawicz, 1990; Bonas, 1991). We inoculated Xcv XV1922
expressing the AvrGf11-106::AvrBs262-574 fusion protein into pepper leaves. We chose Xcv
XV1922 to express the fusion protein because it does not trigger HR in pepper cv. ECW20R due
a natural mutation at avrBs2 gene. The bacterial suspension was infiltrated at 5 x 108 CFU/ml
into the leaf mesophyll. Resistant Bs2 pepper plants specifically recognized AvrBs2 truncated
protein leading to HR 24 h after inoculation (Figure 5-4). Furthermore, no HR was observed in
pepper plants without Bs2 resistant gene (Figure 5-4). This data showed that the first 106 amino
acids were sufficient to translocate AvrGf11-106::AvrBs262-574 fused protein into Bs2 pepper
plants.
Discussion
AvrGf1 was characterized by molecular analysis to understand the role of this protein in
XccAw HR elicitation in grapefruit as originally identified by Rybak et al. (2009) They
determined that AvrGf1 is a homolog of several T3-effectors and it is T3SS-dependent. T3SS
facilitates the translocation of a collection of proteins into plant cells (Buttner and Bonas, 2002).
The translocation signal in the avrBs2 gene of X. c. pv. vesicatoria is located between 1 - 58
amino acid and the region of 62 – 714 amino acids activate Bs2 disease resistance (Mudgett et
al., 2000). In this work, we used AvrBs2 as a sensitive reporter (Guttman et al., 2002; Roden et
al., 2004) to identify if the AvrGf1 is translocated inside the host cell in Bs2 resistance pepper
plants from Xcv strain XV1922 transfomed with the AvrGf11-106::AvrBs262-574 fused protein. The
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AvrBs2/Bs2 report system has been used to identify new translocation effectors (Roden et al.,
2004). The HR induced for the XV1922, expressing the fusion protein, showed that the
translocation signal is present in the first 106 amino acids in the N-terminus. The fact that the
translocation signal is present in the N-terminus is not surprising, since most T3-effectors carry
the translocation signal in the N-terminal, corroborating the results reported by previous studies
(Leach and White, 1996; Mudgett et al., 2002; Roden et al., 2004). Additionally, Rybak et al.
(2009) demonstrated that AvrGf1 is translocated into grapefruit cells in a T3SS-dependent
manner.
The deletion mutagenesis analysis per the N-terminus showed that deletion of 116 amino
acids affect HR activity in pepper plant cv. ECW20. Gathering the data from the
AvrGf1::AvrBs2 translocation study indicate that the first 116 amino acids on the N-terminal
definitely play important roles in protein translocation and and HR induction. In contrast to the
HopG1 in P. s. pv. tomato DC3000, which is a homolog, that does not elicit cell death in
Nicotiana benthamiana (Wei et al., 2007). Additionally, the deletion of only 83 amino acids in
the C-terminal, directly upstream of the stop codon, was shown to be enough to block cell death
elicitation. While, Xcv AvrBs2 protein required, at least, the deletion of 297 amino acids
upstream of the stop codon to lose the ability to trigger HR in Bs2 pepper plants (Mudgett et al.,
2000).
In this work, an Agrobacterium-mediated transient expression protocol was established for
grapefruit to analyze the gene-for-gene interaction between AvrGf1 and the putative R gene
product controlling the HR response in grapefruit. An Agrobacterium-mediated transient
expression in citrus leaves has been reported, which followed the procedure described by Kapila
et al. (1997) and uses vacuum to infiltrate citrus seedling (Duan et al., 1999). However, the aim
127
of that experiment was to show that the expression of pthA pathogenicity gene from Xcc alone
can induce citrus canker symptoms in the absence of the pathogen and no HR. The symptoms
showed by the transient expression are much smaller than the lesions induced by Xcc, thus, this
difference may be because of the relatively inefficient gene transfer (Duan et al., 1999). In
contrast, in the procedure developed here we exclude the necessity of orange seedlings that are
time-consuming and the vacuum chamber. Furthermore, the procedure here demonstrated the
same level of HR elicitation compared with the Xcc wild type strain. Infiltrated plants showed
stable expression of avrGf1 through the HR in young tissue but the same results were not
observed in intermediate and old leaves. Previous attempts to perform Agrobacterium-mediated
transient expression in mature leaves have failed. The inefficiency of this method to function in
old leaves may be because, in general, plant cells have hard and thick cell walls that reduce
access to the bacterium. In addition, this procedure excludes the need to regenerate transformed
cells that are used commonly for functional analysis of gene regulation. The circumvention of
tissue culture is particularly valuable in the case of gene expression of woody trees such as
citrus, which are difficult to regenerate (Ghorbel et al., 1999; Chávez-Bárcenas et al., 2000;
Tucker et al., 2002).
128
Table 5-1. Bacterial strains and plasmids used in the study Desigantion Relevant characteristics Source Strains X. citri subsp. citri 306 Wild type, Asiatic strain, isolated in Brazil,
RifR DPIa
306::avrGf1 306 carrying pHavrGf1, AmpR, KnR, SpcR This study X. campestris pv. campestris XV1922 Pepper race 6, RifR This study
XV1922-1 1922 carrying pUF12, KnR, RifR This study Agrobacterium tumefaciens GV3101 RifR Lahaye, T.b
31::WB5 GV3101 carrying pGWB5, AmpR, RifR This study 31::WB2 GV3101 carrying pGWB2, AmpR, RifR This study 31::Gf1B5 GV3101 carrying pGGf1B5, AmpR, RifR This study 31::Gf1B2 GV3101 carrying pGGf1B2, AmpR, RifR This study Gf1::∆N13 GV3101 carrying p5∆N13, KnR, RifR This study Gf1::5∆N116 GV3101 carrying p5∆N116, KnR, RifR This study Gf1::5∆C7 GV3101 carrying p5∆C7, KnR, RifR This study Gf1::5∆C83 GV3101 carrying p5∆C83, KnR, RifR This study Gf1::2∆N13 GV3101 carrying p2∆N13, KnR, RifR This study Gf1::2∆N116 GV3101 carrying p2∆N116, KnR, RifR This study Gf1::2∆C7 GV3101 carrying p2∆C7, KnR, RifR This study Gf1::2∆C83 GV3101 carrying p2∆C83, KnR, RifR This study Escherichia coli DH5α FrecAϕ80dlacZ∆M15 Invitrogen
pGWB5 Binary vector Lahaye, T. pBS1 BglII::avrBs262-574::HA of Bluescript-KS+/-,
AmpR Mudgettd
pCR799 avrGf1 total sequence in pCR2.1-TOPO, AmpR, KnR
This study
pHAvrGf1 avrGf1 (pCR799) ligated with pHM1 This study
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Table 5-1. Continued Desigantion Relevant characteristics Source pBSNGf1 avrGf11-106 fused to avrBs262-574 of pBS1 This study PUF12 avrGf11-106::avrBs262-574 fusion in pUFR034 This study pEavrGf1 avrGf1 total sequence in pENTR-D, AmpR,
KnR This study
pE∆N13 avrGf1 deleted 13 codon after ATG in pENTR-D, AmpR, KnR
This study
pE∆N116 avrGf1 deleted 116 codon after ATG in pENTR-D, AmpR, KnR
This study
pE∆C7 avrGf1 deleted 7 codon before stop codon in pENTR-D, AmpR, KnR
This study
pE∆C83 avrGf1 deleted 83 codon before stop codon in pENTR-D, AmpR, KnR
This study
pGGf1B5 avrGf1 total sequence in pGWB5, RifR, KnR This study pGGf1B2 avrGf1 total sequence in pGWB2, RifR, KnR This study p5∆N13 avrGf1 deleted 13 codon after ATG in
pGWB5, AmpR, KnR This study
p5∆N116 avrGf1 deleted 116 codon after ATG in pGWB5, AmpR, KnR
This study
p5∆C7 avrGf1 deleted 7 codon before stop codon in pGWB5, AmpR, KnR
This study
p5∆C83 avrGf1 deleted 83 codon before stop codon in pGWB5, AmpR, KnR
This study
p2∆N13 avrGf1 deleted 13 codon after ATG in pGWB2, AmpR, KnR
This study
p2∆N116 avrGf1 deleted 116 codon after ATG in pGWB2, AmpR, KnR
This study
p2∆C7 avrGf1 deleted 7 codon before stop codon in pGWB2, AmpR, KnR
This study
p2∆C83 avrGf1 deleted 83 codon before stop codon in pGWB2, AmpR, KnR
This study
a DPI, Division of Plant Industry of the Florida Department of Agriculture and Consumer Services, Gainesville, FL, USA. b T. Lahaye, Martin-Luther-Universität, Halle, Germany. c BJS, B. J. Staskawicz, University of California, Berkley, CA. d M. B. Mudgett, Stanford University, Stanford, CA.
130
Table 5-2. Oligonucleotides sequence used in this study Gene Primer Name Primer sequence Length avrGf1 HRF1 5’-CGCCAGGAAGGGCCTGCCATG-3’ 21
Figure 5-1. Diagram of AvrGf1 and truncated versions used to test regions of the protein for elicitor activity. A. ATG indicates the site at the N-terminal coding portion was excluded, and the red triangle indicates the point of truncation of the C-terminal coding region. B. Organization of the binary vectors, pGWB2 and pGWB5, used to clone the truncated proteins.
132
Figure 5-2. Agrobacterium-mediated expression of avrGf1 in grapefruit leaf tissue. A Phenotype elicited by Agrobacterium, harboring 31::Gf1B2 plasmid, grown overnight in YEP medium and directly infiltrated into grapefruit leaves (indicate by circle and arrow) and leaf infiltrated with Agrobacterium, carrying the 31::Gf1B2 plasmid, grown under the conditions developed in the section 5 of materials and methods (indicate arrow), 31::Gf1B5 without cell treatment (indicate by circle) and 31::Gf1B5 treated cells; B. Lower panel: Agrobacterim carrying pGWB5 empty vector, Up panel: Agrobacterium tumefaciens strain GV3101; C. Phenotype of the N-terminal mutants Gf1::∆N13 (low) and Gf1::∆N116 (up); D. Upper panel: HR induced by strain containing 31::Gf1B5; lower panel: C-terminal mutant containing Gf1::∆C7. Bacterial suspensions were prepared following the transient expression protocol developed in this study (see Methods and Materials). The plants were photographed 5 days after infiltration.
133
Figure 5-3. Hypersensitive reaction induced by X. citri subsp. citri 306 expressing avrGf1. Xcc wild type strain 306 (indicate by the arrow) and 306::avrGf1 (circled). A. Grapefruit leaf; B. Valencia orange leaf. The bacterial suspension was infiltrated at concentration of 5 x 108 CFU/ml. The plants were photographed 6 days after infiltration.
134
Figure 5-4. Phenotypes elicited on pepper plants cv. ECW20R 24 h after inoculation. A. Xcv: X. campestris pv. vesicatoria wild type strain XV1922; Xcv::A, Xcv::B and Xcv::C: different X. c. pv. vesicatoria transconjugants carrying the pUF12. B. Close-up of the X. c. pv. vesicatoria transconjugants, Xcv::A and Xcv::C.
135
CHAPTER 6 OVERALL SUMMARY AND DISCUSSION
The most aggressive form of Asiatic citrus canker disease is caused by Xanthomonas citri
subsp. citri (Xcc) and designated as an A-strain. Another strain, Xanthomonas fuscans pv. citri
(Xfc) and known as a C-strain, is known also to incite citrus canker-like symptoms in
Key/Mexican lime (Citrus aurantiifolia) and hpersensitive response (HR) in grapefruit (C.
paradisi) (Stall and Civerolo, 1991; Rybak et al., 2009). The pathogenicity and virulence of Xcc
rely on the highly specialized type three secretion system (T3SS) delivery system, which
transports a collection of effector proteins directly inside host cells and thereby amending the
host cell environment for bacterial colonization (Bonas et al., 1991; Fenselau et al., 1992;
Cornelis and van Gijsegem, 2000). Hypersensitive response and pathogenicity (hrp) genes
encode the T3SS system, and they are also chaperones for some effectors in the translocation
process (Chang et al., 2004; Tang et al., 2006). The aim of this study was to characterize the
putative T3-effectors involved in the pathogenicity of Xcc. To accomplish this aim and to further
characterize some of the genes that were identified in the process, the study was separated into
four objectives: I. Identification and characterization of candidate T3-effector genes in Xcc based
on genomic sequencing data by da Silva et al. (2002) using mutagenesis analysis; II. Genetic
characterization of a T3SS-independent HR elicitor activity in Xcc; III. Characterization of hrpW
and the domains, harpin and pectate lyase, in the Xcc pathogenicity; and, IV. Molecular
characterization of the Xfc avrGf1 gene and development of avrGf1 as an efficient transient
expression system in citrus leaves.
The candidate effector genes in this study were selected from genes that were scanned for
the presence of the plant inducible promoter (PIP) Box (TTCGC-N15-TTCGC), which is the
transcriptional signal for HrpX regulator (Wengelnik and Bonas, 1996; Wengelnik et al., 1996a;
136
Wengelnik et al., 1996b), another motif called the - 10 box-like motif (YANNNT; Y: C/T; N:
A/T/C/G), which is localized 30 – 32 bases pair from the PIP box that has been proposed to be
necessary for HrpX regulon, and for genes with sequence similarity to known T3-effectors
(Cunnac et al., 2004). Only two candidate T3-effector genes with homology to known T3-
effectors were found without the PIP Box, avrPphE2, which was previously reported by da Silva
et al. (2002) and xopX, which was identified in another Xanthomonas on the basis of an analysis
of T3SS-dependent extracellular protein secretion (Koebnik et al., 2006). Insertional
mutagenesis was used as the primary research approach in this study to evaluate the contribution
of candidate T3-effector genes of Xcc in pathogenicity. Pathogenicity assays in grapefruit
showed that none of the mutants created in this study had visible effects on citrus canker
phenotype, nor had visible effects on the HR elicitation activity on the non-host species tomato.
At the same time, insertions into hrp genes previously known to control T3SS function (hrpA,
hrpG, and hrpX) resulted in loss of pathogenicity. However, mutations in hrpA, hrpG and hrpX
also failed to result in the loss of the nonhost HR on tomato. Concluding, most of the genes
studied here and shown to be conserved in other Xanthomonas were not found to contribute to
the visible canker symptooms. The presence of the PIP-box also does not define the direct
interaction with pathogencity or T3SS, such as avrPphE or xopX which may be pseudogenes and
which may not be involved in pathogenicity. Therefore, the lack of PIP does not mean PIP-box
does not define direct involvement. Further pathogenicity tests, possibly involving bacterial cell
counts, will be conducted to determine what role, if any, the candidate genes play in citrus
canker.
The insertional and deletion mutagenesis performed in Xcc hrpW domains, harpin and
pectate lyase (PEL), demonstrated that the deletion of harpin plays crucial function on Xcc
137
compatible interaction in grapefruit. The hrpW null mutation is not impaired with the
pathogenicity ability. Probably the phenotype observed with harpin mutant is caused by a change
in protein conformation, thus altering the formation of a protein complex that HrpW and the
other proteins (Alegria et al., 2004). Additionally, attempts to complement the harpin mutant
with full length hrpW genes driven by the native promoter in trans failed several times, although,
when the mutation was replaced in the chromosome, the pathogenicity was recovered. This
inability to complement the harpin mutant might be a dominant negative effect of the defective
protein expressed by the mutant.
The Xcc hrp regulator mutants, 306::ΩhrpX and 306::ΩhrpG, preserved the ability to
induce HR in non-host tomato plant. Thus, we speculate that an unknown T3SS-independent
gene, or genes, controls the biosynthesis of the elicitor. Previously, a study by our group and
involving the characterization of Xfc genes that are involved in incompatible host reactions
revealed a clone designated 450, which, when expressed in hrp- mutant of the tomato pathogen
X. perforans, demonstrated same phenotype in non-host tomato plants as Xcc and Xfc. To
identify the possible candidate(s) for the T3-independent HR phenotype, clone 450 was
subcloned, and a screen was performed in tomato plants, revealing a subclone of 3.0 kb that
triggered an HR when expressed in trans in the X. perforans hrp- mutant. Sequence analysis of
the flanking ends of this subclone revealed sequence similarity for the ends of a region
containing three genes in Xcc, namely, XAC3857, XAC3858 and XAC3859. The search for
homology with these three ORFs did not show any relation with known HR elicitor. BLAST
analysis of the NCBI Genbank indicated that none of the ORF had relatedness to any knonw HR-
related proteins. Further characterization of the three genes will reveal whether one or more of
the genes control the T3SS- independent HR phenotype of Xcc and Xfa.
138
Further experiments are in progess to develop an efficient Agrobacterium-mediated
transient expression procedure in citrus leaf tissue. This system will be used to characterize the
critical domains of avrGf1 gene that are required for the HR in grapefruit and as a reporter
system for candidate T3-effector genes of Xcc and related pathogens. In this study, a deletion
mutagenesis analysis made in the N-terminus and C-terminus revealed that the region between
13 and 116 amino acids (aa) in the N-terminus are required for HR elicitation, and also, the
region 7to 83 aa before the stop codon was required. Moreover, a translocation assay using a
AvrGf11-106::AvrBs262-574 fused protein in Bs2 pepper plants, revealed that the secretion signal
Figure A-3. Nucleotide sequence of HrpW1-109::AvrBs262-574 fused protein. The restriction sites and the start codon of each gene is underline.
142
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BIOGRAPHICAL SKETCH
José Francisco Lissoni Figueiredo was born in the city of Cosmorama, Brazil. During his
undergraduate studies in biomedicine at the University of Barão de Mauá, he started a trainee
program in the sequencing genome project of the Xanthomonas citri subsp. citri and
Xanthomonas campestris pv. campestris. In 2001, he started the Master of Science program at
University of State of São Paulo in the genetics and plant improvement program. After
graduating with his master’s, he was invited by Dr. Frank F. White to work with Xanthomonas
campestris pv. campestris in the Plant Pathology Department at Kansas State University. In
2004, he was admitted in the Plant Pathology Department Ph.D. program at Kansas State
University and transferred to the Plant Pathology Department at University of Florida where he
received his Ph.D. in 2009 conducting a research project on screen pathogenic factors involved
in Xanthomonas citri subsp. citri disease progress.