AN ABSTRACT OF THE THESIS OF
Thomas W. Holman for the degree of Master of Science in Botany and Plant
Pathology presented on August 15, 2012.
Title: Pyrenophora tritici-repentis: Investigation of Factors that Contribute to
Pathogenicity.
Abstract approved:
Lynda M. Ciuffetti
Pyrenophora tritici-repentis (Ptr) is the necrotrophic fungus responsible for
tan spot of wheat (Triticum aestivum). Ptr causes disease on susceptible wheat
cultivars through the production and secretion of host-selective toxins (HSTs). HSTs
are compounds that are only known to be produced by fungi and considered to be
primary determinants of pathogenicity. Infiltration of these toxins into sensitive wheat
elicits the same symptoms as the pathogen, which simplifies investigations of hostpathogen interactions due to exclusion of the pathogen. These characteristics make
HSTs ideal molecules to dissect molecular plant-microbe interactions. Known HSTs
of Ptr include Ptr ToxA (ToxA), Ptr ToxB (ToxB) and Ptr ToxC (ToxC). ToxA is the
most characterized toxin of Ptr, as well as the first proteinaceous HST identified. The
proposed mode-of-action for ToxA includes internalization into sensitive wheat
mesophyll cells, localization to the chloroplast, photosystem perturbations and
elicitation of high amounts of reactive oxygen species (ROS), all of which lead to
necrosis. However, it is still unknown how ToxA is transported to the chloroplast. To
identify additional interacting components involved in ToxA symptom development,
genes were silenced in tobacco plants (Nicotiana benthamiana) using the tobacco
rattle virus (TRV) virus-induced gene-silencing (VIGS) system. Four genes were
identified that potentially could play a role in ToxA-induced cell death: a 40S
ribosomal subunit, peroxisomal glycolate oxidase (GOX), a thiamine biosynthetic
enzyme (Thi1), and the R-gene mediator, Sgt1. Ptr exhibits a complex race structure
determined by the HST(s) produced and the symptom(s) elicited on sensitive wheat
cultivars. Currently, there are eight characterized races and other HSTs and races have
been proposed. Isolate SO3 was discovered in southern Oregon and elicits ToxA-like
symptoms on a wheat differential set, yet lacks the ToxA gene. The transcriptome of
SO3 was sequenced, assembled, and aligned to a ToxA-producing isolate, Pt-1C-BFP,
which will aid in the identification of the protein(s) that may be responsible for these
ToxA-like symptoms. SO3 contains a set of 497 sequences that were not found in the
ToxA-producing isolate Pt-1C-BFP (BFP). These sequences should be further
investigated to identify those that encode small secreted proteins (SSPs) and could
potentially serve as HSTs and pathogenicity factors of SO3.
©Copyright by Thomas W. Holman
August 15, 2012
All Rights Reserved
Pyrenophora tritici-repentis: Investigation of Factors that Contribute
to Pathogenicity
by
Thomas W. Holman
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Presented August 15, 2012
Commencement June 2013
Master of Science thesis of Thomas W. Holman presented on August 15, 2012.
APPROVE
Major Professor, representing Botany and Plant Pathology
Chair of the Department of Botany and Plant Pathology
Dean of the Graduate School
I understand that my thesis will become part of the permanent collection of Oregon
State University libraries. My signature below authorizes release of my thesis to any
reader upon request.
Thomas W. Holman, Author
ACKNOWLEDGEMENTS
I would like to express my gratitude to my major professor, Lynda Ciuffetti,
for providing me the opportunity to gain experience and skills over these past years in
a model laboratory for molecular plant pathology. I would also like to thank my other
committee members, Tom Wolpert, Ken Johnson, and Dave Myrold, for all of their
support and advice throughout this process and helping me stay positive.
To my wife, Nicole, and daughter, Farrah: you are the reasons that I was able
to make it throughout my undergraduate and graduate education. You never let me
give up and always were there to cheer me up with your smiles when I was feeling
down or stressed. Thanks to my grandmother, Glenda Malone, for instilling the love
of plants and nature in me through the weekends in the garden and fishing, as well as
undying support in more ways than she probably knows.
My utmost gratitude goes out to fellow members of the Ciuffetti lab: Viola
Manning, Iovanna Pandelova, Melania Figueroa, Barbara Gvakharia, Ashley Chu and
Genevieve Weber. To Viola, Iovanna, Melania, Gen, and Barbara: thank you for
teaching me how to be a great scientist through example and leadership. I would
never have accomplished this without you and I will never forget that. Ashley, you
made the first 2 years of my time in the lab more fun and educational than I could
have imagined. I wish you the best in your future as a forensic scientist, you deserve
it. Thanks to the members of the Wolpert lab, Jennifer Lorang and Brain Gilbert,
ACKNOWLEDGEMENTS (Continued)
for friendship and continued assistance with writing and experiments.
Erik Rowley, thank you for everything. Throughout our time at Oregon State,
you were always there to bounce experiments off of, share a beer or two, or just hang
out and laugh about anything. I could never forget the role you played in me
completing my degree. You are as instrumental in this completion as my own family.
I would like to express my gratitude to the entire BPP community: faculty,
staff and students. You are the best department of which I could have ever asked to be
a part. Everyone has always treated me as family and went out of their way to help. I
wish nothing but success and happiness for all of you in your future endeavors.
Thanks also goes to Dr. Kirankumar Mysore, Samuel Roberts Noble Foundation, for
providing the library used for silencing experiments and to Dr. Greg Martin, Boyce
Thompson Institute for Plant Research, for providing the Sgt1 silencing construct.
My graduate research was funded primarily by a grant from the National
Science Foundation to LMC (grant number: IOS-0819001), a grant from the
Agricultural Research Foundation to LMC, and teaching assistantships from the
department of Botany and Plant Pathology, Oregon State University. I would like to
acknowledge the Confocal Microscopy Facility of the Center for Genome Research
and Biocomputing and the Environmental and Health Sciences Center, Oregon State
ACKNOWLEDGEMENTS (Continued)
University; this research was also made possible in part by grant number
1S10RR107903-01 from the National Institutes of Health.
CONTRIBUTION OF AUTHORS
Dr. Lynda Ciuffetti assisted with the design and editing of all sections
contained in this thesis. Dr. Genevieve Weber assisted with the design and editing of
chapter 1. Dr. Iovanna Pandelova assisted with the design and editing of chapter 2 and
Viola Manning, M.Sc., assisted with the design and editing of chapter 3.
TABLE OF CONTENTS
Page
Chapter 1:
Introduction…………………………………………………………………………… 1
Tan spot of wheat……………………………………………………………... 1
Host-selective toxins………………………………………………………….. 2
ToxA…………………………………………….…………………………….. 3
Intracellular expression of ToxA sufficient for symptom development………. 8
Ptr race structure and potential pathogenicity factors……………..………. …9
Chapter 2: Virus-induced gene silencing-mediated identification of proteins involved
in ToxA-induced symptom development.…………………………………….. 12
ABSTRACT………………………………………………………………….. 12
INTRODUCTION…………………………………………………………… 13
MATERIALS AND METHODS……………………………………………. 21
Production of tobacco plants...……………………………………….. 21
Bacterial growth and induction………………………………………. 22
VIGS Agrobacterium constructs and experimental design...………… 23
Clone identification…………………………………………………...24
Cell death assessment………………………………………………... 27
Assay to measure ToxA effect on chlorophyll concentration..……….27
RT-PCR……………………………………………………………… 28
Detection of GFP fluorescence in tobacco plants……………………. 29
TABLE OF CONTENTS (Continued)
Page
ToxA detection on tobacco leaves…..………………………….….. 29
RESULTS……………………………………………….…………………. 30
VIGS reveals proteins involved in ToxA symptom development…. 30
Phenotypes of silenced plants and ToxA effects…………………... 30
Quantitative assessment of symptom development………………... 33
Reduction in transcripts correlated with decreased cell death…….. 35
Agroinfection and ToxA expression not inhibited by silencing…… 39
DISCUSSION……………………………………………………………… 42
Chapter 3: Assembly and analysis of the SO3 transcriptome to identify potential
pathogenicity factors of Ptr……………………………………………….. 50
ABSTRACT………………………………………………………………... 50
INTRODUCTION…………………………………………………………. 51
MATERIALS AND METHODS……………………………….…….…….53
Growth of fungi and RNA isolation………………………………...53
Sequencing and sequence quality assessment…………………….. 54
Transcriptome assemblies and alignments………………………… 54
RESULTS……………………………………………………………………56
Assembly statistics.…………………………………………………56
Alignments to BFP genomes………..……………………………... 56
Alignment of SO3 to BFP transcripts reveals unique sequences...… 58
TABLE OF CONTENTS (Continued)
Page
DISCUSSION……………………………………………………………… 63
Chapter 4: General Conclusion…………………………………………………….. 67
Bibliography……………………………………………………………...73
LIST OF FIGURES
Figure
Page
1.1: Three-dimensional structure of ToxA…………………………………………… 5
2.1: Model of plant immunity……………………………………………………….. 16
2.2: Genomic architecture of the tobacco rattle virus …………………………….. 20
2.3: Phytoene desaturase-silenced tobacco…………………………………………. 25
2.4: TRV-VIGS procedure……………...…………………………………………… 31
2.5: Silencing phenotypes of candidate genes. ………………………………….….. 32
2.6: ToxA and GFP symptom development on silenced tobacco…………………… 34
2.7: Chlorophyll assay of tobacco leaves…………………………………………….36
2.8: Reverse transcription polymerase chain reaction results……………………..…38
2.9: GFP fluorescence in tobacco plants……………………………………………. 40
2.10: Western blot to detect ToxA in silenced plants……………………….………. 41
3.1: Percent coverage of SO3 transcripts to the BFP genome………………………. 59
3.2: Percent coverage of SO3 transcripts to the BFP transcriptome……….………... 61
3.3: Summary of filtering steps and candidate SO3 sequences……………………... 62
LIST OF TABLES
Table
Page
1.1: Race structure of Pyrenophora tritici-repentis…………………………………. 10
2.1: Primers and sequences for clone identification and RT-PCR…………………...26
2.2: Chlorophyll concentrations in silenced leaves…………………………………..37
2.3: Summary of ToxA and GFP symptoms observed……………………………… 48
Pyrenophora tritici-repentis: Investigation of Factors that Contribute to
Pathogenicity
Chapter 1
INTRODUCTION
Tan spot of wheat
The fungus Pyrenophora tritici-repentis (Ptr) is the causal agent of tan spot of
wheat (Triticum aestivum L.) (Drechsler, 1923; Singh and Hughes, 2006; Cao et al.,
2009) and is an increasingly important pathogen in wheat-growing regions worldwide,
leading to crop losses up to 50% (Ciuffetti et al., 1998, Oliver et al., 2008). The
fungus has been reported to cause significant damage to wheat in the central plains of
the United States and Canada (Friesen et al., 2006; Tekauz et al., 2004), South
America (Perello et al., 2003; Chu et al., 2008), Australia (Oliver et al., 2008) and
Mexico (Singh et al., 2011). Ptr is a homothallic fungus, which is defined as an
organism that possesses the necessary components to reproduce without the presence
of another individual (Bruggeman et al., 2003). Ptr produces sexual propagules called
ascospores, which overwinter in pseudothecia on wheat stubble in the field and reside
2
in structures called asci. Ascospores compose the primary inoculum for the fungus,
meaning they initiate the infection process in spring. The pathogen also produces
wind-dispersed asexual propagules called conidia. These are the secondary inoculum
and are responsible for disease spread during the growing season (Ellis and Waller,
1976; Weise, 1987). Symptoms in the field include three disease phenotypes:
necrosis, chlorosis (Lamari and Bernier, 1991), and spreading chlorosis (Lamari et al.,
2003). The prevalence and type of the symptoms on wheat is dependent on the isolate
of the fungus, the toxins it produces, and the genotype of wheat. For example, Ptr
race 1 elicits necrosis on cultivars Glenlea and Katepwa but chlorosis on the cultivar
6B365.
Host-selective toxins
Isolates of Ptr are pathogenic on specific wheat cultivars due to the activity of
host-selective toxins (HSTs) produced by the fungus (Wolpert et al, 2002; Martinez,
2004). HSTs are proteins or low molecular weight compounds produced by some
necrotrophic fungi that act as primary determinants of pathogenicity/virulence, which
indicates the toxins typically confer the ability of the pathogen to cause disease and
selectively affect only those genotypes of wheat that are susceptible to the fungus
(Walton, 1996; Markham and Hille, 2001; Friesen et al., 2007). Infiltration of purified
HSTs into sensitive plants provokes the same reaction from the host as inoculation
with the fungus (Wolpert, et al., 2002; Dunkle, 1984; Scheffer, 1983).
3
Ptr is known to produce at least three HSTs. Ptr ToxA (ToxA) (Ciuffetti et
al., 1997; Tuori et al., 2000) and Ptr ToxB (ToxB) (Kim and Strelkov, 2007;
Figueroa-Betts, 2011) are proteinaceous while Ptr ToxC (ToxC) (Lamari and Bernier,
1989), is non-proteinaceous. ToxA is the most characterized HST of Ptr and will be
described in greater detail in the next section. ToxB is a 6.5 kD protein expressed as
an 87 amino acid (aa) pre-protein, which also includes a 23 aa signal peptide
(Martinez et al., 2001; Strelkov and Lamari, 2003; Kim et al., 2010; Andrie and
Ciuffetti, 2010). ToxC has been partially characterized and appears to be a polar, nonionic, and low molecular weight compound (Effertz et al., 2002). While ToxA is a
single copy gene in all isolates tested, ToxB is present in multiple copies, with the
number dependent on the isolate and virulence dependent on copy number (Strelkov et
al., 1999; Martinez et al., 2001; Lamari et al., 2003; Andrie and Ciuffetti, 2010;
Ciuffetti, et al., 2010).
ToxA
ToxA was the first proteinaceous HST to be described (Ballance et al., 1989;
Tomas et al., 1990; Tuori et al., 1995). ToxA encodes a pre-pro-protein with a 23amino acid (aa) N-terminal signal sequence (Ballance et al., 1996; Ciuffetti et al.,
1997) and a 38 aa pro-domain required for proper folding, both are cleaved prior to
secretion of the 13.2 kDa mature ToxA protein (Tuori, et al., 2000). The threedimensional structure of ToxA has been resolved and it is a single-domain protein
with a β-barrel fold that presents a solvent-exposed loop with an arginine-glycine-
4
aspartic acid (RGD) motif (Sarma et al., 2005) (Figure 1.1). This RGD-motif has
been shown to be required for ToxA activity (Meinhardt et al., 2002; Manning et al.,
2008). Site-directed mutagenesis of amino acids within the motif, as well as
coinfiltration of ToxA with RGD-containing inhibitor peptides, resulted in lower toxin
activity that was correlated with the amount of internalized toxin (Manning et al.,
2008). The mechanism behind the internalization of ToxA is unknown, although
microscopic and microarray data (Pandelova et al., 2009; Manning and Ciuffetti,
2005), as well as treatments with endocytosis inhibitors (V. A. Manning and L. M.
Ciuffetti, unpublished), point to receptor-mediated endocytosis as a potential
mechanism. The RGD-motif likely plays a role in internalization via an extracellular
receptor that recognizes this aa sequence (Manning and Ciuffetti, 2005; Manning et
al., 2008; Ciuffetti et al., 2010). The Ciuffetti laboratory also demonstrated that ToxA
is internalized into sensitive wheat mesophyll cells as determined by a proteinase K
protection assay and fluorescent microscopy of GFP-labeled ToxA (Manning and
Ciuffetti, 2005). Another example of a protein that uses an RGD motif for entry is the
IPI-O protein from Phytophthora infestans (Gouget et al., 2006), which disrupts cell
wall-plasma membrane interactions.
HSTs elicit symptoms only on sensitive cultivars and provide fungi with the
ability to confer disease. The ToxA gene and its native promoter were isolated from a
pathogenic tox+ isolate of Ptr, Pt-1C-BFP (BFP), cloned, and transformed into a
5
Figure 1.1. Three-dimensional structure of ToxA.
Three-dimensional structure of Ptr ToxA as determined by X-ray
crystallography. The circle highlights the solvent-exposed RGD-motif
which has been shown to be important for internalization of ToxA into
sensitive wheat cells (Manning and Ciuffetti, 2005). Purple arrows indicate
the beta-sandwich fold. Adapted from ―Structure of Ptr ToxA: an RGDcontaining host-selective toxin from Pyrenophora tritici-repentis.‖ Sarma,
GN, Manning VA, Ciuffetti LM, and Karplus PA. 2005. Plant Cell.
17:3190–3202.
6
nonpathogenic tox- isolate (SD20) (Ciuffetti, et al., 1997). Introduction of the ToxA
gene conferred pathogenicity to the previously nonpathogenic isolate, SD20, on
ToxA-sensitive wheat. These results indicate ToxA is indeed a pathogenicity
determinant and provides the ability to cause disease on sensitive wheat cultivars
(Ciuffetti et al., 1997).
Sensitivity in wheat to ToxA is determined by the presence of the Tsn1 locus
(Lamari and Bernier, 1989; Faris et al., 1996; Anderson et al., 1999; Adhikari et al.,
2009). This is commonly referred to as an inverse gene-for-gene interaction, where
the dominant response is susceptibility to the fungus and sensitivity to the HST
(Ciuffetti et al., 2010; Wolpert et al., 2002). This contrasts with the classical gene-forgene relationships where a protein encoded by a resistance gene interacts with a
pathogen effector, resulting in a hypersensitive response (HR) and subsequent
resistance (Flor, 1971; de Wit, 1992; Jones and Dangl, 2006; de Wit et al., 2009;
Gassmann and Bhattacharjee, 2012). As shown by yeast two-hybrid analyses, Tsn1
does not directly interact with ToxA (Faris et al., 2010); it is unknown exactly how
Tsn1 conditions sensitivity.
ToxA appears to be internalized only in sensitive wheat cells. After ToxA
enters the cell, it localizes to the chloroplasts where it interacts with a chloroplast
protein, ToxA binding protein 1 (ToxABP1). This interaction was verified by yeast
two-hybrid analysis, as well as pull-down assays with biotinylated His-tagged ToxA
and isolated chloroplasts from sensitive wheat (Manning et al., 2007). ToxABP1
contains a lysine-rich region inside a coiled-coil domain, which is similar to
7
phosphotidyl-inositol binding sites shown to be involved in protein-protein
interactions and endocytosis in animal cells (Mason and Arndt, 2004). BLAST
searches to nonredundant databases revealed ToxABP1 homologs with 87% identity
in potato (Solanum tuberosum), 96% in rice (Oryza sativa) and 89% in Arabidopsis
thaliana (Manning et al., 2007). Like their wheat counterpart, these homologs all
possess chloroplast transit peptides (cTPs) for compartmentalization in this organelle
(Emanuelsson et al., 1999; Wang et al., 2004).
The ToxABP1 homolog in A. thaliana, designated THF1, was shown to
localize to chloroplasts and stromules both in vitro by 35S-radiolabeling and in planta
by GFP-fusion (Wang et al., 2004.) Transmission electron microscop was used to
identify a role for THF1 in thylakoid stacking; Arabidopsis Thf1 deletion lines were
without thylakoid membranes, grana, or starch granules (Wang et al., 2004). Thf1
transcripts were also shown to be light-regulated, with greater abundance of transcript
under prolonged exposure to light. ToxA-induced symptom development is also lightdependent (Manning and Ciuffetti, 2005) so ToxA could be manipulating sensitive
cells in a similar photoregulated manner in order to induce cell death.
Wangdi and associates (2010) identified a role for THF1 in the response to
coronatine (COR). COR is produced by the bacterial pathogen Pseudomonas syringae
pv. tomato DC3000 (DC3000) and is also able to induce chlorosis on tobacco
(Nicotiana benthamiana). Tobacco plants silenced for Thf1 were infiltrated with COR
and monitored for disease phenotypes. Silenced plants exhibited a hypersensitive-like
8
response with localized necrosis in the infiltration zone, which indicates THF1 plays a
role in the sensitivity response to the bacterial effector coronatine.
As mentioned previously, ToxABP1 localizes to chloroplasts and stromules.
Stromules are extensions of chloroplasts and known to connect to other plastids,
endoplasmic reticula, nuclei, and plasma membranes (Kwok and Hanson 2004;
Natesan et al. 2005). Therefore, ToxA could interact with ToxABP1 via stromules
and get transported to the chloroplast. After localization of ToxA to the chloroplast, a
light-dependent accumulation of reactive oxygen species (ROS) is produced that
correlates with necrosis, reduction of the chloroplast protein RuBisCo and a decrease
in photosystem I and photosystem II proteins (Manning et al., 2009). Massive
transcriptional reprogramming in wheat, especially in defense-related genes, is also
evident in response to treatment with ToxA (Pandelova et al., 2009). Upregulation of
genes that encode ethylene and jasmonic acid biosynthetic enzymes, components of
the phenylpropanoid pathway, and pathogenesis-related (PR) genes was shown by
microarray analysis upon treatment of sensitive wheat with ToxA.
Intracellular expression of ToxA sufficient for symptom development
In order for ToxA to cause symptoms on sensitive wheat, it must first be
internalized into the cell. However, bypassing this internalization step by expressing
ToxA intracellularly via barley-stripe mosaic virus (BSMV) is sufficient to induce
disease symptoms in insensitive wheat (Manning et al., 2010). Biolistic
bombardment of ToxA into both ToxA-sensitive and -insensitive wheat cells also
9
resulted in cell death, indicating the site-of-action is in both cell types (Manning and
Ciuffetti, 2005). BSMV-mediated internal expression of ToxA in barley (Hordeum
vulgare) also resulted in symptom development despite being a nonhost for Ptr.
Agrobacterium-mediated expression of ToxA in tobacco, a dicotyledonous nonhost of
Ptr, also led to the induction of disease symptoms (Manning et al., 2010). The ability
to internally express ToxA and induce symptoms in a variety of plant genera allows
the utilization of high-throughput genetic approaches to identify components that
interact in the ToxA symptom-development pathway.
Ptr race structure and potential pathogenicity factors
Ptr isolates present a complex race structure that is determined by the
repertoire of HSTs produced by the individual isolates and the wheat cultivars they
affect (Lamari and Bernier, 1995; Strelkov and Lamari, 2003; Ciuffetti et al, 2010;
Pandelova et al., 2012). On sensitive cultivars, ToxA induces necrosis, ToxB induces
chlorosis (Kim et al., 2010; Ciuffetti et al., 2010) and ToxC induces a spreading
chlorosis (Lamari et al., 2003). This race classification system has resulted in eight
well-described races. Table 1.1 shows the HSTs produced by each race and the
symptoms they elicit on a differential set of wheat. A Ptr isolate collected in southern
Oregon, designated SO3 (M. L. Putnam and L. M. Ciuffetti, unpublished data), was
originally considered to be nonpathogenic due to the absence of the ToxA gene
(Ciuffetti et al., 1997). After inoculation of SO3 on the standard wheat differential
set, elicitation of ToxA-like symptoms (necrosis on cultivars Glenlea and Katepwa)
10
Table 1.1. Race structure of Pyrenophora tritici-repentis.
Characterized races of Ptr determined by the toxins they produce and
the symptoms they elicit on the wheat differential set tested.
Differential set includes the cultivars Glenlea, Katepwa, 6B365 and
Salamouni. N (ToxA) = necrosis induced by Ptr ToxA; C (ToxC) =
spreading chlorosis induced by Ptr ToxC; C (ToxB) = chlorosis
induced by Ptr ToxB. R = resistant reaction.
11
and resistant reactions on the other three cultivars in the differential set contrasted with
the previous classification by eliciting a race 2 phenotype. Analyses by Southern and
Western blots reconfirmed that SO3 did not contain the ToxA gene and, therefore,
could not be a member of race 2 based on the current classification system (Ciuffetti et
al., 1997; Andrie et al., 2007). The disease phenotype observed in the absence of
ToxA suggested that the necrotic lesions induced by SO3 on Glenlea and Katepwa
were caused by a previously undescribed HST (Pandelova and Ciuffetti, 2005), which
was tentatively named Ptr ToxD (Manning et al., 2002). The combination of
phenotypic and genotypic evaluations led to the classification of SO3 as a new race,
race 9 (Andrie, 2007). Comparative transcriptomics with SO3, as well as sequencing
and resequencing of pathogenic and non-pathogenic isolates of Ptr, are methods being
utilized by the Ciuffetti laboratory to identify novel secreted proteins in SO3 that
could aid in further characterization of potential new HSTs.
12
Chapter 2
Virus-induced gene silencing-mediated identification of proteins involved
in ToxA-induced symptom development
ABSTRACT
Pyrenophora tritici-repentis (Ptr) is known to produce multiple host-selective
toxins (HSTs), which include Ptr ToxA (ToxA), Ptr ToxB (ToxB) and Ptr ToxC
(ToxC). ToxA is the most characterized toxin of Ptr and was the first proteinaceous
HST identified. Research has shown that ToxA is internalized into mesophyll cells of
wheat cultivars that harbor the ToxA sensitivity gene, Tsn1. The toxin then localizes
to the chloroplast where it interacts with the protein ToxABP1. Subsequent
photosystem perturbations lead to the accumulation of reactive oxygen species (ROS)
and cell death. However, it is unclear how ToxA transits to the chloroplast and if
additional ToxA-interacting proteins are involved in ToxA-induced cell death. To
address these questions, a virus-induced gene silencing (VIGS) approach was utilized.
Defense-related genes from tobacco (Nicotiana benthamiana) were silenced with the
tobacco rattle virus (TRV) VIGS system. This process revealed four genes that could
play a role in ToxA-induced cell death: a 40S ribosomal subunit, peroxisomal
glycolate oxidase (GOX), a thiamine biosynthetic enzyme (Thi1) and the R-gene
13
mediator, Sgt1. These four genes were narrowed to two, GOX and Sgt1, which were
assumed to have the highest likelihood to be involved in ToxA-induced symptom
development due to their roles in photorespiration and defense signaling, respectively.
The discovery of these genes in the ToxA symptom development network further
supports the hypothesis that Ptr takes advantage of defense responses in order to
colonize plant tissue and obtain nutrients, thereby causing extensive crop damage in
the process.
INTRODUCTION
Pyrenophora tritici-repentis (Ptr) is the necrotrophic fungus responsible for
tan spot of wheat (Triticum aestivum L.), which elicits disease on susceptible cultivars
through the production and secretion of host-selective toxins (HSTs) (Lamari and
Bernier, 1989; Oliver et al., 2008). HSTs are compounds only produced by fungi and
are considered to be primary determinants of pathogenicity. The property of the HSTs
to affect only those genotypes that are susceptible to the pathogen and their
requirement by the pathogen to cause disease gives them their ―selective‖ attribute
(Dunkle, 1984; Wolpert et al., 2002). HSTs are ideal compounds to study
susceptibility; these toxins can be purified, infiltrated into sensitive cultivars, and
reproduce the symptoms of disease in the absence of the pathogen. This allows for a
less complicated system to study toxin sensitivity and host-pathogen interactions.
14
Plant pathogens use multiple means to evade host defense responses. PAMPtriggered immunity (PTI) is the result of a plant detecting pathogen-associated
molecular patterns (PAMPs), such as chitin from the cell walls of fungi or flagellin
from bacterial flagella (Zipfel, 2004; Liu et al., 2012). PAMPS are recognized by
particular receptors on the surface of plant cells called pattern recognition receptors
(PRRs) (Jones and Dangl, 2006; de Jonge, et al., 2010). Recognition leads to the
activation of the mitogen-activated protein kinase (MAPK) cascade (Pitzschke et al.,
2009) and the initiation of several defense responses to combat the pathogen, which
include callose deposition on the cell wall to prevent pathogen ingress (Grant et al.,
2006), and expression of phytoalexins (Nürnberger and Lipka, 2005) and micro RNAs
(Navarro et al., 2008).
To evade PTI, many pathogens produce effector proteins to dampen the
plant‘s basal defense responses. This effector-triggered susceptibility (ETS) allows
the microbe to gain nutrition without recognition by the host (Zhang et al., 2010;
Gassmann and Bhattacharjee, 2012). Many plants have evolved a set of genes, called
R-genes, to recognize these effectors and mount a defense response known as effectortriggered immunity (ETI) (Jones and Dangl, 2006). This includes multiple defense
responses and typically culminates in a hypersensitive response (HR) where the plant
sacrifices a portion of its own cells to prevent further colonization by the pathogen
(Zipfel et al., 2004).
Only when a successful avr/R interaction occurs is the plant able to defend
itself; in all other cases the result is disease. This recognition event is known as the
15
gene-for-gene paradigm. Hallmarks of HR include a plethora of plant responses, some
of which include defense gene activation, an increase in cytoplasmic calcium, defense
gene activation, callose deposition, and production of reactive oxygen species, all of
which eventually lead to programmed cell death (Mur, 2008). ETS and ETI typically
reciprocate within pathosystems in an evolutionary arms race as pathogens evolve new
effectors to suppress basal (PTI) immunity and hosts evolve new R-genes that trigger
ETI (Figure 2.1).
The Ptr-wheat pathosystem follows a slightly different pattern from those
mentioned above. Necrotrophic fungi like Ptr infect and kill host tissue and extract
nutrients from the dead cells, so the HR would only exacerbate the disease seen on the
host plant (Shlezinger, 2011). With regard to Ptr, a gene in certain wheat cultivars
confers sensitivity to ToxA, and subsequent susceptibility to the fungus, allowing the
pathogen to necrotize the cells of the host in order to gain nutrition (Wolpert et al.,
2002; Strelkov and Lamari, 2003). This has been regarded as an inverse gene-forgene interaction. Contrary to classical gene-for-gene, inverse gene-for-gene
interactions result in susceptibility when the sensitivity locus and the effector are
present in the host and pathogen, respectively.
Inverse gene-for-gene relationships are not uncommon in regards to
necrotrophic fungi and their hosts. Other interactions that involve an inverse gene-forgene relationship include those of Cochliobolus victoriae and oat (Avena sativa)
(Lorang et al., 2004) as well as Stagnospora nodorum and wheat (Friesen et al., 2008).
These fungi, like Ptr, use HSTs to cause disease. HSTs can be either proteinaceous or
16
Strength of defense response
Strong
PTI
ETS
ETI
ETS
ETI
Hypersensitivity
Effectors
Effectors
R-Avr
R-Avr
Resistance
Weak
PAMPs
Figure 2.1. Model of plant immunity.
The transitions between PAMP-triggered immunity (PTI), effector-triggered
susceptibility (ETS) and effector-triggered immunity (ETI). Plants detect
pathogen-associated molecular patterns (PAMPS) with pattern recognition
receptors and basal defense is initiated, which results in PTI. Pathogens
evolve effectors to repress this defense response and cause disease, which
results in ETS. Plants in turn evolve resistance (R) proteins to recognize
these effectors (Avr), which elicits ETI and typically the hypersensitive
response. Adapted from ―The Plant Immune System.‖ Jones, JD and Dangl,
JL. 2006. Nature. 444:323–329.
17
non-proteinaceous, and Ptr produces both varieties. Ptr ToxA (ToxA) and Ptr ToxB
(ToxB) are both proteinaceous (Ciuffetti et al., 2010), while Ptr ToxC (ToxC) is nonproteinaceous and appears to be a non-ionic, low molecular weight compound (Effertz
et al., 2002).
ToxA is the most characterized HST produced by Ptr. Much research has
been directed at elucidating the site- and mode-of-action of ToxA, which has led to
several discoveries in regards to necrotrophic pathogenicity and the characteristics of
HSTs. The crystal structure of ToxA has been solved and it appears to be a single
domain protein with a β-sandwich fold and contains an aspartic arginine-glycineaspartic acid (RGD)-motif on a solvent-exposed loop (Figure 1.1) (Sarma et al., 2005).
RGD-like peptide motifs are known to be involved in cell attachment to integrin-like
receptors, such as the mammalian proteins vitronectin and fibronectin (Meinhardt et
al., 2002; Manning et al., 2004; Sarma et al., 2005). Virulence proteins from the
malaria parasite Plasmodium falciparum have been shown to use this motif for hosttargeting in blood-stage infections of humans (Bhattacharjee et al.,2006). Sitedirected mutagenesis of this amino acid motif in ToxA resulted in a significant
decrease in ToxA activity. Competition assays with coinfiltration of ToxA and other
RGD peptides in sensitive wheat resulted in a reduction of ToxA symptoms (Manning
et al., 2004). Studies have indicated that some fungal human pathogens utilize RGDmotifs on the cell surfaces of their hosts to facilitate adhesion (Hostetter, 2000).
ToxA also induces defense-related responses in sensitive wheat. Microarray
data has shown ToxA elicits several responses in sensitive wheat that are typically
18
associated with incompatible resistance reactions (Pandelova et al., 2009), which
include the upregulation of ethylene and jasmonic acid synthesis, calcium signaling,
and expression of MAPK signaling components and PR-proteins. These host
reactions are typically those seen in the HR during incompatible pathogen attack and
recognition by an R-protein (Leach and White, 1996). In addition, reductions in
photosystem proteins after ToxA treatment have been observed via Blue native-gel
electrophoresis (Manning et al., 2009) and the previously mentioned microarray
analyses. The current hypothesis for the mode-of-action of ToxA involves an
association with a high affinity receptor on the surface of mesophyll cells in sensitive
wheat cultivars, leading to receptor-mediated endocytosis of ToxA via recognition of
the solvent-exposed arginine-glycine-aspartic acid (RGD)-loop (Ciuffetti et al., 2010).
Following internalization, it is hypothesized that ToxA dissociates from the endosome
and localizes to the chloroplast. Upon reaching this destination, perturbations in the
photosystem complexes and the production of generous amounts of ROS lead to
massive transcriptional reprogramming, all of which lead to necrosis (Ciuffetti et al.,
2010). There are, however, gaps in knowledge of the ToxA mode-of-action including
how it transits to the chloroplast from the plasma membrane and if there are any
additional proteins involved in this process. One hypothesis is that ToxA interacts
with the protein, ToxABP1, via stromules at the plasma membrane and gets
transported to the chloroplast (Manning et al., 2010).
In order to identify additional genes that may play a role in ToxA-induced
symptom development, the bipartite tobacco rattle virus (TRV)-based VIGS system
19
was used for the research presented in this chapter. This system has successfully
identified multiple participants in plant disease resistance (Liu et al., 2002a; Liu et
al.,2002b; Rojas et al., 2012).
VIGS is the process of using a recombinant virus, which contains an insert
with homology to a host gene, to infect a plant. In response to viral infection, the
plant degrades any transcript with homology to the virus via RNA silencing, which
includes transcripts of its own gene products that are homologous to the viral insert
(Baulcombe, 1999; Holzberg et al., 2002; Lu et al., 2003; Burch-Smith et al., 2004).
This post-transcriptional gene silencing method results in a reliable method to silence
genes that impact a phenotype of interest.
TRV-VIGS was used as a forward genetics system to screen a cDNA library
from tobacco (N. benthamiana) (Liu et al. 2002a; Liu et al., 2002b; Anand et al.,
2007; Wangdi et al., 2010). TRV is composed of two molecules, TRV1 and TRV2,
with TRV2 containing a multiple cloning site (MCS) for introduction of a silencing
library (Figure 2.2). Both viral molecules must be present in the same cell to form a
complete virus.
The library used for silencing, which was kindly provided by Dr. Kirankumar
Mysore of the Samuel Roberts Noble Foundation (Ardmore, OK, USA), was
constructed from cDNA synthesized from tobacco plants that were treated with biotic
and abiotic elicitors of plant defense-related genes. As ToxA was shown to induce the
expression of several defense-related genes, an approach that silences genes from this
cDNA library could potentially reveal additional players in ToxA symptom
20
A)
LB
2x35S
RdRp
2x35S
Coat
MP
Riboz.
NOS
RB
B)
LB
MCS Riboz.
NOS
Figure 2.2. Genomic architecture of the tobacco rattle virus.
TRV is a bipartite virus made of two RNA molecules encoding different
proteins. A) TRV1; 2x35S = duplicated 35S promoter from the cauliflower
mosaic virus; RdRp = RNA-dependent RNA polymerase; MP = movement
protein; Riboz = ribozyme; NOS = nopaline synthase terminator; LB = left
border; RB = right border. B) TRV2; Coat = coat protein; MCS = multiple
cloning site. Adapted from ―Role of SCF Ubiquitin-ligase and the COP9
Signalosome in the N Gene–mediated Resistance Response to Tobacco Mosaic
Virus.‖ Liu, Y, Schiff, M, Serino, G, Deng, XW, and Dinesh-Kumar, SP. 2002.
The Plant Cell. 14:1483–1496.
RB
21
development. These genes would be identified by a reduction in ToxA-induced
symptom development after VIGS. Silencing of the genes in the library and
monitoring for subsequent ToxA symptom development led to the identification of
four genes, 40S, Thi1, GOX and Sgt1, that led to a decrease in ToxA symptom
development. These four genes were narrowed to two, GOX and Sgt1, which were
assumed to have the highest likelihood to be involved in ToxA-induced symptom
development due to their roles in photorespiration and defense signaling, respectively.
In conjunction with previous data regarding the site- and mode-of action, it appears
that ToxA could be regulating signaling, protein degradation, and photorespiratory
components to kill host cells. As a necrotroph, Ptr consumes these dead plant cells as
a means of nutrition, which promotes further colonization by the fungus and disease of
the crop.
MATERIALS AND METHODS
Production of tobacco plants
N. benthamiana seeds were placed in a solution of 0.1% agarose and pipetted
directly onto the surface of water-saturated Sunshine professional growing mix
(SunGro Horticulture, Seba Beach, AB Canada) in 4 inch pots and covered to retain
humidity. Pots were placed in a growth chamber at 25°C and 70% humidity. Light
22
intensity was 50 µE s-1 m-2 in a 16 hour photoperiod. Three-week-old plants were
used in silencing experiments.
Bacterial growth and induction
Agrobacterium GV2260 cells containing expression constructs of either TRV1,
ToxA or GFP were streaked on low salt Luria-Bertani agar media (LSLB; Research
Products International Corp, Prospect, IL, USA) supplemented with the appropriate
antibiotics (50 µg/ml kanamycin for constructs with TRV1 or ToxA; 5 µg/ml
tetracycline for the construct with GFP) . Single colonies of each Agrobacterium
construct were picked and grown in 5 ml yeast extract-peptone (YEP) broth (5g NaCl,
10g yeast extract, 10g peptone in 1L ddH2O) overnight at 28°C with shaking
conditions. The next day, 50 µl of the overnight culture was inoculated into 25 ml
YEP and grown overnight under the same conditions. Cells were pelleted by
centrifugation at 6,500 rpm at room temperature and resuspended in 25 ml induction
media consisting of M9 medium, pH 5.2 (6 g Na2HPO4, 3 g KH2PO4, 0.5 g NaCl, 1 g
NH4Cl, 1 ml 1M MgSO4, 0.1 ml 1M CaCl2 and 10 ml 20% glucose) plus 0.1 mM
acetosyringone and 10 mM 2-(N-morpholino)-ethanesulfonic acid (MES), pH 5.2.
The culture was incubated overnight on the bench, spun down, and resuspended in
infiltration media (10 mM MES, pH 5.2, 10 mM MgCl2 and 0.15 mM acetosyringone)
to an O.D.600 of 0.9 for TRV1-expressing cells and 0.5 for ToxA- and GFP-expressing
cells. TRV2-expressing constructs for silencing were streaked onto low-salt Luria
23
Bertani (LSLB) agar medium supplemented with 50 µg/ml kanamycin and grown at
28°C for two days immediately before they were used.
VIGS Agrobacterium constructs and experimental design
TRV1 and TRV2 cDNA library constructs in Agrobacterium GV2260 cells
were graciously provided by Dr. Kirankumar Mysore of the Samuel Roberts Noble
Foundation (Ardmore, OK, USA). TRV1 and TRV2 plasmids were constructed as
described by Liu et al. (2002a). TRV2: GFP (green fluorescent protein) clone was
constructed and transformed into Agrobacterium GV2260 cells by Brian Gilbert
(Wolpert laboratory, Oregon State University, Corvallis, OR, USA). TRV2:SGT1
(suppressor of G2 allele skp1) Agrobacterium clone was a gift from Dr. Greg Martin
(Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, NY, USA).
The plasmid expressing the N- and C-domains of Ptr ToxA was constructed as
described previously (Manning and Ciuffetti, 2010). The plasmid expressing GFP,
pSLJ:GFP, was provided by Dr. J. C. Carrington (Donald Danforth Plant Science
Center, St. Louis, MO, USA). Both the ToxA- and GFP-containing plasmids were
transformed into Agrobacterium GV2260 cells.
Agrobacterium cells expressing TRV1 at a concentration of O.D.600 = 0.9 were
infiltrated into three-week old tobacco plants on the abaxial side of the secondary leaf
with a 1-ml needleless syringe. TRV2-containing Agrobacterium clones, including
the silencing library as well as the phytoene desaturase- (PDS) and GFP-silencing
24
constructs, were picked from LSLB plates with a toothpick and individually pricked
on the adaxial side of the TRV1-infiltrated leaf within the TRV1 infiltration zone.
Plants were then incubated under the same growth chamber conditions listed above for
two weeks to silence the genes. The two-week time period was determined by
observation of the photobleached phenotype displayed by the PDS-silenced control
plants (Figure 2.3). Three plants were inoculated with each clone in the initial VIGS
screenings. During all subsequent experiments, 5 plants were inoculated per clone.
All silencing experiments were repeated at least 3 times with similar results. After
silencing had been determined, ToxA- and GFP-expressing constructs were
agroinfiltrated and symptom development was monitored over seven days. Those
silenced tobacco plants with no ToxA-induced symptoms are candidates for genes
involved in the ToxA symptom development network.
Clone identification
The primer used to sequence the TRV2 plasmids containing the silencing
inserts was TRV2_F1 (Table 2.1). Confirmation of the candidate TRV2 clones was
performed by direct DNA sequencing of the inserts at the Center for Genome
Research and Biocomputing core laboratory (Oregon State University, Corvallis, OR,
USA) followed by BLAST searches to the NCBI database
25
Figure 2.3. Phytoene desaturase-silenced tobacco.
Nicotiana benthamiana plant silenced for the phytoene desaturase gene,
which results in an extensively photobleached phenotype. When
photobleaching is evident, the virus has spread systemically and silencing
of the genes in the library has occurred.
26
Table 2.1. Primers used in clone identification and RT-PCR.
Names and sequences of primers used for clone identification and PCR.
Primer ID:
Sequence:
TRV2_F1
5‘-AGTTTGTACAAAAAAGCAGGC-3‘
Nb40S_F1
5‘-ATGCTTCTGACTTTGGATGAGA-3‘
Nb40S_R1
5‘-CATGGTGGATTGGATGAGA-3‘
NbThi1_F1
5‘-TGGCTCTGCTGGTCTCTCTTG-3‘
NbThi1_R1
5‘-TAGTTGTCTTGCTCGTCATAGTC-3‘
NbGOX_F1
5‘-CTATCATGATTGCACCAACAG-3‘
NbGOX_R1
5‘-CACAAGCTGAGCAACAACAT-3‘
NbSgt1_F1
5‘-CGAACAAGGCCATTGAGTTAC-3‘
NbSgt1_R1
5‗-TCTCCAGCTTCCTCTGCAAT-3‘
18S_F
5‘-GTGACGGGTGACGGAGAATT-3‘
18S_R
5‘-AGACTCATAAAGCCCGGTAT-3‘
27
(http://www.ncbi.nlm.nih.gov/) and to the Dana Farber Cancer Institute N.
benthamiana Gene Index (http://compbio.dfci.harvard.edu/cgibin/tgi/gimain.pl?gudb=n_benthamiana).
Cell death assessment
ToxA-induced symptoms on silenced tobacco plants were compared to those
elicited by the GFP-expressing cells, which served as the negative control for
symptom development. Symptoms were monitored on unsilenced plants
agroinfiltrated with ToxA or GFP as well as negative controls for no silencing. Cell
death was determined visually, photographed (Canon 300D SLR camera), and leaves
scanned on an Epson 1600 scanner (Epson, Long Beach, CA, USA).
Assay to measure ToxA effect on chlorophyll concentration
Cell death was quantified as total chlorophyll in leaves. Sections of tobacco
leaves were taken from agroinfiltration zones using a #6 borer. Ethanol was added
(500 µl of 95% v/v) to each sample separately and tubes incubated in the dark
overnight at room temperature to extract chlorophyll. The ethanol solution containing
the chlorophyll was measured (200 µl) in a spectrophotometer at 654 nm. The total
chlorophyll concentration was calculated as described by Manning et al. (2004).
Concentration values were averaged from 10 independent chlorophyll extractions per
28
silenced clone and 10 per GFP-treated and nonsilenced negative controls. Each
measurement was repeated 5 times with similar results. Statistical significance was
determined with a nonparametric two-tailed t-test from the program GraphPad Prism 5
(GraphPad Software, Inc., La Jolla, CA, USA). All samples were compared to their
GFP controls, as well as to the nonsilenced controls, in the statistical analysis.
RT-PCR
Total RNA was isolated from ToxA- and GFP-agroinfiltrated regions of leaves
silenced for TRV2:Thi1, TRV2:GOX, TRV2:Sgt1 and control TRV2:GFP-silenced
leaves with the Qiagen RNeasy Plant Mini Kit (Maryland, USA). First strand cDNA
synthesis was performed with the Superscript III reverse transcriptase (Invitrogen)
with 5µg starting RNA. The resulting cDNA was amplified via PCR with GoTaq
DNA polymerase (Promega) and primers specific for the candidate clones Thi1, GOX
and Sgt1, as well as the 18S ribosomal gene as an internal control, in both silenced
plants agro-infiltrated with ToxA and unsilenced plants. Thi1 transcripts were detected
with primers NbThi1_F1 and NbThi1_R1. GOX transcripts were detected with
primers NbGOX_F1 and NbGOX_R1 . Sgt1 transcripts were detected with primers
NbSgt1_F1 and NbSgt1_R1. Internal controls with the 18S gene for equal loading of
RNA were verified with the primers 18S_F and 18S_R. Cycling parameters for the
genes were 25 cycles of 30 seconds at 95°C; 30 seconds at 61°C, 56°C, 67°C and
59°C for Thi1, GOX, Sgt1 and 18S, respectfully; 72°C for 30 seconds. Primers were
29
designed to anneal outside the region homologous to the viral insert in TRV2. DNA
was analyzed by size-separation on a 1% agarose gel and stained with ethidium
bromide. All primers used are listed in Table 2.1.
Detection of GFP fluorescence in tobacco
Leaf discs were excised with a #6 borer from GFP-agroinfiltration zones of
silenced and unsilenced tobacco plants. Fluorescence was visualized under 40x
magnification on a Leica MZFL III stereoscope (Buffalo Grove, IL, USA) and images
captured with a CoolSNAP-PRO color camera (RS Photometrics; Tucson, AZ, USA).
ToxA detection in tobacco leaves
Proteins were extracted from 1 cm2 tobacco leaf segments based on a protocol
by Curtis and Wolpert (2002). Protein concentration was quantified via bicinchoninic
acid (BCA) assay using the Pierce BCA assay kit (Rockford, IL, USA), as per the
manufacturer‘s instructions, with bovine serum albumin (BSA) as the standard. Total
protein (80 µg) from tobacco leaves, which had been agroinfiltrated with either ToxA
or GFP, was resolved via sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE). Proteins were then transferred to nitrocellulose membrane and detected
via Western blot with ToxA antisera. A His-tagged ToxA fusion protein was used as a
positive control for antibody detection. Protein from GFP-agroinfiltrated leaves
served as negative controls.
30
RESULTS
VIGS reveals proteins involved in ToxA symptom development
From the cDNA library, 1,153 clones were silenced in tobacco. A schematic of
the experimental protocol is presented in Figure 2.4. The TRV-VIGS screen identified
four genes that encode proteins that could play a role in ToxA-induced symptom
development. These genes include the 40S ribosomal subunit, the chloroplastlocalized THI1, the peroxisomal enzyme GOX, and the defense- associated SGT1.
Phenotypes of silenced and ToxA effects
When a gene is silenced in an organism, the function contributed by its
associated protein is diminished. This loss-of-function can show a visible phenotype
displayed by the plant, as was observed with silencing of the candidate genes in this
study. Each silenced gene revealed a different plant phenotype (Figure 2.5; A-F).
Wild type N. benthamiana plants (Fig. 2.5-A) and those silenced for GFP (Fig. 2.5-B)
were similar with no loss-of-function phenotypes observed. Plants silenced for Thi1
(Fig. 2.5-C) exhibited a high degree of branching along with a mottled appearance in
the foliage. The phenotype associated with tobacco silenced for Sgt1 (Fig. 2.5-D) was
31
A. Silence defense-related genes in
tobacco
B. Internally express ToxA in silenced tobacco and monitor
for symptom development
Yes
C. ToxA
Symptom
No
D. Confirmation of
agroinfection and
ToxA expression
Silenced gene may not
play a role in ToxA
symptom development
Yes
E. Candidate
gene!
No
Silenced gene
affected
agroinfection or
ToxA expression
Figure 2.4. TRV-VIGS procedure.
Outline of steps in silencing genes in the library. A) Genes from the library
are silenced in tobacco with TRV; B) ToxA agroinfiltrated in silenced
tobaccoo; (C) Leaves monitored for symptom development; D) If no symptom
development, GFP fluorescence is checked to confirm agroinfection and a
Western blot is performed to confirm ToxA expression; E) If both detected, the
gene is a potential candidate.
32
A. -TRV
B. TRV:GFP
C. TRV:THI1
E. TRV:40S
D. TRV:SGT1
F. TRV:GOX
Figure 2.5. Silencing phenotypes of candidate genes.
Tobacco plants showing phenotypes from silencing without agroinfiltration of
ToxA or GFP. Panel A shows an unsilenced wild type Nicotiana benthamiana
plant. Panel B shows a tobacco plant silenced for GFP. The wild type (A) and
TRV:GFP plants (B) exhibit similar, nonaffected phenotypes. Panel C shows
tobacco silenced for Thi1. Panel D shows tobacco silenced for Sgt1, panel E
shows tobacco silenced for 40S. Panel F shows tobacco silenced for GOX.
33
that of an extremely stunted growth form and extensive nodal branching. Tobacco
plants silenced for 40S (Fig. 2.5-E) were extremely stunted and showed a high degree
of chlorosis with mottled, crinkled leaves.
The phenotype seen with GOX-silenced
tobacco (Fig. 2.5-F) was also highly mottled in foliar appearance, but included
dwarfed plants with extensive necrosis at shoot tips and in the veins. With the
exception of wild type (-TRV), agroinfiltration of ToxA into all plants shown in
Figure 2.5 showed no symptom development in the infiltration zones and were similar
to those silenced for the same genes and agroinfiltrated with GFP. However, in
nonsilenced tobacco plants agroinfiltrated with ToxA cell death is noticeable in the
infiltration zone compared to those treated with GFP alone (Fig. 2.6). Circles in the
figure indicate the infiltration zones where symptoms would be observed. This
experiment was repeated four times with similar results.
Quantitative assessment of symptom development
The extent of symptom development was monitored not only visually but also
quantitatively by a spectrophotometric chlorophyll assay, in which decreased amounts
of chlorophyll are indicative of the initial processes of cell death. Tobacco plants
silenced for the genes being investigated had higher levels of total chlorophyll present
in the ToxA agroinfiltration zones than non-silenced control plants. This was
comparable to plants agroinfiltrated with GFP, which also had high levels of
34
Non-silenced
-TRV
Silenced
TRV:40S
TRV:GOX
TRV:THI1
TRV:SGT1
ToxA
GFP
Figure 2.6. ToxA and GFP symptom development on silenced tobacco.
Silenced tobacco plants agroinfiltrated with either ToxA (ToxA) or GFP (GFP).
Leaves were harvested and scanned 7 days after agroinfiltration. The leaves in
the top panels were treated with ToxA while the leaves in the bottom panels
were treated with GFP. TRV:40S = tobacco silenced for 40S; TRV:GOX =
tobacco silenced for glycolate oxidase; TRV:THI1 = tobacco silenced for Thi;
TRV:SGT1 = tobacco silenced for Sgt1; -TRV = non-silenced tobacco plants
treated with ToxA or GFP.
35
chlorophyll, as shown in Figure 2.7 and Table 2.2. ToxA-agroinfiltrated, nonsilenced
control leaves had a lower chlorophyll concentration than GFP-agroinfiltrated,
nonsilenced control leaves with a statistically significance of p<0.05. The same
significance was observed with plans silenced for GFP, with ToxA-agroinfiltrated
leaves significantly lower than those agroinfiltrated with GFP (p<0.05). Leaves
silenced for the four candidate genes, however, showed no statistically significant
difference in chlorophyll concentration between ToxA- and GFP-treated leaves and
were significant with a p-value < 0.05.These high chlorophyll concentrations in
silenced leaves treated with ToxA, similar to their GFP-treated counterparts, indicates
that the silencing of these genes inhibited ToxA symptom development.
Reduction in transcripts correlated with decrease in cell death
To ensure that VIGS reduced the transcript levels in silenced plants, RT-PCR
was performed and the transcript levels of the candidate genes in silenced and nonsilenced plants were compared with the 18S gene as an internal control for expression
(Figure 2.8). Primers specific to silenced genes were used to amplify cDNA generated
from total RNA isolated from ToxA agroinfiltration zones of silenced and nonsilenced plants, including control plants silenced for GFP. These primers were
designed with no sequence specificity to the TRV2 insert to ensure that spurious
amplification did not occur. After amplification, transcript levels of silenced genes
were greatly reduced when compared to those of non-silenced plants. cDNA from
36
*
*
Figure 2.7. Chlorophyll assay of tobacco leaves.
Leaves of tobacco plants that were silenced for the candidate TRV2 clones were
measured for chlorophyll content after agroinfiltration with ToxA or GFP.
Samples were taken 7 days post infiltration from within the agroinfiltration
zones. A higher chlorophyll concentration indicates a reduction in ToxA
symptom development while a low chlorophyll concentration indicates a lack of
reduction in ToxA symptom development. Error bars represent standard error
from five biological replicates while asterisks indicate statistical significance
from GFP controls with a p-value <0.05 as determined by a nonparametric twotailed t-test.
37
Table 2.2. Chlorophyll concentrations in silenced leaves.
Chlorophyll concentration (µg/ml) was taken 7 days post infiltration. AgroToxA = total chlorophyll from leaves agroinfiltrated with ToxA; Agro-GFP
= total chlorophyll from leaves agroinfiltrated with GFP; NT = wild type
tobacco, unsilenced and uninfiltrated for baseline tobacco chlorophyll
concentration. Dashes indicate no measurement for that condition.
TRV: TRV:
40S
THI1
TRV: TRV:
GOX SGT1
TRV:
GFP
ToxA
Only
4.04
GFP
Only
Wild
Type
-
-
Agro-ToxA
4.58
11.02
8.04
10.66
3.98
Agro-GFP
4.66
10.84
7.93
11.03
11.04
-
11.45
-
NT
-
-
-
-
-
-
-
12.09
38
Figure 2.8. Reverse transcription polymerase chain reaction.
Reverse transcription polymerase chain reaction (RT-PCR) was performed on
cDNA from silenced and unsilenced tobacco plants with primers specific for
the candidate genes identified through VIGS or 18S control primers. A)
Unsilenced tobacco cDNA amplified with gene-specific primers (left 4 lanes)
and silenced tobacco cDNA amplified with gene-specific primers (right 4
lanes); B) Amplification of cDNA from TRV:GFP-silenced tobacco (virus
only control) with gene-specific primers of candidate clones; C) Wild type
tobacco cDNA (lane 1), cDNA from silenced tobacco plants (lanes 2-5), and
cDNA from TRV:GFP tobacco plants (lane 6) amplified with 18S control
primers. This analysis was repeated three times with similar results.
39
TRV:GFP plants was also amplified with the candidate gene-specific primers to show
that the presence of the virus did not affect gene expression. This resulted in transcript
levels of the candidate genes similar to those from non-silenced tobacco plants. The
18S transcript levels were similar for all silenced leaves and non-silenced samples.
These results indicate that the lack of ToxA symptoms seen on the silenced candidate
clones and higher levels of chlorophyll is correlative with a reduction of transcripts
caused by the TRV-VIGS process.
Agroinfection and ToxA expression not inhibited by silencing
To confirm that the agroinfection process was not affected by silencing, GFP
fluorescence was visualized in silenced plants with fluorescent microscopy (Fig. 2.9).
Plants silenced for Thi1, GOX and Sgt1 showed high levels of fluorescence similar to
non-silenced tobacco plants agroinfiltrated with GFP. TRV:40S and TRV:GFP plants
had lower levels of fluorescence, most likely due to low translation efficiency in the
case of TRV:40S and because TRV:GFP plants are silenced for GFP.
To confirm that silencing did not affect the expression of ToxA, total protein
was isolated from ToxA-agroinfiltrated leaves of silenced plants and ToxA was
detected via Western blot (Fig. 2.10). Protein from silenced plants agroinfiltrated with
40
Agro-GFP
TRV:40S
TRV:GOX
TRV:GFP
TRV:THI1
TRV:SGT1
Figure 2.9. GFP fluorescence in tobacco plants.
Images were taken 7 days post agroinfiltration. GFP fluorescence is indicated
by the green pigment in the panels above. Agro-GFP = unsilenced tobacco
treated only with GFP; TRV:GFP = tobacco silenced for GFP; TRV:40S =
tobacco silenced for 40S; TRV:THI1 = tobacco silenced for Thi1;
TRV:GOX = tobacco silenced for GOX; TRV:SGT1 = tobacco silenced for
Sgt1. Images taken at 400x magnification.
41
40S
T
G
THI1
GOX
SGT1
GFP
T
T G
T
T
G
G
G
-TRV His:ToxA
T
G
+
-20 kD
-17 kD
-15 kD
Figure 2.10. Western blot to detect ToxA in silenced plants.
Total protein from silenced and non-silenced plants agroinfiltrated with
either ToxA or GFP was analyzed by Western blot. T = sample from
tobacco agroinfiltrated with ToxA; G = sample from tobacco agroinfiltrated
with GFP; + = positive control for ToxA antibody; 40S = proteins from
tobacco silenced for 40S; THI1 = proteins from tobacco silenced for Thi1;
GOX = proteins from tobacco silenced for GOX; SGT1 = proteins from
Sgt1-silenced tobacco; GFP = proteins from GFP-silenced tobacco; -TRV
= proteins from non-silenced tobacco; His:ToxA = His-tagged ToxA.
Values on the right indicate molecular mass as determined from a protein
standard.
42
GFP served as negative controls for the ToxA antibody. As a positive control, a
purified fusion peptide of His-tagged ToxA was used (His:ToxA). ToxA from
agroinfiltrated leaves has a molecular mass of 17.3 kD, while His:ToxA has a
molecular mass of 19.7 kD. ToxA was detected in each sample at the location of
agroinfiltration, as well as in the positive control (His:ToxA). All samples from GFPagroinfiltrated leaves were void of ToxA.
DISCUSSION
TRV was chosen for the VIGS vector because of the efficient and systemic
silencing it induces in the host, as well as very mild background viral symptoms. The
TRV vectors used have also been engineered for ease of introducing coding sequences
and Agrobacterium-mediated delivery, which facilitates high-throughput genetic
screens (Liu et al., 2002a). Previous research (Wu et al., 2010) has shown that the
empty TRV vector exacerbates the disease symptoms typically induced by TRV, but
inclusion of an insert of ~250 bp greatly reduces symptom expression. Therefore,
TRV2 containing a fragment of the green fluorescent protein (GFP) was used as a
virus-only control in the silencing experiments (GFP does not have any sequence
similarity to N. benthamiana DNA and, therefore, will not lead to silencing of a host
gene). The use of the TRV2:GFP construct as the negative control greatly reduces
background viral symptoms, thus ensuring the TRV does not interfere with the ToxA
phenotype (Hein et al., 2005; Scofield et al., 2005; Scofield and Nelson, 2009). The
43
inclusion of this control also confirms that the presence of TRV in plants does not
compromise expression of symptoms after agroinfiltration with ToxA.
The 40S ribosomal subunit was implicated as a protein involved in ToxA
symptom development. It is logical to assume that silencing a component of protein
synthesis such as 40S would result in a loss of ToxA symptom due to the reduction in
translation efficiency. As expected, both GFP fluorescence and ToxA protein levels
were also highly reduced in TRV2:40S plants. This reduction in fluorescence and
ToxA protein levels made it difficult to determine if 40S is involved in ToxA
symptom development or if it is due to lowered translation efficiency in the host. The
phenotype exhibited by TRV:40S included severely stunted growth, crinkled leaves
and extensive chlorosis throughout the entire plant, making it difficult to discern ToxA
symptom from the silencing phenotype. Low chlorophyll levels for both ToxA- and
GFP-treated tobacco silenced for 40S were observed when compared to the
chlorophyll concentrations of other silenced candidate clones. Due to the importance
of 40S in overall protein synthesis and the observation of 40S-silenced plants as
extensively chlorotic, TRV:40S was not considered an interacting clone that affects
ToxA-induced cell death.
Thi1, a gene that encodes an enzyme responsible for the formation of the
thiazole ring portion of thiamine, or vitamin B1, in plants (Machado et al., 1996;
Machado et al., 1997), was also identified as a candidate through the TRV-VIGS
screens. The presence of two alternate start codons for Thi1 gives the cell the ability
to shuttle the protein to either the chloroplast or mitochondrion. Use of the first start
44
codon directs THI1 to the chloroplast where it is hypothesized to function in amending
thiamine deficiencies. If the mitochondrion is the preferred organelle, the second start
codon is utilized. Studies show that Arabidopsis thaliana THI1 can partially correct
defects in DNA repair in Escherichia coli, which includes base and nucleotide
excision repair (Machado et al., 1996). The fact that translation primarily ensues using
the first AUG of this protein suggests a high requirement for THI1 in chloroplasts
(Chabregas et al., 2003). Thiamine-related antioxidants have been proposed to play a
role in stress and defense in plants (Rapala-Kozik et al., 2012). Previous research has
also indicated an association of ToxA with ToxABP1, which leads to decreases in
photosystem proteins, induction of ROS, and eventual cell death (Manning and
Ciuffetti, 2005; Manning et al., 2007). However, THI1 resides at the beginning of a
large biosynthetic pathway that is involved in multiple important cellular events
including glycolysis, nucleic acid formation, NADPH and ATP synthesis, and the
pentose phosphate pathway (Rapala-Kozik et al., 2012). This makes it extremely
difficult to pinpoint THI1 as a target of ToxA without considering other possible
downstream effects of silencing this gene. Because of these complications, Thi1 was
not considered a final interacting clone that plays a direct role in ToxA-induced
symptom development. Virus-induced gene silencing of other components in the
thiamine biosynthetic pathway could clarify its role in ToxA-induced cell death, as it
is apparent that ToxA may be affecting a component of the thiamine biosynthetic
cluster to induce cell death.
45
GOX is a peroxisomal enzyme in plants responsible for the conversion of
glycolate to glyoxylate. It was revealed by the VIGS screen as a candidate in ToxA
symptom development. GOX activity in the peroxisome results in the production of
high amounts of hydrogen peroxide (H2O2), which is a powerful ROS. The glyoxylate
produced in this reaction is eventually converted to the amino acids glycine and serine,
which are shuttled to the mitochondria for protein synthesis (Bourguignon et al., 1999;
Rashad et al., 2007).
GOX is the predominant H2O2-generating enzyme in the peroxisome, which is
known to be a source of signaling molecules in plant cells during stress and defense
(Corpas et al., 2001). In the case of resistance of melon to the downy mildew
pathogen Pseudoperonospora cubensis, it is evident that the HR arrests the growth of
the pathogen (Jackson and Taylor, 1996; Heath, 2000). Evidence gathered by Taler et
al. (2004) indicates a role for increased GOX activity. GOX levels were 10 to 20
times more in resistant melon cultivars, leading to high levels of H2O2 during the
defense response. Similar to our results, Rojas et al. (2012) has used TRV-VIGS to
show that GOX mediates nonhost resistance in tobacco to Pseudomonas syringae by
modulating ROS production. Tobacco plants silenced for GOX and inoculated with 3
nonhost bacteria, two Pseudomonas syringae pathovars and one Xanthomonas sp.,
showed a delayed HR and at least a 10-fold increase in bacterial growth.
The same study addressed the role of GOX in ETI- and PAMP-triggered
immunity. Several R-genes and their Avr-gene counterparts were coexpressed in the
leaves of GOX-silenced tobacco plants. In the case of coexpression of Pto:AvrPto, the
46
typical tissue collapse seen with the associated HR was delayed significantly (Rojas et
al., 2012). INF1 from Phytophthora infestans was also transiently expressed with
Agrobacterium in TRV:GOX leaves, similar to this study with ToxA transient
expression. HR associated with INF1 was severely delayed as well. Pathogens
included in these studies are biotrophic organisms, and data indicate a strong role of
ROS generated through GOX as a means of defense against these organisms. In the
case of necrotrophic microbes, however, this would likely not inhibit colonization and
disease. Necrotrophs would likely exploit the attempt at defense and use it to their
advantage, metabolizing the dead cells and inducing further disease. Because of the
propensity to generate high amounts of ROS, its association with chloroplasts via the
peroxisomes, and previous evidence for its role in resistance, GOX should be
investigated for consideration as an interactor in ToxA symptom development.
Sgt1 was identified through the VIGS screen as potentially playing a role in ToxAinduced symptom development. Past research has identified SGT1 as a participant in
multiple defense-related interactions. Arabidopsis SGT1 was found through a
mutational screen for a loss-of-resistance mediated by the R-genes RPP5 and RPP7
(Austin et al., 2002; Tor et al., 2002) and was identified via yeast two-hybrid analysis
as a protein that interacts with RAR1 (Azevedo et al., 2002). The SGT1-RAR1
physical interaction, as well as their association with ubiquitination-related proteins,
indicates the SGT1-RAR1 complex is a signaling molecule that plays a role in protein
degradation (Muskett, 2002; Shirasu et al., 1999; Tornero et al., 2002). Studies in
barley (Hordeum vulgare) have shown, RAR1 functions downstream of pathogen
47
recognition and upstream of H2O2 production (Shirasu et al., 1999). Research using
the TRV-VIGS system in tobacco to silence Sgt1 has also revealed a role for nonhost
resistance to tobacco mosaic virus (TMV) (Liu et al., 2002a), which supports the
hypothesis of proteins involved in non-host resistance to biotrophic pathogens
functioning in susceptibility to necrotrophic fungi. The selection of SGT1 as a
candidate in affecting ToxA-induced symptom development is also strengthened by
the previous TRV-VIGS data presented by Liu et al. (2002a).
There were no clones, however, that resulted in an increase of ToxA activity
after silencing. In addition to the four silenced clones that resulted in no ToxA
symptoms on tobacco, there were 128 clones that resulted in reduced, but not an
absence of ToxA cell death (Table 2.3). These particular clones were not pursued
mostly because their silencing phenotypes were indistinguishable from ToxA
symptoms, so their associations with the induced cell death were not able to be
resolved. These clones have potential as components in the cell death process of
ToxA and future work to sequence these and determine their cellular functions could
provide additional insight into the ToxA site- and mode-of-action.
The structure of the protein encoded by Tsn1, the wheat sensitivity gene for
ToxA, is composed of conserved domains representing a serine/threonine protein
kinase, a nucleotide binding site, and a leucine-rich repeat (S/T PK-NBS-LRR) with
no transmembrane domain demonstrating some similarities to known resistance
proteins (Faris et al., 2010). Lov1, the Arabidopsis functional homolog of the oat
48
Table 2.3. Summary of ToxA and GFP symptoms observed.
Summary of the degree of symptoms observed after agroinfiltration of
ToxA or GFP with all 1,153 genes silenced from the cDNA library.
Strong
Symptoms
ToxA
GFP
1,021
0
Moderate
Symptoms
128
0
No
Symptoms
4
1,153
Total
1,153
1,153
49
(Avena sativa) Vb gene that confers sensitivity to the HST victorin from C. victoriae,
is compositionally homologous to resistance proteins (Lorang et al., 2007). Therefore,
it could be possible that Tsn1 also confers resistance to an unidentified pathogen and
sensitivity to Ptr.
Together, the results from the TRV-VIGS screen point to multiple proteins that
could play a role in ToxA-mediated symptom development. The two proteins from
this study considered as the most promising contributors to ToxA-induced cell death,
GOX and SGT function primarily in photorespiration and defense, respectively. This
correlates with previous biochemical and microarray data that indicates ToxA‘s modeof-action involves ROS induction and defense gene perturbation. These results
suggest that ToxA leads to cell death by tricking the host into ―killing itself‖ through
defense-associated responses such as photoinhibition and subsequent ROS production.
Taken together, these data suggest that ToxA could be manipulating host machinery in
a manner similar to defense. Ptr, as a necrotroph, uses these defense responses to its
advantage by exploiting dead tissue for its lifestyle. Subsequent senescence of the
plant tissue allows the fungus to advance, obtaining nutrition from dead cells (Stone,
2001). Continued analysis of genetic variables helps to bring us closer to a full
understanding of Ptr pathogenicity and elucidation of the site- and mode-of-action of
ToxA.
50
Chapter 3
Assembly and analysis of the SO3 transcriptome for identification of
potential pathogenicity factors of Pyrenophora tritici-repentis
ABSTRACT
Pyrenophora tritici-repentis (Ptr) has a complex race structure dependent upon
host-selective toxin (HST) production and the symptoms elicited on wheat cultivars.
There are eight well described races of Ptr as identified by inoculation on wheat
differentials. The isolation of Ptr from a field in southern Oregon (isolate SO3) led to
the addition of a putative new race, race 9. SO3 is responsible for the elicitation of
ToxA-like symptoms on the ToxA-sensitive cultivars Glenlea and Katepwa, yet
genotypic analyses have revealed that SO3 does not possess the ToxA gene. To
elucidate the HST responsible for disease symptoms, the transcriptome of SO3 was
sequenced and aligned to the genome of the ToxA-producing isolate Pt-1C-BFP (BFP)
to identify expressed genes unique to SO3. Results revealed 497 sequences in the
SO3 transcriptome that are not found in BFP. Sequences that encode low molecular
weight proteins with N-terminal secretion signals are of most interest as potential
51
HSTs, as two of the three HSTs produced by Ptr are small secreted proteins (SSPs).
In addition to SSPs, sequences from SO3 that encode non-ribosomal peptide
synthetases (NRPSs) or polyketide synthases (PKSs) should be evaluated for their
potential role in symptoms elicited by SO3 as these genomic clusters have been shown
to contribute to the pathogenicity of other fungi.
INTRODUCTION
Many pathogenic organisms are able to cause disease through the production
and secretion of effector proteins (Hensel et al., 1998; Grant et al., 2006; Shabob et
al., 2008; de Wit et al., 2009). Fungi other than Pyrenophora tritici-repentis (Ptr)
known to use effectors for pathogenicity include Cladosporium fulvum (tomato leaf
mold) (Hammond-Kosack et al., 1996; van Esse et al., 2007), Fusarium oxysporum
(vascular wilt of tomato) (Rapp, 2005; Houterman et al., 2009) and Stagnospora
nodorum (Stagnospora blotch of wheat) (Friesen et al., 2008; Abeysekara et al., 2009;
Faris et al., 2010), with the latter fungus expressing the host-selective toxin (HST)
SnToxA, which is 99% similar to Ptr ToxA. Various oomycetes, especially in the
genus Phytophthora, have also been extensively studied for production of secreted
proteins, some of which possess RXLR (arginine-any amino acid-leucine-arginine)
amino acid motifs at the N-terminus (Kamoun, 2007; Kale, 2012). These motifs have
52
been hypothesized to play a role in the internalization of the effectors into host cells
through interaction with phosphatidylinositol-3-phosphates (PI3Ps) in the cell
membranes (Tyler, 2009).
To date there are eight well characterized races of Ptr determined by the hostselective toxin(s) produced and the symptoms they elicit on wheat differentials (Table
1.1). The races are differentiated by the expression of all possible combinations of the
three described HSTs and one that does not produce any HSTs. The known HSTs are
ToxA (necrosis), ToxB (chlorosis) and ToxC (spreading chlorosis) (Lamari and
Bernier, 1989). ToxA-sensitive wheat cultivars are susceptible to all races that
produce ToxA. This is the same for ToxB- and ToxC-sensitive cultivars and their
respective races.
An isolate of Ptr, SO3 (previously referred to as EO3), was thought to be a
race 2 isolate due to the ToxA-like symptoms elicited on ToxA-sensitive cultivars,
Glenlea and Katepwa, and resistant reactions on ToxA-insensitive cultivars (race two
isolates only produce ToxA). However, genotypic analysis revealed that SO3 does not
contain the ToxA gene (Andrie et al., 2007). This led to the reclassification of SO3
into a new race, race 9. It was also concluded that phenotypic characterization alone
was insufficient for race designation; genotypic evidence must also be considered.
Comparative transcriptomics is one method by which genes that are shared or
unique between species or isolates can be identified. To determine genes expressed in
SO3 and not the ToxA-producing isolate, BFP, the transcriptomes of SO3 and BFP
53
were sequenced on the Illumina platform and assembled with the program Velvet.
The assembled SO3 transcripts were aligned to the reference genome of BFP, its
mitochondrial genome, and its Velvet-assembled transcriptome to identify proteincoding sequences from SO3 that are not shared with BFP. Filtering alignments
resulted in the generation of a list that contains 497 unique SO3 sequences, some of
which could be responsible for the symptoms induced on Glenlea and Katepwa. This
list should be mined for sequences that encode low molecular weight proteins with Nterminal secretion signals. These characteristics make them prime candidates for hostselective toxins produced by Ptr.
MATERIALS AND METHODS
Growth of fungi and RNA isolation
Conidia of BFP and SO3 were produced as described (Andrie et al., 2007) and
inoculated in quarter-strength PDB (Potato dextrose broth, Difco, Becton, Dickinson
and Company, MD, USA) as well as a modified Fries medium (Tomas and Bockus,
1987) and incubated for 48 hours at 25°C in constant light or dark. RNA was isolated
from mycelia grown under each condition with the RNeasy Plant Mini Kit and oncolumn DNase digestion with the RNase-Free DNase Set following the
manufacturer‘s instructions (Qiagen, Chatsworth, CA, USA). Quality of RNA was
54
assessed with the RNA 6000 nano LabChip kit on the Agilent Bioanalyzer 2100
(Agilent technologies, Inc., Palo Alto, CA, USA) at the Center for Genome Research
and Biocomputing (CGRB) at Oregon State University and quantity determined by a
Nanodrop ND-1000 UV-Vis spectrophotometer (Nanodrop. Thermo Scientific,
Waltham, Massachusetts, USA).
Sequencing and sequence quality assessment
SO3 and BFP samples were used to prepare Illumina libraries as per the
manufacturer's instructions. These libraries were sequenced on an Illumina HiSeq
2000 (Illumina, Inc., San Diego, CA, USA) sequencer with a 50-cycle run in the
Center for Genome Research and Biocomputing (CGRB) at Oregon State University.
Fifty-one base pair (BP) single-end reads were generated, of which the first five
nucleotides were trimmed with a Perl script to remove ambiguous basecalls (Ns),
which resulted in 46-bp reads. The sequence files of the nuclear genome of BFP were
downloaded from the Broad Institute‘s website (www.broadinstitute.org/annotation/
genome/pyrenophora_tritici_repentis.3/MultiDownloads.html). The BFP
mitochondrial genome was also sequenced at the Broad Institute (Cambridge, MA,
USA) and was contained in contigs zero and one.
Transcriptome assemblies and alignments
55
The SO3 and BFP reads generated from the Illumina platform were assembled
with the program Velvet 0.7.55 with a hash length of 31. The SO3 assembly was
supplemented with ~5,000 expressed sequence tags (ESTs), which were sequenced at
the Broad Institute (http://www.broadinstitute.org) with Sanger sequencing methods
(Manning et al., 2012, in review). The BioEdit sequence alignment editor
(http://www.bioedit.co.uk) was used to trim these ESTs for the first 30 nucleotides on
the 5‘ region, which represented vector sequence from pGEM T-Easy (Promega).
The resulting assemblies were aligned to the BFP reference genome and to the
BFP mitochondrial genome with the Blastall program from NCBI
(http://www.ncbi.nlm.nih.gov) and an e-value cutoff of e25. SO3 transcripts were
aligned to the Velvet-assembled BFP transcriptome, again with Blastall from NCBI.
Sequences from the alignments were filtered and binned into groups based on percent
identity generated by Blastall during the alignment and percent coverage of the
contigs. Because introns are present in the BFP reference genome assembly, some
SO3 contigs aligned at multiple locations or with only a small region of the query
sequence having a match. Pivot tables in Microsoft Excel were used to calculate the
percent coverage of each contig by determining the total length of alignment for each
SO3 query sequence at all locations in the reference genome and dividing by the
length of the aligned contig. This value was multiplied by 100 to report the coverage
as a percentage.
56
RESULTS
A bioinformatics approach was used to identify genes unique to SO3 compared
to BFP. One or more of the sequences identified could be responsible for the
symptoms SO3 elicits on Glenlea and Katepwa. The transcriptome of SO3, sequenced
on the Illumina platform and assembled with Velvet, was aligned to the previously
assembled genome of BFP, which includes both the nuclear and mitochondrial
genomes as well as the de novo-assembled transcripts of BFP.
Assembly statistics
The transcriptome assemblies for BFP and SO3 resulted in the construction of
10,668 and 6,229 contiguous sequences, respectively. The BFP assembly contained
~13 Mb of sequence with an N50 of 1,909 bp. The SO3 assembly contained ~8 Mb of
sequence with an N50 of 1,867 bp. The N50 statistic refers to 50% of sequences in the
entire assembly are contained in contigs equal to or larger than this value. The longest
contiguous sequence for BFP was 7,832 bp and the longest for SO3 was 12,296 bp.
Alignments to BFP genomes
57
The BFP transcriptome was aligned to its own reference and mitochondrial
genomes. Of the 10,668 assembled transcripts, 97 (0.009%) did not align to the
reference. Further analysis of these 97 transcripts revealed they were repetitive
sequences and, therefore, may be difficult to assemble in the reference genome. With
such a high degree of similarity between the BFP reference genome and its transcripts,
we can assume the Velvet assembly method is accurate and acceptable to use with
SO3.
The de novo assembly of the SO3 transcriptome was followed by alignment to
the BFP reference genome. The first alignment of the SO3 transcripts to the BFP
nuclear genome resulted in 101 sequences that were not found in BFP and 6,128
sequences that were shared based on the Blastall parameters set. These contigs were
evaluated for % identity as well as for % coverage of the query sequence. Any SO3
contigs that aligned with greater than 98% identity and greater than 95% coverage
were considered true hits to the genomes. These cutoff values were used because
mapping of SO3 ESTs to the reference genome indicated that the ESTs were found to
be > 98% identical (Manning et al., 2012; in review). To make sure our alignment
results signified true similarities and differences between the isolates, percent
coverage values for each alignment were calculated. Filtration of the alignments
based on percent coverage was performed because some alignments displayed high evalues that were not truly representative of the data. This was due to several SO3
transcripts that mapped with 100% identity, yet only 10% of the query was aligned to
the BFP sequence. One example of this occurrence is the SO3 contig NODE_1901,
58
which has a total length of 3,213 nucleotides. This contig aligned to the BFP
reference genome with 100% identity. However, only 565 nucleotides out of 3,213
(17%) were covered in this alignment. Since the percent identity was so high, the evalue was also inflated. Analysis of the alignment by percent coverage helps to reduce
this bias and eliminate false-positive hits.
Categories based on percent coverage values were used to group the SO3
sequences that mapped to the genomes (Fig. 3.1). These include those that aligned
with greater than 100% coverage (275 contigs; 4.5%), between 95%-100% coverage
(5,030 contigs; 82.1%), between 90%-94% coverage (424 contigs; 6.9%), between
80%-89% coverage (237 contigs; 3.9%), between 50%-79% coverage (142 contigs;
2.3%), and those that aligned with less than 50% coverage (20 contigs; 0.3%). The
greater than 100% classification includes 110 SO3 contigs that aligned from 151957% coverage, which is likely due to highly repetitive sequences. After filtering the
6,128 hits to the BFP genomes, SO3 transcripts that aligned with less than 95%
coverage (815), as well as the 101 sequences that did not align at all, comprised the
916 contigs present in SO3 that are not present in the BFP genome.
Alignment of SO3 to BFP transcripts reveals unique sequences
The BFP genome assembly is 95% complete. Aligning the SO3 transcriptome
to the BFP transcriptome could help identify shared genes that are represented in the
unassembled 5%. Also, alignment of the transcripts from one Velvet assembly to the
59
Figure 3.1. Percent coverage of SO3 transcripts to the BFP genome.
The percent coverage values of the SO3 sequences that aligned to the BFP
reference and mitochondrial genomes. The Y-axis denotes the percentage of
the 6,128 SO3 contigs that mapped to BFP. The X-axis represents the bins
of percent coverage values for the contigs.
60
other helps to ensure that the assembly algorithm is reliable and acceptable for both
isolates. The reciprocal transcript BLAST should detect a problem with the assembly
algorithm if there was one present due to the degree of similarity between the two sets
of transcripts given the same similarity between their ESTs. Of the 916 SO3
transcripts aligned to the BFP transcriptome, 822 had a match in the BFP
transcriptome based on the Blastall parameters used while 94 did not align with any
similarity to BFP. The 822 shared contigs were filtered in Microsoft Excel with the
98% identity and 95% coverage parameters. Similar to the hits to the genomes, these
BLAST hits were sorted by percent coverage values that include those with >100%
coverage, those between 95-100% coverage, those between 90-94% coverage, those
between 80-89% coverage, those between 50-79% coverage, and those with < 50%
coverage (Fig. 3.2). There were zero contigs with > 100% coverage after this
alignment. There were 50.5% of the sequences that fell between 95-100% coverage.
Eleven percent of the hits fell in the range of 90-94% coverage, and a similar 11.7%
were included in the classification of 80-89% coverage. The categories of sequences
that aligned between 50-79% coverage and < 50% coverage contained 20.2% and
6.6%, respectively. Based on these results, a putative list of 497 unique SO3
sequences was generated that included those with below 95% coverage and the 94
sequences that didn‘t align to BFP (Fig. 3.3).
61
Figure 3.2. Percent coverage of SO3 transcripts to the BFP transcriptome.
The percent coverage values of the SO3 sequences that aligned to the BFP
transcriptome. The Y-axis denotes the percentage of the 822 SO3 contigs that
mapped to BFP. The X-axis represents the groups of percent coverage values
for the contigs.
62
Figure 3.3. Summary of filtering steps and candidate SO3 sequences.
The SO3 transcriptome BLAST results were sequentially filtered and
aligned based on percent identity and percent coverage. Contigs with <
98% ID and < 95% coverage to the BFP genomes were included in the
subsequent alignment to the BFP transcriptome; 497 sequences were
identified as being unique to SO3 and will be further evaluated.
63
DISCUSSION
Previous work in the Ciuffetti laboratory identified a putative HST from SO3
that has been named Ptr ToxD (Manning et al., 2002; Andrie et al., 2002). A
bioinformatics approach was chosen to identify genes in SO3 that encode novel
proteins that could be responsible for the ToxA-like symptoms elicited by SO3. The
SO3 transcriptome was sequenced, assembled and aligned to the genome and
transcriptome of the isolate, Pt-1C-BFP (BFP). Alignment of the SO3 transcripts to
those from BFP allowed us to exclude the shared contigs and generate a list of
sequences that could be responsible for inducing the ToxA-like symptoms caused by
SO3. However, it is unknown if BFP contains the ToxD gene. ToxD presence in both
BFP and SO3 would prevent us from identifying the gene responsible for the toxin(s)
in SO3. To address this problem, it would be helpful to align SO3 with an isolate that
doesn‘t produce ToxA or ToxD, such as the race 5 isolate DW7.
There are different programs and methods that one could utilize in assembling
a transcriptome, and they all perform the process in a different manner. We used the
assembly program Velvet 0.7.55 (Tang et al., 2012; Piskur et al., 2012), which uses an
algorithm to assemble short reads (< 100 bp) and generate much longer contiguous
sequences. This program was chosen due to the accessibility on the CGRB computer
infrastructure and the ease of application to downstream procedures such as contig
evaluations and alignments. Sequences can then be piped through long read programs,
such as MIRA (Kumar and Baxter, 2010), which further assembles these contigs into
64
larger contiguous sequences. However, this approach was not pursued due to the
outdated nature of the MIRA assembly algorithm and complications with the computer
infrastructure that resulted in inaccessibility to the MIRA directory. Following the
Velvet assemblies, contigs were directly aligned to the genomes and transcripts with
Blastall from NCBI. This procedure builds an index of the query sequences
(assembled contigs) and scans linearly through the reference database to identify
matches. An alternate alignment tool, called BLAT (BLAST-like alignment tool),
does the opposite and builds an index of the database to be searched and then scans
linearly through the query sequences. This program was not available for the
assembly prior to using Velvet, and training the indices for alignment is timeconsuming. Due to these circumstances and being more familiar with NCBI
parameters, Blastall was used as the alignment tool in all of the analyses. However, it
would be useful to test the alternate alignment methods to provide confirmation for the
results obtained in this study.
The transcriptome of BFP was assembled alongside that of SO3 and aligned to
its own reference genome and mitochondrial genome for quality assurance of the
assembly method. Only 97 BFP transcripts (0.009%) did not align to the genomes.
The genomes of BFP were not 100% complete, so it is not surprising that a small
percentage of BFP transcripts did not have a match. The percentage of the total
number of BFP contigs that did not have a match in the reference genome was very
small and should not warrant concern for the assembly.
The alignment of the Velvet-assembled SO3 transcriptome to the BFP
65
reference and mitochondrial genomes yielded a set of 6,128 contigs that mapped and a
set of 101 contigs that did not map. These 101 sequences were the first group of SO3
transcripts unique from BFP. Some of these 6,128 contigs had a high % identity (%
ID) value from the BLAST, yet only 10% of the entire query sequence aligned. This
could lead to some SO3 contigs being considered as legitimate hits when their
alignment qualities are actually low. To circumvent this potential, the percent
coverage value was calculated for each BLAST hit, which resulted in a more reliable
assessment of the contigs‘ alignments. Some SO3 sequences aligned with > 98% ID
at one location in the BFP genome and then < 98% ID at another. These are likely
transcripts of repetitive DNA that are present multiple times in the genome, but
contain mutations or deletions that lower the % ID. These repeats are interesting in
their own right due to the high occurrence of repetitive DNA in pathogenic fungi, in
particular flanking pathogenicity loci (McDonald and McDermott, 1993; Kistler,
1997; van der Does and Rep, 2007).
Through the alignment and filtering steps with BFP, a putative list of 497
transcripts found in the race 9 isolate, SO3, but not in BFP (Fig. 3.3), was generated.
Additional steps are required to understand what role, if any, these unique sequences
play in SO3 pathogenicity. The first of these steps includes identification of open
reading frames with a web-based application, such as ORF-PREDICTOR
(http://proteomics.ysu.edu/tools/OrfPredictor.html). This program generates the
predicted coding sequences as well as a six-frame translation of the nucleotide
sequence. These gene translations will then be submitted to the web-based
66
application, SignalP 4.0 (http://www.cbs.dtu.dk/services/SignalP/), which determines
if the predicted proteins contain N-terminal signal sequences for direction to the
secretory pathway (Peterson et al., 2011). Time did not allow for this step to be
completed, however sequences that do contain a secretion signal are prime candidates
as potential proteinaceous HSTs of SO3.
Next-generation sequencing technologies, such as Illumina and 454
pyrosequencing, have become important and abundant tools in scientific research. By
traversing the genetic architecture of an organism, we can learn more about them than
from observation alone. The assembled transcriptome of SO3 can provide support for
future endeavors to understand the pathogenicity of Pyrenophora tritici-repentis.
Many attributes are characterized; however, many still require resolution. Highthroughput sequencing has proven to be an efficient and effective method to identify
novel sequences and these data will greatly benefit the plant pathology community by
providing expression data for a new race of a devastating pathogen of wheat.
67
Chapter 4
General Conclusions
Pyrenophora tritici-repentis (Ptr) is a necrotrophic fungus in the class
Dothideomycetes, which includes several other devastating plant pathogens such as
Stagnospora nodorum, Cochliobolus victoriae, Mycosphaerella graminicola and
Alternaria brasssicola. Ptr emerged as a devastating pathogen and causal agent of tan
spot of wheat (Triticum aestivum L.) around 1941 and can be responsible for up to
50% yield losses in major wheat growing regions worldwide, especially in areas where
farmers use no-till cultivation practices. In order to improve management practices, it
is beneficial to investigate the molecular and genomic properties of this fungus.
Currently the most practical methods to avert disease is through application of
fungicides and the deployment of resistant wheat cultivars. Gaining a better
understanding of the molecular interactions between host-selective toxins (HSTs) of
Ptr and their targets inside the host may lead to the development of additional resistant
cultivars. This will be important as new races and toxins are discovered for this
pathogen, as could be the case for SO3.
In chapter two, the virus-induced gene silencing (VIGS) approach with tobacco
rattle virus (TRV) facilitated an efficient and reliable analysis of over a thousand
genes for their potential role in symptom development induced by ToxA. The
approach resulted in the identification of four genes that, when silenced, resulted in the
68
loss of ToxA-induced cell death in tobacco (Nicotiana benthamiana). These genes
included 40S, Thi1, GOX and Sgt1. The first genes, 40S and Thi1, were surmised to
have an indirect effect due to their global roles. The genes GOX and Sgt1 encode
proteins that could play a direct role in the symptoms elicited by ToxA and warrant
further investigation.
GOX is the peroxisomal enzyme glycolate oxidase and is responsible for the
oxidation of glycolate to glyoxylate and the subsequent production of hydrogen
peroxide, a toxic reactive oxygen species (ROS). The substrate is exported from the
chloroplast as a product of the Calvin Cycle and photorespiration. It has been shown
that ROS plays a major role in the cell death observed in wheat as a result of ToxA.
Photorespiration is also affected through a reduction in proteins composing the
photosynthetic complex. Another study using TRV-VIGS in tobacco revealed the role
of GOX in non-host resistance pathways to biotrophic pathogens through an
accelerated hypersensitive response (HR) (Rojas et al., 2012). Biotrophic fungi
require association with living host tissue in order to survive; therefore, the killing of
the plant cells during HR restricts the pathogen from further colonization and
pathogenicity. Necrotrophs, however, can utilize the dead cells as a nutritional source,
often using toxins to incite the plant to kill its own cells. One common hypothesis is
necrotrophic fungi utilize the defense responses directed towards biotrophic pathogens
to their advantage, using the necrotized cells for nutrition and further colonization.
Through ToxA activity and eventual transcriptional reprogramming of the host genetic
machinery, it is likely that Ptr is manipulating GOX to produce a higher amount of
69
hydrogen peroxide through increased activity of the enzyme and using it as one means
to cause cell death.
SGT1 is a very interesting candidate in that researchers identified this protein
through loss-of-resistance screens in Arabidopsis thaliana. They also discovered
SGT1 is an interactor with the resistance-associated protein RAR1. The nature of this
interaction, including structural properties and association with ubiquitination-related
proteins, suggest these may be signaling components in defense and possibly play a
role in protein degradation. Interestingly, reduction in photosystem proteins and
changes in signaling pathway activities are both observed in ToxA-treated wheat
(Pandelova et al., 2009). TRV-VIGS in tobacco has also been used to show a role for
SGT1 in nonhost resistance and Pto-mediated resistance to biotrophic organisms. An
accelerated HR upon inoculation or infiltration with various plant pathogens was
observed in Sgt1-silenced plants (Azavedo et al., 2002). Increases in defense
signaling activity and protein degradation properties of SGT1 could lead to the
reduction of important photosynthetic components. Past research has suggested that
protein degradation is a key component in R-gene signaling pathways in various
degrees. Several resistance genes have other roles in the plant besides defense,
therefore making them less expendable. This lack of expendability makes it harder for
the host to use mutation or deletion of these genes in order to evade the pathogen. Ptr
uses the defense responses from these genes, such as ROS production and protein
degradation, to induce cell death and pathogen advancement.
70
However, because we observed this lack of ToxA symptom development in
tobacco after intracellular expression of the toxin does not necessarily infer causation.
It is possible that this observation was a response to an alternate ToxA symptom
development pathway in tobacco, a non-host of Ptr that does not possess the ToxAsensitivity locus Tsn1. To support these results, the respective genes should be
silenced in a ToxA-sensitive wheat cultivar such as Glenlea or Katepwa using the
barley stripe mosaic virus (BSMV)-based VIGS approach. After silencing, ToxA
should be infiltrated into secondary leaves of the silenced wheat and plants monitored
for ToxA symptom development. If GOX and SGT1 truly play a role in sensitivity to
ToxA, there should also be a reduction in ToxA-induced cell death in the pathogen‘s
natural host. Chlorophyll assays, observation of GFP fluorescence, RT-PCR and
Western blots should be used to confirm results, as was done in the tobacco silencing
experiments.
It is just as important to identify new host-selective toxins as it is to understand
the molecular interactions of the ones that are currently described. These new HSTs
could lead to an entirely different epidemic given the appropriate sensitive cultivar,
especially with the abundance of monocultures used in wheat production. The race 9
isolate SO3, which elicits ToxA-like symptoms on the wheat differential similar to
that of race 2, was identified in southern Oregon. However, SO3 does not contain
ToxA. With the recent advancements in high-throughput sequencing technologies,
comparative genomics is becoming a useful method to identify similarities and
differences between species and isolates. Assembly and alignment of the SO3 and
71
BFP transcriptomes revealed a significant number of SO3 genes that were unique from
BFP, one of which could be the gene encoding Ptr ToxD or another new HST.
Bioinformatic analysis was an effective method to identify these genes and will
continue to be important in further dissection of the unique SO3 sequences to discern
those secreted and small enough to be considered HSTs, as most proteinaceous HSTs
are of low molecular mass. Conserved domains could indicate potential roles of the
proteins. An important characteristic of a host-selective toxin is that it is able to
reproduce the symptoms of disease upon infiltration of the purified compound. Genes
from this unique set that meet the above requirements for HSTs should be evaluated
for their role in pathogenicity; proteins encoded by these genes should be purified and
infiltrated into a wheat differential set. This would determine if the symptoms seen
upon inoculation with SO3 can be repeated via infiltration of a purified toxin. If the
protein of interest causes cell death in a comparable manner, this would support the
identification of a new pathogenicity factor of Pyrenophora tritici-repentis, potentially
ToxD.
It is important to remember that BFP could also produce ToxD. If this is the
case, the bioinformatics approach of aligning the SO3 and BFP transcriptomes would
not identify ToxD. Despite this potential pitfall, 497 sequences were discovered in
SO3 that are not found in BFP and one or more of these sequences could be new HSTs
of Ptr. With the identification of new HSTs comes the process of unraveling
interactions between the toxin and the host cells.
72
Tan spot of wheat caused by the necrotrophic fungus Pyrenophora triticirepentis is an economically important disease in major wheat growing regions. As
wheat is consistently one of the top three food crops grown worldwide, gaining a
better understanding of how an important pathogen such as Ptr induces cell death on
susceptible cultivars will lead to a greater food supply in a world where hunger is so
widespread. Data from studies reported in this thesis will be used to supplement
previous knowledge about Ptr and may aid breeders in developing additional resistant
cultivars to combat this disease.
73
Bibliography
Abeysekara, N, Friesen, T, Keller, B, and Faris, J. 2009. Identification and
characterization of a novel host–toxin interaction in the wheat–Stagonospora
nodorum pathosystem. Theoret. App. Gen. 120:117–126.
Adhikari, TB, Bai J, Meinhardt SW, Gurung, S, Myrfield M, Patel J, Ali S,
Gudmestad, NC, and Rasmussen, JB. 2009. Tsn1-mediated host responses to ToxA
from Pyrenophora tritici-repentis. Mol. Plant Microbe Interact. 22:1056–1068.
Anand, A, Vaghchhipawala, Z, Ryu, CM, Kang, L, Wang, K, del-Pozo, O, Martin,
GB, and Mysore, KS. 2007. Identification and characterization of plant genes
involved in Agrobacterium-mediated plant transformation by virus-induced gene
silencing. Mol. Plant Microbe Interact. 20:41–52.
Anderson, JA, Effertz, RJ, Faris, JD, Francl, LJ, Meinhardt, SW, Gill, BS. 1999.
Genetic analysis of sensitivity to a Pyrenophora tritici-repentis necrosis-inducing
toxin in durum and common wheat. Phytopath. 89:293–297.
Andrie, RM, Pandelova, I, and Ciuffetti, LM. 2007. A combination of phenotypic and
genotypic characterization strengthens Pyrenophora tritici-repentis race
identification. Phytopath. 97:694–701.
Andrie, RM, and Ciuffetti, LM. 2011. Pyrenophora bromi, causal agent of brownspot
of bromegrass, expresses a gene encoding a protein with homology and similar
activity to Ptr ToxB, a host-selective toxin of wheat. Mol. Plant Microbe Interact.
24:359–367.
Austin, MJ, Muskett, P, Kahn, K, Feys, BJ, Jones, JD, Parker, JE. 2002. Regulatory
role of SGT1 in early R gene-mediated plant defenses. Science. 295:2077–2080.
Azevedo, C, Sadanandom, A, Kitagawa, K, Freialdenhoven, A, Shirasu, K, SchulzeLefert, P. 2002. The RAR1 interactor SGT1, an essential component of R genetriggered disease resistance. Science 295:2073–2076.
Ballance, GM, Lamari, L, and Bernier, CC. 1989. Purification and characterization of
a host-selective necrosis toxin from Pyrenophora tritici-repentis. Physiol. Mol.
Plant Pathol. 35:203–213.
Ballance GM, Lamari L, Kowatsch R, Bernier CC. 1996. Cloning, expression and
occurrence of the gene encoding the Ptr necrosis toxin from Pyrenophora triticirepentis. Mol. Plant Path. On-line [http://www.bspp.org.uk/mppol/]
74
Baulcombe, D.C. 1999. Fast forward genetics based on virus-induced gene silencing.
Curr. Opin. Plant Biol. 2:109–113.
Bhathal, JS, Loughman, R, and Speijers, J. 2003. Yield reduction in wheat in relation
to leaf disease from yellow (tan) spot and septoria nodorum blotch. Eur. J. Plant
Path. 109:435–443.
Bhattacharjee, S, Hiller, NL. Liolios, K, Win, J, Kanneganti, TD, Young, C, Kamoun,
S, and Haldar, K. 2006. The malarial host-targeting signal is conserved in the irish
potato famine pathogen. PLoS Path. 2:e50.
Bourguignon, J, Rebeille, F, and Douce, R. 1999. Serine and glycine metabolism in
higher plants. In Plant Amino Acids: Biochem. and Biotech. B.K. Singh, ed. New
York: Marcel Dekker. pp. 111–146.
Brosch, G, Ransom, R, Lechner, T, Walton, JD, and Loidl, P. 1995. Inhibition of
maize histone deacetylases by HC toxin, the host-selective toxin of Cochliobolus
carbonum. Plant Cell. 7:1941–1950.
Bruggeman, J, Debets, AJM, Wijngaarden, deVisser, JAGM, and Hoekstra, RF. 2003.
Sex slows down the accumulation of deleterious mutations in the homothallic
fungus Aspergillus nidulans. Genetics. 164:479–485.
Burch-Smith, TM, Anderson, JC, Martin, GB, and Dinesh-Kumar, SP. 2004.
Applications and advantages of virus-induced gene silencing for gene function
studies in plants. Plant J. 39:734–746.
Cao, T, Yong MK, Kav, NV, and Strelkov, SE. 2009. A proteomic evaluation of
Pyrenophora tritici-repentis, causal agent of tan spot of wheat, reveals major
differences between virulent and avirulent isolates. Proteomics. 9:1177–1196.
Caplan, JL, Mamillapalli, P, Burch-Smith, TM, Czymmek, K, and Dinesh-Kumar, SP.
2008. Chloroplastic protein NRIP1 mediates innate immune receptor recognition of
a viral effector. Cell. 132:449–462.
Chabregas, SM, Luche, DD, Farias, LP, Ribeiro, AF, van Sluys, MA, Menck, CFM
and Silva-Filho, MC. 2001. Dual targeting properties of the N-terminal signal
sequence of Arabidopsis thaliana THI1 protein to mitochondria and chloroplasts.
Plant Mol. Biol. 46:639-650.
Chabregas, SM, Luche, DD, Van Sluys, MA, Menck, CFM, and Silva-Filho,MC.
2003. Differential usage of two in-frame translational start codons regulates
subcellular localization of Arabidopsis thaliana THI1. J. Cell Sci. 116:285–291.
75
Ciuffetti LM, Tuori RP, Gaventa JM. 1997. A single gene encodes a selective toxin
causal to the development of tan spot of wheat. Plant Cell. 9:135–144.
Ciuffetti, LM, Francl, LJ, Balance, G.M, Bockus, WW, Lamari, L, Meinhardt, SW,
and Rasmussen JB. 1998. Standardization of toxin nomenclature in the
Pyrenophora tritici-repentis-wheat interaction. Can. J. Plant Path. 4:421-424.
Ciuffetti, LM, Manning, VA, Pandelova, I, Figueroa-Betts, M, and Martinez, JP. 2010.
Host‐selective toxins, Ptr ToxA and Ptr ToxB, as necrotrophic effectors in the
Pyrenophora tritici‐repentis–wheat interaction. New Phytol. 187:911–919.
Corpas, FJ, Arroso, JB, and del Rio, LA. 2001. Peroxisomes as a source of reactive
oxygen species and nitric oxide signal molecules in plant cells. Trends Plant Sci.
4:145-150.
de Jonge, R, van Esse, PH, Kombrink, A, Shinya, T, Desaki, Y, Bours, R, van der
Krol, S, Shibuya, N, Joosten, M, and Thomma, B. 2010. Conserved fungal LysM
effector Ecp6 prevents chitin-triggered immunity in plants. Science. 329:953–955.
de Wit, PJGM. 1992. Molecular characterization of gene-for-gene systems in plantfungus interactions and the application of avirulence genes in control of plant
pathogens. Ann. Rev. of Phytopath. 30:391–418.
de Wit, PJGM, Mehrabi, R, Van Den Burg, HA, and Stergiopoulos, I. 2009. Fungal
effector proteins: past, present and future. Mol. Plant Path. 10:735–747.
de Wit,PJGM, Joosten, M, Thomma, B, and Stergiopoulos. I. 2009. Gene for gene
models and beyond: The Cladosporium fulvum-tomato pathosystem. In Plant
Relationships, ed. Holger B. Deising and K. Esser. The Mycota. Springer Berlin
Heidelberg.
Drechsler, C. 1923. Some graminicolous species of Helminthosporium. Internat. J.
Agric. Res. 24:641-739.
Dunkle, LD. 1984 Factors in pathogenesis. T Kosuge and EW Nester, eds. PlantMicrobe Inter. Macmillan, New York, pp 19-41.
Each, JE and White, FF. 1996. Bacterial avirulence genes. Annu. Rev. Phytopathol.
34:153-176.
Effertz, RJSW, Meinhardt, JA, Anderson, JG, and Francl ,LJ. 2002. Identification of a
chlorosis-inducing toxin from Pyrenophora tritici-repentis and the chromosomal
location of an insensitivity locus in wheat. Phytopath. 92:527–533.
76
Ellis, MB, and Waller, JM. 1976. Pyrenophora tritici-repentis (conidial state:
Drechslera tritici-repentis). Commonw. Mycol. Inst. Descriptions Pathogenic Fungi
Bacteria No. 494.
Emanuelsson, O, Nielsen, H, and von Heijne, G. 1999. ChloroP, a neural networkbased method for predicting chloroplast transit peptides and their cleavage sites.
Protein Sci. 8:978-984.
Faris, JD, Anderson, JA, Francl, LJ, and Jordahl, JG 1996. Chromosomal location of a
gene conditioning insensitivity in wheat to a necrosis-inducing culture filtrate from
Pyrenophora tritici-repentis. Phytopath. 86:459-463.
Faris, JD, Zhang, Z, Lu, H, Lu, S, Reddy, L, Cloutier, S, Fellers, JP, et al. 2010. A
unique wheat disease resistance-like gene governs effector-triggered susceptibility
to necrotrophic pathogens. Proceed. Nat. Acad. Sci. 107:13544–13549.
Figueroa-Betts, M, Manning, VA, Cardwell, KB, Pandelova, I and Ciuffetti, LM.
2011. The importance of the N-terminus for activity of Ptr ToxB, a chlorosisinducing host-selective toxin Produced by Pyrenophora tritici-repentis. Physio.
Mol. Plant Path. 75:138–145.
Flor, HH. 1971. Current status of the gene-for-gene concept. Ann. Rev. Phytopath.
9:275-296.
Friesen, TL, Stukenbrock, EH, Liu, Z, Meinhardt, S, Ling, H, Faris, JD, Rasmussen,
JB, Solomon, PS, McDonald, BA,and Oliver, RP. 2006. Emergence of a new
disease as a result of interspecific virulence gene transfer. Nat. Gen. 38:953–956.
Friesen, TL, Meinhardt, S, and Faris, JD. 2007. The Stagonospora nodorum-wheat
pathosystem involves multiple proteinaceous host-selective toxins and
corresponding host sensitivity genes that interact in an inverse gene-for-gene
manner. Plant J. 51:681–692.
Friesen, TL, Faris, JD, Solomon, PS, and Oliver, RP. 2008. Host‐specific toxins:
effectors of necrotrophic pathogenicity. Cellul. Micro. 10:1421–1428.
Gassmann, W, and Bhattacharjee.S. 2012. Effector-triggered immunity signaling:
from gene-for-gene pathways to protein-protein interaction networks. Mol. PlantMicrobe Interact. 25:862–868.
Gouget A, Senchou V, Govers F, Sanson A, Barre A, Rouge P, Pont-Lezica R, Canut
H. 2006. Lectin receptor kinases participate in protein-protein interactions to
77
mediate plasma membrane-cell wall adhesions in Arabidopsis. Plant Physiol.
140:81–90.
Grant, SR, Fisher, EJ, Chang, JH, BM, Mole, and Dangl. JL. 2006. Subterfuge and
manipulation: type III effector proteins of phytopathogenic bacteria. Ann. Rev.
Micro. 60:425–449.
Hammond-Kosack, KE, Silverman, P, Raskin, I, and Jones, J. 1996. Race-Specific
Elicitors of Cladosporium fulvum induce changes in cell morphology and the
synthesis of ethylene and salicylic acid in tomato plants carrying the corresponding
Cf disease resistance gene. Plant Phys. 110:1381–1394.
Heath, MC. 2000. Hypersensitive response-related cell death. Plant Mol. Biol.
44:321-334.
Hensel, M, Shea, JE, Waterman, SR, Mundy, R, Nikolaus, T,Banks, G, VazquezTorres, A, Gleeson, C, Fang, FC, and Holden, DW. 1998. Genes encoding putative
effector proteins of the type III secretion system of Salmonella pathogenicity island
2 are required for bacterial virulence and proliferation in macrophages. Mol. Micro.
30:163–174.
Holzberg, S, Brosio, P, Gross, C, and Pogue, GP. 2002. Barley stripe mosaic virusinduced gene silencing in a monocot plant. Plant J. 30:315–327.
Hostetter, MK. 2000. RGD-mediated adhesion in fungal pathogens of humans, plants
and insects. Curr. Opin. in Micro. 3:344–348.
Houterman, PM, Ma, L, van Ooijen, G, de Vroomen, MJ, Cornelissen, BJC, Takken,
FLW, and Rep, M. 2009. The effector protein Avr2 of the xylem-colonizing fungus
Fusarium oxysporum activates the tomato resistance protein I-2 intracellularly.
Plant J. 58:970–978.
Jackson, AO, and Taylor, CB. 1996. Plant–microbe interactions: life and death at the
interface. Plant Cell. 8:1651–1668.
Jones, JD., and Dangl, JL. 2006. The Plant Immune System. Nature 444:323–329.
Kamoun, S. 2007. Groovy times: filamentous pathogen effectors revealed. Curr. Opin.
in Plant Bio. 10:358–365.
Rashad, K, Niessen, M, Thiruveedhi, K, Bari, R, Hirsch, HJ, Rosenkranz, R, Stäbler,
N, Schönfeld, B, Kreuzaler, F, and Peterhänsel, C. 2007. Chloroplastic
photorespiratory bypass increases photosynthesis and biomass production in
Arabidopsis thaliana. Nature Biotech. 25:593–599.
78
Kent, W. 2002. BLAT—The BLAST-like alignment tool. Genome Research 12:656–
664.
Kim, YM, and Strelkov, SE. 2007. Heterologous expression and activity of Ptr ToxB
from virulent and avirulent isolates of Pyrenophora tritici-repentis. Can. J. Plant
Pathol. 29:232–242.
Kim, YM, Bouras, N, Kav, NNV, and Strelkov, SE. 2010. Inhibition of photosynthesis
and modification of the wheat leaf proteome by Ptr ToxB: a host-specific toxin
from the fungal pathogen Pyrenophora tritici-repentis. Proteomics. 106:2911–
2926.
Kistler, HC. 1997. Genetic diversity in the plant-pathogenic fungus Fusarium
oxysporum. Phytopath. 87:474–479.
Kumar, S, and Blaxter, ML. 2010. Comparing de novo assemblers for 454
transcriptome data. BMC Genomics. 11:571.
Kwok, EY, and Hanson, MR. 2004. Stromules and the dynamic nature of plastid
morphology. J. Microsc. 214:124-137.
Lamari, L, and Bernier, CC. 1989. Toxin of Pyrenophora tritici-repentis: hostspecificity, significance in disease, and inheritance of host reaction. Phytopath.
79:740-744.
Lamari, L, and Bernier, CC. 1989. Evaluation of wheat lines and cultivars to tan spot
(Pyrenophora tritici-repentis) based on lesion type. Can. J. Plant Pathol. 11:284290.
Lamari, L, Sayoud, R, Boulif, M, and Bernier, CC. 1995. Identification of a new race
in Pyrenophora tritici-repentis: implications for the current pathotype classification
system. Can. J. Plant Pathol. 17:312-318.
Lamari, L, Strelkov, SE, Yahyaoui, A, Orabi, J, and Smith, RB. 2003. The
Identification of two new Races of Pyrenophora tritici-repentis from the host
center of diversity confirms a one-to-one relationship in tan spot of wheat.
Phytopath. 93:391–396.
Liu, T, Zixu, L, Chuanjun, S, Yunfei, H, Zhifu, H, Ji, S, Fangfang, F, et al. 2012.
Chitin-induced dimerization activates a plant immune receptor. Science. 336: 1160–
1164.
79
Liu, Y, Schiff, G. Deng, SXW, and Dinesh-Kumar, SP. 2002a. Role of SCF ubiquitinligase and the COP9 signalosome in the N gene–mediated resistance response to
tobacco mosaic virus. Plant Cell Online 14:1483–1496.
Liu, Y, Michael, S, Rajendra, M, and Dinesh‐Kumar, SP. 2002b. Tobacco Rar1,
EDS1 and NPR1/NIM1 like genes are required for N‐mediated resistance to
tobacco mosaic virus. Plant J. 30:415–429.
Lorang, JM, Carkaci-Salli, N, and Wolpert, TJ. 2004. Identification and
characterization of victorin sensitivity in Arabidopsis thaliana. Mol. Plant Microbe
Interact. 17:577–582.
Lu, R, Martin-Hernandez, AM, Peart, JR, Malcuit, I, and Baulcombe, DC. 2003.
Virus-induced gene silencing in plants. Methods. 30:296–303.
Machado, CR, de Oliveira, RC, Boiteux, S, Praekelt, UPA, and Menck, CF. 1996.
Thi1, a thiamine biosynthetic gene in Arabidopsis thaliana, complements bacterial
defects in DNA repair. Plant Mol. Biol. 31:585–593
Machado, CR. Praekelt, UM, Costa de Oliveira, RL, Barbosa, ACC, Byrne, KL,
Meacock, PA, and Menck, CF. 1997. Dual role for the yeast THI4 gene in thiamine
biosynthesis and DNA damage tolerance. J. Mol. Biol. 273:114-121.
Manning, VA, Pandelova, I, and Ciuffetti, LM. 2002. A race for a novel host-selective
toxin. Phytopath. 92:S51.
Manning, VA, Andrie, RM,. Trippe, AF, and Ciuffetti, LM. 2004. Ptr ToxA requires
multiple motifs for complete activity. Mol. Plant-Microbe Interact. 17:491–501.
Manning, VA, and Ciuffetti, LM. 2005. Localization of Ptr ToxA produced by
Pyrenophora tritici-repentis reveals protein import into wheat mesophyll cells.
Plant Cell Online. 17:3203–3212.
Manning, VA, Hardison, LK, and Ciuffetti, LM. 2007. Ptr ToxA interacts with a
chloroplast-localized protein. Mol. Plant-Microbe Interact. 20:168–177.
Manning, VA, Hamilton, SM, Karplus, PA, and Ciuffetti, LM. 2008. The Arg-GlyAsp-containing, solvent-exposed loop of Ptr ToxA is required for internalization.
Mol. Plant-Microbe Interact. 21:315–325.
Manning, VA, Chu, AL, Steeves, JE, Wolpert, TJ, and Ciuffetti, LM. 2009. A hostselective toxin of Pyrenophora tritici-repentis, Ptr ToxA, induces photosystem
changes and reactive oxygen species accumulation in sensitive wheat. Mol. PlantMicrobe Interact. 22:665–676.
80
Manning, VA, Chu, AL, Scofield, SR, and Ciuffetti, LM. 2010. Intracellular
expression of a host‐selective toxin, ToxA, in diverse plants phenocopies silencing
of a ToxA‐ interacting protein, ToxABP1. New Phytol. 187:1034–1047.
Manning, VA, Pandelova, I, Dhillon, B Wilhelm, LJ, Goodwin, SB, Berlin, AM,
Figueroa, M, Freitag, M, Hane, JK, Henrissat, B, Holman, WH, Kodira, CD,
Martin, J, Oliver, RP, Robbertse, B, Schackwitz, W, Schwartz, DC, Spatafora, JW,
Turgeon, BG, Yandava, C, Young, S, Zhou, S, Zeng, Q, Grigoriev, IV, Ma, LJ,
Ciuffetti, LM. 2012. Comparative genomics of a plant-pathogenic fungus,
Pyrenophora tritici-repentis, reveals transduplication and the impact of repeat
elements on pathogenicity and population divergence. Genes, Genomes, Genetics.
(in review).
Markham, JE, and Hille, J. 2001. Host-selective toxins as agents of cell death in plant–
fungus interactions. Mol. Plant Path. 2:229–239.
Martinez, JP, Oesch, NW, and Ciuffetti, LM. 2004. Characterization of the multiplecopy host-selective toxin gene, ToxB, in pathogenic and nonpathogenic isolates of
Pyrenophora tritici-repentis. Mol. Plant-Microbe Interact. 17:467-474.
Mason, JM, and Arndt, KM. 2004. Coiled-coil Domains: stability, specificity, and
biological implications. Chembiochem. 5:170-176.
McDonald, BA, and McDermott, JM. 1993. Population genetics of plant pathogenic
fungi. BioScience 43:311–319.
Mehrabi, R,. Bahkali, AH, Abd-Elsalam, KA, Moslem, M, M‘Barek, SB, Gohari, AM,
Jashni, MK, Stergiopoulos, I, Kema, GHJ, and de Wit, PJGM. 2011. Horizontal
gene and chromosome transfer in plant pathogenic fungi affecting host range.
FEMS Micro. Rev. 35:542–554.
Meinhardt, SW. Cheng, W, Kwon, CY, Donohue, CM, and Rasmussen, JB. 2002.
Role of the arginyl-glycyl-aspartic motif in the action of Ptr ToxA produced by
Pyrenophora tritici-repentis. Plant Physiol. 130:1545–1551.
Montalbini, P and Buchanan, BB. 1974. Effect of a rust infection on
photophosphorylation by isolated chloroplasts. Phys. Plant Path. 4:191-196.
Mur, LAJ, Kenton, P, Lloyd, AJ, Ougham, H, and Prats, E. 2008. The hypersensitive
response; the centenary is upon us but how much do we know? J. Exp. Bot.
59:501–520.
81
Muskett, PR, Kahn, K, Austin, MJ, Moisan, LJ, Sadanandom, A, et al. 2002.
Arabidopsis RAR1 exerts rate-limiting control of R gene-mediated defenses against
multiple pathogens. Plant Cell. 14:979–92.
Natesan, SK, Sullivan, JA, and Gray, JC. 2005. Stromules: A characteristic cellspecific feature of plastid morphology. J. Exp. Bot. 56:787-797.
Navarre, DA, and Wolpert, TJ. 1995. Inhibition of the glycine decarboxylase
multienzyme complex by the host-selective toxin victorin. Plant Cell Online.
7:463–471.
Navarre, DA, and Wolpert, TJ. 1999. Victorin Induction of an apoptotic/senescence–
like response in oats. Plant Cell Online. 11:237–249.
Navarro, L, Florence, J, Kinya, N, He, SY, and Voinnet, O. 2008. Suppression of the
microRNA pathway by bacterial effector proteins. Science. 321:964–967.
Nürnberger, T, and Volker L. 2005. Non-host resistance in plants: new insights into an
old phenomenon. Mol. Plant Path. 6:335–345.
Oliver, RP, Lord, M, Rybak, K, Faris, JD, and Solomon, PS. 2008. Emergence of tan
spot disease caused by toxigenic Pyrenophora tritici-repentis in Australia is not
associated with increased deployment of toxin-sensitive cultivars. Phytopath.
98:488–491.
Oliver, RP, Friesen, TL, Faris, JD, and Solomon, PS. 2012. Stagonospora nodorum:
from pathology to genomics and host resistance. Ann. Rev. Phytopath. 50:2.1-2.21.
Pandelova, I, and Ciuffetti, LM. 2005. A proteomics-based approach for identification
of the ToxD gene. Fungal Gen. Newsl. 52(Suppl.):133.
Pandelova, I, Betts, MF, Manning, VA, Wilhelm, LJ, Mockler, TC, and Ciuffetti, LM.
2009. Analysis of transcriptome changes induced by Ptr ToxA in wheat provides
insights into the mechanisms of plant susceptibility. Mol. Plant. 2:1067–1083.
Peart, JR, Rui, L, Sadanandom, A, Malcuit, I, Moffett, P, Brice, DC, Schauser, L, et
al. 2002. Ubiquitin ligase-associated protein SGT1 is required for host and
nonhost disease resistance in plants. Proceed. Nat. Acad. Sciences. 99:10865–
10869.
Perello, A, Moreno, V, Simon, MR, and Sisterna, M. 2003. Tan spot of wheat
infection at different stages of crop development and inoculum type. Crop Prot.
22:157–169.
82
Petersen, T, Nordahl, SB, von Heijne, G, and Nielsen, H. 2011. SignalP 4.0:
discriminating signal peptides from transmembrane regions. Nature Methods.
8:785–786.
Pitzschke, A, Schikora, A, and Hirt, H. 2009. MAPK cascade signaling networks in
plant defense. Curr. Opin. Plant Bio. 12:421–426.
Rapala-Kozik, M, Wolak, N, Kujda, M, and Banas, AK. 2012. The upregulation of
thiamine (vitamin B1) biosynthesis in Arabidopsis thaliana seedlings under salt
and osmotic stress conditions is mediated by abscisic acid at the early stages of this
stress response. BMC Plant Bio. 12:2.
Rapp, P. 1995. Production, regulation, and some properties of lipase activity from
Fusarium oxysporum f. sp. vasinfectum. Enzy. and Microb. Techno. 17 (9) :832–
838.
Reumann, S, and Weber, APM. 2006. Plant peroxisomes respire in the light: some
gaps of the photorespiratory C2 cycle have become filled–others remain. BBA-Mol.
Cell Res. 1763:1496–1510.
Rojas, CM, Senthil-Kumar, M, Wang, K, Ryu, CM, Kaundal, A, and Mysore, KS.
2012. Glycolate oxidase modulates reactive oxygen species–mediated signal
transduction during nonhost resistance in Nicotiana benthamiana and Arabidopsis.
Plant Cell Online. 24:336–352.
Sarma GN, Manning VA, Ciuffetti LM, Karplus PA. 2005. Structure of Ptr ToxA: an
RGD-containing host-selective toxin from Pyrenophora tritici-repentis. Plant Cell.
17:3190–3202.
Scheffer, RP, Nelson, RR, and Ullstrup, AJ. 1967. Inheritance of toxin production and
pathogenicity in Cochliobolus carbonum and Cochliobolus victoriae. Phytopath.
57:1288-1291.
Scheffer, RP. 1983. Toxins as chemical determinants of plant disease. JM Daly, BJ
Deverall, eds, Toxins and Plant Pathogenesis. Academic Press, New York, pp 1-40.
Shabab, M, Shindo, T, Gu, C, Kaschani, F, Pansuriya, T, Chintha, R, Harzen, A,
Colby, T, Kamoun, S, and van der Hoorn, R. 2008. Fungal effector protein AVR2
targets diversifying defense-related Cys proteases of tomato. Plant Cell Online.
20:1169–1183.
Shirasu, K, Lahaye, T, Tan, MW, Zhou, F, Azevedo, C, and Schulze-Lefert, P. 1999.
A novel class of eukaryotic zinc-binding proteins is required for disease resistance
signaling in barley and development in C. elegans. Cell. 99:355–66.
83
Shiv, KD. 2012. Oomycete and fungal effector entry, a microbial trojan horse. New
Phytol. 193:874–881.
Shlezinger, N, Minz, A, Gur, Y, Hatam, I, Dagdas, YF, Talbot, NJ, and Sharon, A.
2011. Anti-apoptotic machinery protects the necrotrophic fungus Botrytis cinerea
from host-induced apoptotic-like cell death during plant infection. PLoS Path.
7:e1002185.
Singh, PK, and Hughes, GR. 2006. Genetic similarity among isolates of Pyrenophora
tritici-repentis, causal agent of tan spot of wheat. J. Phytopath. 154:178–184.
Singh, PK, Duveiller, E, Singh, RP, Braun, HJ, Snape, JW, Šíp, V, et al. 2011.
Evaluation of CIMMYT germplasm for resistance to leaf spotting diseases of
wheat. Proceedings from the 8th International Wheat Conference+ BGRI 2010
Technical Workshop, St. Petersburg, Russia, 30 May-4 June 2010., 47:S102–S108.
Stone, JK. 2001. Necrotroph. Encyclopedia of Plant Pathology, ed. Maloy, OC and
Murray, TD. 2:676-677. New York: Wiley.
Strelkov, SE, Lamari, L, and Balance, GM. 1999. Characterization of a host-specific
protein toxin (Ptr ToxB) from Pyrenophora tritici-repentis. Mol. Plant-microbe
Interact. 12:728–732.
Strelkov, SE, and Lamari, L. 2003. Host-parasite interactions in tan spot (Pyrenophora
tritici-repentis) of wheat. Can. J. Plant. Pathol. 25:339-349.
Sweat, TA, and Wolpert, TJ. 2007. Thioredoxin H5 is required for victorin sensitivity
mediated by a CC-NBS-LRR gene in Arabidopsis. Plant Cell Online. 19:673–687.
Taler, D, Galperin, M, Benjamin, I, Cohen, Y, and Kenigsbuch, D. 2004. Plant eR
genes that encode photorespiratory enzymes confer resistance against disease. Plant
Cell Online. 16:172–184.
Tang, JD, Perkins, AD, Sonstegard, TS, Schroeder, SG, Burgess, SC, and Diehl, SV.
2012. Short-read sequencing for genomic analysis of the brown rot fungus
Fibroporia radiculosa. App. Environ. Micro. 78:2272–2281.
Tekauz, A, Mueller, E, Stulzer, M, and Schultz, D. 2004. Leaf spot diseases of winter
wheat in Manitoba in 2003. Can. Plant Dis. Surv. 83:73–74.
Tomas, A, and Bockus, WW. 1987. Cultivar-specific toxicity of the culture filtrates of
Pyrenophora tritici-repentis. Phytopath. 77:1337-1340.
84
Tomas, A, Feng, GH, Reeck, GR, Bockus, WW, and Leach, JE. 1990. Purification of a
cultivar-specific toxin from Pyrenophora tritici- repentis, causal agent of tan spot of
wheat. Mol. Plant Microbe Interact. 3:221–224.
Tornero, P, Merritt, P, Sadanandom, A, Shirasu, K, Innes, RW, Dangl, JL. 2002.
RAR1 and NDR1 contribute quantitatively to disease resistance in Arabidopsis, and
their relative contributions are dependent on the R gene assayed. Plant Cell.
14:1005–1015.
Tor, M, Gordon, P, Cuzick, A, Eulgem, T, Sinapidou, E, et al. 2002. Arabidopsis
SGT1b is required for defense signaling conferred by several downy mildew
resistance genes. Plant Cell. 14:993–1003.
Tuori, RP, Wolpert, TJ, and Ciuffetti, LM. 2000. Heterologous expression of
functional Ptr ToxA. Mol. Plant-Microbe Interact. 13:456–464.
Tyler, BM. 2009. Entering and breaking: virulence effector proteins of oomycete plant
pathogens. Cell. Micro. 11:13–20.
van Esse, HP, Bolton, MD, Stergiopoulos, I, de Wit, PJGM, and Thomma, BPHJ.
2007. The chitin-binding Cladosporium fulvum effector protein Avr4 is a virulence
factor. Mol. Plant-Microbe Interact. 20:1092–1101.
Walton, J D. 1996. Host-selective toxins: agents of compatibility. Plant Cell. 8:1723–
1733.
Wang, Q, Sullivan, RW, Kight, A, Henry, RL, Huang, J, Jones, AM, and Korth, KL.
2004. Deletion of the chloroplast-localized thylakoid formation1 gene product in
Arabidopsis leads to deficient thylakoid formation and variegated leaves. Plant
Physiol. 136:3594-3604.
Wangdi, T, Uppalapati, SR, Nagaraj, S, Ryu, CM, Bender, CL, and Mysore, KS. 2010.
A virus-induced gene silencing screen identifies a role for thylakoid formation1 in
Pseudomonas syringae pv tomato symptom development in tomato and
Arabidopsis. Plant Physiol. 152:281–292.
Weise, MV. 1987. Compendium of Wheat Diseases. 2nd ed. The American
Phytopathological Society, St. Paul, MN.
Wolpert, TJ, Dunkle, LD, and Ciuffetti, LM. 2002. Host-selective toxins and
avirulence determinants: what‘s in a name? Annu. Rev. Phytopath. 400:251-285.
85
Wu, C, Jia, L, and Goggin, F. 2011. The reliability of virus-induced gene silencing
experiments using tobacco rattle virus in tomato is influenced by the size of the
vector control. Mol. Plant Path. 12:299–305.
Zhang, J, Lu, H, Li, X, Cui, YL, Wen, C, Tang, X, Su, Z, and Zhou, J. 2010. Effectortriggered and pathogen-associated molecular pattern-triggered immunity
differentially contribute to basal resistance to Pseudomonas syringae. Mol. PlantMicrobe Interact. 23:940–948.
Zipfel, C, Robatzek, S, Navarro, L, Oakeley, EJ, Jones, JD, Felix, G, Boller, T. 2004.
Bacterial disease resistance in Arabidopsis through flagellin perception. Nature.
428:764–767.