Brassica napus (L.) contributes over $19 billion dollars each year to the Canadian economy. However, yields are constantly threatened by Sclerotinia sclerotiorum (Lib) de Bary, the fungus responsible for Sclerotinia stem rot. To date, there are no global RNA profiling data or gene regulatory analyses of plant tissues directly at the main site of foliar infection in the B. napus-S. sclerotiorum pathosystem. Using RNA sequencing and a gene regulatory analysis, I discovered putative transcriptional regulators of biological processes associated with the tolerant phenotype of B. napus cv. Zhougyou821 including subcellular localization of proteins, pathogen detection, and redox homeostasis. Functional characterization of Arabidopsis mutants identified a number of genes that contribute directly to plant defense to S. sclerotiorum. Together this research amounts to the expansion of our understanding of the B. napus-S. sclerotiorum pathosystem and a valuable resource to help protect B. napus crops from virulent pathogens such as S. sclerotiorum. / October 2016

Septoria nodorum blotch is a disease of wheat caused by the necrotrophic fungus Parastagonospora nodorum. In the wheat-P. nodorum pathosystem, recognition of pathogen-produced necrotrophic effectors (NEs) by dominant host genes leads to host cell death, which allows the pathogen to gain nutrients and proliferate. To date, nine host gene-NE interactions have been reported in this pathosystem. Among them, the Snn2-SnTox2 interaction has shown to be important in both seedling and adult plant susceptibility. A saturated genetic linkage map was developed using a segregating population of recombinant inbred lines and a high-resolution map was then developed using F2 plants derived from a cross between the SnTox2-insensitive wheat line BR34 and the SnTox2-sensitive line BG301. Over 10,000 gametes were screened for high-resolution mapping and the Snn2 gene was delineated to a genetic interval of 0.10 cM that corresponds to a physical segment of approximately 0.53 Mb on the short arm of wheat chromosome 2D. A total of 27 predicted genes present in this region and thirteen of them were identified as strong candidates. Seven EMS-induced Snn2-insensitive mutants were generated for gene validation. Results of this study provide the foundation for cloning of Snn2. The host sensitivity gene Snn1, which confers sensitivity to SnTox1, was previously cloned. Here, allelic diversity of Snn1 was studied to identify causal polymorphisms, and to develop markers useful for marker assisted selection (MAS). Twenty-seven coding sequence haplotypes that correspond to 21 amino acid haplotypes were identified. Three SNPs were identified as the possible mutations that caused the insensitive allele in wild emmer to become the sensitive allele in domesticated wheat. In addition, four SNPs that changed the sensitive allele into insensitive alleles were identified. SNP-based markers that could detect three of those SNPs were developed. Results of this study help to increase our knowledge in wheat-NE interactions and host sensitivity gene evolution. / USDA – Agricultural Research Service / National Institute of Food and Agriculture

No description available.

A plant's response to pathogen infection is tailored dependent on infection strategy. Successful plant pathogens employ various infection strategies to avoid or reduce plant defense responses for the establishment of host compatibility. Autophagy is a non-selective degradation pathway conserved in eukaryotic organisms, which has been implicated in the regulation of cell survival or cell death, depending on cell type and stimulus. In <i>Arabidopsis thaliana</i>, an autophagic response has been reported to be activated during nutrient deprivation. Cellular contents, such as cytoplasm and organelles, are sequestered into double-membraned autophagosomes and delivered to the vacuole for degradation; degradative products, such as amino acids, are released back into the cell and reutilized to maintain cellular function. In this study, the response of the autophagy pathway was investigated in <i>A. thaliana</i> leaf tissues upon biotrophic <i>Erysiphe cichoracearum</i> and hemibiotrophic <i>Colletotrichum higginsianum</i> infections. Expression of some autophagy genes was induced in <i>A. thaliana</i> at 9 days post infection with <i>E. cichoracearum</i> and, 3 and 5 days post infection with <i>C. higginsianum</i>. Using a transgenic <i>A. thaliana</i> plant line over expressing autophagosome associated protein autophagy-8e (<i>ATG8e</i>) conjugated to green fluorescent protein (GFP) (<i>ATG8e-GFP</i>), confocal analysis revealed that autophagosomes specifically accumulated at the infection sites during <i>E. cichoracearum</i> and <i>C. higginsianum</i> invasions. These results indicate that the plant autophagic pathway responds to an interaction between <i>A. thaliana</i> and fungal pathogens. None of the defense signaling molecules including salicylic acid, jasmonic acid, ethylene, hydrogen peroxide and nitric oxide consistently triggered expression of autophagy genes. The insensitivity to defense signaling molecules and the delayed induction of autophagy genes compared to expression of pathogenesis-related genes suggest that the activation of this pathway does not contribute to host resistance responses during the infection process. In <i>A. thaliana</i> mutants, <i>atg4a/b, atg5-1, atg9-1</i> and <i>atg9-6</i> deficient for the autophagic response, virulence of <i>E. cichoracearum</i> was retarded whereas pathogenesis of <i>C. higginsianum</i> was accelerated. Taken together, these data suggest that the autophagy pathway is a potential host susceptibility factor for pathogen infection, possibly involved in establishing/facilitating biotrophy in <i>A. thaliana</i>.

A plant's response to pathogen infection is tailored dependent on infection strategy. Successful plant pathogens employ various infection strategies to avoid or reduce plant defense responses for the establishment of host compatibility. Autophagy is a non-selective degradation pathway conserved in eukaryotic organisms, which has been implicated in the regulation of cell survival or cell death, depending on cell type and stimulus. In <i>Arabidopsis thaliana</i>, an autophagic response has been reported to be activated during nutrient deprivation. Cellular contents, such as cytoplasm and organelles, are sequestered into double-membraned autophagosomes and delivered to the vacuole for degradation; degradative products, such as amino acids, are released back into the cell and reutilized to maintain cellular function. In this study, the response of the autophagy pathway was investigated in <i>A. thaliana</i> leaf tissues upon biotrophic <i>Erysiphe cichoracearum</i> and hemibiotrophic <i>Colletotrichum higginsianum</i> infections. Expression of some autophagy genes was induced in <i>A. thaliana</i> at 9 days post infection with <i>E. cichoracearum</i> and, 3 and 5 days post infection with <i>C. higginsianum</i>. Using a transgenic <i>A. thaliana</i> plant line over expressing autophagosome associated protein autophagy-8e (<i>ATG8e</i>) conjugated to green fluorescent protein (GFP) (<i>ATG8e-GFP</i>), confocal analysis revealed that autophagosomes specifically accumulated at the infection sites during <i>E. cichoracearum</i> and <i>C. higginsianum</i> invasions. These results indicate that the plant autophagic pathway responds to an interaction between <i>A. thaliana</i> and fungal pathogens. None of the defense signaling molecules including salicylic acid, jasmonic acid, ethylene, hydrogen peroxide and nitric oxide consistently triggered expression of autophagy genes. The insensitivity to defense signaling molecules and the delayed induction of autophagy genes compared to expression of pathogenesis-related genes suggest that the activation of this pathway does not contribute to host resistance responses during the infection process. In <i>A. thaliana</i> mutants, <i>atg4a/b, atg5-1, atg9-1</i> and <i>atg9-6</i> deficient for the autophagic response, virulence of <i>E. cichoracearum</i> was retarded whereas pathogenesis of <i>C. higginsianum</i> was accelerated. Taken together, these data suggest that the autophagy pathway is a potential host susceptibility factor for pathogen infection, possibly involved in establishing/facilitating biotrophy in <i>A. thaliana</i>.

Incompatible plant/pathogen interactions are often manifested as the hypersensitive response (HR), characterised by host cell death and rapid tissue collapse at the site of attempted infection. A key early response during the HR is the generation of reactive oxygen species (ROS), such as the superoxide anion ( 0; -) and hydrogen peroxide (H20 2), in an oxidative burst. The ROS produced during the oxidative burst have been implicated as cellular signalling molecules for the induction of defences responses including hypersensitive cell death. Increasing evidence exist that the free radical, nitric oxide (NO) also acts as a signalling molecule in plants during plant/pathogen interactions. The generation of NO in response to bacterial challenge, and the potential signalling pathways involved in H20 2- and NO-induced defence responses in Arabidopsis were therefore investigated Arabidopsis suspension cultures were found to generate elevated levels of NO and undergo cell death analogous to HR seen in planta, in response to challenge by avirulent bacteria. Using NO donors, elevated levels of NO were found to be sufficient to induce cell death independently of ROS, but not the expression of the defence-related genes PAL or GST. The NO-induced cell death was sensitive to inhibitors of RNA processing and protein synthesis, suggesting that NO-induced cell death is a form of programmed cell death (PCD), requiring the expression of at least one gene. However, the source of NO production by Arabidopsis remains to be elucidated, but appears to be independent of nitric oxide synthase-like activity. Pharmacological studies using specific inhibitors of mammalian mitogen activated protein kinase (MAPK) signalling cascades, and guanylate cyclase, the enzyme responsible for the production of second messenger cyclic guanosine monophosphate (cGMP), suggest that a MAPK signalling cascade acts downstream or independently of the oxidative burst to initiate H20 2-induced defence responses, while NO-induced cell death requires the production of cGMP in Arabidopsis. A number of studies have attempted to establish whether PCD induced during the HR in plants is similar to apoptotic cell death of anin1al cells. The key executioners of apoptosis in animal cells are caspases. NO was found to induce caspase-like activity in Arabidopsis cells, while a specific inhibitor of caspase-l blocked harpin-, H20 2- and NO-induced cell death. A characteristic of apoptosis is chromatin condensation and DNA fragmentation into nucleosomal fragments. Chromatin condensation was observed in Arabidopsis cells treated with the NO donor Roussin's black salt, but no DNA fragmentation was found in DNA extracted from cells treated with harpin, H20 2 or NO. In addition, random DNA degradation indicative of necrosis was found in DNA extracted from cells following avirulent bacterial challenge.

Investigations into plant-pathogen interactions have provided us with several models underlying the genetic basis of host resistance in plants. In the past decade, tens of resistance genes have been isolated from numerous crop and model plant species and these form a few distinct classes when classified by domain structure, the majority being nucleotide-bindingsite- leucine-rich-repeat (NBS-LRR) genes. The NBS-LRR family consists of two sub-families based on the N-terminal domain: the coiled-coil (CC) NBS-LRRs and the Toll Interleukin Receptor homology domain (TIR) NBS-LRRs. The potential of these genes for future and current agricultural breeding programs has driven a large number of studies exploring the members of these gene families in the genomes of a variety of crop species. In the present study I focused on the NBS-LRR family in the allohexaploid wheat genome and obtained a comprehensive set of Triticeae NBS-LRR homologues using a combination of data-mining approaches. As starting point I detected conserved motifs in the dataset, finding all six previously characterized in the core-NBS domain of other plant NBS-LRRs. Phylogenetic analysis was performed to study relationships between the Triticeae NBS-LRR family and the 25 CC-NBS-LRR (CNL) R genes identified to date. I found the Triticeae CNL family to be highly divergent, containing ancient clade lineages, as seen in all angiosperm 120 taxa previously studied, and found a number of “ancient” dicotyl R genes grouped with Triticeae clades. The evolution of recent NBS-LRR gene duplications in the Triticeae was studied at the hand of two modes of duplication - firstly individual gene duplications yielding paralogous loci and secondly gene duplication by allopolyploidy. Current models of NBS-LRR family evolution predict that functional divergence occurs after gene duplication. An alternative is that divergence takes place at allele level, followed by a locus duplication that fixes heterozygosity in a single haplotype by unequal recombination. I investigated this hypothesis by studying the evolution of gene duplicates in two different contexts – paralogous duplications in the diploid barley genome and homeologous duplications in the allohexaploid genome of wheat. Nonsynonymous to synonymous substitution rate ratios were estimated for paralogous gene duplications in three recently diverged NBS-LRR clades. All pairwise comparisons yielded Ka:Ks ratios strongly indicative of purifying selection. Given that R gene mediated resistance is inherited qualitatively rather than quantitatively, I interpret this as evidence that even closely related paralogous copies (90-95% identity) should have independent recognition specificities maintained by purifying selection. Homeologous duplications were studied in allohexaploid wheat (AABBDD) using a section of the go35 NBS-LRR gene (2L) of the B and D diploid donor species of wheat. Numerous synonymous substitutions distinguished the B and D genome copies, with an absence of nonsynonymous substitutions. In contrast, single unique nonsynonymous substitutions were found in four out of five polyploid wheat go35 alleles, indicating that selection pressure was indeed relaxed across the homeolocus. Recent studies on polyploid genomes have shown that duplicated resistance genes are far more likely to be eliminated than highly transcribed genes such as tRNAs and rRNAs. These results are in agreement with the view that functional divergence takes place before duplication for NBS-LRR genes, as the loci duplicated by polyploidy appear not to evolve under purifying selection, as I found for the paralogous loci investigated. / Dissertation (MSc)--University of Pretoria, 2008. / Genetics / unrestricted

Exserohilum turcicum is the hemibiotrophic causal agent of Northern leaf blight of maize and sorghum. Despite the global importance of this yield-limiting pathogen, knowledge regarding genes contributing to disease development and race-specificity is limited. Therefore, this study aimed to identify genes involved in host colonization during biotrophic and necrotrophic phases of infection, as well as race-specific differences in gene expression. RNAseq of maize seedlings inoculated with a race 13N or 23N E. turcicum isolate was conducted to identify genes contributing to fungal pathogenicity, and expression was validated for four effector candidates. A population genetic study was undertaken of isolates from maize and sorghum to select isolates for sequencing of three putative effectors. Fungal biomass positively correlated with the percentages of E. turcicum reads mapped and indicated a lifestyle switch from biotrophy to necrotrophy between 7 and 13 dpi. Transcriptome sequencing enabled identification of cell wall degrading enzymes, peptidase-encoding genes, secondary metabolite biosynthesis genes and candidate effectors likely contributing to the pathogenicity of E. turcicum. Profiling of Ecp6 and candidate effector SIX13-like revealed increased expression at 5 and 7 dpi compared to 2 and 13 dpi. Evidence of host specificity was obtained from microsatellite haplotypes and sequencing of SIX13-like. Identification of candidate effector SIX13-like is consistent with the colonization of E. turcicum through the xylem of susceptible hosts and possibly indicates specificity of E. turcicum to either maize or sorghum. This study identified E. turcicum genes putatively involved in pathogenicity and describes a hypothetical model of the E. turcicum – maize interaction. / Thesis (PhD)--University of Pretoria, 2019. / The financial assistance of the National Research Foundation (NRF South Africa, grant unique numbers 85847, 88785, 92762 and 93671) toward this research is hereby acknowledged. Opinions expressed and conclusions arrived at, are those of the authors and are not necessarily to be attributed to the NRF. / Plant Science / PhD / Unrestricted

The interaction between Arabidopsis and its natural downy mildew pathogen, Hyaloperonospora arabidopsidis (Hpa), provides a model for understanding how oomycetes colonize plants. Hpa is a model organism for many highly destructive oomycete pathogens and transcriptomics of this interaction have been well-documented. However, the material in these studies has been derived from infected leaves that contain a mix of pathogen-proximal and pathogen-distal plant cells. The most direct interactions between Arabidopsis and Hyaloperonospora arabidopsidis occur in haustoriated cells- where the pathogen can secrete effectors and acquire nutrients needed for successful colonization and reproduction. These cells are difficult to isolate due to their limited number and ephemeral nature. I have developed a method to isolate the translatome (i.e., mRNAs associated with ribosomes) of pathogen-proximal cells. This method utilizes translating ribosome immuno-purification technology (TRAP), regulated by both pathogen-responsive and tissue-specific promoters, to isolate mRNAs that are being translated in pathogen-proximal cells. Compared to "bulk" transcriptomics of material isolated from homogenized leaves, this method will enrich for transcripts that are differentially expressed, and translated, in pathogen-proximal cells. From this method, RNA was isolated in amount and quality sufficient for sequencing. This sequencing data will enable the discovery of plant genes that may be manipulated by the pathogen to suppress defense responses and extract nutrients. / Ph. D. / The interactions between plants and the pathogens that feed on them are complex and at times difficult to study. Among the many different types of plant pathogens, oomycetes (a class of fungus-like organisms) are especially destructive. Using Arabidopsis and its natural downy mildew pathogen, Hyaloperonospora arabidopsidis (Hpa) as model for understanding how oomycetes colonize plants, I hope to learn more about plant-pathogen interactions. Hpa is a model organism for many highly destructive oomycete pathogens and several aspects of this interaction have been well-documented. However, the material in these studies has been derived from infected leaves that contain a mix of plant cells that are both in direct contact with the pathogen, or from uninfected areas of the plant. The most direct interactions between Arabidopsis and Hpa occur in cells that have been invaginated with a pathogen feeding structure called a haustorium. These cells are difficult to isolate due to their limited number and ephemeral nature. I have developed a method to isolate the translatome (i.e., mRNAs that are being translated by and are associated with ribosomes) of pathogen-proximal cells. This method utilizes translating ribosome immuno-purification technology (TRAP), regulated by both pathogen-responsive and tissue-specific promoters, to isolate mRNAs that are being translated in pathogen-proximal cells. Compared to “bulk” transcriptomics of material isolated from homogenized leaves, this method will enrich for transcripts that are differentially expressed, and translated, in pathogen-proximal cells. From this method, RNA was isolated in amount and quality sufficient for sequencing. This sequencing data will enable the discovery of plant genes that may be manipulated by the pathogen to suppress defense responses and extract nutrients.

Upon detection of a pathogen, plants initiate specific signaling events designed to prevent host colonization and pathogen proliferation. Appearance of the hypersensitive response (HR), a type of programmed cell death signifies activation of active defenses in response to a one-to-one recognition of host, Resistance or R gene, and pathogen, avirulence or avr gene, encoded products. Turnip crinkle virus (TCV), however, has been shown to suppress the ability of Col-0 Arabidopsis thaliana plants to produce the HR in response to an avirulence factor. The extent of suppression was quantified by measuring cellular electrolyte leakage resulting from programmed cell death. Interestingly, cellular ion leakage levels were significantly lower in TCV-infected plants when challenged with bacteria expressing either of two bacterial effectors avrRpt2 or avrRpm1, suggesting that TCV can suppress the HR to a range of HR-inducing avirulence factors. In order to determine the viral component(s) responsible for mediating this suppression, each of the five TCV open reading frames (ORFs) was tested using an Agrobacterium tumefaciens-mediated transient expression assay in Nicotiana benthamiana. Though sequencing of the five TCV clones revealed mutations in the p28, p88, and p9 clones, Agro infiltration of an HR-inducing system in conjunction with individual TCV ORFs, or combinations of, was used to gather data to determine the role each may possess in the suppression phenotype. Full-length TCV was also expressed in the presence of AvrPto/Pto to establish suppression phenotype in Nicotiana. To assay for suppression of cell death in a heterologous system, both the mutant and wild-type clones were also tested in yeast for cell-death suppression induced by hydrogen peroxide exposure.