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No description available.

Oomycetes, such as Phytophthora sojae, are plant pathogens that employ protein effectors that enter host cells to facilitate infection. Plants may overcome infection by recognizing pathogen effectors via intracellular receptors (R proteins) that form part of their defense system. Entry of some effector proteins into plant cells is mediated by conserved RxLR motifs in the effectors and phosphoinositides (PIPs) resident in the host plasma membrane such as phosphatidylinositol 3-phosphate (PtdIns(3)P). Recent reports differ regarding the regions on RxLR effector proteins involved in PIP recognition. To clarify these differences, I have structurally and functionally characterized the P. sojae effector, avirulence homolog-5 (Avh5). Using NMR spectroscopy, I demonstrate that Avh5 is helical in nature with a long N-terminal disordered region. Heteronuclear single quantum coherence titrations of Avh5 with the PtdIns(3)P head group, inositol 1,3-bisphosphate (Ins(1,3)P2), allowed us to identify a C-terminal lysine-rich helical region (helix 2) as the principal lipid-binding site in the protein, with the N-terminal RxLR (RFLR) motif playing a more minor role. Furthermore, mutations in the RFLR motif slightly affected PtdIns(3)P binding, while mutations in the basic helix almost abolished it. Avh5 exhibited moderate affinity for PtdIns(3)P, which increased the thermal stability of the protein. Mutations in the RFLR motif or in the basic region of Avh5 both significantly reduced protein entry into plant and human cells. Both regions independently mediated cell entry via a PtdIns(3)P-dependent mechanism. My findings support a model in which Avh5 transiently interacts with PtdIns(3)P by electrostatic interactions mainly through its positively charged helix 2 region, providing stability to the protein during RFLR-mediated host entry. / Ph. D.

Disease outbreaks caused by oomycetes can be catastrophic. The first part of this dissertation describes development of a system to identify potential new and durable resistance (R) genes against P. sojae in soybean germplasm. We developed a system to screen soybean germplasm for genes that recognize core Phytophthora sojae RXLR effectors that are conserved within the pathogen species and essential for virulence. R genes that recognize these effectors will likely be effective and durable against diverse P. sojae isolates. We developed a system to deliver individual P. sojae effectors by Type III secretion into soybean using the bacterium Pseudomonas, and we screened 12 core effectors on a collection of 30 G. max lines that likely contain new resistance genes against P. sojae. We identified candidate R genes against 10 effectors. Genetic segregation ratios from crosses indicated that three of these genes have a simple inheritance pattern and would be amenable to breeding into elite cultivars. The second part of the dissertation involves use of a model plant-oomycete system to study the genetic basis of susceptibility to oomycete diseases. We compared host transcriptomes from a resistant and a susceptible infection of Arabidopsis thaliana by the downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa). We identified five gene clusters with expression patterns specific to the susceptible interaction. Genes from each cluster were selected and null mutants were tested for altered susceptibility to virulent Hpa. Most A. thaliana null mutants showed enhanced disease susceptibility, suggesting their involvement in pattern-triggered immunity (PTI). A knockout mutant in the AtGcn5 gene was completely resistant to Hpa Emco5 suggesting that the gene/protein is necessary for Hpa to successfully colonize the plant. This study provided new molecular insights into plant-oomycete interaction and revealed a plant gene that could potentially be engineered to provide enhanced resistance to oomycete pathogens. / Ph. D.

Estimated at approximately $43 billion annually, the cultivated soybean Glycine max (L.) Merr., is the second most valuable crop in the United States. Soybeans account for 57% of the world oil-seed production and are utilized as a protein source in products such as animal feed. The value of a soybean crop, measured in seed quality and quantity, is negatively affected by biotic and abiotic stresses. This research is focused on resistance to biotic disease stress in soybean. In particular, we are working on the Phytophthora soja (P. sojae) and Soybean Mosaic Virus (SMV) systems. For each of these diseases, we are working to develop superior soybean germplasm that is resistant to the devastating economic impacts of pathogens. The majority of this research is focused on screening for novel sources of P. sojae resistance with core effectors to identify resistance genes (R-genes) that will be durable under field conditions. Four segregating populations and two recombinant inbred line (RIL) populations have been screened with core effectors. Effector-based screening methods were combined with pathogen-based phenotyping in the form of a mycelium-based trifoliate screening assay. One RIL population has been screened with virulent P. sojae mycelium. Disease phenotyping has generated a preliminary genetic map for resistance in soybean accession PI408132. The identification of novel R-genes will allow for stacking of resistance loci into elite G. max cultivars. The second project covered in this dissertation describes the validation of the SMV resistance gene Rsv3. Utilizing a combination of transient expression and homology modeling; we provide evidence that Glyma14g38533 encodes Rsv3. / Ph. D.

Pathogens colonize their hosts by releasing molecules that can enter host cells. A biotrophic oomycete plant pathogen, Phytophthora sojae harbors a superfamily of effector genes whose protein products enter the cells of the host, soybean. Many of the effectors contain an RXLR-dEER motif in their N-terminus. More than 400 members belonging to this family have been previously identified using a Hidden Markov Model. Amino acids flanking the RXLR motif have been utilized to identify effector proteins from the P. sojae secretome, despite the high level of sequence divergence among the members of this protein family. I present here machine learning methods to identify protein candidates that belong to a particular class, such as the effector superfamily. Converting the flanking amino acid sequences of RXLR motifs (or other candidate motifs) into numeric values that reflect their physical properties enabled the protein sequences to be analyzed through these methods. The methods evaluated include Support Vector Machines and a related spherical classification method that I have developed. I also approached the effector prediction problem by building functional linkage networks and have produced lists of predicted P. sojae effector proteins. I tested the best candidate through gene gun bombardment assays using the beta-glucuronidase reporter system, which revealed that there is a high likelihood that the candidate can enter the soybean cells. / Ph. D.

Titre de l'écran-titre (visionné le 2 octobre 2023) / Le soya (Glycine max [L.] Merr.) est une culture de plusieurs milliards de dollars principalement utilisée pour l'industrie de l'élevage, l'alimentation humaine et aussi le biocarburant. Le Canada se classe au quatrième rang en termes de superficie, et cette dernière augmente chaque année. Avec l'augmentation de la superficie cultivée, les problèmes de ravageurs et de maladies ont également augmenté. Le pourridié phytophthoréen (PRR) est l'une des maladies les plus importantes du soya. L'oomycète Phytophthora sojae est l'agent pathogène responsable du PRR et représente chaque année un milliard de dollars de pertes pour l'industrie nord-américaine du soya. Ce pathogène attaque la plante à tous ses stades de croissance, entraînant souvent le besoin de semer de nouveau et même la perte complète de la récolte. Le moyen le plus efficace de réduire les pertes économiques causées par le PRR est d'utiliser des cultivars résistants à P. sojae. Ces cultivars possèdent des gènes de résistance (Rps) et, jusqu'à ce jour, 33 gènes Rps ont été répertoriés grâce à l'utilisation de marqueurs génétiques. Toutefois, seul le gène Rps11 a été cloné et identifié. Récemment, notre laboratoire a identifié des gènes candidat qui seraient possiblement des gènes Rps. Pour confirmer la fonction de ces gènes, il faut être en mesure de les exprimer par une plante sensible via la transformation génétique. Différentes méthodes telles que la transformation médiée par Agrobacterium ou la transformation biolistique génèrent des plants de soya génétiquement modifiés à partir de cals ou d'autres explants. Pourtant, ces techniques demandent beaucoup de main-d'œuvre et de temps, avec un faible taux d'efficacité. Lors de l'étude des gènes exprimés dans les systèmes racinaires, la technique de transformation par Rhizobium rhizogenes est idéale. Cet organisme, par le transfert d'un ADN aux cellules de la plante, cause une prolifération de fines racines « hairy root ». Cette méthode de transformation génétique a été régulièrement appliquée pour étudier la biologie des racines ou bien pour la production de métabolites secondaires, mais rarement pour étudier les interactions hôte-pathogène. Dans ce travail, la technique de transformation hairy-root a été optimisée pour caractériser la fonctionnalité des gènes candidats Rps contre P. sojae par des essais biologiques dans des systèmes hydroponiques. Dans un premier temps, nous avons testé plusieurs souches de Rhizobium rhizogenes et différentes méthodes d'inoculation pour optimiser le procédé qui donnerait le nombre maximum de racines transformées. En tant que gène rapporteur, RUBY a été sélectionné pour ses propriétés non destructives et son expression facilement identifiable dans les racines transformées. Afin d'optimiser la cassette d'expression pour les gènes candidat, la fonction de six promoteurs de soya a également été validée. Cinq promoteurs sur six ont présenté une expression du gène rapporteur dsRed2 supérieure au promoteur le plus couramment utilisé soit le CaMV35S. Pour les besoins de ces travaux, le promoteur du gène de l'ubiquitine a été choisi pour la conception de la cassette d'expression servant à héberger les gènes candidat Rps. À la suite de l'expression de ces gènes candidats dans les racines transformées, il devenait alors possible de tester la fonction de ces derniers par bioessai hydroponique. / Soybean (Glycine max [L.] Merr.) is a multi-billion-dollar crop primarily used to produce animal feed, food, and fuel. Soybean ranks fourth in terms of area, and the area under its cultivation is increasing every year. With the increase in cultivated areas, pest and disease problems have also increased. Phytophthora root rot (PRR) is one of the most important diseases of soybeans. The oomycete Phytophthora sojae is the pathogen responsible for the disease and accounts forbillion in losses annually for the North American soybean industry. This pathogen attacks the plant at all stages of growth, often leading to replanting or complete loss of the crop. The most effective way to reduce the economic losses caused by PRR is to use cultivars resistant to P. sojae (Rps) genes. Approximately 33 Rps genes has been identified. Many of these 33 Rps genes are linked to genetic markers, but only Rps11 has been identified and cloned. Our laboratory has identified putative sequences of Rps genes, and the functional characterization of these sequences must go through the transformation of soybeans. Different methods, such as Agrobacterium-mediated transformation or biolistic transformation, generate genetically modified soybean plants from callus or other explants. Yet, these techniques are labor-intensive and time-consuming, with a low efficiency rate. When studying genes expressed in root systems, the Rhizobium rhizogenes-mediated hairy root transformation technique is ideal. Hairy root transformation has been regularly applied to study root biology or secondary metabolite production, but rarely to study host-pathogen interactions. In this work, the hairy root transformation technique was optimized to characterize the functionality of candidate Rps genes against P. sojae by bioassays in hydroponic systems. First, we tested several strains of Rhizobium rhizogenes and different inoculation methods to identify the procedure that would yield the maximum number of transformed roots. As a reporter gene, RUBY was selected for its non-destructive properties and easily identified expression in transformed roots. We also validated the function of six soybean promoters using the optimized hairy root transformation system. Five of six promoters expressed the dsRed2 reporter gene better than the positive control CaMV35S promoter. Finally, the best promoter was used to design an expression cassette for candidate Rps gene from previously identified sequences, which can be used for functional characterization by hydroponic phenotyping. In conclusion, this optimized hairy root transformation will provide a rapid and reliable method to validate the function of putative Rps genes in the soybean- P. sojae interaction.

La pourriture phytophthoréenne causée par Phytophthora sojae est une des principales maladies affectant la culture du soya au Canada. Les moyens de lutte actuellement utilisés sont le traitement des semences à l’aide de fongicides et l’utilisation de cultivars possédant une résistance partielle multigénique ou un gène majeur de résistance (Rps). Dans ce contexte, les semenciers souhaitent pouvoir caractériser leurs lignées quant à leur niveau de résistance/susceptibilité face aux différentes races de P. sojae. Ce projet avait ainsi pour objectif d'exploiter une méthode d'inoculation permettant de discriminer de façon claire, rapide et reproductible, la réponse des lignées à P. sojae en lien avec la virulence des races. Nos résultats ont démontré qu'une infection par zoospores dans un système de culture hydroponique présentait plusieurs avantages par rapport à d’autres techniques d’inoculation utilisées. Cette méthode permet non seulement d’identifier les cultivars porteurs de gènes de résistance Rps, mais aussi potentiellement de mettre en évidence les cultivars à résistance racinaire.