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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.

L'objectif de ce projet visait à déterminer si le silicium (Si), reconnu pour son effet prophylactique contre les agents biotrophes, pouvait protéger le soja contre Phytophthora sojae, un oomycète hemi-biotrophe. Le défi initial consistait à développer une méthode d'inoculation qui reproduisait le processus d'infection naturelle tout en permettant aux plants d'absorber le Si. Dans un premier temps, l'inoculation de zoospores dans une solution hydroponique a permis de générer des infections reproductibles conséquentes avec les réponses phénotypiques attendues. Dans un deuxième temps, l'ajout de Si a permis de réduire significativement l'incidence de la maladie tout en augmentant le rendement des plants. Cet effet était surtout manifeste sur les cultivars possédant un niveau de résistance contre P. sojae ou sur ceux absorbant plus de Si. Nous proposons ainsi une nouvelle méthode d'inoculation de P. sojae représentative de l'infection naturelle et démontrons que le Si peut servir pour la lutte contre cet oomycète.