The recently-emerged field of chemical genomics is centered on the use of small molecules to perturb biological systems as a means of investigating their function. In order to employ this approach for the study of plant-pathogen interactions, I established an assay in which Arabidopsis thaliana seedlings are grown in liquid media in 96-well plates. Inoculation of these seedlings with a virulent strain of the bacterial phytopathogen Pseudomonas syringae resulted in macroscopic bleaching of the cotyledons of these seedlings. This symptom was used as the basis for high-throughput chemical genomic screens aimed at identifying small molecules that protect Arabidopsis seedlings from infection. One of the first chemicals identified through this screen was the sulfanilamide compound sulfamethoxazole (Smex). This compound was later shown to also reduce the susceptibility of both Arabidopsis and wheat to infection by the fungal pathogen Fusarium graminearum, suggesting a broad spectrum of activity. More detailed investigations of Smex indicated that the protective activity of this compound did not derive from antimicrobial effects, and that this activity was not executed through common defence-related signalling pathways. The folate biosynthetic pathway enzyme dihydropteroate synthase is a known target of sulfanilamides, and it does appear to contribute to Smex-induced disease resistance, albeit in a folate-independent manner. In order to identify downstream mediators of Smex activity, I initiated two forward genetic screens intended to recover mutants with altered sensitivity to Smex in a seedling growth assay. Interestingly, while these screens yielded mutants with striking Smex sensitivity phenotypes, disease resistance phenotypes were not altered. Gene expression profiling of Arabidopsis tissues treated with Smex prior to bacterial inoculation suggested that this compound generally affects lipid signalling. Altogether, it is evident that Smex elicits a complex set of responses in Arabidopsis with apparently non-overlapping phenotypic outputs.

The recently-emerged field of chemical genomics is centered on the use of small molecules to perturb biological systems as a means of investigating their function. In order to employ this approach for the study of plant-pathogen interactions, I established an assay in which Arabidopsis thaliana seedlings are grown in liquid media in 96-well plates. Inoculation of these seedlings with a virulent strain of the bacterial phytopathogen Pseudomonas syringae resulted in macroscopic bleaching of the cotyledons of these seedlings. This symptom was used as the basis for high-throughput chemical genomic screens aimed at identifying small molecules that protect Arabidopsis seedlings from infection. One of the first chemicals identified through this screen was the sulfanilamide compound sulfamethoxazole (Smex). This compound was later shown to also reduce the susceptibility of both Arabidopsis and wheat to infection by the fungal pathogen Fusarium graminearum, suggesting a broad spectrum of activity. More detailed investigations of Smex indicated that the protective activity of this compound did not derive from antimicrobial effects, and that this activity was not executed through common defence-related signalling pathways. The folate biosynthetic pathway enzyme dihydropteroate synthase is a known target of sulfanilamides, and it does appear to contribute to Smex-induced disease resistance, albeit in a folate-independent manner. In order to identify downstream mediators of Smex activity, I initiated two forward genetic screens intended to recover mutants with altered sensitivity to Smex in a seedling growth assay. Interestingly, while these screens yielded mutants with striking Smex sensitivity phenotypes, disease resistance phenotypes were not altered. Gene expression profiling of Arabidopsis tissues treated with Smex prior to bacterial inoculation suggested that this compound generally affects lipid signalling. Altogether, it is evident that Smex elicits a complex set of responses in Arabidopsis with apparently non-overlapping phenotypic outputs.

Biotrophic plant pathogens lead to huge crop losses and they have great economical drawbacks on wheat and barley production. These pathogens rely on formation of haustoria and transfer of effector proteins into the host cells for generating disease. The main role of effector proteins is to disable plant defense mechanisms. Effector proteins contain N-terminal signal peptides and they have little sequence similarity between each other. It is vital to detect as many effector proteins as possible to understand infection and disease formation processes of biotrophic plant pathogens. To this end, sequencing of pathogen genomes are being emerged, the data will be invaluable for identifying the candidate effectors in terms of biological and biochemical roles in infection and more. There are some bioinformatics based methods available that can be utilized to classify and distinguish effectors from other pathogenic genes. It is important to understand how candidate effectors can be searched from Expressed Sequence Tags or transcriptome sequences. Hereby, our attempt is to present a pipeline in establishing a methodology. As a consequence, here we propose new candidate effectors. In plant-pathogen interactions also miRNAs are too proving to be an important factor which cannot be neglected. During disease infection, expression levels of miRNAs are varying which in turn may be a proof of miRNA regulation of pathogen genes. Therefore, cross-kingdom RNA interference may take place between plant and pathogen. We have tested plant pathogens for possible plant miRNA availability and found their most likely targets with in the pathogen genes.

Verticillium is a soil-borne fungus that is one of the world's foremost plant pathogens. Commercial plant grafting suggests that resistance occurs in the root; this conflicts with decades of research indicating that resistance occurs in the stem. The goal of this thesis work was to use an alternative approach to determine the location of resistance by expressing the Ve1 gene using organ-specific promoters. Promoter sequences for the stem-specific gene, Ribulose 1,5-bisphosphate carboxylase oxygenase small chain 2A (Rbsc2A), and root-specific gene, Tobacco Mosaic Virus Induced (TMVi) were taken from the Sol Genomics Network (SGN) database, cloned into constructs with the Ve1 gene and susceptible tomato germplasm was transformed using Agrobacterium tumefaciens. Preliminary results suggest that resistance may not be localized and expression of the Ve1 gene in either the root or the stem is sufficient to develop whole plant resistance to the Verticillium pathogen.

No description available.

The cereal seed plays a crucial role in society – both in the “food as medicine” paradigm, but also in food security. It is the starch and proteins present in the seed that lend it importance in these dissimilar anthropomorphic activities. This thesis investigation first characterized the post-translational processing of the potential diabetogen, wheat globulin-3. Globulin-3-like peptides were observed primarily in the embryo. These peptides varied significantly in their molecular masses and isoelectric points, as determined by two dimensional electrophoresis and immunoblotting. Five major polypeptide spots were sequenced by mass spectrometry, allowing for the development of a model of the post-translational events contributing to the globulin-3 processing profile. Three separate investigations of starch granules from different cereal species were performed. In the first series of experiments, pathogen-susceptible maize kernels were injected with either conidia of the fungal pathogen Fusarium graminearum or sterile water controls. Proteins in the desiccated fungal remnants on the surface of the kernels as well as in the endosperm and embryo tissues of the control and infected kernels were isolated and these proteomes were sequenced using tandem mass spectrometry. Approximately 250 maize proteins were identified. These proteins were classified into functional categories. There was an increased representation of defense proteins in the both the embryo and endosperm tissues of infected maize samples. The proteome of the fungal remnants was composed of 18 proteins. Several of these proteins were categorized as being involved in the metabolism of plant-sourced molecules, or in stress response. The second series of experiments detail the investigation of commercially prepared rice and maize starches using tandem mass spectrometry. The majority of identified proteins, in both rice and maize samples, were involved in either carbohydrate metabolism or storage. Markers for seed maturity and for starch mobilization were also documented. Finally, the third series of experiments investigated the non-host proteomes present in commercially-prepared starches. Non-host proteins from a variety of species, including Homarus americanus were found in the starch samples. This documentation of H. americanus proteins in these starch samples may have food safety implications with regards to shellfish allergies.

An interaction was recently detected between the potyviral protein, genome-linked (VPg) and the Arabidopsis thaliana translation initiation factor eIF(iso)4E (Wittmann et al., 1997). / Here, experiments were undertaken to address biological aspects of the VPg-eIF4E interaction. First, coimmunoprecipitation experiments performed with purified recombinant proteins have shown that VPg not only associates with eIF4E, as was previously published, but also with the larger eIF4F complex, of which eIF4E is a subunit. These results were confirmed by ELISA-type binding assays. It was also shown that there is no direct interaction between VPg and the other subunit of eIF4F, namely eIF4G. Finally, with the same experimental system, it was shown that the presence of eIF4G does not influence the binding affinity of VPg and eIF4E. / The interaction of VPg with the plant translation apparatus suggests that potyviral infection may alter the host protein expression profile. This hypothesis was investigated with the use of a protoplast system. We have shown that the global rates of protein synthesis in protoplasts transfected with an infectious TuMV cDNA clone dropped shortly after transfection, by as much as an estimated 70%. Recovery to normal levels occurred within 48 hours. / Evidence was obtained that the interaction between VPg and eIF4E is instrumental in this transient down-regulation of protein expression: protoplasts transfected with a mutant TuMV cDNA clone, the VPg of which has no affinity for eIF4E, failed to exhibit the drop in protein synthesis observed with the wild-type clone.

Turnip mosaic potyvirus (TuMV) is a single-stranded RNA plant virus. One of its proteins, the cylindrical inclusion (CI) protein, was hypothesized to interfere with host transcription via interaction with histone H3 protein. Interaction between CI and histone H3 was previously observed in Dr. Fortin's laboratory. Based on previous studies that demonstrated the importance of the H3 tail domain in gene regulation and chromosome arrangement, it was hypothesized that CI would interact with the tail rather than the globular domain. The objective of this project was to identify which histone H3 domains CI protein interacts with. The full-length, globular, and tail domains of histone H3 DNA were expressed in E. coli and purified. Based on in vitro interaction experiments, the CI protein was observed to interact with the globular domain of histone H3.

Turnip mosaic virus (TuMV) infects a variety of crops, worldwide, including the economically relevant Brassicacea family. It was previously demonstrated that TuMV infection in tobacco protoplasts leads to an overall decrease of host protein. However, it remains unclear whether this phenomenon is due to the repression of plant gene transcription during the infection period or due to viral inhibition of host translation. In this study, quantification of various transcripts and protein products from infected tobacco was performed via real-time RT-PCR and ELISA. In comparison to the gamma-tubulin endogenous control, gene expression for the tobacco H3, HSP70 and granule-bound starch synthase was affected by TuMV infection with time. / Tobacco protein accumulation in whole leaf tissues was also significantly affected by increase of virus particles.

Thesis (Ph. D.)--University of Alberta, 2009. / Title from pdf file main screen (viewed on June 29, 2009). "A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy Plant Science, Department of Agricultural, Food and Nutritional Science, University of Alberta." Includes bibliographical references.