| Downy mildew of cucumber (Cucumis sativus) is caused by the obligate oomycete, Pseudoperonospora cubensis, and the research described in the dissertation provides new insight on the transcriptional regulation within the pathogen through the mechanism of alternative splicing and investigates the transcriptional changes of the host genes within a resistant and susceptible interaction. Previously, the genome of cucumber and P. cubensis as well transcriptome of a compatible interaction between the plant and the pathogen were sequenced. In addition, one gene from P. cubensis was known to be alternatively spliced, but the breadth of alternative splicing across the transcriptome was unknown. Through the work described in this thesis, we investigated the breadth of alternative splicing across the entire transcriptome of P. cubensis over the time course of infection. We found P. cubensis genes are frequently spliced and have intron retention as the most common mechanism of alternative splicing with some evidence for the retention of the 5' or 3' end of the exon but no evidence for exon skipping. Furthermore, we found that alternative splicing occurred in genes encoding several types of proteins, including the effectors, which impact pathogenicity and virulence. In some cases, the frequency of alternative splicing was found to correlate with developmental stages of the pathogen and thus alternative splicing might play a role in regulating transcript abundance and availability during development.;Advances in sequencing and bioinformatics also contributed to our work in advancing the knowledge of the transcriptomic changes in a resistant (PI 197088) or susceptible (Vlaspik) plant during an infection time course. Our work shows that while P. cubensis is able to enter the resistant plant leaf, it is not able to sporulate; in contrast, the pathogen is able to grow and sporulate in the susceptible host, Vlaspik. To investigate the transcriptional changes underlying resistance, we identified the differentially expressed genes between the resistant and susceptible plant lines over the time course of infection. We found that the resistant plant responded earlier to the pathogen, as demonstrated by a higher number of differentially expressed genes at earlier time points compared to the susceptible plant. In addition, we found changes in genes encoding proteins with functions in hormone-related processes, nutrition, and transportation that might indicate a role for some of these genes in initiating or responding to the resistance response in cucumber. Beyond identifying differentially expressed transcripts, we identified small RNAs in the host and pathogen, as small RNAs have a role in modifying gene expression. We found novel miRNAs in the pathogen and known and novel miRNA in the host and predicted potential targets for each miRNA within the cucumber transcriptome. Some of these miRNAs may have a role in mediating the response of the plant to the pathogen. In the future, work will be done to validate the roles of candidate resistance-associated genes and to validate the presence and role of miRNA in both the host and the pathogen. Some of this future work will involve incorporating other "omics" methods including metabolomics and proteomics in order to get a more complete understanding of the molecular changes in the plant during infection. Finally, strong candidates for resistance could be validated using the proposed in planta methods, which includes the development of transgenic cucumbers. |
