A proteomic investigation of Phytophthora species using mass spectrometry and reverse genetics
Savidor, A.  2008.  Ph.D. Dissertation, .

Abstract Organisms in the genus Phytophthora are agriculturally and ecologically important plant pathogens, although understudied. Phytophthora was first brought into human awareness with the identification of P. infestans as the culprit for the Irish potato famine in the mid eighteen hundreds. Since then, over 80 other Phytophthora species have been identified, many of which infect a large variety of crops world wide with devastating results. Traditionally, much of the work aimed at controlling Phytophthora diseases involved applied research, including plant breeding for resistance to Phytophthora, or treatment of the field with different chemicals. However, only limited success was achieved using these strategies, as different Phytophthora species were able to overcome such challenges due to their highly adaptive and biologically unique nature. In recent years there has been a marked increase in molecular work on Phytophthora. This increase is evident not only from increased funding by agencies such as the National Science Foundation (NSF), but also from the type of research that was applied to Phytophthora for the first time. New and exciting cutting edge technologies were successfully used in the study of different Phytophthora species, including microarray analysis, siRNA gene silencing, and Solexa transcriptome analysis. The first Phytophthora species to have their genomes sequenced were P. sojae and P. ramorum at 2004. Since then the genomes of two more Phytophthora species- P. capsici and P. infestans, were also sequenced. In fact, P. capsici was the first eukaryotic organism to be sequenced using the traditional Sanger sequencing method in combination with the new 454 pyrosequencing platform. Availability of Phytophthora genome sequences provided us with the basis necessary for a proteomic investigation of these organisms. The study presented here represents the first large scale proteomic study of any Phytophthora species. Using mass spectrometry and available or newly developed bioinformatic tools we measured the proteomes of different asexual Phytophthora life stages. We also measured the protein complement of P. capsici infected tomato plants, the so called “interactome”, in order to gain an insight into the biological processes occurring in the pathogen during infection, and in the plant in response to the pathogen. We also used data from these proteomic experiments as a part of a novel approach aimed at improving the genome annotation of those Phytophthora species. Finally, we used different molecular techniques, including a reverse genetic technique called Targeted Induced Local Lesions in Genome (TILLING), to begin to characterize some of the protein targets identified in those experiments. The accumulated data from all our experiments identified certain molecular processes, metabolic and others, that may explain the success of Phytophthora as a plant pathogen. The data from these experiments provides a platform on which future experiments can be based on to further characterize these interesting organisms.