Identification of virulence factors is a necessary first step to study the mechanisms and evolution of pathogenicity. We propose to compare the type III disease effector proteins from a phylogenetically diverse set of plant pathogenic Pseudomonads chosen to represent the most broad distribution of the species possible. P. syringae is an ideal model organism to study the distribution and evolution of type III effectors. Type III effectors interact with cellular host targets, and modulate host defense responses or metabolism in a manner conducive to pathogen proliferation. Most of the P. syringae effectors have not yet been characterized, and we remain naive as to the collective diversity of host cellular functions they manipulate. Although the functions of P. syringae effector proteins during disease remain poorly understood, they can often be monitored via their phenotypes following in planta expression of a given type III effector. Bacterial pathogens of both animals, such as Salmonella spp., Yersinia spp., Shigella spp. and pathogenic E. coli also rely on type III secretion systems for pathogenesis. Thus, our results will inform studies of these bacteria and their animal hosts, including humans. Because P. syringae is pathogenic on a variety of distantly related plant hosts, many of which can be genetically manipulated, our system has advantages over models of type III pathogenesis of animals, which generally focus only on strains pathogenic on phylogenetically related mammalian hosts. We have devised and implemented a high throughput Fluorescence Activated Cell Sorter (FACS) based experimental approach to capture all of the type III effectors from the genome of any given P. syringae isolate. We chose 13 P syringae isolates that are pathogens of an evolutionarily diverse set of host plants. We intend to sieve through these 13 genomes to describe their suites of type III effectors. We further intend to begin dissection of their effects on host cell biology, using the easily manipulated Arabidopsis plant as a model where appropriate. This proposal is highly interdisciplinary, drawing on methodologies and expertise in microbiology, bioinformatics and plant-pathogen interactions in conjunction with high throughput bacterial cell sorting using the FACS. Our work will broadly impact the understanding of a widely distributed pathogenicity mechanism that affects both human health and agriculture. Additionally, our work may have relevance in biodefense with respect to basic understanding of pathogenesis in bacteria that can potentially be weaponized.
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