UC Davis

Both plants and animals are impacted by diverse biotic threats. To limit disease, plants use protein receptors to recognize and respond to pathogen protein epitopes or effectors. Pathogens have evolved strategies to circumvent recognition to proliferate and cause disease. Pathogens can also persist on non-hosts, leading to reservoir populations and subsequent costly outbreaks. Despite considerable resources focused on understanding the interactions between pathogens and model organisms, we lack considerable knowledge in how the natural diversity of bacterial pathogens, particularly Gram-positive actinobacteria, impact plant immune perception, colonization, and disease susceptibility. Using a combination of comparative genomics, genetics, and biochemistry, I leveraged natural genetic variation to understand the evolution of pathogen epitopes and elucidate a driver of pathogen evasion in a Gram-positive actinobacteria. Pathogen recognition and receptor signaling is crucial in host-pathogen interactions, but most studies use a single pathogen epitope and thus, the impact of multi-copy epitopes on pathogen outcomes is unknown. Through comparative genomics of thousands of plant-associated bacterial genomes, I characterized the naturally-evolved bacterial epitope landscape and their impact on pathogen outcomes. I revealed that natural variation was constrained yet experimentally testable and both epitope sequence and copy number variation altered pathogen-immune outcomes. Through genetic and biochemical analyses, I uncovered a mechanism for pathogen immune evasion, intrabacterial antagonism, where a non-immunogenic epitope blocks perception of immunogenic forms encoded in a single genome. One such intrabacterial antagonist, cold shock protein CspB, was conserved in actinobacteria including Clavibacter, a genus comprised of several crop pathogens including tomato, potato, wheat, and corn. As a non-model system, I developed a genetic toolkit to manipulate Clavibacter and test the role of CspB in blocking immune perception of one host species, tomato. While I was able to build and validate the genetic tools through deletion of several critical virulence genes, I was unable to generate a null mutant of the cspB gene in C. michiganensis, likely due to its high GC-content between 73-78%. Instead, I validated our intrabacterial antagonism model though a combination of biochemical assays and genetic transfer of cspB to another foliar pathogen of tomato, Pseudomonas syringae pathovar tomato DC3000. I show via bacterial titers that expression of antagonist cspB blocked perception of other native encoded immunogenic cold shock proteins in a receptor-dependent manner. Collectively, I revealed a mechanism for immune evasion and showcased the importance of analyzing all epitope copies within a genome. I also provided evidence that Gram-positive actinobacteria interface with the plant immune system, a paradigm previously put into question due to insufficient evidence. Finally, I developed a genetic toolkit which may aid in characterizing other genotypic-phenotypic outcomes in the non-model bacterium. While my research has shown that we can leverage natural genetic variation to generate hypotheses and understand their impact on phenotypic outcomes, major questions remain in the evolution, functional biology, and signaling in plant-microbe interactions, which is addressed in the final chapter. Findings from the research questions posed may provide critical insights for subsequent advancements in bioengineering for disease resistance.