This project concerns the mechanisms by which adaptation of bacterial plant pathogens to the host environment contributes to control of gene expression. The central hypothesis is that stress or nutrient limitation encountered by the bacterial pathogen upon host infection results in metabolic changes that in turn lead to expression of genes required for disease progression. Understanding the steps that lead to virulence gene expression is important for eventual control of disease progression in economically important plants. This program also focuses on integrating research and teaching by offering the opportunity for both graduate and undergraduate students to acquire research experience and proficiency in techniques associated with bacterial genetics and quantitative analysis of transcriptional regulation. Graduate students will be integral to developing these research projects. Undergraduate students will also contribute to this project, with special emphasis on increasing research access to members of underrepresented groups. The training of undergraduate students will not only prepare them for advanced studies, but contribute to the development of a competitive STEM workforce.
Bacterial plant pathogens encounter stress or nutrient limitation when they colonize host tissues. A "stringent response" ensues in which global changes in gene activity occur, favoring expression of genes that contribute to survival (for plant pathogens, this includes virulence genes). The stringent response depends on production of "alarmones", such as the phosphorylated guanosine nucleotides commonly referred-to as (p)ppGpp. This project addresses the hypothesis that purine salvage pathways are mobilized during the stringent response. The enzyme xanthine dehydrogenase (Xdh) is critical for purine salvage, and it biases the pathway towards formation of guanine nucleotides. The first aim of this project is to address the hypothesis that (p)ppGpp is a ligand for a transcription factor that represses the gene encoding Xdh in the soil bacterium Streptomyces coelicolor. Using both S. coelicolor and the plant pathogen Agrobacterium tumefaciens, the genes encoding (p)ppGpp synthetases will be inactivated to address the role of (p)ppGpp in xdh gene activity. DNA binding by purified transcription factors and the ability of (p)ppGpp to attenuate DNA binding will also be determined. Xdh is also responsible for producing urate, and another aim of this project is to address the hypothesis that urate produced during the stringent response in turn functions as a diffusible messenger to induce expression of genes under control of the transcription factor PecS, which was originally identified as a master regulator of virulence gene expression. Expression of pecS and other confirmed PecS target genes will be examined upon inactivation of Xdh enzyme activity as well as in strains deleted for the xdh gene to determine the role of urate as a ligand in vivo. Gene expression analyses in bacterial strains deleted for the pecS gene will also be implemented to compare the PecS regulons in A. tumefaciens and S. coelicolor. Thus, a fundamental hypothesis to be tested is that urate produced during the stringent response functions as a diffusible messenger and as a ligand for PecS, establishing a novel connection between the stringent response and the regulation of virulence genes by plant pathogens.
