Evaluation of the expression of water stress-responsive \u3ci\u3ePseudomonas syringae\u3c/i\u3e genes during plant infection and in the presence of low osmotic versus low matric potential in culture

The purpose of these studies was to characterize bacterial gene expression changes within the intercellular plant environment during infection with Pseudomonas syringae pv. tomato DC3000 as well as during water stress in culture. Our goals were to measure gene expression of the osmoresponsive proU promoter during early plant infection, and to measure specifically osmotically and matrically-induced genes during upshock with NaCl or the nonpermeating compound PEG8000 which sequesters water, at -1 MPa. To accomplish our first goal of measuring gene expression in planta we employed proU-inaZ and proU-uidA reporter gene fusions, both of which were found to be limited due to their dependence for normalization on the recovery of culturable bacterial cells. To overcome this limitation, direct measurement of bacterial RNA transcripts was made using quantitative RT-PCR (qRT-PCR). Use of this technique was predicated on successful extraction of sufficient bacterial RNA from infected plant leaves for qRT-PCR analysis. We developed and optimized bacterial RNA extraction from infected plants, and used this RNA in downstream expression measurements during an early infection timecourse of 0, 4, 6, and 8 hours after plant infection. We found that levels of osmoresponsive proU, opuC, and asnB transcripts increased in infected plants for both pathogenic and avirulent strains, but that water stress-responsive transcripts increased more for avirulent bacteria. This increase was maintained for proU , but opuC and asnB transcripts were transiently expressed. Bacteria respond to water stress in leaves, and this water stress may be sustained, as suggested by proU expression. Due to the nature of the opuC and asnB genes, which function in osmoprotectant uptake and synthesis, respectively, they may be transiently expressed until osmoadaptation occurs, when the need for additional osmoprotectant compounds decreases. This increased water stress in planta was associated with the timing of the plant cell hypersensitive response, induced by the introduction of the avirulence genes avrRpm1 and avrRpt2 into Col-O, but only by avrRpm1 in the Col-O mutant derivative ndr1-1. For our second goal, we studied in vitro adaptation to low water potential shocks due to osmotic or matric stress using microarrays. We contrasted expression of genes during these two types of water stress and found that expression of genes for osmoprotectant uptake and synthesis (OpuC, NAGGN, and Trehalose) were induced more by matric stress than osmotic stress. Water stress has been implicated in plant defense against avirulent pathogens, making study of the mechanisms of adaptation and gene expression interesting to further knowledge of how water availability changes contribute to plant defenses