New approaches to access the P. syringae transcriptome in planta.
B. KVITKO (1), A. Lovelace (1), A. Smith (1) (1) University of Georgia, U.S.A.

Detailed characterization of P. syringae genetic regulation in planta has been difficult to achieve partially due to the vast differences in the ratio of pathogen to host RNA. We have developed two complementary strategies to facilitate in planta P. syringae transcriptional analysis. For measuring the global P. syringae transcriptome, we have developed an approach in which bacteria are physically separated from leaf tissue in an mRNA stabilizing buffer. This allows us to retain the integrity of the in planta bacterial transcriptome while vastly reducing the amount of recovered host RNA. The recovered P. syringae mRNA is then sequenced to determine alterations in gene expression under various test conditions. To support our global RNAseq approach with we are also optimizing a QRT strategy for use with inoculated leaf tissue. In our strategy, host-derived bacterial RNA concentrations are determined using P. syringae-specific 16s primers, and test gene expression levels are then measured against a panel of rigorously validated reference genes.  These strategies will facilitate the facile and robust analysis of P. syringae transcriptional regulation under a variety of conditions and provide us with a clearer understanding of pathogen-host interactions.

Abstract Number: P6-145
Session Type: Poster