Analysis of the in planta Transcriptome Expressed by the Corn Pathogen Pantoea stewartii subsp. stewartii via RNA-Seq

Pantoea stewartii subsp. stewartii is a bacterial phytopathogen that causes Stewart's wilt disease in corn. It uses quorum sensing to regulate expression of some genes involved in virulence in a cell density-dependent manner as the bacterial population grows from small numbers at the initial infection site in the leaf apoplast to high cell numbers in the xylem where it forms a biofilm. There are also other genes important for pathogenesis not under quorum-sensing control such as a Type III secretion system. The purpose of this study was to compare gene expression during a high-density in planta infection versus a low-density pre-inoculum liquid culture and in a high-density culture grown on agar medium to identify genes specifically expressed in planta that may also be important for colonization and/or virulence. RNA was purified from each sample type to determine the transcriptome via RNA-Seq using Illumina sequencing of cDNA. Fold gene expression changes in the high-density in planta data set in comparison to the two in vitro grown samples were determined and a list of the most differentially expressed genes was generated to elucidate genes important for plant association. Quantitative reverse transcription PCR (qRT-PCR) was used to validate expression patterns for a select subset of genes. Analysis of the transcriptome data via gene ontology revealed that bacterial transporters and systems active under low oxygen tensions appear to play a critical role for P. stewartii as it colonizes and causes wilt disease in corn plants. Overall design: Duplicate samples of wild-type P. stewartii were obtained for RNA-Seq after a 10 day in planta corn infection, pre-inoculum liquid culture in RM medium, and a high cell density plate culture