Investigation of Pantoea stewartii Quorum-Sensing Controlled Regulators and Genes Important for Infection of Corn

Bacteria interact with their eukaryotic hosts using a variety of mechanisms that range from being beneficial to detrimental. This dissertation focuses on Pantoea stewartii subspecies stewartii (P. stewartii), an endosymbiont in the corn flea beetle gut that causes Stewart's wilt disease in corn. Gaining insights into the interactions occurring between this bacterial pathogen and its plant host may lead to informed intervention strategies. This phytopathogen uses quorum sensing (QS) to coordinate cell density-dependent gene expression and successfully colonize corn leading to wilt disease. Prior to the research presented in this dissertation, the QS master regulator EsaR was shown to regulate two major virulence factors of P. stewartii, capsule production and surface motility. However, the function and integration of EsaR downstream targets in P. stewartii were still largely undefined. Moreover, only a draft genome of a reference strain of P. stewartii was publicly available for researchers, limiting bioinformatics and genome-scale genetic approaches with the organism. The work described in this dissertation has now addressed these important issues.

The function of two EsaR direct targets, LrhA and RcsA, was explored (Chapter Two) and the existence of integration in the regulation between them was discovered (Chapters Two and Four). RcsA and LrhA are transcription factors controlling capsule production and surface motility in P. stewartii, respectively. In Chapter Two, the RcsA and LrhA regulons were investigated using RNA-Seq. This led to the discovery of a potential regulatory interaction between them that was confirmed by qRT-PCR and transcriptional gene fusion assays. The involvement of LrhA in surface motility and virulence was also established in this project. A direct interaction between LrhA and promoter of rcsA was defined in Chapter Four. Additional direct regulatory targets of LrhA were also identified.

A project to generate a complete assembly of the P. stewartii genome (Chapter Three) enabled more thorough genome-wide analysis and revealed the existence of a previous unknown 66-kb region in the P. stewartii genome believed to contain genes important for motility and virulence. In addition, completion of the genome sequence permitted genes for two distinctive Type III secretion systems, used for interactions with corn or the corn flea beetle, to be placed on two mega-plasmids. Furthermore, the complete genome sequence facilitated a Tn-Seq approach (Chapter Five). Tn-Seq is a potent tool used to identify bacterial genes required for certain environmental test conditions. This project is a pioneering utilization of a Tn-Seq analysis in planta to investigate genes important for colonization and survival of P. stewartii within its corn host. It was discovered that OmpC and Lon are important to in planta growth and OmpA plays a role in plant virulence.

In conclusion, these studies have broadened our understanding about the role of the QS regulon and other genes important for the pathogenesis of this phytopathogen. This knowledge may now be applied toward the development of future disease intervention strategies against P. stewartii and other wilt-disease causing plant pathogens.