Transcriptome of Erwinia Amylovora Signal Transduction Networks

NON-TECHNICAL SUMMARY: Fire blight, caused by a plant bacterial pathogen, is a particularly devastating disease for the apple and pear fruit industry, in large part because of lack of effective control measures driven by the development of resistance to streptomycin in the bacterial pathogen population. There are also significant continuing concerns over the potential impact of agricultural use of antibiotics on human health. Further, apple exports contribute to the health of the overall U.S. economy and have been very crucial to the apple industry. However, most U.S. apple cultivars are not allowed into Asian markets due to concerns over their susceptibility to this plant bacterial pathogen. In addition, this plant bacterial pathogen, which is closely related to mammalian enterobacteria such as Escherichia coli, could serve as a model for understanding the basic mechanisms of how bacteria cause disease that can lead to new insights into understanding mammalian pathogens. In bacterial pathogens, a group of genes, called two-component signal transduction systems (TCSTs), play critical roles in sensing and responding to environmental conditions and in virulence and, as such, are potential targets for antibacterial intervention strategies. Thus, understanding the genetics and molecular mechanisms of bacterial pathogenesis can be expected to greatly enhance the likelihood of developing novel methods of control. However, most TCST genes in this plant bacterial pathogen are currently not well understood. Further, genome-wide comprehensive studies involving TCST systems are relatively limited in plant pathogenic bacteria, and most importantly, comparative studies between plant and mammalian pathogens are lacking. Therefore, there is a critical need to understand the molecular mechanisms of bacterial pathogenesis and how this bacterial plant pathogen responds to its environment which, in turn will lead to the development of alternative control measures of fire blight. In this proposal, we will study a group of TCST genes in this bacterial plant pathogen which, based on our preliminary data, play important roles in virulence and survival. We will employ bacterial genetics, genomic and bioinformatics tools to dissect signal transduction events and determine which gene(s) are turned on or off by certain regulatory genes and how each gene is connected to each other. Critical target genes will be identified and further characterized. Our studies could eventually lead to alternative control procedures, thus reducing crop losses and enhancing profitability for farmers, and increasing food safety and security. Potential beneficiaries include apple growers, distributers, and ultimately, the consumers. Knowledge gained in this study might also be broadly applicable to other bacterial species and in control of animal/human diseases.

<P>

APPROACH: Currently, the most rapid progress in understanding of two-component signal transduction systems (TCSTs) is being made by combining traditional molecular genetic approaches with genome-scale analysis. Our studies are based on results of our preliminary data using genetic, functional genomic and bioinformatic approaches to infer key regulatory networks involved. We propose to apply these cutting-edge technologies to dissect the complex signaling networks on a genome-wide scale for one of the most important plant bacterial pathogens, Erwinia amylovora. To accomplish these goals, we have generated TCST mutants and will determine the role of TCST genes through virulence and phenotypic assays by comparing mutants and wild type strains, and identify target genes under stress conditions using an oligo-array containing all the E. amylovora genes. Gene expression data will be statistically analyzed and differentially regulated genes will be verified using qPCR or GFP-promoter assay by flow cytometry. These microarray data will then be used to establish gene regulatory networks (GRNs) using bioinformatics tools and computational modeling. This project is also designed to advance our understanding of TCSTs in related plant and mammalian pathogens through comparative genomics studies. Finally, selected critical target genes and their functions will be characterized and compared in both E. amylovora and Escherichia coli, thus extending our knowledge to two closely-related plant and mammalian microorganisms. This work will stimulate research in molecular pathogenesis and fundamental gene regulation studies. Research and education activities will be integrated into the proposed project through training of undergraduates, graduate students, and postdoctoral research associates. All findings will be disseminated through peer-reviewed journals and presentations at scientific conferences.