Publication no. M-2004-0701-01R

Genome-Wide Identification of Plant-Upregulated Genes of Erwinia chrysanthemi 3937 Using a GFP-Based IVET Leaf Array. Shihui Yang (1), Nicole T. Perna (2), Donald A. Cooksey (1), Yasushi Okinaka (3), Steven E. Lindow (4), A. Mark Ibekwe (5), Noel T. Keen (1), and Ching-Hong Yang (6). (1) Department of Plant Pathology, University of California, Riverside 92521, U.S.A.; (2) Department of Animal Health and Biomedical Sciences, University of Wisconsin-Madison 53706, U.S.A.; (3) Graduate School of Biosphere Science, Hiroshima University, Kagamiyama 1-4-4, Higashi-hiroshima, 739-8528, Japan; (4) Department of Plant and Microbial Biology, University of California, Berkeley 94720, U.S.A.; (5) United States Department of Agriculture–Agricultural Research Service George E. Brown Jr. Salinity Lab., Riverside, CA 92507, U.S.A., (6) Department of Biological Sciences, University of Wisconsin-Milwaukee 53211, U.S.A. MPMI 17:999-1008. Submitted 22 December 2003. Accepted 3 May 2004. Copyright 2004 The American Phytopathological Society.

A green fluorescent protein-based in vivo expression technology leaf array was used to identify genes in Erwinia chrysanthemi 3937 that were specifically upregulated in plants compared with growth in a laboratory culture medium. Of 10,000 E. chrysanthemi 3937 clones, 61 were confirmed as plant upregulated. On the basis of sequence similarity, these were recognized with probable functions in metabolism (20%), information transfer (15%), regulation (11%), transport (11%), cell processes (11%), and transposases (2%); the function for the remainder (30%) is unknown. Upregulated genes included transcriptional regulators, iron uptake systems, chemotaxis components, transporters, stress response genes, and several already known or new putative virulence factors. Ten independent mutants were constructed by insertions in these plant-upregulated genes and flanking genes. Two different virulence assays, local leaf maceration and systemic invasion in African violet, were used to evaluate these mutants. Among these, mutants of a purM homolog from Escherichia coli (purM::Tn5), and hrpB, hrcJ, and a hrpD homologs from the Erwinia carotovorum hrpA operon (hrpB::Tn5, hrcJ::Tn5, and hrpD::Tn5) exhibited reduced abilities to produce local and systemic maceration of the plant host. Mutants of rhiT from E. chrysanthemi (rhiT:: Tn5), and an eutR homolog from Salmonella typhimurium (eutR::Tn5) showed decreased ability to cause systemic inva sion on African violet. However, compared with the wild-type E. chrysanthemi 3937, these mutants exhibited no significant differences in local leaf maceration. The pheno type of hrpB::Tn5, hrcC::Tn5, and hrpD::Tn5 mutants further confirmed our previous findings that hrp genes are crucial virulence determinants in E. chrysanthemi 3937. Additional keywords: oxidative stress, phosphotransferase system, transport of oligogalacturonides, type III secretion system.