|Ibekwe, Abasiofiok - Mark|
Submitted to: Molecular Plant Microbe Interactions
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/3/2004
Publication Date: 7/20/2004
Citation: Yang, S., Perna, N.T., Coksey, D.A., Okinaka, Y., Lindow, S.E., Ibekwe, A.M., Keen, N.T., Yang, C.H. 2004. Genome-wide identification of plant-up-regulated genes of erwinia chrysanthemi 3937 using a gfp based ivet leaf array. Molecular Plant Microbe Interactions. 2004. 17(9):999-1008. Interpretive Summary: Microarray gene expression technology is one of the emerging technologies that can be used to identify gene expression profiles of organisms in different environments. However, several limitations, e.g., low amount of available RNA from target cells and the presence of contaminating organisms in samples, still deter the usage this modern technology. In this study we used green fluorescent protein based technology to identify certain genes that may be expressed in host or host-specific environment that may cause certain diseases. Many factors that may cause major diseases when plants are infected by bacteria are associated with the production of certain enzyme. Our data showed that bacteria can use very complex systems of communications and the mode of communications are highly affected by environmental factors such as temperature, pH, iron levels, growth phase, and population density.
Technical Abstract: A green fluorescent protein (GFP) based IVET (in vivo expression technology) leaf array was used to identify genes in Erwinia chrysanthemi 3937 that were specifically up-regulated in plants, as compared to growth in a laboratory culture medium. Sixty-one of 10,000 E. chrysanthemi 3937 clones were confirmed as plant up-regulated. 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%), transposases (2%); the function for the remainder (30%) is unknown. Up-regulated 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-up-regulated 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 (purM::Tn5), hrpB, hrcJ,and a hrpD homologs from the E. 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 a eutR (eutR::Tn5) showed decreased ability to cause systemic invasion on African violet. However, compared to the wild-type E. chrysanthemi 3937, these mutants exhibited no significant differences in local leaf maceration. The phenotype of hrpB::Tn 5, hrcC::Tn5 and hrpD::Tn5 mutants further confirmed our previous findings that hrp genes are crucial virulence determinants in E. chrysanthemi 3937.