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Institute of Agricultural & Environmental Research Tennessee State University |
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Seminar Series Abstract
Genetic Analysis of Soft rot Pathogenesis Korsi Dumenyo and Paul Agyemang Tennessee State University Institute of Agricultural & Environmental Research The soft rot Erwinia, mainly comprising E. carotovora subspecies, E. chrysanthemi cause soft rot disease in many plants and blackleg disease of potato. These gram negative bacteria produce and secret a battery of extracellular plant cell wall degrading enzymes with which they macerate the host tissues. These enzymes are isoforms of pectinases including pectate lyase (Pel), polygalacturonase, (Peh), Pectin lyase, (Pnl), Pectin methyl esterase (Pme); and Cellulases (Cel), and Protease (Prt). The expression of some of these genes along with some regulatory genes is regulated by host extracts. An array of bacterial genes has been reported to be expressed in response to host signals. However, the full catalogue of genes, involved in the pathogenesis of soft rot Erwinia is unknown. In the published genome data of E. carotovora subspecies atroseptica, over 27% (1232 out of 4491) of protein coding genes are predicted to encode hypothetical proteins (proteins of unknown functions) and some of these could be involved in pathogenesis as well. Transposon mutagenesis, in which transposable elements are randomly inserted in many points in the bacterial chromosome, is a powerful and successful strategy used for genetic analysis of bacteria. We have been using transposon mutagenesis to study pathogenicity in E. carotovora subsp. carotovora (Ecc) strain 71. Our objective is to identify and characterize virulence genes of Ecc. AC5006N3, a spontaneous nalidixic acid resistant mutant of Ecc strain AC5006 (lacZ) was isolated and mutagenized using E. coli S17-1λpir as the donor of mini-Tn5 Km containing promoter-less lacZ reporter gene. The kanamycin- and nalidixic acid-resistant transconjugants were screened for deficiency and overproduction of extracellular protease on nutrient-gelatin agar medium. Three protease hyper producing mutants and four deficient mutants were selected and are being studied. The mutants are being analyzed for their levels of other extracellular enzymes, tissue maceration, expression of LacZ reporter and response to host signals. The altered levels of extracellular enzymes in these mutants suggest they have transposon insertion in genes that regulate extracellular enzyme production and possibly pathogenicity. Understanding the roles of these genes in pathogenesis could lead to better soft rot disease management.
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