Identification of dspEF, hrpW, and hrpN loci and characterization of the hrpNEp gene in Erwinia pyrifoliae

초록

Erwinia pyrifoliae, the causal pathogen of shoot blight in the Asian pear tree (Pyrus pyrifolia cv. Singo), is host-specific and endemic to Korea. To identify the genes associated with the hypersensitive response (HR) and pathogenicity, a genomic library of E. pyrifoliae WT3 was constructed, and the cosmid clone Escherichia coli (pCEP33) was selected. Sequence analysis of 19.7-kb pCEP33 determined disease-specific (dsp) region homolog and approximately 40% of the hrp genes, which included hrpW, hrpN<inf>Ep</inf>, hrpV, hrpT, hrcC, hrpG, hrpF, and partial hrpE homologs, with respect to the cluster of Erwinia amylovora. Additionally, two open reading frames, ORFD and ORFE, were found downstream of the dspEF region. The results of the sequence analysis showed that the pCEP33 did not contain any hrp regulatory genes or most of the genes encoding components of the Hrp protein secretion system. The hrpN<inf>Ep</inf> gene of E. pyrifoliae contained five intergenic nucleotide fragment insertions (INFIs) and produced the HR elicitor protein harpin <inf>Ep</inf>, with a molecular mass of approximately 44kDa. The purified HrpN<inf>Ep</inf> protein elicited faster and stronger HR when infiltrated into tobacco leaves than did HrpN<inf>Ea</inf> from E. amylovora. To observe the role of the hrpL gene in the expression of HrpN<inf>Ep</inf>, the pEL2 containing hrpL was used to transform E. coli (pCEP33). Expression of HrpN<inf>Ep</inf> in E. coli (pCEP33 + pEPL2) was detected with an immunoblot using antiserum raised against HrpN<inf>Ep</inf>, indicating a role of hrpL gene in enhancing the expression of HrpN<inf>Ep</inf>. © The Phytopathological Society of Japan and Springer-Verlag 2005.

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