[Type III secretion system in Se9R can recognize and secret harpin in Erwinia amylovora]

Bacterial pathogens of both animals and plants use type III secretion machines to secret virulence proteins. The signal that leads to tye type III secretion is encoded as stem loop structure in the messenger RNA. Harpin, produced by Erwinia anylovora is secreted via type III secretion. Erwinia carotovora Se9R and Erwinia amylovora belong to the same genus and they use type III secretion machines to secret virulence proteins. We used PCR to amplify hrpN from pCPP430 which harbors hrpN gene cluster, and cloned it to pGEM-T vector to get pWGF1, which was then chemically transformed into DH5 alpha (pCPP430hrpN-) and electrotransformed into Se9R. DH5 alpha (pCPP430hrpN-/pWGF1) induced hypersensitive response on tomato leaf and Se9R(pWGF1) showed significantly reduced virulence than Se9R on Chinese cabbage leaf. Western-blotting analysis of the CFEP of Se9R(pWGF1) showed production of harpin protein. These results suggest the type III secretion machine in Se9R can recognize secretion singal in the gene of harpin in Erwinia amylovora and secret it as a biologically native form.