ERWINIA-CHRYSANTHEMI HARPIN(ECH) - AN ELICITOR OF THE HYPERSENSITIVE RESPONSE THAT CONTRIBUTES TO SOFT-ROT PATHOGENESIS

Mutants of the soft-rot pathogen Erwinia chrysanthemi EC16 that are deficient in the production of the pectate lyase isozymes PelABCE can elicit the hypersensitive response (HR) in tobacco leaves, The hrpN(Ech) gene was identified in a collection of cosmids carrying E, chrysanthemi hrp genes by its hybridization with the Erwinia amylovora hrpN(Ea) gene. hTpN(Ech) appears to be in a monocistronic operon, and it encodes a predicted protein of 340 amino acids that is glycine-rich, lacking in cysteine, and highly similar to HrpN(Ea) in its C-terminal half. Escherichia coli DH5 alpha cells expressing hrpN(Ech) from the lac promoter of pBluescript II accumulated HrpN(Ech) in inclusion bodies, The protein was readily purified from cell lysates carrying these inclusion bodies by solubilization in 4.5 M guanidine-HC1 and reprecipitation upon dialysis against dilute buffer, HrpN(Ech) suspensions elicited a typical HR in tobacco leaves, and elicitor activity was heat-stable, Tn5-gusA1 mutations were introduced into the cloned hrpN(Ech) and then marker-exchanged into the genomes of E, chrysanthemi strains AC4150 (wild type), CUCPB5006 (Delta pelABCE), and CUCPB5030 (Delta pelABCE outD : :TnphoA), hrpN(ech) :: Tn5-gusA1 mutations in CUCPB5006 abolished the ability of the bacterium to elicit the HR in tobacco leaves unless complemented with an hrpN(Ech) subclone, An hrpN(Ech)::Tn5-gusA1 mutation also reduced the ability of AC4150 to incite infections in witloof chicory leaves, but it did not reduce the size of lesions that did develop, Purified HrpN(Ech) and E chrysanthemi strains CUCPB5006 and CUCPB5030 elicited HR-like necrosis in leaves of tomato, pepper, African violet, petunia, and pelargonium, whereas hrpN(Ech) mutants did not, HrpN(Ech) thus appears to be the only HR elicitor produced by E, chrysanthemi EC16, and it contributes to the pathogenicity of the bacterium in witloof chicory.