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 de ficient in the production of the pectate lyase isozymes PelABCE can el icit the hypersensitive response (HR) in tobacco leaves, The hrpN(Ech) gene was identified in a collection of cosmids carrying E, chrysanthe mi hrp genes by its hybridization with the Erwinia amylovora hrpN(Ea) gene. hTpN(Ech) appears to be in a monocistronic operon, and it encode s 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. E scherichia coli DH5 alpha cells expressing hrpN(Ech) from the lac prom oter of pBluescript II accumulated HrpN(Ech) in inclusion bodies, The protein was readily purified from cell lysates carrying these inclusio n 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, T n5-gusA1 mutations were introduced into the cloned hrpN(Ech) and then marker-exchanged into the genomes of E, chrysanthemi strains AC4150 (w ild type), CUCPB5006 (Delta pelABCE), and CUCPB5030 (Delta pelABCE out D : :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 muta tion also reduced the ability of AC4150 to incite infections in witloo f chicory leaves, but it did not reduce the size of lesions that did d evelop, Purified HrpN(Ech) and E chrysanthemi strains CUCPB5006 and CU CPB5030 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, chrys anthemi EC16, and it contributes to the pathogenicity of the bacterium in witloof chicory.