Molecular characterization and expression of the Erwinia carotovora hrpN(Ecc) gene, which encodes an elicitor of the hypersensitive reaction

The nucleotide sequence of hrpN(Ecc) DNA, cloned from Erwinia carotovora subsp. carotovora strain Ecc71, reveals a coding region of 1,068 bp which matches the size of hrpN(Ecc) transcripts, hrpN(Ecc) is predicted to encode a glycine-rich protein of approximately 36 kDa. Like the elicitors of the hypersensitive reaction (HR) produced by E. chrysanthemi (Harpin(Ech)) and E. amylovora (Harpin(Ea)), the deduced 36-kDa protein does not possess a typical signal sequence, but it contains a putative membrane-spanning domain, In Escherichia coli strains overexpressing hrpN(Ecc), the 36-kDa protein has been identified as the hrpN(Ecc) product by Western blot analysis using anti-Harpin(Ech) antibodies. The 36-kDa protein fractionated from E. coli elicits the HR in tobacco leaves. Moreover, a HrpN(-) and RsmA(-) double mutant (RsmA = regulator of secondary metabolites) does not produce this 36-kDa protein or elicit the HR, although this strain, like the RsmA(-) and HrpN(+) bacteria? overproduces extracellular enzymes and macerates celery petioles. These observations demonstrate that hrpN(Ecc) encodes the elicitor of the HR, designated Harpin(Ecc). The levels of hrpN(Ecc), transcripts are affected in both RsmA(+) and RsmA(-) strains by media composition and carbon sources, although the mRNA levels are substantially higher in the RsmA(-) strains, The expression of hrpN(Ecc) in Ecc71 is cell density dependent and is activated by the quorum-sensing signal, N-(3-oxohexanoyl)-L-homoserine lactone (OHL). By contrast, hrpN(Ecc) expression in an RsmA(-) strain is independent of cell density, and substantial expression occurs in the absence of OHL. The effects of cultural conditions and the occurrence of putative cis-acting sequences, such as consensus sigma(54) promoters and an hip promoter upstream of the transcriptional start site, indicate that the production of Harpin(Ecc), in wild-type RsmA(+) E. carotovora subsp. carotovora is tightly regulated. These observations, taken along with the finding that the HR is caused by RsmA(-) mutants but not by RsmA(+) strains (Cui et al., 1996, Mol. Plant-Microbe Interact, 9:565-573), strongly support the idea that the inability of the wild-type pectolytic E. carotovora subsp. carotovora to elicit the HR is due to the lack of a significant level of Harpin(Ecc) production.