Molecular characterization and expression of the Erwinia carotovora hrpNEcc gene, which encodes an elicitor of the hypersensitive reaction.

The nucleotide sequence of hrpNEcc DNA, cloned from Erwinia carotovora subsp. carotovora strain Ecc71, reveals a coding region of 1,068 bp which matches the size of hrpNEcc transcripts. hrpNEcc is predicted to encode a glycine-rich protein of approximately 36 kDa. Like the elicitors of the hypersensitive reaction (HR) produced by E. chrysanthemi (HarpinEch) and E. amylovora (HarpinEa), 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 hrpNEcc, the 36-kDa protein has been identified as the hrpNEcc product by Western blot analysis using anti-HarpinEch 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 hrpNEcc encodes the elicitor of the HR, designated HarpinEcc. The levels of hrpNEcc 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 hrpNEcc in Ecc71 is cell density dependent and is activated by the quorum-sensing signal, N-(3-oxohexanoyl)-L-homoserine lactone (OHL). By contrast, hrpNEcc 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 hrp promoter upstream of the transcriptional start site, indicate that the production of HarpinEcc 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 HarpinEcc production.