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
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 σ54 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.
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