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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 σ⁵⁴ promoters and an hrp 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.