title: Molecular characterization and expression of the Erwinia carotovora hrpNEcc gene, which encodes an elicitor of the hypersensitive reaction.
abstract: 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.
authors.affiliation: Department of Plant Pathology, University of Missouri, Columbia 65211, USA.
authors.forename: A
authors.initials: A
authors.lastname: Mukherjee
authors.forename: Y
authors.initials: Y
authors.lastname: Cui
authors.forename: Y
authors.initials: Y
authors.lastname: Liu
authors.forename: A K
authors.initials: AK
authors.lastname: Chatterjee
mesh_terms: D015107:4-Butyrolactone / Q000031:analogs & derivatives / Q000378:metabolism; D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis / Q000235:genetics*; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D003470:Culture Media; D004885:Erwinia / Q000235:genetics* / Q000472:pathogenicity; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010935:Plant Diseases / Q000382:microbiology; D010947:Plants, Toxic; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins / Q000096:biosynthesis; D012097:Repressor Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana / Q000382:microbiology; D014158:Transcription, Genetic; D014675:Vegetables / Q000382:microbiology
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D003470:Culture Media; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; C095468:RsmA protein, Erwinia carotovora; C075462:harpin protein, Erwinia amylovora; C087295:HrpZ protein, Pseudomonas syringae; C030654:N-(3-oxohexanoyl)-3-aminodihydro-2(3H)-furanone; D015107:4-Butyrolactone