Identification of regulators of hrp/hop genes of Erwinia carotovora ssp. carotovora and characterization of HrpLEcc (SigmaLEcc), an alternative sigma factor

Abstract

Summary Erwinia carotovora ssp. carotovora (Ecc) possesses hrpN(Ecc)[hrp = gene for hypersensitive reaction (HR) and pathogenicity], the structural gene for Harpin(Ecc), the inducer of the HR-like response and genes for the type III secretion system. In Ecc, RsmA, an RNA-binding protein responsible for the accelerated decay of RNA species, tightly controls the expression of the Hrp regulon. We document here that Ecc strain 71 possesses several presumed Hrp regulators: HrpX(Ecc), HrpY(Ecc), HrpS(Ecc), and HrpL(Ecc). Nucleotide sequence data indicate that the regulatory genes occur as a cluster. The data also suggest that HrpX(Ecc) is a putative sensor kinase, HrpY(Ecc) is the cognate response regulator, and HrpS(Ecc) is an NtrC-like regulator. hrpL(Ecc) consists of a 543-bp open reading frame (ORF), which encodes a c. 21-kDa protein product. This protein shares significant homology with members of the extracytoplasmic function (ECF) subfamily of alternative sigma factors, including SigE. To examine the regulatory role of hrpL(Ecc), we constructed by marker exchange an HrpL(Ecc) (-) derivative of an RsmA(-) strain. The levels of transcripts of hrcC(Ecc), a gene for the type III secretion system, and hrpN(Ecc) were lower in the RsmA(-)HrpL(Ecc) (-) mutant compared to its RsmA(-)HrpL(Ecc) (+) parent. The RsmA(-)HrpL(Ecc) (-) strain, like the RsmA(-)HrpL(Ecc) (+) parent, caused maceration of celery petioles and produced extracellular pectinases, cellulase, and protease, indicating that the alternative sigma factor is not required for the type I and type II secretion systems, for tissue maceration, or for the production of proteins secreted by these pathways. However, the RsmA(-)HrpL(Ecc) (-) strain, unlike the RsmA(-)HrpL(Ecc) (+) strain, did not elicit the HR in tobacco leaves. In addition, the RsmA(-)HrpL(-) strain failed to cause symptoms in Arabidopsis thaliana. Genetic and biochemical data revealed that Harpin(Ecc) is required for symptom production in Arabidopsis, but is not sufficient by itself to cause necrosis. These observations raise the possibility that another 'effector' protein of Ecc, secreted by the type III system, acts in conjunction with Harpin(Ecc) to trigger responses resulting in cell death in Arabidopsis.