Regulation of Long-Chain N -Acyl-Homoserine Lactones in Agrobacterium vitis

Homologs of quorum-sensing luxR and luxI regulatory genes, avsR and avsI , were identified in Agrobacterium vitis strain F2/5. Compared to other LuxI proteins from related species, the deduced AvsI shows the greatest identity to SinI (71%) from Sinorhizobium meliloti Rm1021. AvsR possesses characteristic autoinducer binding and helix-turn-helix DNA binding domains and shares a high level of identity with SinR (38%) from Rm1021. Site-directed mutagenesis of avsR and avsI was performed, and both genes are essential for hypersensitive-like response (HR) and necrosis. Two hypothetical proteins (ORF1 and ORF2) that are positioned downstream of avsR-avsI are also essential for the phenotypes. Profiles of N -acyl-homoserine lactones (AHLs) isolated from the wild type and mutants revealed that disruption of avsI , ORF1, or ORF2 abolished the production of long-chain AHLs. Disruption of avsR reduces long-chain AHLs. Expression of a cloned avsI gene in A. tumefaciens strain NT1 resulted in synthesis of long-chain AHLs. The necrosis and HR phenotypes of the avsI and avsR mutants were fully complemented with cloned avsI . The addition of synthetic AHLs (C 16:1 and 3-O-C 16:1 ) complemented grape necrosis in the avsR , avsI , ORF1, and ORF2 mutants. It was determined by reverse transcriptase PCR that the expression level of avsI is regulated by avsR but not by aviR or avhR , two other luxR homologs which were previously shown to be associated with induction of a tobacco hypersensitive response and grape necrosis. We further verified that avsR regulates avsI by measuring the expression of an avsI :: lacZ fusion construct.