Biosynthesis of the phytotoxin coronatine (COR) in Pseudomonas syringae pv. glycinea PG4180 is regulated by temperature at the transcriptional level. A 3.4-kb DNA fragment from the COR biosynthetic gene cluster restored temperature-regulated phytotoxin production to Tn5 mutants defective in COR production. Nucleotide sequence analysis of this fragment revealed three genes, corS, corP, and corR, which encode a modified two-component regulatory system consisting of one sensor protein, CorS, and two response regulator proteins, CorP and CorR. Although only one response regulator, CorR, had a DNA-binding domain, the phosphatereceiving domains of both response regulator proteins were highly conserved. Transcriptional fusions of the corP and corR promoters to a promoterless glucuronidase gene (uidA) indicated that these two genes are expressed constitutively at 18 and 28؇C. In contrast, a corS::uidA fusion exhibited the temperature dependence previously observed for COR biosynthetic promoters and exhibited maximal transcriptional activity at 18؇C and low activity at 28؇C. Furthermore, glucuronidase activity for corS::uidA was decreased in corP, corR, and corS mutants relative to the levels observed for PG4180(corS::uidA). This difference was not observed for corP::uidA and corR::uidA transcriptional fusions since expression of these fusions remained low and constitutive regardless of the genetic background. The three regulatory genes functioned in a P. syringae strain lacking the COR gene cluster to achieve temperature-dependent activation of an introduced COR biosynthetic promoter, indicating that this triad of genes is the primary control for COR biosynthesis and responsible for thermoregulation. Our data suggest that the modified two-component regulatory system described in this study might transduce and amplify a temperature signal which results in transcriptional activation of COR biosynthetic genes.

We conclude that the full potential of RNAi in SPW is affected by the presence of nucleases. Therefore, for future application in crop protection, it is necessary constantly to provide new dsRNA and/or protect it against possible degradation in order to obtain a higher RNAi efficacy. © 2016 Society of Chemical Industry.

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The gene coding for the phaseolotoxin-insensitive ornithine carbamoyltransferase (OCTase) from Pseudomonas syringae pv. phaseolicola has been cloned and sequenced. The gene has a deduced coding capacity for a polypeptide with a calculated Mr of 36,520 daltons. Comparison of the amino acid sequence of the OCTase enzymes encoded by the P. aeruginosa argF and the Escherichia coli argI and argF genes with the deduced sequence of the newly identified gene shows that 79 amino acid residues are strictly conserved in all four polypeptides; among these 7 out of 9 residues are involved in enzyme function. Of three amino acid regions that have been implicated in substrate binding or catalysis, two are strictly conserved, and the third involved in carbamoylphosphate binding differs. This correlates well with published data showing that phaseolotoxin competes for the carbamoylphosphate binding site in the phaseolotoxin-sensitive OCTases. We propose that the gene be named argK.