Abstract
Pseudomonas syringae pv. syringae B728a, causal agent of brown spot on bean, is an economically important plant pathogen that utilizes extracellular signaling to initiate a lifestyle change from an epiphyte to a pathogen. LuxR regulatory proteins play an important role in the transcriptional regulation of a variety of biological processes involving two-component signaling, quorum sensing, and secondary metabolism. Analysis of the B728a genome identified 24 LuxR-like proteins, three of which are encoded by salA, syrF, and syrG located adjacent to the syringomycin gene cluster. The LuxR-like proteins encoded by these three genes exhibit a domain architecture that places them in a subfamily of LuxR-like proteins associated with regulation of secondary metabolism in B728a. Deletion mutants of salA, syrF, and syrG failed to produce syringomycin and displayed reduction of virulence on bean. The transcriptional start sites of salA, syrG, and syrF were located 63, 235, and 498 bp upstream of the start codons, respectively, using primer extension analysis. The predicted -10/-35 promoter regions of syrF and syrG were confirmed using site-directed mutagenesis and GFP reporters that showed conserved promoter sequences around the -35 promoter region. Overexpression analysis and GFP reporters identified SyrG as an upstream transcriptional activator of syrF, where both SyrG and SyrF activate promoters of syringomycin biosynthesis genes. This study shows that syrG and syrF encode important transcriptional regulators of syringomycin biosynthesis genes.
Highlights
P. syringae pv. syringae (Pss) B728a [1, 2] is an aggressive plant pathogen of bean that causes brown spot
It was observed that all three regulatory proteins are exclusively found in P. syringae genomospecies 2, with the C-terminal region being highly conserved with regulatory protein SyrG (S1 Fig)
Homologs of these LuxR-like proteins were found in the genome of Pss B301D displaying a high degree of sequence conservation compared to Pss B728a
Summary
P. syringae pv. syringae (Pss) B728a [1, 2] is an aggressive plant pathogen of bean that causes brown spot. The proteins that are encoded by salA (Psyr_2601), sylA (Psyr_1702), syrG (Psyr_2602), syrF (Psyr_2607), syrR (Psyr_2575), Psyr_2045, Psyr_2578, Psyr_3767, Psyr_4266, and Psyr_4278 exhibit domain architecture that is typical of the fourth subfamily of LuxR proteins, which is the second largest group of LuxRs found in the Pss B728a genome These transcriptional regulators seemingly play a key role in the regulation of genes associated with secondary metabolism, pathogenicity, and virulence of Pss B728a. It is hypothesized that the LuxR-like protein SyrG is involved in the regulation of genes essential for the pathogenic lifestyle of Pss B728a while under transcriptional control of SalA This hypothesis was tested utilizing phenotypic characterization and quantitative real-time PCR analysis in an effort to identify new components of the SyrG regulon. It is important to further define these transcriptional regulators in order to fully understand the complex nature of SalA, SyrG, and SyrF regulons in regards to virulence and the plant-pathogen interaction
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