Characterization of a Novel Negative Regulator of Toxoflavin Production, <i>ntpR</i>, in the Plant Pathogen <i>Burkholderia glumae</i> that Causes Bacterial Panicle Blight of Rice

Abstract

Burkholderia glumae is the major causal agent of bacterial panicle blight of rice, a disease that can cause significant yield losses. This bacterial pathogen produces a number of virulence factors that contribute to disease development. The phytotoxin, toxoflavin is required for full virulence and is the most important virulence factor in B. glumae. The known regulatory cascade of toxoflavin production involves the TofI-TofR quorum-sensing system, the transcriptional activators ToxJ and ToxR, and the toxoflavin biosynthesis and transport genes. Transposon mutagenesis of toxoflavin-producing B. glumae strains generated a number of mutants with increased toxoflavin production. Several of these mutants contained independent insertions in a gene encoding a putative LysR-type transcriptional regulator. This gene, named ntpR for negative regulator of toxoflavin production LysR, was confirmed to be a negative regulator of toxoflavin production since these and other generated ntpR mutants produced significantly more toxoflavin than their parent strains. ntpR mutants also produced less EPS and lipase and were more virulent on rice than their parent strains suggesting that ntpR also regulates EPS and lipase production and virulence on rice. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and RNA sequencing (RNA-Seq) and comparative transcriptome analyses indicated that ntpR regulation likely occurs at the last step in the known regulatory cascade of toxoflavin production; however, it is not known if this regulation is direct or indirect. RNA-Seq analyses also identified 96 genes that were differentially expressed in an ntpR mutant compared to its parent strain, including other transcriptional regulators that may be regulated by ntpR. The collective results from these studies are significant in that a novel regulatory component in B. glumae, ntpR, that influences toxoflavin, lipase, and EPS production, was identified. In addition, the probable location of ntpR regulation within the known regulatory cascade of toxoflavin production and a large number of genes that are also potentially regulated by ntpR were identified. These findings add to our knowledge of the currently known regulatory mechanisms of toxoflavin production and of the regulatory mechanisms in B. glumae.