Pseudomonas syringae Two-Component Response Regulator RhpR Regulates Promoters Carrying an Inverted Repeat Element

Abstract

The two-component system RhpRS was identified in Pseudomonas syringae as a regulator of the genes encoding the type III secretion system and type III effector proteins (together called the T3 genes). In the absence of the sensor kinase RhpS, the response regulator RhpR represses the induction of the T3 gene regulatory cascade consisting of hrpRS, hrpL, and the T3 genes in a phosphorylation-dependent manner. The repressor activity of RhpR is inhibited by RhpS, which presumably acts as a phosphatase under the T3 gene inducing conditions. Here, we show that RhpR binds and induces its own promoter in a phosphorylation-dependent manner. Deletion and mutagenesis analyses revealed an inverted repeat (IR) element, GTATC-N(6)-GATAC, in the rhpR promoter that confers the RhpR-dependent induction. Computational search of the P. syringae genomes for the putative IR elements and Northern blot analysis of the genes with a putative IR element in the promoter region uncovered five genes that were upregulated and two genes that were downregulated in an RhpR-dependent manner. Two genes that were strongly induced by RhpR were assayed for the IR element activity in gene regulation and, in both cases, the IR element mediated the RhpR-dependent gene induction. Chromatin immunoprecipitation assays indicated that RhpR binds the promoters containing a putative IR element but not the hrpR and hrpL promoters that do not have an IR element, suggesting that RhpR indirectly regulates the transcriptional cascade of hrpRS, hrpL, and the T3 genes.

A Novel Signal Transduction Pathway that Modulates rhl Quorum Sensing and Bacterial Virulence in Pseudomonas aeruginosa

“…For some sensor kinases, the phosphatase activity may be the critical function in vivo [42]–[45]. Interestingly, the BfmS homologue in Pseudomonas syringae , RhpS, has also been shown to be a negative regulator of its cognate regulator RhpR in our previous studies [44], [45].…”

Regulation of type III secretion system in Pseudomonas syringae

“…In P. syringae, RhpRS is the best-studied master regulatory TCS for the T3SS genes Deng et al, 2010Deng et al, , 2014Zhou et al, 2016;Xie et al, 2019). The rhpR and rhpS genes are located in the same operon, where the rhpR gene encodes a response regulator and rhpS encodes a cognate sensor kinase.…”

“…Over the past 10 years, multiple important T3SS regulators have been identified and characterized (Deng et al, 2010;Jovanovic et al, 2011;Deng et al, 2014;Jovanovic et al, 2014Jovanovic et al, , 2017Waite et al, 2017;Xie et al, 2019). The expression of the T3SS genes is activated by the alternative sigma factor HrpL at different levels including transcription, stability and activity Hendrickson et al, 2000;Hutcheson et al, 2001;Lam et al, 2014;Waite et al, 2017;Li et al, 2019b).…”

“…2B, process 8 and 9 and Table 1) (Chatterjee et al, 2003;Fishman et al, 2018). Histidine kinase RhpS is responsible for sensing changes in external nutrient conditions and thus directly represses the transcription of the downstream hrpRS operon by the cognate regulator RhpR (Deng et al, 2009(Deng et al, , 2010(Deng et al, , 2014. The detailed regulation of RhpRS by T3SS will be discussed in the following section.…”

“…hrpAHrpA HrpA upregulates expression of hrpL, hrcC and hrpRS in planta (Ortiz-Martin et al, 2010b). RhpR functions as a negative regulator of hrpRS by directly binding with an imperfect inverted repeat element at the 5 0 terminal of the hrpRS upstream region(Deng et al, 2010(Deng et al, , 2014Xie et al, 2019). P-RhpR also plays a role in lon activation) rpoN RpoN Alternative sigma factor RpoN (σ 54 ) is required for hrpL induction…”