Negative Autogenous Control of the Master Type III Secretion System Regulator HrpL inPseudomonas syringae

Abstract

The type III secretion system (T3SS) is a principal virulence determinant of the model bacterial plant pathogen Pseudomonas syringae. T3SS effector proteins inhibit plant defense signaling pathways in susceptible hosts and elicit evolved immunity in resistant plants. The extracytoplasmic function sigma factor HrpL coordinates the expression of most T3SS genes. Transcription of hrpL is dependent on sigma-54 and the codependent enhancer binding proteins HrpR and HrpS for hrpL promoter activation. hrpL is oriented adjacently to and divergently from the HrpL-dependent gene hrpJ, sharing an intergenic upstream regulatory region. We show that association of the RNA polymerase (RNAP)-HrpL complex with the hrpJ promoter element imposes negative autogenous control on hrpL transcription in P. syringae pv. tomato DC3000. The hrpL promoter was upregulated in a ΔhrpL mutant and was repressed by plasmid-borne hrpL. In a minimal Escherichia coli background, the activity of HrpL was sufficient to achieve repression of reconstituted hrpL transcription. This repression was relieved if both the HrpL DNA-binding function and the hrp-box sequence of the hrpJ promoter were compromised, implying dependence upon the hrpJ promoter. DNA-bound RNAP-HrpL entirely occluded the HrpRS and partially occluded the integration host factor (IHF) recognition elements of the hrpL promoter in vitro, implicating inhibition of DNA binding by these factors as a cause of negative autogenous control. A modest increase in the HrpL concentration caused hypersecretion of the HrpA1 pilus protein but intracellular accumulation of later T3SS substrates. We argue that negative feedback on HrpL activity fine-tunes expression of the T3SS regulon to minimize the elicitation of plant defenses.

Regulation of type III secretion system in Pseudomonas syringae

“…A recent study revealed that HrpL imposes autogenous control via the shared intergenic region with hrpJ , whose promoter is indispensable for this effect (Fig. B, process 1 and Table ) (Waite et al ., ). By forming an hrpJ promoter‐RNA polymerase‐HrpL complex, the putative HrpRS‐binding element and integration host factor recognition elements in the hrpL promoter are occluded, thus generating negative control of hrpL transcription (Waite et al ., ).…”

“…B, process 1 and Table ) (Waite et al ., ). By forming an hrpJ promoter‐RNA polymerase‐HrpL complex, the putative HrpRS‐binding element and integration host factor recognition elements in the hrpL promoter are occluded, thus generating negative control of hrpL transcription (Waite et al ., ). This feedback control can help to minimize the activation of plant immune systems and allow the pathogen to evade host recognition (Waite et al ., ).…”

“…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). A deeper understanding of T3SS regulation will provide knowledge of the mechanism of P. syringae pathogenesis, which will in turn reveal new approaches for controlling this notorious phytopathogen.…”

Diversity of extracytoplasmic function sigma (σECF) factor‐dependent signaling in Pseudomonas

“…The GacSA two‐component system is required for hrpL , rpoN and hrpR‐hrpS expression (Chatterjee et al ., ), although how plant signals are perceived and transduced into the Hrp regulatory network remains still poorly understood. Importantly, σ HrpL negatively regulates its own transcription (Waite et al , ), which is not a common feature of Pseudomonas σ ECF factors which usually promote their own expression (Chevalier et al , ). It has been proposed that this negative feedback on σ HrpL activity may fine‐tune T3SS production to minimize the elicitation of plant defenses (Waite et al , ).…”

“…Importantly, σ HrpL negatively regulates its own transcription (Waite et al , ), which is not a common feature of Pseudomonas σ ECF factors which usually promote their own expression (Chevalier et al , ). It has been proposed that this negative feedback on σ HrpL activity may fine‐tune T3SS production to minimize the elicitation of plant defenses (Waite et al , ).…”