Negative Autogenous Control of the Master Type III Secretion System Regulator HrpL in Pseudomonas syringae.

Milija Jovanovic,Jörg Schumacher,Mark Bennett,Christopher Waite,Martin Buck

doi:10.1128/mbio.02273-16

Abstract

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.

Highlights

The type III secretion system (T3SS) is a principal virulence determinant of the model bacterial plant pathogen Pseudomonas syringae
We show that hrpL expression is subject to negative autogenous control (NAC), mediated via HrpL binding at the hrpJ promoter
The relative levels of activity of the hrpL promoter (PhrpL) across various regulatory mutant strains were compared using a transcriptional green fluorescent protein (GFP) fusion construct encompassing the intergenic region shared between hrpL and hrpJ (Fig. 1a)

Summary

Introduction

The type III secretion system (T3SS) is a principal virulence determinant of the model bacterial plant pathogen Pseudomonas syringae. Many plant-pathogenic bacteria utilize the needle-like T3SS to inject virulence factors into host plant cells to suppress defense signaling. We propose that the master regulator of its entire T3SS gene set, HrpL, downregulates its own expression to minimize elicitation of plant defenses. Revealing such conserved regulatory strategies will inform future antivirulence strategies targeting plant pathogens. Comprising over 50 disease-causing pathovars, many of which infect valuable crops such as tomato, bean, and rice, Pseudomonas syringae is the most highly developed model for T3SS-dependent plant pathogenesis and evolution of host specificity [6,7,8]. The P. syringae pv. tomato DC3000 pathovar (here DC3000) enters Arabidopsis thaliana leaves through wounds or stomata and replicates within the apoplast, causing chlorosis and necrotic lesions [8, 9]

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