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Journal of Integrative Agriculture
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Polymyxin B inhibits T3SS gene expression via WspR in Pseudomonas syringae pv. actinidiae

Yudi Wang1, 2*, Mingming Yang1, 3*, Xianwei Xie1, 2*, Bobo Zhao1, 2, Jinfang Zhou1, 2, Yuqing Yang1, 2, Yao Wang1, 2, Xihui Shen1, 2#, Lili Huang1, 3#

1 State Key Laboratory for Crop Stress Resistance and High-Efficiency Production, Northwest A&F University, Yangling 712100, China

2 College of Life Sciences, Northwest A&F University, Yangling 712100, China

3 College of Plant Protection, Northwest A&F University, Yangling 712100, China

 Highlights 

l Polymyxin B suppresses the expression of T3SS genes through WspR in Pseudomonas syringae pv. actinidiae.

l The TetR family transcription factor PsrA directly binds to the hrpL promoter and positively regulates hrpL transcription and Psa virulence.

l WspR interacts with PsrA, inhibiting its binding to the hrpL promoter.

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摘要  

猕猴桃细菌性溃疡病(Kiwifruit bacterial cankerKBC)是由丁香假单胞菌猕猴桃致病变种(Pseudomonas syringae pv. actinidiaePsa)引起的毁灭性病害,严重威胁猕猴桃产业的发展。型分泌系统(Type III secretion systemT3SS)是Psa关键的毒力因子,然而,其表达调控的分子机制仍不完全清楚。多粘菌素B1能够显著抑制PsaT3SS相关基因的表达,但其作用机制尚未阐明。环二鸟苷酸(cyclic diguanosine monophosphatec-di-GMP)是细菌中重要的第二信使,主要由含GGDEF结构域的二鸟苷酸环化酶(diguanylate cyclaseDGC)合成。本研究以Psa M228菌株为研究对象,旨在鉴定T3SS的调控因子,解析多粘菌素B抑制T3SS表达的分子机制。本研究筛选并鉴定了Psa中的GGDEF结构域蛋白PSA_1379(命名为WspR)。结构域分析显示,WspRN端的REC结构域和C端的GGDEF结构域组成。体外酶活检测表明,WspR具有DGC活性,能够催化c-di-GMP的合成。基于对wspR基因的缺失、回补及表型分析,发现WspR负调控PsaT3SS基因表达和致病能力。进一步研究发现,多粘菌素B能够显著诱导wspR的表达,提高胞内c-di-GMP水平,并通过WspR抑制T3SS相关基因的转录。通过细菌双杂交、GST pull-down 和细胞内共定位分析,发现WspR可与TetR家族转录调控因子PsrA互作。启动子活性、凝胶阻滞(EMSA)和微量热泳动(MST)实验发现,PsrA能够直接与T3SS主调控因子hrpL的启动子区结合,从而正向调控hrpL转录。致病性检测也证明PsrA正向调控Psa的致病力。进一步的启动子结合分析表明,WspRc-di-GMP可有效抑制PsrAhrpL启动子的结合,在分子水平上揭示了“WspR–PsrA–hrpL的转录调控通路。综上所述,本研究鉴定并解析了Psa中二鸟苷酸环化酶WspR的酶学特性及其在毒力调控中的功能,揭示了多粘菌素B通过WspR抑制T3SS表达的分子机制,首次提出了WspR–PsrA–hrpL构成的T3SS转录调控新通路。本研究加深了对Psa环境信号感知与毒力调控机制的理解,为猕猴桃细菌性溃疡病的新型防治策略的开发提供了理论基础。



Abstract  

Kiwifruit bacterial canker (KBC), caused by Pseudomonas syringae pv. actinidiae (Psa), severely threatens the kiwifruit industry. The type III secretion system (T3SS) is a key virulence factor in Psa, but the regulatory mechanisms remain poorly understood. Polymyxin B1, the main component of polymyxin B, inhibits T3SS gene expression in Psa, yet its underlying mechanism is unclear. Cyclic diguanosine monophosphate (c-di-GMP), a crucial bacterial second messenger, is synthesized by diguanylate cyclases (DGCs) containing a GGDEF domain. In this study, we identified and characterized PSA_1379 (WspR), a GGDEF domain-containing protein in Psa. Biochemical assays demonstrated that WspR exhibits DGC activity. Virulence assays showed that WspR negatively regulates Psa virulence. RT-qPCR analyses revealed that polymyxin B induces wspR expression. Additionally, polymyxin B upregulates intracellular c-di-GMP levels and inhibits the expression of T3SS genes through WspR. Bacterial two-hybrid and GST pull-down assays confirmed that WspR interacts with the transcription factor PsrA. Both WspR and c-di-GMP inhibit the binding of PsrA to the promoter of the T3SS master regulator hrpL, thereby suppressing PsrA-mediated transcriptional activation of hrpL and ultimately repressing T3SS gene expression. This study provides new insights into Psa virulence regulation and suggests potential targets for KBC control through the WspR-c-di-GMP pathway.

Keywords:  Pseudomonas syringae pv. actinidiae       type III secretion system              diguanylate cyclase              virulence  
Online: 10 February 2026  
Fund: 

This work was supported by the National Key Research and Development Program (2022YFD1400200), National Natural Science Foundation of China (32330004 to Shen Xi-hui, 32170130 to Wang Yao and 32102283 to Yang Ming-ming). 

About author:  #Correspondence Xihui Shen, Tel: +86-29-87092820, E-mail: xihuishen@nwsuaf.edu.cn; Lili Huang, E-mail: huanglili@nwsuaf.edu.cn * These authors have contributed equally to this work.

Cite this article: 

Yudi Wang, Mingming Yang, Xianwei Xie, Bobo Zhao, Jinfang Zhou, Yuqing Yang, Yao Wang, Xihui Shen, Lili Huang. 2026. Polymyxin B inhibits T3SS gene expression via WspR in Pseudomonas syringae pv. actinidiae. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2026.02.022

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