Two-Component Signaling System RegAB Represses Pseudomonas syringae pv. actinidiae T3SS by Directly Binding to the promoter of hrpRS

doi:10.1016/j.jia.2024.09.028

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Two-Component Signaling System RegAB Represses Pseudomonas syringae pv. actinidiae T3SS by Directly Binding to the promoter of hrpRS

Mengsi Zhang^{1, 2*}, Mingming Yang^{2, 3*}, Xiaoxue Zhang^{1, 2}, Shuying Li^{1, 2}, Shuaiwu Wang^{1, 2}, Alex Muremi Fulano⁴, Yongting Meng^{1, 2}, Xihui Shen^{1, 2}, Lili Huang^{2, 3#}, Yao Wang^{1, 2#}

¹ College of Life Sciences, Northwest A&F University, Yangling 712100, China.

²State Key Laboratory for Crop Stress Resistance and High-Efficiency Production, Northwest A&F University, Yangling 712100, China.

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

⁴Department of Plant Science and Crop Protection, University of Nairobi, Nairobi, Kenya

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摘要

丁香假单胞菌猕猴桃致病变种（Pseudomonas syringae pv. actinidiae, Psa）引起的猕猴桃溃疡病是一种毁灭性的细菌病害，生物型3 Psa的主要致病因子为三型分泌系统（T3SS）。双组分信号系统（two component signaling systems, TCSs）可通过感知环境信号调控细菌毒力，但双组分信号系统RegAB如何调控T3SS的表达还未见报道。本研究通过缺失突变体构建、致病相关表型测定、生物信息学及转录组数据分析、启动子活性和实时荧光定量PCR测定、EMSA和MST等对RegAB调控T3SS的分子机制进行解析。结果表明，双组分系统RegAB负向调控Psa M228对寄主植物猕猴桃的致病性和非寄主植物本氏烟的超敏反应，RNA-seq数据显示RegA可调控细菌分泌系统、病原菌与植物互作、能量代谢等多种细胞过程，其中T3SS基因表达差异显著。启动子活性和实时荧光定量PCR试验表明，相比于野生型菌株M228，在组氨酸激酶regA及其下游调控蛋白regB缺失突变体中T3SS相关基因的表达及菌株致病性都显著增加。进一步研究发现RegA可直接与hrpR的启动子结合抑制hrpR/S基因的转录，调控Psa M228 T3SS基因表达从而降低其在宿主猕猴桃上的致病性和非宿主本氏烟上的超敏反应，且RegA对hrpR的转录调控依赖于其磷酸化。磷酸化的RegA (RegA^D64E)与hrpR启动子的结合能力增强，抑制T3SS表达更为显著。总之，本研究发现了一组尚未被报道过可以调控T3SS的双组分系统RegAB，并阐述了其通过直接作用于HrpRS调控T3SS表达进而影响Psa致病性的分子机制，拓宽了RegAB的功能，并为猕猴桃溃疡病防治提供了新的作用靶点。

Abstract

Kiwifruit bacterial canker, caused by Pseudomonas syringae pv. actinidiae (Psa), is a significant threat to the kiwifruit industry. The two-component signaling systems (TCSs) play a crucial role in regulating the virulence of Pseudomonas syringae (P. syringae), yet their specific function in Psa remains largely unclear. In this study, we found that disrupting the TCS RegAB (encoded by Psa_802/Psa_803) resulted in a notable increase in the pathogenicity of Pseudomonas syringae pv. actinidiae M228 (Psa M228) in host plant and hypersensitive reaction (HR) in nonhost plant. Through comparative transcriptome analysis of the Psa M228 wild-type strain and the regA mutant, we identified the pivotal role of RegA/B in controlling various physiological pathways, including the Type III secretion system (T3SS), a key determinant of Psa virulence. Additionally, we discovered that the RegA does have binding sites in the promoter region of the hrpR/S, and the transcriptional level of the hrpR and other T3SS-related genes increased in the regA deletion strain relative to the Psa M228 wild-type. The DNA-binding affinity of RegA, and therefore the repressor function, is enhanced by its phosphorylation. Our findings unveil the function of TCS RegAB and the regulatory mechanism of T3SS by RegAB in Psa, highlighting the diverse functions of the RegAB system.

Keywords: Pseudomonas syringae pv. actinidiae Type III secretion system (T3SS) Two-component signaling system RegAB hrpR/S

Online: 26 September 2024

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

About author: Mengsi Zhang, Tel: +86-18838964968, E-mail: 188838964968@163.com; Mingming Yang, Tel: +86-13260828693, E-mail: yangming862@126.com; #Correspondence Yao Wang, Tel: +86-13488215263, E-mail: wangyao@nwafu.edu.cn; Lili Huang, Tel: +86-13991351090, E-mail: huanglili@nwsuaf.edu.cn * equal contribution.

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Mengsi Zhang, Mingming Yang, Xiaoxue Zhang, Shuying Li, Shuaiwu Wang, Alex Muremi Fulano, Yongting Meng, Xihui Shen, Lili Huang, Yao Wang. 2024. Two-Component Signaling System RegAB Represses Pseudomonas syringae pv. actinidiae T3SS by Directly Binding to the promoter of hrpRS. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2024.09.028

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