猕猴桃溃疡病菌中PilZ结构域类蛋白在致病力和运动性上的功能研究  

Identification and characterization of PilZ domain-containing proteins in pathogenic-ity and motility of Pseudomonas syringae pv.actinidiae

作　　者：马嘉冰 杨明明 赵颖嘉 谭云晓 常邦帅 王娜娜 黄丽丽[1,2] MA Jiabing;YANG Mingming;ZHAO Yingjia;TAN Yunxiao;CHANG Bangshuai;WANG Nana;HUANG Lili(State Key Laboratory of Crop Stress Biology for Arid Areas,Yangling 712100,China;College of Plant Protection,Yangling 712100,China;College of Life Sciences,Yangling 712100,China;College of Natural Resources and Environment,Northwest A&F University,Yangling 712100,China)

机构地区：[1]旱区作物逆境生物学国家重点实验室,杨凌712100 [2]西北农林科技大学植物保护学院,杨凌712100 [3]西北农林科技大学生命科学学院,杨凌712100 [4]西北农林科技大学资源环境学院,杨凌712100

出　　处：《植物病理学报》2024年第4期756-768,共13页Acta Phytopathologica Sinica

基　　金：国家重点研发计划资助(2022YFD1400200);国家自然科学基金(32100148)。

摘　　要：含PilZ结构域蛋白是目前最大的一类c-di-GMP受体,但该类受体在猕猴桃溃疡病菌(Pseudomonas syringae pv.actinidiae,Psa)中的功能及其作用机制都未见报道,揭示其在Psa中的功能及调控机制,为猕猴桃溃疡病的防治提供新思路。首先对Psa M228进行全基因组分析及序列比对,对PilZ结构域蛋白进行鉴定及c-di-GMP结合保守位点分析;同源重组法构建单基因缺失突变体,叶盘真空渗透、软琼脂平板和生长曲线测定法分别测定突变体与野生型的致病力、运动性及生长速率差异;qRT-PCR测定T3SS和鞭毛相关基因的转录量分析PSA_2195是否对Psa的致病力和运动性存在转录调控。结果显示Psa M228中共有8个含PilZ结构域蛋白,其中PSA_2195和PSA_1975没有与c-di-GMP结合的保守基序RxxxR和(D/N)xSxxG。8个含PilZ结构域蛋白存在功能分化,与野生型M228相比,ΔPsa_1116、ΔPsa_2195、ΔPsa_2203、ΔPsa_762、ΔPsa_4490和ΔPsa_4763的致病力显著减弱,ΔPsa_2195、ΔPsa_762、ΔPsa_1116和ΔPsa_3989的泳动和群集运动能力显著减弱,生长速率之间没明显差异。其中,PSA_2195通过在转录水平上调控鞭毛合成、T3SS等相关基因影响Psa的运动性和致病性。总而言之,本研究对Psa M228中的8个含PilZ结构域蛋白功能进行了系统研究,并重点揭示了PSA_2195调控运动性和致病性的分子机制。PilZ domain-containing proteins are the largest known receptors of second messenger c-di-GMP in bacteria,but the functions and underlying mechanisms have not been reported in Pseudomonas syringae pv.actinidiae(Psa).To reveal the contribution and regulation mechanism of PilZ domain-containing proteins to the pathogenicity of Psa and to provide new ideas for controlling kiwifruit bacterial canker.Firstly,genome analysis and sequence alignment of Psa M228 were performed to identify the PilZ domain-containing proteins of Psa and analyze the conserved c-di-GMP binding motif.Then,homologous recombination was used to construct deletion mutants,and the pathogenicity,motility and growth between mutants and wild type were determined by leaf discs vacuum infiltration,soft agar plate assays and growth curve measurment,respectively.The transcripts of pathogenicity-and motility-relative genes in WT and△Psa_2195 mutant were measured by qRT-PCR.The results show that there are eight PilZ domain-containing proteins in Psa M228,among them PSA_2195 and PSA_1975 have neither a conserved RxxxR motif nor a(D/N)xSxxG binding motif that binds to c-di-GMP.The pathogenicity of△Psa_1116,△Psa_2195,△Psa_2203,△Psa_762,△Psa_4490 and△Psa_4763 were significantly reduced.Deletion of PSA_1116 and PSA_2195,PSA_3989,PSA_762 affected swimming motility and swarming motility,respectively.The growth curve of all mutants are no significant difference with wild type M228.Among all PilZ domain proteins,PSA_2195 regulates the transcription of flagella genes flgA,filE and T3SS genes.Taking together,our research revealed the function of eight PilZ domain-containing proteins in regulating pathogenicity and motility of Psa and the simple molecular mechanisms of PSA_2195.

关 键 词：猕猴桃溃疡病菌 PilZ结构域 致病力 运动性 

分 类 号：S432.1[农业科学—植物病理学]