猕猴桃细菌性溃疡病菌T3SS关键效应蛋白基因致病功能

doi:10.3864/j.issn.0578-1752.2022.03.007

摘要/Abstract

摘要： 目的由丁香假单胞菌猕猴桃致病变种（Pseudomonas syringae pv. actinidiae,Psa）引起的猕猴桃细菌性溃疡病是全球猕猴桃产业最具毁灭性的病害。病原细菌主要通过III型分泌系统（type III secretion system,T3SS）将多种效应蛋白（T3SS effector,T3SE）注入寄主植物细胞,进而促进病菌侵染和致病。本研究旨在解析Psa基因组中T3SE的信息并对其T3SS和T3SE的致病功能进行系统分析,为溃疡病菌致病机制的研究和防治策略的制定提供依据。方法利用marker-free同源重组基因敲除技术获得M228菌株的T3SS功能缺陷突变体ΔhrcS和ΔhrcC,观察突变体在寄主上的致病力,同时检测突变体诱导本氏烟产生细胞坏死的情况;随后利用从Pseudomonas-Plant Interaction数据库下载的T3SE数据库,本地BLAST多序列比对构建强、弱致病菌株M228和M227的T3SE库,并对二者的T3SE基因信息进行比对分析;另外,获得M228菌株T3SE单、多效应子突变菌株20株及2株HopR1基因回补菌株（共涉及19个T3SE）,并将各突变体室内有伤接菌猕猴桃枝条,系统评价各突变体致病力变化并进行统计分析。结果通过对Psa的hrcS和hrcC基因进行突变,证明T3SS是其在寄主上致病以及非寄主上过敏性坏死反应（HR）所必需的。通过数据库同源比对,发现在强毒株系和弱毒株系中有31个T3SE基因具有100%的同源性,选取一些基因进行缺失突变,发现hopM1/avrE1和hopR1是Psa重要的毒性因子,且二者不存在功能冗余。另外,单独敲除avrPto5或avrRpm1均能提高Psa致病力。在缺失A-F-E基因簇和avrPto5的菌株中,敲除hopM1/avrE1和hopR1也分别导致Psa的致病力显著下降;而同时敲除hopM1/avrE1、hopR1、avrPto5和A-F-E基因簇导致病菌完全丧失致病力。结论HopM1/AvrE1与同家族HopR1均为Psa重要致病因子,且独立于其他效应子发挥作用;avrPto5和avrRpm1基因缺失可以增强Psa的致病力。

关键词: 猕猴桃溃疡病, 丁香假单胞菌猕猴桃致病变种, 效应蛋白, III型分泌系统, 致病力

Abstract: 【Objective】 Pseudomonas syringae pv. actinidiae (Psa), the causal agent of bacterial canker of kiwifruit, is the most devastating pathogen in global kiwifruit production. The pathogenic bacteria secrete a series of effectors (T3SEs) into host cell to promote infection and pathogenesis by the type III secretion system (T3SS). The objective of this study is to analyze the T3SEs information in Psa genome and systematically evaluate the pathogenicity function of T3SS and T3SEs, and to provide the basis evidence for the research of the pathogenic mechanism and the establishment of the control strategies.【Method】 By marker-free homologous recombination knockout technique, the M228 deficiency mutants of T3SS, ΔhrcS and ΔhrcC, were obtained for inoculating on host to evaluate the pathogenicity and injecting on Nicotiana benthamiana to observe the cell death response. Then, based on the T3SEs database downloaded from Pseudomonas-Plant Interaction, the T3SEs library of strong pathogenicity M228 and weak pathogenicity M227 was separately constructed against the database by local BLAST multiple sequence alignment program, and then the T3SEs information between them was compared. Moreover, 20 T3SE single- and poly-genetic mutants from M228 and 2 HopR1-genetic complementing mutants were constructed, involving 19 T3SEs, and then the mutants were wound-inoculated on kiwifruit canes for assessing and statistical analyzing the pathogenicity.【Result】 T3SS was proved to be essential for Psa pathogenicity on host and hypersensitive response (HR) on non-host by the hrcS and hrcC mutants, separately. Further the BLAST results against database showed there were almost 31 complete T3SE genes and their sequences were displayed 100% similarity between the strong pathogenicity strain and attenuated strain. Then, some T3SE genes were selected for deletion mutants. The results showed that hopM1/avrE1 and hopR1 genes were essential for Psa pathogenicity and had no function redundant with each other. In addition, the avrPto5- and avrRpm1-deletion mutant could in turn increase the Psa pathogenicity. Based on avrPto- and T3SE group (cluster A, E and F) deletion mutant, single- or poly-genetic mutant of hopR1 and hopM1/avrE1 could still separately lead to a significant decrease in Psa pathogenicity. However, simultaneous deletion of hopM1/avrE1, hopR1, avrPto5 and A-F-E cluster resulted in complete loss of pathogenicity.【Conclusion】 HopR1 and its homologous family HopM1/AvrE1, which don’t have a redundant function independent with others, are the unique key pathogenicity factors in Psa, but AvrPto5-and AvrRpm1-deletion can enhance Psa pathogenicity.

Key words: bacterial canker of kiwifruit, Pseudomonas syringae pv. actinidiae (Psa), effector, T3SS, pathogenicity

张晋龙,赵志博,刘巍,黄丽丽. 猕猴桃细菌性溃疡病菌T3SS关键效应蛋白基因致病功能[J]. 中国农业科学, 2022, 55(3): 503-513.

ZHANG JinLong,ZHAO ZhiBo,LIU Wei,HUANG LiLi. The Function of Key T3SS Effectors in Pseudomonas syringae pv. actinidiae[J]. Scientia Agricultura Sinica, 2022, 55(3): 503-513.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2022.03.007

https://www.chinaagrisci.com/CN/Y2022/V55/I3/503

图/表 7

表1

敲除和回补突变体信息汇总"

突变体名称 The name of mutants	详细信息 Related detail information
ΔhrcC	T3SS结构基因hrcC功能缺陷突变体 hrcC-deletion generating the T3SS-deficient mutant
ΔhrcS	T3SS结构基因hrcS功能缺陷突变体hrcS-deletion generating the T3SS-deficient mutant
ΔO	T3SE hopM1/avrE1缺失突变体T3SE hopM1/avrE1-deletion mutant
ΔM	T3SE hopR1缺失突变体T3SE hopR1-deletion mutant
ΔOM	T3SE hopM1/avrE1+hopR1缺失突变体T3SE hopR1-deletion mutant based on ΔO background
ΔMO	T3SE hopR1+hopM1/avrE1缺失突变体T3SE hopM1/avrE1-deletion mutant based on ΔM background
ΔM-C-hopR1	T3SE hopR1突变体基础上回补hopR1的回补菌株T3SE hopR1-complement mutant based on ΔM background
ΔOM-C-hopR1	T3SE hopR1+hopM1/AvrE1突变体基础上回补hopR1的回补菌株T3SE hopR1-complement mutant based on ΔOM background
ΔA	表2中对应T3SE基因簇A缺失突变体T3SE cluster A-deletion mutant listing on Table 2
ΔF	表2中对应T3SE基因簇F缺失突变体T3SE cluster F-deletion mutant listing on Table 2
ΔE	表2中对应T3SE基因簇E缺失突变体T3SE cluster E-deletion mutant listing on Table 2
ΔAF	表2中对应T3SE基因簇A和F缺失突变体T3SE cluster A- and F-deletion mutant listing on Table 2
ΔAFE	表2中对应T3SE基因簇A、F和E全敲除缺失突变体T3SE cluster A-, F- and E-deletion mutant listing on Table 2
ΔAFEL	在ΔAFE突变体的背景下敲除avrPto5获得突变体avrPto5-deletion mutant based on ΔAFE background
ΔAFELM	在ΔAFEL突变体的背景下敲除hopR1获得突变体hopR1-deletion mutant based on ΔAFEL background
ΔAFELO	在ΔAFEL突变体的背景下敲除hopM1/avrE1获得突变体hopM1/avrE1-deletion mutant based on ΔAFEL background
ΔAFELOM	在ΔAFEL突变体的背景下同时敲除hopM1/avrE1和hopR1获得突变体 hopM1/avrE1- and hopR1-deletion mutant based on ΔAFEL background
ΔOMN	在ΔOM突变体基础上敲除avrRpm1获得突变体avrRpm1-deletion mutant based on ΔOM background

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表2

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位置 Locus	T3SE	ICMP 18884		伴侣分子 Chaperon		基因敲除标识符 Symbol of gene deletion
	HopY1	IYO_000845
Cluster A	HopQ1	IYO_003525				A
	HopD1	IYO_003530
	AvrD1	IYO_003570
	AvrB4-l	IYO_003600
	HopAYl	IYO_003610
	HopX3	IYO_003635		ShcF
	HopAW1	IYO_003657
	HopBB1-2	IYO_003675		ShcF
	HopAF1	IYO_003680
	HopAO2	IYO_003720
	HopBB1-1	IYO_003727		ShcF
	HopS2	IYO_004052		ShcS2
	HopI1	IYO_005160
CEL/Cluster O	HopN1	IYO_006735		ShcN
	HopAA1-1	IYO_006745
	HopM1	IYO_006760		ShcM		O
	AvrE1	IYO_006770		ShcE		O
	AvrRpm1	IYO_008065				N
Cluster E	HopZ5	IYO_008282				E
Cluster E	HopH1	IYO_008285				E
	HopAM1-2	IYO_008385
	HopAE1	IYO_012225
	HopAZ1	IYO_018555
	AvrPto5	IYO_020425				L
	HonAM1-1	IYO_023205
Cluster F	HopAI1	IYO_023980				F
	HopAH1	IYO_023985
	HopAG1	IYO_023990
Cluster M	HopR1	IYO_024150				M
Cluster M	HopW1	IYO_024160
	HopF2	IYO_024217		ShcF
	HopAS1	IYO_027420
	HopA1	IYO_028375		ShcA
	HopZ3	IYO_029045		Tir
	AvrRpm2	IYO_029288		ShcF
Plasmid	HopAV1	IYO_029710
	HopAA1-2	IYO_029780-795
	HopAU1	IYO_029795
	存在的基因 Present		差异的基因 Variable		不完整的基因 Incomplete