小麦条锈菌效应蛋白Hasp83在条锈菌致病性中的功能分析

doi:10.3864/j.issn.0578-1752.2023.05.005

摘要/Abstract

摘要：

【背景】条锈病是小麦上的重大病害，由条形柄锈菌小麦专化型（Puccinia striiformis f. sp. tritici，Pst）侵染引起。条锈菌是活体营养型寄生真菌，在侵染过程中形成吸器，通过吸器从寄主植物汲取营养。同时，吸器分泌效应蛋白调控寄主免疫，促进侵染过程。【目的】明确条锈菌效应蛋白的功能及其作用机理，为揭示条锈菌的致病机制打下基础。【方法】比较分析条锈菌夏孢子、芽管和吸器转录组，获得在吸器诱导表达的分泌蛋白基因Hasp83，在本氏烟叶片细胞中瞬时表达观察是否能抑制由BAX引起的细胞坏死；利用qRT-PCR分析该基因在条锈菌侵染小麦不同阶段的表达水平。借助荧光假单胞菌Ⅲ型分泌系统和寄主诱导的基因沉默（host-induced gene silencing，HIGS）分析Hasp83在条锈菌侵染过程中的功能。利用酵母双杂交系统筛选小麦中与Hasp83互作的蛋白，免疫共沉淀技术进一步在烟草细胞中共表达验证Hasp83及其候选靶标蛋白的互作。【结果】Hasp83开放阅读框全长522 bp，编码173个氨基酸，蛋白N端1—29位氨基酸为信号肽，无保守结构域，在本氏烟叶片细胞中瞬时表达能抑制由BAX引起的细胞坏死。qRT-PCR分析显示，Hasp83在条锈菌侵染时期上调表达；利用细菌Ⅲ型分泌系统在小麦水源11品种中瞬时表达Hasp83能够抑制荧光假单胞菌引起的胼胝质积累，在接种条锈菌无毒性小种CYR23后，瞬时表达Hasp83株系产生的活性氧积累面积和过敏性坏死面积相比对照减少19.35%—38.62%；利用HIGS技术在接种条锈菌毒性小种CYR31的小麦水源11中沉默Hasp83，发现条锈菌的产孢量、菌丝长度、菌丝扩展面积和吸器数目减少，致病力降低。酵母双杂交结果表明，效应蛋白Hasp83与其小麦中候选靶标过敏性坏死诱导蛋白Tahir1互作。免疫共沉淀进一步证明Hasp83及其候选靶标Tahir1存在互作。【结论】条锈菌效应蛋白Hasp83可抑制寄主由非致病细菌和无毒性条锈菌生理小种引起的小麦防卫反应，增强病原菌的致病力。

关键词: 小麦条锈病, 吸器, 效应蛋白, 转录组分析, 靶标蛋白鉴定

Abstract:

【Background】Wheat stripe rust is a serious disease on wheat, which is caused by Puccinia striiformis f. sp. tritici (Pst). Pst is an obligate biotrophic fungus, which can form haustorium during infection and absorb nutrients from the host plants via haustorium. Moreover, Pst secretes effectors through haustorium to regulate host immunity and promotes the infection process.【Objective】The objective of this study is to clarify the function and mechanism of Pst effectors, and to reveal the pathogenicity mechanism of Pst.【Method】By comparing the transcriptome of Pst urediospore, germinated tube and haustorium, Hasp83 encoding secreted protein was identified to be significantly induced in haustorium, and whether it could inhibit the cell death caused by BAX on Nicotiana benthamiana leaves was observed through Agrobacterium-mediated transient expression. qRT-PCR was used to detect the expression level of Hasp83 during different Pst infection stages in wheat. The type Ⅲ secretion system (T3SS) of Pseudomonas fluorescens EtHan and host induced gene silencing (HIGS) were carried out to investigate the function of Hasp83 during Pst infection. The yeast two-hybrid (Y2H) system was used to screen the proteins interacting with Hasp83 in wheat, and co-inmunoprecipitation (Co-IP) assay was used to further verify the interaction by co-expressing Hasp83 and its candidate target proteins in N. benthamiana cells.【Result】The open reading frame (ORF) of Hasp83 is 522 bp, encoding 173 amino acids. Hasp83 contains no conserved domain, and the N-terminal 1-29 amino acids encode a signal peptide, which could inhibit the cell death caused by BAX on N. benthamiana leaves through Agrobacterium-mediated transient expression. qRT-PCR analysis revealed that Hasp83 was up-regulated during Pst infection in wheat. Transient expression of Hasp83 in wheat via T3SS could inhibit callose accumulation caused by P. fluorescens, and lead to 19.35%-38.62% decrease of reactive oxygen species (ROS) accumulation area and necrotic cell area caused by the avirulent Pst race CYR23 in wheat. Silencing of Hasp83 by HIGS in Suwon11 wheat leaves infected with the virulent Pst race CYR31 significantly reduced pathogenicity of Pst compared to controls, resulting in less urediospore sporulation, shorter infection hyphal length, smaller infection area, and decreased haustorium number. Y2H result showed that the effector Hasp83 interacted with wheat hypersensitive-induced reaction (HIR) protein, Tahir1. The interaction between Hasp83 and Tahir1 was further confirmed by Co-IP assay in N. benthamiana.【Conclusion】Pst effector Hasp83 can suppress wheat immunity caused by the non-pathogenic bacteria and avirulent Pst, and enhance the pathogenicity of Pst.

Key words: wheat stripe rust, haustorium, effector, transcriptome analysis, identification of target protein

王建锋, 成嘉欣, 舒伟学, 张艳茹, 王晓杰, 康振生, 汤春蕾. 小麦条锈菌效应蛋白Hasp83在条锈菌致病性中的功能分析[J]. 中国农业科学, 2023, 56(5): 866-878.

WANG JianFeng, CHENG JiaXin, SHU WeiXue, ZHANG YanRu, WANG XiaoJie, KANG ZhenSheng, TANG ChunLei. Functional Analysis of Effector Hasp83 in the Pathogenicity of Puccinia striiformis f. sp. tritici[J]. Scientia Agricultura Sinica, 2023, 56(5): 866-878.

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用途 Use	引物名称 Primer name	引物序列 F Primer sequence F (5′-3′)	引物序列 R Primer sequence R (5′-3′)
分泌系统载体构建 Construction of secretion system vector	pEDV6-Hasp83^∆sp	GGGGACAAGTTTGTACAAAAAAGCAGG CTTCATGCATAAAGCCCTGAACC	GGGGACCACTTTGTACAAGAAAGCTGGGTC TCCTCGATGTGAAATACC
沉默载体构建 Construction of silent vector	γ-Hasp 83-1	TTAATTAACGGAAACTCATCTGTCAATCA	GCGGCCGCGAGGAAAGGGAAAATGGAGA
沉默载体构建 Construction of silent vector	γ-Hasp 83-2	TTAATTAATCCATTACCTTGTCGTTCTCA	GCGGCCGCATACCGCCAGTCGTCCTTA
酵母载体构建 Construction of yeast-expression vector	pGBKT7-Hasp83^∆sp	CGGAATTCATGCATAAAGCCCTGAACC	CGGGATCCTTATCCTCGATGTGAAATACC
酵母载体构建 Construction of yeast-expression vector	pGADT7-Tahir1	CGGAATTCATGGGTGGGGTACTTGGT	CGGGATCCTCAAACCATCCTGCCCT
瞬时表达载体构建 Construction of transient overexpression vector	pGR106-Hasp83^∆sp	GGCATCGATATGCATAAAGCCCTGAACC	CATGCTGACTTATCCTCGATG
	pBinGFP2-Hasp83^∆sp	GACGAGCTGTACAAGGGTACCATGCATAAA GCCCTGAACC	TCTAGTTCATCTAGAGGATCCTCCTCGATGTGAAATACC
	pICH86988-Tahir1-HA	TTACAATTATCGATACAATGTACCCATACGA CGTCCCAGACTACGCTATGGGTGGGGTACTTGGT	CTCATTAAAGCAGGACAAGCAACCATCCTGCCCTGGA
实时荧光定量PCR qRT-PCR	qHasp83-1	CGGAAACTCATCTGTCAATCA	GAGGAAAGGGAAAATGGAGA
	qHasp83-2	TCCATTACCTTGTCGTTCTCA	ATACCGCCAGTCGTCCTTA
	Pst EF	TTCGCCGTCCGTGATATGAGACAA	ATGCGTATCATGGTGGTGGAGTGA
	Ta EF	TGGTGTCATCAAGCCTGGTATGGT	ACTCATGGTGCATCTCAACGGACT