甜菜孢囊线虫抑制寄主免疫反应的潜在效应蛋白全基因组筛选与鉴定

doi:10.1016/j.jia.2023.09.028

Journal of Integrative Agriculture ›› 2025, Vol. 24 ›› Issue (1): 196-208.DOI: 10.1016/j.jia.2023.09.028

甜菜孢囊线虫抑制寄主免疫反应的潜在效应蛋白全基因组筛选与鉴定

收稿日期:2023-06-06 接受日期:2023-08-11 出版日期:2025-01-20 发布日期:2025-01-07

Genome-wide identification and characterization of putative effectors in Heterodera schachtii that suppress plant immune response

Ke Yao¹, Menghan Zhang¹, Jianjun Xu², Deliang Peng¹, Wenkun Huang¹, Ling’an Kong^{1, 3}, Shiming Liu¹, Guangkuo Li^2#, Huan Peng^{1, 2#}

¹State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China
²Key Laboratory of Integrated Pest Management on Crop in Northwestern Oasis, Institute of Plant Protection, Xinjiang Academy of Agricultural Sciences/Scientific Observing and Experimental Station of Korla, Ministry of Agriculture and Rural Affairs, Urumqi 830013, China
³Zhongyuan Research Center, Chinese Academy of Agricultural Sciences, Xinxiang 453000, China

Received:2023-06-06 Accepted:2023-08-11 Online:2025-01-20 Published:2025-01-07
About author:Ke Yao, E-mail: 17794539779@163.com; #Correspondences Huan Peng, Tel/Fax: +86-10-62815576, E-mail: penghuan@caas.cn; Guangkuo Li, Tel: +86-991-4524641, E-mail: lgk0808@163.com
Supported by:
This research was supported by the Open Fund of the Key Laboratory of Integrated Pest Management on Crop in Northwestern Oasis, Ministry of Agriculture and Rural Affairs of China (KFJJ202101), the National Key R&D Program of China (2021YFD1400100), the National Natural Science Foundation of China (31972247), the Tianchi Talent Introduction Program in Xinjiang Uygur Autonomous Region, China and the Science and Technology Innovation Project of the Chinese Academy of Agricultural Sciences.

摘要/Abstract

摘要：

甜菜孢囊线虫（Heterodera schachtii）是重要的植物病原线虫，曾给世界甜菜生产具有毁灭性的破坏。2015年在我国新疆首次发现了甜菜孢囊线虫的危害，对我国甜菜生产安全造成了严重威胁，迫切需要开展相关研究。本研究基于甜菜孢囊线虫的基因组和转录组数据，筛选能够抑制寄主免疫反应的潜在效应蛋白，为解析甜菜孢囊线虫的致病分子机制提供理论依据。采用SignalP 5.0和TMHMM2.0等生物信息学分析方法，从甜菜孢囊线虫基因组筛选出有信号肽无跨膜结构域的潜在效应子序列，在此基础上，筛选出在植物寄生线虫保守存在，但与自由生活线虫无同源性的蛋白（e值<1.0 e^-5且覆盖率≥30.0%）的序列，根据甜菜孢囊线虫各发育阶段的基因表达量差异，随后筛选出在甜菜孢囊线虫二龄幼虫和寄生阶段上调表达的序列（差异倍数log2>2且e值≤0.01）。最后过滤掉功能已知的植物线虫效应蛋白序列（e值<1.0 e^-10），剩余序列作为潜在的效应蛋白进行下一步分析。将上述候选效应蛋白基因序列构建到植物表达载体pYBA1132中，采用本氏烟草瞬时表达系统，对候选效应蛋白是否能激发或抑制Bax和GPA2/RBP1诱导的细胞死亡进行筛选。采用qRT-PCR技术检测候选效应蛋白异源表达对植物免疫相关基因PR1、PR2、WRKY12和PI1转录的影响。采用原位杂交方法分析候选效应蛋白在甜菜孢囊线虫中的表达定位。采用生物信息学的方法从甜菜孢囊线虫基因组中筛选出51个新的候选效应蛋白。利用烟草瞬时表达系统，对37个成功克隆的候选效应蛋白基因进行功能验证，筛选出13个能够抑制Bax引起的细胞死亡的候选效应蛋白，其中9个效应蛋白能够抑制GPA2/RBP1诱导的坏死反应。qRT-PCR分析发现，13个候选效应蛋白基因的瞬时表达均能引起寄主植物PR1、PR2、WRKY12和PI1防御基因表达量的显著下调。原位杂交结果表明，13个候选效应蛋白的表达位点均位于甜菜孢囊线虫的食道腺细胞。本研究从甜菜孢囊线虫基因组中筛选到了13个能够抑制植物免疫应答的新效应蛋白，它们均可以通过抑制寄主抗性相关基因的表达，促进线虫寄生。上述研究结果将为进一步研究甜菜孢囊线虫与寄主的互作提供理论依据。

Abstract:

The sugar beet cyst nematode (Heterodera schachtii) is one of the most destructive pathogens in sugar beet production, which causes serious economic losses every year. Few molecular details of effectors of H. schachtii parasitism are known. We analyzed the genome and transcriptome data of H. schachtii and identified multiple potential predicted proteins. After filtering out predicted proteins with high homology to other plant-parasitic nematodes, we performed functional validation of the remaining effector proteins. 37 putative effectors of H. schachtii were screened based on the Nicotiana benthamiana system for identifying the effectors that inhibit plant immune response, eventually determines 13 candidate effectors could inhibit cell death caused by Bax. Among the 13 effectors, nine have the ability to inhibit GPA2/RBP1-induced cell death. All 13 effector-triggered immunity (ETI) suppressor genes were analyzed by qRT-PCR and confirmed to result in a significant downregulation of one or more defense genes during infection compared to empty vector. For in situ hybridization, 13 effectors were specifically expressed and located in esophageal gland cells. These data and functional analysis set the stage for further studies on the interaction of H. schachtii with host and H. schachtii parasitic control.

Key words: sugar beet cyst nematode , Heterodera schachtii , effector-triggered immunity , hypersensitive response , effector

Ke Yao, Menghan Zhang, Jianjun Xu, Deliang Peng, Wenkun Huang, Ling’an Kong, Shiming Liu, Guangkuo Li, Huan Peng. 甜菜孢囊线虫抑制寄主免疫反应的潜在效应蛋白全基因组筛选与鉴定[J]. Journal of Integrative Agriculture, 2025, 24(1): 196-208.

Ke Yao, Menghan Zhang, Jianjun Xu, Deliang Peng, Wenkun Huang, Ling’an Kong, Shiming Liu, Guangkuo Li, Huan Peng. Genome-wide identification and characterization of putative effectors in Heterodera schachtii that suppress plant immune response[J]. Journal of Integrative Agriculture, 2025, 24(1): 196-208.

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甜菜孢囊线虫抑制寄主免疫反应的潜在效应蛋白全基因组筛选与鉴定

Genome-wide identification and characterization of putative effectors in Heterodera schachtii that suppress plant immune response

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