中国农业科学 ›› 2019, Vol. 52 ›› Issue (24): 4518-4526.doi: 10.3864/j.issn.0578-1752.2019.24.007

• 植物保护 • 上一篇    下一篇

葡萄溃疡病菌外泌蛋白LtGH61A的致病力及基因表达模式

彭军波,李兴红,张玮,周莹,黄金宝,燕继晔()   

  1. 北京市农林科学院植物保护环境保护研究所/北方果树病虫害绿色防控北京市重点实验室,北京100097
  • 收稿日期:2019-06-24 接受日期:2019-07-18 出版日期:2019-12-16 发布日期:2020-01-15
  • 通讯作者: 燕继晔
  • 作者简介:彭军波,E-mail:pjb169961@163.com。
  • 基金资助:
    北京市青年拔尖个人项目(2016000021223ZK29);北京市农林科学院院储备项目(KJCX20170412)

Pathogenicity and Gene Expression Pattern of the Exocrine Protein LtGH61A of Grape Canker Fungus

JunBo PENG,XingHong LI,Wei ZHANG,Ying ZHOU,JinBao HUANG,JiYe YAN()   

  1. Institute of Plant and Environment Protection, Beijing Academy of Agriculture and Forestry Sciences/Beijing Key Laboratory of Environmental Friendly Management of Diseases and Pests of North China Fruits, Beijing 100097
  • Received:2019-06-24 Accepted:2019-07-18 Online:2019-12-16 Published:2020-01-15
  • Contact: JiYe YAN

摘要:

【目的】由葡萄座腔菌属(Botryosphaeria)真菌引起的溃疡病是葡萄生产上的重要病害之一,在国内多个葡萄产区发生,严重威胁葡萄的产量及品质。本研究对葡萄溃疡病菌(Lasiodiplodia theobromae)中一个假定外泌蛋白LtGH61A进行功能分析,为进一步解析葡萄溃疡病菌的致病机理及病害防控提供理论依据。【方法】通过SignalP 4.0预测LtGH61A蛋白的信号肽;氨基酸序列同源比对结合基因功能注释,预测LtGH61A蛋白的功能;通过酵母互补试验分析LtGH61A蛋白的外泌特性;采用实时荧光定量PCR(qRT-PCR)分析LtGH61A在病原菌营养菌丝及侵染寄主不同阶段的表达量;借助RNA干涉(RNAi)对LtGH61A进行表达抑制;通过葡萄枝干离体接种试验,分析LtGH61A蛋白对葡萄溃疡病菌致病力的影响。通过比较菌落直径,分析LtGH61A蛋白对葡萄溃疡病菌菌丝生长速率的影响。【结果】信号肽预测表明LtGH61A蛋白N端具有一个长度为18个氨基酸的信号肽;基因功能注释推定LtGH61A的编码产物属于糖苷水解酶61(GH61)家族,能酶解纤维素类物质;互补蔗糖酶外泌缺陷型酵母YTK12结果表明,LtGH61A蛋白的信号肽具有外泌活性,能够引导酵母YTK12蔗糖酶的外泌;与营养菌丝阶段相比,LtGH61A的表达量在病原菌侵染阶段显著升高,并且在接种后48 h高达营养菌丝阶段的19倍;通过RNAi试验及qRT-PCR验证,获得了2个LtGH61A表达量明显降低的阳性转化子,分别命名为RNAi-LtGH61A1和RNAi-LtGH61A2;葡萄枝干离体接种试验结果显示,与野生型CSS-01s相比,RNAi-LtGH61A1和RNAi-LtGH61A2转化子在葡萄枝干形成的病斑长度显著变短,约为野生型CSS-01s的55%,表明LtGH61A影响葡萄溃疡病菌的致病力;菌落直径比较显示,与野生型CSS-01s相比,RNAi-LtGH61A1和RNAi-LtGH61A2转化子的菌落直径变小,约为野生型85%,表明LtGH61A影响葡萄溃疡病菌的菌丝生长速率。【结论】LtGH61A影响葡萄溃疡病菌的致病力及生长速率;LtGH61A蛋白能够分泌至胞外;LtGH61A在病原菌侵染阶段的表达量显著升高,推测其通过发挥自身酶活功能,破坏寄主植物组织,从而促进病原菌的侵染。

关键词: 葡萄溃疡病菌, 外泌蛋白, 致病力, 表达模式, 实时荧光定量PCR, RNA干涉

Abstract:

【Objective】Grape canker disease, caused by Botryosphaeria genus fungi, occurs in a wide range of grape-producing areas in China and seriously threatens the yield and quality of grape. The objective of this study is to analyze the function of a hypothetical exocrine protein, LtGH61A, in grape canker fungus Lasiodiplodia theobromae, and to lay a foundation for in-depth analysis of the pathogenic mechanism and disease control of grape canker fungus.【Method】The signal peptide of LtGH61A protein was predicted by SignalP 4.0. The function of LtGH61A protein was predicted by the homologous comparison and functional annotation. The exocrine characteristic of LtGH61A protein was analyzed by yeast complementary experiment. The quantitative real-time PCR (qRT-PCR) was used to analyze the expression of LtGH61A in vegetative hyphae and different infection processes. The expression of LtGH61A was inhibited through RNA interference (RNAi). The effect of LtGH61A protein on the pathogenicity of L. theobromae was analyzed by in vitro inoculation test of grape shoots. The effect of LtGH61A protein on the hyphal growth rate of L. theobromae was analyzed by comparing the colony diameter.【Result】Amino acid sequence analysis predicts that the N-terminal of the LtGH61A protein contains a signal peptide with a length of 18 amino acids. The gene function annotation suggests that LtGH61A belongs to glycoside hydrolase family 61 (GH61) and can degrade cellulose as a substrate. Yeast complementary experiments showed that the signal peptide of LtGH61A protein could guide the secretion of invertase of yeast YTK12. Compared with the vegetative hyphae, the expression of LtGH61A was increased significantly at the infectious stages, and the mRNA accumulation of LtGH61A at 48 h post inoculation was 19 times of that in the vegetative hyphae. Moreover, RNAi lines were constructed for LtGH61A and two lines RNAi-LtGH61A1 and RNAi-LtGH61A2 were confirmed by qRT-PCR. The results of in vitro inoculation test of wild-type and RNAi transformants on wounded grape shoots showed that the lesion length caused by both RNAi-LtGH61A1 and RNAi-LtGH61A2 was significantly shorter than that of wild type (WT) CSS-01s, which was about 55% of WT, indicating that LtGH61A affected the pathogenicity of L. theobromae. The colony diameter comparison showed that compared with WT, the colony diameter of RNAi-LtGH61A1 and RNAi-LtGH61A2 transformants became smaller, about 85% of WT, indicating that LtGH61A affected the hyphal growth rate of L. theobromae.【Conclusion】LtGH61A affects the pathogenicity and hyphal growth of grape canker pathogen. LtGH61A protein can be secreted outside the cell. The expression level of LtGH61A during infectious stages is significantly increased, suggesting that LtGH61A can destroy the host plant tissue by exerting its own enzyme activity function, thus promoting pathogen infection.

Key words: grape canker fungus, exocrine protein, pathogenicity, expression pattern, qRT-PCR, RNA interference (RNAi)