分泌型RNase T2家族蛋白在球孢白僵菌突破昆虫免疫防御过程中的作用

批准号：

32072485

项目类别：

面上项目

资助金额：

58.0 万元

负责人：

张永军

依托单位：

西南大学

学科分类：

生物防治

结题年份：

2024

批准年份：

2020

项目状态：

已结题

项目参与者：

张永军

关键词：

致病性昆虫病原真菌作用靶标免疫逃避分泌性T2家族核糖核酸酶

项目摘要

结项摘要

张永军的其他基金

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中文摘要

分泌型RNase T2广泛分布于生物体，独立于胞内RNA加工参与核糖核酸清除、宿主细胞免疫、植物自交不亲和、细胞膜稳定性等细胞学过程。然而，分泌型RNase T2是否参与病原菌与宿主互作，尚不明晰。申请人在解析昆虫病原真菌球孢白僵菌细胞表面蛋白与环境及宿主互作关系时发现，两个分泌型RNase T2蛋白BbTrv和BbRNT2分布于细胞表面。其中BbTrv受昆虫血细胞和氧化胁迫诱导，可分泌至胞外并对昆虫血细胞产生毒性，抑制免疫反应。该发现为解析分泌型RNase T2参与病原菌逃避宿主免疫反应的机制提供了重要线索。本项目拟结合菌株遗传操作及异源表达蛋白，系统解析两个RNase T2蛋白与病原菌突破昆虫免疫防御的关系及作用方式，鉴定两个蛋白作用于宿主细胞的靶标并解析其作用机制。项目的开展将为揭示病原真菌-宿主昆虫互作机制提供新线索，同时，研究结果将有望为改良和提升真菌生防效果提供理论依据。

英文摘要

Secreted RNase T2 proteins are widespread family of RNases found in every organism examined thus far, which are independent of cellular RNA processing and participate in vital cellular functions such as salvage of RNAs, defense against pathogens, self-incompatibility, membrane permeability or stability, and likely other cellular processes. However, it is unknown whether secreted RNase T2 proteins of pathogens are involved in pathogen-host interaction. Insect fungal pathogens adopt a variety of morphological switch to adapt to varied environment and host. Two secreted RNase T2 proteins, BbTrv and BbRNT2 were found to present in cell surface in Beauveria bassiana when we revealed the cell surface proteins from different mophological cells in this fungal pathogen. One of the protein-encoding genes, BbTrv was induced by insect haemolymph and oxidative stress. Besides distribution of cell surface, the coding protein also secreted into substrate to to supress the insect immune response due to its cytotoxicity to hemocyte, which provide an important cue for reveal role of the secreted RNase T2 in supressing immune defence of the host insect. In this project, we would systematically reveal contribution of the two RNase T2 proteins to pathogenicity and breaking immune response in B. bassiana by using of genetically modifiticated fungal strains and the protein expressed in vitro. And then, the mechanism of the two RNase T2 proteins that act on the host cell will be invesigated by identified the targeted proteins in host cells that are interated with fungal RNase T2 proteins. The goals of the project are to expant understanding of secreted RNase T2 protein in microbiol pathogens. Another objective is to expect to provide a novel cue for understanding mechanism of interaction between the fungal pathogen and host insect. Furthoremore, we hope to provide a theoretical basis for improvement of fungal the fungal agent using genetic engineering.

病原体分泌的RNase T2蛋白在不同的病原体-宿主互作中扮演了不同的角色。然而，大多真菌病原体分泌的RNase T2蛋白在其与宿主互作的关系及其作用机制尚不明晰。课题组研究发现，昆虫生防真菌球孢白僵菌与环境及宿主互作过程中分泌两个RNase T2蛋白，命名为BbRNT2和BbTrv，其中后者的基因表达受昆虫血淋巴和氧化胁迫诱导，可分泌至胞外并对昆虫血细胞产生毒性。项目基于该基础，结合基因表达分析及菌株遗传操作系统研究了两个RNase T2蛋白在病原真菌与昆虫互作过程的作用，发现两个蛋白均参与菌株氧化胁迫反应及侵染致病。分别敲除单个基因不影响菌株生长、氧化胁迫敏感性及毒力，而同时敲除两个基因则降低了菌株生长，显著增强了菌株氧化敏感性并降低菌株毒力，表明二者存在功能互补性。过表达单个基因和两个基因，均促进了菌株生长，增强氧化耐受性，并激活昆虫免疫反应引起昆虫快速死亡。然而，过表达菌株在虫体内增殖低于亲本菌株，昆虫血细胞却显著减少，该现象与菌株毒力提高和易逃避免疫反应不一致，推测过表达菌株可能分泌过量的BbRNT2和BbTrv引起昆虫细胞毒性并过度激活免疫反应导致昆虫死亡。利用酵母表达蛋白验证发现，两个蛋白均依赖于核糖核酸酶活性引起昆虫细胞毒性，并激活体液免疫反应。以草地贪夜蛾sf9细胞为模式探究两蛋白与昆虫细胞互作，发现两蛋白可快速聚集于昆虫细胞膜并进入细胞引起细胞破裂，推测其可能分别与昆虫细胞膜互作而聚集并进入细胞。为验证该推测，利用蛋白互作等策略分离鉴定了与BbRNT2和BbTrv互作的sf9细胞膜蛋白，分别为一个ABC转运蛋白SfABCG和Innexin 7类蛋白Sfinx。采用RNAi转染沉默两个膜蛋白基因，发现分别沉默两个基因显著降低了sf9细胞对BbRNT2和BbTrv的敏感性，表明与特定昆虫细胞膜蛋白结合是两个RNase T2蛋白引起细胞毒性必需的。项目研究结果揭示了昆虫病原真菌分泌的RNase T2蛋白在其与昆虫互作的关系及作用机制，为菌株改良提供了目的基因和理论支撑。