层出镰刀菌分泌蛋白阿魏酸酯酶(FpESD)的鉴定及功能研究  

Identification and Functional Analysis of the Secretory Protein Feruloyl Esterase(FpESD)in Fusarium proliferatum

作　　者：王艺潼 李海博 王雨晴 吴志宏 高贻宙 WANG Yi-Tong;Li Hai-Bo;WANG Yu-Qing;WU Zhi-Hong;GAO Yi-Zhou(School of Biological and Chemical Engineering,Zhejiang University of Science and Technology,Hangzhou 310023,China)

机构地区：[1]浙江科技大学生物与化学工程学院,杭州310023

出　　处：《农业生物技术学报》2025年第2期386-399,共14页Journal of Agricultural Biotechnology

基　　金：浙江科技学院人才引进启动经费(F701103M09);浙江省教育厅一般科研项目(Y202351867)。

摘　　要：层出镰刀菌(Fusarium proliferatum)寄主范围广泛、为害严重,但对于其致病机制的认识尚不清晰。效应蛋白在病原真菌侵染过程中发挥重要作用,其预测、筛选及鉴定对探究病原真菌与寄主的互作机理具有重要意义。本研究基于层出镰刀菌的转录组测序结果,通过信号肽鉴定、跨膜域筛选、半胱氨酸数量分析及病原宿主互作数据库(Pathogen Host Interactions database,PHI-base)分析等流程,筛选出43个候选效应蛋白。针对其中1个候选效应蛋白阿魏酸酯酶FpESD(esterase D)展开了功能研究,通过酵母系统验证FpESD的分泌特性,结果显示,其N端20个氨基酸编码的信号肽具有分泌活性。通过同源重组法构建FpESD基因敲除突变体(ΔFpESD),并利用回补实验获得回补菌株(ΔFpESD-C),对其生长速率、产孢量和孢子长度进行测定,结果表明FpESD基因的缺失显著降低了菌株的生长速率和产孢量(P<0.05),敲除突变体分生孢子的长度明显变短。此外,在环境胁迫实验中,ΔFpESD菌株对CaCl_(2)、MgCl_(2)及刚果红的敏感性显著降低(P<0.05),表明FpESD参与调控层出镰刀菌对环境胁迫的响应。通过平板接种法测定ΔFpESD菌株对苜蓿(Medicago sativa)的致病力,结果显示,ΔFpESD菌株侵染后,苜蓿幼苗的病情指数显著低于野生型(P<0.05),表明FpESD正调控层出镰刀菌的致病性。本研究结果有助于解析层出镰刀菌的致病性分子机制,同时为层出镰刀菌效应蛋白的深入研究提供了思路。Fusarium proliferatum is a phytopathogenic fungus with a wide host range and serious damage,but its pathogenic mechanism is not well understood.Effectors play an important role in the infection process of phytopathogenic fungi.The prediction,screening,and identification of effectors are of great importance in exploring the mechanism of host-pathogen interaction.In this study,based on the transcriptome sequencing results of F.proliferatum,43 putative effectors were screened by signal peptide identification,transmembrane domain screening,cysteine number analysis,and Pathogen Host Interactions database(PHI-base)analysis.Functional studies were carried out on one of the candidate effectors,feruloyl esterase FpESD.Yeast system validation revealed that the signal peptide encoded by the N-terminal 20 amino acids of FpESD possesses secretion activity.The homologous recombination strategy was used to generate the FpESD knockout mutant(ΔFpESD),and the complementary strain(ΔFpESD-C)was obtained through complementation experiments.Growth rate measurements,spore yield assessments,and spore length analysis demonstrated that the deletion of the FpESD gene significantly reduced fungal growth rate and conidia production(P<0.05),with shorter conidia length observed for the knockout mutant.Additionally,environmental stress tests revealed thatΔFpESD exhibited significantly reduced sensitivity to CaCl_(2),MgCl_(2),and Congo red(P<0.05),indicating that FpESD was involved in regulating the response to environmental stress.Pathogenicity assays using plate inoculation showed that the disease index of alfalfa(Medicago sativa)seedlings infected byΔFpESD was significantly lower than that of the wild-type strain(P<0.05),suggesting that FpESD positively regulated the pathogenicity of F.proliferatum.This study helps to elucidate the pathogenic molecular mechanism of F.proliferatum,and also provides ideas for the in-depth study of F.proliferatum effectors.

关 键 词：层出镰刀菌 效应蛋白 阿魏酸酯酶 致病性 

分 类 号：S432.1[农业科学—植物病理学]