辣椒炭疽菌中分泌蛋白找寻及其致病性分析

Secreted proteins and pathogenicity of Colletotrichum truncatum

摘要:

背景 辣椒是我国重要的经济作物之一，主要种植区域涉及云南、贵州和四川等地。云南辣椒产业在全国占据着较大的种植份额，近些年辣椒炭疽病对辣椒生产及产业发展造成严重威胁。由平头炭疽菌(Colletotrichum truncatum)引起的辣椒炭疽病是危害云南辣椒生产的重要病原真菌之一，相关基础研究落后于云南辣椒发展布局。植物病原真菌中分泌蛋白是其侵染寄主植物过程中发挥定殖、致病等作用的重要“武器”，然而，目前学术界尚缺乏对于平头炭疽菌中分泌蛋白的预测及其特性的研究报道，严重制约着新型绿色药剂作用靶标的开发利用。目的 明确平头炭疽菌中分泌蛋白数量、理化性质及其致病性、拮抗作用。方法 针对真菌分泌蛋白所具有的核心特征属性，采用软件SignalP v6.0、ProtComp v9.0、TMHMM v2.0、BIG-PI Fungal predictor和TargetP v2.0等生物信息学工具对平头炭疽菌中15 830条蛋白质序列进行预测、理化性质分析，运用SMART软件进行保守结构域分析，并采用室内接种、平板对峙法对其致病性、拮抗作用分别进行测定。结果 C. truncatum中分泌蛋白数量为96个，占总蛋白质数量的0.60%，其氨基酸数量在94-521 aa之间，均为亲水性蛋白，亲水性氨基酸残基数量最多的是苯丙氨酸、谷氨酰胺、丝氨酸、甘氨酸，疏水性氨基酸残基数量最多的是亮氨酸。仅有4个分泌蛋白具有明显的保守结构域，包括2个蛋白含有LysM结构域，2个蛋白具有GH结构域；病菌接种辣椒果实3-5 d后发病，致病性较强，生防菌株S1对病原菌抑菌率高于70%，拮抗活性较好。结论 为开发基于S1菌株的辣椒炭疽病靶向生物防治策略提供了理论依据，为云南辣椒炭疽菌分泌蛋白的功能研究奠定了坚实基础，也为进一步开展辣椒炭疽病菌致病机制解析提供了重要的数据支撑。

Abstract:

Background Pepper (Capsicum annuum) is an important economic crop in China, mainly planted in Yunnan, Guizhou, and Sichuan provinces. The pepper industry in Yunnan occupies a large proportion in China. In recent years, pepper anthracnose has posed a serious threat to pepper production and future development of this industry. Pepper anthracnose caused by Colletotrichum truncatum, a pathogenic fungus, causes harm to the production of pepper in Yunnan, while the related basic research lags behind the development strategy of pepper in this province. Secreted proteins of plant pathogenic fungi are important “weapons” that play a role in the colonization and infection in host plants. However, few studies have reported the prediction and characteristics of secreted proteins of C. truncatum, which seriously restricts the development and utilization of targets of new green biocontrol agents.Objective To clarify the quantity, physicochemical properties, pathogenicity, and antagonistic effects of secreted proteins in C. truncatum.Methods On the basis of the characteristics of fungal secreted proteins, We employed bioinformatics tools including SignalP v6.0, ProtComp v9.0, TMHMM v2.0, BIG-PI Fungal predictor, and TargetP v2.0 to predict and analyze the physicochemical properties of 15 830 protein sequences from C. truncatum, SMART software was used for conserved domain analysis, and indoor inoculation and plate confrontation methods were used to determine their pathogenicity and antagonistic effects.Results The number of secreted proteins in C. truncatum is 96, accounting for 0.60% of the total protein count. The secreted proteins had the lengths ranging from 94 to 521 aa. They were hydrophilic proteins, with phenylalanine, glutamine, serine, and glycine being the hydrophilic residues with the largest number and leucine being the hydrophobic residue with the largest number; After 3-5 days of inoculation with the pathogen, the chili fruit develops disease with strong pathogenicity. The biocontrol strain S1 has an inhibition rate of over 70% against the pathogen and good antagonistic activity.Conclusion This provides a theoretical basis for the development of targeted biocontrol strategy for pepper anthracnose based on S1 strain, especially in reducing chemical fungicide dependence and promoting sustainable control in the field. The findings provide important data support for further analysis of the pathogenic mechanism of C. truncatum and lay a solid theoretical foundation for further research on the functions of the secreted proteins of C. truncatum in Yunnan.