植物病原真菌纤维素酶与植物互作的分子机制

Plant pathogenic fungi produce cellulases. However, little information is involved in cellulase as an elicitor in plant-pathogen interaction. An endocellulase (EG1) isolated from Rhizoctonia solani induced cell death in maize, tobacco and Arabidopsis leaves, and transcription of three defense-marker genes in maize and tobacco and 10 genes related to defense responses in maize. Moreover, it also induced accumulation of reactive oxygen species, medium alkalinization, Ca2+ accumulation and ethylene biosynthesis of suspension-cultured tobacco cells. Using analysis of elicitor responses for studying elicitor-plant interaction along with the mutational analysis and the PVX expression system, we demonstrated that the EG1 was an elicitor but its elicitor activity was independent of its enzymatic activity. How plant cells recognize EG1 is not understood. We hypothesize that the elicitor activity of the EG1 may be based on the specific recognition between the EG1 elicitor and a possible potential protein receptor in plants. To further understand molecular mechanism of interaction between EG1 and plant and to demonstrate the EG1 elicitor to be an MAMP (Microbe-Associated Molecular Pattern), a mutant library from maize B73 will be constructed by Mutator (Mu) transposon. From the mutant library, the possible potential protein receptor will be isolated using MuTail-PCR. Using analysis of yeast two-hybrid, bimolecular fluorescence complementation, binding between EG1 and mammalian COS-7 Cells overexpressing receptor and coimmunoprecipitation, we will demonstrate their binding between the EG1 and the possible potential protein receptor. With technology of gene silencing, complementation and overexpression, we will further demonstrate the biological function of the possible potential protein receptor and the interaction between the EG1 and the possible potential protein receptor. To examine cellular distribution of the possible potential protein receptor, we will fuse it with green fluorescent protein and be then transformed into onion and epidermal tobacco cells for its transient expression. This project will provide a new mechanism of interaction between fungal cellulase and plants.

玉米纹枯病菌的一种45家族内切纤维素酶EG1能够诱导植物细胞过敏性死亡、植物防卫反应基因表达、活性氧产生、培养基碱化、钙离子积累、乙烯合成等植物防卫反应，表现为典型的激发子功能，并且它的激发活性与酶活性没有关系。为了进一步理解真菌纤维素酶EG1与植物互作的分子机制，证明纹枯病菌纤维素酶EG1是一种MAMP分子，需要分离和鉴定它的受体蛋白。本项目以玉米与纹枯病菌为研究体系，利用玉米Mu转座子插入突变和MuTail-PCR技术，分离纹枯病菌纤维素酶EG1的受体蛋白基因；利用酵母双杂交、双分子荧光互补、动物细胞表达与结合、免疫沉淀技术，证明真菌纤维素酶EG1与玉米受体蛋白的互作关系；利用受体蛋白基因沉默、互补和超表达技术，进一步证明受体蛋白的生物学功能；利用受体蛋白在洋葱和烟草细胞的瞬时表达，确定受体蛋白的亚细胞定位。研究为植物与病原真菌纤维素酶互作的新机制提供证据。

本项目以玉米纹枯病菌Rhizoctonia solani内切纤维素酶EG1为对象，对其PAMP的活性结构域以及与植物互作蛋白进行了研究。主要研究结果：（1）为了找到EG1发挥PAMP活性的结构域，对EG1野生型及突变型的6个保守区域进行了突变。其中突变第六个保守区后，WT-M具有较高的催化活力，可以引起植物组织坏死而不能提高植物防卫反应标志基因的表达量结果证实了该保守区为EG1发挥PAMP活性的关键区域。（2）在保留其结构域完整独立的基础上对EG1基因从N端及C端进行不同长度的截短。烟草瞬时表达结果表明，截短体RsEG1-1能引起烟草叶片坏死。Rseg1基因经过截短、突变后利用烟草瞬时表达和酵母稳定表达以及体外接种实验等一系列研究证明第223~227位氨基酸所在的肽段（GCSRK）为该酶激发功能必不可缺的关键区段，而双点突变证明第224位半胱氨酸和第226位精氨酸为该酶激发功能关键位点。（3）以EG1基因cDNA为诱饵构建酵母双杂交诱饵载体，筛选玉米cDNA文库，经过对阳性克隆的报告基因检测、DNA测序和BLAST比对分析，确定与WT相互作用的蛋白。本实验筛选到的与WT相互作用的阳性克隆共有53个，进一步分析有48个阳性克隆分别属于43种不同的蛋白编码基因。（4）本研究利用玉米纹枯病菌内切纤维素酶EG1的野生酶和催化位点D32突变酶D32A接种玉米叶片，转录组分析发现EG1诱导玉米多种基因（特别与植物防卫相关的基因）的上调表达，野生酶和突变酶差异表达基因分别为2318和2248。（5）用Pull-down 分析EG1与玉米互作蛋白，从玉米膜蛋白中发现有15个蛋白和3个富含LRR的跨膜蛋白与EG1有潜在互作关系。用TMHMM预测三个膜蛋白结构，发现这三个蛋白由细胞内区、跨膜区、胞外区三部分组成。（6）利用VIGS技术对本氏烟中的22个LRR-RLK和LRR-RLP基因进行沉默，确定EG1与它们的互作关系。结果显示该22个LRR-RLK/RLP受体均不于EG1互作，它们可能不是EG1的受体蛋白。项目上述结果为深入研究EG1与植物互作的分子机制提供了重要线索。