棘孢木霉MYB36转录因子抗链格孢菌的分子调控机制

Trichoderma asperellum is an excellent biocontrol strain with broad spectrum of antifungal properties. We had identified a transcription factor MYB36 from T. asperellum that is strongly induced by stimulus of poplar leaf wither Alternaria alternata. Additionally, the transformed poplar (Populus davidiana × P. alba var. pyramidalis) overexpressing MYB36 showed highly improved resistance to A. alternata. Based on these studies, we will further investigate the mechanism of T. asperellum against A. alternata in this project. The T. asperellum strains that overexpression (Ta-Hi) and knockout/inhibited expression (Ta-In) of MYB36 will be generated for study. The resistant property to A. alternata conferred by MYB36 and hydrolytic enzyme activities and products of metabolism regulated by MYB36 will be studied by comparison the strains among Ta-Hi, Ta-In and Ta-Wt (wild type T. asperellum). RNA-Seq is used for identification of the target genes regulated by MYB36. The target genes directly regulated by MYB36 and cis-acting element bound by MYB36 in gene expression regulation will be determined by using ChIP-Seq experiments. Co-immunoprecipitation (CoIP) and yeast two hybrid are used in identification the proteins interact with MYB36, and the expression regulation activity affected by protein interaction of MYB36 will be further studied. These studies together will reveal the molecular mechanism of T. asperellum against A. alternata.

棘孢木霉是对植物病原真菌有广谱抗性的生防菌。我们从棘孢木霉中分离了强烈响应杨树叶枯病病原菌链格孢菌刺激的转录因子MYB36，将其转化山新杨转基因植株也显示出优良的抗叶枯病能力，提示其在棘孢木霉中起调控抗链格孢菌的作用。在此基础上拟研究MYB36抗链格孢菌机制。分别培育出MYB36基因过表达Ta-Hi木霉菌株以及敲除/抑制表达Ta-In木霉菌株，用野生型木霉Ta-Wt作对照，分析MYB36的抗链格孢菌能力，及其对抗菌相关水解酶及代谢产物的调控；用链格孢菌分泌物分别诱导Ta-Hi、Ta-In和Ta-Wt菌株，通过RNA-Seq鉴定MYB36的靶基因；利用ChiP-Seq鉴定MYB36直接调控的靶基因及其所结合的顺式作用元件；通过Co-IP与酵母双杂交研究MYB36的互作蛋白，并进一步研究蛋白互作对MYB36转录活性的调控。通过以上研究揭示棘孢木霉抗链格孢菌的分子机制。

木霉转录因子如何调控其抗病基因的表达来提高抗杨树叶枯病链格孢菌能力的研究，为木霉菌在杨树叶枯病上的生防应用提供理论依据。本研究以棘孢木霉Tas653野生型（Ta-Wt）为材料，采用原生质体介导法获得过表达Ta-Hi、敲除Ta-Kn和抑制表达Ta-In型转基因木霉共60株。与Ta-Wt相比，Ta-Hi对链格孢菌Aa1004抑制率提高32%，发酵滤液使Aa1004菌丝膨大变形甚至破裂，还提高氧化应激相关酶活性和基因表达量来提高其生防能力；1/2 Ta-Kn发酵滤液对Aa1004抑制率降低32%，Aa1004次生代谢物诱导后Ta-Kn的S50、C820和P120基因下调-3.43、-8.1和-2.43倍导致抗氧化能力降低。比较Aa1004次生代谢物诱导后Ta-Wt和Ta-Kn的转录组发现TaMYB36调控的下游抗病相关转录因子有5类71个，为MYB、bZIP、bHLH、真菌特异转录因子（FS TFs）和锌指转录因子（Zn TFs）；以TaMYB36为中心的酵母单杂交筛选到6个DNA序列（5个已知生防相关和一个未知序列）包含9个顺式作用元件。其中，“TGTGACATAG”（已知）和“TCCCATCGAT”（未知新序列）核心基序分别为“ACAT”和“AGCT”，凝胶迁移和染色质免疫共沉淀分别在体外与体内验证TaMYB36可与“ACAT”和“AGCT”结合。此外，酵母双杂交验证TaMYB36蛋白无自激活活性。RNA-seq筛选数量多且表达量高的差异基因进行RT-qPCR，将结果与RNA-seq相符且启动子区包含“ACAT”和“AGCT”的差异基因分为TaMYB36直接调控的转录因子5个（bZIP61.5，MYB38.1，ZF83.7，ZF67.4和ZF58.0）和直接调控的生防基因20个（5个P450，4个蛋白激酶，4个水解酶和7个转运蛋白）。最终表明TaMYB36直接调控bZIP61.5，MYB38.1，ZF83.7，ZF67.4和ZF58.0来间接调控抗病，同时直接调控细胞色素P450家族和ABC转运蛋白家族基因来应对病原菌毒素和过氧化胁迫，通过直接调控蛋白激酶来传递抗病信号，通过直接调控水解酶的表达来拮抗Aa1004。本项目明确了TaMYB36转录因子调控棘孢木霉抗链格孢菌的作用，揭示其抗链格孢菌的分子机制。