假禾谷镰孢菌有性生殖及其调控的分子基础

Fusarium pseudograminearum, a heterothallic fungal pathogen newly found, caused wheat crown rot and wheat head blight. Our preliminary data showed that the relatively abundant mature perithecia could be generated naturally in the field, which is rare in nature abroad and unknown whether the connection with the variation of pathogenicity and the epidemics of wheat head blight. Therefore, we propose to investigate the host ranges systematically and detected the pathogenicity on wheat; The collection of perithecia in the field and capture of ascospores in the air would be applied to link the dynamic of discharge and epidemics of wheat head blight. The transcriptome during the time course of perithecia formation would be performed using high fertile strains to explore the gene expression profiles and putative RNA editing events. Meanwhile, the function of the mating type genes and another two genes related to sexual development would be evaluated through knockout strategy by the analysis of several characteristics including mutants’ morphology, pathogenicity, fertility, and differential expression of genes under different mutant background compared to the wild-type. Further analysis of the targets regulated by products of the mating types and the related genes in sexual stage as the transcription factors would be conducted to generate the network of regulation during sexual development. Given these points above would enhance understanding of significance and mechanism of the sexual reproduction of Fusarium pseudograminearum and conduct the effective control of the increasing emergence diseases.

假禾谷镰孢菌引起小麦茎基腐和赤霉病，我国新近发现，发生面积和为害程度连年加重。该菌为异宗配合子囊菌，前期研究发现，与国外报道的子囊壳少见相反，田间子囊壳形成的数量多且育性高，但该有性生殖特性在致病性变异与流行中的作用还不清楚。本项目拟系统分离该菌田间不同寄主的分离物，测定对小麦的致病性，明确寄主范围；通过田间采集子囊壳和空中捕捉子囊孢子，明确子囊孢子释放动态与赤霉病流行的关系；通过室内有性杂交筛选高育性菌株，利用转录组测序分析子囊壳发育过程的基因表达谱和RNA编辑；通过敲除交配型基因及其它有性发育相关基因，从突变体表型、致病性、育性、基因表达差异、回补和与禾谷镰孢菌交叉互补等方面，分析基因功能，并进一步揭示交配型基因产物和有性生殖相关转录因子的互作靶标，了解有性生殖调控网络。上述研究将有助于深入了解假禾谷镰孢菌有性生殖规律和作用，为该菌引起的小麦茎基腐和赤霉病的有效防控提供理论指导。

假禾谷镰孢引起小麦茎基腐病和赤霉病，在我国小麦灌浆期就产生成熟的子囊壳，育性高，与国际上报道的不同，其在遗传分化上的意义有待揭示。.采用Split-maker方法敲除5个MAT基因(MAT1-1-1，MAT1-1-2，MAT1-1-3，MAT1-2-1，MAT1-2-3)，3个在MAT-1位点，2个在MAT-2位点。与野生型比较，其中△MAT1-1-1、△MAT1-1-2和△Mat1-2-3表型没有明显改变，△Mat1-1-3和△Mat1-2-1有多方面显著缺陷。酵母双杂显示，Mat1-1-1与Mat1-2-1和Mat1-1-3互作；Mat1-2-1还与Mat1-2-3和Mat1-1-3互作。MAT基因在侵染阶段均显著上调表达。.△MAT1-1-3和△Mat1-2-1菌落生长慢，气生菌丝缺乏，菌丝顶端分叉增多且密集，有角变现象；在CMC中产孢量显著降低，在PDA、SNA固体培养基上却显著提高。接种大麦叶片和小麦胚芽鞘以及土壤接种表明△Mat1-1-3致病性下降，但伤口接种没有差异，突变体附着孢侵染有缺陷。△Mat1-1-3对H2O2和SDS耐受性增强，一定浓度的NaCl和Sorbitol胁迫反而促进菌落生长，CR能恢复扇形突变。室内杂交，△Mat1-1-3子囊壳的形成早（15天），数量大，但没子囊孢子。自身启动子驱动的野生型MAT1-1-3基因回补了△Mat1-1-3突变表型。对Mat1-1-3-FLAG pull-down验证，Mat1-1-3能与FPSE_06440编码的线粒体ATP合成酶互作。.△Mat1-2-1丝融合现象鲜见。菌丝体的表面疏水性显著下降。△Mat1-2-1在CMC中产孢量显著降低，但孢子长度极显著增加；△Mat1-2-1对大麦叶片和小麦胚芽鞘致病性显著下降，侵染菌丝在胚芽鞘细胞生长畸形，扩展受阻。△Mat1-2-1对H2O2和SDS敏感，对NaCl、KCl耐受性显著增加，Sorbitol和CR能显著促进突变体气生菌丝生长。MAT1-2-1定位于细胞核。.优化了室内诱导子囊壳产生条件，Sachs培养基，加水稻茎杆，22℃对峙培养，黑光灯和冷白光灯交替光照，60 d左右；获得高育性菌株，KF86 × KF73产生大量子囊壳和成熟子囊孢子，产生子囊壳的时间比国外报道提前1个月左右。.本研究为进一步假禾谷镰孢致病性分化和流行传播规律奠定了基础。