稻曲病菌致病相关基因UvSUN2的鉴定及其功能研究

Rice false smut, caused by Ustilaginoidea virens, is a common grain disease of rice around the world, with both quantitative and qualitative losses, and has been observed in severe in recent years. However, the studies on the molecular mechanisms involved in infection processes and pathogensis of the U. virens are few. Thus, to shed light on the pathogenic mechanism of the U. virens, we carried out ATMT of the wild-type U. virens, and obtained 15600 hygromycin- resistant transformants. In screening experiments, one of the mutants, B20, was identified as a nonpathogenic mutant, with completely incapable of causing false smut balls on susceptible rice. B20 exhibited retarded vegetative growth of mycelia and decreased sensitivity to H2O2 stresses compared to wild type strain. Southern blot analysis showed that a single T-DNA integration event occurred in the genome of the mutant. Because of the encoding a protein with similarity to the SUN proteins, which appears restricted to ascomycetes, we named the T-DNA-tagged gene UvSUN2. In this project, we will present the identification and functional characterization of the novel pathogenic-related gene UvSUN2. Based on the nucleic acid and amino acid sequence analysis, firstly, the identification of morphogensis and virulence in the knockout mutant and the complementary mutant, will be carried out to reveal the biological function of UvSUN2 gene in the development process, combined with homologous genes of other ascomycetes complementation experiments, as well as the the temporal and spatial expression patterns of the gene during the developmment and infection processes of the wild-type U. virens, and the observation of subcellular localization. Moreover, to study on the effect of UvSUN2 during infection, the colonization and the expansion of EGFP-labeled knockout mutant will be observed compared with EGFP-labeled wild-type strain after inoculation. Additionally, RNA sequence will be used to analyse the differentially expressed genes associated with UvSUN2 during infection. All above, this study is therefore of great significance to clarify the molecular pathogenic mechanism of the U. virens. And the expected results will provide the molecular targets for developing new anti-U. virens drugs, and offer the theoretical basis to the durable disease control strategies and to the breeding rice for resistant to rice false smut.

稻曲病菌引起的稻曲病是水稻生产上重要致灾病害，但目前对该病菌的致病机制却鲜有报道。申请人前期筛选获得一株T-DNA单拷贝插入突变体，与野生型相比表现出致病力完全丧失、生长速率下降、耐氧胁迫性增强等性状。序列分析表明突变基因UvSUN2编码产物与子囊菌特有的SUN蛋白家族同源。本项目在克隆突变基因全长基础上，拟通过获得敲除突变体及回补菌株，结合其它真菌同源基因互补等实验手段，鉴定该基因的功能；利用荧光蛋白标记，观察野生型和突变体在水稻组织内的侵染、扩展情况以及该蛋白的亚细胞定位；对比不同生长阶段、不同培养条件以及侵染过程中该基因的表达模式；转录组测序找寻野生型和突变体在接种水稻后的差异表达基因等方法，阐明基因在致病过程中的作用机制。研究结果不仅对揭示UvSUN2决定稻曲病菌致病性的机理具有意义，而且对SUN蛋白家族基因的功能揭示也有参考价值，此外也可为防治药物开发和分子抗病育种提供新的靶标。

稻曲病菌可侵染水稻引起水稻稻曲病，该病害的发生会严重影响水稻产量和稻米品质。随着气候变暖、氮肥施用量增加、杂交水稻大面积推广等因素的影响，稻曲病已逐渐成为水稻重要病害之一，明确致病机制是有效防控病害发生的关键。然而稻曲病菌致病机制研究进展缓慢，存在初侵源不明确、侵染过程不清晰等问题，许多关于稻曲病发病过程研究尚处推测阶段。因此，克隆和鉴定关键的致病相关基因，并以此为突破口，在分子水平解析稻曲病菌致病机制，对推动稻曲病菌致病机制研究具有重要价值。本项目在前期研究基础上，1）克隆了稻曲病菌中两个含SUN domain的蛋白，对蛋白的结构域和进化进行了系统分析；2）用同源双交换、基因沉默和Crispr-cas9系统等方法进行了基因沉默及敲除，UvSUN2沉默突变体致病性与T-DNA插入突变体一致，而UvUTH1沉默突变体表现为致病性增强；3）基因表达模式分析表明UvSUN2在稻曲病菌侵染早期发挥作用，UvUTH1参与了分生孢子的萌发；4）酵母双杂交筛选到两个蛋白的互作蛋白，分别为3个和9个。本项目研究了稻曲病菌致病相关基因UvSUN2及其含同源结构域的UvSUN1蛋白，并对它们的功能进行了深入的分析，研究结果可为杀菌剂的创制提供潜在的分子靶标，为抗病品种选育提供可靠的理论依据，还将在分子水平加深对稻曲病菌致病机制的理解。