bHLH家族关键转录因子UvBHLH6对稻曲病菌致病过程的调控机制研究

Rice false smut, caused by Ustilaginoidea virens, is an destructive panicle disease in China, which seriously threaten the quality of rice production. bHLH transcription factor (TF) is widely involved in the regulation of carbohydrate, lipid and other substances metabolic processes. The mechanism of this family in the development and pathogenic processes of U. virens is still unclear. Previous study has shown that UvBhlh6 was essential for the maintenance of the virulence in U. virens. It is speculated that UvBHLH6 play an important role in the interaction between U. virens and rice by regulating the nutrients utilization. In this project, the UvBHLH6 gene deletion mutants were obtained by CRISPR-Cas9 system, and then analysis the differences between ΔUvbhlh6 and the wild type strain in the invasion to rice panicle. The reasons why ΔUvbhlh6 could not form smut balls were explained by analyzing the differences in carbohydrate and lipid metebolism. The downstream target genes of UvBhlh6 will be analyzed by RNA-Seq, ChIP-Seq and EMSA, subsequently some important target genes’ biological functions will be analyzed by gene deletion. The results of this study will reveal the molecular basis of bHLH TFs in U. virens, and broaden the understanding of pathogenic mechanism of U. virens and its interaction mechanism with rice.

由稻曲病菌侵染引起的水稻稻曲病严重威胁水稻质量和安全，是我国水稻生产的重要穗部病害。bHLH转录因子参与调控真核生物糖类、脂质等物质代谢过程，该家族转录因子在稻曲病菌致病过程的作用机制尚不清楚。前期研究发现bHLH家族转录因子UvBHLH6的基因缺失突变体接种水稻穗部后不能形成稻曲球，推测UvBHLH6主要通过调控营养物质的吸收利用进而参与稻曲病菌和水稻的互作过程。本项目以UvBHLH6为研究对象，通过CRISPR-Cas9技术获得基因敲除突变体，分析ΔUvbhlh6与野生型在侵染水稻穗部时的差异，通过糖类、脂类等物质代谢差异分析突变体不能形成稻曲球的原因；结合RNA-Seq、ChIP-Seq和EMSA等寻找UvBhlh6的下游靶标基因，并分析部分重要靶标基因的生物学功能。本研究结果有助于揭示bHLH转录因子调控稻曲病菌致病过程的分子机制，扩宽对稻曲病菌致病机理及其与水稻互作机制的认识。

稻曲病是我国水稻生产的重要穗部病害，严重威胁稻米产量和品质。稻曲病菌Ustilaginoidea virens的致病机制具有一定的特异性。bHLH转录因子作为真核生物中高度保守的转录因子家族，广泛参与调控糖类、脂质等物质代谢过程，本项目研究bHLH家族关键转录因子UvBHLH6在稻曲病菌侵染过程的作用机制。UvBHLH6的基因缺失突变体在稻曲病菌菌丝生长和分生孢子产生方面与野生型无显著差异，但接种水稻穗部后不能形成稻曲球，UvBHLH6主要参与调控稻曲病菌的致病过程。表达谱分析显示UvBHLH6在接种水稻穗部后3天时表达量最高，该基因可能在接种水稻初期发挥主要作用。bHLH结构域的作用特点之一是可以与自身或其它bHLH蛋白形成同源或异源二聚体，结合于下游靶标基因启动子区的同一位点或不同位点，共同调控下游靶标基因的表达。通过酵母双杂交实验和双分子荧光互补实验，表明稻曲病菌中UvBhlh6与UvBhlh1、UvBhlh4，UvBhlh6和UvBhlh7均存在相互作用。UvBHLH1的基因缺失突变体致病能力减弱，UvBHLH7的基因缺失突变体致病能力丧失，转录因子UvBHLH1和UvBHLH7也参与调控稻曲病菌致病过程。上述结果表明bHLH转录因子UvBhlh1、UvBhlh6和UvBhlh7可能通过形成同源、异源二聚体或多聚体共同参与稻曲病菌致病过程的调控。本项目研究结果是对稻曲病菌bHLH转录因子调控机制的探索，对于揭示转录因子在稻曲病菌致病过程的协同调控网络有重要参考价值。