水稻灌浆转录因子RISBZ1和RPBF调控稻曲球形成的分子机理

Rice false smut (RFS) disease occurs more and more frequently in recent years, and study on RFS becomes an active area of plant pathology. As RFS ball is the typical symptom of this disease, uncovering the mechanism of the RFS ball formation will provide important insight into the pathogenesis of RFS. Previously, we investigated the microscopic and transcriptional responses of rice to the pathogen, and found that the ovaries of pathogen (Ustilaginoidea virens)-infected flowers were interrupted in fertilization and grain-filling. Intriguingly, a set of rice grain-filling-related genes, such as key transcription factors RISBZ1 and RPBF, and genes encoding seed storage proteins or related to grain starch synthesis, were activated in U. virens-infected flowers. Moreover, knocking-out of RISBZ1 resulted in significantly reduced susceptibility of rice to RFS, with less number of RFS balls. These data lead to a hypothesis that U. virens may hijack rice grain filling system to acquire abundant nutrients for the formation of RFS ball. In this proposal, the roles of RISBZ1 and RPBF in RFS ball formation will be thoroughly explored using methods of gene knock-out and gene silence. Detailed temporal and spatial expression patterns of RISBZ1, RPBF and their target genes, including genes encoding seed storage proteins and genes related to grain starch synthesis, will be characterized in U. virens-infected rice flower. To track the products of grain-filling-related genes during infection, accumulation patterns of rice seed storage proteins and starch granules will be examined. The outcome of the proposed research will clarify the molecular mechanism of the formation of RFS ball regulated by RISBZ1 and RPBF, will reveal a novel mechanism of U. virens hijacking rice nutrient reservoir, and will provide gene candidates for engineering RFS resistance in rice.

稻曲病的发生日益严重，成为植物病理学研究的一个热点。稻曲球是稻曲病的典型症状，解析稻曲球的形成机理将深入认识稻曲病的发病机制。我们前期研究发现，稻曲病菌侵染水稻颖花阻断其开花受精，但能激活灌浆关键转录因子RISBZ1、RPBF及其调控的种子储存蛋白基因和籽粒淀粉合成相关基因的表达，而且RISBZ1的敲除突变体对稻曲病的敏感性显著下降、稻曲球形成数目减少，说明稻曲病菌可能通过劫持水稻灌浆系统获取营养形成稻曲球。在此基础上，本项目拟通过基因敲除和基因沉默方法深入分析水稻灌浆转录因子RISBZ1和RPBF在稻曲球形成中的作用，研究RISBZ1、RPBF及其靶基因被稻曲病菌激活的时期与组织，明确感病颖花中灌浆基因相关产物的去向，以期阐明RISBZ1和RPBF调控稻曲球形成的分子机理，揭示稻曲病菌劫持水稻营养库的新机制，为创制稻曲病抗性材料提供基因资源。

稻曲球的形成是稻曲病唯一可见症状，也是稻曲病毒素危害的源头，明确其形成机制对解析稻曲病发病机理有重要意义。前期研究发现，稻曲病菌侵染的水稻颖花开花相关基因的表达被抑制、开花授粉过程被阻断，但感病颖花中大量灌浆相关基因被激活，例如水稻种子储存蛋白基因、籽粒淀粉合成相关基因、灌浆过程中关键转录因子RISBZ1和RPBF等。本项目研究发现，当稻曲病菌接种于雄性不育水稻材料时，灌浆相关基因RISBZ1和RPBF等的表达也被激活，并产生成熟稻曲球。非靶代谢组分析表明，感病水稻接种稻曲病菌后9天颖花样品的代谢谱发生明显改变，且与灌浆过程引起的代谢谱变化相似，暗示稻曲病菌侵染颖花可能模拟水稻灌浆并促进灌浆相关代谢产物的合成。继而，我们在不同水稻背景中敲除RISBZ1和RPBF基因，通过稻曲病人工接种发现，与野生型相比，突变体中稻曲球的形成数目显著减少，说明RISBZ1和RPBF对稻曲病菌成功侵染并形成稻曲球是必需的。risbz1和rpbf突变体籽粒的粒长离宽与野生型相比没有显著差异，但垩白度明显增加，说明RISBZ1和RPBF基因突变可引起水稻籽粒灌浆物质的累积发生改变。启动子-GUS报告基因分析发现，RISBZ1及其靶基因的表达在浆片中受稻曲病菌诱导表达。进而，利用浆片转录组筛选鉴定到浆片特异表达基因nc33734，通过克隆其启动子，构建了RISBZ1 和RPBF组织特异性沉默转基因水稻，为创制稻曲病抗病新材料提供了宝贵基础。此外，利用组织细胞学和花器官突变体分析发现，雄蕊是稻曲病菌成功定殖水稻花器形成稻曲球所必需的，揭示了该病原菌独特的致病策略。稻曲病菌致病因子挖掘及功能分析发现，Uv1261和UvCBP1等效应子对稻曲病菌毒力有重要贡献。有趣的是，其中UvGH18.1突变导致稻曲病菌丧失形成稻曲球的能力，说明稻曲病菌可能通过UvGH18.1劫持水稻营养库供自身生长及稻曲球形成，但其具体作用机制有待进一步研究。