水稻AGO18抗水稻重要病毒（RSV和RBSDV）机制研究

Small RNAs mediated RNAi plays important roles both in gene expression and antiviral defense in many organisms. AGO proteins, the heart of RNAi machinery, incorporate one strand of a duplex to form a RNA induced silencing complex (RISC) to regulate the stability/translatability of target RNAs or to mediate methylation of target DNA sequences. In Arabidopsis AGO1 is a major effector of antiviral RNAi, AGO1 binds viral siRNAs and mediates the degradation of viral RNAs. Infection of many viruses can elevate the miR168 level to down-regulate AGO1, thereby nullifying this layer of host defense. Rice is one of the most important food crops. However, rice production and consequently food sustainability is under the constant threat of emerging and reemerging viral diseases. Rice viral pathogens are genetically diverse and many highly pathogenic viruses such as Rice stripe Tenuivirus (RSV, a negative-stranded RNAs) and Rice black-streaked dwarf virus (RBSDV, a double-stranded RNAs) are transmitted persistently by arthropod vectors. Because of the global circulation of these vectors and lack of virus resistance germplasms, the incidence and severity of rice viral diseases in many rice-growing regions are unpredictable. Developing new and effective strategies to control infection by multiple viruses for a crop is of paramount importance for human food sustainability. The rice genome encodes 19 AGOs. How do different AGOs regulate antiviral RNAi in rice is not known. We found that rice AGO18, a new AGO clade in monocots, is specifically induced by infections of two different viruses under natural infection conditions with viruliferous insect vectors. Loss-of-function ago18 mutant abolishes, whereas over-expression of AGO18 increases, the AGO1 antiviral activity to both of RSV and RDV (rice dwarf virus, genome with 12 dsRNAs). AGO18 competes with AGO1 for binding miR168, resulting in elevated levels of AGO1 in the infected plants to enable antiviral defense. Genetic studies reveal that the antiviral function of AGO18 depends on its activity to sequester certain miRNA escaping of repression of the rice AGO1 essential for antiviral RNAi. Our findings reveal a novel mechanism AGO1 homeostasis regulation by AGO18 for antiviral defense and have significant implications in understanding the evolutionary amplification of RNA silencing mechanisms and in developing novel antiviral strategies.. In addition to miR168, AGO18 binds several other miRNAs including miR528, miR159. The question is whether the binding of AGO18 to these miRNAs in viral infected rice is also involved in antiviral defense? Rice viruses are genetically diversified, whether AGO18 displays resistance to RBSDV, another severe pathogen of rice and maize? Understanding of AGO18 resistance to RBSDV is of significant importance to rice breeding. Lastly, whether over-expression of AGO18 affects rice agro-traits? is there a possibility that AGO18 could be used in antiviral breeding?

RNA沉默通路广泛存在于真核生物中，是重要的抗病毒机制。水稻条纹病毒（RSV）和水稻黑条矮缩病毒（RBSDV）基因组分别为单链、负链RNA或双链RNA，均由灰飞虱介体以持久性方式传播，严重影响水稻的产量。我们前期的研究发现RNA沉默是水稻抗病毒的重要途径，AGO18和AGO1发挥重要作用。ago18突变体高度感病，而AGO18过表达高度抗病。其抗病机制是病毒侵染诱导AGO18高表达，AGO18进而竞争性结合miR168，使AGO1表达升高并结合病毒vsiRNA降解病毒RNA，从而增强水稻抗性。本申请是在此基础上，深入研究：1）AGO18抗病毒机制，在RSV侵染水稻中AGO18结合的其它miRNAs是否参与抗病毒免疫? 2）AGO18是否可以抵御水稻和玉米重要病毒如RBSDV？ 3）过量表达AGO18是否影响水稻生长、发育以及结实？如果没有是否可以用作广谱抗病毒基因用于育种。

水稻条纹病毒（RSV）、水稻黑条矮缩病毒（RBSDV）和水稻矮缩病毒（RDV）是严重威胁水稻等禾本科作物高产和稳产的重要病毒病原，当前栽培的品种和育种材料尚无高抗这几种病毒基因资源。我们的研究发现，RSV侵染或者病毒外壳蛋白（CP）过表达显著激活水稻茉莉酸（JA）信号通路以及该通路的关键转录因子（JAMYB），该转录因子激活AGO18的表达，AGO18通过与AGO1竞争性结合miR168和miR528从而释放AGO1以及miR528的靶基因抗坏血酸氧化酶（AO）。AGO1通过结合病毒源siRNA形成沉默复合体降解病毒基因组，而AO的上调表达提高细胞间隙ROS水平，从而增强水稻抗病毒功能，而且这抗性具有广谱性，可以高抗水稻条纹病毒RSV和RBSDV。该研究首次揭示了JA信号通路与RNAi信号通路协同参与水稻抗病毒防御的分子机制，发现了JAMYB作为响应JA信号通路的转录因子参与水稻AGO18的转录调控，揭示了水稻中独特的RSV CP作为效应蛋白介导的抗病毒分子机制。进一步对miR528转录调控研究发现SPL9特异性激活miR528的转录，RSV编码的NS3蛋白具有促进内源miRNA加工的功能。. 此外，研究还发现，植物激素乙烯负调控水稻对水稻矮缩病毒（RDV）的抗性。