小麦Argonaute 1d介导的白粉菌致病路径解析

Plant pathogens can secrete small RNAs (sRNAs) into host cells to “hijack” the host Argonaute 1 (AGO1) protein, so as to selectively silence host immunity genes and achieve their infection. Based on such a mode of action, changing the pathogen sRNA target sites on the host genes can avoid pathogen attack on the host immune system, and hence enhance the resistance of the host. Previously, we found that the loss of function of the AGO1d-B gene in a tetraploid wheat mutant increased its resistance to the wheat powdery mildew (Blumeria graminis tritici, Bgt), suggesting that a similar AGO1d-hijacking may exist in wheat powdery mildew pathogenicity. In this proposal, we plan to take the advantage of the synthesized AGO1d antibody to pull-down the sRNAs enriched after Bgt infection wheat leaf cells and compare them with the genome sequence of Bgt. Target genes of sRNAs will be predicted using the availabe wheat D genome sequences. We will then study whether Bgt sRNAs regulate targets through clevage or translation repression. Using the tetraploid wheat ago1d-B mutant and transgenic wheat overexpressing sRNAs or target genes with and without target sites, we will determine genetically the effectiveness of AGO1d mediated powdery mildew pathogenicity of wheat and explore the potential of this system for wheat disease resistance breeding.

植物病原菌通过向宿主体内释放Small RNA (sRNA)，“劫持”宿主Argonaute 1 (AGO1) 蛋白，可以实现对宿主免疫基因干扰，使宿主感病。利用此种作用方式，通过改变病原菌sRNA宿主靶向基因位点，可以使宿主抗病性增强。前期发现，四倍体小麦AGO1d-B基因突变后，其抗病性明显增强，暗示AGO1d可能是小麦白粉菌“劫持”的对象，小麦中可能存在类似的重要免疫机制，但尚未有研究。本项目拟在此基础上，利用已测序的小麦白粉菌基因组信息和已在制备的AGO1d抗体，明确接菌后应答且与小麦AGO1d结合的白粉菌sRNAs；结合小麦供体种D基因组草图预测sRNA的靶基因，并检验确定sRNA对靶基因的作用方式；利用ago1d-B突变体和六倍体中过量表达sRNA、突变和非突变靶位点靶基因材料，阐明小麦AGO1d介导的小麦白粉菌致病机制，并探索该机制在小麦抗病育种中的应用。

植物病原菌通过向宿主体内释放Small RNA (sRNA)，“劫持”宿主Argonaute 1 (AGO 1) 蛋白，可以实现对宿主免疫基因干扰，使宿主感病。利用此种作用方式，通过改变病原菌sRNA宿主靶向基因位点，可以使宿主抗病性增强。小麦中可能存在类似的重要免疫机制，但尚未有研究。本研究围AGO1d基因开展了以下六个方面的工作，并获得了较好的发现：1) 小麦AGO1包含AGO1a/AGO1b/AGO1c/AGO1d四个分支，每个分支分别包含A/B/D三个拷贝，相似性在92%以上；2) AeAGO1d受白粉菌诱导，AeAGO1a/AeAGO1b/AeAGO1c变化不明显，AGO1d-A/B均受白粉菌诱导，且AGO1d-B受诱导程度强于AGO1d-A；3) AGO1d-B突变株系在苗期的抗病性不变，在成株期的抗病性增强；4)；初步纯化了AGO1d抗体；5)通过白粉菌侵染后sRNA测序，发现一批12 h 表达（0 h 不表达）的白粉菌sRNAs；6)靶基因预测，发现bgt-miR-1的靶基因为TraesCS2A02G365500.1，注释为csAtPR5基因，应答白粉菌侵染；bgt-miR-2的靶基因为TraesCS1B02G024400.1和TraesCS3D02G316700.2，分别注释为FYVE zinc finger domain containing protein和receptor protein kinase。本研究首次从小麦白粉菌中鉴定到2个bgt-sRNA，并初步预测其在小麦中的靶基因分别为csAtPR5基因、锌指结构蛋白和受体蛋白激酶，初步解析了小麦白粉菌sRNA调控植物宿主靶基因的机理，为小麦抗病遗传育种提供重要的信息。