RNA结合蛋白MdYTP2在苹果抗褐斑病应答中的功能和作用机制解析

The northwestern arid area is the largest apple production region in China. Apple Marssonina leaf blotch, caused by the fungus M. coronaria, is an important leaf disease threating apple production in this region. A new pathway for apple Marssonina leaf blotch disease resistance breeding is to identify and mutate susceptibility (S) genes that play negative roles in defense against the invading pathogen by genome editing. Previously we found transgenic apple plants overexpressing MdYTP2 become more susceptible to M. coronaria infection compared with non-transgenic control. At the meantime, transgenic plants also have significantly lower expression level for COI1 gene which is a key component in jasmonic acid (JA) signaling pathway, indicating MdYTP2 might compromise apple defense by suppressing JA signal transduction. However, whether MdYTP2 also plays certain roles in other defense-related pathways and its working mechanism are still unclear. The applicants will characterize the defense pathways that MdYTP2 is involved in by performing RNA-Seq on MdYTP2-overexpressing apple plants that we already made and MdYTP2-silencing apple plants that will be constructed in this project after being infected by M. coronaria fungus. Thereafter, the applicants will utilize yeast two-hybrid screening and RNA binding protein immunoprecipitation together with high throughput RNA sequencing (RIP-Seq) to identify the interacting proteins and target RNAs of MdYTP2 during apple defense process against M. coronaria. The regulatory mechanism of MdYTP2 on its interacting proteins and its RNA targets will be determined as well. The essential binding sites of MdYTP2 with its targets will also be analyzed by site-directed gene mutagenesis. The outcome of this project will broaden our knowledge on the molecular function of MdYTP2 in apple defense reactions and settle theoretical basis for our future apple Marssonina leaf blotch resistance breeding with genome editing.

西北旱区是我国最大的苹果产区，褐斑病是危害我国旱区苹果产业的重要叶片病害，挖掘苹果褐斑病感病基因，通过基因编辑使之功能丧失是抗褐斑病育种的新途径。课题组前期发现，过表达RNA结合蛋白MdYTP2使苹果褐斑病抗性降低，并发现MdYTP2可能通过抑制茉莉酸信号传导负调控苹果褐斑病抗性，但MdYTP2是否也影响其它抗病途径及其作用机理尚不清楚。该项目拟以MdYTP2过表达和待构建的干扰表达苹果植株为材料，通过转录组测序解析MdYTP2调控的抗病途径；利用酵母双杂交和RNA结合蛋白免疫沉淀联合高通量测序鉴定MdYTP2的互作蛋白及靶标RNA，分析MdYTP2对互作蛋白和靶标RNA的影响，并通过基因定点突变探明MdYTP2与互作蛋白及靶标RNA结合的关键位点。研究结果将进一步明确MdYTP2在苹果抗褐斑病应答中的功能及作用机制，为利用基因编辑技术改良苹果褐斑病抗性奠定理论基础。

本研究利用RNA-seq技术明确了MdYTP2在苹果抗病应答过程中主要调控苹果中的植物-病原菌互作和类黄酮代谢途径相关基因表达；本研究还对苹果mRNA的m6A甲基化组进行了研究，发现了苹果mRNA的m6A甲基化特征，且其与番茄中的m6A有相同的“URUAY”motif；本研究还发现MdYTP2与MdARD4存在相互作用，并解析了MdARD4在植物抗逆及调控番茄果实成熟中的功能；通过联合RNA-seq、MeRIP-seq和RIP-seq等技术，本研究鉴定到MdYTP2的一系列靶标RNA，也明确了MdYTP2对靶标RNA的调控作用，MdYTP2对部分靶标RNA起稳定作用，而对部分靶标RNA起加速降解作用；另外，MdYTP2蛋白YTH结构域中的8个氨基酸位点是决定其与含m6A修饰mRNA结合的重要位点。. 除申报书中所涉及的研究内容外，本项目还研究了MdYTP2及其同源基因MdYTP1对干旱胁迫下苹果WUE的调控功能、MdYTP2的互作蛋白MdARD4调控番茄果实成熟和抵御低温胁迫的功能。取得的结果如下：（1）在长期干旱胁迫条件下，MhYTP2转基因植株比野生型植株具有更高的WUE和更强的抗旱性。干旱胁迫下，MhYTP2转基因植株的净光合速率和根系吸水能力增强，且MhYTP2超量表达可激活乙烯信号转导途径和引起ABA含量增加，继而导致叶片气孔开张度减小，从而实现植株蒸腾速率的降低。（2）长期干旱条件下，MhYTP1超量表达转基因植株比野生型植株的净光合速率和WUE均显著提高，在干旱处理40和80天后，超量表达MhYTP1提高了苹果植株的ABA含量，增加了叶片的气孔密度，降低了气孔开张度。表明MhYTP1通过提高植物内源ABA水平而调控叶片气孔密度及开张度，从而提高苹果在长期干旱条件下的WUE与抗旱性。（3）鉴定了苹果ARD家族的MdARD1、MdARD2和MdARD4 三个成员。其中MdARD4在苹果果实成熟过程中显著上调，同时该基因也受低温胁迫高度诱导表达。在番茄‘Micro-Tom’中异源表达MdARD4后发现，MdARD4超表达能够通过提高乙烯和类胡萝卜素含量参与乙烯和类胡萝卜素信号通路，加速番茄果实成熟。此外，MdARD4还能够提高番茄的抗氧化能力，增强番茄的低温抗性。