类激酶受体MdBAK1负调控苹果对真菌病害抗性的分子机理解析

Apple ring rot was one of the most destructive and prevalent diseases in China that severely destroyed apple production. So far, the mechanism on apple disease resistance towards the casual fungi was still elusive. MdBAK1 was a receptor-like kinase located at the cell membrane that played pivotal role in regulating plant growth, development and disease resistance. In the preliminary experiment, we cloned the orthologues of Arabidopsis BAK1 in apple, which was named as MdBAK1-1 and MdBAK1-2 respectively. It was unexpectedly that the apple callus cells with MdBAK1 overexpressed showed significantly decreased disease resistance towards the apple ring rot casual fungi. It was very interesting to find that MAPK kinase activation was nearly abolished under the pathogen infection compared with the high stimulation of MAPK signaling in wild type apple callus infected by the pathogen. .The applying project was aimed to identify the components involved in suppressing MAPK signaling as well as the apple disease resistance. We would firstly employ immuneprecipitation technique to precipitate MdBAK1 complex from the apple callus with MdBAK1 overexpression, which would be subject for ESI/MS to identify the components of MdBAK1 protein complex, which may be implied for interacting with MdBAK1 in regulating apple disease resistance. Meanwhile, phosphoprotein characterization strategy would also be initiated to identify the proteins whose phosphorylation was regulated by MdBAK1 as MdBAK1 has a strong kinase activity and phosphorylation was one important method for MdBAK1 controlling signaling in interior cells. And in this way, we try to identify the components that may interfere with apple disease resistance toward the pathogenic fungi but did not interact with MdBAK1. The encoding genes of those proteins revealed by above two methods would be transformed into apple to investigate their roles in apple disease resistance, their relationship with MdBAK1 in disease resistance signaling. The candidates that may mediate MdBAK1 suppressing MAPK signaling would also be pursued. The research would provide the information on the signaling network triggered by apple ring rot casual fungi and laid the foundation for unraveling the disease resistance mechanism for the devastating disease.

苹果轮纹病是我国苹果产业发展中的重要病害，对轮纹病的抗病机制，至今仍知之甚少。MdBAK1为苹果中一重要的类激酶受体，预试验中，将MdBAK1于苹果中超表达，显著降低了苹果对轮纹病等真菌病害的抗性，并且，在病菌侵染下，代表植物基础抗性的MAPK级联反应信号激活水平显著低于野生型。本研究拟以MdBAK1负调控苹果抗病过程的信号转导为研究中心，首先应用免疫沉淀与质谱鉴定技术，研究参与MdBAK1负调控苹果抗病的MdBAK1互作蛋白；然后应用磷酸化肽段富集与磷酸化蛋白组学研究技术，确定参与MdBAK1调控苹果抗性，但不与MdBAK1直接互作的，MdBAK1信号途径中的其他成员；并把以上两种方法获得蛋白的编码基因转入苹果，研究它们在苹果抗病中的作用，在抗病信号转导中与MdBAK1的关系，以及可能对MAPK级联反应进行调控的分子机制。本研究将为理解苹果对轮纹病菌产生抗性的分子机制提供基本研究资料。

BAK1是植物中一重要类激酶受体，参与调控植物激素信号转导与植物先天免疫反应。苹果愈伤组织细胞中过表达MdBAK1基因，显著促进了苹果轮纹病菌的扩展，并抑制病菌侵染对苹果MAPK激酶活性。本项目因此为基础，首先建立了苹果原生质体细胞瞬时表达研究技术体系。应用该研究技术体系，发现MdBAK1-1是在先天免疫反应中与MdFLS2形成免疫蛋白复合体的主要基因。虽然在苹果愈伤细胞中过量表达，MdBAK1-1促进了轮纹病菌扩展，但是，MdBAK1-1苹果植株中过量表达在促进自发免疫反应的同时，显著提高了苹果果实对轮纹病菌的抗性。我们的研究显示，发生免疫反应的MdBAK1-1过表达植株中，激活了细胞中类Caspase信号途径。.重要结果：.（1）AtUBQ10或MdUBQ10的启动子是能够驱动目标基因在苹果原生质体细胞中进行稳定与高效表达的启动子。而应用CaMV 35S启动子，大部分参试基因不能获得稳定与有效表达。.（2）MdBAK1-1是苹果细胞中与MdFLS2形成免疫复合体的主要基因。而另一同源基因MdBAK1-2则与MdFLS2的相互作用较弱。MdBAK1与MdFLS2之间的亲和力弱于MdBAK1与AtFLS2之间的亲和力。 .（3）MdBAK1-1过量表达提高了苹果果实对轮纹病菌的抗性，MdBAK1-1可能是苹果对轮纹病菌抗性的候选基因。.（4）在MdBAK1-1过表达植株中，免疫反应激活了细胞中保守类Caspase信号途径。免疫反应所活化的蛋白水解酶能够特异性水解MdPARP2、AtPARP1。MdBAK1可能通过调控caspase信号途径，调控植物细胞内的免疫与抗病反应。.（5）鉴定到包括MDP0000213640在内的185个MdPARP2的蛋白复合体的组分。