苹果树腐烂病菌AGO蛋白介导的致病相关small RNAs及其调控机理

Apple is an important pillar industry for improving the farmer income in the north of China, especially in the west area. However, the apple tree valsa canker, caused by the weakly parasitic pathogen Valsa mali, seriously restricts the development of the industry, and has been the key objects of prevention and treatment in apple production. Little information about the molecular pathogenic mechanisms is still the primary limitation for disease management. So, speeding up the exploration of pathogenic mechanisms of the pathogen has become extremely urgent, and it will be significant for formulating the strategies of effective disease control. Based on our genome and transcriptome data, large-scale screening of pathogenicity-related genes has been performed through pathogenicity identification of knockout mutants. And significantly changed virulence of V. mali was found in AGO knockout mutants, inferring the important roles of small RNAs in the pathogenicity of V. mali. Thus, in this project, we aim to identify the pathogenic related sRNAs from the wild type V. mali and AGO mutants using the specific antibodies of AGO proteins through immunoprecipitation technology; predict their target genes based on the genome and transcriptome data of V. mali; confirm the regulation interactions between sRNAs and their target genes and explore the regulation mechanisms of key pathogenicity-related sRNAs. Our results will give insight into the mechanisms of sRNA-mediated regulation during Valsa mali infection. Moreover, the information will provide theoretical basis for developing new control strategies of the disease, and it is of great significance to ensure the apple production safety in China.

苹果是我国北方尤其是西部地区农民增收致富的重要支柱产业，而由弱寄生性苹果黑腐皮壳属真菌侵染树干引起的苹果树腐烂病严重制约该产业的发展，一直是生产上的重点防治对象。但对病菌的分子致病机理却知之甚少。因而，加快解析病菌致病机理将对病害有效防治策略的制定意义重大。为此，基于腐烂病菌全基因组和转录组信息，课题组开展大规模基因敲除挖掘致病基因，发现AGO蛋白突变体致病力显著变化，推测其介导的small RNAs（sRNAs）在病菌致病过程中发挥重要作用。本项目拟在前期研究发现的基础上，分别对野生型和突变体材料AGO蛋白介导的致病相关sRNAs进行分离鉴定；利用病菌全基因组和转录组信息进行靶标基因预测；对关键致病相关sRNAs进行靶标基因验证及调控机理解析，为全面揭示sRNAs调控苹果树腐烂病菌致病机理奠定基础，进而为病害防治方法建立和新策略制定提供理论依据，对确保我国苹果安全生产具有重要意义。

苹果是我国北方尤其是西部地区农民增收致富的重要支柱产业，而由弱寄生性苹果黑腐皮壳属真菌侵染树干引起的苹果树腐烂病严重制约该产业的发展，一直是生产上的重点防治对象。但对病菌的致病机理却知之甚少，加快解析病菌致病机理将对病害有效防治策略的制定意义重大。本项目在前期研究的基础上，分别对苹果树腐烂病菌野生型菌株及受病菌侵染的树皮组织的small RNAs进行了分离鉴定，发现苹果树腐烂病菌存在NAT-siRNA、micro-like RNAs（milRNAs）及disiRNA等多种sRNAs分子。进一步分析发现，苹果树腐烂病菌共存在57个milRNAs。通过生物信息学分析和实时荧光定量获得了在病菌侵染过程中显著差异表达的milRNAs。进而通过降解组测序技术鉴定到45 milRNAs的靶标基因。这些milRNAs与靶标基因形成复杂的调控网络，包括DNA转录、蛋白转运、蛋白水解、蛋白翻译、信号转导、胞内运输、外泌、囊泡转运以及能量转化等生物学过程。为了明确milRNA的生物学功能，对17个milRNA前体进行过表达，明确了其对病菌生长、繁殖体形成及致病力的影响。其中，Vm-milRNA16过表达之后并未影响菌落形态和生长速率，但致病力显著降低。检测发现，该基因在病菌侵染过程中持续下调表达，其调控的四个候选靶标基因菌在侵染过程中显著上调。对四个靶标基因进行敲除发现缺失突变体致病力均显著降低。在此基础上，进一步证实了Vm-milRNA16对外泌蛋白VM1G_10102表达的调控关系，并对VM1G_10102的互作蛋白进行了筛选，初步明确了其作用机理。同时，分析了在病菌侵染过程中显著下调表达的Vm-milR37的功能，揭示了其主要通过调控谷胱甘肽过氧化物酶的表达来影响菌株的生长、繁殖体形成及致病力。此外，揭示了在病菌侵染过程中，特别是侵染早期显著上调表达的Vm-milR34的生物学功能，发现其能够通过调控exportin 1-like蛋白的表达从而影响病菌的生长、繁殖体形成及致病力。上述结果为全面揭示sRNAs调控苹果树腐烂病菌致病机理奠定了重要基础，进而为病害防治方法建立和新策略制定提供理论依据。

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