Vm-milR21调控苹果黑腐皮壳侵染致病的功能和机理

Function and mechanism study of Vm-milR21 in the infection of apple tree Valsa canker pathogen

摘要:

苹果黑腐皮壳(Valsa mali)引起的腐烂病是苹果最具毁灭性的枝干病害。微小核糖核酸(microRNA-like RNAs, milRNAs)在真菌生长发育、侵染致病和胁迫响应等过程中发挥重要作用。前期研究发现Vm-milR21在病菌侵染阶段特异性下调表达，推测其可能参与V. mali的侵染致病过程。【目的】本文对Vm-milR21的功能进行研究。【方法】制备Vm-milR21前体过表达和沉默转化株，评价不同转化株与野生型菌株表型差异。进而，通过实时荧光定量聚合酶链反应(quantitative real time polymerase chain reaction, qRT-PCR)和共转化技术验证Vm-milR21与其潜在靶标Vm-03494之间的靶向调控关系。在此基础上，构建Vm-03494的基因敲除突变体，并鉴定突变体表型。【结果】与野生型菌株相比，过表达转化株的菌丝生长速率以及对叶片和枝条的致病力均大幅降低，而沉默转化株无明显变化。Vm-milR21能够序列特异性地抑制Vm-03494表达。相较于野生型菌株，Vm-03494敲除突变体菌丝生长速率和致病力均显著降低。【结论】Vm-milR21能够调控靶标基因Vm-03494表达参与病菌侵染过程。研究结果丰富了对真菌milRNAs功能的认知，同时为全面解析V. mali的致病机理提供了新的理论依据。

Abstract:

Apple Valsa canker caused by Valsa mali is a destructive disease of apple trees. MicroRNA-like RNAs (milRNAs) play an important role in fungal growth and development, pathogenicity, and stress responses. Our previous study had demonstrated that the expression of Vm-milR21 was significantly down-regulated in the infection, suggesting Vm-milR21 might be involved in the pathogenicity of V. mali. [Objective] To reveal the role of Vm-milR21. [Methods] The strains with overexpression and silencing of Vm-milR21 precursor were constructed, and the phenotypic characteristics were compared between different transformants and the wild type. Furthermore, the targeting relationship between Vm-milR21 and Vm-03494 was verified by Quantitative real time polymerase chain reaction (qRT-PCR) and co-transformation. On this basis, the mutant with the knockout of Vm-03494 was constructed and the phenotype of the mutant was characterized. [Results] Compared with the wild type, the transformant overexpressing Vm-milR21 precursor presented slow mycelial growth and weakened pathogenicity, while the silencing of Vm-milR21 did not cause significant changes. Vm-milR21 inhibited the expression of Vm-03494 in a sequence-specific manner. Compare with the wild type, the mutant without Vm-03494 presented slow mycelial growth and diminished pathogenicity. [Conclusion] Vm-milR21 plays a role in the infection of V. mali by regulating the expression of Vm-03494. The results enrich the understanding of the functions of fungal milRNAs and provide a new theoretical basis for comprehensively deciphering the pathogenesis of V. mali.