香蕉枯萎病菌Argonaute基因功能分析及其调控小RNA发生

尖孢镰刀菌古巴专化型Fusarium oxysporum f. sp. cubense是威胁香蕉生产的重要土传病原真菌,其中4号生理小种(Foc4)能感染几乎所有的栽培品系。Argonaute蛋白(AGO)介导的RISC复合体在RNAi干扰中起到重要作用。Foc4含有两个进化上高度保守的AGO蛋白,本研究利用同源重组技术获得了Δago1、Δago2和Δago1/2基因缺失突变体,并对其突变体的生物学表型、非生物胁迫、致病力及小RNA发生进行分析。与Foc4相比,缺失突变体不影响菌丝的营养生长,但是分生孢子产量下降,对巴西蕉的致病力明显减弱。其次,在hpRNA诱导的基因沉默中,AGO1主要负责产生siRNA介导基因沉默。最后,sRNA测序显示,缺失突变体的sRNA长度分布和5°-端首位碱基出现频率都发生了改变,并且都能产生自身特异性miRNAs。另外,本研究还鉴定到不依赖AGOs形成的miRNA。这些结果说明Foc4的AGOs在调控产孢量、非生物胁迫、致病力,以及小RNA发生中发挥作用。本研究为揭示Foc4小RNA发生的分子遗传机制奠定理论基础。

Functional analysis of the Argonaute genes encoded in Fusarium oxysporum f. sp. cubense race 4 for biogenesis of small RNAs

Fusarium oxysporum f. sp. cubense (Foc) is an important soil-borne pathogenic fungus leading to a serious threat to the production of banana in the world. The Foc race 4 (Foc4) attacking almost all Cavendish cultivars is currently noteworthy in global banana production. The biological roles of the RNA interfering (RNAi) pathway in the pathogenicity of Foc4 remain unknown. The Argonaute protein (AGO) mediated gene silencing plays a critical role in RNA interference (RNAi) pathway. Foc4 have two AGO proteins containing three typical domains. In this study, Δago1, Δago2 and Δago1/2 gene-knockout mutants of Foc4 were obtained by split-marker homologous recombination technique. The biological phenotypes including the vegetative growth, the amount of sporulation, abiotic stress response and pathogenicity of Foc4 and its knockout mutants were tested. The knockout mutants did not affect the vegetative growth, but their sporulation quantity and the virulence to Cavendish banana were significant reduced when AGOs were deleted as compared with wild type Foc4. Meanwhile, hpRNAi-FCC1 is able to induced endogenous FCC1 reporter gene silencing, resulting in clear phenotypes evaluating the AGOs function in RNAi pathway, and the AGO1 played a critical role in the hpRNA mediated gene silencing process in Foc4. sRNA deep sequencing data showed that the sRNA data characterization were markedly changed as compared with Foc4. miRNA could be generated depending on individual, alternative or joint AGOs, indicating the Foc4 AGOs with overlapped and redundant function in small RNA biogenesis. In addition, AGOs-independent miRNA also was identified in the sequencing data. These results indicated that Foc4 AGOs not only play key roles in conidiation, environmental responses and fungal pathogenicity, but also perform a function in generation of sRNAs.