Small RNA-Based Antiviral Defense in the Phytopathogenic Fungus Colletotrichum higginsianum.
Small RNA-Based Antiviral Defense in the Phytopathogenic Fungus Colletotrichum higginsianum.
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DOI：
10.1371/journal.ppat.1005640
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发表时间：
2016-06
期刊：
影响因子：
6.7
通讯作者：
Carrington JC
中科院分区：
文献类型：
--
作者：
Campo S;Gilbert KB;Carrington JC
尽管真菌王国包含超过300万种，但人们对真菌中RNA沉默的生物学作用知之甚少。炭疽菌属由真菌种类组成，对世界范围内广泛的作物物种具有致病性。为了研究RNA沉默在子囊菌真菌Colletotrichum higginsianum中的作用，通过靶向基因替换产生了影响3种RNA依赖性RNA聚合酶（RDR）、2种dicer样（DCL）和2种Argonaute （AGO）蛋白基因的敲除突变体。在复杂或最小培养基上生长对任何突变株的营养生长没有影响。而Δdcl1、Δdcl1Δdcl2双突变株和Δago1菌株在分生孢子和分生孢子形态上存在严重缺陷。分析了亲本和突变株的总RNA转录本和小RNA群体。在Δdcl1， Δdcl1Δdcl2和Δago1菌株中观察到对这两个RNA群体的最大影响，其中先前未表征的dsRNA分枝病毒[称为炭疽杆菌higginsianum非节段dsRNA病毒1 (ChNRV1)]被抑制。系统发育分析清楚地表明，尽管基因组是非片段性的，但ChNRV1与片段性Partitiviridae家族成员之间存在密切关系。与AGO1相关的小rna的免疫沉淀显示含有5 ' u的病毒siRNA的丰富负载。经ChNRV1处理后的希金丝酵母亲本株和Δdcl1突变株的孢子形态缺陷主要由ChNRV1引起。基于这些结果，我们提出了在C. higginsianum中涉及ChDCL1和ChAGO1的RNA沉默可能作为一种抗病毒机制。炭疽病菌由一组不同的真菌病原体组成，它们攻击全世界3000多种植物物种。了解控制真菌发育和致病性的潜在机制可能会为作物病害管理和控制提供更有效和可持续的方法。在大多数生物体中，RNA沉默是控制内源性和外源性RNA的重要机制。RNA沉默利用Dicer （DCL）蛋白产生的小调控分子（小RNA），并通过Argonaute （AGO）效应蛋白发挥其功能。在这里，我们研究了RNA沉默机制在真菌炭疽杆菌中的作用，通过在编码RNA沉默成分的基因中产生缺失。在Δdcl1、Δdcl1Δdcl2和Δago1菌株的分生孢子和分生孢子形态上都观察到严重的缺陷。转录本和小rna分析显示，一种未表征的dsRNA病毒持续感染希金丝芽孢杆菌。结果表明：(1)该病毒在Δdcl1、Δdcl1Δdcl2和Δago1株中被去抑制，(2)引起分生和孢子突变表型。我们的研究结果表明，C. higginsianum采用RNA沉默作为抗病毒机制来抑制病毒及其衰弱效应。
Even though the fungal kingdom contains more than 3 million species, little is known about the biological roles of RNA silencing in fungi. The Colletotrichum genus comprises fungal species that are pathogenic for a wide range of crop species worldwide. To investigate the role of RNA silencing in the ascomycete fungus Colletotrichum higginsianum, knock-out mutants affecting genes for three RNA-dependent RNA polymerase (RDR), two Dicer-like (DCL), and two Argonaute (AGO) proteins were generated by targeted gene replacement. No effects were observed on vegetative growth for any mutant strain when grown on complex or minimal media. However, Δdcl1, Δdcl1Δdcl2 double mutant, and Δago1 strains showed severe defects in conidiation and conidia morphology. Total RNA transcripts and small RNA populations were analyzed in parental and mutant strains. The greatest effects on both RNA populations was observed in the Δdcl1, Δdcl1Δdcl2, and Δago1 strains, in which a previously uncharacterized dsRNA mycovirus [termed Colletotrichum higginsianum non-segmented dsRNA virus 1 (ChNRV1)] was derepressed. Phylogenetic analyses clearly showed a close relationship between ChNRV1 and members of the segmented Partitiviridae family, despite the non-segmented nature of the genome. Immunoprecipitation of small RNAs associated with AGO1 showed abundant loading of 5’U-containing viral siRNA. C. higginsianum parental and Δdcl1 mutant strains cured of ChNRV1 revealed that the conidiation and spore morphology defects were primarily caused by ChNRV1. Based on these results, RNA silencing involving ChDCL1 and ChAGO1 in C. higginsianum is proposed to function as an antiviral mechanism. Colletotrichum sp. comprises a diverse group of fungal pathogens that attack over 3000 plant species worldwide. Understanding the underlying mechanisms that govern fungal development and pathogenicity may enable more effective and sustainable approaches to crop disease management and control. In most organisms, RNA silencing is an important mechanism to control endogenous and exogenous RNA. RNA silencing utilizes small regulatory molecules (small RNAs) produced by proteins called Dicer (DCL), and exercise their function though effector proteins named Argonaute (AGO). Here, we investigated the role of RNA silencing machinery in the fungus Colletotrichum higginsianum, by generating deletions in genes encoding RNA silencing components. Severe defects were observed in both conidiation and conidia morphology in the Δdcl1, Δdcl1Δdcl2, and Δago1 strains. Analysis of transcripts and small RNAs revealed an uncharacterized dsRNA virus persistently infecting C. higginsianum. The virus was shown (1) to be de-repressed in the Δdcl1, Δdcl1Δdcl2 and Δago1 strains, and (2) to cause the conidiation and spore mutant phenotypes. Our results indicate that C. higginsianum employs RNA silencing as an antiviral mechanism to suppress viruses and their debilitating effects.