Analysis of RNA silencing suppressor activities of viral proteins in a chestnut blight fungus system

转录后基因沉默（Post - transcriptional gene silencing， PTGS）是一种序列特异性的RNA降解系统，最早是作为植物的共抑制和RNA介导的病毒抗性而在植物中被发现的，现在认为它普遍存在于从真菌到哺乳动物的真核生物中。双链RNA是触发PTGS的关键分子，被RNaseIII型核酸酶消化为22-25个核苷酸（siRNA， PTGS的标志），引导RISC （RNA诱导沉默复合体，AGO家族成员为主要成分）靶向RNA并导致RNA降解。通过几条证据线，PTGS被概念化为一种反病毒宿主防御机制。作为它的反击策略，植物和动物病毒也编码PTGS抑制子，它们的作用方式似乎不同。它们大多来源于正、负链RNA或DNA病毒。在本项目中，dsRNA病毒在丝状真菌、栗子寄生菌和动物中进行了抗ptgs活性测定。检测的蛋白包括原型次病毒CHV1-EP713、两种植物呼肠孤病毒、双子叶植物侵染伤口肿瘤病毒（WTV）和单子叶植物侵染水稻矮病毒（RDV）编码的蛋白，后两种病毒也侵染虫（动物）。1)已知一些RNA沉默抑制因子可增强异源病毒的复制。在这里，根据呼肠孤病毒科新建立的属成员MYRV1的水平评估了不同病毒来源的候选蛋白的抑制活性。本实验发现，源自原次病毒（CHV1）的木瓜蛋白酶样蛋白酶p29可提高MYRV1的复制和垂直传播水平，表明p29具有RNA沉默抑制活性，而未发现源自原次病毒（CHV1）的p40具有增强同源病毒复制的活性。研究了侵染植物和昆虫的呼肠孤病毒（水稻矮化植物呼肠孤病毒和伤口肿瘤病毒）编码的病毒蛋白。然而，这些蛋白没有发现MYRV1复制的升高。2)尝试制备抗潮霉素基因（HygR）沉默的转基因C寄生品系。为此，在本构启动子的控制下，用HygR对C寄生球质体进行转化。用带内含子间隔子的HygR反向重复对转基因系进行双重转化。在200多个独立的转化子中，大约有10-20个表现出Hyg易感性，这可能是因为HygR的dsRNA形式触发了RNA沉默。这些分离株被用于RNA沉默抑制试验，其中检查上述候选蛋白是否能够将其表型恢复为HygR。少

Post transcriptional gene silencing(PTGS), first discovered in plants independently as cosuppresion and RNA-mediated virus resistance, is a sequence-specific RNA degradation system which is now believed to be ubiquitous in eukaryotic organisms from fungi to mammals. Double-stranded RNA is key molecules triggering PTGS and being digested by RNaseIII type nuclease to 22-25 nucleotides (siRNA, a hall mark of PTGS) which guide RISC (RNA inducing silencing complex, AGO family member being a major component) to target RNA and lead to the degradation of the RNA. PTGS is conceptualized as an anti-virus host defense mechanism by several lines of evidence. As its counterattack strategy plant and animal viruses as well encode PTGS suppressors which seem to act in different ways. Most of them are derived from positive and negative strand RNA or DNA viruses. Here in this project dsRNA viruses were subjected to anti-PTGS activity assay in a filamentous fungus, Cryphonectria parasitica (the chestnut … More blight fungus) and animal. Proteins tested included those encoded by the prototype hypovirus, CHV1-EP713, two plant reoviruses, dicot plant-infecting wound tumor virus(WTV) and monocot plant-infecting rice dwarf virus(RDV), the latter two of witch are also insect (animal)-infecting.1)Some RNA silencing suppressors are known to enhance replication of heterologous viruses. Here suppressive activities of candidate proteins of different virus origins were evaluated based on the levels of MYRV1, a member of a newly established genus within the family Reoviridae. By this assay the papain-like proteinase p29 derived from the prototype hypovirus (CHV1), was shown to elevate the replication and vertical transmission levels of MYRV1, indicative the RNA silencing suppressive activity of p29, while no such activity by p40 of CHV1, reported to augment the homologous virus replication, was not found. Viral proteins encoded by plant- and insect-infecting reoviruses, i.e., rice dwarf phytoreovirus and wound tumor virus were tested. However, no elevation of MYRV1 replication was found by those proteins.2)Trangenic C parasitica lines silenced for Hygromycin resistance gene(HygR) were attempted to be generated. To this end, C parasitica spheroplasts were transformed with HygR under the control of constitutive promoter. Resulting transgenic lines were doubly transformed with an inverted repeat of HygR with an intron spacer. Out of more than 200 independent transformants, approximately 10-20 showed Hyg succeptible, probably because of RNA silecing triggered by the dsRNA form of HygR. These isolates were being used for RNA silencing suppressor assay in which the candidate proteins as above are examined for the ability to reverting their phenotype to HygR. Less