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

转录后基因沉默（PTGS）是一种序列特异性的RNA降解系统，广泛存在于从真菌到哺乳动物的真核生物中。双链RNA是引发PTGS的关键分子，被RNaseIII型核酸酶消化为22-25个核苷酸（siRNA，PTGS的霍尔标记），引导RISC（RNA诱导沉默复合物，AGO家族成员是主要成分）靶向RNA，导致RNA降解。PTGS被概念化为一种抗病毒宿主防御机制，有几条证据。作为其反击策略，植物和动物病毒也编码PTGS抑制子，其似乎以不同的方式起作用。它们大多数来源于正链和负链RNA或DNA病毒。在本项目中，dsRNA病毒在丝状真菌Cryphonectria parasitica（栗疫病菌）中进行抗PTGS活性测定。 ...更多信息 枯萎病真菌）和动物。所测试的蛋白包括由原型低病毒CHV 1-EP 713、两种植物呼肠孤病毒（双子叶植物感染的伤口肿瘤病毒（WTV）和单子叶植物感染的水稻矮缩病毒（RDV））编码的蛋白，后两者也是昆虫（动物）感染的。在此，基于MYRV 1（呼肠孤病毒科内新建立的属的成员）的水平来评价不同病毒来源的候选蛋白的抑制活性。通过该测定，来自原型低病毒（CHVl）的木瓜蛋白酶样蛋白酶p29显示出提高MYRVl的复制和垂直传播水平，表明p29的RNA沉默抑制活性，而没有发现CHVl的p40的这种活性，据报道增加同源病毒复制。对水稻矮缩呼肠孤病毒和创伤瘤病毒进行了检测。2）尝试构建潮霉素抗性基因（HygR）沉默的转基因C parasitica品系。为此，在组成型启动子的控制下，用HygR转化寄生线虫原生质球。将所得转基因系用具有内含子间隔子的HygR的反向重复进行双重转化。在超过200个独立转化体中，大约10-20个显示Hyg可成功，可能是因为HygR的dsRNA形式触发的RNA沉默。这些分离物用于RNA沉默抑制子测定，其中检查上述候选蛋白质将其表型恢复为HygR的能力。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