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

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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