Exploration of the yadokari/yadonushi nature of YkV3 and RnMBV3 in the original host and a model filamentous fungus

The yadokari/yadonushi nature is a recently discovered virus lifestyle; “yadokari” refers to the ability of capsidless positive-sense (+) RNA viruses (yadokariviruses) to utilize the capsids of phylogenetically distant double-stranded RNA (dsRNA) viruses possibly as the replication site, while “yadonushi” refers to the ability of dsRNA viruses to provide capsids to yadokariviruses. This virus–virus interaction, however, has been only studied with limited pathosystems. Here, we established a new study model with a capsidless (+)RNA yadokarivirus YkV3 (family Yadokariviridae) and its capsid donor RnMBV3 (family Megabirnaviridae) in the original host fungus Rosellinia necatrix and a model filamentous fungal host Cryphonectria parasitica. YkV3 has a simple genome structure with one open reading frame of 4305 nucleotides encoding a single polyprotein with an RNA-dependent RNA polymerase and a 2A-like self-cleavage peptide domain. Reverse genetics of YkV3 in R. necatrix showed that YkV3 tolerates a nucleotide substitution in the extreme 5′-terminus. The insertion of two termination codons immediately downstream of the 2A-like cleavage site abolished YkV3 viability, suggesting the importance of the C-terminal portion of the polyprotein of unknown function. Transfection of RnMBV3 and YkV3 into an RNA silencing-deficient mutant Δdcl2 of C. parasitica showed the replication competency of both viruses. Comparison between the wild-type and Δdcl2 strains of C. parasitica in virus accumulation suggested that RnMBV3 and YkV3 are susceptible to RNA silencing in C. parasitica. Taken together, we have established a platform to further explore the yadokari/yadonushi nature using genetically manipulable host fungal and virus strains.