A mycovirus-encoded homologue of plant viral movement proteins is not functional in movement complementation assays in plants

Valsa mali negative strand RNA virus 1 (VmNSRV1), a fungal virus infecting Valsa mali and belonging to the Phenuiviridae family, was recently shown to be capable of naturally infecting plants. Its RNA2 encodes a homologue of a plant virus movement protein (MP) that can complement MP-deficient virus vectors and localizes to plasmodesmata, allowing natural cross-kingdom infection. To determine whether this property is a common feature among mycoviruses possessing distant MP homologues or an isolate-specific trait, we investigated Trichoderma gamsii cogu-like virus 1 (TgClV1), a Phenuiviridae member in the Bocivirus genus detected in Trichoderma gamsii. Structural analysis guided by AlphaFold confirmed that the RNA2-encoded open reading frame (ORF) contains a conserved seven beta-strand module characteristic of the 30 kDa MP superfamily, making it a putative MP-like protein (MP-L). To assess its functional role, we tested whether TgClV1's putative MP (TgCl-MPl) could complement movement-deficient plant viruses, employing tomato apex necrosis virus (ToANV), alfalfa mosaic virus (AMV), and potato virus X (PVX) MP-deficient vectors. While tobacco mosaic virus-MP successfully complemented cell-to-cell movement in trans in all these systems, TgCl-MPl did not. Additionally, localization studies of TgClV1-MPl-GFP fusions revealed that, unlike TMV-MP and VmNSRV1-MP, TgClV1-MPl did not specifically target plasmodesmata, a key characteristic of functional viral MPs. Finally, direct inoculation of Nicotiana benthamiana-either via mechanical application of purified TgClV1 or via natural inoculation through TgClV1-infected Trichoderma gamsii associated with plant roots-failed to establish infection. In conclusion, our findings do not support the hypothesis that TgClV1-MPl functions as a plant virus movement protein. The evolutionary implications of this observation are discussed.