A tomato fruit blotch viral replicon defines minimal requirements for cell autonomous replication and identifies functional RNA4-encoded movement and silencing suppression activities

Tomato fruit blotch virus (ToFBV) is an emerging multipartite positive-sense RNA virus associated with blotchy symptoms on tomato fruits and classified within the genus Blunervirus (family Kitaviridae). Despite its increasing agricultural relevance, the study of ToFBV has been hindered by the lack of mechanical transmissibility and the difficulty in reproducing infections under controlled conditions. In this work, we report a preliminary step toward the development of the first infectious agroclone system for ToFBV, based on full-length cDNA copies of its four genomic RNAs. We demonstrate that the cloned viral genome is capable of initiating cell autonomous replication in Nicotiana benthamiana, as indicated by the accumulation of negative-sense RNA intermediates in infiltrated tissues. To further validate the system, RNA3 was engineered to express GFP, enabling visualization of infection foci and confirming active viral replication in both N. benthamiana and tomato. Functional assays of RNA4-encoded proteins demonstrated that it encodes a movement protein capable of complementing movement-deficient viral vectors and a putative suppressor of post-transcriptional gene silencing (PTGS). Together, these results establish a versatile reverse genetics platform for ToFBV, providing new insights into the replication and functional organization of blunerviruses and enabling future studies on virus-host interactions, pathogenicity, and control strategies.