A novel ToBRFV cDNA full-length infectious clone provides insights on virus host range and inoculation strategies

Tomato brown rugose fruit virus (ToBRFV) is an emerging virus threatening tomato cultivation worldwide. To facilitate research on ToBRFV, we constructed a full-length clone of the ToBRFV strain PV1241 (designated pJL89-ToBRFV) using Gibson Assembly. Agro-inoculation of the infectious clone caused systemic infection in different host plants, such as Nicotiana benthamiana, N. tabacum ‘Samsun nn’ and Solanum lycopersicum ‘Linda’. Virions were produced in inoculated plants that were identical to those derived from the wild-type strain. Extracted virions were infectious on different host plants demonstrating that the infectious clone is able to produce viral RNA and viral protein in those host plants. A host range study was conducted using both agro-inoculation and mechanical inoculation using pJL89-ToBRFV-infected leaf material where Physalis floridana and Claytonia perfoliata were identified as hosts of ToBRFV. This infectious clone is an important tool for ToBRFV research and may be used to investigate virus-host interactions, for standardized infection assays of known or new hosts, for optimizing and standardizing resistance screens and for identifying virus control agents and/or attenuated virus strains.