Integrating the tomato chlorosis virus with the tobacco rattle virus facilitates the direct infection of tomato.

Tomato chlorosis virus (ToCV) threatens tomato production, resulting in substantial yield losses. An efficient inoculation system for ToCV can accelerate the isolation and utilization of tomato-resistant genetic resources. However, infectious ToCV clones cannot directly infect tomato plants. In this study, we cloned the full-length ToCV genome, including RNA1 and RNA2 molecules, and integrated their corresponding DNA fragments separately into the less virulent tobacco rattle virus-based pTRV2GW vector. Agrobacterium suspensions carrying pTRV1, pTRV2GW-RNA1, and pTRV2GW-RNA2 plasmids were mixed in equal amounts as the ToCV infectious clones and introduced into tomato cotyledons via a needleless syringe. RT-PCR analysis detected RNA1 ORF3 and RNA2 ORF10 after infection, and the ToCV capsid protein was detected via western blotting. Transmission electron microscopy revealed the viral aggregates of ToCV in infected leaves. Plants with successful infection displayed typical disease symptoms, including stunted growth, yellow leaves, increased anthocyanin accumulation on the abaxial side, reduced photosynthetic and antioxidant capacities, decreased fruit size, and impaired fruit maturation. The ToCV infectious clones were applied to susceptible tomato cultivars and resistant wild species, and only susceptible cultivars developed typical disease symptoms. Western blotting showed lower viral accumulation in resistant species than in susceptible cultivars. Thus, integrating the ToCV genome with the less virulent TRV enables direct infection of tomato by ToCV, accelerating the breeding of ToCV-resistant tomato and creating a feasible strategy to establish highly efficient viral infectious clones in other systems.