The necrogenic strain N5 of tomato mosaic virus (ToMV-N5) causes systemic necrosis in tomato cultivar Hezuo903. In this work, we mapped the viral determinant responsible for the induction of systemic necrosis. By exchanging viral genes between N5 and a non-necrogenic strain S1, we found that movement protein (MP) was the determinant for the differential symptoms caused by both strains. Compared with S1 MP, N5 MP had an additional ability to increase virus accumulation, which was not due to its functions in viral cell-to-cell movement. Actually, N5 MP, but not S1 MP, was a weak RNA silencing suppressor, which assisted viral accumulation. Sequence alignment showed that both MPs differed by only three amino acid residues. Experiments with viruses having mutated MPs indicated that the residue isoleucine at position 170 in MP was the key site for MP to increase virus accumulation, but also was required for MP to induce systemic necrosis in virus-infected tomato plants. Collectively, the lethal necrosis caused by N5 is dependent on its MP protein that enhances virus accumulation via its RNA silencing suppressor activity, probably leading to systemic necrosis responses in tomato plants.

Interspecific exchange of RNA1 or RNA2 between the cucumoviruses cucumber mosaic virus (CMV) and tomato aspermy virus (TAV) was reported to be non-viable in plants previously. Here we investigated viability of the reassortants between CMV and TAV in Nicotiana benthamiana plants by Agrobacterium-mediated viral inoculation. The reassortants were composed of CMV RNA1 and TAV RNA2 plus RNA3 replicated in the inoculated leaves, while they were defective in viral systemic movement at the early stage of infection. Interestingly, the reassortant containing TAV RNA1 and CMV RNA2 and RNA3 infected plants systemically, but produced RNA4A (the RNA2 subgenome) at an undetectable level. The defect in production of RNA4A was due to the 1a protein encoded by TAV RNA1, and partially restored by replacing the C-terminus (helicase domain) in TAV 1a with that of CMV 1a. Collectively, exchange of the replicase components between CMV and TAV was acceptable for viral replication, but was defective in either directing transcription of subgenomic RNA4A or facilitating viral long-distance movement. Our finding may shed some light on evolution of subgenomic RNA4A in the family Bromoviridae.

In this study, the insert length, location within the coat protein-encoding gene, and sequence orientation of the target fragment were optimized to construct an efficient virus-induced gene silencing (VIGS) system in melon using a Begomovirus solanumdelhiense vector. Existing systems are mostly RNA viruses, requiring in vitro synthesis of viral strands that are prone to degradation, although they exhibit high infectivity and stability in cucurbit hosts and ease of manipulation. This vector was selected for its more stable genome structure and these advantages. The melon phytoene desaturase (CmPDS), a key gene of carotenoid biosynthesis, was selected as a reporter gene to evaluate the effects of the VIGS system. Our results revealed that the melon leaves in all the VIGS treatments exhibited a typical photobleaching phenotype at 21 days post-inoculation. Moreover, reverse transcription quantitative real-time PCR revealed a significant reduction in the mRNA levels of PDS in melon. The highest silencing efficiency (lowest PDS mRNA levels) was achieved by the VIGS vector harboring a 165 bp CmPDS fragment at the 3′ end of the AV1. These findings not only establish a more efficient VIGS protocol for melon but also provide a foundation for developing novel virus-based silencing tools applicable to functional genomics and cucurbit crop improvement, particularly for traits requiring precise gene expression modulation such as disease resistance and fruit quality.