Transgenically expressed T-Rep of Tomato yellow leaf curl Sardinia virus acts as a trans-dominant-negative mutant, inhibiting viral transcription andreplication

We have previously shown that transgenic expression of a truncated C1 gene of Tomato yellow leaf curl Sardinia virus (TYLCSV), expressing the first 21 0 amino acids of the replication-associated protein (T-Rep) and potentially coexpressing the C4 protein, confers resistance to the homologous virus in Nicotiana benthamiana plants. In the present study we have investigated th e role of T-Rep and C4 proteins in the resistance mechanism,, analyzing cha nges in virus transcription and replication. Transgenic plants and protopla sts were challenged with TYLCSV and the related TYLCSV Murcia strain (TYLCS V-ES[1]). TYLCSV-resistant plants were susceptible to TYLCSV-ES[1]; moreove r, TYLCSV but not TYLCSV-ES[1] replication was strongly inhibited in transg enic protoplasts as well as in wild-type (wt) protoplasts transiently expre ssing T-Rep but not the C4 protein. Viral circular single-stranded DNA (css DNA) was usually undetectable in transgenically and transiently T-Rep-expre ssing protoplasts, while viral DNAs migrating more slowly than the cssDNA w ere observed. Biochemical studies showed that these DNAs were partial duple xes with the minus strand incomplete. Interestingly, similar viral DNA form s were also found at early stages of TYLCSV replication in wt N. benthamian a protoplasts. Transgenically expressed T-Rep repressed the transcription o f the GUS reporter gene up to 300-fold when fused to the homologous (TYLCSV ) but not to the heterologous (TYLCSV-ES[1]) Cl promoter. Similarly, transi ently expressed T-Rep but not C4 protein strongly repressed GUS transcripti on when fused to the CI promoter of TYLCSV. A model of T-Rep interference w ith TYLCSV transcription-replication is proposed.