Selection of target sequences as well as sequence identity determine the outcome of RNAi approach for resistance against cotton leaf curl geminivirus complex

Cotton leaf curl disease is caused by a geminivirus complex that involves multiple distinct begomoviruses and a disease-specific DNA satellite, cotton leaf curl Multan betasatellite (CLCuMB), which is essential to induce disease symptoms. Here we have investigated the use of RNA interference (RNAi) for obtaining resistance against one of the viruses, Cotton leaf curl Multan virus (CLCuMV), associated with the disease. Three hairpin RNAi constructs were produced containing either complementary-sense genes essential for replication/pathogenicity or non-coding regulatory sequences of CLCuMV. In transient assays all three RNAi constructs significantly reduced the replication of the virus in inoculated tissues. However, only one of the constructs, that targeting the overlapping genes involved in virus replication and pathogenicity (the replication-associated protein (Rep), the transcriptional activator protein and the replication enhancer protein) was able to prevent systemic movement of the virus, although the other constructs significantly reduced the levels of virus in systemic tissues. In the presence of CLCuMB, however, a small number of plants co-inoculated with even the most efficient RNAi construct developed symptoms of virus infection, suggesting that the betasatellite may compromise resistance. Further analyses, using Rep gene sequences of distinct begomoviruses expressed from a PVX vector as the target, are consistent with the idea that the success of the RNAi approach depends on sequence identity to the target virus. The results show that selection of both the target sequence, as well as the levels of identity between the construct and target sequence, determine the outcome of RNAi-based resistance against geminivirus complexes.