Mapping and identification of cassava mosaic geminivirus DNA-A and DNA-B genome sequences for efficient siRNA expression and RNAi based virus resistance by transient agro-infiltration studies

•Maximum siRNA accumulated from 3 to 7 day after agroinfiltration of RNAi construct.•A gap of 3 days between agro-infiltration and virus inoculation was critical.•Maximum resistance was obtained by targeting AC2 ORF, in addition to AC1 and AC4.•In the ACMV DNA-B component targeting BC1 gave the best results.•The 5′-half of AC3 and AC4 ORFs yielded more siRNA than the corresponding 3′-half.

Geminiviruses are among the most serious pathogens of many economically important crop plants and RNA interference (RNAi) is an important strategy for their control. Although any fragment of a viral genome can be used to generate a double stranded (ds) RNA trigger, the precursor for generation of siRNAs, the exact sequence and size requirements for efficient gene silencing and virus resistance have so far not been investigated. Previous efforts to control geminiviruses by gene silencing mostly targeted AC1, the gene encoding replication-associated protein. In this study we made RNAi constructs for all the genes of both the genomic components (DNA-A and DNA-B) of African cassava mosaic virus (ACMV-CM), one of the most devastating geminiviruses causing cassava mosaic disease (CMD) in Africa. Using transient agro-infiltration studies, RNAi constructs were evaluated for their ability to trigger gene silencing against the invading virus and protection against it. The results show that the selection of the DNA target sequence is an important determinant for the amount of siRNA produced and the extent of resistance. The ACMV genes AC1, AC2, AC4 from DNA-A and BC1 from DNA-B were effective targets for RNAi-mediated resistance and their siRNA expression was higher compared to other RNAi constructs. The RNAi construct targeting AC2, the suppressor of gene silencing of ACMV-CM gave highest level of resistance in the transient studies. This is the first report of targeting DNA-B to confer resistance to a bipartite geminivirus infection.