The development of SACMV-resistant cassava using pathogen-derived resistance strategies, targeting the DNA - a component

Taylor, Sarah Helen
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South African cassava mosaic virus (SACVM) is a cassava mosaic disease- causing virus, belonging to the genus Begomovirus of the Geminiviridae family. It is a bipartite geminivirus, having two circular single-stranded DNA components, designated DNA-A and DNA-B. The AC1 open reading frame on the DNA-A component encodes for the Rep protein, which is absolutely required for virus replication. Plant virus silencing strategies exploit the use of the viralderived sequences to confer resistance towards that particular virus, a concept termed pathogen derived resistance (PDR). PDR resistance strategies used to engineer resistance towards geminiviruses include the expression of viral proteins, the use of naturally occurring defective interfering molecules which delay and attenuate infection symptoms, the sequence specific degradation of viral transcripts in the host cytoplasm by inducing RNA silencing with sense, antisense or invert-repeat transgenes and the use of double-stranded RNA molecules to direct methylation of virus promoter sequences. In this study SACMV AC1-derived sequences in sense and invert-repeat transgene constructs were used to induce RNA silencing against SACMV infection in Nicotiana benthamiana. The replication of SACMV DNA-A was reduced by almost 100% in two N. benthamiana lines expressing untranslatable SACMV AC1 sense transcripts compared to antisense and control lines. Two size classes of transgene derived siRNAs were isolated from unchallenged leaf tissue; one size class being unique, amongst the sense lines, to the replication knockdown lines. Similarly, a sense arm sequence-modified invert-repeat construct resulted in almost 100% SACMV replication reduction in six transgenic lines. This construct was produced using a technique for the production of stable RNA silencing constructs. Unmodified, spliceable intron invert-repeat transgene constructs transcribed to produce hairpin RNA molecules of 572-nt and 193-nt arm length, resulted in SACMV replication knockdown of 60 and 80% respectively. Towards the production of SACMV resistant cassava cultivars, TMS60444 FEC and MTAI 16 cotyledons were transformed with an invert-repeat RNA silencing construct, however regeneration of transformed plants failed. Cassava transformation and regeneration is inherently difficult and it has been proposed here that a minimal cassette technology-based method can be applied to achieve transformation of cassava with the RNA silencing constructs that showed significant SAMCV silencing in this study.