Cassava mosaic disease resistant cassava

The cassava was modified for resistance to Cassava mosaic disease (CMD) by introducing an RNA interference cassette that targets African cassava mosaic virus (ACMV) common region. The production of hairpin RNA by the host cells trigger an RNAi response that is expected to cause methylation of the viral DNA to prevent replication and/or expression of the viral genome. Due to conservation between CR sequences in ACMV and East african cassava mosaic virus, the modified cassava is expected to resistance to both viruses, which are causal agents of CMD. A selectable marker, Escherichia coli hygromycin B phosphotransferase, was additionally included for hygromycin selection during transformation.

Cassava cultivar TMS60444

pCAMBIA1300

RNA interference cassette
The modified cassava contains an RNA interference (RNAi) cassette designed to target African cassava mosaic virus common region (CR). Transcription is initiated from the Cauliflower mosaic virus (CaMV) 35S promoter and terminates at the CaMV 35S terminator. The transcript contains two segments (sense and antisense) of CR separated by a plant synthetic intron. Post-transcription, the intron functions as a loop and allows the sense and antisense segments of CR base pair, forming hairpin RNA (hpRNA). The hpRNA acts as double stranded RNA (dsRNA), which triggers an RNAi response and the host cell machinery will target the CR region of the viral genome. Since the CR region is not transcribed, the RNAi response will not be directed towards viral transcripts. However, it is thought that the complementarity between the siRNA and the viral DNA triggers methylation to prevent replication and/or expression of the viral genome.

Note:
- The CR sequence corresponds to almost the entire sequence of the common region with core elements of the leftward promoter and the adjacent 86 bp sequence with core elements of the rightward promoter.
- The segment of CR corresponds to positions 21 to 277 of the ACMV DNA 1 (DNA A) (GenBank accession NC_001467).
- The source of the AC1 and AV1 sequences is ACMV strain West Kenyan 844.
- Due to the RNAi response, no proteins are expected to be translated from the RNAi cassette's transcript.

Selectable marker
Transcription of Escherichia coli hygromycin B phosphotransferase is under transcriptional control of the CaMV 35S promoter and Agrobacterium tumefaciens nopaline synthase terminator.