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) replication associated disease AC1 and capsid protein (AV1). The production of hairpin RNA by the host cells trigger an RNAi response that is expected to target viral transcripts and prevent viral replication and thus further infection. Due to conservation between AC1 and AV1 sequences in ACMV and East african cassava mosaic virus, the modified cassava is expected to resistant to both viruses, which can cause CMD. A selectable marker, Escherichia coli hygromycin B phosphotransferase, was additionally included for hygromycin selection during transformation.

pCAMBIA1300

RNA interference cassette
The modified cassava contains an RNA interference (RNAi) cassette designed to target African cassava mosaic virus replication associated gene (AC1) and capsid protein (AV1). 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 AC1 separated by a plant synthetic intron. Following the AC1 portion, the transcript contains another synthetic intron and then AV1 antisense and sense sequences separated another synthetic intron. Post-transcription, the introns between the sense and antisense sequences function as loops, allowing the sense and antisense segments of AC1 and AV1 to base pair, forming hpRNA. Two hpRNA structures separated by a synthetic intron are expected to be formed: one corresponding to the AC1 segment and one corresponding to the AV1 segment. The hpRNA acts as double stranded RNA (dsRNA), which triggers an RNAi response and the host cell machinery will target AC1 and AV1 viral transcripts for degradation.

Note:
- The segment of AC1 corresponds to position 1690 to 1844 of the ACMV DNA 1 (DNA A) (GenBank accession AJ427910).
- The segment of AV1 corresponds to position 492 to 647 of the ACMV DNA 1 (DNA A) (GenBank accession AJ427910).
- The source of the AC1 and AV1 sequences is ACMV strain Nigeria-Ogo.
- Due to the RNAi response, no protein translation is expected to occur from the transcript produced from the RNAi cassette.

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

RNA interference
An RNAi response is an anti-viral response triggered by the recognition of dsRNA. Host DICER recognizes dsRNA, cleaving the dsRNA into small interfering RNA (siRNA), roughly 21-23 bp long (size is host dependent). The siRNA is then bound by ARGONAUTE family proteins, which unwind the duplex, leaving a single strand of the siRNA, and activating the RISC complex. The RISC complex targets transcripts with homology to the siRNA and degrades them.