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. 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 sequences in ACMV and East african cassava mosaic virus, the modified cassava is expected to resistant to both viruses, which are the 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 replication associated gene (AC1). 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. Post-transcription, the intron functions as a loop and allows the sense and antisense segments of AC1 to 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 AC1 viral transcripts for degradation.

Note:
- The segment of AC1 corresponds to positions 1690 to 1844 of the ACMV genome (GenBank accession AJ427910).
- The source of the AC1 sequence is ACMV strain Nigeria-Ogo.
- The AC1 sequence is conserved between ACMV and East african cassava mosaic virus.
- Due to the RNAi response, no protein is expected to be translated from the RNAi cassette.

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