Cassava brown streak disease (CBSD) threatens food and economic security for smallholder farmers throughout East and Central Africa, and poses a threat to cassava production in West Africa. CBSD is caused by two whitefly-transmitted virus species: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) (Genus: Ipomovirus, Family Potyviridae). Although varying levels of tolerance have been achieved through conventional breeding, to date, effective resistance to CBSD within East African cassava germplasm has not been identified. RNAi technology was utilized to integrate CBSD resistance into the Ugandan farmer-preferred cassava cultivar TME 204. Transgenic plant lines were generated expressing an inverted repeat construct (p5001) derived from coat-protein (CP) sequences of CBSV and UCBSV fused in tandem. Northern blots using probes specific for each CP sequence were performed to characterize 169 independent transgenic lines for accumulation of CP-derived siRNAs. Transgenic plant lines accumulating low, medium and high levels of siRNAs were bud graft challenged with the virulent CBSV Naliendele isolate alone or in combination with UCBSV. Resistance to CBSD in the greenhouse directly correlated to levels of CP-derived siRNAs as determined by visual assessment of leaf and storage root symptoms, and RT-PCR diagnosis for presence of the pathogens. Low expressing lines were found to be susceptible to CBSV and UCBSV, while medium to high accumulating plant lines were resistant to both virus species. Absence of detectable virus in the best performing p5001 transgenic lines was further confirmed by back-inoculation via sap or graft challenge to CBSD susceptible Nicotiana benthamiana and cassava cultivar 60444, respectively. Data presented shows robust resistance of transgenic p5001 TME 204 lines to both CBSV and UCBSV under greenhouse conditions. Levels of resistance correlated directly with levels of transgene derived siRNA expression such that the latter can be used as predictor of resistance to CBSD.

Artificial miRNAs (amiRNA) were generated targeting conserved sequences within the genomes of the two causal agents of Cassava brown streak disease (CBSD): Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Transient expression studies on ten amiRNAs targeting 21nt conserved sequences of P1(CBSV and UCBSV), P3(CBSV and UCBSV), CI(UCBSV), NIb(CBSV and UCBSV), CP(UCBSV) and the un-translated region (3'-UTR) were tested in Nicotiana benthamiana. Four out of the ten amiRNAs expressed the corresponding amiRNA at high levels. Transgenic N. benthamiana plants were developed for the four amiRNAs targeting the P1 and NIb genes of CBSV and the P1 and CP genes of UCBSV and shown to accumulate miRNA products. Transgenic plants challenged with CBSV and UCBSV isolates showed resistance levels that ranged between ∼20-60% against CBSV and UCBSV and correlated with expression levels of the transgenically derived miRNAs. MicroRNAs targeting P1 and NIb of CBSV showed protection against CBSV and UCBSV, while amiRNAs targeting the P1 and CP of UCBSV showed protection against UCBSV but were less efficient against CBSV. These results indicate a potential application of amiRNAs for engineering resistance to CBSD-causing viruses in cassava.

Summary: Cassava mosaic disease (CMD) and cassava brown streak disease (CBSD) are the two most important viral diseases affecting cassava production in Africa. Three sources of resistance are employed to combat CMD: polygenic recessive resistance, termed CMD1, the dominant monogenic type, named CMD2, and the recently characterized CMD3. The farmer‐preferred cultivar TME 204 carries inherent resistance to CMD mediated by CMD2, but is highly susceptible to CBSD. Selected plants of TME 204 produced for RNA interference (RNAi)‐mediated resistance to CBSD were regenerated via somatic embryogenesis and tested in confined field trials in East Africa. Although micropropagated, wild‐type TME 204 plants exhibited the expected levels of resistance, all plants regenerated via somatic embryogenesis were found to be highly susceptible to CMD. Glasshouse studies using infectious clones of East African cassava mosaic virus conclusively demonstrated that the process of somatic embryogenesis used to regenerate cassava caused the resulting plants to become susceptible to CMD. This phenomenon could be replicated in the two additional CMD2‐type varieties TME 3 and TME 7, but the CMD1‐type cultivar TMS 30572 and the CMD3‐type cultivar TMS 98/0505 maintained resistance to CMD after passage through somatic embryogenesis. Data are presented to define the specific tissue culture step at which the loss of CMD resistance occurs and to show that the loss of CMD2‐mediated resistance is maintained across vegetative generations. These findings reveal new aspects of the widely used technique of somatic embryogenesis, and the stability of field‐level resistance in CMD2‐type cultivars presently grown by farmers in East Africa, where CMD pressure is high.

Cassava brown streak disease (CBSD) presents a serious threat to cassava production in East and Central Africa. Currently, no cultivars with high levels of resistance to CBSD are available to farmers. Transgenic RNAi technology was employed to combat CBSD by fusing coat protein (CP) sequences from Ugandan cassava brown streak virus (UCBSV) and Cassava brown streak virus (CBSV) to create an inverted repeat construct (p5001) driven by the constitutive Cassava vein mosaic virus promoter. Twenty-five plant lines of cultivar TME 204 expressing varying levels of small interfering RNAs (siRNAs) were established in confined field trials (CFTs) in Uganda and Kenya. Within an initial CFT at Namulonge, Uganda, non-transgenic TME 204 plants developed foliar and storage root CBSD incidences at 96-100% by 12 months after planting. In contrast, 16 of the 25 p5001 transgenic lines showed no foliar symptoms and had less than 8% of their storage roots symptomatic for CBSD. A direct positive correlation was seen between levels of resistance to CBSD and expression of transgenic CP-derived siRNAs. A subsequent CFT was established at Namulonge using stem cuttings from the initial trial. All transgenic lines established remained asymptomatic for CBSD, while 98% of the non-transgenic TME 204 stake-derived plants developed storage roots symptomatic for CBSD. Similarly, very high levels of resistance to CBSD were demonstrated by TME 204 p5001 RNAi lines grown within a CFT over a full cropping cycle at Mtwapa, coastal Kenya. Sequence analysis of CBSD causal viruses present at the trial sites showed that the transgenic lines were exposed to both CBSV and UCBSV, and that the sequenced isolates shared >90% CP identity with transgenic CP sequences expressed by the p5001 inverted repeat expression cassette. These results demonstrate very high levels of field resistance to CBSD conferred by the p5001 RNAi construct at diverse agro-ecological locations, and across the vegetative cropping cycle.