Douglas Miano

The VIRCA Project: virus resistant cassava for Africa.

The VIRCA (Virus Resistant Cassava for Africa) project is a collaborative program between the Donald Danforth Plant Science Center, USA the National Crops Resources Research Institute, Uganda and the Kenya Agricultural Research Institute, Kenya. VIRCA is structured to include all aspects of the intellectual property, technology, regulatory, biosafety, quality control, communication and distribution components required for a GM crop development and delivery process. VIRCAs goal is to improve cassava for resistance to the viral diseases cassava brown streak disease (CBSD) and cassava mosaic disease (CMD) using pathogen-derived RNAi technology, and to field test, obtain regulatory approval for and deliver these products to small landholder farmers. During Phase I of the project, proof of concept was achieved by production and testing of virus resistant plants under greenhouse and confined field trials in East Africa. In VIRCA Phase II, two farmer-preferred varieties will be modified for resistance to CBSD and CMD, and lead events identified after molecular and field screening. In addition to delivery of royalty-free improved planting materials for farmers, VIRCA capacity building activities are enhancing indigenous capability for crop biotechnology in East Africa.

A Virus-Derived Stacked RNAi Construct Confers Robust Resistance to Cassava Brown Streak Disease

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.

Field Level RNAi-Mediated Resistance to Cassava Brown Streak Disease across Multiple Cropping Cycles and Diverse East African Agro-Ecological Locations

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.

Differential response of cassava genotypes to infection by cassava mosaic geminiviruses

Highlights • Cassava genotypes respond differently to infection by cassava mosaic geminiviruses.• Cassava mosaic disease resistant loci prompt recovery from systemic infection.• CMD symptoms are directly correlated with contents of viral DNA and virus specific small RNAs.• CMD infected plants abundantly accumulate 21–24 nt of virus specific small RNAs.• VsRNAs heterogeneously map the entire virus genome in both polarities.

Immunohistochemical localization of Cassava brown streak virus and its morphological effect on cassava leaves

The localization of Cassava brown streak virus (CBSV) in cassava (Manihot esculenta) leaf tissues was determined and cellular morphological changes in CBSV-infected tissues were evaluated. CBSV-symptomatic leaves were screened with CBSV-specific primers using reverse-transcriptase polymerase chain reaction. Immunohistochemical reactions showed precipitation in CBSV-infected but not CBSV-free tissues, demonstrating successful localization of CBSV. Microscopic inspection showed significantly larger (P < 0.001) midribs in CBSV-infected compared with control (uninfected) leaves. Viral accumulation occurred in middle and lower but rarely in young upper leaves. This immunohistochemical method for virus localization will be invaluable for efficient screening of CBSV and for breeding resistant cassava.

Status of Begomovirus Research and Management in Kenya

Viruses belonging to the genus Begomovirus (family: Geminiviridae) infect different crops in different parts of the world, resulting in great economic losses. In Kenya, begomoviruses have been reported to infect important cultivated crops such as cassava, sweet potatoes, and tomatoes and noncultivated plant Deinbollia borbonica, a perennial weed. Apart from begomoviruses infecting cassava, those infecting other crops have not been fully characterized, and their distribution within the country has not been established. This paper describes the current status of begomoviruses in Kenya, management strategies employed, and research gaps that need to be addressed.