RNAi-derived field resistance to Cassava brown streak disease persists across the vegetative cropping cycle

Abstract

Cassava brown streak disease (CBSD) is currently considered the most important disease of cassava in East Africa and a significant threat to food security in the region. 1 CBSD is caused by two distinct virus species: Cassava brown streak virus (CBSV) and Ugandan Cassava brown streak virus (UCBSV), both of which belong to the genus Ipomovirus, family Potyviridae, and possess a ssRNA genome of messenger sense.2,3 Both viruses are transmitted by whiteflies (Bemisia tabaci), resulting in CBSDsymptomatic plants carrying infections by one or both viral species. Since its re-emergence in Uganda in 2004, 4 CBSD has become prevalent in East Africa, with new outbreaks of the disease resulting from dissemination of infected cassava planting materials and high whitefly vector populations. 4,5 Genetically engineered resistance to CBSD was reported recently in tobacco and cassava plants under greenhouse conditions 6,7 and in cassava in a confined field trial (CFT) in Uganda. 8 Two RNAi constructs targeting UCBSV were tested in the field: p718 consisting of an 894 bp inverted repeat sequence (nts 208-1101) of a truncated version of the full-length UCBSV coat protein (CP), and p719 consisting of a 397 bp portion (nts 208-604) of the UCBSV CP N-terminal.7 Tissue culturederived cassava plants of cv 60444 shown to be accumulating transgenically derived UCBSV CP specific siRNAs, were planted in the field and proved to be highly resistant to whitefly transmitted UCBSV. Fourteen transgenic lines were tested, seven of each of the two constructs. Across all 14 lines, greater than 98% of the plants (n = 60) remained free of UCBSV 11 months after planting (MAP), as determined by RT-PCR. Protection against the non-targeted CBSV was observed in two transgenic lines of the p718 construct (carrying the full length CP sequence). This was most striking in line p718-001 in which 54 out of 60 plants (90%) remained CBSD symptom free and RT-PCR negative for Keywords: cassava brown streak disease, RNAi-mediated resistance, cassava, transgenic, confined field trial, UgandaAbbreviations: CBSD, Cassava brown streak disease; CBSV, Cassava brown streak virus; CFT, confined field trial; CMD, cassava mosaic disease; CP, coat protein; MAP, months after planting; NaCRRI, National Crops Resources Research Institute; RT-PCR, reverse transcription polymerase chain reaction; siRNA, small interfering RNA; ssRNA, single-stranded RNA; UCBSV, Uganda Cassava brown streak virus a confined field trial was established to determine durability of RNai-mediated resistance to cassava brown streak disease (cBsD). stem cuttings were obtained from field-grown cassava plants of cv 60444 transgenic for construct p718, consisting of an 894 bp inverted repeat sequence from the Ugandan cassava brown streak virus (UcBsV) coat protein.Plants were established from three transgenic lines previously shown to provide complete resistance to UcBsV and differing levels of protection to the non-homologous virus species cassava brown streak virus (cBsV), and grown for 11 months. cBsD sympto...

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

“…These qualities make TME 204 an ideal target for improvement within the VIRCA project, which aims to bring CMD- and CBSD-resistant planting material to smallholder farmers in East Africa (Taylor et al, 2012). We previously reported field resistance to CBSD generated from an inverted repeat construct carrying the CP of UCBSV (p718) (Ogwok et al, 2012; Odipio et al, 2014). While high levels of resistance to the homologous virus species was achieved in p718 transgenic plants, resistance to CBSV was restricted to two lines that accumulated CP-derived siRNAs at very high levels (Ogwok et al, 2012).…”

“…Storage roots were weighed on a digital scale and each root was sliced transversely along their length into five pieces. Each slice was visually assessed for presence and severity of CBSD necrosis on a 1–5 scale (Ogwok et al, 2012; Odipio et al, 2014), adapted from that reported by Hillocks et al (1996). Storage root CBSD incidence was expressed as (a) percentage of plants per line that showed CBSD symptoms in their storage roots and (b) number of roots per line showing CBSD symptoms expressed as a percentage of total number of roots harvested.…”

Recent Biotechnological Advances in the Improvement of Cassava

“…In cassava improvement, hpRNA and antisense silencing procedures have been employed mostly in virus control [52][53][54][55][56]. An indirect approach where the hpRNA is used to knockdown the expression of V-ATPase A, an enzyme that provides force for many transport processes, has been used to control whitefly vectors of CMGs and CBSVs [57,58].…”

Genetic Engineering and Sustainable Crop Disease Management: Opportunities for Case-by-Case Decision-Making

“…While consumers may be hesitant to eat transgenic papaya containing a viral coat-protein gene, they may be surprised to know that they are eating complete virus particles in fruit harvested from non-transgenic, infected trees, including fruit from cross-protected trees. RNAi provides control of other destructive viruses of crops, including the viral complexes that attack cassava in East Africa [22,82], soybean [83], and summer squash [84], and others [47,85].…”