Transgenic RNA interference (RNAi)‐derived field resistance to cassava brown streak disease

Abstract

Cassava brown streak disease (CBSD), caused by the Ipomoviruses Cassava brown streak virus (CBSV) and Ugandan Cassava brown streak virus (UCBSV), is considered to be an imminent threat to food security in tropical Africa. Cassava plants were transgenically modified to generate small interfering RNAs (siRNAs) from truncated full-length (894-bp) and N-terminal (402-bp) portions of the UCBSV coat protein (ΔCP) sequence. Seven siRNA-producing lines from each gene construct were tested under confined field trials at Namulonge, Uganda. All nontransgenic control plants (n = 60) developed CBSD symptoms on aerial tissues by 6 months after planting, whereas plants transgenic for the full-length ΔCP sequence showed a 3-month delay in disease development, with 98% of clonal replicates within line 718-001 remaining symptom free over the 11-month trial. Reverse transcriptase-polymerase chain reaction (RT-PCR) diagnostics indicated the presence of UCBSV within the leaves of 57% of the nontransgenic controls, but in only two of 413 plants tested (0.5%) across the 14 transgenic lines. All transgenic plants showing CBSD were PCR positive for the presence of CBSV, except for line 781-001, in which 93% of plants were confirmed to be free of both pathogens. At harvest, 90% of storage roots from nontransgenic plants were severely affected by CBSD-induced necrosis. However, transgenic lines 718-005 and 718-001 showed significant suppression of disease, with 95% of roots from the latter line remaining free from necrosis and RT-PCR negative for the presence of both viral pathogens. Cross-protection against CBSV by siRNAs generated from the full-length UCBSV ΔCP confirms a previous report in tobacco. The information presented provides proof of principle for the control of CBSD by RNA interference-mediated technology, and progress towards the potential control of this damaging disease.

Characterization of Brown Streak Virus–Resistant Cassava

“…Our virus inoculation method resulted in 100% infection rates in the susceptible 60444 scions in all experiments. Infection rates obtained by the top grafting method are more consistent and reproducible compared to other inoculation methods reported to date (Maruthi et al, 2005;Mohammed et al, 2012;Ogwok et al, 2012).…”

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

“…8 Farmers propagate cassava vegetatively from field-derived stem cuttings, not from tissue culture-derived plantlets. Demonstrating proof of concept for resistance to CBSD across the vegetative cropping cycle is an essential step within the product development process to develop and deliver resistant planting materials for farmers in East Africa.…”

“…A field trial was therefore established to evaluate efficacy of the RNAi-mediated resistance to CBSD through a typical vegetative propagation cycle. Woody stem cuttings measuring 25 to 30 cm in length were obtained from plants within the CFT harvested in November 2011, as previously described by Ogwok et al 8 Three lines transgenic for p718 (p718-001, p718-002, and p718-005) that provided complete resistance to UCBSV and differing levels of protection to CBSV in the original field experiment were re-planted, in addition to stakes of the nontransgenic control cv 60444 and the CBSD tolerant cv TMS30572. Stem cuttings of the highly susceptible Ugandan farmer-preferred cv TME204 were also obtained from an experimental field at the National Crops Resources Research Institute (NaCRRI).…”

“…The plants were grown with no agrochemical or fertilizer applications and with hand weeding performed as necessary. Plants were visually assessed on a monthly basis for CBSD and CMD incidence and severity, and for the number of adult whiteflies (B. tabaci) on the underside of the uppermost five fully open leaves, as described previously by Ogwok et al 8 Plants were harvested at 11 MAP.…”

“…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.…”