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

Raj Deepika Chauhan,Anton Bua,Hannington Obiero,Nigel J. Taylor,Muhammad Ilyas,Peter Raymond,Theresia Munga,Titus Alicai,John Odipio,Jude Aleu,Henry Wagaba,Douglas Miano,Mark E. Halsey,Geoffrey Okao-Okuja,Getu Beyene

doi:10.3389/fpls.2016.02060

Abstract

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.

Highlights

Cassava brown streak disease (CBSD) is a major constraint to cassava (Manihot esculenta) production in sub-Saharan Africa
Transgenic lines expressing low, medium and high levels of small interfering RNAs (siRNAs) derived from p5001 were evaluated by challenge with Cassava brown streak virus (CBSV) and Ugandan Cassava brown streak virus (UCBSV) using a bud grafting procedure for virus inoculation under greenhouse conditions (Wagaba et al, 2013)
Cassava cultivar TME 204 was genetically modified to express siRNAs derived from the inverted repeat construct p5001 consisting of fused coat protein (CP) sequences from CBSV and UCBSV (Chauhan et al, 2015; Beyene et al, 2016a)

Summary

Introduction

Cassava brown streak disease (CBSD) is a major constraint to cassava (Manihot esculenta) production in sub-Saharan Africa. Viruses that cause CBSD are transmitted semi-persistently by the whitefly vector Bemisia tabaci (Maruthi et al, 2005; Mware et al, 2009) and can occur as single or dual infections (Mbanzibwa et al, 2011). CBSD was geographically restricted to the lower coastal regions of East Africa (Hillocks et al, 2001) before being confirmed as present in Uganda in the mid-2000s (Alicai et al, 2007). The rapid spread of CBSD to reach epidemic proportions in East and Central Africa has been linked to a “super-abundance” of whitefly vectors and associated with the evolving environmental and climatic conditions occurring within the region (Jeremiah et al, 2015)

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