Engineering virus resistant transgenic cassava: the design of long hairpin RNA constructs against South African cassava mosaic virus

ABSTRACT \nCassava is currently the second most important source of carbohydrates on the African \ncontinent. In the last two decades, cassava crops have been severely affected by \noutbreaks of cassava mosaic disease (CMD). South African cassava mosaic virus \n(SACMV) has been associated with CMD outbreaks in the Mpumalanga province. \nAdvances in post-transcriptional gene silencing (PTGS) technology have provided \npromising new strategies for the engineering of virus resistance in plants. Inverted repeat \n(IR) constructs are currently the most potent inducers of PTGS, however, these constructs \nare inherently unstable. The purpose of this study was to develop IR constructs with an \nimproved stability for the efficient induction of PTGS in plants. Two mismatched \ninverted repeat constructs, one targeting the SACMV BC1 open reading frame, the other \ntargeting the Maize streak virus (MSV) AC1 open reading frame, were successfully \ncreated. Sodium bisulfite was used to deaminate cytosine residues on the sense arm of the \nconstructs. The resulting number of GT mismatches was seemingly sufficient to stabilize \nthe linear conformation of the IR constructs, as they were efficiently propagated by E.coli \nDH5!, and subsequently behaved like linear DNA molecules. Furthermore, it was found \nthat the number of mismatches on the BC1 construct (17.5%) was ideal, as the \nsubsequent stability of the predicted RNA hairpin was not affected. Due to the higher \nnumber of mismatches on the AC1 construct (23.5%), it was found that the loop region of \nthe RNA hairpin was marginally destabilized. Despite this, long stretches of stable \ndsRNA were still produced from the AC1 IR construct, and is likely to induce PTGS. \nInterestingly, it was observed that the mismatched IR constructs, although still replicated \nin E.coli, were marginally destabilized in Agrobacterium. Therefore, it was deduced that \nthe stability of a mismatched IR construct may be influenced by the particular \nintracellular environment of an organism. Due to the recalcitrance of cassava to \ntransformation, a model plant system, Nicotiana benthamiana, was used to screen \nconstructs for toxicity, stability, and efficiency of PTGS induction. Agrobacteriummediated \ntransformation and regeneration of N. benthamiana was optimized, and 86% \ntransformation efficiency was achieved when using leaf disk explants. It was found that \nthe addition of an ethylene scrubber, potassium permanganate, substantially increased the \nrate of regeneration by reducing the frequency of hyperhydritic plants. Transgene \niv \nintegration was confirmed by PCR amplification of the hptII gene in the T-DNA region. \nTransgene expression was confirmed by screening for GUS and GFP reporter genes. No \ntoxic responses to the transgene have been observed thus far. Studies are currently \nunderway to confirm the stability of the mismatched IR constructs in N. benthamiana. \nPAGE Northern blotting is being done, as the detection of siRNAs derived from the \ntransgene will confirm that constructs are functional. In addition, infectivity assays are \nunderway to determine the efficacy of BC1 knockdown by a stably integrated construct. \nDue to the enhanced stability of mismatched IR constructs, they may be an appealing \nalternative to currently available intron-spliced, or exact matched hairpin systems.