Cassava is a very important crop in poverty alleviation in sub-Saharan Africa (SSA), and it contributes to 60% of the daily calorie intake. One major constrain to cassava cultivation is cassava mosaic disease (CMD) (Legg et al., 2015; Thresh & Cooter, 2005). CMD is caused by bipartite cassava begomoviruses species including African cassava mosaic virus (ACMV). African cassava mosaic virus consists of a single-stranded circular bipartite genome comprising of DNA-A and DNA-B. DNA-A has six open reading frame coding for four complementary sense genes that is AC1 to AC4 and two virion sense genes AV1 and AV2 (Vanitharani et al, 2005). AC1 codes for the (Rep) 358 amino acids (aa) protein, and Rep is essential for replication initiation. AC2 codes for a 135 aa protein called (TrAP) which is essential for transcriptional activation as well as suppression of post-transcriptional gene silencing. AC3 codes for (REn) a 134 aa protein essential for replication enhancement. AC4 functions as a suppressor of RNA silencing (VSR) and symptom modulator. AV1 codes a coat protein of 258 aa and AV2 encodes a pre-coat protein (Vanitharani et al., 2004; Vanitharani et al., 2005). DNA-B has 2 open reading frames coding for a complementary sense gene (BC1) and a virion sense gene (BV1). BV1 encodes a nuclear shuttle protein (NSP) and BC1 encodes a cell to cell movement protein (MP) (Varma and Malathi, 2003). Plants have a natural defence mechanism called RNA silencing which protects them from virus invasion (Bologna and Vionnet, 2014; Ribeiro et al., 2006; Zvereva and Pooggin, 2012). RNA silencing results in sequence specific inhibition of transcription or translation (Balcombe, 1999). Post transciptional gene silencing (PTGS) plays a major role in plant defence against viral pathogen invasion (Balcombe, 1999; Waterhouse et al., 2001). The process is dependent on the recognition of foreign double stranded RNA (dsRNA). Ribonuclease III like enzymes called Dicer then processes dsRNA to short interfering RNA (siRNA) (Bologna and Vionnet, 2014; Hamilton et al., 2002). Subsequently, one strand of the siRNA then acts as guide strand for RNA induced silencing complex degradation of homologous RNA molecules (Arreger et al., 2012; Hammond et al., 2000; Tang et al., 2003).
27 Several studies in cassava or other related plants which can be affected by geminiviruses have shown that introduction of full-length or truncated segments of the different geminivirus genes induces varying levels of viral tolerance (Hong and Stanley, 1996; Norris et al., 1996; Ntui et al., 2015; Vanderschuren et al., 2007). Significant levels of resistance to ACMV have been reported in transgenic plants expressing antisense mRNA of Rep, TrAP and REn proteins (Zheng et al., 2005). Increased resistance has also been reported in transgenic plants expressing sense and antisense RNA homologous to Sri Lankan cassava mosaic virus (SLCMV) AV1/AV2 overlapping region (Ntui et al., 2015), and transgenic plants expressing sense and antisense RNA homologous to ACMV-[NG:Ogo:90] AC1 (Vanderschuren et al., 2009). Constructs with self complementary sense and antisense strands forming inverted repeat (IR) are more efficient PTGS inducers as opposed to either sense or antisense strategies (Wang et al, 2008; Waterhouse et al, 1998). Expression of the IR contruct would result in formation of a hairpin RNA (hpRNA) which is recognised as being double stranded thereby efficiently triggering PTGS (Aregger et al., 2013; Duan et al., 2012).
High-throughput gene silencing vectors such as pHELLSGATE and pHANNIBAL which facilitate insertion of the transgene by Gateway recombination in the sense and antisense orientation whilst being separated by a sliceable intron are commonly used in designing IR constructs (Helliwell and Waterhouse, 2003; Wesley et al., 2001). However, large introns in generic vectors like pHellsgate often result in T-DNA deletion and recombination (Nakano et
al., 2005). Taylor et al. (2012) from our laboratory developed a method to avoid the use of
large introns by replacing the intron with a few bases (spacer) and stabilizing the hairpin, by introducing mismatches in the sense arm of the IR hairpin construct using bisulfite treatment. Amplification of the converted fragment would result in uracil being replaced by thymine causing base mismatches. The use of mismatched constructs to induce PTGS in tobacco for virus resistance was successfully reported (Taylor et al., 2012).
In this chapter transgenic cassava was evaluated for resistance/tolerance to CMD. The transgenic plants were from two transformation events with either a mismatched (AMM4) or non-mismatched (CMM6) Africa cassava mosaic virus-[Nigeria:Ogorocco;1990] (ACMV- [NG:Ogo:90]) IR construct derived from stacking the overlapping AC1/AC4 (2437-2572 nt) and AC2/AC3 (1297-1479 nt) viral sequences, were screened. The ACMV-[NG:Ogo:90] derived (non-mismatched construct) was constructed using the Gateway method where the sense and antisense arms are separated by a 800 bp pdk intron; and the mismatched transgene by bisulfite treatment for introduction of mismatches (C to T) in the sense arm of the identical
28 ACMV-[NG:Ogo:90] IR sequence (Rey et al., 2015; Taylor et al., 2012). CMM6 and AMM4 transgenic lines were derived from previous transformation of the model cassava cultivar cv.60444.
The current study evaluates CMM6 and AMM2 lines for viral resistance or tolerance to ACMV-[NG:Ogo:90] by monitoring symptoms, viral replication and plant height in comparison with untransformed cv.60444. Symptoms were evaluated using a 0-3 scale similar to the one described by Ntui et al. (2015). The current study evaluates if there is a correlation between the symptoms and the viral load as this helps in defining whether the plants are tolerant or resistant. For resistance/tolerance trials in our laboratory we adopted definitions for virus resistance, tolerance or susceptibility from (Bengyella and Rey, 2014; Lapidot and Friedman, 2002): resistant plants show no symptoms as a result of no virus replication; tolerance is defined as the presence of mild symptoms due to low virus levels; and susceptibility refers to the presence of severe symptoms due to uncontrolled virus replication. Small interfering RNA generation was also evaluated to determine if tolerance/resistance correlated with the siRNAs being produced. The infectivity trials were done in growth facilities and the green-house with controlled environmental conditions. Resistance or tolerance has been shown to differ when conditions vary greatly, such as temperature, viral dosage and light intensity (Chellappan et al., 2005; Szittye et al., 2003; Vanderschuren et al., 2009). The tuber yields of tolerant lines from this trial were evaluated to determine if tolerance resulted in less yield loss of the storage roots, and also if insertion of the transgene would interfere with root formation.
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2.1.1 Specific Aims
The aim of this research was to subject selected transgenic non-mismatched CMM6 lines 2, 3, 5, 6 and 7, and corresponding mismatched AMM2 lines 30, 41, 44, 52, 53 and 54 lines to reproducible trials and evaluate for response to virus challenge and select potential resistant/tolerant lines for further larger GM trials in future.
Steps in achieving our aim
i. To confirm successful integration of transgenes in previously transformed lines
ii. Micro-propagation of CMM6 and AMM2 transgenic cv.60444 lines and acclimatization for (6 weeks)
iii. Agro-infection of transgenic lines with ACMV-[NG:Ogo:90] infectious virus clones iv. Plant evaluations for virus symptom severity, plant height and viral load at 14, 36, 56
and 365 days post inoculation (dpi) v. Tuber yield evaluation at 365 dpi
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