Developing Cucumber mosaic virus (CMV) resistant transgenic chilli (Capsicum annum) through RNAi strategy.

Cucumber mosaic virus is major constraints to the production of chilli and other vegetable crops worldwide. The virus is known to possess widest host range of any known plant virus. The pathogen-derived resistance to the disease is partially successful resulting in only partial or very narrow spectrum of resistance to the virus. The aim of this work was to efficiently engineer resistance to CMV in chilli, through RNA silencing by the expression of dsRNA of CMV genes. Similar approach with the expression of CMV replicase gene attempted by Ntui et al (2014). Also dsRNA of a defective replicase CMV gene was expressed in transgenic potato (Ntui et al., 2013). Double stranded RNA (dsRNA) derived from viral sequences induces gene silencing and can serve as an important tool for lowering the viral titre in plants. Hence, in the present study, a dsRNA containing partial inverted repeat fragments of coat protein (cp), viral replicase or RNA dependent RNA polymerase (2a) and RNA silencing suppressor (2b) of CMV was used to directly deliver to laminal cells by mechanical inoculation and found to restrict the virus spread as evidenced by northern assay. The same dsRNA was over expressed in chilli using CaMV35S promoter in pBI121 backbone. About 25 T0 Chilli cv. G4 putative transformants over expressing dsRNA were developed by Agrobacterium mediated method using cotyledons as explants. Of these 20 transformants showed amplification corresponding to 364 bp region in PCR analysis by using gene specific primers. The PCR confirmed transformants were further analysed by reverse transcriptase –PCR and southern analysis. The introduction of CMV-specific dsRNA derived from the partial replicase, CP and 2b genes into chilli resulted in moderate resistance unlike the previous report (Ntui et al., 2014) in tomato with three categories of plants: complete resistance, highly resistant plants, and susceptible plants. However in transgenic potato and tobacco containing the defective gene, no susceptible plants were observed (Ntui et al 2013).The key feature of RNAi-mediated resistance is the production of short dsRNA fragments known as “short interfering RNAs (siRNAs) of 21 - 25 bp in length. The artificially expressed dsRNA activates the plant’s PTGS machinery even in the absence of the virus. During PTGS, the strand of siRNA complementary to the target RNA becomes incorporated into the RNA-induced silencing complex (RISC), which is responsible for the actual RNA degradation of the homologous target RNA (Hammond et al 2001). Of the transgenic lines analyzed only one (Line no 5) did show moderate symptoms compared to the control for CMV infection. We could thus suggest that the presence of siRNA prior to challenge by CMV plays a vital role in the resistance of transgenic lines to the virus. Plants that produced detectable levels of siRNA were resistant to viral infections. Therefore, the northern analysis of the plants for the virus load during validation experiment with cucumber showed less signal intensity in dsRNA treated plants compared to the controls. Earlier reports showed the accumulation of Si RNA was used to predict the degree of resistance which is an advantage of RNAi-mediated strategy (Kalantidis 2002). Cucumber mosaic virus (CMV)-contains the 2b protein which inhibits post-transcriptional gene silencing (PTGS). The 2b genes of CMV are targeted for silencing has lead to reduced infection as against control probably inhibiting the 2b protein from its role of silencing suppressor. The progenies of single copy event showed moderate resistance to CMV in the present study. However Ntui etal 2014 reported siRNA accumulation and not the transgene copy was responsible for the symptoms. Together, our results demonstrate that the resistance of chilli against CMV infection can be achieved by expression of multiple target genes of CMV