Host delivered RNAi mediated silencing of acetylcholinesterase (Ace-1) gene for gram pod borer resistance.

HOST-DELIVERED RNAi-MEDIATED SILENCING OF Ace-1 GENE FOR GRAM POD BORER RESISTANCE Gram pod borer (Helicoverpa armigera) is a polyphagous insect pest of a large range of field crops, like tomato, cotton, pigeonpea, chickpea, sorghum, cowpea etc., and is responsible for a huge loss in terms of quality and quantity of agricultural products. In India it causes 25-40% yield loss per annum in pigeonpea and chickpea alone. Hence, yield loss due to pod damage caused by H. armigera is the most important constraint to pulse production. Chemical insecticides are partially ineffective as larvae rapidly bore into pods/seeds after hatching. Conventional breeding for imparting H. armigera resistance is yet to be successful due to lack of resistant gene pool in the cultivated germplasm. Transgenic route for imparting pod borer resistance is considered the best alternative for pod borer resistance. Bt transgenic strategy for insect pest management has shown spectacular success in replacing chemical pesticides in a range of crops. However, sustainability of Bt transgenic crops is a major concern due to resistance development by insects. Hence, a novel strategy of host-delivered RNAi-mediated silencing of H. armigera gene, acetylcholinesterase (Ace1), was employed for imparting resistance. RNA-mediated gene silencing in plants is a collective manifestation of broadly two different functionsdefence against molecular parasites like viruses, transposons and introduced transgenes by either post-transcriptional or transcriptional gene silencing (collectively called RNA interference or RNAi), and orchestration of developmental stages by regulation of endogenous gene expression at post-transcriptional level by microRNA (miRNA). RNAi-mediated gene silencing is now the preferred gene knockout tool for functional genomics, stress resistance and qualitative crop improvement for robustness and efficacy, RNAi-mediated gene knockdown has been successfully applied in entomology to investigate insect RNAi mechanisms and gene function, and develop insect resistant transgenics. Efficacy of host-delivered RNAi (HD-RNAi) strategy has been demonstrated successfully for protection from southern cotton rootworm and cotton bollworms by introducing dsRNA targeting insect genes in maize and cotton, respectively. Hence, HD-RNAi strategy was employed to silence Ace-1 for imparting H. armigera resistance in transgenic tobacco. Partial Ace1cDNA sequence of 2364 nt long, with an ORF of 1875, was retrieved from GenBank. A region/sequence of 643 nt corresponding to 1258-1900 cDNA sequence of Ace1, having least homology with acetylcholinesterase sequence of off-target organisms, was selected as target site for development of inverted repeat (IR) gene construct. The targeted Ace1 cDNA region was RT-PCR amplified from H. armigera total RNA using two specially designed primer pairs for its ligation in sense and antisense orientation. Potato granule-bound starch synthase (GBSS) 13 intron of 105 nt (with sequence of flanking exons) was PCR amplified from potato genomic DNA. The inverted repeat Ace1 gene construct (IR-Ace1) was developed by ligation of the cDNA fragment in sense and antisense orientation, intervened by GBSS intron, onto the backbone of pUC19 vector. The IR-Ace1 fragment was re-ligated onto the plnat binary vector, pBI121, under the transcriptional control of CaMV 35S promoter to get the Ace1 Hd-RNAi vector cassette, pBI121::IR-Ace1 for Agrobacterium mediated transformation of tobacco (Nicotiana tabacum L.) var. Petit Havana. A total 31 putative transformants were generated after cocultivation of 320 leaf disc explants. Fourteen Ace1 RNAi tobacco transgenic lines were obtained after screening of putative transformants by PCR and RT-PCR using nptII-specific primers. Five out of 14 nptII positive Ace1 RNAi tobacco transgenic lines possessed high to moderate level of resistance against H. armigera larvae based on detached leaf insect bioassay. The highest mortality rate of 44% was observed in transgenic tobacco fed larvae compared to that control (nontransformed) tobacco fed larvae. Semi-quantitative PCR revealed many fold reduction in steady state level of Ace1 in larvae fed on selected transgenic tobacco leaves compared to that in control tobacco leaf fed larvae. The selected tobacco transgenic lines carried two to three copies of IR-Ace1. puk Qyh cs/kd izfrjksf/krk gsrq Ace&1 thu dh vfrFks;&forfjr vkj ,u , vkbZ &ehfM,VsM lkbySafla x