DsRNA-mediated Vital Genes Silencing of Fruit Fly, Bactrocera dorsalis (Hendel) for Producing RNAi Plants

Abstract

Background: Bactrocera dorsalis (Hendel), the oriental fruit fly, is the world's most damaging pest, infesting 30 to 100 percent of important fruits and vegetables. Because of its polyphagous, multivoltine nature and unexposed developmental stages, it is extremely difficult to control. So far, no fruit fly resistant guava germplasm has been reported anywhere in the world. RNAi approach for controlling fruit flies in fruit crops especially in guava can provide an attractive alternative. Objectives of the Study: Investigating the possibility of vital genes, ecr (ecdysone receptor) and rpl19 (a ribosomal protein L19) for dsRNA based RNAi study via feeding bacteria expressing corresponding dsRNA to fruit fly. Further, transformation of ecr-RNAi construct to guava for producing RNAi plants. Main Body: The dsRNA expression strategy based on Escherichia coli was used to investigate its potential in controlling B. dorsalis by targeting its two vital genes, ecr and rpl19.  Effects of feeding E. coli, HT115 (DE3) expressing dsRNA of Bdecr and Bdrpl19 with artificial diet to maggots of B. dorsalis resulted in severe mortality and deformities in treated maggots, emerged pupae and adults. Total mortality (including deformity) of maggots, pupae and adult fruit flies was 86.3 % and 87.9% and was highest in 700 µl (200x of 3.5x108 cells) dsRNAs of Bdecr and Bdrpl19 bacterial treatment respectively, compared to 350 and 200 µl bacterial treatments. In emerged adults, severe developmental defects such as melanisation and deformities of maggots and pupae, lack of wings, underdeveloped abdomen/absence of complete abdomen, absence of legs, severely curled wings, malformed legs, and incomplete eclosion, as well as suppression of these target gene expression, were observed. To mobilise the RNAi cassette, an RNAi construct was created using the same ECR gene of B. dorsalis and standardised in planta (floral drop) transformation of Allahabad safeda guava cultivar. A total of eight RNAi guava plants (T1) germinated from seeds of transformed fruits (T0) were confirmed using genomic DNA-PCR with gene and vector specific primer combinations. Short Conclusion: The study provides a proof of concept of feasibility to silence two potential genes by feeding bacteria expressing dsRNA in all developmental stages of B. dorsalis to step further to perform RNAi based fruit fly control in several fruit crops specifically guava as the case in the present study.