Transgenic banana plants expressing small interfering RNAs targeted against viral replication initiation gene display high-level resistance to banana bunchy top virus infection

Abstract

The banana aphid-transmitted Banana bunchy top virus (BBTV) is the most destructive viral pathogen of bananas and plantains worldwide. Lack of natural sources of resistance to BBTV has necessitated the exploitation of proven transgenic technologies for obtaining BBTV-resistant banana cultivars. In this study, we have explored the concept of using intron-hairpin-RNA (ihpRNA) transcripts corresponding to viral master replication initiation protein (Rep) to generate BBTV-resistant transgenic banana plants. Two ihpRNA constructs namely ihpRNA-Rep and ihpRNA-ProRep generated using Rep full coding sequence or Rep partial coding sequence together with its 59 upstream regulatory region, respectively, and castor bean catalase intron were successfully transformed into banana embryogenic cells. ihpRNA-Rep-and ihpRNA-ProRepderived transgenic banana plants, selected based on preliminary screening for efficient reporter gene expression, were completely resistant to BBTV infection as indicated by the absence of disease symptoms after 6 months of viruliferous aphid inoculation. The resistance to BBTV infection was also evident by the inability to detect cDNAs coding for viral coat protein, movement protein and Rep protein by RT-PCR from inoculated transgenic leaf extracts. Southern analysis of the two groups of transgenics showed that ihpRNA transgene was stably integrated into the banana genome. The detection of small interfering RNAs (siRNAs) derived from the ihpRNA transgene sequence in transformed BBTV-resistant plants positively established RNA interference as the mechanism underlying the observed resistance to BBTV. Efficient screening of optimal transformants in this vegetatively propagated non-segregating fruit crop ensured that all the transgenic plants assayed were resistant to BBTV infection.

Modifying Bananas: From Transgenics to Organics?

“…The accessible gene pool available for GM is significantly broader than that available to conventional banana breeders and, as a result, genes for resistance to BBTV and BXW have already been identified [44][45][46][47]. Further, genes from M. balbisiana are available for use in genetic modification, as there is no possibility of transferring BSV genes [48].…”

“…The majority of these studies have been reports from a glasshouse evaluation and therefore, it is difficult to determine whether they only represent experimental projects, or whether they are part of a program to progress to field trial, and ultimately, to general release. The most targeted traits have been pest and disease resistance, abiotic stress tolerance, and fruit ripening, and these have included resistance to BBTV using RNAi (RNA interference) [44,59], resistance to Fusarium wilt race 1 using anti-apoptosis genes [60], RNAi [61] or a defensin gene [62], resistance to nematodes using plant-derived or synthetic peptides [63], and the over-expression of a banana-derived WRKY transcription factor to alter abiotic responses [64]. These reports, in addition to a number of others, have used a range of banana genes, RNAi constructs derived from banana pathogens, or genes not derived from bananas.…”

Banana bunchy top disease (BBTD) symptom expression in banana and strategies for transgenic resistance: A review

“…Two Indian research groups have claimed success. Shekhawat et al (2012) have published an account of tests that showed that transformed 'Rasthali' (AAB genome, syn. 'Silk') did not develop symptoms when exposed to aphids carrying BBTV.…”

Biochemical Characterization of Compatible Plant Virus Interaction: A Case Study with Bunchy Top Virus-Banana Host-Pathosystem

“…Grand Nain, Virupakshi (Hill banana), Robusta, Nendran, Rasthali, Poovan, Ney Poovan, Monthan and Red Banana are severely affected by BBTD. There is no resistance gene source available in germplasm of bananas and plantains (Shekhawat et al, 2012). The BBTV affected plants show intermittent dark green dots, dash, streaks of variable length on leaf sheath, midrib, leaf veins and petioles of infected plants.…”