Silencing of Coat Protein Gene Using IhpRNA Develops Resistance to Banana Bract Mosaic Virus in Musa Acuminata (AAA) cv. Grand Naine

Abstract

Banana bract mosaic virus (BBrMV), transmitted by aphids, is a major threat to banana cultivation, causing substantial economic losses. This study focuses on the development of BBrMV-resistant lines of banana cv. ‘Grand Naine’ by silencing viral coat protein (CP) gene using RNA interference (RNAi) strategy. To achieve this, an intron hairpin RNA (ihpRNA) construct containing a 326 bp fragment of the CP gene was designed using the pSTARLING vector. Identification of a Dicer substrate within the CP gene facilitated the prediction of small interfering RNAs (siRNAs) through Custom Dicer-Substrate siRNA analysis. The absence of viral silencing suppressors was validated using the VsupPred tool. Cloning of the sense and antisense fragments of the CP gene into the pSTARLING vector, flanking the cre intron, was confirmed through PCR analysis. Subsequently, the NotI fragment comprising the ubiquitin promoter, ubiquitin intron, sense fragment inserts, cre intron, antisense strand insert, and tumour morphology locus (tmL) terminator was transferred to the Agrobacterium tumefaciens binary vector pART27. Embryogenic calli were transformed with the ihpRNA-CP cassette, and regenerated plantlets were screened for complete cassette integration using PCR. Northern hybridization confirmed the production of siRNAs against coat protein mRNA. Upon exposure to virulent aphids carrying BBrMV, the transformed lines exhibited no disease symptoms. Additionally, reverse transcription quantitative PCR (RT-qPCR) demonstrated the absence of BBrMV, with transformed lines resembling healthy, non-inoculated controls both morphologically and in terms of coat protein gene expression. This RNAi-based approach showcases the successful creation of BBrMV-resistant banana lines, presenting a promising strategy for combating the virus's detrimental effects on banana cultivation.