Effectiveness of RNA interference against black Sigatoka in transgenic dessert banana

Abstract

Black Sigatoka (BS) caused by Mycosphaerella fijiensis [M. fijiensis] is a worldwide foliar disease constraint of banana and plantain, leading to 30-50% yield losses. Control of BS through spraying with mineral oil-based fungicide formulations is the most common method on commercial farms however, fungicide cost is very prohibitive to the smallholder farmers who depend on banana for food and income generation. The aim of this study was to develop a dessert banana cultivar resistant to BS through the transgenic host delivered RNA interference (RNAi) approach. Embryogenic cell suspensions (ECS) of dessert banana cultivar Sukali Ndizi were modified with transformation constructs for production of double stranded RNA (dsRNA) scripts that silence essential cellular genes in M. fijiensis. Each of two hairpin constructs used were designed to silence two M. fijiensis genes. Construct ‘A’ targeted RNA polymerase and actin-related protein (ARP 2/3) while Construct ‘B’ targeted Coatomer Z and Guanosine triphosphatase (GTPase). A total of 228 transgenic lines were generated for screening against BS with 123 containing construct ‘A’ and 105 containing construct ‘B’. Regenerated transgenic banana plants were evaluated in a potted trial with the experimental plants inoculated with pure M. fijiensis fragmented mycelium isolate under screen-house conditions. Assessment of resistance to BS involved monitoring Stage of Disease (SOD) and Diseased Leaf Area (DLA) as correlates of disease progression on the inoculated banana leaves over 6 weeks. Significant differences (p<0.001) were observed among genotypes/lines for the SOD and DLA parameters for all data points over the evaluation period. PCR analysis indicated presence of the RNAi constructs in the BS partially resistant lines with unique amplicon sizes for the ‘A’ and ‘B’ constructs. Lower quantities of M. fijiensis cDNA compared to banana cDNA were observed based on fragment intensity as assessed by Reverse Transcriptase PCR (RT-PCR). From this study, four transgenic banana lines; A187, A480, B106, B200, with partial resistance to BS were selected and recommended for field evaluation.