Deciphering the role of fungal calmodulin gene using Host-mediated gene silencing approach in apple

Abstract Premature leaf fall caused by Marssonina coronaria is one of the most destructive diseases of apple in India. In this study, host induced gene silencing approach was exploited to develop resistance to this disease in an apple cultivar ‘Red Chief’. Calmodulin gene (CaM) having its role in fungal differentiation, development and pathogenicity was selected as target. hpRNAi construct was prepared from the conserved off target free partial gene sequence of CaM and used for transformation trials. Upto 6% kanamycin resistant shoots were obtained on selective medium having 5–6 mg/l kan after 7 weeks of coculture. In PCR analysis of 13 RNAi putative transformants, 10 lines were found positive with CaM and nptII gene specific primers and six lines showed hybridization signal. Semi qRT-PCR revealed variable levels of transgene expression among RNAi lines which seems to be related to copy number of integrated gene. In vitro detached leaf assay revealed lesion development and disease progression in wild type after 5 dpi but not visible in five CaM RNAi lines. Microscopic examination of infected control leaves showed fully developed, septate mycelium, and conidia along with necrosis of whole tissue while three transformants showed reduced growth and differentiation of fungus and in rest three, hyphal development and necrosis were strongly restricted. We conclude that trafficking of dsRNA/ siRNA from apple plants to pathogen might have triggered the down regulation of fungal CaM gene which confirms that deciphering the role of CaM through HIGS lead to resistance to Marssonina blotch in apple.