Engineering an Agro-Infectious Apple Stem Grooving Virus Clone for Virus-Induced Gene Silencing (VIGS) in Plants

Abstract

Apple stem grooving virus (ASGV) is an important pathogen with a broad host range, infecting both monocot and dicot species, making it a promising candidate for virus-induced gene silencing (VIGS) applications in functional genomics. In this study, we developed an infectious cDNA clone of an ASGV apple isolate and demonstrated its infectivity across 10 diverse plant species from families’ including Solanaceae, Fabaceae, Cucurbitaceae, and Amaranthaceae. The clone also successfully infected woody hosts such as Citrus limon and Malus domestica, and resulted in systemic infection through back-inoculation in Cucumis sativus, thereby fulfilling Koch's postulates. To engineer a VIGS vector, we inserted a duplicated coat protein gene minimal promoter sequence, into the 3′ untranslated region (UTR) of the viral genome, which exhibited full promoter activity confirmed by GUS expression assays. Cloning of the phytoene desaturase (PDS) gene initially caused plasmid instability, which was resolved by introducing a stop codon mutation in ORF1. The resulting vector carrying a partial PDS insert induced a silencing phenotype in cucumber and was further validated in Phaseolus vulgaris, where effective gene silencing was achieved. The result demonstrates the utility of ASGV as a functional VIGS platform, enabling gene functional analysis in economically important crop species, including both monocots and dicots.