Rescue of a plant cytorhabdovirus as versatile expression platforms for planthopper and cereal genomic studies

: Plant viruses have been used as rapid and cost-effective expression vectors for heterologous protein expression in genomic studies. However, delivering large or multiple foreign proteins in monocots and insect pests is challenging. Here, we recovered a recombinant plant cytorhabdovirus, Barley yellow striate mosaic virus (BYSMV), for use as a versatile expression platform in cereals and the small brown planthopper (SBPH, Laodelphax striatellus) insect vector. We engineered BYSMV vectors to provide versatile expression platforms for simultaneous expression of three foreign proteins in barley plants and SBPHs. Moreover, BYSMV vectors could express the c. 600-amino-acid β-glucuronidase (GUS) protein and a red fluorescent protein stably in systemically infected leaves and roots of cereals, including wheat, barley, foxtail millet, and maize plants. Moreover, we have demonstrated that BYSMV vectors can be used in barley to analyze biological functions of gibberellic acid (GA) biosynthesis genes. In a major technical advance, BYSMV vectors were developed for simultaneous delivery of CRISPR/Cas9 nuclease and single guide RNAs for genomic editing in Nicotiana benthamiana leaves. Taken together, our results provide considerable potential for rapid screening of functional proteins in cereals and planthoppers, and an efficient approach for developing other insect-transmitted negative-strand RNA viruses.