Rescue of the first alphanucleorhabdovirus entirely from cloned complementary DNA: An efficient vector for systemic...

Recent reverse genetics technologies have enabled genetic manipulation of plant negative-strand RNA virus (NSR) genomes. Here, we report construction of an infectious clone for the maize-infecting Alphanucleorhabdovirus maydis, the first efficient NSR vector for maize. The full-length infectious clone was established using agrobacterium-mediated delivery of full-length maize mosaic virus (MMV) antigenomic RNA and the viral core proteins (nucleoprotein N, phosphoprotein P, and RNA-directed RNA polymerase L) required for viral transcription and replication into Nicotiana benthamiana. Insertion of intron 2 ST-LS1 into the viral L gene increased stability of the infectious clone in Escherichia coli and Agrobacterium tumefaciens. To monitor virus infection in vivo, a green fluorescent protein (GFP) gene was inserted in between the N and P gene junctions to generate recombinant MMV-GFP. Complementary DNA (cDNA) clones of MMV-wild type (WT) and MMV-GFP replicated in single cells of agroinfiltrated N. benthamiana. Uniform systemic infection and high GFP expression were observed in maize inoculated with extracts of the infiltrated N. benthamiana leaves. Insect vectors supported virus infection when inoculated via feeding on infected maize or microinjection. Both MMV-WT and MMV-GFP were efficiently transmitted to maize by planthopper vectors. The GFP reporter gene was stable in the virus genome and expression remained high over three cycles of transmission in plants and insects. The MMV infectious clone will be a versatile tool for expression of proteins of interest in maize and cross-kingdom studies of virus replication in plant and insect hosts.