Fig. 1

Assembly and functionality of the new ALSV vectors pALSV-RNA1u and pALSV-RNA2u. A Sequence of the USER cassette that was introduced into ALSV-RNA2L5R5. The cassette allows for rapid and efficient insertion of one or more target gene fragments. In turn, this allows for VIGS studies with single or multiple gene targets, for example, as part of a PDS co-silencing approach. B Cloning strategy for the generation of the new VIGS vectors pALSV-RNA1u and pALSV-RNA2u. The USER cassette detailed above was introduced into ALSV-RNA2L5R5 between MP and VP25 to create pEALSR2L5R5u. Then, the full ALSV expression cassettes from pALSR1 and pALSR2L5R5u were sub-cloned into the binary vector pCAMBIA1300ΔHygRu. C Schematic diagram of the USER cloning of a single target gene fragment as part of a single-gene silencing approach. D Schematic diagram of the USER fusion reaction where two fragments—an LaPDS fragment and a target gene fragment—are simultaneously fused and cloned as part of a PDS co-silencing approach. Abbreviations: P35S, 35S promoter from the cauliflower mosaic virus (CaMV); PRO-co, protease cofactor; HEL, NTP-binding helicase; Vpg, viral protein genome-linked; C-PRO, cysteine protease; POL, RNA-dependent RNA polymerase; MP, movement protein; VP25, VP20 and VP24, capsid proteins; Tnos, nopaline synthase terminator from A. tumefaciens; LB and RB, T-DNA left and right borders; KanR, kanamycin kinase (resistance gene). Dashed boxes represent the ALSV-RNA1 and ALSV-RNA2 polyprotein-coding sequences. Solid, grey boxes represent the USER cassette and associated overhangs. The solid, black boxes represent custom, USER-compatible overhangs