Cell-to-cell and systemic movement of recombinant green fluorescent protein-tagged turnip crinkle viruses

Abstract

To facilitate analyses of turnip crinkle virus (TCV) cell-to-cell and systemic movement, we created a series of recombinant viruses expressing green fluorescent protein (GFP) either as substitutions of coat protein (CP) sequences or as fusions to movement proteins (MPs). Constructs were used to inoculate leaves of Arabidopsis seedlings. TCV carrying its two native MPs and GFP fused near the start of CP translation (GFP DeltaCP) resulted in cell-to-cell movement manifested by the expansion of fluorescent foci on inoculated leaves. GFP fusions to either MP were inactive for movement. However, TCV carrying the p9-GFP fusion, which expresses a functional p8 gene, could be complemented for cell-to-cell movement by coinoculation with virus carrying native p9 but mutant for p8. This same coinoculation combination also lead to systemic spread of GFP fluorescence to noninoculated leaves, as the complementing virus carries native CP. Complementation for systemic movement of virus carrying GFP DeltaCP constructs was achieved by inoculation onto transgenic plants expressing TCV CP. GFP-tagged TCV movement was detected throughout the plant, including the inflorescence stem, cauline leaves, flowers, siliques, and substructures such as organ primordia and meristematic regions. The recombinant viruses described herein provide (1) genetic information relevant to define regions of TCV that can, or cannot, be manipulated by insertion of foreign coding sequences and (2) a set of tools to allow the study of viral cell-to-cell and long-distance movement in the model plant system Arabidopsis.