CHARACTERIZATION OF CUCUMBER MOSAIC-VIRUS .4. MOVEMENT PROTEIN AND COAT PROTEIN ARE BOTH ESSENTIAL FOR CELL-TO-CELL MOVEMENT OF CUCUMBER MOSAIC-VIRUS

cDNA clones of cucumber mosaic virus (CMV) RNA 3 were modified to expr ess the jellyfish green fluorescent protein (GFP) in place of the 3a m ovement protein (MP) or coat protein (CP), as fusions to the N (GFP-3a ) or C (3a-GFP) terminus of the MP or from a separate open reading fra me as pari of tricistronic RNAs 3. CMV RNA transcripts containing the individual modified RNAs 3 were unable to infect either Nicotiana taba cum or Nicotiana benthamiana systemically. Infection, as measured by c onfocal microscopy of GFP fluorescence, generally was limited to one t o three epidermal cells at each inoculation site, Limited cell-to-cell movement, but not systemic movement, could be detected by complementa tion involving expression of MP and CP from two different RNA 3 constr ucts, each also expressing GFP. Infection involving RNA 3 expressing t he GFP-3a fusion showed bright granules of variable size distributed p redominantly and nonuniformly throughout the cytoplasm and, to a lesse r extent, associated with the cell wall in single fluorescent cells, w hile infections expressing the 3a-GFP fusion showed bright, punctate f luorescence associated only with the cell wall. Infected cells express ing either 3a-GFP or free GFP showed a halo of less bright, fluorescen t, neighboring cells, indicating limited movement of GFP. The initiall y infected cells also allowed movement of 10-kDa fluorescent dextran t o the neighboring halo cells, while infection did not spread, suggesti ng different requirements for movement of either MP or dextran versus RNA. (C) 1997 Academic Press.