The p20 gene product of Citrus tristeza virus accumulates in the amorphousinclusion bodies

Citrus tristeza virus (CTV) has 10 3' open reading frames (ORFs) of unknown function except for the two coat proteins. The highest produced subgenomic RNAs are those of the major coat protein gene (p25) and the 3' most genes, p20 and p23. The proteins from three ORFs, p25, p27, and p20, were examine d in the yeast two-hybrid assay for the interactions between themselves and to one another. The p20 protein exhibited a high affinity for itself, sugg esting that it might aggregate in infected cells. The cytopathology of CTV infections includes characteristic paracrystalline and amorphous inclusions in the phloem elements of infected citrus. Polyclonal antiserum raised aga inst the bacterial expressed p20 gene product detected a protein of similar to 22-23 kDa, which accumulated to relatively high levels in CTV-infected citrus, but not in healthy citrus. Immunogold localization using antibodies to p20 protein showed strong and specific labeling of the amorphous inclus ion bodies present in CTV-infected cells. Mesophyll protoplasts of Nicotian a benthamiana transfected with a CTV mutant containing the green fluorescen t protein (GFP) ORF fused in-frame to the 3' end of p20 protein ORF express ed high levels of GFP. The fusion protein was concentrated in one specific area in the cytoplasm and lacked an organized shape. Accumulation of high l evels of p20 protein in infected tissue, specific localization of the p20-G FP fusion protein, immunolocalization of p20 protein into amorphous inclusi ons, and strong homologous p20 protein-p20 protein interactions in the yeas t-two-hybrid assay suggest that the p20 protein of CTV is a major component of the amorphous inclusion bodies present in CN-infected cells. (C) 2000 A cademic Press.