Detection of the nonstructural p21 protein of beet yellows closterovirus in vivo with polyclonal antibodies to the bacterially expressed protein

In the positive-sense RNA genome of beet yellows closterovirus (BYV), the 3 '-terminal ORF 8 codes for a nonstructural protein of 21 kDa. The cDNA of O RF 8 was inserted into an E. coli expression vector pQE-30 under the contro l of bacteriophage T5 promoter. The recombinant protein having an N-termina l tag of six histidines, was overexpressed in bacteria, purified, and used for antiserum production in rabbits. Using the IgG fraction from a polyclon al anti-p21 serum preadsorbed with the extract of healthy Tetragonia expans a plants, the p21 was detected in BYV-infected plants. Analysis of subcellu lar fractions of BYV-infected T. expansa indicated that the bulk of p21 is in the fractions of cytoplasm and cell walls. The p21 was readily detected by immunoblotting in the BYV-infected plant samples taken 20 to 120 days po st inoculation, thus indicating steady accumulation of the protein in the B YV infection and/or its stability.