Subcellular fractionation and analyses of the PMV replicase proteins isolated from millet plants and assay for RNA products generated by the PMV RNA-dependent-RNA polymerase (RdRp). (A) Mock-inoculated and PMV-infected leaves were harvested and subjected to differential centrifugation to isolate the cell wall, nuclei and chloroplasts (P1), membranes (P30, P44, P100), and soluble proteins (S100). Proteins were separated by SDS-PAGE, transferred to nitrocellulose membrane, and probed with rabbit polyclonal antiserum against the C-terminal half of p48 (p48C). (B) The P44 (44,000 × g) fraction isolated from PMV-infected millet plants probed with p48C-derived antiserum (upper panel) or CP-specific antiserum (lower panel). The molecular weight markers are indicated in kDa, and the predicted forms of the PMV RdRp encoded proteins (p48 and p112) are shown, including the 48C (~29 kDa) derivative and its putative dimer, trimer, and multimeric forms. (C) The P44 fraction from PMV-infected plants was assayed for in vitro RdRp activity measured by incorporation of [32P]-UTP into the associated PMV RNAs. The products were analyzed on TBE-agarose gels followed by transfer to nylon membranes and exposure to X-ray film. The single stranded- (ss) and double-stranded (ds) genomic (gRNA) and subgenomic (sg) RNAs are indicated.