Replicase motif mutations to the conserved domain (CD) affect PMV replication in protoplasts and plants. (A) Foxtail millet protoplasts were transfected with transcripts of alanine replicase mutants (Fig. 4) and harvested 40 hours postinoculation. The RNA was extracted and separated on 1% agarose gels, blotted, and probed for PMV accumulation with a 32P-labelled cDNA that detects genomic (g) and subgenomic (sg) RNA. Proteins were separated via SDS-PAGE and probed with rabbit polyclonal antiserum against the SPMV coat protein (CP) (bottom). (B) Proso millet plants were mechanically co-inoculated with SPMV and either PMV or the replicase mutants. The blots were probed for PMV, as described for panel A, and then hybridized with a 32P-labelled SPMV cDNA. (C) Proso millet plants at 1-month postinoculation with PMV+SPMV or the replicase motif mutants (P399A, C335A, D363A, Y330A, and F313A) plus SPMV.