FIG. 6.

Effects of manipulating MP and CP expression in agroinfiltrated tobacco and transfected barley protoplasts. (A) BMV capsid protein expressed in tobacco after agroinfiltration. A slice of a 4 to 12% denaturing protein gel is shown. MPKO and CPKOI indicate that plasmid pBR3 contains a mutation of the translation initiation codon from AUG to CUG. The BMV CP gene has a second initiation codon, which allows expression of a smaller amount of a truncated protein. CPKOII has two mutations that altered the first two initiation codons (codons 1 and 8) to CUGs. The lane marked with “M” denotes a protein molecular mass standard of 20 kDa, and the lane marked with “C” denotes a mock-inoculated control. (B) Northern blot analysis of the RNAs produced by agroinfiltration in tobacco by RNAs with mutations affecting either CP or MP expression from pBR3. dpi, days postinfiltration. The quantifications were performed by adjusting the amount of RNA3 or RNA4 in the wild-type sample each day to 100%. The amount of the comparable RNAs in plants infiltrated with CPKOII or MPKO were normalized to that in the wild-type sample. (C) Effects of the MPKO and CPKOII mutations on BMV RNA accumulation in barley protoplasts. The presence or absence of plasmids encoding wild-type BMV RNAs is denoted with a +. The MPKO or the CPKO substituted for wild-type RNA3 is shown by the names of the mutant construct. hpi, hours postinfiltration. The amount of BMV RNA3 and RNA4 were normalized to the amount present in the protoplasts transfected with wild-type BMV RNAs. (D) Accumulations of the BMV RNAs in tobacco protoplasts transfected with the three wild-type BMV RNAs or wild-type RNA1, RNA2, and CPKOII.