Fig 3.

A summary of RNA recombinants generated from replication initiated on B3 mutants of (+)- and (−)-strand inocula. N. benthamiana plants were coinfiltrated with a mixture of agroconstructs containing B1(+) and B2(+) and each B3 mutant in either (+)- or (−)-strand orientation. Open boxes represent the 3′ untranslated region (UTR) of B3, and black boxes represent the coat protein ORF. Stippled and crosshatched boxes represent the 3′ UTRs of B1 and B2, respectively. An asterisk in the B3 3′ UTR represents the location of the engineered mutation. Numbers above each recombinant represent the 3′ nucleotides of B1 or B2 recombined with the 3′ end of B3. For example, in recombinant GCI-4, 221(B2) indicates that a 3′ 221-nt region from B2 was recombined with the 3′ end of B3. The dotted boxes in the columns labeled (+) RNA inoculum and (−) RNA inoculum show the overlapped and commonly shared homologous sequences between RNAs of B3 and B1 or B2. CCA denotes the last three nucleotides in the 3′ end of the BMV RNA. The length is the number of nucleotides (nt) in a given recombinant. The recombination frequency (%) was determined based on the total number of clones sequenced. B3R-α and B3R-β represent homologous and nonhomologous recombinants (see text for details). Naming of recombinants is as follows (for example). GCI, GUA mutant, correct orientation (i.e., plus strand), inoculated leaves; GRI GUA mutant, reverse orientation (i.e., minus strand), inoculated leaves; K, Δknob; P, 5′Psk. Recombinant progeny isolated at 4 dpi were amplified from total RNA using RT-PCR and sequenced after subcloning into pGEM-T vector (see Materials and Methods).