Figure 1

Schematic representation of full-length cDNA clones of BPMV RNA1 and BPMV RNA2 vector (pG7R2V). (A) Schematic representation of BPMV RNA1 cDNA constructs used for generation of infectious transcripts. Plasmids pGHopR1 and pCRHanR1 contain full-length RNA1 cDNA of two strains of BPMV (K-Hop and K-Han, respectively) inserted downstream of a modified T7 promoter and cloned in pGEM Teasy and pCR2.1 TOPO, respectively [23]. Vertical lines indicate cleavage sites in the polyproteins; the designations of the mature proteins (protease co-factor, Co-pro; helicase, Hel; genome-linked protein, VPg; protease, Pro; and RNA-dependent RNA polymerase RdRp) are written above the relevant coding regions. For transcription, pGHopR1 and pCRHanR1 are linerized with Sal I (lanes HopR1Sal I and HanR1Sal I, respectively, Figure 1C) or, preferably for better yield, with Sal I and Not I (see corresponding lanes, first two lanes on the right, Figure 1C). (B) Genome organization of BPMV RNA2 and vector construction strategy. BPMV RNA2 is translated into two overlapping carboxy coterminal polyproteins. CR, RNA2 replication cofactor; MP, movement protein; L-CP, large coat protein; and S-CP, small coat protein. The vector pG7R2V [12] contains a GFP fragment (ΔGFP) inserted between the coding regions of MP and L-CP and also contains additional restriction sites (Bam HI and Msc I) for cloning of target gene sequences (TGS). A target gene can be cloned as a Bam HI-Msc I fragment in Bam HI-Msc I digested pGG7R2V vector. Alternatively, the target gene can be blunt-end ligated into Msc I-digested pGG7R2V. In constructing the vector, the Q/M cleavage site sequence between MP and L-CP (the dipeptide QM plus flanking sequences) was duplicated. A T7 RNA polymerase promoter sequence was engineered upstream the modified full length RNA2 cDNA and cloned into plasmid pGEM T easy to generate pG7R2V. The plasmid pG7R2V can be linearized by digestion with Sal I prior to transcription (lane G7R2Vsal I; Figure 1C). (C). Restriction digestion of recombinant plasmids containing RNA1 cDNA (HopR1 and HanR1) and recombinant RNA2 (G7R2GFP, G7R2Nod22 and G7R2V) with Sal I or Sal I and Not I restriction enzymes and visualized after electrophoresis on 1.0% agarose gel and staining with ethidium bromide.