Figure 3

One-step assembly by homologous recombination in yeast of a ternary Yeast- Escherichia coli - Agrobacterium tumefaciens shuttle vector and simultaneous cloning of an ACLSV FL-cDNA to generate an infectious agroinoculable full-length viral cDNA clone. (A, B, C) Schematic representation of the strategy used to generate the four PCR fragments from which the construct is assembled by homologous recombination. The primers used are indicated by blue arrows near the respective PCR templates and are listed in Table 1. Regulatory elements are indicated by arrows of various colors. LB: T-DNA left border; 35S P: CaMV 35S promoter; 35S ter: CaMV 35S terminator; RB: T-DNA right border; ColE1: E. coli ColE1 origin of replication; oriV: A. tumefaciens oriV origin of replication; KanR: kanamycin resistance gene; 2μ Ori: yeast 2μ origin of replication; TRP1: yeast TRP1 selection gene. (A) Map of pBin61 from which are amplified the ~10.7 kbp fragment (PCR2, dark blue, carrying the 35S ter, the RB, the ColE1 and OriV replication signals and the KanR selection marker) and the ~2 kbp fragment (PCR4, yellow, carrying the LB and the 35S promoter) using respectively primer pairs ACPCR2F/PCR2R and PCR4F/ACPCR4R. (B) Map of Yeast-pBS70T used to amplify the ~2 kbp fragment (PCR3, red, carrying the yeast 2μ Ori-TRP1) using the PCR3F/PCR3R primer pair. (C) Genomic organization of ACLSV and position of the ACPCR1F/FLAC3 primer pair used to amplify the FL-viral cDNA (PCR1, light blue). (D) Non-denaturing 0.8% agarose gel electrophoresis of the four purified overlapping PCR products. (E) Schematic representation of the recombinant YEA-ACLSV ternary plasmid carrying the ACLSV FL-cDNA obtained by assembly by homologous recombination of the four fragments. The position of Eco RI sites used for restriction mapping of the plasmid is shown. (F) Non-denaturing 0.8% agarose gel electrophoresis of 5 independently obtained recombinant plasmids showing the expected Eco RI digestion pattern.