Rapid Construction of Complex Plant RNA Virus Infectious cDNA Clones for Agroinfection Using a Yeast-E. coli-Agrobacterium Shuttle Vector

Abstract

The availability of infectious full-length clone is indispensable for reverse genetics studies of virus biology, pathology and construction of viral vectors. However, for RNA viruses with large genome sizes or those exhibiting inherent cloning difficulties, procedure to generate biologically active circular DNA (cDNA) clones can be time-consuming or technically challenging. Here we have constructed a yeast-Escherichia coli-Agrobacterium shuttle vector that enables highly efficient homologous recombination in yeast for assembly of Agrobacterium compatible plant virus clones. Using this vector, we show that infectious cDNA clones of a plant negative-stranded RNA virus, sonchus yellow net rhabdovirus, can be rapidly assembled. In addition, one-step assembly of infectious clones of potato virus Y in yeast, either with or without intron, was readily achieved from as many as eight overlapping DNA fragments. More importantly, the recovered yeast plasmids can be transformed directly into Agrobacterium for inoculation, thereby obviating the E. coli cloning steps and associated toxicity issues. This method is rapid, highly efficient and cost-effective and should be readily applicable to a broad range of plant viruses.

Keywords: agroinfection; infectious cDNA clone; potato virus Y; potyvirus; rhabdovirus; sonchus yellow net virus; yeast homologous recombination; yeast-E.coli-Agrobacterium shuttle vector.

Figure 1

Construction of a yeast-Escherichia coli-Agrobacterium tumefaciens shuttle vector: (a) Schematic diagram illustrating construction of the shuttle plasmid pCB301-2μ-HDV. A fragment containing the yeast 2μ replication origin and TRP1 selection marker gene was amplified from the yeast plasmid pGBK-T7 with primers TRP/R and 2μ-ori/F and then inserted into the pCB301-HDV Agrobacterium binary vector through the AfeI restriction site. p35S², doubled CaMV 35S promoter; (b) Transient expression of eGFP in agroinfiltrated leaf patches. Nicotiana benthamiana leaves were infiltrated with Agrobacterium mixtures carrying the pCB301-2μ-eGFP or pCB301-eGFP plasmid and the TBSV p19 suppressor plasmid. The infiltrated leaves were visualized under a fluorescence microscope at 36 h post infiltration; (c) Total proteins extracted from infiltrated leaf patches were analyzed in a Western blot with an eGFP-specific antibody. TRP1: tryptophan selection maker gene; OriV: RK2 replication origin; TrfA: replication initiator protein; HDRz: HDV ribozyme; tNos: Nos terminator; 2μ ori: yeast 2μ replication origin; KanR: kanamycin resistance gene; LB: left border sequence; RB: right border sequence; eGFP: enhanced green fluorescence protein.

Figure 2

Construction of cDNA clones by homologous recombination (HR) in yeast for recovery of recombinant sonchus yellow net virus (SYNV): (a) Diagram depicting the SYNV antigenomic RNA. Six viral genes (N, P, sc4, M, G and L) are flanked by the 5′ leader (le) and 3′ trailer (tr) sequences; (b) Schematic representation of the yeast HR-based cloning strategy. In the upper panel, four overlapping SYNV cDNA fragments (A, 3584 bps; B, 3637 bps; C, 3370 bps; D, 3439 bps) and the linearized vector pCB301-HDV-2μ are shown. After assembly, viral cDNA is positioned precisely between the 35S promoter (35S²) and HDV ribozyme (HDRz). The lower panel shows the assembly scheme for the tandem arrayed expression cassettes of the tomato bushy stunt virus p19, tobacco etch virus HC-Pro and barley stripe mosaic virus γb viral suppressors of RNA silencing. The overlapping regions are depicted by gray shadow boxes, with the number of overlapping nucleotides indicated. Note that all of the fragments in the figure are not drawn exactly to scale; (c) SalI and NotI double digestion of ten recombinant plasmids. The positions of the 10,000, 6000, 3000 and 1500 bps size marker (M) are indicated to the left of the panel; (d) Symptoms of N. benthamiana plant infected by recombinant SYNV at 35 days post infiltration. Healthy: an uninfected plant; (e) Total proteins extracted from systemically infected leaf tissues were analyzed by Western blotting using an antibody against SYNV virions. The Coomassie blue-stained Rubisco larger submit (Rub L) is shown as loading control.

Figure 3

One-step assembly of intron-less and intron-containing potato virus Y (PVY) infectious cDNA clones by homologous recombination in yeast: (a) PVY genome structure. The 5’ viral protein genome-linked (vpg) and the 3′ polyadenylated tail [(A)n] are depicted. The positions of two inserted introns are indicated on the top of the genome; (b) Schematic representation of the cloning strategies for yeast homologous recombination-mediated assembly of the PVY intron-less (upper panel) and intron-containing (lower panel) full-length clones. The intron-less PVY clone was assembled by co-transformation of yeast cells with the three overlapping PVY cDNA fragments (A, 3311 bps; B, 3359 bps; C, 3259 bps) and the appropriately linearized vector pCB301-HDV-2μ. For the intron-containing clone, the B fragment is divided into three fragments (B1, B2 and B3) by insertion of the two inserted intron (IV and II). The overlapping regions are depicted by gray boxes, with the number of overlapping nucleotides indicated. Note that all of the fragments in the figure are not drawn exactly to scale; (c) Recombinant plasmids were digested with BamHI and PstI and the products were separated on 0.8% agarose gels. The positions of the 10,000, 6000, 3000 and 1500 bp size markers (M) are indicated to the left of the panel; (d) Nicotiana tabacum plants systemically infected with the intron-less or intron-containing PVY infectious clone were photographed at 15 days post infiltration. Healthy: an uninfected plant; (e) Total proteins extracted from PVY infected plants were analyzed by Western blotting using an antibody against the PVY CP protein. The Coomassie blue-stained Rubisco large subunit (Rub L) is used as a loading control.

Figure 4

Construction of an eGFP-tagged PVY intron-containing infectious cDNA clone: (a) Schematic representation of the eGFP insertion; (b) Symptoms and eGFP expression of PVY-intron-GFP and PVY-intron infected N. tabacum plants. The upper panels show symptomatic plants photographed at 15 days post infiltration and the lower panels show eGFP expression in systemically infected leaf tissues observed by fluorescence microscope; (c) Total proteins extracted from systemically infected plants or un-infiltrated control plants were analyzed by Western blotting using an antibody against the PVY coat protein (CP). The Coomassie blue-stained Rubisco large subunit (Rub L) is showed as a loading control.