Positive-Selection and Ligation-Independent Cloning Vectors for Large Scale in Planta Expression for Plant Functional Genomics

Abstract

Transient expression is an easy, rapid and powerful technique for producing proteins of interest in plants. Recombinational cloning is highly efficient but has disadvantages, including complicated, time consuming cloning procedures and expensive enzymes for large-scale gene cloning. To overcome these limitations, we developed new ligation-independent cloning (LIC) vectors derived from binary vectors including tobacco mosaic virus (pJL-TRBO), potato virus X (pGR106) and the pBI121 vector-based pMBP1. LIC vectors were modified to enable directional cloning of PCR products without restriction enzyme digestion or ligation reactions. In addition, the ccdB gene, which encodes a potent cell-killing protein, was introduced between the two LIC adapter sites in the pJL-LIC, pGR-LIC, and pMBP-LIC vectors for the efficient selection of recombinant clones. This new vector does not require restriction enzymes, alkaline phosphatase, or DNA ligase for cloning. To clone, the three LIC vectors are digested with SnaBI and treated with T4 DNA polymerase, which includes 3' to 5' exonuclease activity in the presence of only one dNTP (dGTP for the inserts and dCTP for the vector). To make recombinants, the vector plasmid and the insert PCR fragment were annealed at room temperature for 20 min prior to transformation into the host. Bacterial transformation was accomplished with 100% efficiency. To validate the new LIC vector systems, we were used to coexpressed the Phytophthora AVR and potato resistance (R) genes in N. benthamiana by infiltration of Agrobacterium. Coexpressed AVR and R genes in N. benthamiana induced the typical hypersensitive cell death resulting from in vivo interaction of the two proteins. These LIC vectors could be efficiently used for high-throughput cloning and laboratory-scale in planta expression. These vectors could provide a powerful tool for high-throughput transient expression assays for functional genomic studies in plants.

IRDL Cloning: A One-Tube, Zero-Background, Easy-to-Use, Directional Cloning Method Improves Throughput in Recombinant DNA Preparation

“…By contrast, conventional restriction digestion-ligation, Gateway, and LIC all require multiple cloning procedures. Although the TA cloning method could rapidly clone PCR products, preparation of the TA vector involves multiple steps and the final TA-generated construct typically possesses no directionality, resulting in difficulties in selection [24]. TOPO cloning methods also perform rapid cloning, but lack directionality.…”

“…Since cells containing the gyrA462 mutation and cells containing the F′ episome carried a ccdA gene encoding for an antidote to the ccdB -expressing toxic protein, they are not sensitive to its ccdB -killing activity [28]. The ccdB and ccdA of F′ episome expressed a toxin-antitoxin construct that has been extensively used in the construction of various plasmids with positive selection [9], [24], [25], [29], [30], including commercial Gateway plasmids. Besides the ccdB lethal marker, other lethal genes can be used as positive selection marker, e.g., the cell lysis-related genes from phages, Mu, X174, and Q [28]; the plasmid-encoding toxin, e.g., RelE, PemK/Kid, Doc, ShoB, Zor, and MazF [31], [32], [33], [34]; and other lethal genes, e.g., Hlg1 [35], RCSB [28] and barnase [27].…”

Ligation independent cloning: an efficient tool for high-throughput cloning and modular cDNA assembly

“…This technique for cloning is much faster, simpler and more efficient than the 'restriction-ligation' method. Many other researchers use this method to generate vectors with genes of their interest [11][12][13][14] . Some improvements have also been made to facilitate its application.…”

Ectopic Expression of Capsicum-Specific Cell Wall Protein Capsicum annuum Senescence-Delaying 1 (CaSD1) Delays Senescence and Induces Trichome Formation in Nicotiana benthamiana

“…Subcellular localization of CaSD1-soluble modified form of green fluorescent protein (smGFP) or smGFP Both pMBP1:CaSD1-smGFP (pBI121-Modified) and pMBP1: smGFP were constructed using the ligation-independent cloning method (Oh et al, 2010) and transformed into Agrobacterium tumefaciens strain GV2260. The transformed cells were cultured overnight in YEP medium at 30°C with shaking (200 rpm), centrifuged, and resuspended in infiltration buffer containing 10 mM MES (pH 5.5)/10 mM MgCl 2 .…”