Construction of full-length cDNA infectious clones of Chilli veinal mottle virus.

Chilli veinal mottle virus (ChiVMV), a member of the genus Potyvirus in the family Potyviridae, causes severe diseases and poses a great threat to solanaceous crops. Reverse genetics technology is an efficient tool to facilitate the study of virus biology and pathogenicity. However, the construction of an infectious cDNA clone of ChiVMV is yet to be reported. In this study, full-length cDNA infectious clones of ChiVMV and GFP-tagged ChiVMV were constructed using yeast homologous recombination for the first time. These infectious clones were able to successfully infect host plants (Nicotiana benthamiana, Nicotiana tabacum and Solanum lycopersicum) by Agrobacterium-mediated infiltration and cause vein banding and leaf curling symptoms. Mutations were introduced to pChiVMV-GFP to investigate the role of key amino acids in ChiVMV 6K2. The results showed that substitution mutants of leucine (L9, 11) to alanine acid (A), tryptophan (W15) to alanine acid (A), and glycine (G29, 33) to valine acid (V) reduced the viral accumulation and the mutant clones were unable to induce the symptoms in N. benthamiana plants. Taken together, these infectious clones we developed will be effective tools for future studies of the function of viral factors encoded by ChiVMV and the interactions between ChiVMV and its different host plants.

High-Throughput DNA Assembly Using Yeast Homologous Recombination.

Yeast homologous recombination is a reliable, low-cost, and efficient method for DNA assembly. Using homology regions as short as 24 base pairs, constructs of up to 12 unique parts can be assembled into a diverse range of vectors. The simplicity and robustness of this protocol make it amenable to laboratory automation and high-throughput operations. Here we describe a high-throughput protocol to generate DNA parts through PCR, assemble them into a vector via yeast transformation, and "shuttle" the resulting plasmid constructs into E. coli for storage and propagation. Though this protocol is intended for high-throughput workflows, it can be easily adapted for bench-scale DNA assembly.

Direct cloning and heterologous expression of natural product biosynthetic gene clusters by transformation-associated recombination.

Heterologous expression of natural product biosynthetic gene clusters (BGCs) is a robust approach not only to decipher biosynthetic logic behind natural product (NP) biosynthesis, but also to discover new chemicals from uncharacterized BGCs. This approach largely relies on techniques used for cloning large BGCs into suitable expression vectors. Recently, several whole-pathway direct cloning approaches, including full-length RecE-mediated recombination in Escherichia coli, Cas9-assisted in vitro assembly, and transformation-associated recombination (TAR) in Saccharomyces cerevisiae, have been developed to accelerate BGC isolation. In this chapter, we summarize a protocol for TAR cloning large NP BGCs, detailing the process of choosing TAR plasmids, designing pathway-specific TAR vectors, generating yeast spheroplasts, performing yeast transformation, and heterologously expressing BGCs in various host strains. We believe that the established platforms can accelerate the process of discovering new NPs, understanding NP biosynthetic logic, and engineering biosynthetic pathways.

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

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.

Efficient Assembly of DNA Using Yeast Homologous Recombination (YHR).

The assembly of multiple DNA parts into a larger DNA construct is a requirement in most synthetic biology laboratories. Here we describe a method for the efficient, high-throughput, assembly of DNA utilizing the yeast homologous recombination (YHR). The YHR method utilizes overlapping DNA parts that are assembled together by Saccharomyces cerevisiae via homologous recombination between designed overlapping regions. Using this method, we have successfully assembled up to 12 DNA parts in a single reaction.

Use of homologous recombination in yeast to create chimeric bovine viral diarrhea virus cDNA clones

Abstract The open reading frame of a Brazilian bovine viral diarrhea virus (BVDV) strain, IBSP4ncp, was recombined with the untranslated regions of the reference NADL strain by homologous recombination in Saccharomyces cerevisiae, resulting in chimeric full-length cDNA clones of BVDV (chi-NADL/IBSP4ncp#2 and chi-NADL/IBSP4ncp#3). The recombinant clones were successfully recovered, resulting in viable viruses, having the kinetics of replication, focus size, and morphology similar to those of the parental virus, IBSP4ncp. In addition, the chimeric viruses remained stable for at least 10 passages in cell culture, maintaining their replication efficiency unaltered. Nucleotide sequencing revealed a few point mutations; nevertheless, the phenotype of the rescued viruses was nearly identical to that of the parental virus in all experiments. Thus, genetic stability of the chimeric clones and their phenotypic similarity to the parental virus confirm the ability of the yeast-based homologous recombination to maintain characteristics of the parental virus from which the recombinant viruses were derived. The data also support possible use of the yeast system for the manipulation of the BVDV genome.

Use of homologous recombination in yeast to create chimeric bovine viral diarrhea virus cDNA clones.

The open reading frame of a Brazilian bovine viral diarrhea virus (BVDV) strain, IBSP4ncp, was recombined with the untranslated regions of the reference NADL strain by homologous recombination in Saccharomyces cerevisiae, resulting in chimeric full-length cDNA clones of BVDV (chi-NADL/IBSP4ncp#2 and chi-NADL/IBSP4ncp#3). The recombinant clones were successfully recovered, resulting in viable viruses, having the kinetics of replication, focus size, and morphology similar to those of the parental virus, IBSP4ncp. In addition, the chimeric viruses remained stable for at least 10 passages in cell culture, maintaining their replication efficiency unaltered. Nucleotide sequencing revealed a few point mutations; nevertheless, the phenotype of the rescued viruses was nearly identical to that of the parental virus in all experiments. Thus, genetic stability of the chimeric clones and their phenotypic similarity to the parental virus confirm the ability of the yeast-based homologous recombination to maintain characteristics of the parental virus from which the recombinant viruses were derived. The data also support possible use of the yeast system for the manipulation of the BVDV genome.

Flexible and Versatile Strategy for the Construction of Large Biochemical Pathways.

Synthetic pathways and circuits have been increasingly used for microbial production of fuels and chemicals. Here, we report a flexible and versatile DNA assembly strategy that allows rapid, modular, and reliable construction of biological pathways and circuits from basic genetic parts. This strategy combines the automation-friendly ligase cycling reaction (LCR) method and the high-fidelity in vivo yeast-based DNA assembly method, DNA assembler. Briefly, LCR is used to preassemble basic genetic parts into gene expression cassettes or to preassemble small parts into larger parts to reduce the number of parts, in which many basic genetic parts can be reused. With the help of specially designed unique linkers, all preassembled parts will then be directly assembled using DNA assembler to build the target constructs. As proof of concept, three plasmids with varying sizes of 13.4, 24, and 44 kb were rapidly constructed with fidelities of 100, 88, and 71%, respectively. The yeast strain harboring the constructed 44 kb plasmid was confirmed to be capable of utilizing xylose, cellobiose, and glucose to produce zeaxanthin. This strategy should be generally applicable to any custom-designed pathways, circuits, or plasmids.

Full-length infectious clone of a low passage dengue virus serotype 2 from Brazil

Full-length dengue virus (DENV) cDNA clones are an invaluable tool for many studies, including those on the development of attenuated or chimeric vaccines and on host-virus interactions. Furthermore, the importance of low passage DENV infectious clones should be highlighted, as these may harbour critical and unique strain-specific viral components from field-circulating isolates. The successful construction of a functional Brazilian low passage DENV serotype 2 full-length clone through homologous recombination reported here supports the use of a strategy that has been shown to be highly useful by our group for the development of flavivirus infectious clones and replicons.

A pathogenicity determinant maps to the N-terminal coat protein region of the Pepino mosaic virus genome.

Pepino mosaic virus (PepMV) poses a worldwide threat to the tomato industry. Considerable differences at the genetic level allow for the distinction of four main genotypic clusters; however, the basis of the phenotypic outcome is difficult to elucidate. This work reports the generation of wild-type PepMV infectious clones of both EU (mild) and CH2 (aggressive) genotypes, from which chimeric infectious clones were created. Phenotypic analysis in three solanaceous hosts, Nicotiana benthamiana, Datura stramonium and Solanum lycopersicum, indicated that a PepMV pathogenicity determinant mapped to the 3'-terminal region of the genome. Increased aggression was only observed in N. benthamiana, showing that this factor is host specific. The determinant was localized to amino acids 11-26 of the N-terminal coat protein (CP) region; this is the first report of this region functioning as a virulence factor in PepMV.

Insertion and stable expression of Gaussia luciferase gene by the genome of bovine viral diarrhea virus.

As a tool to address selected issues of virus biology, we constructed a recombinant cDNA clone of bovine viral diarrhea virus (BVDV) expressing Gaussia luciferase (Gluc) reporter gene. A full-length genomic cDNA clone of a non-cytopathic BVDV isolate was assembled by recombination in yeast Saccharomyces cerevisiae. The Gluc gene was inserted between the N(pro) and Core protein coding regions by recombination. The cDNA transcribed in vitro was infectious upon transfection of MDBK cells, resulting in reporter gene expression and productive virus replication. The rescued viruses were stable for 15 passages in cell culture, maintaining the replication kinetics, focus size and morphology similar to those of the parental virus. Expression and correct processing of the reporter protein were also maintained, as demonstrated by Gluc activity. These results demonstrate that genes up to 555 bp are simply assembled by a single step in yeast recombination and are stably expressed by this cDNA clone.

Yeast homologous recombination cloning leading to the novel peptides ambactin and xenolindicin.

Heterologous production of GameXPeptide A (1), as well as of the novel peptide natural products ambactin (2) and xenolindicins A-C (3 a-c), was achieved by using the "overlap extension PCR-yeast homologous recombination" (ExRec) method. ExRec cloning is based on the ability of yeast to assemble overlapping DNA fragments into functional plasmids. Here we used this technique to clone a total of 15 biosynthesis gene clusters from Photorhabdus and Xenorhabdus with sizes of up to 45 kb. The structures of the novel compounds 2 and 3 a, which were produced in Escherichia coli, were elucidated by detailed MS and bioinformatics analysis, and additionally confirmed by their chemical synthesis.

A universal cloning method based on yeast homologous recombination that is simple, efficient, and versatile.

Cloning by homologous recombination (HR) in Saccharomyces cerevisiae is an extremely efficient and cost-effective alternative to other methods of recombinant DNA technologies. Unfortunately, it is incompatible with all the various specialized plasmids currently used in microbiology and biomedical research laboratories, and is therefore, not widely adopted. In an effort to dramatically improve the versatility of yeast gap-repair cloning and make it compatible with any DNA plasmid, we demonstrate that by simply including a yeast-cloning cassette (YCC) that contains the 2-micron origin of replication (2µm ori) and the ura3 gene for selection, multiple DNA fragments can be assembled into any DNA vector. We show this has almost unlimited potential by building a variety of plasmid for different uses including: recombinant protein production, epitope tagging, site-directed mutagenesis, and expression of fluorescent fusion proteins. We demonstrate the use in a variety of plasmids for use in microbial systems and even demonstrate it can be used in a vertebrate model. This method is remarkably simple and extremely efficient, plus it provides a significant cost saving over commercially available kits.