Roger Thilmony

PhiC31 recombination system demonstrates heritable germinal transmission of site-specific excision from the Arabidopsis genome.

BackgroundThe large serine recombinase phiC31 from broad host range Streptomyces temperate phage, catalyzes the site-specific recombination of two recognition sites that differ in sequence, typically known as attachment sites attB and attP. Previously, we characterized the phiC31 catalytic activity and modes of action in the fission yeast Schizosaccharomyces pombe.ResultsIn this work, the phiC31 recombinase gene was placed under the control of the Arabidopsis OXS3 promoter and introduced into Arabidopsis harboring a chromosomally integrated attB and attP-flanked target sequence. The phiC31 recombinase excised the attB and attP-flanked DNA, and the excision event was detected in subsequent generations in the absence of the phiC31 gene, indicating germinal transmission was possible. We further verified that the genomic excision was conservative and that introduction of a functional recombinase can be achieved through secondary transformation as well as manual crossing.ConclusionThe phiC31 system performs site-specific recombination in germinal tissue, a prerequisite for generating stable lines with unwanted DNA removed. The precise site-specific deletion by phiC31 in planta demonstrates that the recombinase can be used to remove selectable markers or other introduced transgenes that are no longer desired and therefore can be a useful tool for genome engineering in plants.

The Bxb1 recombination system demonstrates heritable transmission of site-specific excision in Arabidopsis

BackgroundThe mycobacteriophage large serine recombinase Bxb1 catalyzes site-specific recombination between its corresponding attP and attB recognition sites. Previously, we and others have shown that Bxb1 has catalytic activity in various eukaryotic species including Nicotiana tabacum, Schizosaccharomyces pombe, insects and mammalian cells.ResultsIn this work, the Bxb1 recombinase gene was transformed and constitutively expressed in Arabidopsis thaliana plants harboring a chromosomally integrated attP and attB-flanked target sequence. The Bxb1 recombinase successfully excised the target sequence in a conservative manner and the resulting recombination event was heritably transmitted to subsequent generations in the absence of the recombinase transgene. In addition, we also show that Bxb1 recombinase expressing plants can be manually crossed with att-flanked target transgenic plants to generate excised progeny.ConclusionThe Bxb1 large serine recombinase performs site-specific recombination in Arabidopsis thaliana germinal tissue, producing stable lines free of unwanted DNA. The precise site-specific deletion produced by Bxb1 in planta demonstrates that this enzyme can be a useful tool for the genetic engineering of plants without selectable marker transgenes or other undesirable exogenous sequences.

NetVenn: an integrated network analysis web platform for gene lists

Many lists containing biological identifiers, such as gene lists, have been generated in various genomics projects. Identifying the overlap among gene lists can enable us to understand the similarities and differences between the data sets. Here, we present an interactome network-based web application platform named NetVenn for comparing and mining the relationships among gene lists. NetVenn contains interactome network data publically available for several species and supports a user upload of customized interactome network data. It has an efficient and interactive graphic tool that provides a Venn diagram view for comparing two to four lists in the context of an interactome network. NetVenn also provides a comprehensive annotation of genes in the gene lists by using enriched terms from multiple functional databases. In addition, it allows for mapping the gene expression data, providing information of transcription status of genes in the network. The power graph analysis tool is integrated in NetVenn for simplified visualization of gene relationships in the network. NetVenn is freely available at http://probes.pw.usda.gov/NetVenn or http://wheat.pw.usda.gov/NetVenn.

The OsGEX2 Gene Promoter Confers Sperm Cell Expression in Transgenic Rice

Expression control elements (i.e., promoters) are crucial components required for the genetic engineering of plants, but relatively few well-characterized organ-specific promoters are available. We have characterized the rice Gamete Expressed 2 (OsGEX2) gene promoter in transgenic rice plants. The OsGEX2 gene (Os09g25650) is homologous to Arabidopsis GEX2, a gene that exhibits expression specifically in the gametic cells of Arabidopsis pollen. The OsGEX2 gene transcript was only detected in rice pollen (or mixed tissues containing pollen) and not found in other organs or tissues. Transgenic rice plants containing an OsGEX2 promoter fused to a GUSPlus reporter gene displayed cell type-specific β-glucuronidase enzyme activity localized within the sperm cells of mature rice pollen. This expression pattern was clearly distinct from the uniform reporter gene activity observed in the pollen vegetative cell of transgenic rice carrying the rice Pollen Specific 1 promoter. The sperm cell reporter gene activity in OsGEX2–GUSPlus transgenic plants correlated well with the native gene transcript levels, suggesting that the cis-regulatory elements necessary for this specificity are present in the 1.9-kb OsGEX2 promoter fragment. The OsGEX2 promoter with its cell type-specific expression will be a useful tool for precisely controlling sperm cell gene expression in rice and potentially other cereal crop plants.

pGPro1, a Novel Binary Vector for Monocot Promoter Characterization

Promoter analyses can be compromised by interactions between the test promoter and those driving the expression of other genes within the same construct. Our laboratory is engaged in identifying and characterizing promoters that will be useful in controlling transgene expression in monocot crops. For this purpose, we have built the pGProl binary vector construct, a pGreen derived binary vector that contains a useful multiple cloning region allowing the creation of transcriptional or translational fusions to agusA::enhanced Green Fluorescent Protein bifunctional reporter gene. The pGProl vector has many features that make it an efficient tool forAgrobacterium-mediated transformation, and most importantly, its design minimizes the potential impact the promoter/selectable marker gene cassette has on the fidelity of the expression controlled by the test promoter. We demonstrate that the pGProl T-DNA is functional in both transient expression assays in onion cells and transgenic rice plants. This new binary vector will be a useful tool for characterizing promoter function in transgenic monocot plants.

PIECE 2.0: an update for the plant gene structure comparison and evolution database

PIECE (Plant Intron Exon Comparison and Evolution) is a web-accessible database that houses intron and exon information of plant genes. PIECE serves as a resource for biologists interested in comparing intron–exon organization and provides valuable insights into the evolution of gene structure in plant genomes. Recently, we updated PIECE to a new version, PIECE 2.0 (http://probes.pw.usda.gov/piece or http://aegilops.wheat.ucdavis.edu/piece). PIECE 2.0 contains annotated genes from 49 sequenced plant species as compared to 25 species in the previous version. In the current version, we also added several new features: (i) a new viewer was developed to show phylogenetic trees displayed along with the structure of individual genes; (ii) genes in the phylogenetic tree can now be also grouped according to KOG (The annotation of Eukaryotic Orthologous Groups) and KO (KEGG Orthology) in addition to Pfam domains; (iii) information on intronless genes are now included in the database; (iv) a statistical summary of global gene structure information for each species and its comparison with other species was added; and (v) an improved GSDraw tool was implemented in the web server to enhance the analysis and display of gene structure. The updated PIECE 2.0 database will be a valuable resource for the plant research community for the study of gene structure and evolution.

The wheat HMW-glutenin 1Dy10 gene promoter controls endosperm expression in Brachypodium distachyon

The grass species Brachypodium distachyon has emerged as a model system for the study of gene structure and function in temperate cereals. As a first demonstration of the utility of Brachypodium to study wheat gene promoter function, we transformed it with a T-DNA that included the uidA reporter gene under control of a wheat High-Molecular-Weight Glutenin Subunit (HMW-GS) gene promoter and transcription terminator. For comparison, the same expression cassette was introduced into wheat by biolistics. Histochemical staining for β-glucuronidase (GUS) activity showed that the wheat promoter was highly expressed in the endosperms of all the seeds of Brachypodium and wheat homozygous plants. It was not active in any other tissue of transgenic wheat, but showed variable and sporadic activity in a minority of styles of the pistils of four homozygous transgenic Brachypodium lines. The ease of obtaining transgenic Brachypodium plants and the overall faithfulness of expression of the wheat HMW-GS promoter in those plants make it likely that this model system can be used for studies of other promoters from cereal crop species that are difficult to transform.

Accurate measurement of transgene copy number in crop plants using droplet digital PCR.

Genetic transformation is a powerful means for the improvement of crop plants, but requires labor- and resource-intensive methods. An efficient method for identifying single-copy transgene insertion events from a population of independent transgenic lines is desirable. Currently, transgene copy number is estimated by either Southern blot hybridization analyses or quantitative polymerase chain reaction (qPCR) experiments. Southern hybridization is a convincing and reliable method, but it also is expensive, time-consuming and often requires a large amount of genomic DNA and radioactively labeled probes. Alternatively, qPCR requires less DNA and is potentially simpler to perform, but its results can lack the accuracy and precision needed to confidently distinguish between one- and two-copy events in transgenic plants with large genomes. To address this need, we developed a droplet digital PCR-based method for transgene copy number measurement in an array of crops: rice, citrus, potato, maize, tomato and wheat. The method utilizes specific primers to amplify target transgenes, and endogenous reference genes in a single duplexed reaction containing thousands of droplets. Endpoint amplicon production in the droplets is detected and quantified using sequence-specific fluorescently labeled probes. The results demonstrate that this approach can generate confident copy number measurements in independent transgenic lines in these crop species. This method and the compendium of probes and primers will be a useful resource for the plant research community, enabling the simple and accurate determination of transgene copy number in these six important crop species.

Transgene autoexcision in switchgrass pollen mediated by the Bxb1 recombinase.

BackgroundSwitchgrass (Panicum virgatum L.) has a great potential as a platform for the production of biobased plastics, chemicals and energy mainly because of its high biomass yield on marginal land and low agricultural inputs. During the last decade, there has been increased interest in the genetic improvement of this crop through transgenic approaches. Since switchgrass, like most perennial grasses, is exclusively cross pollinating and poorly domesticated, preventing the dispersal of transgenic pollen into the environment is a critical requisite for the commercial deployment of this important biomass crop. In this study, the feasibility of controlling pollen-mediated gene flow in transgenic switchgrass using the large serine site-specific recombinase Bxb1 has been investigated.ResultsA novel approach utilizing co-transformation of two separate vectors was used to test the functionality of the Bxb1/att recombination system in switchgrass. In addition, two promoters with high pollen-specific activity were identified and thoroughly characterized prior to their introduction into a test vector explicitly designed for both autoexcision and quantitative analyses of recombination events. Our strategy for developmentally programmed precise excision of the recombinase and marker genes in switchgrass pollen resulted in the generation of transgene-excised progeny. The autoexcision efficiencies were in the range of 22-42% depending on the transformation event and assay used.ConclusionThe results presented here mark an important milestone towards the establishment of a reliable biocontainment system for switchgrass which will facilitate the development of this crop as a biorefinery feedstock through advanced biotechnological approaches.

Novel sulI binary vectors enable an inexpensive foliar selection method in Arabidopsis

BackgroundSulfonamide resistance is conferred by the sul I gene found on many Enterobacteriaceae R plasmids and Tn21 type transposons. The sul I gene encodes a sulfonamide insensitive dihydropteroate synthase enzyme required for folate biosynthesis. Transformation of tobacco, potato or Arabidopsis using sul I as a selectable marker generates sulfadiazine-resistant plants. Typically sul I-based selection of transgenic plants is performed on tissue culture media under sterile conditions.FindingsA set of novel binary vectors containing a sul I selectable marker expression cassette were constructed and used to generate transgenic Arabidopsis. We demonstrate that the sul I selectable marker can be utilized for direct selection of plants grown in soil with a simple foliar spray application procedure. A highly effective and inexpensive high throughput screening strategy to identify transgenic Arabidopsis without use of tissue culture was developed.ConclusionNovel sul I-containing Agrobacterium binary vectors designed to over-express a gene of interest or to characterize a test promoter in transgenic plants have been constructed. These new vector tools combined with the various beneficial attributes of sulfonamide selection and the simple foliar screening strategy provide an advantageous alternative for plant biotechnology researchers. The set of binary vectors is freely available upon request.