A dual-site gateway cloning system for simultaneous cloning of two genes for plant transformation

- Rapid and efficient cloning of two ORFs on a binary vector with the commonly used ORF entry clones (attL1-ORF-attL2).
- The moderate Pnos was used to provide a constitutive expression and to direct the simultaneous expression of both ORFs.
- As an N-terminal tag, six kinds of fluorescent protein and seven kinds of epitope are available in this system.
- Construction of all the eight tagging patterns of N- or C-terminal fragments of YFP on a single T-DNA is possible.
- Transgenic A. thaliana lines expressing both cis-Golgi and TGN markers can be used to explore Golgi localized proteins.

Analyses of the subcellular localization of proteins and protein-protein interaction networks are essential to uncover the molecular basis of diverse biological processes in plants. To this end, we have created a Gateway cloning-compatible vector system, named dual-site (DS) Gateway cloning system to allow simple cloning of two expression cassettes in a binary vector and to express them simultaneously in plant cells. In the DS Gateway cloning system, (i) a moderate constitutive nopaline synthase promoter (Pnos), which is much suitable for localization analysis, is used to guide each expression cassette, (ii) four series of vectors with different plant resistance markers are established, (iii) N-terminal fusion with 6 fluorescent proteins and 7 epitope tags is available, (iv) both N- and C-terminal fusions with split enhanced yellow fluorescent protein (EYFP) are possible for efficient detection of protein-protein interactions using a bimolecular fluorescence complementation (BiFC) assay. The usefulness of the DS Gateway cloning system has been demonstrated by the analysis of the expression and the subcellular localization patterns of two Golgi proteins in stable expression system using A. thaliana, and by the analyses of interactions between subunits of coat protein complex II (COPII) both in transient and stable expression systems using Japanese leek and A. thaliana, respectively. The DS Gateway cloning system provides a multipurpose, efficient expression tool in gene function analyses and especially suitable for investigating interactions and subcellular localization of two proteins in living plant cells.