A dual-site gateway cloning system for simultaneous cloning of two genes for plant transformation
Introduction
All physiological processes including DNA replication, protein trafficking, and different metabolic and signaling pathways require coordinated expression of genes and a huge network of protein functions and interactions. Innovative systems designed to deliver two or more expression cassettes simultaneously could facilitate the dissection of the complex network of protein functions and interactions. Plant vector systems enabling the cloning of two or more expression cassettes on a single T-DNA and deliver them simultaneously to the plant genome provide a technical advance in multiplication of transgenes over other methods, i.e., crossing between individual transgenic plants, co-transformation of plants, and re-transformation of established transgenic plants (Buntru et al., 2013, Hecker et al., 2015, Kimura et al., 2013). Such plant vector systems minimize the time and labor, reduce gene dosage effects, eliminate independent segregation of introduced genes in next generations, and enable the use of only one selection marker to screen the transformants.
In research areas using transgenic plants, promoter selection is one of the key factors for successful expression of transgenic genes and obtaining appropriate transgenic plants for various research purposes. For coordinated expression of two or more genes, two strategies are available when designing expression vectors: either using different promoters to drive the expression of transgenes (diverse promoter strategy) or using the same promoter for all multiple genes (repetitive promoter strategy). The diverse promoter strategy may be useful in pyramiding multiple transgenes for trait improvement, but finding different promoters with identical activities is extremely difficult (Peremarti et al., 2010). In contrast, although in some cases it has been linked with gene silencing issues due to sequence homology, the repetitive promoter strategy often works effectively and there are several studies reported the successful use of same promoter to guide two or more transgene expressions (Hecker et al., 2015, Naqvi et al., 2009, Paine et al., 2005, Zhu et al., 2008). Constitutive promoters are frequently used for the repetitive promoter strategy to ensure expression of transgenes in most cell types, most stages of development, in a wide range of plant species. The cauliflower mosaic virus 35S promoter (P35S) is the most prevalently used among constitutive promoters with high expression levels of transgenes in many plants (Benfey and Chua, 1990, Odell et al., 1985). However, strong overexpression by P35S may lead to mislocalization of investigated protein(s) and the enhancer sequence exists in P35S may alter the expression levels of nearly located transgenes (Yoo et al., 2005). Furthermore, the frequent methylation of P35S may cause transcriptional inactivation (Matzke and Matzke, 1995, Stam et al., 1997) or gene silencing in stable transformants (Chalfun-Junior et al., 2003, Curradi et al., 2002, Dong and von Arnim, 2003, Mishiba et al., 2005), especially when used repetitively for multi-gene constructs (Kimura et al., 2013, Mishiba et al., 2005). Such problems associated with the strong activity of P35S can be avoided by using a less active promoter, i.e., a promoter that provides a moderate level of constitutive expression such as nopaline synthase promoter (Pnos) (Bevan et al., 1983). The Pnos showed a 20–30 fold lower expression levels (Harpster et al., 1988, Sanders et al., 1987) and much lower methylation frequency (Mishiba et al., 2005) when compared with P35S. Although some binary vectors enabling two-gene constitutive expression have been developed, they were equipped with diverse promoters (Ghareeb et al., 2016, He et al., 2016) or two P35S (Hecker et al., 2015), which limits their usage in experiments requiring similar and moderate expression of the tested two genes. Thus, there is a need for an effective cloning system for two-gene construction utilizing a moderate constitutive promoter such as Pnos to simultaneously drive the transgenes. Besides promoters, other sequences are also known to affect expression of transgenes. Several reports have showed the use of matrix attachment regions (MARs) to increase overall expressions of transgenes (Allen et al., 2000, Han et al., 1997, Petersen et al., 2002, Verma et al., 2005) and reduce the variations in expressions levels among transformants (Cheng et al., 2001, Mlynarova et al., 1994, Petersen et al., 2002, Spiker and Thompson, 1996).
In recent years, bimolecular fluorescence complementation (BiFC) has been extensively used for identification and visualization of protein complex formation and protein interactions in living organisms including plants [Reviewed in: (Kodama and Hu, 2012, Kudla and Bock, 2016, Miller et al., 2015, Tanaka et al., 2012)]. However, improper fusion orientation of amino- (N-) or carboxyl-(C-) terminal fragments of a fluorescent protein, e.g. enhanced yellow fluorescent protein (EYFP), may affect the protein binding or block the targeting information of that protein (Kudla and Bock, 2016). To this end, trying all the eight combinations of fusion patterns for the two tested proteins is highly recommended even necessary in some cases (Bhat et al., 2006, Kamigaki et al., 2016, Nishimura et al., 2015).
Here, we created the dual-site (DS) Gateway cloning system for simple assembly and cloning of two transgenes driven by the moderate Pnos on a single T-DNA. The DS Gateway cloning system permits the N-terminal labeling of tested proteins with various tags (6 fluorescent proteins and 7 epitope tags) providing a suitability for subcellular localization analysis and co-precipitation experiments. The DS Gateway cloning system also enables both N- and C-terminal labeling of tested proteins with N- or C-terminal fragments of EYFP (n/cYFP) with all possible tagging patterns for analysis of protein-protein interactions by the BiFC assay. The DS Gateway cloning system provides a choice for selection of transformants by 4 different plant resistance markers (kanamycin resistance (Kmr), hygromycin resistance (Hygr), BASTA resistance (BASTAr) and Tunicamycin (Tunicar)). We confirmed the stable co-expression and subcellular localization of two soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in Arabidopsis thaliana. Furthermore, we detected the interaction of two cage subunits of coat protein complex II (COPII) by a BiFC assay in both transient expression system using Japanese leek and stable expression system using A. thaliana. Taken together, the newly developed DS Gateway cloning system should be an efficient multipurpose two-gene expression system in plant research with multiple applications, e.g., analysis of co-localization patterns of transgenes and investigation of protein-protein interactions by BiFC or co-immunoprecipitation.
Section snippets
Plasmid manipulation and bacterial strains
Plasmid manipulations were performed according to standard molecular procedures (Sambrook and Russell, 2001). KOD-Plus-Neo DNA polymerase (Toyobo, Osaka, Japan) was used to make amplified products with blunt ends. Nucleotide sequences of adaptors and primers are listed in Table S1. In all cloning steps, plasmids were confirmed by digestion with restriction enzymes and sequencing. Escherichia coli strains One Shot® ccdB survival™ 2T1R (Thermo Fisher Scientific, Kanagawa, Japan) or DH5α (Toyobo)
DS gateway cloning system for expression of two ORFs with two distinct N-terminal fusions
We developed the DS Gateway cloning system to enable a rapid cloning of two ORFs in a binary vector with the commonly used entry clones (attL1-ORF-attL2) and to allow constitutive expression using the Pnos. The N-terminal fusion with diverse tags (fluorescent proteins and epitope tags) is available in this system. The DS Gateway cloning system uses two types of vectors, dual-site binary (DSB) vectors and destination donor (DD) vectors. Fig. 1 shows a line-up of DSB vectors (pGWB6xxx-MD8-Pnos)
Conclusions
In this study, we demonstrated the construction and validation of a versatile vector system named “DS Gateway cloning system” that facilitates the cloning of two genes simultaneously on a single T-DNA by LR reactions with commonly used entry clones (attL1-ORF-attL2) accumulated in the research community. The binary vectors of four selection marker-series (Kmr, Hygr, BASTAr, Tunicar) can be used for various plant transformation experiments. The two cloned genes are constitutively expressed by
Acknowledgements
This work was supported by KAKENHI Grant from Japan Society for the Promotion of Science (JSPS) [Grant-in-Aid for Scientific Research (C) No. JP26440157 to SM and No. JP15K07109 to TN]. We thank Roger Y. Tsien (University of California, San Diego) for provision of mRFP1 clone.
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