Development of Gateway Binary Vector Series with Four Different Selection Markers for the Liverwort Marchantia polymorpha

We previously reported Agrobacterium-mediated transformation methods for the liverwort Marchantia polymorpha using the hygromycin phosphotransferase gene as a marker for selection with hygromycin. In this study, we developed three additional markers for M. polymorpha transformation: the gentamicin 3'-acetyltransferase gene for selection with gentamicin; a mutated acetolactate synthase gene for selection with chlorsulfuron; and the neomycin phosphotransferase II gene for selection with G418. Based on these four marker genes, we have constructed a series of Gateway binary vectors designed for transgenic experiments on M. polymorpha. The 35S promoter from cauliflower mosaic virus and endogenous promoters for constitutive and heat-inducible expression were used to create these vectors. The reporters and tags used were Citrine, 3×Citrine, Citrine-NLS, TagRFP, tdTomato, tdTomato-NLS, GR, SRDX, SRDX-GR, GUS, ELuc(PEST), and 3×FLAG. These vectors, designated as the pMpGWB series, will facilitate molecular genetic analyses of the emerging model plant M. polymorpha.


Introduction
Bryophytes, including liverworts, mosses, and hornworts, are the earliest diverging group of extant land plants. Liverworts are a key lineage for comparative genomic studies aimed at elucidating the fundamental and diverse gene regulatory networks in land plants [1][2][3]. The liverwort Marchantia polymorpha L. is a widespread dioecious species that is easily cultivated in laboratory conditions. Previous research on Marchantia has focused on the genomes of organelles in cultured cells [4,5] and its Y chromosome [6]. Recently, the whole genome shotgun sequencing project of M. polymorpha was initiated under the Community Sequencing Program at the Joint Genome Institute (DOE-JGI: http://jgi.doe.gov/sequencing/why/CSP2008/ mpolymorpha.html). Our research group has been developing M. polymorpha as an experimental model organism, because it occupies a critical evolutionary position for studies on specific molecular and cellular developmental processes in detail [7].
We previously reported an efficient Agrobacterium-mediated transformation system using sporelings of the liverwort M. polymorpha. Hundreds of transformants carrying T-DNAs randomly inserted in the genome were obtained from a single sporangium [8]. An Agrobacteriummediated transformation method has also become available using thallus explants in M. polymorpha, providing opportunities for genetic transformation without preparing spores [9]. Recently, these methods have been simplified so that all of the procedures can be conducted in a Petri dish [10,11]. These methods for genetic transformation have been used for functional molecular studies of M. polymorpha [12][13][14][15][16][17][18][19][20][21][22][23][24]. Furthermore, a method for homologous recombination-mediated gene targeting has been established for M. polymorpha-this method uses positive/negative selection in combination with an efficient Agrobacterium-mediated transformation system [25]. To date, however, only the hygromycin phosphotransferase gene (hpt) has been available for use as a marker for Agrobacterium-mediated transformation in M. polymorpha. Introduction of a transgene into pre-existing transgenic lines is an important method for analyses of transgene function and the localization of its product. For this purpose, additional selection markers that differ from those in pre-existing transgenic plants are required.
Here we describe three markers that have been developed for M. polymorpha transformation: the gentamicin 3'-acetyltransferase gene (aacC1) [26] for selection with gentamicin; a mutated acetolactate synthase gene (mALS) for selection with the sulfonylurea herbicide, 2-chloro-N-[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)aminocarbonyl]-benzenesulfonamide (chlorsulfuron; CS); and the neomycin phosphotransferase II (nptII) gene for selection with G418. Based on these marker genes, we have designed and constructed a series of Gateway binary vectors, the pMpGWB series, for transgenic experiments on M. polymorpha. Using a combination of different selection markers, multiple constructs can be introduced into M. polymorpha by a single transformation procedure. These pMpGWB vectors will facilitate transgenic research on the model plant M. polymorpha.

Vector construction
All the primers used in this study are listed in S1 Table. The PCR-amplified regions and ligation junctions were confirmed by sequence analysis for all vectors.
Acetolactate synthase (ALS) is a key enzyme in the biosynthesis of the branched-chain amino acids leucine, isoleucine, and valine. Sulfonylurea herbicides, e.g. CS, bind reversibly to the ALS-FAD-thiamine pyrophosphate-Mg 2+ -decarboxylated pyruvate complex and also compete for the second pyruvate binding site in ALS [29]. Mutagenesis at these sites in ALS confers tolerance to sulfonylurea herbicides. For example, mALSs containing mutations at P197H, R198S, and W574L conferred resistance to CS in Arabidopsis [30]. To generate a CS-resistance marker gene for use in M. polymorpha, its ALS sequence was mutagenized to contain corresponding mutations (P207S/R208S/W582L) to those described above, as follows. First, M. and for Challenging Exploratory Research (26650095 to T.K.); by a research fellowship from the JSPS for young scientists (21-764 to M.U., 24-7049 to K.In). URL for Kakenhi: http://www.jsps.go.jp/english/egrants/index.html. This work was also funded by the Asahi Glass Foundation (http://www.af-info.or.jp/en/ index.html) and the SUNTORY Foundation for Life Sciences (http://www.sunbor.or.jp/egs/index.html) to K.Is. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. polymorpha ALS cDNA was amplified by RT-PCR with the primer set ALS-P1 and ALS-P2 using first-strand cDNA synthesized from a 10-day-old thallus. The resultant ALS cDNA was cloned into the pENTR/D-TOPO vector (Life Technologies, Gaithersburg, MD, USA). A point mutation at W582L in the ALS sequence was introduced by PCR-based site-directed mutagenesis with the primer pair ALS-W582L-F and ALS-W582L-R, using the ALS cDNA plasmid as the template. The PCR product was digested with DpnI, and the digested PCR product was transformed into Escherichia coli competent cells (DH5α). The mutation in ALS was confirmed by sequencing the resultant plasmid. The mALS was prepared by repeating the same procedure with the primer set ALS-P207S-R208S-F and ALS-P207S-R208S-F using the W582L-mutated ALS as a template. The mALS cDNA was cloned into pCAMBIA1300 to replace the hpt gene, generating the plasmid pCAMBIA1300-mALS.
The vector for expression of TagRFP-LTI6b was constructed as follows: the GFP-LTI6b coding sequence was PCR-amplified using the primers GFP-LTI6b_GW_L1 and GFP-LTI6b_ GW_R1 and cloned into pENTR/D-TOPO. The GFP coding sequence flanked by two NotI sites in this plasmid was replaced with the TagRFP-coding sequence with two similarly flanking NotI sites, which was PCR-amplified using the primers pENTRD_NotI_TagRFP_F and TagRFP_No-tI_R. This plasmid was used for LR recombination with pMpGWB103 to generate pMpGWB 103-TagRFP-LTI6b.
To construct the vector for expression of SP-GFP-HDEL, the SP-GFP-HDEL-coding sequence [33] was PCR-amplified using the primers SP_Lc and HDEL_R and cloned into pENTR/D-TOPO. The resulting vector was used for LR recombination with pMpGWB303 to generate pMpGWB303-SP-GFP-HDEL.
To construct the vectors for expression of tdTomato-NLS and GUS under the endogenous ELONGATION FACTOR1α promoter ( pro EF), the 1,729-bp promoter sequence of MpEF1α [34] was amplified by PCR using the primers MpEF-P_L1 and MpEF-P_R1 and cloned into pENTR/D-TOPO. The resulting vector was used for LR recombination with pMpGWB216 and pMpGWB404 to generate pMpGWB216-proEF and pMpGWB404-proEF, respectively.

Agrobacterium-mediated transformation of M. polymorpha
Transformation of M. polymorpha was performed as described previously [8]. For simultaneous transformation with multiple vectors, the respective Agrobacterium cultures were mixed at a 1:1 ratio. Half-strength Gamborg's B5 medium containing 0.5 g/L MES and 1% (w/v) agar and supplemented with hygromycin B (Wako Pure Chemical Industries, Osaka, Japan), gentamicin sulfate (Nacalai Tesque, Kyoto, Japan), CS (kindly provided by Dupont, Wilmington, DE, USA), or G418 (Nacalai Tesque) was used to select transformants. The stock solution of CS (50 mM) was prepared with DMSO.

GUS staining
Histochemical staining of GUS was performed as described previously [13,35].

Nucleotide accession numbers and distribution of pMpGWBs
The complete nucleotide sequences of the pMpGWBs have been deposited in the GenBank/ EMBL/DDBJ databases under the accessions nos. LC057442 to LC057589. pMpGWB plasmids can be obtained from Addgene (www.addgene.org; plasmid numbers 68062 and 68555-68701).

Selection of transformants using gentamicin, CS, and G418
To explore the possibility of using gentamicin, CS, and G418 to select transformed M. polymorpha, we first investigated whether these substances suppressed plant growth. M. polymorpha gemmae were grown on agar medium containing various concentrations of gentamicin, CS, and G418. Thalli showed severe growth defects when grown on medium containing 50 mg/l or higher concentrations of gentamicin, 0.05 μM or higher concentrations of CS, and 2.5 mg/l or higher concentrations of G418 (S1 Fig).
To facilitate the selection of transformants on medium containing these antibiotics/herbicide, chimeric marker gene cassettes were introduced into the binary vector pGWB400 [31]. The backbone of this vector was pPZP221, harboring ColE1 and pVS1 plasmid origins for replication in E. coli and Agrobacterium, respectively [36]. In the case of hygromycin selection, the nopaline synthase gene promoter did not drive expression of the hpt marker gene as strongly as did the CaMV 35S promoter in M. polymorpha. Consequently, the nopaline synthase gene promoter resulted in a lower transformation efficiency. To ensure efficient expression of the resistance genes in M. polymorpha, a double-enhancer version of the CaMV 35S promoter ( pro 35S×2) in the pCAMBIA vector (http://www.cambia.org/daisy/cambia/585), which was derived from the pPZP vector [36,37], was used to drive the expression of the marker genes. Thus, the constructed binary vectors were pMpGWB100, pMpGWB200, pMpGWB300, and pMpGWB400, carrying pro 35S×2-driven hpt, aacC1, mALS, and nptII, respectively (S2 Fig).

Gateway binary vectors pMpGWBs
In functional genetic studies, the construction of fusion genes is valuable for analyses of expression, protein localization, and protein-protein interactions in vivo. We have constructed Gateway binary vectors (pMpGWBs) for M. polymorpha based on the four binary vectors pMpGWB100, pMpGWB200, pMpGWB300, and pMpGWB400. The pMpGWB vectors allow genes to be fused to a variety of reporters and tags through a simple and uniform procedure using Gateway cloning technology [38]. The structure of the pMpGWBs is shown in Fig 3. The pMpGWB100 series carry the hygromycin-resistance marker ( pro 35S×2:hpt: ter 35S). Similarly, the pMpGWB200, pMpGWB300, and pMpGWB400 series carry the gentamicin-resistance marker ( pro 35S×2:aacC1: ter 35S), the CS-resistance marker ( pro 35S×2:mALS: ter 35S), and the G418-resistance marker ( pro 35S×2:nptII: ter 35S), respectively. All markers were placed in the tail-to-tail orientation in relation to the gene cloned by the LR reaction.
The last two digits of the plasmid names indicate the structures around the Gateway cassette that are common to the pMpGWBs. For example, pMpGWB101, pMpGWB201, pMpGWB301, and pMpGWB401 are simple cloning vectors with neither promoters nor reporters/tags. These vectors can be used in various experiments, for example, for complementation of mutations with genomic fragments. pMpGWB102, pMpGWB202, pMpGWB302, and pMpGWB402 carry a standard CaMV 35S promoter ( pro 35S) for constitutive expression in plants. pMpGWB103, pMpGWB203, pMpGWB303, and pMpGWB403 carry the endogenous EF1α promoter ( pro EF), which drives constitutive expression in M. polymorpha plants [34]. pMpGWB132, pMpGWB232, pMpGWB332, and pMpGWB432 carry the endogenous MpHSP17.8A1 promoter ( pro HSP) for heat-inducible expression in M. polymorpha [35].
Fusion genes of promoter-GUS are usually used in histochemical and quantitative expression analyses, while promoter-luciferase fusions are useful for monitoring transcriptional responses in living cells. pMpGWB104, pMpGWB204, pMpGWB304, and pMpGWB404 are suitable for promoter analysis with the GUS reporter gene. pMpGWB131, pMpGWB231, pMpGWB331, and pMpGWB431 contain a luciferase gene from click beetle (Photinus pyralis) [39] fused to the mouse ornithine decarboxylase PEST sequence at the C-terminus. The ELuc (PEST) fusion gene was constructed to produce a promoter-luciferase assay with enhanced response dynamics [40]. pMpGWB115-116, pMpGWB215-216, pMpGWB315-316, and pMpGWB415-416 contain sequences encoding fluorescent proteins, Citrine or tdTomato, fused to a short amino acid sequence that contains the nuclear localization signal sequence (PKKKRKV) from the SV40 large T antigen [41]. These vectors were designed for monitoring promoter activity in planta. For promoter analysis, one would assume that the doubleenhancer version of the CaMV 35S promoter, which drives marker genes in these pMpGWB vectors, might affect promoter activity in the same T-DNA. However, this does not appear to be the case in M. polymorpha as shown previously [13,35].
Multiple fusion types are available for most reporters/tags. There are vectors for constitutive expression, driven by the CaMV 35S promoter, of proteins fused with reporters at the C-terminus ( pro 35S/C) or at the N-terminus ( pro 35S/N). There are vectors for expression of C-terminal fusion proteins driven by their own promoters (No pro/C). There are also vectors for the endogenous EF1α promoter-driven expression of proteins that are fused with a reporter at the C-terminus ( pro EF/C) or at the N-terminus ( pro EF/N), and those for the endogenous MpHSP17.8A1 promoter-driven expression of proteins that are fused with a reporter at the Cterminus ( pro HSP/C).
The vectors pMpGWB117-119, pMpGWB217-219, pMpGWB317-319, and pMpGWB417-419 were designed for expressing transcriptional regulators tagged with the modified EAR motif plant-specific repression domain (SRDX; amino acid sequence LDLDLELRLGFA), which shows strong repression activity, at the C-terminus [48]. A chimeric repressor that is produced by fusing a transcription factor to SRDX suppresses the target genes of the transcription factor dominantly over the activity of endogenous and functionally redundant transcription factors [48]. pMpGWB120-122, pMpGWB220-222, pMpGWB320-322, and pMpGW B420-422 were designed for expressing transcriptional regulators tagged with both SRDX and GR at the C-terminus. Using these constructs, the activity of the chimeric transcriptional repressor can be made conditional on the presence of steroid ligands.

Simultaneous transformation with multiple constructs
To test the performance of these vectors, we made two types of binary vectors: one designed to express a plasma membrane-localized fluorescent protein, TagRFP-LTI6b [49], under the control of pro EF using pMpGWB103; and the other designed to express an endoplasmic reticulum-localized fluorescent protein, SP-GFP-HDEL [32], under the control of pro EF using pMpGWB303. These binary vectors were separately introduced into Agrobacterium cells, and the respective transformant clones were used together to transform M. polymorpha sporelings. Sporelings co-cultured with the mixed Agrobacterium culture were subjected to hygromycin selection first, and then, to CS selection. Approximately 30% of transformants that passed the first hygromycin selection were resistant also to CS. Transgenic plants that survived selection with both hygromycin and CS were found to express the two proteins at their predicted subcellular locations (Fig 4A). We also examined triple and quadruple transformation using the two plasmids used above plus pro EF-introduced pMpGWB216 (for expression of tdTomato-NLS) or pro EF-introduced pMpGWB404 (for expression of GUS), or both. A portion (10-20%) of transformants that passed the first hygromycin selection showed resistance to either of the following combinatorial second selections: (i) hygromycin, gentamicin, and CS; (ii) hygromycin, CS, and G418; or (iii) all the four antibiotics/herbicide. As expected, expression of all transgenes in quadruple transformants was observed at predicted locations ( Fig 4B).
Thus, the pMpGWB series are capable of driving proper expression. In addition, the four kinds of selection markers available in the pMpGWB system should provide options for transformation with multiple constructs simultaneously.

Conclusion
We have developed a series of binary vectors for transformation of M. polymorpha. Four kinds of selection markers are now available for transgenesis of M. polymorpha. The pMpGWBs constructed in this study will be useful for efficient cloning in transgenic research, and will be particularly powerful for the construction of fusion genes and simultaneous introduction of multiple constructs. We have used various reporters and tags suitable for Marchantia research in these pMpGWBs.
In Arabidopsis, the Gateway vector series developed by Nakagawa et al. have been widely used and have accelerated the construction of transgenes [31,32,50]. It should be noted that the pMpGWBx00 binary vectors have unique HindIII/SacI sites next to the nopaline synthase terminator sequence ( ter NOS). Consequently, the HindIII/SacI fragments in the pGWB and R4pGWB series [31,32,50] that harbor Gateway cassettes and reporter/tag genes can be transferred into pMpGWBs by a single-step cloning procedure. Therefore, various Gateway cassettes and reporter/tags in the pGWB and R4GWB series (i.e., sGFP, G3GFP, mRFP, 6×His, 3×HA, 4×Myc, 10×Myc, GST, T7, and TAP) can be easily transferred into the pMpGWB series as needed. Similarly, the original Gateway cassette and reporters/tags in pMpGWBs (Citrine, 3×Citrine, Citrine-NLS, tdTomato, tdTomato-NLS, GR, SRDX, SRDX-GR, ELuc(PEST), and 3×FLAG) can be readily transferred into pGWBs. Although the pMpGWBs were developed for transgenesis experiments in M. polymorpha, they could also be used for other plants in which the pro 35S×2-driven selection markers are effective, such as monocots.