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

Abstract

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.

Fig 1. Transformation of M. polymorpha using pMpGWB vectors developed in this study.

Sporelings co-cultivated with A. tumefaciens strain GV2260 containing the binary vector pMpGWB100 (A), pMpGWB200 (C), pMpGWB300 (E), pMpGWB400 (G), or no binary vector (B, D, F, and H) at 7 d after transfer onto selection medium containing 10 mg/l hygromycin (Hyg; A, B), 100 mg/l gentamicin (Gen; C, D), 0.5 μM chlorsulfuron (CS; E, F), or 5 mg/l G418 (G, H). Insets show magnified view of antibiotic/herbicide-resistant transformants. Bars = 1 mm.

Fig 2. Comparison of transformation efficiencies of M. polymorpha among four vectors with different selection markers and selective antibiotics/herbicide.

Hyg, hygromycin; Gen, gentamicin; CS, chlorsulfuron. Values are means ± SD from three independent experiments.

Fig 3. Schematic illustrations of pMpGWBs.

A. Outline of pMpGWBs (pMpGWB101-136, pMpGWB201-236, pMpGWB301-336, and pMpGWB401-436 containing selection markers for Hyg, Gen, CS, and G418, respectively). Markers were placed in tail-to-tail orientation in relation to genes cloned by LR reaction. B. Structures of “Gateway” regions shown in (A). Only general structures are shown for vectors harboring fusion construct; i.e., no promoter and C-fusion (No pro/C), CaMV 35S promoter and C-fusion (_pro35S/C), CaMV 35S promoter and N-fusion (_pro35S/N), EF promoter and C-fusion (_proEF/C), EF promoter and N-fusion (_proEF/N), MpHSP17.8A1 promoter and C-fusion (_proHSP/C). C. Reporters and tags used in pMpGWBs illustrated in (B). Vector number corresponds to combination of reporter/tag and fusion type. Citrine, synthetic yellow fluorescent protein; 3×Citrine, three repeats of Citrine fluorescent protein; TagRFP, tag red fluorescent protein; tdTomato; tandem dimer Tomato fluorescent protein; GR, ligand-binding domain of rat glucocorticoid receptor; SRDX, modified EAR motif plant-specific repression domain with strong repression activity; SRDX-GR, SRDX fused with GR; 3×FLAG, three repeats of FLAG tag; GUS, β-glucuronidase; ELuc(PEST), Emerald Luc (luciferase from click beetle) fused with PEST sequence; Citrine-NLS, Citrine fused with nuclear localization signal sequence; tdTomato-NLS, tdTomato fused with nuclear localization signal sequence. RB, right border; LB, left border; sta, region conferring stability in Agrobacterium; rep, broad host-range replication origin; bom, cis-acting element for conjugational transfer; ori, ColE1 replication origin. Cm ^r, chloramphenicol-resistance marker (chloramphenicol acetyl transferase) used to select bacteria; aadA, spectinomycin-resistance marker (Spc ^r) used to select bacteria; ccdB, negative selection marker.

Fig 4. Simultaneous transformation with multiple pMpGWB vectors.

A. Two binary vectors, pMpGWB103-TagRFP-LTI6b and pMpGWB303-SP-GFP-HDEL, were used simultaneously for Agrobacterium-mediated sporeling transformation with double selection. B. Four binary vectors, pMpGWB103-TagRFP-LTI6b, pMpGWB216-proEF, pMpGWB303-SP-GFP-HDEL, and pMpGWB404-proEF, were used simultaneously for transformation with quadruple selection. Separate and merged images for fluorescence from TagRFP or TagRFP/tdTomato, GFP, and chlorophyll autofluorescence of thallus epidermal cells and a micrograph for GUS staining in a representative line from each transformation are shown. Bars = 10 μm for fluorescence; 1 cm for GUS.