Development of an R4 dual-site (R4DS) gateway cloning system enabling the efficient simultaneous cloning of two desired sets of promoters and open reading frames in a binary vector for plant research

<div><p>Vast numbers of proteins work cooperatively to exert their functions in various cells. In order to understand the functions and molecular mechanisms of these proteins in plants, analyses of transgenic plants that concomitantly express two protein-coding genes are often required. We developed a novel Gateway cloning technology-compatible binary vector system, the R4 dual-site (R4DS) Gateway cloning system, which enables the easy and efficient cloning of two desired sets of promoters and open reading frames (ORFs) into a binary vector using promoter and ORF entry clones. In this system, C-terminal fusions with 17 kinds of tags including visible reporters and epitope tags are available for each ORF, and selection by four kinds of resistance markers is possible. We verified that the R4DS Gateway cloning system functioned well in <i>Arabidopsis thaliana</i> by observing the expression and localization patterns of fluorescent proteins fused with organelle-targeting signals and driven by stomatal-lineage specific promoters. We also confirmed that the two cloning sites in the R4DS Gateway cloning system were equivalent and independently regulated. The results obtained indicate that the R4DS Gateway cloning system facilitates detailed comparisons of the expression patterns of two promoters as well as co-localization and interaction analyses of two proteins in specific cells in plants.</p></div

Expression and intracellular localization of G3GFP and TagRFP fused with peroxisome- or plastid-targeting signals in transformed <i>A</i>. <i>thaliana</i> with different combinations of promoters.

<p>(A, B) Structural diagram of the binary constructs with peroxisome-targeting signals (constructs 2 and 7). (C, D) Fluorescent images of the leaf epidermis of <i>A</i>. <i>thaliana</i> transformed with construct 2 (C) or 7 (D). (E, F) Structural diagram of the binary constructs with plastid-targeting signals (constructs 3 and 8). (G, H) Fluorescent images of the leaf epidermis of <i>A</i>. <i>thaliana</i> transformed with construct 3 (G) or 8 (H). Different developmental stages in stomatal lineages are indicated by arrows (meristemoids), stars (GMCs), asterisks (immature GCs), or arrowheads (mature GCs). In meristemoids, only the expression of the fluorescent protein directed by P_MUTE was observed, while both signals were detected in later stages. GFP, signal of G3GFP; RFP, signal of TagRFP; Overlay, overlay of GFP and RFP; DIC Merged, differential interference contrast (DIC) merged with GFP and RFP; Boxed Area, enlargement of the boxed area in the overlay. Scale bars = 10 μm.</p

Illustration of ten R4pGWB64xx-MD8 constructs carrying Pro1:ORF1-tag1-Pro2:ORF2-tag2 and confirmation of structures by restriction digestion.

<p>(A) Structure of P_MUTE:ORF1-G3GFP-P_SDD1:ORF2-TagRFP constructed with R4pGWB6450-MD8 and R4pDD659-MD8 (binary clones 1–5). (B) Structure of P_MUTE:ORF1-TagRFP-P_SDD1:ORF2-G3GFP constructed with R4pGWB6459-MD8 and R4pDD650-MD8 (binary clones 6–9). (C) Structure of P_SDD1:Mt-TagRFP-P_MUTE:Mt-G3GFP constructed with R4pGWB6459-MD8 and R4pDD650-MD8 (binary clone 10). The positions of <i>Hin</i>dIII sites are indicated and the sizes of restriction fragments are shown in kilobase pairs (kbp). (D) Binary clones 1–10 were digested by <i>Hin</i>dIII and electrophoresed on 1.5% agarose gel. Lanes 1–10 show binary clones 1–10. Lane M shows the DNA ladder marker. The positions of 10, 5, 3, 2, 1, 0.5, and 0.1 kbp are indicated. P_MUTE, <i>MUTE</i> promoter; P_SDD1, <i>SDD1</i> promoter; Mt, mitochondria-targeting signal; PTS2, peroxisome-targeting signal type 2; Pt, plastid-targeting signal.</p

Outline for cloning two sets of promoters and ORFs in the R4DS Gateway cloning system.

<p>(A) The 1st LR reaction. In the 1st LR reaction (tripartite LR reaction), Pro2 and ORF2 are linked and cloned into an R4DD vector to make R4pDD6xx-MD8-Pro2:ORF2. The dashed arrow indicates that established R4pDD6xx-MD8-Pro2:ORF2 is used further for the 2nd LR reaction. (B) The 2nd LR reaction. In the 2nd LR reaction (quadripartite LR reaction), Pro1 and ORF1 are linked and cloned into an R4DSB vector together with the Pro2:ORF2-tag2 constructed by the 1st LR reaction to make the binary clone R4pGWB6xxx-MD8-Pro1:ORF1-Pro2:ORF2 with two independent expression cassettes. MD8, the matrix attachment region; B1, <i>att</i>B1; B2, <i>att</i>B2; B4, <i>att</i>B4; B5, <i>att</i>B5; B6, <i>att</i>B6; L1, <i>att</i>L1; L2, <i>att</i>L2; L4, <i>att</i>L4; L5, <i>att</i>L5; L6, <i>att</i>L6; R1, <i>att</i>R1; R2, <i>att</i>R2; R4, <i>att</i>R4; R5, <i>att</i>R5; R6, <i>att</i>R6; <i>ccd</i>B, Control of Cell Death B as a negative selection marker for bacteria; Cm^r, chloramphenicol resistance; Marker, plant selection marker; Pro1, promoter1; Pro2, promoter2; LB, left border; RB, right border; Tnos, nopaline synthase terminator; <i>aadA</i>, the gene for spectinomycin resistance (Spc^r) in bacteria; <i>bla</i>, gene for ampicillin resistance (Amp^r) in bacteria. Arrows under ORF1-B2-tag1 and ORF2-B2-tag2 indicate expression. Figures are not drawn to scale.</p

Line-up of R4DSB vectors (R4pGWB6xxx-MD8) and R4DD vectors (R4pDD6xx-MD8).

<p>(A) Structural diagrams of the four no tag-type R4DSB vectors: R4pGWB6401-MD8, R4pGWB6501-MD8, R4pGWB6601-MD8, and R4pGWB6701-MD8. Backbone and restriction sites were shown in R4pGWB6401-MD8. The only difference between these four vectors is the selection marker for plants. LB, left border; RB, right border; <i>sta</i>, the region conferring stability in <i>Agrobacterium tumefaciens</i>; <i>rep</i>, broad host range replication origin; <i>bom</i>, cis-acting element for conjugational transfer; <i>ori</i>, ColE1 replication origin; Pnos, nopaline synthase promoter; Tnos, nopaline synthase terminator. (B) Structural diagram of the no tag-type R4DD vector, R4pDD601-MD8. (C) Structural diagram of tag fusion-type R4DSB vectors (R4pGWB6xxx-MD8). These vectors have the same structure as the no tag-type vector R4pGWB6x01 represented in A, except for a tag downstream of <i>att</i>R2. (D) Structural diagram of tag fusion-type R4DD vectors (R4pDD6xx-MD8). These vectors have the same structure as no tag-type R4pDD601-MD8 represented in B, except for a tag downstream of <i>att</i>R2. (E) Tags carried in R4pGWB6xxx-MD8 and R4pDD6xx-MD8. Figures in A-D are not drawn to scale.</p

Expression and intracellular localization of G3GFP and TagRFP fused with different combinations of mitochondria-, peroxisome-, and plastid-targeting signals in transformed <i>A</i>. <i>thaliana</i> with different promoters.

<p>(A-C) Structural diagram of the binary constructs 4, 5, and 9 with different targeting signals. (D-F) Fluorescent images of the leaf epidermis of <i>A</i>. <i>thaliana</i> transformed with construct 4 (D), 5 (E), or 9 (F). Different developmental stages in stomatal lineages are indicated by arrows (meristemoids), stars (GMCs), asterisks (immature GCs), or arrowheads (mature GCs). In meristemoids, only the expression of the fluorescent protein directed by P_MUTE was observed, while both signals were detected in later stages. GFP, signal of G3GFP; RFP, signal of TagRFP; Overlay, overlay of GFP and RFP; DIC Merged, differential interference contrast (DIC) merged with GFP and RFP; Boxed Area, enlargement of the boxed area in the overlay. Scale bars = 10 μm.</p