A modular plasmid assembly kit for multigene expression, gene silencing and silencing rescue in plants

Abstract

The Golden Gate (GG) modular assembly approach offers a standardized, inexpensive and reliable way to ligate multiple DNA fragments in a pre-defined order in a single-tube reaction. We developed a GG based toolkit for the flexible construction of binary plasmids for transgene expression in plants. Starting from a common set of modules, such as promoters, protein tags and transcribed regions of interest, synthetic genes are assembled, which can be further combined to multigene constructs. As an example, we created T-DNA constructs encoding multiple fluorescent proteins targeted to distinct cellular compartments (nucleus, cytosol, plastids) and demonstrated simultaneous expression of all genes in Nicotiana benthamiana, Lotus japonicus and Arabidopsis thaliana. We assembled an RNA interference (RNAi) module for the construction of intron-spliced hairpin RNA constructs and demonstrated silencing of GFP in N. benthamiana. By combination of the silencing construct together with a codon adapted rescue construct into one vector, our system facilitates genetic complementation and thus confirmation of the causative gene responsible for a given RNAi phenotype. As proof of principle, we silenced a destabilized GFP gene (dGFP) and restored GFP fluorescence by expression of a recoded version of dGFP, which was not targeted by the silencing construct.

Figure 1. Overview of multi-level construct assembly.

The basis for the toolkit is a library of Level I functional modules, consisting of promoters (Prom), amino-terminal tags (N tag), genes of interest (GOI), carboxy-terminal tags (C tag), terminators (Term) as well as miscellaneous modules (Misc). Removal of type IIS sites can be facilitated by creation of L0 fragments for mutagenesis (see Figure S3). Level I modules are assembled into a Level II vector backbone by BsaI cut-ligation to construct synthetic genes. Up to 5 LII synthetic genes are combined into a LIII vector backbone by BpiI cut-ligation to create a LIII binary plasmid. Higher level constructs can be created by sequentially using LII and LIII vector backbones. For instance, LIII multigene assemblies are combined together in a LII backbone to obtain LIV plasmids, while LIV multigene assemblies are again ligated into LIII backbones to create LV constructs. BsaI and BpiI cut-ligations are used in succession to assemble the inserts of one level with the backbone of the next one. Backbone vectors for each level carry a ccdB negative selection marker, as well as a different antibiotic resistance, allowing for easy selection of correctly assembled constructs. Level 0 vectors use ampicillin resistance (Amp^R), Level I vectors gentamicin (Gen^R), LII cloning-only vectors (Amp^R), LII binary vectors spectinomycin (Sp^R) and Level III binary vectors kanamycin (Kan^R).

Figure 2. Assembly of Level II, III and IV plasmids.

A) LI modules and LI dummies (LI dy) are fused with compatible BsaI overhangs into a LII backbone vector by BsaI cut-ligation. B) To create a LIII binary vector for plant expression up to five LII synthetic genes (LII 1–2, LII 2–3, LII 3–4, LII 4–5 and LII 5–6) or suitable LII dummies (LII dy) are combined with compatible BpiI overhangs into a LIII backbone by BpiI cut-ligation. C) A LIV plasmid is constructed from LIII multigene assemblies combined with LI dy fragments, which carry compatible BsaI overhangs, into a LII backbone by BsaI cut-ligation. LI fusion sites are named A to G, while LII fusion sites are numbered from 1 to 6. Triangles indicate the orientation of the restriction sites relative to the BsaI and BpiI recognition sites.

Figure 3. Construction of custom binary vector backbones for promoter and gene of interest analysis.

Pre-assembled binary constructs are created with Esp3I-lacZ dummies. The desired LI modules are assembled together with a LI dy-Esp3I-lacZ A-B fragment instead of a specific LI A-B promoter module or a LI dy-Esp3I-lacZ C-D fragment instead of a specific LI C-D GOI module into a LII backbone by BsaI cut-ligation. The resulting LII assembly is combined with inserts from other LII constructs into the LIII final (LIII fin) backbone by BpiI cut-ligation. Into the resulting LIII binary vectors LI A-B or LI C-D modules can be directly inserted by a combined BsaI+Esp3I cut-ligation. Alternatively the LIII binary vectors can be further refined in a second step, by addition of a ccdB dummy fragment (LI dy-Esp3I-ccdB C-D) via Esp3I cut-ligation. The final version of the pre-assembled LIII binary vector backbone can be combined with custom LI A-B or LI C-D modules in one BsaI cut-ligation using ccdB based negative selection.

Figure 4. Construction of plasmids for RNAi mediated gene silencing.

A) A target sequence containing LI C-D fusion sites and a LI intron element are combined into a LII 1–2 RNAi vector backbone by BsaI cut-ligation. The chosen BsaI overhangs enable a one-step assembly of a hairpin construct containing the intron element between two copies of the LI C-D target sequence. When expressed, this creates an intron-spliced hairpin RNA, which is effective in gene silencing.

Figure 5. In planta expression of LIII constructs.

A) Binary plasmids LIII Tri-Color and LIII Tri-Color (neo) were constructed from LII F 1–2 NES-mCherry or LII F1–2 NES-mCherry (neo), LII R 3–4 NLS-CFP, LII F 5–6 Plastid-YFP assemblies and LII insulator fragments LII ins 2–3 and LII ins 4–5 by BpiI cut-ligation into a LIII vector backbone. All LI modules contained in the final LIII construct are depicted under the plasmid map. B) Confocal laser scanning microscopic (CLSM) images of transformed plants. LIII Tri-Color was expressed in N. benthamiana leaves and L. japonicus roots by Agrobacterium mediated transformation. LIII Tri-Color (neo) was used to generate stable transgenic A. thaliana lines. p35S = cauliflower mosaic virus 35S promoter; pUbi = L. japonicus polyubiquitin promoter; NES = nuclear export signal; NLS = nuclear localization signal; 35S-T = cauliflower mosaic virus 35S terminator; HSP-T = heat shock protein terminator of A. thaliana; nos-T = nopaline synthase terminator; neo = neomycin resistance cassette; Plas-M. = Plastid Marker (plastid localized protein of L. japonicus). Scale bars = 25 µm.

Figure 6. GFP silencing and complementation.

A) Schematic representation of plasmids LIII dGFP, LIII dGFP+RNAi and LIII dGFP+RNAi+re-dGFP. B) Expression of destabilized GFP (dGFP) was assayed in N. benthamiana two days after A. tumefaciens mediated transformation. The larger stereomicroscopic pictures depict 5×5 mm² leaf sections, while the inlay pictures show confocal scans of single epidermal cells (scale bar = 75 nm). For image acquisition the same settings were used for all samples. Co-expression of dGFP together with the RNAi construct targeting dGFP mRNA completely silences dGFP expression. A recoded version of dGFP (re-dGFP) is immune to silencing and able to restore strong dGFP fluorescence despite expression of the RNAi construct. Addition of P19 is able to suppress dGFP silencing, but only partly: expression of dGFP together with the RNAi construct is weaker and spottier than expression of dGFP alone.