A Highly Efficient Agrobacterium-Mediated Method for Transient Gene Expression and Functional Studies in Multiple Plant Species

Abstract

Although the use of stable transformation technology has led to great insight into gene function, its application in high-throughput studies remains arduous. Agro-infiltration have been widely used in species such as Nicotiana benthamiana for the rapid detection of gene expression and protein interaction analysis, but this technique does not work efficiently in other plant species, including Arabidopsis thaliana. As an efficient high-throughput transient expression system is currently lacking in the model plant species A. thaliana, we developed a method that is characterized by high efficiency, reproducibility, and suitability for transient expression of a variety of functional proteins in A. thaliana and 7 other plant species, including Brassica oleracea, Capsella rubella, Thellungiella salsuginea, Thellungiella halophila, Solanum tuberosum, Capsicum annuum, and N. benthamiana. Efficiency of this method was independently verified in three independent research facilities, pointing to the robustness of this technique. Furthermore, in addition to demonstrating the utility of this technique in a range of species, we also present a case study employing this method to assess protein-protein interactions in the sucrose biosynthesis pathway in Arabidopsis.

Keywords: agro-infiltration; protein-protein interaction; subcellular localization; transient expression.

Figure 1

Schematic Representation of the Timeline Required for the Generation of Transiently Transformed Plants Using Agro-Infiltration. Briefly, Agrobacterium was streaked onto YEB agar plates containing acetosyringone and antibiotics. After washing, the transformed agrobacteria in infiltration buffer were injected into a plant leaf. Transiently transformed plants for all of the species mentioned could be obtained within 2–3 days after a single hour of infiltration.

Figure 2

The High-Efficiency Agro-Infiltration of Arabidopsis Leaves. (A)Agrobacterium was streaked on YEB-induced medium, diluted with infiltration medium, and injected into leaves of Arabidopsis. (B) Mitochondria-targeted citrate synthase with C-terminal GFP (CYS4-GFP) was infiltrated into Arabidopsis leaves using the standard protocol described in this paper. (C) Agro-infiltration protocol with transformed plants kept in darkness for 2 days. (D) Agro-infiltration protocol without the addition the 0.005% Silwet L-77. (E) Transformation efficiency of wild-type Arabidopsis plants. Dark method, CYS4-GFP was infiltrated into Arabidopsis leaves and plants were kept in the dark for 2 days. Light method, CYS4-GFP was infiltrated into Arabidopsis leaves without 0.005% Silwet L-77 and plants were kept in the light. New method, CYS4-GFP was infiltrated into Arabidopsis leaves using the standard protocol described in this paper. Old method, CYS4-GFP was infiltrated into Arabidopsis leaves using a previously published method (Mangano et al., 2014). The transformation efficiency was calculated from eight different replicates. (F) Both agrobacteria strains, GV3101 and AGL1, were transformed into Arabidopsis leaves. Agro-infiltration of AGL1 into Arabidopsis leaves was achieved with high efficiency agro-infiltration of leaves.

Figure 3

The High-Efficiency Agro-Infiltration of the Leaves of Different Plant Species. The standard Arabidopsis transient transformation protocol described above was used without modifications for the transformation of Brassica oleracea(A), Capsella rubella(B), Thellungiella salsuginea(C), Thellungiella halophila(D), Solanum tuberosum(E), Capsicum annuum(F), and Nicotiana benthamiana(G).

Figure 4

Efficiency and Case Study of the Transient Transformation Method for Assessing Protein–Protein Interactions. (A) The transformation efficiency achieved using AGL1 agrobacteria for the transient transformation of the species described. Data presented are mean ± SD (n = 8). (B) Transient expression of the Arabidopsis sucrose phosphate synthase 1 (AtSPS) gene in Arabidopsis leaves. (C) Transient expression of free GFP in Arabidopsis leaves as a control. (D) Using the transiently transformed Arabidopsis leaves to analyze protein–protein interactions by AP-MS.

Figure 5

Subcellular Co-localization of Four AtSPS1 Interactors. (A) AtSPS2F-mCitrine/AtSPS1-mCherry. (B) AtCYT1-mCitrine/AtSPS1-mCherry. (C) AtRHM1-mCitrine/AtSPS1-mCherry. (D) 14-3-3OMEGA-mCitrine/AtSPS1-mCherry. The panels from left to right show the mCitrine fluorescence, mCherry fluorescence, autofluorescence, blank, and the merged image, respectively. AtSPS2F (AT5G11110) is an isoform of sucrose phosphate synthase. AtCYT1 (AT2G39770) is GDP-mannose pyrophosphorylase/mannose-1-pyrophosphatase. AtRHM1 (AT1G78570) is a UDP-L-rhamnose synthase. 14-3-3OMEGA (AT1G78300) is a 14-3-3 protein.

Figure 6

Protein–Protein Interactions Between Four Candidate Gene Proteins and AtSPS1 Assayed by Bimolecular Fluorescent Complementation (BiFC). (A) AtSPS2F-NE/AtSPS1-CE. (B) AtCYT1-NE/AtSPS1-CE. (C) AtRHM1-NE/AtSPS1-CE. (D) 14-3-3OMEGA-NE/AtSPS1-CE. NE is the N-terminus of the split mCitrine, CE is the C-terminus of the split mCitrine. The panels from the left to right show the BiFC fluorescence, auto-fluorescence, blank, and the merged image, respectively.