AGROBEST: an efficient Agrobacterium-mediated transient expression method for versatile gene function analyses in Arabidopsis seedlings

Abstract

Background: Transient gene expression via Agrobacterium-mediated DNA transfer offers a simple and fast method to analyze transgene functions. Although Arabidopsis is the most-studied model plant with powerful genetic and genomic resources, achieving highly efficient and consistent transient expression for gene function analysis in Arabidopsis remains challenging.

Results: We developed a highly efficient and robust Agrobacterium-mediated transient expression system, named AGROBEST (Agrobacterium-mediated enhanced seedling transformation), which achieves versatile analysis of diverse gene functions in intact Arabidopsis seedlings. Using β-glucuronidase (GUS) as a reporter for Agrobacterium-mediated transformation assay, we show that the use of a specific disarmed Agrobacterium strain with vir gene pre-induction resulted in homogenous GUS staining in cotyledons of young Arabidopsis seedlings. Optimization with AB salts in plant culture medium buffered with acidic pH 5.5 during Agrobacterium infection greatly enhanced the transient expression levels, which were significantly higher than with two existing methods. Importantly, the optimized method conferred 100% infected seedlings with highly increased transient expression in shoots and also transformation events in roots of ~70% infected seedlings in both the immune receptor mutant efr-1 and wild-type Col-0 seedlings. Finally, we demonstrated the versatile applicability of the method for examining transcription factor action and circadian reporter-gene regulation as well as protein subcellular localization and protein-protein interactions in physiological contexts.

Conclusions: AGROBEST is a simple, fast, reliable, and robust transient expression system enabling high transient expression and transformation efficiency in Arabidopsis seedlings. Demonstration of the proof-of-concept experiments elevates the transient expression technology to the level of functional studies in Arabidopsis seedlings in addition to previous applications in fluorescent protein localization and protein-protein interaction studies. In addition, AGROBEST offers a new way to dissect the molecular mechanisms involved in Agrobacterium-mediated DNA transfer.

Keywords: Agrobacterium; Arabidopsis; Gain-of-function; Gene expression; Innate immunity; Transient transformation.

Figure 1

Transient transformation assays in different Arabidopsis ecotype/genotypes. Four-day-old Arabidopsis seedlings were infected with Agrobacterium strain C58C1(pTiB6S3ΔT)^H carrying pBISN1, which was pre-incubated in AB-MES (pH5.5) supplemented with 200 μM acetosyringone (AS) to induce vir gene expression. Seedlings were co-cultivated with pre-induced A. tumefaciens cells with final OD₆₀₀ = 0.02 in the MS medium (1/2 MS, 0.5% sucrose (w/v), pH 5.5) containing 50 μM AS and determined for transient GUS expression levels by overnight GUS staining (A) and quantitative GUS activity (B) at 3 dpi. The GUS activity obtained from Col-0 seedlings was set to 100% and that of Ws, efr-1, and fls2 is relative to that of Col-0. Data are mean ± SD GUS activity from two biological replicates. Similar results were obtained from at least two independent experiments. Values significantly different from that obtained with Col-0 are denoted (*P = 0.058 by Student’s t test).

Figure 2

Optimization of Agrobacterium pre-culture and infection media for efficient transient expression efficiency. Four-day-old Arabidopsis efr-1 seedlings infected with Agrobacterium C58C1(pTiB6S3ΔT)^H carrying pBISN1 were grown in various pre-culture and co-cultivation media to test their effects on transient GUS expression efficiency measured by GUS staining and quantitative GUS activity. (A) Various pre-culture and infection media in the absence or presence of vir gene inducer AS at the indicated concentration. (B) Effect of factors in AB-MES medium on increased transient expression efficiency. (C) Effect of AB salts, pH and buffering systems on transient GUS expression efficiency. Data for relative quantitative GUS activity are mean ± SD of 3 independent experiments. Values significantly different from that infected by ABM50 (A) or condition 1 (B and C) are denoted (*P < 0.05, **P < 0.01, ***P < 0.005 by Student’s t test).

Figure 3

Transient transformation of the Arabidopsis seedlings by various Agrobacterium strains. Four-day-old Arabidopsis Col-0 and efr-1 seedlings infected with different Agrobacterium stains carrying pBISN1 by ABM50 or ABM-MS (named as AGROBEST) were compared by GUS staining (A), quantitative GUS activity (B), and root length (C) at 3 days post-inoculation (dpi). Data for quantitative GUS activity are mean ± SD of at least 4 biological replicates from 2 independent experiments. Values significantly different from that infected with wild-type C58 are denoted (*P < 0.05, **P < 0.01 by Student’s t test). Data for root length measurement are mean ± SEM of 4-6 biological replicates from 2–4 independent experiments. Statistics was analyzed by ANOVA and means annotated with the same letter (a-c) are not significantly different; those with different letters are significantly different (P < 0.05). Seedlings grown in the same co-cultivation medium without Agrobacterium infection are indicated (MOCK).

Figure 4

AGROBEST enables high transient expression levels in Col-0. Four-day-old Arabidopsis seedlings were infected with Agrobacterium strain C58C1(pTiB6S3ΔT)^H carrying pBISN1 (A and C) or 35S::LUC2(B), and transient expression activity was determined at 3 dpi. (A) Transient GUS expression efficiency of Col-0 and efr-1 seedlings by AGROBEST, FAST and Marion et al. methods. Data for quantitative GUS activity are mean ± SD of 3 biological replicates. Values significantly different from those obtained with Col-0 by AGROBEST are denoted (*P < 0.05 by Student’s t test). (B) Transient luciferase expression efficiency of Col-0 and efr-1 seedlings by AGROBEST, FAST and Marion et al. methods. Seedlings infected by C58ΔvirB2 carrying 35S::LUC2 were used as a background control and those without Agrobacterium infection are indicated as MOCK. Luciferase activity of Col-0 obtained by AGROBEST was set to 100% and that of others is relative to activity of Col-0 by AGROBEST. Data are mean ± SD of 3 biological replicates. Values significantly different from those obtained with Col-0 by AGROBEST are denoted (**P < 0.01, by Student’s t test). (C) Transient GUS expression efficiency of Col-0, AvrPto transgenic line, and efr-1 by AGROBEST. For dexamethasone (DEX) treatment, 3-d-old seedlings were treated with 10 μM DEX for 1 day and the following 3 days infected by the AGROBEST method. Quantitative GUS activity from DEX-induced Col-0 seedlings by AGROBEST was set to 100% and that of others is relative to activity of DEX-induced Col-0 seedlings with AGROBEST. Data are mean ± SD GUS activity from 4 repeats (2 biological repeats from each of 2 independent experiments). Values significantly different from that obtained with Col-0 are denoted (**P < 0.01 by Student’s t test). Seedlings grown in the same co-cultivation medium without Agrobacterium infection are indicated (MOCK).

Figure 5

Impact of seedling age and infection time on transient expression. (A and B) Different ages of Arabidopsis efr-1 seedlings were infected with C58C1(pTiB6S3ΔT)^H carrying pBISN1 by the AGROBEST method and analyzed for GUS activity (A) and morphologic features (B) at different dpi.

Figure 6

Impact of Timentin treatment on transient GUS expression efficiency. Four-day-old Arabidopsis efr-1 seedlings were infected with Agrobacterium C58C1(pTiB6S3ΔT)^H carrying pBISN1 by the AGROBEST method at 1 or 2 dpi before Timentin treatment. GUS staining was performed at 0 to 3 days after Timentin treatment.

Figure 7

Transient transformation events in different organs and cell types. (A-D) Four-day-old (A and C-D) or 7-d-old (B)Arabidopsis efr-1 seedlings were infected with C58C1(pTiB6S3ΔT)^H carrying pBISN1 by the AGROBEST method and analyzed for GUS staining. GUS staining was detected in true leaves (B, indicated by asterisk), cotyledons (A and B), main roots near lateral initiation site (C), and elongation zone (D). (E-L) Confocal microscopy of 4-day-old Arabidopsis efr-1 seedlings infected with C58C1(pTiB6S3ΔT)^H carrying various vectors for transient expression of indicated fluorescent proteins by the ABM200 method. Fluorescence signals for 35S::Venus-intron or 35S::NLS-RFP were detected in cotyledons (E and F). Venus-intron signals were detected in different types of cells, including epidermal cells (G), guard cells (H), mesophyll cells (I) of cotyledon, and root epidermal cells (J). (K) Subcellular localization of Venus-intron and NLS-RFP by co-infection of 2 Agrobacterium strains expressing 35S::Venus-intron or 35S::NLS-RFP. (L) Protein–protein interaction by BiFC of nYFP-ASK1 and TIR1-cYFP. Images show fluorescence alone (K) and/or merged with bright field (E, F, J and L) or chloroplast fluorescence (G-I). Scale bars are 2 mm (A and B), 0.5 mm (C and D), 100 μm (E, F and J), 50 μm (L) and 20 μm (G-I and K). BiFC, bimolecular fluorescence complementation.

Figure 8

Monitoring Arabidopsis circadian rhythm by transient expression of GIGANTEA::luciferase (GI::LUC2). Four-day-old Arabidopsis efr-1 seedlings were infected with Agrobacterium C58C1(pTiB6S3ΔT)^H carrying a vector (pCAMBIA1390) or p1390-GI-LUC2 by the AGROBEST method for 3 days in a 16-h/8-h light/dark cycle (75 μmol m^-2 s^-1), then transferred to 1/2 MS liquid medium in the presence of 100 μM Timentin and 0.5 mM luciferin and grown under continuous light at 40 μmol m^-2 s^-1 for up to 5 days. The GI::LUC2 transgenic Arabidopsis plant (TP) cultured in identical conditions without Agrobacterium infection was a positive control. Real-time bioluminescence signals were photographed and the luciferase intensity is shown as mean ± SEM from 12 seedlings expressing GI::LUC2. Similar results were obtained from at least 3 independent experiments. The white and gray regions indicate subjective light and dark periods, respectively.

Figure 9

Transient expression of MYB75 increases anthocyanin accumulation. Four-day-old Arabidopsis efr-1 seedlings were infected with Agrobacterium C58C1(pTiB6S3ΔT)^H carrying a vector (pCAMBIA1390), 35S::MYB75, 2X35S::MYB75, super::MYB75, or super::gusA-intron by the AGROBEST method. At 3 dpi, co-cultivation medium was replaced with MS medium containing 100 μM Timemtin for additional incubation for 3 days. qRT-PCR of relative expression of MYB75(A) and CHS(B) with representative data shown with mean ± SD from 3 technical repeats. Similar results were obtained from three independent experiments. Zeiss inverted microscopy of anthocyanin accumulation in seedlings (upper panels) and cotyledons (lower panels) and quantification (C). Data for anthocyanin content are mean ± SD from 4 repeats (2 biological repeats from each of 2 independent experiments, 20–30 seedlings for each biological repeat), Values significantly different from that obtained with vector are denoted (**P < 0.01, by Student’s t test).

Figure 10

Transient expression of MYB75 increases anthocyanin accumulation in Col-0 seedlings. Four-day-old Arabidopsis Col-0 seedlings were infected with Agrobacterium C58C1(pTiB6S3ΔT)^H carrying a vector (pCAMBIA1390), 2X35S::MYB75, or super::gusA-intron by the AGROBEST method. At 3 dpi, co-cultivation medium was replaced with MS medium containing 100 μM timemtin for additional incubation for 3 days. qRT-PCR of relative expression of MYB75(A) and CHS(B) with representative data shown with mean ± SD from 3 technical repeats. Similar results were obtained from three independent experiments. Zeiss inverted microscopy of anthocyanin accumulation in seedlings (upper panels) and cotyledons (lower panels) and quantification (C). Data for anthocyanin content are mean ± SD from 3 independent experiments (20–30 seedlings for each biological repeat, 3 biological repeats for each independent experiment). Values significantly different from those obtained with vector are denoted (**P < 0.01, by Student’s t test).