使用根癌农杆菌在<em>NahG</em>拟南芥植株中进行瞬时表达分析

Pepe Cana-Quijada; Eduardo R. Bejarano; Rosa Lozano-Durán; Tábata Rosas-Díaz

doi:10.21769/BioProtoc.2894

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Peer-reviewed

Transient Expression Assay in NahG Arabidopsis Plants Using Agrobacterium tumefaciens

使用根癌农杆菌在NahG拟南芥植株中进行瞬时表达分析

PC Pepe Cana-Quijada

Eduardo R. Bejarano

RL Rosa Lozano-Durán

Tábata Rosas-Díaz email

发布: 2018年06月20日第8卷第12期 DOI: 10.21769/BioProtoc.2894 浏览次数: 9972

评审: Zhibing LaiAraceli Castillo GarrigaShunping Yan

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Abstract

Agrobacterium-mediated transient expression has greatly contributed to research in molecular plant biology but has low efficiency and inconsistency in Arabidopsis thaliana (Arabidopsis). Here, we describe a simple, efficient and fast protocol to make transient gene expression in NahG Arabidopsis plants using Agrobacterium tumefaciens. This protocol has been successfully used to assess protein sub-cellular localization and accumulation, enzyme activity, and protein-protein interaction. In addition, this assay overcomes the use of Nicotiana benthamiana plants as a surrogate system for transient gene expression assays. Finally, the use of this protocol does not require complex inoculation methods or specific growth conditions, and can be used with different Agrobacterium strains with similar results.

Keywords: NahG Arabidopsis (NahG拟南芥)

Agrobacterium (农杆菌)

Transient expression (瞬时表达)

Transient transformation (瞬时转化)

Protein accumulation (蛋白质积累)

Background

Agrobacterium tumefaciens (hereafter referred to as Agrobacterium)-mediated transient transformation assays have greatly contributed to research in molecular plant biology. These methods have many advantages over the laborious and time-consuming stable transformation approaches including, among others, a higher efficiency, simplicity, and fast, consistent results when the transient transformation assays are carried out in Nicotiana benthamiana. On the other hand, these assays are inefficient and lack robustness when carried out in the model plant Arabidopsis thaliana (hereafter referred to as Arabidopsis), forcing Arabidopsis researchers to use N. benthamiana as a heterologous system, which entails obvious limitations and might generate misleading results.

Many efforts have been made in the past to increase the efficacy of Agrobacterium-mediated transient transformation in Arabidopsis (reviewed in Krenek et al., 2015). Recently, we described a protocol to perform transient gene expression using NahG Arabidopsis plants, overcoming previous limitations (Rosas-Díaz et al., 2017). Using Arabidopsis NahG plants, which contain low levels of salicylic acid (SA) due to the expression of an SA hydroxylase from the bacterium Pseudomonas putida (Lawton et al., 1995), we have been able to obtain high accumulation of marker proteins such as GUS and GFP, and carry out sub-cellular localization and protein-protein interaction experiments. Remarkably, this protocol for transient expression can be used with, at least, three widely used Agrobacterium strains, LBA4404, GV3101 and C58C1. In summary, this protocol shows that expression of the NahG transgene greatly enhances the efficiency of Agrobacterium-mediated transformation in rosette leaves in Arabidopsis, enabling the routine use of this technique in the model plant.

Materials and Reagents

Materials
1. Soil mix or substrate such as Compo Sana^® Universal Ligera (COMPO, TSUSTPROF25L)
2. Plant pots such as Desch vol 11 (Desch Plantpak, catalog number: 1055278 )
3. Seed tray–40 cavities
4. Plant trays
5. Cling film
6. Syringes 1 ml or 2 ml
7. Tissue paper
8. Petri dishes
9. Falcon tubes
10. Eppendorf tubes
11. Pipette tips
12. Sterile toothpicks
13. Syringe filter 0.22 µm
Biological materials
1. Arabidopsis thaliana NahG seeds (Lawton et al., 1995)
2. Agrobacterium tumefaciens strain (LBA4404, GV3101 or C58C1) carrying a binary vector with the gene of interest
Reagents
1. Sterile deionized water
2. Glycerol (CARLO ERBA Reagents, catalog number: 453752 )
3. NaCl (AppliChem, catalog number: 121659.1210 )
4. Tryptone (Biolife, catalog number: 412290 )
5. Yeast Extract (AppliChem, catalog number: 403687.1210 )
6. Bacteriological agar (MICROKIT, catalog number: BCB006+ )
7. MES (2-(N-morpholino) ethanesulfonic acid) (Sigma-Aldrich, catalog number: M2933 )
8. MgCl₂ (AppliChem, catalog number: 131396.1210 )
9. DMSO (Sigma-Aldrich, catalog number: M81802 )
10. Acetosyringone (Sigma-Aldrich, catalog number: D134406 )
11. Rifampicin (Duchefa Biochemie, catalog number: R0146.0005 )
12. Tetracycline (Sigma-Aldrich, catalog number: T3383 )
13. Gentamycin (Sigma-Aldrich, catalog number: G3632 )
14. Spectinomycin (Duchefa Biochemie, catalog number: S0188.0005 )
15. Kanamycin (Sigma-Aldrich, catalog number: K4378 )
16. LB medium (see Recipes)
17. 1 M MES (see Recipes)
18. 1 M MgCl₂ (see Recipes)
19. 0.1 M Acetosyringone (see Recipes)
20. Infiltration solution (see Recipes)
21. Antibiotics solution (depending on construct and Agrobacterium strain, see Recipes)

Equipment

Sterile Erlenmeyer flasks
Plant growth chamber capable of sustaining 21 °C under short-day conditions (8 h light/16 h dark) with 140-150 µmol m^-2 sec^-1 light intensity (Radiber SA, catalog number: AGP-1400 )
Spectrophotometer capable of OD₆₀₀ measurements such as Shimadzu UV-1601 (Shimadzu, catalog number: 206-67001-34 )
Automatic P1000, P200 and P20 micropipettes
Incubator set at 28 °C such as Incubator D-6450 Hanau (Heraeus Instruments, model: D-6450 )
Incubator shaker capable of sustaining 28 °C and 180 rpm such as New Brunswick^TM I26 (Eppendorf, New Brunswick scientific, model: I26, catalog number: M1324-0000 )
Centrifuge for 50 ml tubes such as Rotofix 32A (Hettich, catalog number: 1206-01 )
Autoclave

Procedure

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Cana-Quijada, P., Bejarano, E. R., Lozano-Durán, R. and Rosas-Díaz, T. (2018). Transient Expression Assay in NahG Arabidopsis Plants Using Agrobacterium tumefaciens. Bio-protocol 8(12): e2894. DOI: 10.21769/BioProtoc.2894.