MYB3R4 and IMPA3 co-expression in tobacco

Co-Expression Assay in Tobacco (Nicotiana benthamiana) Leaves

1. Transform the binary vectors into the Agrobacterium tumefaciens strain GV3101. The P19 silencing suppressor (Vargason et al., 2003) in a binary vector is also separately transformed into GV3101;
2. Streak out GV3101 harboring each of the binary vectors as well as the GV3101 strain containing the assistant plasmid P19, and grow them on LB medium containing appropriate antibiotics depending on the binary vectors used; The concentrations of the antibiotics are: Gentamycin (100 μg/ml), Rifampicin (25 μg/ml), Spectinomycin (50 μg/ml), Kanamycin (50 μg/ml).
3. Take a single colony of GV3101 harboring each binary vector or the assistant plasmid P19, culture them separately in 5 ml LB medium supplemented with antibiotics;
4. Grow the agrobacteria at 28℃ for ~24 h with 220 rpm shaking;
5. Take 1 ml of culture and dilute in 100 ml fresh LB supplemented with the same antibiotics as well as Acetosyringone (200 μM);
6. Incubate at 28℃ for 8-12 h with 220 rpm shaking;
7. Centrifuge at 3,100 g (Beckman Avanti J-26 XPI, Rotor JA-14 at 4,500 rpm) for 10 min to collect the agrobacterium cells;
8. Remove the supernatant, then add 10 ml infiltration buffer and pipette up and down to resuspend the cells.
9. Mix the agrobacterium suspension containing each of the expression vectors with a suspension of the strain containing P19 by diluting each strain to appropriate concentrations in the infiltration buffer. The final concentration of agrobacteria containing the expression vector should be OD₆₀₀ = 1.0, and the final concentration in the mixture of P19-containing agrobacteria should be OD₆₀₀ = 0.7.
10. Keep the agrobacterium suspensions at 28℃ for 3 hours without shaking;
11. Identify smooth and healthy leaves on 15- to 20-day old tobacco plants. Press your index finger on the top surface of the selected leaves, and infiltrate the leaves from the lower epidermis using 1 ml syringe;
12. Grow the infiltrated tobacco plants for 2-3 days at 22℃ with 16 h light and 8 h dark.
13. Observe the fluorescence signals under a confocal microscope. Laser excitations were 488 nm for GFP and 543 nm for mCherry;
14. Measure the fluorescence intensity in Fiji (image J).

Infiltration buffer: 10 mM MES, pH 5.6; 10 mM MgCl₂; 200 μM Acetosyringone

Reference:

Vargason, J. M., Szittya, G., Burgyán, J., and Hall, T. M. T. (2003). Size selective recognition of siRNA by an RNA silencing suppressor. Cell 115, 799–811.

How to cite: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

1. Yang, W. (2021). Co-expression assay in tobacco (Nicotiana benthamiana) leaves. Bio-protocol. bio-protocol.org/xxx.
2. Yang, W., Cortijo, S., Korsbo, N., Roszak, P., Schiessl, K., Gurzadyan, A., Wightman, R., Jönsson, H., and Meyerowitz, E. (2021). Molecular mechanism of cytokinin- activated cell division in Arabidopsis. Science 371, 1350-1355. doi: 10.1126/science.abe2305.