Standard molecular biology methods

2.3.1.1 Agarose gel electrophoresis

Products resulting from PCR or plasmid restriction reactions were separated and visualized by agarose gel electrophoresis. Prior to loading, samples were combined with 6 x loading dye and gels were prepared. BioReagent Agarose (Sigma Aldrich) was diluted in 1 x TAE buffer and melted in microwave to a final concentration of 1 %. The gel was cast and a comb was inserted to create loading pockets.

Once the gel solidified, it was submerged in an electrophoresis tank filled with 1 x TEA. The comb was removed and the DNA-samples were loaded. Separation was conducted under 125 V for 45 minutes.

The gel was incubated for 10 minutes in EtBr solution to enable its binding to DNA strands prior to fluorescence visualization under UV-light.

2.3.1.2 Measurement of DNA and RNA concentrations

Thermo Scientific™ NanoDrop 2000 was used to quantify and assess the purity of nucleic acids. 2µl of DNA (plasmid) or RNA was used for measurement at a wave length of 260 nm. The optimal ratio for the sample purity of DNA is OD260/OD280≈1.8 and for RNA is OD260/OD2801.9~2.0 and OD230/OD260≈2.4.

2.3.1.3 Golden Gate cloning

The Golden Gate technique is a system for the generation of recombinant plasmids where the vector plasmid and the fragment to be inserted are designed in a way that both molecules carry a restriction site for one specific type IIs endonuclease, e.g. BpiI (Engler et al. 2009). The principle of the method is based on the ability of the enzyme to cleave outside the recognition sequence. DNA ends of the DNA fragment of interest can be designed to be flanked by a type IIs restriction site such that digestion of the fragments removes the enzyme recognition sites and generates overhang ends complementary to the overhang ends of the digested vector. Once the wanted DNA is restricted, there is no need to extract or separate products since most of the DNA is restricted. Thus, it is not necessary to separate restriction and ligation processes. Instead both are performed in one restriction-ligation step (Table 1). Plasmids were mixed with PCR products in 1:6 molar ratios, with plasmid amount of approximately 150 ng. The reaction was incubated for an hour at 37 °C. Aliquot of 10 µL was used for E.coli transformation.

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COMPONENT CONCENTRATION VOLUME / ΜL

pB-CRISPR-AT2S3pGFP 150 ng/µL 1

PCR product 35 ng/µL 1

ATP 50 mM 0.2

BpiI 10 U/µL 0.5

T4 DNA ligase 5 U/µL 1

Green Buffer 10 x 2

H2O Up to 20 μL

Table 2 Polymerase Chain Reaction program for thermocycler

PROGRAM STEP TEMPERATURE / °C TIME / MIN NUMBER OF CYCLES

PCR program for cloning

Initial denaturation 98 1 1

Denaturation 98 0.25 35

Annealing 60 0.5

Elongation 72 1.5/

Final elongation 72 10

2.3.1.4 GATEWAY

cloning

The plasmids used for Dual-luciferase assay in Arabidopsis protoplast were constructed using the Invitrogen Gateway^TM Technology (Thermo Fisher Scientific, USA) (Katzen 2007). The method is based on the sequence specific recombination system of phage λ.

In the first step, sequences for recombination are fused to the gene of interest by PCR, enabling the exchange with the donor plasmids cassette using BP clonase II. In the second step, donor plasmid containing region of interest is exchanged with Gateway^TM cassette of the destination vectors pUBQ10GWHAS7 and pBGWL7. For the BP and LR reactions equimolar ratios of PCR fragment or plasmids were mixed with the kit-provided enzyme and incubated for 2 hours. The plasmids were transformed into E. coli DH5α.

2.3.1.5 Transformation of Escherichia coli

Transformation of competent Escherichia coli cells (DH5α) was performed by heat shock method (Hanahan 1983). Cells were incubated for 30 minutes on ice, when a plasmid generated by ligation reaction or Golden Gate cloning was added. Cells were incubated for 30 more minutes on ice followed by heat shock at 42 °C for 90 seconds. After cooling off on ice for 3 minutes, cells resuspended in 800 µL of dYT medium were incubated for 45 minutes at 37 °C. Cells were harvested by centrifugation for

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1 minute at 13 000 rpm and plated on LB plate containing the selective antibiotic. Plates were incubated overnight at 37 °C.

2.3.1.6 Transformation of Agrobacterium tumefaciens by electroporation

Agrobacterium tumefacies electrocompetent cells (GV3101) were transformed according to (Mattanovich et al. 1989) and incubated on ice for 30 minutes. Meanwhile, cuvettes were washed with distilled water, 70 % ethanol and 100 % ethanol and left to dry. Approximately 100 ng of plasmid was added to the bacteria and incubated for 30 minutes on ice. The Gene Pulser (BioRAD) was set to 2.5 kV, 200 Ω and 25 µF. The cuvette was filled with the cells and electroporation under described condition was conducted for 5 seconds. Immediately 1 mL of YEB medium was added and cells were incubated for two hours at 29 °C and 220 rpm. Cells were harvested by centrifugation for one minute at 13 000 rpm and plated on YEB plates containing selective antibiotics. Plates were incubated for two days at 29 °C.

2.3.1.7 Plasmid extraction from E. coli and A. tumefaciens

Prior to plasmid extraction, bacteria were grown over night at 37 °C (E. coli) in dYT medium or at 29 °C (A. tumefacies) in YEB medium with respective antibiotics. We used NucleoSpin® Plasmid (Macherey-Nagel) for plasmid extraction according to manufacturer manual. Concentration of plasmid was measured by NanoDrop 2000 (peqLab).

2.3.1.8 Transformation of Arabidopsis thaliana using the floral dip method

The Agrobacterium-mediated stable transformation of Arabidopsis was performed as previously described (Clough and Bent 1998). Plants were grown at long day (LD) conditions (16h photoperiod, 22 °C, 100-120 photons m^-2 sec^-1 and 65 % rel. humidity) for approximately four weeks before the first flowers were cut. It took another week for second flowers to grow.

In the meantime, Agrobacterium tumefacies, containing the plasmid of interest, was prepared.

Glycerol-stored bacteria were grown on YEB plates on selective antibiotics for two to three days at 29

°C before transferring to liquid media. After two days, 5 mL aliquot of the first liquid culture was inoculated in 400 mL of fresh media and grown over night at 29 °C and 220 rpm. The second liquid culture was harvested by centrifugation for 20 minutes at 6000 rpm/RT. The pellet resuspended in 5

% sucrose solution with 0.02 % Silwet L-77. OD600 of the solution was adjusted to 0.8 mL^-1

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Arabidopsis inflorescences were dipped in the solution for 20 seconds. Plants were covered by a plastic hood and placed in the climate chamber overnight. The following day, hood was removed and plants were grown for a month at LD conditions to allow seed production.

2.3.1.9 Extraction of genomic DNA

Leaf samples from were drilled in 300 µL of DNA extraction buffer, followed by centrifugation for 10 min RT/13000. The supernatant was transferred to a new tube and mixed with an equal volume isopropanol and centrifuged at the same conditions as before. The supernatant was removed with a pipette and the pellet was rinsed with 200 µL of 70 % ethanol. The supernatant was removed and the pellet was dried at 37 °C. When dry, it was resuspended in 100 µL of sterile water and incubated for 10 min at 65 °C. After centrifugation circa 80 µL of the supernatant was transferred to a new tube and stored at -20 °C.

Table 3 DNA extraction buffer

COMPONENT FINAL CONCENTRATION

Tris-HCl pH7.5 200 mM

NaCl 250 mM

EDTA 25 mM

SDS 0.5 % (w/v)