1 Polymerase chain reaction(PCR)

(1) To the reaction system, upstream and downstream primers, template DNA, 2xPhenta Max Master Mix(P525-01, Vazyme) and ddH2O are added proportionally;

(2) Cycling Conditions: Step 1: 95℃, 30s; Step 2: 95℃, 15s; Step 3: Primer melting point minus 3 degrees, 15s; Step 4: 72℃, 1kb for 30s; Step 5: Go to Step2, repeat for 35 cycles; Step 6: 72℃, 5min; Step 7: 4℃, infinite.

2 Plasmid Transformation (using DH5α/Fast-T1 competent cell)

(1) Place the bacterial liquid on ice for 8min to melt;

(2) Mix 50μL bacterial liquid with around 100ng plasmid and place on ice for 25min;

(3) 42℃ metal bath heat hit 45s;

(4) Place the mixture on the ice again for 2-3min;

(5)Add 500-700μL Autoclaved bacterial culture medium, shake on the shaker for 1h (37℃, 220rpm).

3 Plasmid Transformation(using GV3101(pSoup-P19) competent cell)

(1) Place the bacterial liquid on ice for 5min to melt;

(2) Mix 50μL bacterial liquid with around 100ng plasmid and place on ice for 5min;

(3) Add the mixture to the pre-colded electroporation cuvettes;

(4) Electric hit in the Bio-Red MicroPulser;

(5) Add 500-700μL Autoclaved bacterial culture medium, shake on the shaker for 2h (28℃, 220rpm).

4 Construction of Plasmids by Homologous Recombination

(1) To the reaction system, insert fragments, linearized vector, 2x seamless cloning mix(D7010S, Beyotime), ddH₂O are added proportionally;

(2) Reaction Condition: 50℃, 15min;

5 Plasmid Mini extraction

Using FastPure Plasmid Mini Kit(DC201, Vazyme).

6 Colony PCR

(1) Pick the monoclonal with the pipet tip;

(2) Streak on a blank bacterial culture plate using the pipet tip with colony on;

(3) Add the remaining bacteria on the pipet tip to the pre-prepared PCR system as the template;

(4) Cycling Conditions: Step 1: 95℃, 5min; Step 2: 95℃, 15s; Step 3: Primer melting point minus 3 degrees, 15s; Step 4: 72℃, 1kb for 30s; Step 5: Go to Step2, repeat for 35 cycles; Step 6: 72℃, 5min; Step 7: 4℃, infinite.

7 DNA agarose gel electrophoresis

(1) Configure 30ml 1% agarose solution, heat and melt, add 3μL 10000x dye EB;

(2) Add 10x loading buffer with around 100 ng plasmid in each well;

(3) 110V electrophoresis for 30min;

(4) Fluorescence chromogenic photography.

8 DNA Gel Extraction and DNA product purification

Using FastPure Gel DNA Extraction Mini Kit(DC301-01, Vazyme)

9 Competent cells preparation (DH5α/Fast-T1/STABLE/GV3101(pSoup-P19) competent cell)

(1) Spread competent cells onto the bacterial culture plate;

(2) Take single clone in 3mL liquid medium for small amount amplification;

(3)1:100 inoculation of bacterial solution from shaking tube into 1000ml shaking bottle with 200mL liquid medium for bulk amplification;

(4) Cultivate the bacteria on the shaker until the OD600 of the bacterial solution is about 1.0-1.2 andto 0.5-0.7;

(5) Centrifuge the bacterial solution at 4℃, 5000xg for 5min, and collect all the bacteria into a 50mL centrifuge tube in several times;

(6) Gently resuspend the bacteria with 40 ml of pre-colded 10% glycerol and centrifuge again(4℃, 5000xg for 5min);

(7) Repeat the operation(6) again;

(8) Resuspend the bacteria with 16mL of 10% glycerol and dispense 50ul per tube into 1.5mL centrifuge tubes, freeze in liquid nitrogen and transfer to -80℃ refrigerator.

1 Transient transformation of tobacco by Agrobacterium tumefaciens

(1) Configuring the transient expression buffer;

(2) Cultivate Agrobacterium on a shaker until the OD₆₀₀ of the bacterial solution is about 1.0-1.2 and record the OD₆₀₀ value of the bacterial solution;

(3) Centrifuge the bacteria solution at 3600rpm for 10min at 26°C and discard the supernatant;

(4) Resuspend the Agrobacterium with 10mM MgCl₂ solution;

(5) Centrifuge the bacteria solution at 3600rpm for 10min at 26°C and discard the supernatant;

(6) Resuspend the Agrobacterium with an amount of transient expression buffer, making the OD₆₀₀ of the solution to 0.8;

(7) Leave the above bacteria solution in the dark for 3h;

(8) Draw up the above bacterial solution with a 1mL syringe to inject tobaccos.

2 Plant RNA Extraction

Using Plant total rna extraction kit(DP432, TIANGEN)

3 RT-qPCR

(1) Genomic DNA removal: The RNA template was mixed proportionally with 4x gDNA wiper Mix(R323-01, Vazyme) and ddH2O;

(2) Reaction Condition: 42℃, 2min;

(3) RT-PCR: Mix the above mixture with 5x HiScript III qRT SuperMix(R323-01, Vazyme) proportionally;

(4) Reaction Conditions: Step1: 37°C, 15min; Step2: 85°C, 5s;

(5) qPCR: Mix the above reaction solution as cDNA template with qPCR primers, 2x ChamQ Universal SYBR qPCR Master Mix(Q711-02, Vazyme), and ddH2O proportionally.

(6) Cycling Conditions: Step1: 95°C, 30s; Step2: 95°C, 10s; Step3: 60°C, 30s; Step 4: Go to Step2, repeat for 40 cycles; Step5: Measuring melting curves;

4 Plant protein Extraction

(1) Plant tissues were taken and cryomilled in liquid nitrogen;

(2) Plant tissue powder was resuspended with Extraction buffer (25mg tissue/200uL) and placed on ice for 15min;

(3) Centrifuge at 4℃ at 12000rpm for 10min and take the supernatant.

5 Measurement of plant protein concentration(BCA method)

Using BCA Protein Concentration Measurement Kit(P0009,Beyotime)

6 Western blot

(1) SDS-PAGE electrophoresis: the appropriate amount of target protein is separated by SDS-PAGE;

(2) Put PVDF membrane into methanol solution for 5min, making it activated;

(3) After electrophoresis, remove the SDA-PAGE gel, and stack the PVDF membrane, gel and filter paper in the transfer buffer in order, taking care not to create bubbles;

(4) Place the filter paper-membrane-glue-filter paper sandwich into the Bio-Red Tans-Blot, and remove bubbles with a roller;

(5) Procedure: 20V, 1h;

(6) After the membrane transfer was completed, take out the PVDF membrane,and washed it with PBST on a shaker for 3 times, 10min each time;

(7) Add 5% skim milk powder (PBST dissolved W/V %) and block for 1h;

(8) Add primary antibody (5 ml, 2000 times dilution, diluent can choose 5%BSA or biyuntian primary antibody dilution) and incubate at 4°C overnight. After that, wash it with PBST for 3 times, 10min each;

(9) Add secondary antibody (5 ml, 1:500 dilution, diluent can choose 5%BSA or 5% skim milk powder), incubate at room temperature for 1h. After that, wash it with PBST for 3 times, 10min each;

(10) Incubate PVDF membrane with SuperSignal West Pico PLUS buffer(34577, Thermo Scientific);

(11) Use Bio-Rad chemiluminescence instrument for imaging.

7 Coomassie Blue staining

Using Coomassie Blue Fast Staining Kit(P0017, Beyotime)

8 Tobacco cultivation

Culture Conditions: Daytime: 25℃, 80%sunlight, 16h; Night: 20℃, 0%sunlight, 8h.

1 GUS staining assay

(1) Dissolve the X-Gluc dry powder(SL7160, Coolaber) in X-Gluc solvent(SL7160, Coolaber) to prepare a 50x GUS dyeing concentrate;

(2) Dilute the 50x GUS staining concentrated solution with GUS staining buffer(SL7160, Coolaber) to prepare GUS staining solution.

(3) The prepared plant materials were soaked in GUS staining solution and incubated at 25-37°C for 1 hour to overnight(based on the strength of the transformed gene promoter and the tenderness of the material and the thickness of the cuticle);

(4) Leaves were decolored 2-3 times in 70% ethanol until the negative control material was white;

(5) Under the naked eye or microscope, the blue dots on the white background are the GUS expression sites.

1 Plant reactive oxygen species detection

Using Plant Reactive Oxygen Species (ROS) ELISA Kits(MM-0724O1, MEIMIAN)

1 Light-induced bacterial suicide experiment

(1) Cultivate engineered bacteria on a shaker until the OD₆₀₀ of the bacterial solution reaches 0.6;

(2) Dilute the bacterial solution 10⁵ times with liquid medium, and then spread plates, adding 50μL of the bacterial solution to each plate;

(3) Invert the plates on an insulated foam board, and place it out under sunlight(the light intensity will be record by an illuminometer). In order to control the plate temperature not higher than 30℃, place ice packs under the foam board and use a infrared thermometer gun to monitor the temperature.

(4) After illumination, incubat the bacterial culture plates at 28℃ in the dark for 48-72h. Finally, take pictures of plates using a multifunctional imager and count the number of bacterial colonies.

2 Directed evolution

(1) Use the software tbtools to extract 40bp DNA sequences upstream of all Agrobacterium genes and counted the frequency of codons;

(2) Use arbitrary degenerate primer to mutate the bases from -2 to -7 and from -32 to -37, GFPuv is used as the reporter gene. After transformation, E. coli are cultured and the plasmids are extracted to obtain the mutant library;

(3) Transform Agrobacterium by electric shock using the mixed plasmids. then spread plates and incubat them at 28℃ for 48h. After 48h, observe the brightness of colonies, and high brightness colonies need to be cultured in the liquid medium before plasmid extraction for sequencing.

1 Protein Fluorescent Analysis

(1) Plant proteins were extracted and the total protein concentration was adjusted at 2.5ug/ul. The total protein concentration can be adjusted according to the results of the pre-experiment to ensure that the fluorescence intensity is measured in the linear region where fluorescence is directly correlated with protein level.

(2) Protein extracts were added to a 96-well plate and the fluorescence intensity of the protein extracts was measured using a Thermo Fisher Varioskan Flash Multiple Reader at an excitation wavelength of 485 nm (12 nm slit) and an emission wavelength of 535 nm.

(3) Relative GFP fluorescence values were calculated by dividing the background autofluorescence. Fluorescence intensity was measured in the linear region where fluorescence directly correlates with protein levels.