Silwet l 77

In this study, all the vectors were electroporated and transferred into Agrobacterium tumefaciens strain GV3101 (Hellens et al., 2000 (link)). Agrobacterium tumefaciens cells containing overexpressing and promoter truncation variants were harvested by centrifugation at 5,000×g for 8 min and resuspended in 5% sucrose solution to a final OD of 0.5. By using the floral dip method (Steven and Andrew, 1998 (link)), the shoot apex of Arabidopsis mir159ab double mutant plants was dipped into a bacterial suspension supplemented with 0.05% Silwet (Silwet L-77, Sigma). Seeds were germinated on agar plates containing Murashige and Skoog basal medium as well as antibiotics in order to select transformants from the population. Transformants were detected and transplanted into the soil after 7 to 10 days of growth. The photographs of all plants were taken with the help of DSLR EOS 70D (Canon) using scale as reference. Then, the size of a rosette, lamina length, lamina width, and leaf area of plants were measured with ImageJ (version 1.8.0) software.

All vectors were transformed into Agrobacterium tumefaciens strain GV3101 by electroporation (Hellens et al., 2000 (link)) and then transformed into the Arabidopsis mir159ab double mutant by using the floral dip method (Steven and Andrew, 1998 (link)). A. tumefaciens cells containing nested miRNA variants were harvested by centrifugation at 5000 × g for 8 min and resuspended in 5% sucrose solution to a final OD of 0.5. The shoot apex of mir159ab plants were dipped into a bacterial suspension supplemented with 0.05% Silwet (Silwet L-77, Sigma). Seeds were grown on agar plates containing Murashige and Skoog basal medium and antibiotics to pick transformants. Transformants were identified and transplanted into the soil after 7–10 days of growth.

Pto DC3000 COR‐ bacteria were streaked out from glycerol stock on fresh King's B media plates containing 1% agar and 50 μg/ml rifampicin and 50 μg/ml kanamycin. Bacteria were collected from plates with a sterile pipette tip and resuspended in water containing 0.04% Silwet L77 (Sigma Aldrich, St. Louis, USA) to an OD₆₀₀ = 0.2 (10⁸ cfu/ml). The bacterial suspension was sprayed on 4‐ to 5‐week‐old plants, which were subsequently covered with lids for 3 days. Three leaf discs per sample from different plants were collected in microfuge tubes and ground with a tissue lyser (Qiagen, Düsseldorf, Germany). Serial dilutions were plated on LB agar before counting colonies. For inducing resistance with GLV2 peptides, 4‐ to 5‐week‐old plants were infiltrated with 1 μM GLV2 and incubated for 24 h. Subsequently, a suspension of Pseudomonas syringae pv. tomato DC3000 bacteria was prepared as above to an OD₆₀₀ = 0.0002 (10⁵ colony forming units per mL) and syringe infiltrated into pre‐treated leaves. Two days after inoculation, samples were collected as described above.

Transgenic Arabidopsis conditionally expressing avrPto under control of a dexamethasone-inducible promoter [68 (link)] were sprayed with 20 μM dexamethasone (cat. no. D1756, Sigma-Aldrich) in 0.1 % ethanol with 0.01 % Silwet L-77 (cat. no. VIS-01, Lehle Seeds) to induce gene expression. Leaves were infiltrated twice with 10 μM myristic acid analog Alk12, 6 h after induction and 6 h before sampling. Tissue was collected 30 h after induction and stored at −80 °C until further processing.

Seedlings were grown on half-strength MS medium for five days. Reconstitution of aequorin was performed in vivo by spraying seedlings with 10 µM coelenterazine and followed by incubation at 21°C in the dark for 12–16h. For surfactant treatment, 0.01% or 0.1% of silwet L-77 (Sigma) was added to the coelenterazine solution. For Ca²⁺ inhibitor treatments, rice roots were treated with different concentrations of GdCl₃, LaCl₃, neomycin and thapsigargin, respectively for 30min before 0.25M NaCl and 1mM H₂O₂ treatment. Treatments and aequorin luminescence imaging were performed at room temperature using a ChemiPro HT system as described previously (Jiang et al., 2013 (link)). The recording was started about 5 s prior to treatment and luminescence images were acquired for 3min. For the analysis of time courses of increase in [Ca²⁺]_i, each exposure time was 30 s and the images were taken continuously for several minutes. To avoid the interference of chloroplast auto-fluorescence signal in the aequorin luminescence imaging, all the treatments were performed in the complete darkness. To record the chloroplast auto-fluorescence signal, seedlings were first exposed to strong light for 1min. After that, the light was turned off and the chloroplast auto-fluorescence was recorded. WinView/32 and Meta Morph 7.7 were used to analyse recorded luminescence images.