Chitin azure

Laminarin (from Laminaria digitata), Chitin azure, trans-cinnamic acid, and salicylic acid were purchased from Sigma (St. Louis, MO, USA). Curdlan was purchased from Takeda Chemical Industries, Ltd. (Osaka, Japan). Other materials and chemicals were all analytical grade, and purchased from Sinopharm Chemical Reagents Co. Ltd. (Shanghai, China).

At the first trifoliate stage, two weeks after planting, the seedlings were used for an experiment with technical grade chitin azure, a chitinase substrate (Sigma-Aldrich, cat# C3020). A modified stem-cut method was used to administer the chitin treatment^{28 (link)}. For each genotype, nine seedlings were treated with chitin and nine were with buffer in each experiment. We conducted three experiments. In half of the chitin treatment, the stem-cuts received 0.5 mL of 100 µmol/L chitin in sodium phosphate (15 mmol/L, pH 6.5); and in controls, stem-cuts received 0.5 mL of sodium phosphate buffer (15 mmol/L, pH 6.5) only. The stem-cuts were placed in 2-ml Eppendorf tubes containing either chitin suspension or phosphate buffer. After the treatment suspensions were depleted, all stem-cuts were transferred to 50-ml tube containing 25 ml distilled water and incubated in a growth chamber maintained at 22.5 °C, 16 h light (85 µmol m⁻² s⁻¹) and 8 h dark photoperiod. Leaf samples were harvested at 12 and 24 h post-treatment. Collected leaves were immediately placed in liquid nitrogen and stored at − 80 °C for RNA isolation.