Actigard 50wg

Actigard 50WG^® was obtained from Syngenta (Fargo, ND, USA) as a water-dispersible granular formulation containing 50% active ingredient. cis-jasmone was obtained from Sigma-Aldrich (St. Louis, MO, USA) as a soluble liquid formulation with 85% purity. Azadirachtin^® (extracts from neem), as a 1.2% emulsifiable concentration, was obtained from Gowan Company (Yuma, AZ, USA). For both laboratory and field experiments, the chemicals were applied at concentrations of 0.75 ml/l (Actigard), 0.5 ml/l (cis-jasmone) and 2.88 ml/l (Azadirachtin), the concentrations recommended by manufacturers for agricultural use.

RCSCs were treated for four hours with different compounds: 500 µM diproline, 250 µM S-methyl 1,2,3-benzothiadiazole-7-carbothioate (BTH) formulated as Actigard^® 50WG (Syngenta; Basel, Switzerland), 1 µM riboflavin (Alfa Aesar; Massachusetts, Ward Hill, USA), 100 µM azelaic acid (Sigma-Aldrich; Missouri, Saint Louis, USA) and 20 mM dehydroascorbic acid (DHA - Sigma-Aldrich; Missouri, Saint Louis, USA). Per sample, 40 mL of cells were treated with 20× solutions. All other downstream steps were done as described in Section 4.3.2. Diproline (Cyclo(l-Pro-l-Pro)) was synthesized starting from H-Pro-OMe·HCl (Apollo Scientific; Bredbury, UK) and Boc-Pro-OH (Sigma-Aldrich; Missouri, Saint Louis, USA) with a procedure modified from Campbell et al. (2009) [62 (link)]. DMF was used as solvent, OxymaPure (ChemPUR; Karlsruhe, Germany) was added as additive and the final compound was purified with a preparative HPLC. The equipment used was an Agilent 1100 Series system (Agilent; Santa Clara, California, USA) with a Supelco Ascentis C18 column (21.2 mm × 150 mm, 5 µm) (Sigma-Aldrich; Missouri, Saint Louis, USA).