Ethephon

The rubber trees were cultivated at the experimental farm of the Chinese Academy of Tropical Agricultural Sciences (Danzhou, Hainan, China), and the study was approved by the experimental farm of the Chinese Academy of Tropical Agricultural Sciences. Field experiments were performed using mature, seven-year-old virgin (unexploited) rubber trees (Clone Reyan 7-33-97) that had never been tapped. Stimulation assays with exogenous ethephon were carried out according to the method previously described [44 , 45 (link)]. Briefly, 0.5% (w/w) ethephon (Sigma-Aldrich, USA) in water was applied to the bark below the half-spiral of the tapping cut. Water was used as the mock stimulation for the control samples. The trunks of the treated trees were wrapped with black plastic film around the tapping cut and stimulated for 8, 24 or 48 h, after which they were tapped for latex early in the morning on the same day. Each sample included three independent biological replicates, and each replicate comprised the latex collected from six trees.

All-trans-farnesol, all-trans-geranylgeraniol, farnesyl diphosphate, geranylgeranyl diphosphate, dexamethasone, fosmidomycin, methyl-jasmonate, ethephon and mevalonolactone were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). Cellulase RS was obtained from Yakult Pharmaceutical Industry (Tokyo, Japan), and mevinolin was a kind gift from Drs. M. Greenspan and A.W. Alberts (Merck Sharp and Dohme, Rahway, NJ, USA). Before use, the lactone forms of mevinolin and mevalonolactone were converted into their free salt forms, as described by [28 (link)]. 1-deoxy-d-xylulose (DX) was obtained from AlsaChim (Illkirch Graffenstaden, France). (R,S)-[2-¹⁴C]mevalonolactone was purchased from Amersham. Analytical-grade solvents were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France), Carlo Erba (Val-de-Reuil, France), or Fischer Scientific France (Illkirch-Graffenstaden).

Pepper plants were treated with ethephon (Sigma-Aldrich Korea, Korea), which promotes ethylene release, or AVG (Sigma-Aldrich Korea, Korea), which inhibits ethylene biosynthesis. The treatments were performed as described previously (Bak et al., 2019 (link)), with minor modifications. To increase ethylene emission by plants, each four-weeks-old healthy pepper plant was sprayed homogeneously with 5 ml of 0.1 mg/l ethephon. ethephon-treated plants were used in aphid preference tests and for ethylene analysis 2 h after the treatment. To inhibit ethylene release, 5 ml of 0.25 mM AVG was sprayed homogeneously on each four-weeks-old pepper plants inoculated with CMV-GTN 2 weeks previously. AVG-treated plants were used in aphid preference tests and for ethylene analysis 24 h after the treatment. Control plants (mock-treated) were sprayed with distilled water.

Sodium salicylate (Wako, Osaka, Japan), methyl jasmonate (Wako) and ethephon (Sigma‐Aldrich, St Louis, MO, USA), an ET generator, were used as phytohormones. Acetylsalicylic acid (Ac‐SA) (Nacalai Tesque, Kyoto, Japan), 3,5‐dichloroanthranilic acid (DCA) (Tokyo Chemical Industry, Tokyo, Japan), 2,6‐dichlorisonicotianic acid (INA) (Wako) and acibenzolar‐S‐methyl (benzothiadiazole, BTH) (Wako) were used as structural or functional analogs of SA. All phytohormones and chemicals were diluted with water or dimethyl sulfoxide (DMSO). The B. distachyon seedlings or detached leaves were sprayed, soaked or soil‐drenched with a chemical solution containing 0.04% (v/v) Tween 20 and incubated for 24 or 48 h at 23°C. Droplets of chemical solution adhering to the leaf surface were wiped off before the following experiments.

Sodium salicylate (Wako, Osaka, Japan), methyl jasmonate (Wako), and ethephon (Sigma-Aldrich), an ethylene generator, were used. The phytohormones were dissolved in dimethyl sulfoxide (DMSO), then diluted with distilled water to prepare 1 mM solutions (final DMSO concentration is 0.1%), which were supplemented with 0.04% (v/v) tween-20. Four-week-old A. thaliana plants grown on soil were sprayed with the phytohormone solutions and incubated for 48 h at the same growth condition. Then, the detached rosette leaves were prepared and inoculated with R. solani PDA plugs as described above [24 (link)]. For the soil inoculation method, 10-day-old A. thaliana seedlings grown on 1/2MS were sprayed with the phytohormones solutions and kept in the same growth condition for 48 h. Then, seedlings were transplanted in the R. solani-inoculated soil as described before.

Seeds of Cherry belle with uniform size and plumpness were put into a 9 cm diameter petri dish lined with three pieces of filter paper and wetted with 4 ml deionized water. Three days after imbibition, the seeds were treated with deionized water or solutions including 100 μM abscisic acid (ABA, Sigma A1049), 100 μM 6-Benzylaminopurine (6-BA, Sigma B3274), 100 μM Gibberellic acid (GA₃, Sigma G7645), 50 μM alpha-Naphthaleneacetic acid (NAA, Sigma N0640), and 50 μM Ethephon (Sigma 45473). At least 300 seeds were used for each treatment. Root samples were collected 12 and 24 h after treatment, immediately frozen in liquid nitrogen, and stored at −80 °C for further use.

Exogenous phytohormone treatments were performed as described in Nahar et al. [63 (link)] with necessary modifications. Briefly, the rice plants were individually sprayed with 100 μM MeJA (Sigma-Aldrich, St. Louis, MO, USA), 200 μM SA (Sigma-Aldrich, St. Louis, MO, USA), and 500 μM ethephon (Sigma-Aldrich, USA) until runoff to induce JA, SA, and ET treatments, respectively. Rice plants sprayed with 0.1% ethanol (v/v) served as a control for the MeJA and ethephon treatments. Rice plants sprayed with water served as a control for the SA treatments. Plant samples were collected at 1 and 3 days post-hormonal treatments, frozen immediately with liquid nitrogen, and stored at −80 °C until further use. The experiment was performed with 3 independent biological replicates.
Treatments with PD98059 and Genistein were peformed as described in Bjornson et al. [58 (link)] with necessary modifications. Breifely, the rice plants were individually sprayed with a mixture of PD98059 (Sigma-Aldrich, St. Louis, MO, USA) and Genistein (Sigma-Aldrich, St. Louis, MO, USA) of 40 µM concentration dissolved in 0.5% (v/v) dimethyl sulfoxide (DMSO) to induce MAPK inhibition. Rice plants sprayed with only 0.5% (v/v) DMSO served as a control for the experiment. The experiment was performed with 3 independent biological replicates.