Hordenine And Methyl Jasmonate Case Study

Chemicals
Hordenine and methyl jasmonate were purchased from Tokyo Chemical Industry (Tokyo, Japan). Dimethyl sulfoxide (DMSO) was obtained from Sigma-Aldrich (Tokyo, Japan).

Plant materials
Two-year-old seedlings of Vitis vinifera cv. Koshu were cultivated in perlite:vermiculite (1:1) soil in a growth chamber (11.8 Wm-2 for 14 h in a day) at 27 °C. Seedlings of Fragaria×ananassa cv. Nyoho were grown in 55% peat moss, 10% perlite, 5% vermiculite, and 30% decomposed granite soil at 25 ºC in a greenhouse under natural light condition.
Arabidopsis thaliana wild-type (Col-0), SA-insensitive mutant npr1-5 (CS3724) (Zypfel et al. 2004) and JA-insensitive mutant atmyc2 (SALK_039235) (Lorenzo et al. 2004) were used. Seeds of each Arabidopsis were
2016) was performed to evaluate whether hordenine suppresses strawberry anthracnose caused by Colletotrichum gloeosporioides. Leaves were detached from strawberry seedlings. Three detached leaflets were sprayed with 600 μL of 1 mM hordenine. Water or DMSO was used in control experiments. After the leaflets were air-dried overnight, the center of each leaflet was punctured with a sterile needle of 1 mm diameter. C. gloeosporioides inoculum was incubated on a potato dextrose agar plate (PDA, Becton and Dickinson, MA) at 25 °C for 10 days. C. gloeosporioides agar plugs (4 mm diameter) were prepared by using a cork borer, and one plug was placed on the wound of the leaflet that was turned upside down. The leaflets were incubated at 25 °C in a plastic box containing moistened paper towel under light/dark condition (11.8 Wm-2 for 14 h in a day). On day 7 post incubation, lesion diameter on each leaf was measured to determine anthracnose severity. Means of the lesion diameters from two independent experiments were
DMSO was used in a control experiment. The seedlings were incubated at 22 ºC for 24 h in an incubator (11.8 Wm-2 for 16 h in a day).

Treatment of grape leaf disks with methyl jasmonate
Leaf disks (15 mm diameter) were prepared from grapevine seedlings as described above. One mM, 100 μM or 10 μM methyl jasmonate was dropped on leaf disks (80 μL/disk). The disks were incubated on moistened filter paper in Petri dishes at 25 ºC for 24 h. Water or ethanol was used in control experiments.

RNA isolation
Hordenine-treated grape leaf disks or Arabidopsis rosette leaves and methyl jasmonate-treated grape leaf disks were collected 24 h after the treatment. The leaves were homogenized in a mortar containing liquid nitrogen using a pestle. The pulverized samples were transferred to a microtube. Total RNA isolation from the pulverized samples was performed with a Fruit-mate for RNA Purification (Takara, Otsu, Japan) and then with a NucleoSpin RNA Plant (Takara) according to the manufacturer’s