Ten pairs of plants from two comparable genotypes were used in each experiment. In the no-choice test, 20 aphids were released on each 30-day-old WT, coi1, and Ri17 plant. Inoculated plants were cultivated separately for 48?hr in growth chambers set at a 16?hr (22��C �C24��C) light/8?hr (16��C �C19��C) dark photoperiod. The numbers of aphids (adults?+ nymphs) on each plant were counted. Ten plants from each genotype were used in each experiment. Both the choice and no-choice tests were repeated three times. Error bars denote?��SEM. According to Student��s t test, asterisks indicate statistically significant differences compared with WT (**p?< 0.01). Seedlings (7?days old) from WT, Ri17, and Ri55 grown on MS plates with or without 25?��M MeJA were stretched on plates containing CYC202 order
1% agarose. Root lengths were determined using a Digimizer (MedCalc Software bvba). The Itraconazole
relative root length percentage for each genotype on MS plates with 25?��M MeJA was calculated relative to its control treated without MeJA (Figure?2D). Thirty plants from each genotype were used in each experiment. All experiments were repeated three times. Error bars denote?��SEM. The Affymetrix microarray assay with jasmonate or wounding treatment utilized the JAV1 RNAi line Ri17 versus WT after treatment for various time periods. The detailed analysis is provided in the Supplemental Experimental Procedures. Quantitative real-time PCR analyses were performed with the Real Master Mix (SYBR Green I; Takara) using the ABI 7500 Real-Time PCR System (see Supplemental Experimental Procedures). The target genes were amplified using their respective primer pairs (Table S1). The experiments were repeated five times. Error bars denote?��SEM. In Figure?7A, 10-day-old Arabidopsis plants expressing the Myc-tagged JAV1 (Myc-JAV1) were treated with the proteasome inhibitor MG132 (30?��M; Sigma-Aldrich) in DMSO for 1?hr followed by continuous treatment with 100?��M MeJA for 0, 15, 30, 45, and Selleckchem Verteporfin
60?min. The seedlings treated with DMSO acted as a mock control. Total protein samples were extracted from treated seedlings for immunoblot analysis with anti-Myc antibody (Sigma-Aldrich) to determine the Myc-JAV1 protein level, following standard procedures ( Xu et?al., 2002). The actin level was detected by anti-Actin antibody (Sigma-Aldrich) and served as a loading control. In Figure?6I, 10-day-old Arabidopsis seedlings of WT::pVSP-GUS ( Zheng et?al., 2006) and OX1::pVSP-GUS were treated with 100?��M MeJA at the indicated time. The OX1::pVSP-GUS plant is described in Supplemental Experimental Procedures. In Figure?6J, 21-day-old Arabidopsis seedlings of WT::pVSP-GUS and OX1::pVSP-GUS were wounded and left for 30?min and 60?min before staining.