Cis-12-Oxo-Phytodienoic Acid Stimulates Rice Defense Response to a Piercing-Sucking Insect  Hui-Min Guo, Hai-Chao Li, Shi-Rong Zhou, Hong-Wei Xue, Xue-Xia Miao  Molecular Plant  Volume 7, Issue 11, Pages 1683-1692 (November 2014) DOI: 10.1093/mp/ssu098 Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 1 Rice AOC Is Induced by Brown Planthopper (BPH) Infestation and AOC Overexpression Enhances Resistance to BPH. (A) qRT–PCR analysis revealed higher expression of AOC in RHT (a BPH-resistant variety) than in two BPH-sensitive varieties, TN1 and ZH11. Leaf sheaths of 6-week-old rice plants were analyzed, and data are presented as means ± SD. Experiments were repeated three times, and the expression level of AOC in ZH11 was set as 1.0. (B) qRT–PCR analysis showed induced expression of AOC under BPH infestation. Leaf sheaths of 6-week-old ZH11 plants infested with BPH were analyzed, and data are presented as means ± SD. Experiments were repeated three times, and the expression level at 0h was set as 1.0. (C) qRT–PCR analysis confirmed the enhanced expression of AOC (left panel) or OPR3 (right panel) in plants transformed with p35S::AOC or p35S::OPR3. Leaf sheaths of 6-week-old ZH11, AOCox, or OPR3ox plants were analyzed, and data are presented as means ± SD. Experiments were repeated three times, and the expression level of AOC or OPR3 in ZH11 was set as 1.0. (D, E) Phenotypic observations of rice plants at the tillering stage (D) or rice stems (E) showed that AOC overexpression enhanced resistance to BPH. Rice stems or whole plants of ZH11 and second-generation AOCox or OPR3ox plants at the tillering stage were infested with 15 third-instar BPH nymphs for 15 d (n = 6). (F) Rice seedling mortality of ZH11, AOCox, and OPR3ox plants after infestation with eight second-instar nymphs for 7 d. Each line had 30 replications. Seedling mortality was evaluated according to a nine-grade standard (Table 1). Molecular Plant 2014 7, 1683-1692DOI: (10.1093/mp/ssu098) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 2 Overexpression of AOC Enhances Rice Antibiosis to Brown Planthopper (BPH) Mainly by Reducing the Feeding Activity and Survival Rate. (A) Honeydew excretion of BPH feeding on ZH11, AOCox, and OPR3ox plants at the four-leaf stage. Size of the honeydew area and intensity of the honeydew color, which correspond to BPH feeding activity, were observed (left panel) or measured (right panel) after feeding by five third-instar nymphs for 2 d. Data are presented as means ± SD (n = 5). Student's t-test revealed significant differences (** P < 0.01, compared to ZH11). (B) Population growth rates of BPH feeding on ZH11, AOCox, and OPR3ox plants at the four-leaf stage. A cohort of 20 pre-weighed, first- to second-instar nymphs of BPH was placed on a plant at the four-leaf stage. The nymphs were re-weighed after infestation for 7 d. Data are presented as means ± SD (n = 3). Student's t-test revealed significant differences (* P < 0.05, compared to ZH11). (C) Survival tests for BPH feeding on ZH11, AOCox, and OPR3ox plants. Twenty first-instar nymphs were placed on each 6-week-old plant and covered with a cylindrical mylar cage. The status (alive or dead) was recorded daily for 10 d. Data are presented as means ± SD (n = 3). Significantly fewer surviving BPHs per plant were found on AOCox plants than on ZH11 after infestation for 2 d. Student's t-test revealed significant differences (* P < 0.05, compared to ZH11). Molecular Plant 2014 7, 1683-1692DOI: (10.1093/mp/ssu098) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 3 AOC or OPR3 Overexpression Enhances Resistance to Striped Stem Borer (SSB). (A) Observation of damage to ZH11, AOCox, and OPR3ox plants at the seedling stage showed that overexpression of AOC or OPR3 resulted in similar resistance against chewing SSB. Plants were individually infested with three first-instar larvae for 7 d and then observed (n = 3). (B, C) Observation ((B) bar = 0.5cm) and measurement (C) of SSB larval growth after infesting ZH11, AOCox, and OPR3ox plants at the seedling stage for 7 d. First-instar larvae were used to infest plants, and data are presented as means ± SD (n = 3). Student's t-test revealed significant differences (* P < 0.05, ** P < 0.01, compared to ZH11). Molecular Plant 2014 7, 1683-1692DOI: (10.1093/mp/ssu098) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 4 OPDA Stimulates the Rice Resistance to Brown Planthopper (BPH). (A) Concentrations of JA, JA-Ile, and OPDA in leaf sheaths of ZH11, AOCox, and OPR3ox plants at the tillering stage after BPH infestation for 12h. Data are presented as means ± SD (n = 3). Student's t-test revealed significant differences (* P < 0.05, ** P < 0.01, compared to ZH11). (B) Observations of seedling growth (above) and measurement of BPH mass after feeding (below) when treated with different concentrations of JA, JA-Ile, or OPDA and after 5 or 8 d of BPH infestation. ZH11 seedlings at the two-to-three-leaf stage were infested with 25 first-instar BPH nymphs. Experiments were repeated three times, and representative images are shown. Data are presented as means ± SD (n = 3). Duncan's multiple-range test revealed significant differences (* P < 0.05, ** P < 0.01, compared to ZH11). (C) Survival rate of BPH fed plants treated with OPDA (50 μM), JA (500 μM), or JA-Ile (50 μM). Twenty second-instar nymphs were placed on each plant and covered with a cylindrical mylar cage. BPH status (alive or dead) was checked 5 d after infestation. Data are presented as means ± SD (n = 3). Student's t-test revealed significant differences (* P < 0.05, ** P < 0.01, compared to control). (D) qRT–PCR analysis of AOC expression (left; the expression level of AOC without treatment was set as 1.0) and measurement of OPDA concentrations (right) under treatment with JA (500 μM) or JA-Ile (50 μM). Leaf sheaths of ZH11 plants were analyzed, and data are presented as means ± SD. Experiments were repeated three times. Molecular Plant 2014 7, 1683-1692DOI: (10.1093/mp/ssu098) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 5 AOC Stimulates Brown Planthopper (BPH) Resistance by Regulating Multiple Genes. (A) Comparative analysis of up-regulated (left) and down-regulated (right) genes in AOCox, OPR3ox, and both. (B) qRT–PCR analysis confirmed that gene expressions of Os07g47960, Os01g42210, and Os12g29690 were significantly enhanced under AOC expression. Data are presented as means ± SD (n = 3). Leaf sheaths of 6-week-old ZH11, AOCox, and OPR3ox plants were analyzed, and data are presented as means ± SD. Experiments were repeated three times. (C) qRT–PCR analysis revealed that expressions of Os07g47960, Os01g42210, and Os12g29690 were especially induced by OPDA (50 μM) but not by JA (500 μM) or JA-Ile (50 μM). Leaf sheaths of 6-week-old ZH11 plants were analyzed, and data are presented as means ± SD (n = 3). Molecular Plant 2014 7, 1683-1692DOI: (10.1093/mp/ssu098) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 6 OPDA Treatment Improves the Resistance of Radish Seedlings to Green Peach Aphid Myzus persicae. (A) Survival rates of aphids under treatment with OPDA (50 μM). Ten gravid aphids were placed on 1-week-old radish seedlings for 1 d then removed, and the number of surviving offspring was recorded daily for 5 d. Data are presented as means ± SD (n = 3). Student's t-test revealed significant differences (* P < 0.05, ** P < 0.01, compared to control). (B) Measurement of the aphid mass revealed a significant reduction under OPDA treatment (50 μM). Data are presented as means ± SD (n = 3). Molecular Plant 2014 7, 1683-1692DOI: (10.1093/mp/ssu098) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions