JAV1 Controls Jasmonate-Regulated Plant Defense Po Hu, Wu Zhou, Zhiwei Cheng, Meng Fan, Lei Wang, Daoxin Xie  Molecular Cell  Volume 50, Issue 4, Pages 504-515 (May 2013) DOI: 10.1016/j.molcel.2013.04.027 Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 Isolation of JAV1 (A) Genetic screening of ∼20,000 primary transgenic plants identified a hairpin RNA transgenic line, HR-43. HR-43 exhibited obvious resistance to B. cinerea at 7 days after spray inoculation, whereas other transgenic plants developed severe disease symptoms. See also Figure S1. (B and C) Quantitative real-time PCR analysis of At3g22160 (JAV1) transcripts in the indicated MeJA-treated (B) or wound-treated (C) plant at the indicated time points. Error bars denote ±SEM (n = 5). (D) Sequences of JAV1. A unique VQ motif was shown in a red box. (E) Confocal images show that JAV1-GFP is specifically targeted to the nucleus. DAPI fluorescence confirms the location of the nuclei. Scale bars = 10 μm. See also Figure S2. Molecular Cell 2013 50, 504-515DOI: (10.1016/j.molcel.2013.04.027) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 Characterization of the JAV1 RNAi Transgenic Lines (A) The transcript levels of the JAV1 gene significantly decreased, whereas other VQ domain-containing genes were unaffected, in the transgenic lines Ri17 and Ri55. The relative transcript levels were determined by quantitative real-time PCR. The data for three VQ motif-containing genes (At1g80450, At2g41010, and At4g15120), which are marked by black stars in Figure S2A, have been representatively shown. Error bars denote ±SEM (n = 5). (B) The transcript levels of the JAV1 gene were significantly decreased in eight additional transgenic JAV1 RNAi lines. Error bars denote ±SEM (n = 5). (C) JAV1 is not involved in jasmonate-mediated plant root growth. Seedlings from WT and JAV1 RNAi transgenic lines Ri17 and Ri55 were grown for 7 days on MS plates without (upper panel) or with (lower panel) 25 μM MeJA. Scale bars = 5 mm. (D) Relative root length of the plants indicated in (C). Error bars denote ±SEM (n = 3). (E) Phenotypes of the flowers, inflorescence, and seeds of WT and Ri17 are representatively shown. Molecular Cell 2013 50, 504-515DOI: (10.1016/j.molcel.2013.04.027) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 JAV1 Controls Jasmonate-Mediated Plant Defense against B. cinerea (A) The phenotypes of WT, coi1-1, and two JAV1 RNAi transgenic plants (Ri17 and Ri55) 7 days after spray inoculation with B. cinerea or water (CK). (B) Relative fresh weight of the plants is indicated in (A) (mean ± SEM; n = 3; statistics by t test; **p < 0.01). (C) Disease severity of the plants indicated in (A). The brown and red bars indicate percentage of leaves with severe disease symptoms; the yellow or green bars indicate weak symptoms or no visible symptoms. (D) Survival percentage of the plants indicated in (A) (mean ± SEM; n = 3; statistics by t test; **p < 0.01). (E) Necrotic lesion area in each leaf described in (F) (mean ± SEM; n = 3; statistics by t test; **p < 0.01). (F) The phenotypes of the leaves from WT, coi1, Ri17, and Ri55 at 72 hr after drop inoculation with 5 μl spore suspension of B. cinerea or with water (CK). Scale bars = 2 mm. Molecular Cell 2013 50, 504-515DOI: (10.1016/j.molcel.2013.04.027) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 4 JAV1 Regulates Jasmonate-Mediated Plant Defense against Insects (A) Representative rosette leaves of WT, coi1-1, Ri17, and Ri55 after feeding without (CK) or with S. exigua. Scale bar = 2 mm. See also Figure S3. (B) Representative S. exigua larvae taken from rosette leaves of indicated plants at the start (0 days), 4 days, and 6 days. Scale bar = 2 mm. See also Figure S3. (C) Average weight of each S. exigua larva after 2, 4, and 6 days of feeding (mean ± SEM; n = 5; statistics by t test; *p < 0.05; **p < 0.01). See also Figure S3. (D) Survival percentage of plants of each genotype at the start (0 days), 10 days, and 20 days after inoculation with B. impatiens (mean ± SEM; n = 3; statistics by t test; *p < 0.05; **p < 0.01). See also Figure S3. (E) Effect of JAV1 on the preference of aphids in the two-choice test (mean ± SEM; n = 3; statistics by t test; **p < 0.01). (F) Effect of JAV1 on aphid development in the no-choice test. Aphid numbers on each plant were counted at 2 days postinoculation with 20 aphids (mean ± SEM; n = 3; statistics by t test; **p < 0.01). Molecular Cell 2013 50, 504-515DOI: (10.1016/j.molcel.2013.04.027) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 5 Genome-wide Transcriptional Analysis of the JAV1 RNAi Plants in Response to Wounding or Jasmonate Treatments (A and B) Hierarchical clustering display of expression ratios from wound-treated Ri17 versus wound-treated WT (A) and MeJA-treated Ri17 versus MeJA-treated WT (B) seedlings. Each matrix point shows the natural log intensity of one probe between WT and Ri17 at the indicated time points. Green squares represent a mean decrease in index, and red squares indicate a mean increase. Index values range from −3 to 3 (log2), as shown on the color scales at the bottom of the figure (see Supplemental Experimental Procedures). Defense: genes involving in defense response; Lipid: genes involved in lipid metabolism; Transcription: genes regulating transcription. See also Tables S3 and S4 and Figure S4. (C and D) Quantitative real-time PCR analysis of transcript levels of the defense-related genes in WT and JAV1 RNAi plants (Ri17 and Ri55) at the indicated time points after wounding (C) or MeJA treatment (D). Error bars denote ±SEM. Molecular Cell 2013 50, 504-515DOI: (10.1016/j.molcel.2013.04.027) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 6 JAV1 Acts as a Negative Controller of Plant Defense (A) The relative level of JAV1 transcripts in the WT and the JAV1 overexpression lines OX1 and OX2. Error bars denote ±SEM. (B and C) The phenotypes (B) and disease severity (C) of WT, OX1, and OX2 at 7 days after spray inoculation with B. cinerea or with water (CK). In (C), the brown and red bars indicate percentage of leaves with severe disease symptoms; the yellow or green bars indicate weak symptoms or no visible symptoms. (D) Necrotic lesion area in each leaf from WT, OX1, and OX2 at 72 hr after drop inoculation with 5 μl spore suspension of B. cinerea (mean ± SEM; n = 3; statistics by t test; **p < 0.01). (E) Photographs of representative rosette leaves of WT, OX1, and OX2 after feeding without (CK) or with S. exigua. Scale bar = 2 mm. (F) Average weight of each S. exigua larva after 6 days of feeding (mean ± SEM; n = 5; statistics by t test; **p < 0.01). (G and H) Quantitative real-time PCR analysis of transcript levels of VSP1, PDF1.2, and THI2.1 in WT, OX1, and OX2 at the indicated time points after MeJA induction (G) or wounding treatment (H). Error bars denote ±SEM. (I) Histochemical staining of GUS activity in the 10-day-old seedlings upon MeJA treatment. WT::pVSP-GUS, the WT Arabidopsis plants transgenic for the GUS reporter under control of the VSP1 endogenous promoter (pVSP-GUS) (Zheng et al., 2006); OX1::pVSP-GUS, the Arabidopsis plants overexpressing JAV1 and transgenic for pVSP-GUS by genetic crossing of the OX1 with the WT::pVSP-GUS line. (J) Histochemical staining of GUS activity in the 21-day-old seedlings upon wounding treatment. Red arrows representatively indicate the mechanically damaged tissue. Molecular Cell 2013 50, 504-515DOI: (10.1016/j.molcel.2013.04.027) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 7 JAV1 Degradation is Induced by Jasmonate or Wounding via the 26S Proteasome Pathway in a COI1-Dependent Manner (A) Immunoblot analysis of Arabidopsis seedlings expressing Myc-JAV1 that were treated with the proteasome inhibitor MG132 or DMSO (mock) for 1 hr, followed by continuous treatment with 100 μM MeJA at the indicated time points. Actin levels were served as loading control. (B) GUS activity in Arabidopsis plants expressing JAV1-GUS (WT::JAV1-GUS) decreased upon MeJA treatment. MG132 attenuated the MeJA-induced reduction of GUS activity. (C) MeJA treatment decreased GUS activity in WT::JAV1-GUS plants but not in the coi1-1 mutant plants expressing JAV1-GUS (coi1::JAV-GUS). (D) Wounding treatment decreased the GUS activity in WT::JAV1-GUS plants but not in the coi1::JAV1-GUS plants. Lower panel: enlarged leaves from each genotype; red arrows indicate the mechanically damaged tissue. See also Figure S5. Molecular Cell 2013 50, 504-515DOI: (10.1016/j.molcel.2013.04.027) Copyright © 2013 Elsevier Inc. Terms and Conditions