Volume 9, Issue 12, Pages 1620-1633 (December 2016) Identification of Methylosome Components as Negative Regulators of Plant Immunity Using Chemical Genetics  Shuai Huang, Aruna Balgi, Yaping Pan, Meng Li, Xiaoran Zhang, Lilin Du, Ming Zhou, Michel Roberge, Xin Li  Molecular Plant  Volume 9, Issue 12, Pages 1620-1633 (December 2016) DOI: 10.1016/j.molp.2016.10.006 Copyright © 2016 The Author Terms and Conditions

Figure 1 Isolation of Ro 8-4304 that Suppresses the Autoimmune Phenotypes of chs3-2D. (A) Schematic of the primary screen. Seeds were spotted into 96-well screening plates and vernalized for 2 days at 4°C. Compounds were added and plants were allowed to grow in a growth chamber for another 14 days before phenotyping. (B) The chemical structure of Ro 8-4304. (C) The growth of plants on half-strength MS medium at 16°C in the presence or absence of Ro 8-4304 at a final concentration of 15 μM. The photograph was taken when plants were 18 days old. Bar, 1 cm. (D) Relative fresh weight of plants in (C). Error bars represent mean ± SD (n = 3 with six plants each). Stars indicate statistical difference (Student's t-test, p < 0.001). The experiment was repeated three times with similar results. (E) PR gene expression of plants in (C). Plants were grown at 16°C in the presence or absence of 15 μM Ro 8-4304 for 3 weeks before RNA was isolated. (F) Chemical feeding assay. A bacterial suspension containing 15 μM Ro 8-4304 or water was used in this assay. Final inoculum was diluted to an OD600 = 0.001 in 10 mM MgCl2. Letters indicate statistical difference (p < 0.01, ANOVA). Error bars represent means ± SD (n = 5). The experiment was repeated three times with similar results. (G) Growth of Hpa Noco2 spores on Col-0 and chs3-2D plants. Plants were sprayed with Ro 8-4304 at a final concentration of 15 μM 1 day prior to Hpa Noco2 infection. Growth of oomycete spores was quantified 1 week after infection. The experiment was repeated three times with similar results. Molecular Plant 2016 9, 1620-1633DOI: (10.1016/j.molp.2016.10.006) Copyright © 2016 The Author Terms and Conditions

Figure 2 Map-Based Cloning of rim1-1. (A) Map position of rim1-1. Representative BAC clones are indicated. (B) Gene structure of AT5G62290, the mutation site of rim1-1 (icln-1), and the insertion site icln-2 are indicated. (C) Genomic DNA sequence alignment between icln-1 and ICln. The icln-1 mutation is highlighted in red. Star indicates the stop codon change. (D) The growth of Col-0, chs3-2D, rim1-1 chs3-2D and 35S::ICln-GFP in rim1-1 chs3-2D (two independent homozygous T2 lines are shown). Plants were grown on half-strength MS or 15 μM Ro 8-4304 plates. The photograph was taken when the plants were 3 weeks old. Bar, 1 cm. (E) PR gene expression analysis of plants shown in (D). ACT7 serves as a loading control. Molecular Plant 2016 9, 1620-1633DOI: (10.1016/j.molp.2016.10.006) Copyright © 2016 The Author Terms and Conditions

Figure 3 Characterization of the icln Single Mutants. (A) Morphology of Col-0, icln-1, and icln-2 plants. The photograph was taken when the plants were 4 weeks old. Bar, 1 cm. (B) Fresh weight of plants in (A). Error bars represent means ± SD (n = 6). (C) Morphology of Col-0, icln-1, and icln-2 plants at 16°C in the presence (+) or absence (−) of 15 μM Ro 8-4304. The photograph was taken when the plants were 3 weeks old. Bar, 1 cm. (D) Fresh weight of plants in (C). Error bars represent mean ± SD (n = 6 with five plants each). The experiment was repeated once with a similar result. (E–H) Gene expression analysis of Col-0, icln-1, and icln-2 plants. Total RNA was isolated from 3-week-old soil-grown plants. Letters indicate statistical difference (p < 0.01, ANOVA). Error bars represent means ± SD (n = 3). (I) The growth of Hpa Noco2 on the indicated genotypes. Two-week-old soil-grown seedlings were sprayed with Hpa Noco2 conidiospores at a final concentration of 105 spores per mL of water. The growth of the spores was quantified 1 week after infection. Letters indicate statistical difference (one-way ANOVA followed by Bonferroni post-test; p < 0.01). Error bars represent means ± SD (n = 5). The experiment was repeated three times with similar results. (J) Effect of Ro 8-4304 on Hpa Noco2 growth on indicated genotypes. Ten-day old plants were sprayed with Ro 8-4304 at a final concentration of 15 μM 1 day prior to Noco2 infection. Water was used as negative control. Growth of spores was quantified 1 week after infection. Letters indicate statistical difference (one-way ANOVA followed by Bonferroni post-test; p < 0.01). Error bars represent means ± SD (n = 5). (K) The growth of Pst DC3000 on Col-0, icln-1, and icln-2 plants. Error bars represent means ± SD (n = 5). Letters indicate statistical difference (one-way ANOVA followed by Bonferroni post-test; p < 0.01). Bacterial inoculum was diluted to an OD600 = 0.001 in 10 mM MgCl2. Molecular Plant 2016 9, 1620-1633DOI: (10.1016/j.molp.2016.10.006) Copyright © 2016 The Author Terms and Conditions

Figure 4 Two-Electrode Voltage Clamp Analysis of AtICln in Xenopus Oocytes. (A and B) Whole-cell currents were recorded on oocytes injected with AtICln mRNA and water as the control. The holding potential was −20 mV, and currents were elicited by stepping pulses to voltages between −140 to +60 mV in 20 mV increments. (C) I–V plot for the currents in AtICln and control oocytes. Error bars are the SEM of eight independent oocytes. Molecular Plant 2016 9, 1620-1633DOI: (10.1016/j.molp.2016.10.006) Copyright © 2016 The Author Terms and Conditions

Figure 5 AtICln Associates with SmD3b and PRMT5 In Planta, and at Least SmD3b Is Required for Sensitivity to Ro 8-4304. (A) The growth of Col-0, chs3-2D, icln-1, icln-2, icln-1 chs3-2D, prmt5, smd3b, and smd3b chs3-2D on half-strength MS medium in the absence or presence of 15 μM Ro 8-4304 at 16°C. The photograph was taken when the plants were 3 weeks old. Inset: an enlarged view of smd3b chs3-2D. Bar, 1 cm. (B) PR gene expression of plants in (A). ACT7 serves as a loading control. (C and E) AtICln interacts with SmD3b (C) and PRMT5 (E) in a split luciferase assay in N. benthamiana. (D and F) AtICln-Flag co-immunoprecipitates with SmD3b-HA (D) and PRMT5-HA (F) in N. benthamiana. Star indicates a nonspecific band. Molecular Plant 2016 9, 1620-1633DOI: (10.1016/j.molp.2016.10.006) Copyright © 2016 The Author Terms and Conditions

Figure 6 Single Mutant Analysis of smd3b and prmt5-1. (A) The splicing pattern of CHS3 determined using RT–PCR. The CHS3 transcript was amplified using primers AS-F and AF-R for 41 cycles. An equal amount of cDNA was used for each reaction. ACT7 serves as a loading control. Numbers below indicate the percentage of the alternatively spliced form versus total transcript of CHS3 normalized to ACT7. RNA template was used as a negative control, where no PCR amplification was observed. (B) The growth morphology of Col-0, icln-1, icln-2, prmt5-1, and smd3b. The photograph was taken when the plants were 3 weeks old. Bar, 1 cm. (C) Fresh weight of plants in (B). Error bars represent means ± SD (n = 6). (D) The growth of Hpa Noco2 on plants shown in (D). Letters indicate statistical difference (one-way ANOVA followed by Bonferroni post-test; p < 0.01). Error bars represent means ± SD (n = 5). (E and F) RT–PCR analysis of PR gene expression of plants in (B). Error bars represent means ± SD (n = 3). Molecular Plant 2016 9, 1620-1633DOI: (10.1016/j.molp.2016.10.006) Copyright © 2016 The Author Terms and Conditions