1. Plant TRAF Proteins Regulate NLR Immune Receptor Turnover
Shuai Huang, Xuejin Chen, Xionghui Zhong, Meng Li, Kevin Ao, Jianhua Huang, Xin Li 
Cell Host & Microbe 
Volume 19, Issue 2, Pages (February 2016)
DOI: /j.chom
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2. Cell Host & Microbe 2016 19, 204-215DOI: (10.1016/j.chom.2016.01.005)
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3. Figure 1 Characterization and Positional Cloning of muse13-1
(A) Morphology of 4-week-old Col-0, snc1, mos2 snc1 npr1, and muse13-1 mos2 snc1 npr1 plants. Bar, 1 cm.
(B) PR1 and PR2 gene expression in 2-week-old plate-grown seedlings of indicated genotypes. Equal amounts of cDNA were used for RT-PCR analysis. ACT7 serves as loading control.
(C) Sporulation of oomycete Hpa Noco2 on the indicated plants. Bars represent means ± SD (n = 5). Letters indicate statistical difference (p < 0.001, one-way ANOVA, Tukey’s post hoc test).
(D) Morphology of 4-week-old Col-0, snc1, SALK_ (AT1G03770), snc1 SALK_117958, SALK_ (AT1G04200), snc1 SALK_056393, SALK_ (AT1G04300), snc1 SALK_026088, SALK_ (AT1G04450), snc1 SALK_128174, SAIL_723_C07 (AT1G04670), and snc1 SAIL_723_C07. Bar, 1 cm.
(E) Fresh weight of plants as shown in (D). Error bars represent means ± SD (6 plants per genotype; ∗∗∗ indicates significance p < 0.001, one-way ANOVA analysis between snc1 and double mutants).
(F) Allelism test between muse13-1 and SALK_ Morphology of 4-week-old Col-0, snc1, SALK_026088, an F1 plant from a cross between SALK_ and muse13-1 mos2 snc1 npr1, and muse13-1 mos2 snc1 npr1 plants. snc1 is semi-dominant in defense but recessive in morphology (heterozygous SNC1/snc1 plants are WT like), while mos2 and npr1 are recessive. In the F1 plant, snc1, mos2, and npr1 are all heterozygous; thus, they should be WT in morphology if muse13-1 and SALK_ complement each other. Severely dwarfed F1 progeny observed here suggest failed complementation between muse13-1 and SALK_ (both are recessive), which reduces the growth of heterozygous snc1/SNC1.
(G) Gene structure of AT1G Mutation sites of muse13-1 and muse13-2 are shown.
(H) DNA alignment of muse13-1 and MUSE13 where the muse13-1 mutation occurs. Corresponding amino acids are shown below. Star indicates the muse13-1 mutation site, leading to Arg683 to stop codon change.
(I) Protein structure of MUSE13. The TRAF domain is indicated as a gray box. The arrows indicate muse13-1 and muse13-2 mutation sites. See also Table S1 and Figures S1 and S2.
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4. Figure 2 MUSE13 Functions Redundantly with MUSE14
(A) Plant morphology of 4-week-old Col, snc1, muse13-2, muse14-1, and muse13-2 muse14-1 (m.m.) double mutant plants. Bar, 1 cm.
(B) RT-PCR analysis of PR gene expression in 4-week-old soil-grown plants. Error bars represent means ± SD (n = 3). Letters indicate statistical difference (p < 0.001, one-way ANOVA, Tukey’s post hoc test).
(C) Sporulation of Hpa Noco2 on the indicated genotypes. Error bars represent means ± SD (n = 5). Letters indicate statistical difference (p < 0.001, one-way ANOVA, Tukey’s post hoc test). See also Figures S3–S6.
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5. Figure 3
The Autoimmunity of muse13-2 muse14-1 Partially Depends on SNC1
(A) Morphology of 4-week-old Col-0, muse13-2 muse14-1 (m.m.) and two suppressor mutants (9-1 and 84-1) identified in the m.m. double mutant background. Bar, 1 cm.
(B) Schematic structure of SNC1 protein. Mutation sites of snc1, 9-1, and 84-1 are indicated.
(C) Plant morphology of the indicated genotypes. snc1-r1 is a null allele of snc1. Bar, 1 cm.
(D) RT-PCR analysis of PR gene expression in 2-week-old plate-grown seedlings of the indicated genotypes. Error bars represent means ± SD (n = 3). Letters indicate statistical difference (p < 0.01, one-way ANOVA, Tukey’s post hoc test).
(E) Hpa Noco2 sporulation on the indicated genotypes. Error bars represent means ± SD (n = 5). Letters indicate statistical differences (p < 0.01, one-way ANOVA, Tukey’s post hoc test).
(F–H) Growth of Pst DC3000 (AvrRpt2) (F), Pst DC3000 (AvrRps4) (G), and Psm ES4326 (H) on the indicated genotypes. Error bars represent means ± SD (n = 5). Letters indicate statistical difference (p < 0.01, two-way ANOVA). Bacterial inoculum was diluted to an OD600 = in 10mM MgCl2. See also Figure S7.
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6. Figure 4 MUSE13 Is Involved in Protein Turnover of SNC1 and RPS2
(A) Morphology of 4-week-old Col-0, snc1, muse13-2, muse14-1, eds1-2, muse13-2 muse14-1 (m.m.), and m.m. eds1-2 plants. Bar, 1 cm.
(B) RT-PCR analysis of PR1 and PR2 gene expression in 4-week-old soil-grown plants of the indicated genotypes.
(C) Sporulation of Hpa Noco2 on plants of the indicated genotypes. Error bars represent means ± SD (n = 5). Letters indicate statistical difference (p < 0.001, one-way ANOVA followed by Tukey’s post hoc test).
(D) RT-PCR analysis of SNC1 expression in the indicated genotypes. Total RNA was isolated from 2-week-old plants grown on half strength MS media. Expression of SNC1 was normalized to ACT7. Error bars represent means ± SD (n = 3). Letters indicate statistical difference (p < 0.01, one-way ANOVA followed by Tukey’s post hoc test).
(E) Immunoblot analysis of SNC1 protein levels in the indicated genotypes. Total protein was extracted from 4-week-old soil-grown plants. Equal loading was shown by Ponceau S staining. Relative SNC1 band intensity is indicated above (normalized to loading control and relative to Col-0).
(F) Immunoblot analysis of RPS2-HA protein levels in WT and muse13-2 muse14-1 (m.m.) background. Relative RPS2-HA band intensity is shown below (normalized to loading control and relative to RPS2-HA parent line).
(G) RT-PCR analysis of RPS2 expression in the indicated genotypes. Error bars represent means ± SD (n = 3). Letters indicate statistical difference (p < 0.01, one-way ANOVA followed by Tukey’s post hoc test).
(H) Immunoblot analysis of RPS2-HA protein levels in WT and muse13-2 muse14-1 eds1-2 (m.m. eds1-2) background. Relative RPS2-HA band intensity is shown below (normalized to loading control and relative to RPS2-HA parent line). Vertical line: bands are from the same blot with relevant treatments placed together. See also Figure S7.
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7. Figure 5
Overexpression of MUSE13-GFP Leads to Decreased NLR Protein Levels
(A) Plant morphology of 4-week-old Col-0, snc1, and two independent MUSE13-GFP transgenic lines in snc1 background (T1-5 and T1-6). Bar, 1 cm.
(B) Fresh weight of plants shown in (A). Bars represent means ± SD (n = 6). Letters indicate statistical difference (p < 0.01, one-way ANOVA followed by Tukey’s post hoc test).
(C) Immunoblot analysis of MUSE13-GFP expression in T1-5 and T1-6 transgenic lines. Relative MUSE13-GFP band intensity is shown below (normalized to loading control, relative to T1-5).
(D) SNC1 protein levels in the indicated genotypes. Relative SNC1 band intensity is quantified below (normalized to loading control, relative to WT).
(E) RT-PCR analysis of PR1 and PR2 gene expression in 2-week-old plate-grown seedlings of the indicated genotypes. Error bars represent means ± SD (n = 3). Letters indicate statistical difference (p < 0.01, one-way ANOVA followed by Tukey’s post hoc test).
(F) Immunoblot analysis of MUSE13-GFP and RPS2-HA where MUSE13-GFP was transformed into RPS2-HA line. One transgenic line (T1-2) was shown. Relative RPS2-HA band intensity is shown below (normalized to loading control, relative to RPS2-HA parent line). See also Figure S7.
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8. Figure 6 MUSE13-GFP Associates with CPR1-FLAG and SNC1 In Planta
(A) Immunoprecipitation of MUSE13-GFP by CPR1-FLAG in N. Benthamiana. MUSE13-GFP input bands were only obvious after long exposure time.
(B) Immunoprecipitation of MUSE13-GFP by CPR1-FLAG in Arabidopsis plants transformed with the two transgenes. Star indicates MUSE13-GFP degradation product. CoIP carried out with protein A beads without anti-FLAG served as negative control.
(C) MG132 stabilizes MUSE13-GFP. Total protein was extracted from 2-week-old plate-grown MUSE13-GFP transgenic Arabidopsis seedlings. Equal amount of protein was incubated at room temperature in the presence or absence of 100 μM MG132 for the indicated minutes. MUSE13-GFP was visualized by immunoblot with anti-GFP.
(D) Immunoprecipitation of SNC1 by MUSE13-FLAG-ZZ in Arabidopsis. SNC1 was detected using an α-SNC1 antibody (Li et al., 2010). The low molecular weight bands represent degradation products of MUSE13-FLAG-ZZ. The difference in band intensity of the degradation products compared with (B) is likely caused by differential immunoprecipitation efficiency using different antibodies. CoIP carried out with protein A beads without anti-FLAG served as negative control. Vertical line: samples were run on the same blot with relevant treatments placed together.
(E and F) Immunoprecipitation of snc1-GFP (E) and SNC1(aaa)-GFP (F) by MUSE13-FLAG-ZZ in N. Benthamiana. snc1-GFP (E) input was only obvious after long exposure time. See also Figure S7.
Cell Host & Microbe  , DOI: ( /j.chom )
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9. Figure 7
MUSE13-GFP Associates with RPS2-FLAG-ZZ In Planta and It Also Self-Associates
(A) Immunoprecipitation of MUSE13-GFP by RPS2-FLAG-ZZ in N. Benthamiana.
(B) Immunoprecipitation of MUSE13-GFP by MUSE13-FLAG-ZZ in N. Benthamiana. See also Figure S7.
Cell Host & Microbe  , DOI: ( /j.chom )
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