P-Loop-Dependent NLR SNC1 Can Oligomerize and Activate Immunity in the Nucleus  Fang Xu, Yu Ti Cheng, Paul Kapos, Yan Huang, Xin Li  Molecular Plant  Volume 7, Issue 12, Pages 1801-1804 (December 2014) DOI: 10.1093/mp/ssu097 Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 1 The P-Loop-Dependent NLR SNC1 Can Oligomerize in Both Cytosol and Nuclear Compartments, and Its Nuclear Accumulation Is Essential for Its Defense Activation. (A) SNC1(aaa)–GFP P-loop mutant protein level in nine randomly selected transgenic lines. Total protein of 2-week-old plate-grown plants were extracted and subjected to Western blot using an anti-GFP antibody. The transgenic lines of pSNC1::SNC1(aaa)–GFP were randomly selected. pSNC1::SNC1–GFP T1-2 plants, shown as in Supplemental Figure 2, were used as positive control. (B) Four-week-old soil-grown plants of wild-type (WT), snc1, and homozygous transgenic plants from two independent pSNC1::SNC1–GR–3×HA T1 lines. Plants were mock-treated or DEX-treated when they were 2 weeks old. The pictures were taken 2 weeks after DEX treatment. (C) Subcellular fractionation of DEX-treated SNC1–GR–3×HA transgenic plants. Plant tissue was collected at 0, 4, and 8h after DEX treatment. N, nuclear fraction; ΔN, nuclear-depleted fraction. PEPC and Histone H3 were used as cytosolic and nuclear fraction controls, respectively. (D) Time course of PR1 and PR2 expression in 4-week-old soil-grown SNC1–GR–3×HA transgenic plants treated with DEX. The expression of PR1 and PR2 was normalized to that of ACTIN1. The value of each genotype was then compared to that of non-transgenic WT. Bars represent means of three replicates ± SD. (E) Time course of SNC1 expression in 4-week-old soil-grown SNC1–GR–3×HA transgenic plants treated with DEX. The expression of SNC1 was normalized to that of ACTIN1. The value of each genotype was then compared to that of non-transgenic WT. Bars represent means of three replicates ± SD. (F) Quantification of H.a. Noco2 sporulations on the plants of the indicated genotypes. Two-week-old seedlings were mock- or DEX-treated 24h before H.a. Noco2 (100 000 spores ml−1) inoculation. Bars represent means of four replicates ± SD. (G) Cytosolic IP of SNC1–3×FLAG-ZZ transgenic plants in Col background. The cytosolic fraction of SNC1–3×FLAG-ZZ plants was subjected to IP with anti-FLAG agarose beads. The input and elution fraction were then detected using Western blot probed with anti-SNC1 antibody. ‘+’ indicates the samples incubated with anti-FLAG agarose beads while ‘−’ indicates the samples incubated with protein A agarose beads, which were not conjugated with anti-FLAG antibody and served as the negative control. Anti-PEPC was used as a cytosolic marker while anti-H3 was used as a nuclear marker. ‘**’ indicates SNC1-FLAG-ZZ and ‘*’ represents endogenous SNC1. (H) Nuclear IP of tissue collected from N. benthamiana co-expressing SNC1–3×FLAG and SNC1–3×HA. The nuclear protein from leaves co-infiltrated with Agrobacterium containing pCambia1300–35S::SNC1–3×FLAG and pCambia1300::35S-SNC1–3×HA, respectively, were subjected to IP with anti-FLAG agarose beads. The input and elution fractions were then detected using Western blot probed with anti-FLAG or anti-HA antibody. ‘+’ indicates the samples incubated with anti-FLAG agarose beads, while ‘−’ indicates the samples incubated with protein A agarose beads, which were not conjugated with anti-FLAG antibody and served as the negative control. Molecular Plant 2014 7, 1801-1804DOI: (10.1093/mp/ssu097) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions