1. 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 (December 2014)
DOI: /mp/ssu097
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

2. 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 ( 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, DOI: ( /mp/ssu097)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions