Extended Data Fig. 5: The EDS1-PAD4-ADR1s module is involved in mekk1 cell death.
From: Assembly of helper NLR resistosome clusters upon activation of a coiled-coil NLR
a, Expression of ADR1 or ADR1-L2 in adr1/L1/L2 restores the cell death triggered by silencing MEKK1. The plant images were taken 3 weeks after inoculation with Agrobacterium carrying the indicated VIGS vectors in two independent p35S::gADR1-HA (1 & 2) and p35S::ADR1-L2-HA (3 & 4) transgenic lines in adr1/L1/L2 (top). Scale bar, 1 cm. Protein expression of transgenic lines is shown by immunoblotting using an α-HA antibody (upper panel) with CBB staining of RBC as the loading control (bottom panel). b, Expression of EDS1 in eds1-2 restores the cell death triggered by silencing MEKK1. Two lines of transgenic plants carrying the p35S::EDS1-HA (1 & 2) in eds1-2 are shown. Scale bar, 1 cm. Protein expression in transgenic lines is shown at the bottom. c, The adr1/L1/L2, eds1-2, and pad4-1 mutants suppress cell death and H2O2 accumulation triggered by RNAi-MEKK1. Quantification of relative trypan blue-stained cell death (top) and 3,3′-diaminobenzidine (DAB)-stained H2O2 accumulation (bottom) is presented for Fig. 2b. Images in Fig. 2b were converted to grayscale, and the mean value of the area was scored by ImageJ. Data are shown as means ± SEM with RNAi-Ctrl (-) in WT set to 1.0 (n = 5). d, The adr1/L1/L2 and summ2 mutants augment the fresh weight of mekk1. The quantification was performed for Fig. 2d. Data are shown as mean ± SEM (n = 5). e, The adr1/L1/L2/ mutant suppresses the elevated expression of PR1 and PR2 in mekk1. RT-qPCR was performed with the detached leaves from Fig. 2d. The expression of PR1 and PR2 was normalized to the expression of UBQ10. The data are shown as mean ± SD (n = 3). f, The growth defects of mpk4 are reduced in adr1/L1/L2/mpk4 and mekk2/mpk4 mutants. Three-week-old plants grown on 1/2 MS medium with the indicated genotypes are shown. Scale bar, 0.5 cm. g, The adr1/L1/L2 and mekk2 mutants augment the fresh weight of mpk4. The quantification was performed with the detached leaves from (f). Data are shown as mean ± SEM (n = 5). h, The adr1/L1/L2 mutant suppresses the elevated expression of PR1 and PR2 in mpk4. RT-qPCR was performed with the detached leaves from (f). The expression of PR1 and PR2 was normalized to the expression of UBQ10. Data are shown as mean ± SD (n = 3). i, SUMM2ac-triggered cell death and ion leakage are attenuated in the N. benthamiana eds1 mutant. SUMM2 or SUMM2ac was expressed in WT and eds1 N. benthamiana plant leaves. Cell death was documented under UV light at 24 hpi and 42 hpi (top). At 18 hpi, 1 cm-diameter leaf discs were collected and immersed in water for the ion leakage assay. Conductivity was measured at 6 hr and 24 hr after immersing. The data are shown as three repeats (n = 15, bottom). Box plots show the first and third quartiles as bounds of box, split by the medians (lines), with whiskers extending to the minimum and maximum values. j, Arabidopsis EDS1-PAD4, but not EDS1-SAG101, restores SUMM2ac-triggered cell death in the N. benthamiana epss mutant. DEX::SUMM2ac-HA and ADR1-L1-TagRFP were co-expressed in the N. benthamiana epss mutant with either a vector control (-), EDS1-FLAG and PAD4-FLAG, or EDS1-FLAG and SAG101-FLAG. Two days post Agrobacterium infiltration, plants were treated with 50 μM DEX for 6 hr, and images were captured using a confocal laser scanning microscope (left bottom, Scale bar, 10 μm). Cell death was imaged 24 hpi under UV light (left top). Quantification of relative cell death is shown on the right. Images were converted to grayscale, and the mean intensity of the inoculated area was measured using ImageJ, with background signal from the non-inoculated area subtracted. Data represent means ± SEM, normalized to SUMM2ac/ADR1-L1-TagRFP (set to 1.0, n = 6). k, SUMM2ac induces cell death independently of NRG1. Agrobacterium carrying DEX::SUMM2ac-HA (top) or DEX::RBA1-HA (bottom) was inoculated into WT and nrg1-1 N. benthamiana leaves. After 30 hr, the leaves were treated with 50 µM DEX for 24 hr, and cell death was documented under UV light. RBA1, but not SUMM2ac-induced cell death, was reduced in the nrg1-1 mutant. In immunoblots (a, b), RBCs were run on the same gels and used as loading controls. Statistical analysis was performed using one-way ANOVA followed by the Tukey test (c, d, e, g, h, j) and a two-tailed Student’s t-test (i). Experiments were repeated three times with similar results. For gel source data, see Supplementary Fig. 1.
