Extended Data Fig. 6: The EDS1-PAD4-ADR1s module functions downstream of MEKK2 and SUMM2.
From: Assembly of helper NLR resistosome clusters upon activation of a coiled-coil NLR
a, Plant dwarfism and growth defects triggered by overexpressing MEKK2 in WT are alleviated in the adr1/L1/L2 mutant. A total of 32 and 34 independent primary (T1) transgenic plants carrying p35S::MEKK2-HA in WT or adr1/L1/L2 were characterized, respectively. Four-week-old plants representing different levels of dwarfism labeled with the percentage of the total plants are shown (top). Scale bar, 1 cm. Protein expression of MEKK2-HA in transgenic plants with CBB staining for RBC as the loading control is shown on the bottom. b, c, The cell death and H2O2 accumulation triggered by overexpressing MEKK2 in WT are alleviated in the adr1/L1/L2 mutant. Detached leaves from (a) were stained by trypan blue for cell death and DAB for H2O2 accumulation. Scale bar, 0.5 cm (b). Images were converted to grayscale, and the mean value of the area was scored by ImageJ. Quantification data are shown as means ± SEM with WT set to 1.0 (n = 5). d, The adr1/L1/L2 mutant suppresses the PR gene expression triggered by overexpressing MEKK2. RT-qPCR was performed with the detached leaves from (a), and the expression of PR1 and PR2 was normalized to UBQ10. The data are shown as mean ± SD (n = 3). e, f, Plant growth defects triggered by overexpressing MEKK2 are alleviated in eds1-2. A total of 42 and 48 independent T1 transgenic plants carrying p35S::MEKK2-HA in WT and eds1-2 were characterized, respectively. Four-week-old plants representing different levels of dwarfism labeled with the percentage of total plants are shown in (e). Scale bar, 1 cm. Protein expression in transgenic plants is shown in (f). Total proteins were immunoblotted using an α-HA antibody (top panel). CBB staining for RBC is shown as the loading control (bottom panel). g, Protein expression of SUMM2ac-HA in transgenic plants. Total proteins were isolated from plants in Fig. 2e and immunoblotted using an α-HA antibody (top). CBB staining for RBC is shown as the loading control (bottom). h, The adr1/L1/L2 mutant suppresses the PR gene expression triggered by overexpressing SUMM2ac. RT-qPCR analysis was performed with the detached leaves from Fig. 2e, and the expression of PR1 and PR2 was normalized to UBQ10. The data are shown as the mean ± SD (n = 3). i, Plant growth defects triggered by overexpressing ADR1-HA are similar in WT and summ2-8. A total of 32 and 34 independent T1 transgenic plants carrying p35S::gADR1-HA in WT and summ2-8 were characterized, respectively. Two representative three-week-old plants with growth defects are shown (top). Scale bar, 1 cm. Protein expression in transgenic plants is shown in the bottom. j, Plant growth defects triggered by expressing HopAI1 are alleviated in the adr1/L1/L2 and summ2-8 mutants. A total of 55, 26, and 36 independent T1 transgenic plants carrying p35S::HopAI1-HA in WT, adr1/L1/L2, and summ2-8 were characterized, respectively. Three-week-old plants representing different levels of dwarfism labeled with the percentage of total plants are shown (top). Scale bar, 1 cm. Total proteins in transgenic plants were immunoblotted using an α-HA antibody with CBB staining for RBC as the loading control (bottom). In immunoblots (a, f, g, i, j), 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, h). Experiments were repeated three times with similar results. For gel source data, see Supplementary Fig. 1.
