Extended Data Fig. 7: SUMM2ac promotes the association between EDS1/PAD4 and ADR1-L1.
From: Assembly of helper NLR resistosome clusters upon activation of a coiled-coil NLR
a, b, SUMM2 is in close proximity to EDS1 and PAD4, but not ADR1-L1, with the Förster resonance energy transfer (FRET)-fluorescence lifetime imaging (FLIM) assays. The indicated protein pairs, SUMM2-GFP and EDS1-mCherry, PAD4-mCherry, or ADR1-L1-mCherry, were expressed in Arabidopsis WT protoplasts for 16 hr, and FRET-FLIM was visualized using a confocal laser scanning microscope. Localization of the SUMM2-GFP and EDS1-mCherry/PAD4-mCherry/ADR1-L1-mCherry is shown in the first column (green) and second column (red), respectively (a). The lifetime (τ) distribution (third column), and apparent FRET efficiency (fourth column) are presented as pseudo-color images according to the scale. The GFP mean fluorescence lifetime (τ) values were statistically analyzed and are shown as mean ± SEM (n = 15) (b). Scale bar, 10 µm. c, EDS1-PAD4 interaction in WT and adr1/L1/L2. PAD4-FLAG or a vector control (-) was coexpressed with EDS1-HA in Arabidopsis WT or adr1/L1/L2 protoplasts. Total proteins were immunoprecipitated with α-FLAG affinity beads and immunoblotted by an α-FLAG or α-HA antibody (top two panels). Proteins before immunoprecipitation are shown as input controls (bottom two panels). d, SUMM2 associates with EDS1 and PAD4, but not SAG101. A vector control (-) or SUMM2-HA was co-expressed with EDS1-FLAG, as well as PAD4-FLAG or SAG101-FLAG in Arabidopsis protoplasts. Total proteins were immunoprecipitated with α-HA affinity beads and immunoblotted with an α-FLAG or α-HA antibody (top two panels). Proteins before immunoprecipitation are shown as input controls (bottom two panels). e, SUMM2ac promotes the association of EDS1-PAD4 with ADR1-L1 in FRET-FLIM assays. PAD4-GFP/EDS1-FLAG were co-expressed with ADR1-L1-mCherry in the presence of SUMM2 or SUMM2ac in Arabidopsis protoplasts for 16 hr, and FRET-FLIM was visualized using a confocal laser scanning microscope. Localization of the PAD4-GFP and ADR1-L1-mCherry is shown in the first column (green) and second column (red), respectively. The lifetime (τ) distribution (third column), and apparent FRET efficiency (fourth column) are presented as pseudo-color images according to the scale. The GFP mean fluorescence lifetime (τ) values are presented in Fig. 2i. f, SUMM2ac promotes the association of EDS1-PAD4 with ADR1-L1 in Co-IP assays. ADR1-L1-FLAG was co-expressed with a vector (-) or EDS1-HA/PAD4-HA in the presence or absence of DEX::SUMM2ac-GFP or DEX::SUMM2-GFP in N. benthamiana. Two days post-infiltration of Agrobacterium carrying different constructs, SUMM2 was induced by 50 μM DEX treatment for 4 hr, and proteins were extracted and subjected to immunoprecipitation with α-HA affinity beads, followed by immunoblotting using an α-FLAG or α-HA antibody (top two panels). Proteins before immunoprecipitation are shown as input controls (bottom three panels). g, MEKK2 promotes the association of EDS1-PAD4 with ADR1-L1 in Co-IP assays. ADR1-L1-FLAG was co-expressed with a vector (-) or EDS1-HA/PAD4-HA with or without MEKK2-MYC in Arabidopsis WT protoplasts. Total proteins were immunoprecipitated with α-HA affinity beads and immunoblotted by an α-FLAG or α-HA antibody (top two panels). Proteins before immunoprecipitation are shown as input controls (bottom two panels). h, HopAI1 promotes the association of EDS1-PAD4 with ADR1-L1 in Arabidopsis protoplasts. ADR1-L1-FLAG was co-expressed with a vector (-) or EDS1-HA/PAD4-HA with or without HopAI1-MYC in Arabidopsis protoplasts. Total proteins were immunoprecipitated with α-HA affinity beads and immunoblotted by an α-FLAG or α-HA antibody (top two panels). Proteins before immunoprecipitation are shown as input controls (bottom three panels). i, SUMM2ac exhibits reduced interaction with PAD4 compared to SUMM2. PAD4-FLAG was co-expressed with SUMM2-HA or SUMM2ac-HA in Arabidopsis WT protoplasts. Total proteins were immunoprecipitated with α-HA affinity beads and immunoblotted by an α-FLAG or α-HA antibody (top two panels). Proteins before immunoprecipitation are shown as input controls (bottom two panels). j, SUMM2ac promotes EDS1-PAD4 oligomerization in the presence of ADR1-L1. EDS1-GFP, PAD4-GFP, and ADR1-L1-FLAG were co-expressed with or without SUMM2ac-HA in Arabidopsis protoplasts. Proteins were analyzed by BN-PAGE or regular SDS-PAGE and immunoblotted with an α-GFP, α-FLAG, or α-HA antibody. In immunoblot (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 (b). Experiments were repeated three times with similar results. For gel source data, see Supplementary Fig. 1.
