Extended Data Fig. 9: The active SUMM2 induces the formation of ADR1-L1 puncta before the occurrence of cell death in plants.

a, Self-association of ADR1-L1. The ADR1-L1-nYFP and ADR1-L1-cYFP were co-expressed in N. benthamiana for two days by Agrobacterium infiltration, and YFP signals were detected using a confocal laser scanning microscope. FM4-64 was used as a PM marker. Fluorescence intensities were measured along the dotted line (bottom). Scale bar, 25 μm (left). Quantification of co-localization between ADR1-L1-GFP and FM4-64 in N. benthamiana epidermal cells using Mander’s overlap coefficient. Data are shown as mean ± SD with each dot representing an individual cell (n = 16). b, SUMM2^ac stimulates ADR1-L1-YFP to shift from a uniformly to discontinuously distributed PM localization along the cell periphery under the confocal microscope. The ADR1-L1-nYFP and ADR1-L1-cYFP were co-expressed with the vector control (-), DEX::SUMM2-HA, or DEX::SUMM2^ac-HA in N. benthamiana leaves for two days, and the images were taken using confocal laser scanning microscopy at 4 hr post 50 μM DEX induction. The top panel shows single-plane images, and the bottom panel shows maximum projections from 16 images with a z-step of 1 µm. Scale bar, 25 μm. c, SUMM2^ac induces the immobile ADR1-L1-YFP puncta. Time-lapse dynamics analysis with the leaves from (b) shows that ADR1-L1-YFP puncta are static with a period of 75 s. Scale bar, 25 μm. d, Complementation of ADR1-L1-TagRFP in adr1/L1/L2 restores cell death triggered by silencing MEKK1. Plant images were taken three weeks after inoculation with Agrobacterium carrying the indicated VIGS vectors (Ctrl or MEKK1) in two independent p35S::ADR1-L1-TagRFP-HA 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 with CBB staining of RBC as the loading control (bottom). 1, 2, 3, and 4 correspond to plants on the top. e, SUMM2^ac-induced ADR1-L1-TagRFP puncta formation depends on EDS1 and PAD4 in N. benthamiana. ADR1-L1-TagRFP was co-expressed with DEX::SUMM2^ac-HA in N. benthamiana. Two days post-infiltration, SUMM2^ac was induced by 50 μM DEX treatment, and the RFP signal was observed with confocal laser scanning microscopy 5 hr after DEX treatment. Scale bar, 25 μm. f, SUMM2^ac induces ADR1-L1-TagRFP puncta independently of NRG1. Agrobacterium carrying DEX::SUMM2^ac-HA and p35S::ADR1-L1-TagRFP was inoculated into WT and nrg1-1 N. benthamiana leaves. After 30 hr, the leaves were treated with 50 µM Dex for 6 hr, and images were taken using confocal laser scanning microscopy. Scale bar, 10 μm. g, Formation of ADR1-L1-TagRFP puncta occurs before cell death. ADR1-L1-TagRFP was co-expressed with DEX::SUMM2^ac-HA in N. benthamiana. Two days post-infiltration, SUMM2^ac was induced by 50 μM DEX treatment, and the RFP signal was observed with confocal laser scanning microscopy at the indicated time shown as maximum projections from 18 images with a z-step of 1 µm (top panel). Scale bar, 25 μm. Cell death was documented over time under UV light (bottom panel). h, The number and size of ADR1-L1-TagRFP puncta increase progressively over time. The diameter (Ø) of ADR1-L1-TagRFP puncta from (g) was measured at the indicated time points in an area of 400 μm² using ImageJ and divided into groups of ≤ 0.4 μm and > 0.4 μm. Data are presented as mean ± SEM (n = 30). i, Active SUMM2 does not alter the PM localization pattern of BRI1. BRI1-GFP and ADR1-L1-TagRFP were co-expressed with DEX::SUMM2^ac-HA in N. benthamiana. Two days post-infiltration, SUMM2^ac was induced by 50 μM DEX, and the images were taken from the same cell right before (-, top) or 4 hr after DEX treatment (SUMM2^ac, bottom) using a confocal laser scanning microscopy. The left panel shows single-plane images, and the right panel shows maximum projections from 16 images with a z-step of 1 µm. Scale bar, 25 μm. j, ADR1-L1-TagRFP puncta are observed in the mekk1 mutant. Leaves of two-week-old soil-grown transgenic lines of 35S::gADR1-L1-TagRFP in WT or mekk1 were imaged using a confocal laser scanning microscope (scale bar, 10 μm, bottom panel) with the plant phenotypes shown (top panel). k, RNAi-MEKK1-induced ADR1-L1-GFP puncta clusters depend on EDS1 and PAD4 in Arabidopsis. Two-week-old transgenic lines expressing pADR1-L1::gADR1-L1-GFP in the eds1-2, pad4-1, or WT background were inoculated with Agrobacterium carrying RNAi-MEKK1. After two weeks, leaf samples were imaged using a Leica TCS SP8 laser scanning confocal microscope (top) or a Leica DMi8 Infinity TIRF microscope (bottom). ADR1-L1-GFP puncta or puncta clusters were observed in WT but not in eds1-2 or pad4-1, as indicated by arrowheads or dotted lines. For each genotype, ten independent fields of view were examined, with similar distribution patterns were observed. Scale bar, 10 μm (top) and 1 μm (bottom). l, Expression of HopAI1 induces ADR1-L1-GFP punctate signals at the cell periphery. Leaves from four-week-old WT Arabidopsis plants were hand-inoculated with Agrobacteria containing pADR1-L1::gADR1-L1-GFP with or without (-) DEX::HopAI1-HA at OD₆₀₀ = 2.0. Two days post Agrobacterium infiltration, 50 μM DEX was treated. Fluorescence signals were examined at six days post-infiltration using a confocal laser scanning microscope. Scale bar, 10 μm. In immunoblot (d), RBCs were run on the same gels and used as loading controls. Statistical analysis was performed using a two-tailed Student’s t-test (h). Experiments were repeated three times with similar results. For gel source data, see Supplementary Fig. 1.

Source data