Fig. 1: PAT5 and PAT9 control PTI response triggered by eATP.

a Identification of PAT5 and PAT9 tryptic peptides as a substrate of P2K1 kinase by KiC assay. b Ligand-induced calcium influx. 5-day-old seedlings were treated with 100 μM ATP for 15 min. RLU, relative luminescence units. Error bars indicate ±SEM; n = 8 seedlings; *p < 0.05, **p < 0.01, P-values indicate significance relative to Col-0 and were determined by one-sided ANOVA with unpaired, two-tailed Student’s t test. c ROS production was measured in leaf disks after treatment with 200 μM ATPγS for 30 min. Leaf disks were taken from WT (Col-0), pat5, pat9, and pat5/9 double mutants, or their complemented lines PAT5 (NP::ATPAT5-HA/Atpat5) and PAT9 (NP::ATPAT9-HA/Atpat9). Error bars indicate ±SEM; n = 12 leaf disks; *p < 0.05, **p < 0.01, P-values indicate significance relative to Col-0 with ATPγS treatment and were determined by one-sided ANOVA with unpaired, two-tailed Student’s t test. d MAPKs activation of Arabidopsis leaf disks that treated with 200 μM ATPγS for the times indicated. Coomassie Brilliant Blue (CBB) staining of protein was used as loading control. e PAT5 and PAT9 negatively mediated bacterial invasion. Arabidopsis seedlings with the indicated genotype (x axis) were flood-inoculated with Pst. DC3000 bacteria and bacterial growth determined by plate counting 3 days post inoculation. Error bars indicate ±SEM; n = 12 (biological replicates); means with different letters are significantly different (p < 0.05); P-values indicate significance relative to Col-0 and were determined by one-sided ANOVA with multiple comparisons and adjusted using Benjamini–Hochberg post-test. Box extends from the 25th to the 75th percentile, whiskers denote minima and maxima (Boxplots, Col-0: max = 7.76; min = 5.88; center = 6.82; Q2 (25%) = 6.45; Q3 (75%) = 7.43, pat5: max = 7.76; min = 5.45; center = 6.62; Q2 (25%) = 6.13; Q3 (75%) = 7.05, pat9: max = 6.97; min = 5.3; center = 6.47; Q2 (25%) = 6.1; Q3 (75%) = 6.71, pat5/9: max = 6.55; min = 5; center = 5.9; Q2 (25%) = 5.49; Q3 (75%) = 6.21, PAT5-1: max = 7.7; min = 5.59; center = 7.05; Q2 (25%) = 6.76; Q3 (75%) = 7.25, PAT5-2: max = 7.64; min = 5.85; center = 7; Q2 (25%) = 6.51; Q3 (75%) = 7.15, PAT9-1: max = 7.72; min = 5.6; center = 7.09; Q2 (25%) = 6.58; Q3 (75%) = 7.47, PAT9-2: max = 7.7; min = 5.78; center = 7; Q2 (25%) = 6.27; and Q3 (75%) = 7.47). Experiments were repeated three times with similar results. f, g Ligand triggers PAT5 and PAT9 phosphorylation after treated with 200 μM ATP, 1 μM flg22, or 50 μg/ml chitin in their complemented lines PAT5 and PAT9. Lambda protein phosphatase (Lambda PP, − and +) was added to release phosphate groups. CBB was used as loading control. All experiments were repeated and analyzed three times with similar results.