Fig. 2: Parakletos negatively regulates plant immune signaling.

a, b ROS production in response to flg22 (1 μM) in Nicotiana benthamiana plants transiently expressing Parakletos, or in silenced plants, measured as total relative light units (RLU). c Elevated second ROS peak in response to 0.1 µM flg22 in Parakletos-silenced plants. d Expression of NbRBOHB (normalized to EF-1α) in Parakletos-silenced plants after infiltration (12 hpi) with flg22 (1 µM) relative to controls. e, f Aequorin (AEQ) was transiently expressed along with Parakletos for over-expression studies or transiently expressed alone in 3-4 week-old silenced plants to monitor cytosolic Ca²⁺ bursts in response to flg22 (1 μM). g–i Relative expression levels of (g) ICS1 (12hpi), (h) PR1(12 hpi) and (i) PTI5 (45 minutes post infiltration) (normalized to EF-1α) were measured in N. benthamiana leaves in which Parakletos was transiently expressed or silenced, with or without flg22 treatment. j, Tetraallelic detection of mutations sizes in CRISPR/Cas9 generated parakletos-KO (knock-out) potato lines (L19, L24, and L32). k, ROS production during the response to flg22 (8 μM) in parakletos-KO potato plants and Désirée-WT (background control). l parakletos-KO potato plants and Désirée-WT were subjected to nitroblue tetrazolium (NBT) staining 2 days post-infection with Phytophthora infestans. m–o Effects of dual Parakletos and CAS silencing in N. benthamiana. m, ROS levels determined in 3–4-week-old silenced plants induced with 1 μM flg22. n, Cytosolic Ca²⁺ bursts in response to flg22 (1 μM). o, Lesion diameter (mm) in the Phytophthora infestans infection assays. Data were analyzed by two-tailed Student’s t-tests (a–i, k,l) or one-way ANOVA followed by Tukey’s test (m-o). Exact p (a-i, k,j) or adjusted p (m-o) values were shown in figures. Box plots display individual data points for a–i, k–o (a–c, e, f, n = 12; d, g–I, n = 4; k, n = 8; l, n = 24; m, n = 12; n, n = 8; o, n = 12, n=biological replicates). The centerline in box plots indicate medians, the + sign indicates the mean, the box borders delimit the lower and upper quartiles, and the whiskers show the highest and lowest data points. All experiments were conducted at least two to three times with similar results. Source data are provided as a Source Data file.