Extended Data Fig. 9: MtLICK1 interact with MtRBOHs to regulate plant immunity.

a,b,c, qRT-PCR was used to detect mRNA levels of immunity marker genes in transformed plants WT (R108) and Mtlick1/2 at 2 days, post S. meliloti 1021 inoculation. MtEF-1 was used as the reference gene. (3 biological repetitions per group). d-i, qRT-PCR was used to detect mRNA levels of immunity marker genes (MtPR4, MtPR10 and MtWRKY) in transformed plants Mtlick1/2/MtLICK1^WT, Mtlick1/2/MtLICK1^3A, Mtlick1/2/MtLICK1^3D, Mtlick1/2/MtLICK2^WT, Mtlick1/2/MtLICK2^3A, and Mtlick1/2/MtLICK2^3D at 3 days, post S. meliloti 1021 inoculation. MtEF-1 was used as the reference gene. (3 biological repetitions per group). Data are mean ± SEM. Statistical analysis was performed using one-way ANOVA (Tukey test). Different letters indicate significant difference (P < 0.05). j, ROS production following treatment of wild-type (WT) R108 and Mtlick1/2 root segments with 0.4 mg/mL peptidoglycan (PGN) over 40 min. WT-Mock, n = 17; Mtlick1/2-Mock, n = 20; WT-PGN, n = 21; Mtlick1/2-PGN, n = 18). Data are mean ± SEM. Each plant analyzed is plotted as dots and triangle respectively. Statistical analysis was performed using Two-sided Student’s t-test compared to WT-PGN. RLU, relative luminescence unit. k, ROS production following treatment of wild-type (WT) R108 and Mtlick1/2 root segments with 0.4 mg/mL PGN derived from S. meliloti 1021(WT(R108) Mock, n = 21; WT(R108) PGN, n = 20; Mtlick1/2 Mock, n = 19; Mtlick1/2 PGN, n = 17). Data are mean ± SEM. Each plant analyzed is plotted as dots and triangle respectively. RLU, relative luminescence unit. l,m,n, R. solanacearum soil-drenching inoculation assays in Medicago truncatula. In l, the results are presented as disease progression, showing the average wilting symptoms on a scale from 0 to 4. Healthy plants (score 0), leaf curling (score 1), wilting petioles (score 2) and leaf chlorosis (score 3) and whole plant collapse (score 4). (WT (R108), n = 36; Mtlick1, n = 36; Mtlick2, n = 36; Mtlick1/2, n = 34). m, shows the survival analysis of data in l, Data are mean ± SEM. Statistical analysis was performed using Two-sided Student’s t-test compared to wild-type plants (R108). n, Phenotype of plant survival 14 days post-inoculation with R. solanacearum. Scale bar, 2 cm. o, DAB staining of 7-day Medicago roots pretreated with R. solanacearum for 3 h. Scale bars, 0.5 mm. The below histograms show a statistical analysis of the relative staining intensity based on three repeated DAB staining assay data. The relative staining intensity was detected by using Image J software (WT (R108), n = 11; Mtlick1, n = 11; Mtlick2, n = 11; Mtlick1/2, n = 11; each plant analyzed is plotted as black dot, triangle, box and cross respectively). Data are mean ± SEM. Statistical analysis was performed using Two-sided Student’s t-test compared to WT (R108). p, GFP-MtLICK1/2 and MtRBOHB/C-HA were transiently co-expressed in N. benthamiana leaves and Co-IP assay was performed with a-GFP antibodies. GFP-MtLICK1/2 and HA or MtLTI6b-GFP (low temperature–induced protein 6B, PM marker) and MtRBOHB/C-HA or GFP and MtRBOHB/C-HA were transiently co-expressed in N. benthamiana leaves serve as control. q, Number of infection events at 7 dpi with S. meliloti 1021-LacZ (Sm) in wild type (R108) plants with H₂O₂ treatment. The plants, without H₂O₂ treatment, were used as control. (0 mM, n = 20; 1 mM, n = 20; 4 mM, n = 20). Data are mean ± SEM. Each plant analyzed is plotted as dots, triangles and circles respectively. Statistical analysis was performed using one-way ANOVA (Tukey test). Different letters indicate significant difference (P < 0.05). r, Number of nodules and bumps at 14 dpi with S. meliloti 1021 (Sm) in wild type (R108) plants with H₂O₂ treatment. The plants, without H₂O₂ treatment, were used as control. (0 mM, n = 20; 1 mM, n = 20; 4 mM, n = 20). Data are mean ± SEM. Each plant analyzed is plotted as dots, circles, triangles, crosses and boxes respectively. Statistical analysis was performed using Two-sided Student’s t-test. For a-p, the experiments were repeated three times with similar results. For q and r, the experiments were repeated two times with similar results.

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