Fig. 2

Exogenous NAD(P)⁺-induced local defense responses in the lecrk-VI.2 mutants and binding of NAD(P)⁺ to LecRK-VI.2. a–c Exogenous NAD(P)⁺-induced local expression of PR1 a, PR2 b, and PR5 c in the wild-type (WT), lecrk-VI.2, and dorn1-3 plants. Leaves of 4-week-old soil-grown plants were infiltrated with 0.2 mm NAD⁺, 0.4 mm NADP⁺ or H₂O. The infiltrated leaves were collected 20 hr later. Total RNA was extracted and subjected to qPCR analysis. Expression levels were normalized against the constitutively expressed UBQ5. Data represent the mean ± SD of three biological replicates. Asterisks denote significant differences between the induction in the mutants and that in the wild type (*p < 0.05, **p < 0.01, ***p < 0.001; two-way ANOVA with Sidak’s test). Upper line: induction by NADP⁺; lower line: induction by NAD⁺. d Exogenous NADP⁺-induced local resistance in the wild-type, lecrk-VI.2, and dorn1-3 plants. Leaves of 4-week-old soil-grown plants were infiltrated with 0.4 mm NADP⁺ or H₂O. Five hr later, the infiltrated leaves were inoculated with a Psm suspension (OD₆₀₀ = 0.001). Three d later, eight leaves were collected to examine the growth of the pathogen. Data represent the mean ± SD of eight biological replicates. Asterisks denote significant differences between the induction in the mutants and that in the wild type (***p < 0.001; two-way ANOVA with Sidak’s test). e Binding of ³²P-NAD⁺ to recombinant MBP, MBP-eLecRK-VI.2, and MBP-eDORN1 proteins. Approximately 5 μg recombinant proteins were used for each binding assay. Data represent the mean ± SD of three experiments. Different letters denote significant differences (p < 0.05; one-way ANOVA with Tukey’s test). f Saturation binding assay for LecRK-VI.2. Recombinant MBP-eLecRK-VI.2 proteins were incubated with the indicated concentrations of ³²P-NAD⁺ for 30 min. Free NAD⁺ was removed by washing. Data were plotted as a specific binding. The dissociation constant (Kd) was calculated by one site specific binding saturation model using GraphPad Prism 7 (www.graphgpad.com). The experiment was repeated four times with similar results and results from a representative experiment were presented. g Competitive binding assay for LecRK-VI.2. Samples containing 250 nm of ³²P-NAD⁺ in the presence of 100 nm to 1 mm of unlabeled nucleotides were assayed for specific binding of ³²P-NAD⁺. Data were plotted as a specific binding with SD of three experiments. The 50% inhibition concentration (IC₅₀) values were calculated in GraphPad Prism 7 using the one site Fit logIC₅₀ competition model. h Binding of ³²P-NAD⁺ to the microsomal fractions of the wild-type (WT) and lecrk-VI.2-2 mutant plants. Samples containing 250 nm ³²P-NAD⁺ in the absence (total) or presence of 1000-fold unlabeled (cold) NAD⁺ or NADP⁺ were assayed for binding of ³²P-NAD⁺. Data represent the mean ± SD of three experiments. Different letters denote significant differences (p < 0.05; one-way ANOVA with Tukey’s test)