Fig. 1: Pit2 interacts with Pit1 in planta and suppresses Pit1-mediated cell death. | Nature Communications

Fig. 1: Pit2 interacts with Pit1 in planta and suppresses Pit1-mediated cell death.

From: An NLR paralog Pit2 generated from tandem duplication of Pit1 fine-tunes Pit1 localization and function

Fig. 1

A In vivo interaction of full-length Pit1 and Pit2. The indicated tagged proteins were transiently expressed in N. benthamiana. Co-IP was performed and proteins were detected by Western blot. Ponceau staining of Rubisco serves as a loading control. Data was repeated three times with similar results. B The indicated proteins were transiently expressed in N. benthamiana. GUS serves as a negative control. Cell death was photographed at 3 dpi. C Activation of OsRac1 by Pit1 and Pit2 using Raichu-OsRac1 FRET in vivo. Color scale of emission ratio images (left) in rice protoplasts co-expressing Raichu-OsRac1 represents the level of OsRac1 activation. Scale bars, 5 μm. The bar graph (right) indicates statistical analysis of OsRac1 activation. Bars = mean ± s.d. (n  =  3 biological replicates). D The indicated combinations of proteins were transiently expressed in N. benthamiana leaves. Cell death was photographed at 3 dpi. E Cell death activity of indicated proteins in rice protoplasts. The indicated constructs were co-transfected with a luciferase reporter vector. Luciferase activity was measured 40 h after transfection. Relative luciferase activity (GUS = 1) is shown. Bars = mean ± s.d. (n  =  3 biological replicates). F Rice protoplasts from K59 WT or Pit1 KO were co-transfected with RNAi constructs against Pit2 and the luciferase reporter vector. Luciferase activity was measured 40 h after transfection. Relative luciferase activity (Mock = 1) is shown. Bars = mean ± s.d. (n  =  3 biological replicates). The asterisks indicate significant differences determined by one-way (C and E with Tukey’s test) or two-way (F with Šídák’s test) ANOVA. **P < 0.01, ***P < 0.001, ****P < 0.0001, ns indicates no significant difference. G Infection assays of Pit1 and Pit2 KO plants with the M. oryzae race 007.0. Photographs were taken at 7 dpi. Scale bar, 5 mm. H Biomass of M. oryzae was measured by qPCR and normalized with rice endogenous OsUbq. Relative infection ratio (K59 = 1) is shown. Bars = mean ± s.d. (n = 3 independent lines). Different letters indicate significant differences determined by one-way ANOVA (with Tukey’s test) (P < 0.05). Source data are provided as a Source Data file.

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