Fig. 4: Pit1 L301 and C416 are important for OsRac1 activation and plasma membrane localization. | Nature Communications

Fig. 4: Pit1 L301 and C416 are important for OsRac1 activation and plasma membrane localization.

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

Fig. 4

A, B Monitoring OsRac1 activation by amino acid-substituted Pit1 (A) and Pit2 (B) mutants using Raichu-OsRac1 FRET in vivo. Statistical analysis of OsRac1 activation by Raichu-OsRac1 with normalized emission ratios of Venus to CFP. PFW, Pit1 L301P C416F G479W. Bars represent the mean ± s.d. (n  =  3 biological replicates). The asterisks indicate significant differences determined by one-way ANOVA (with Tukey’s test) (****P < 0.0001). C Localization of Pit1, Pit2, and the indicated amino acid substitution mutants in rice protoplasts. Protoplasts were co-transfected with the indicated Venus-tagged fluorescent constructs and the plasma membrane marker FLS2-mCherry. Scale bars, 5 μm. This experiment was repeated three times with similar results. D Cell fractionation assay showing the localization of Pit1, Pit2, and the indicated mutants; the HA-tagged proteins were transiently expressed in N. benthamiana. Western blot was performed with anti-HA, anti-cAPX (cytosolic marker), and anti-H+ATPase (plasma membrane marker) antibodies. M(3x) is three times enrichment relative to T or S. T, S, and M indicate total extract, soluble fraction, and microsomal fraction, respectively. Ponceau staining of Rubisco served as a loading control. This experiment was repeated three times with similar results. E Quantification of localization of Pit1, Pit1 PFW, Pit2 and Pit2 LCG. One hundred transfected cells were counted under a microscope. PM, plasma membrane. Cyt, Cytosol. Source data are provided as a Source Data file.

Back to article page