Fig. 3: The designer NLR receptor RGA5HMA5 activates RGA4-dependent plant cell death upon recognizing the noncorresponding MAX effector AVR-PikD. | Nature Communications

Fig. 3: The designer NLR receptor RGA5HMA5 activates RGA4-dependent plant cell death upon recognizing the noncorresponding MAX effector AVR-PikD.

From: The synthetic NLR RGA5HMA5 requires multiple interfaces within and outside the integrated domain for effector recognition

Fig. 3

a Y2H assays show the interaction of RGA5-HMA5 with AVR-PikD but not with AVR-Pia. b Pull-down assays show specific interaction of HA-AVR-PikD with MBP-RGA5-HMA5 but not with MBP-RGA5-HMA. HA-AVR-PikD, HA-AVR-Pia, MBP- RGA5-HMA5, and MBP-RGA5-HMA proteins were individually expressed in E. coli. Fusion proteins in different combinations were visualized by immunoblotting with the anti-HA and anti-MBP antibodies. The experiment was repeated thrice with similar results. c MST analyses show the dissociation constants (Kd) between the RGA5-HMA or RGA5-HMA5 domain and the effectors. The Kd were calculated and error bars represent the means ± SD from data from three independent biological replicates. d Coimmunoprecipitation assays showing the interaction of RGA5HMA5 with AVR-PikD in N.benthamiana. Co-IP proteins were detected by using anti-HA and anti-GFP antibodies, respectively. The experiment was repeated thrice with similar results. e Representative leaf images showing specific cell death in N. benthamiana leaves after coinfiltration of the A. tumefaciens strains carrying RGA5HMA5 (fused with HA) with RGA4 (fused with Flag) and AVR-PikD (fused with GFP), or RGA5 (fused with HA) with RGA4 (fused with Flag) and AVR-Pia or RGA4. Images were taken three days after the infiltrations under the UV light. HR index was scored based on representative pictures as previously reported18. Twenty biological replicates were used in each group. Three independent groups in different colors were labeled in box plots. Differences among the samples were assessed by Tukey’s HSD test (p < 0.01). f The LUC activity in rice protoplasts after transfection with different vector combinations. RGA4 was set as the positive control, and the empty vectors were served as the negative control. Significant differences with empty vector samples are labeled with an asterisk and assessed by Dunnett’s HSD test (p < 0.01). The assays were repeated three times with similar results.

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