Fig. 8: RxLR23KM impairs the interaction of ERD15La and NbNAC68a/b/c to trigger plant defense against P. capsici infection.
A, B pBinGFP2:RxLR23KM, pBinGFP2:NbERD15La, pBinGFP2:NbNAC68a/b/c, pBinGFP2:NbERD15La + pBinGFP2:NbNAC68a/b/c, pBinGFP2:RxLR23KM + pBinGFP2:NbERD15La, pBinGFP2:RxLR23KM + pBinGFP2:NbNAC68a/b/c, pBinGFP2:RxLR23KM + pBinGFP2:NbERD15La + pBinGFP2:NbNAC68a/b/c, or pBinGFP2:GFP was transiently expressed in N. benthamiana leaves and subsequently inoculated with P. capsici zoospores at 24 hpi. A Representative lesions were taken under UV irradiation at 48 hpi (n = 30 samples). Mean diameter of lesions was measured at 48 hpi. Values are presented as mean ± SD. B Relative biomass of P. capsici was measured by qPCR at 48 hpi. Pcβ-actin and NbEF1α were identified as the most suitable reference genes for normalization. The ratio in the leaves inoculated with GFP was assigned to value of 1.0. Values are presented as mean ± SD, n = 3 independent experiments. C Expected protein sizes were detected by western blot. Protein loading is indicated by Ponceau S. D RxLR23KM attenuates interaction of ERD15La and NbNAC68a/b/c. NbNAC68a/b/c-mCherry and NbERD15La-GFP were transiently co-expressed in N. benthamiana leaves along with increasing amounts of RxLR23KM-FLAG or GST-FLAG. The ratio of NbNAC68a/b/c or RxLR23KM immunoprecipitated by NbERD15La to their expression quantity was assigned to value 1.0 when NbNAC68a/b/c or NbERD15La with RxLR23KM or GST were co-expressed into leaves in a 1:1:0.5 ratio. Equal protein loading is indicated by Ponceau S. E RxLR23KM impairs NbNAC68a/b/c interaction with NbERD15La by LCI. MBP-nLUC and GST-cLUC were used as controls. RxLR23KM and fusion proteins were co-expressed in leaves. Images were obtained at 2 days. LCI was evaluate interaction of NbERD15La with NbNAC68a/b/c in presence or absence of RxLR23KM. Relative luminescence units (RLUs) were used to measure the luminous intensity. Values are presented as mean ± SD, n = 3 independent experiments. F RxLR23KM weakens binding affinity of NbERD15La with NbNAC68a/b/c by MST. Values are presented as mean ± SD, n = 3 independent experiments. The data were analyzed by ANOVA one-way comparison followed by least significant difference (LSD) test. Different letters above the bars indicate a significant difference at p < 0.05. The experiments were repeated three times with similar results. Source data are provided as a Source Data file.
