Tomato TFT1 Is Required for PAMP-Triggered Immunity and Mutations that Prevent T3S Effector XopN from Binding to TFT1 Attenuate Xanthomonas Virulence
Figure 9
XopN promotes TARK1 and TFT1 binding in N. benthamiana.
(A-E) BiFC analysis of TARK1/TFT1 interactions in N. benthamiana leaves in the presence of wild type and mutant XopN protein. Leaves were hand-infiltrated with a suspension (6×108 CFU/mL total) of three A. tumefaciens strains expressing different fusion proteins: (A) GUS-6His+TARK1-SCFP3Ac+TFT1-VenusN; (B) XopN-6His+TARK1-SCFP3Ac+TFT1-VenusN; (C) XopN(L64A,L65A)-6His+TARK1-SCFP3Ac+TFT1-VenusN; (D) XopN(S688A)-6His+TARK1-SCFP3Ac+TFT1-VenusN; (E) XopN-(L64A,L65A,S688A)-6His+TARK1-SCFP3Ac+TFT1-VenusN. Cells were visualized by confocal microscopy at 48 HPI at 63X. White bar = 40 µm. (F) GST-TFT1 affinity purification of TARK1 and XopN in vitro. GST or GST-TFT1 was incubated with N. benthamiana leaf extracts containing TARK1-HA ± vector, XopN-6His or mutant XopN-6His (i.e. XopN(L64A,L65A)-6His, XopN(S688A)-6His, or XopN(L64A,L65A,S688A)-6His). Proteins were purified using glutathione sepharose and analyzed by immunoblot analysis using anti-His, anti-HA, and anti-GST sera. Protein input levels are shown on the left. GST and GST-TFT1 pull-downs are shown on right. Expected protein MW: GST = 28 kDa; GST-TFT1 = 56.5 kDa; TARK1-HA = 67.9 KDa; XopN(L64A,L65A)-6His, XopN(L64A,L65A,S688A)-6His and XopN-6His = 78.7 kDa. +, protein expressed; −, vector control. STD, molecular weight standard.