Tomato TFT1 Is Required for PAMP-Triggered Immunity and Mutations that Prevent T3S Effector XopN from Binding to TFT1 Attenuate Xanthomonas Virulence
Figure 8
XopN L64,L65 motif and S688 are required for TFT1 binding and XopN-dependent virulence.
(A) Pull-down analysis of TFT1-HA and XopN(L64A,L65A,S688A)-6His, XopN(L64A,L65A)-6His, or XopN-6His in N. benthamiana. Leaves were infiltrated with a mixed suspension of A. tumefaciens expressing TFT1-HA (4×108 CFU/mL) and A. tumefaciens expressing XopN(L64A,L65A)-6His, XopN(L64A,L65A,S688A)-6His or XopN-6His (4×108 CFU/mL). After 48 h, protein was extracted, purified by Ni+ affinity chromatography, and then analyzed by protein gel blot analysis using anti-His and anti-HA sera. Expected protein MW: TFT1-HA = 29.3 kDa; XopN(L64A,L65A)-6His, XopN(L64A,L65A,S688A)-6His and XopN-6His = 78.7 kDa. +, protein expressed; −, vector control. STD, molecular weight standard. (B) BiFC analysis of XopN/TFT1 interactions in N. benthamiana. Leaves were hand-infiltrated with a suspension (8×108 CFU/mL total) of two A. tumefaciens strains expressing different fusion proteins (XopN-nYFP+TFT1-cCFP; L64A,L65A-nYFP+TFT1-cCFP; S688A-nYFP+TFT1-cCFP; L64A,L65A,S688A-nYFP+TFT1-cCFP; or GUS-nYFP+TFT1-cCFP) and then visualized by confocal microscopy at 48 HPI at 63X. White bar = 25 µm. (C) Growth of Xcv ΔxopN (vector), Xcv ΔxopN (L64A,L65A,S688A-HA), Xcv ΔxopN (L64A,L65A-HA), or Xcv ΔxopN (XopN-HA) strains in susceptible VF36 tomato leaves. Leaves were hand-infiltrated with a 1×105 CFU/mL suspension of bacteria. Number of bacteria in each leaf was quantified at 0 and 8 DPI. Data points represent mean CFU/cm2 ± SD of three plants. Different letters at day 8 indicate statistically significant (one-way analysis of variance and Tukey's HSD test, P<0.05) differences between the samples. Vector = pVSP61. Analysis was repeated at least three times. (D) Phenotype of tomato leaves inoculated with the strains described in (C). Leaves were photographed at 12 DPI. Similar phenotypes were observed in 3 independent experiments.