TFT1 associates with the C-terminal domain of XopN.
(A) Schematic of XopN protein and various deletion mutants. Wild type and mutant xopN were cloned into pXDGATcy86(GAL4-DNA binding domain) to create DBD-XopN fusion proteins: XopN, XopN 1–733; N, 1–349; C, 344–733; M4, 222–733; M5, 1–604; and M6, 1–514. Numbering refers to amino acid residues in wild type XopN (733 amino acids). (B) TFT1 interaction with XopN mutant proteins in yeast. Yeast strain AH109 carrying pXDGATcy86 containing vector, XopN, N, C, M3, M4, M5 and M6 were independently transformed with the following PREY constructs: pGADT7(GAL4 activation domain) alone (Vector) or pGADT7 containing TFT1. Strains were spotted on nonselective (SD-LT) and selective (SD-LTH) media ± 1 mM 3-amino-1,2,4-triazole and then incubated at 30°C for 3d. (C) Pull-down analysis of TFT1-HA and XopN-6His, XopN1–349-6His, or XopN345–733-6His in N. benthamiana extracts. N. benthamiana leaves were inoculated with a suspension of 6×108 CFU/mL of A. tumefaciens co-expressing TFT1-HA and XopN-6His, XopN1–349-6His, or XopN345–733-6His. 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-6His = 78.7 kDa; XopN1–349-6His = 38.0 kDa; XopN345–733-6His = 42.0 kDa; +, protein expressed; −, vector control. STD, molecular weight standard. (D) Pull-down analysis of TFT1-HA and XopN-6His or XopN1–604-6His in N. benthamiana extracts. N. benthamiana leaves were hand-inoculated with a mixed suspension of 1×108 CFU/mL of A. tumefaciens expressing TFT1-HA and 4×108 CFU/mL of XopN-6His or XopN1–604-6His. Samples were processed as described in (C). Expected protein MW: TFT1-HA = 29.3 kDa; XopN-6His = 78.7 kDa; XopN1–604-6His = 64.9 kDa; +, protein expressed; −, vector control. STD, molecular weight standard.