Journal: PLoS Pathogens
Article Title: Perturbations of the ZED1 pseudokinase activate plant immunity
doi: 10.1371/journal.ppat.1007900
Figure Lengend Snippet: (A) Schematic showing ZAR1 domain truncation boundaries. Subdomains of the central nucleotide-binding region are labeled as described by Wang et al [ , ]: NBD, nucleotide-binding domain; HD1, helical domain 1; and WHD, winged helix domain. (B) Confirmation of expression for each of the three ZAR1 constructs shown in panel A. A western blot of yeast cell lysates probed with serum raised against the LexA DNA-binding domain (top) is compared with Ponceau S staining of the total protein in each lane (bottom). (C) Interactions between ZAR1 bait constructs and 11 PBL preys were assessed in the absence and presence of HopZ1a and/or ZED1 alleles integrated at the ho locus of strains EGY48 ( MAT α ) or RFY206 ( MAT A ), corresponding to Y2H (interaction scheme A), Y3H (interaction schemes B), and Y4H (interaction scheme C) assays (see ). The prey array layout shown at right represents PBL interactions with ZAR1 ΔCC in the presence of both ZED1 and HopZ1a wt . The colours of the labels at each array position (white or black) are determined by the relative interaction strength (see ; Materials and Methods).
Article Snippet: Simultaneous expression of bait-fusion, prey-fusion and integrated FLAG-tagged effectors in diploid cells carrying bait, prey and reporter plasmids is demonstrated in similar western blots shown in panel C of . Primary antibodies against HA (Roche), the LexA DNA-binding domain (Sigma) and FLAG (Sigma), as well as HRP-conjugated secondary antibodies (Cell Signalling) were all applied as 1:10,000 dilutions in TBST (50 mM Tris-Cl, pH 7.5; 150 mM NaCl; 0.05% Tween-20) with 3% powdered skim milk.
Techniques: Binding Assay, Labeling, Expressing, Construct, Western Blot, Staining
![(A) Schematic showing ZAR1 domain truncation boundaries. Subdomains of the central nucleotide-binding region are labeled as described by Wang et al [ , ]: NBD, nucleotide-binding domain; HD1, helical domain 1; and WHD, winged helix domain. (B) Confirmation of expression for each of the three ZAR1 constructs shown in panel A. A western blot of yeast cell lysates probed with serum raised against the <t>LexA</t> <t>DNA-binding</t> domain (top) is compared with Ponceau S staining of the total protein in each lane (bottom). (C) Interactions between ZAR1 bait constructs and 11 PBL preys were assessed in the absence and presence of HopZ1a and/or ZED1 alleles integrated at the ho locus of strains EGY48 ( MAT α ) or RFY206 ( MAT A ), corresponding to Y2H (interaction scheme A), Y3H (interaction schemes B), and Y4H (interaction scheme C) assays (see ). The prey array layout shown at right represents PBL interactions with ZAR1 ΔCC in the presence of both ZED1 and HopZ1a wt . The colours of the labels at each array position (white or black) are determined by the relative interaction strength (see ; Materials and Methods).](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_4424/pmc06634424/pmc06634424__ppat.1007900.g005.jpg)