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. 2019 Aug 29;10:1027. doi: 10.3389/fpls.2019.01027

Figure 2.

Figure 2

EXO70B1 associates with AvrPtoB. (A) EXO70B1 interacts with AvrPtoB in a Y2H assay. The coding sequences of AvrPtoB, AvrPtoBF479A, and AvrPtoB1–410 were fused to the Gal4 DNA binding domain (BD), and the coding sequence of EXO70B1 was fused to the Gal4 transactivation domain (AD). As a positive control, tomato Pto was also fused to the AD. Different pairs of constructs were cotransformed into AH109. A 10-μl suspension (OD600 = 0.5) of each cotransformant was dropped onto synthetic dropout (SD) medium lacking Leu and Trp and SD medium lacking Ade, His, Leu, and Trp. Photographs were taken after 5 days of incubation. (B) Amino acids 1–307 of AvrPtoB are necessary for the interaction with EXO70B1 in a Y2H assay. Different truncated fragments of AvrPtoB (1–205, 1–307, 1–387, and 308–553) were fused to the BD, and EXO70B1 and TN2 were fused to the AD. Different pairs of constructs were cotransformed into AH109. Yeast cells containing the indicated plasmids were spotted onto SD-Leu-Trp and SD-Ade-His-Leu-Trp. Photographs were taken after 5 days of incubation. (C) EXO70B1 interacts with AvrPtoB in a GST-pull-down assay. E. coli-expressed GST, GST-AvrPtoB, GST-AvrPtoBF479A, and GST-AvrPtoB1–410 were incubated with plant-expressed EXO70B1-GFP in pull-down assays. After incubation for 4 h at 4°C, the beads were washed three times, and 5 × SDS sample buffer was added. Then, 20 μl of the samples was utilized to detect the precipitated EXO70B1-GFP by anti-GFP antibody, and 10 μl of the samples was used to detect the levels of GST, GST-AvrPtoB, GST-AvrPtoBF479A, and GST-AvrPtoB1–410 by anti-GST antibody. Arrows indicate the positions of the corresponding proteins. (D) EXO70B1 interacts with AvrPtoB in a Co-IP assay. EXO70B1-GFP was co-expressed with AvrPtoB-Myc in N. benthamiana leaves. After 48 h, leaves were treated with or without 50 μM of MG132 for 3 h. Total protein was extracted and subjected to immunoprecipitation of AvrPtoB by anti-Myc antibody. Proteins were analyzed in an immunoblot using anti-GFP and anti-Myc antibodies. These experiments were repeated three times with similar results.