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. 2017 Mar 22;3(3):e1601528. doi: 10.1126/sciadv.1601528

Fig. 4. Endocytic motifs at VirE2 C terminus interacted with AP2M, and ap2m mutations attenuated tumorigenesis.

Fig. 4

(A) VirE2 C-terminal tail interacted with AP2M. VirE2 C-terminal tail fused onto GST (GST-VirE2C) was used to conduct an in vitro pull-down assay, with AP2M cargo-binding domain fused onto MBP (MBP-AP2MC). The pull-down (upper panel) and 20% of input (lower panels) fractions were analyzed by Western blot. Free MBP and MBP-AP2MC fusion protein are asterisked. (B) Double mutation at the dual endocytic motifs eliminated the interaction between the VirE2 C-terminal tail and AP2M cargo-binding domain. The pull-down (upper panel) and 20% of input (lower panels) fractions were analyzed by Western blot. MBP-AP2MC fusion is asterisked. (C) A. thaliana ap2m-1 and ap2m-2 mutations attenuated tumorigenesis in the root transformation assay. (D) Quantification of the frequency of tumor formation, which is given as a percentage above each column (with ±SE). The quantification was based on the number of tumors formed on 120 root segments. **P < 0.01 (unpaired Student’s t test). PD, pulled down; IB, immunoblot.