The RC4 Domain of PbrS-RNase Is Critical for Interaction with PbrActin1. The RC4 Domain of PbrS-RNase Is Critical for Interaction with PbrActin1. (A) Dissection of the functional domains of PbrS7-RNase required for interaction with PbrActin1 in Y2H assays. The wedge indicates the gradient dilution at 1, 0.1, and 0.01. A positive interaction between PbrS-RNase and actin was confirmed by a filter assay. The interaction strength was quantified based on β-galactosidase activity. The experiments were repeated four times, with similar results. Asterisks indicate significant statistical difference between wild-type PbrS7-RNase and P156A mutation as evaluated by ANOVA and Tukey’s test: **P < 0.01; error bars indicate se. (B) Sequence alignment of PbrS7-RNase, PbrS34-RNase, Aspergillus niger ACTIBIND, and human RNASET2. The black line represents the RC4 domain of PbrS-RNase. The red box represents the sequence of ACTIBIND identified as binding to actin. (C) Superimposition of the structures of the RNase binding actin domains of PbrS7-RNase (blue), PbrS34-RNase (green), ACTIBIND (purple), and RNASET2 (yellow). The ACTIBIND protein (Protein Data Bank entry 3d3z) was used as a crystal structure model. Jianqing Chen et al. Plant Cell 2018;30:1023-1039 ©2018 by American Society of Plant Biologists