TY - DATA T1 - BcMkk1 impedes BcRim15 phosphorylation mediated by BcSch9. PY - 2018/09/13 AU - Yanni Yin AU - Sisi Wu AU - Chaonan Chui AU - Tianling Ma AU - Huixian Jiang AU - Matthias Hahn AU - Zhonghua Ma UR - https://plos.figshare.com/articles/figure/BcMkk1_impedes_BcRim15_phosphorylation_mediated_by_BcSch9_/7086578 DO - 10.1371/journal.ppat.1007285.g005 L4 - https://ndownloader.figshare.com/files/13029905 KW - PAS KW - cell wall integrity KW - mitogen-activated protein kinase KW - MAPK kinase BcMkk 1 KW - BcMkk 1 KW - kinase BcRim 15 KW - EH KW - controls OA production KW - MAPK kinase KW - Botrytis cinerea KW - virulence KW - Pro 40 homolog BcPro 40 KW - oxalic acid biosynthesis KW - cell wall KW - CWI N2 - (A) The interactions of BcRim15 with BcMkk1 and BcSch9 were verified by the Y2H assays. Serial concentrations of yeast cells were drop-plated on SD-Leu-Trp-His plates. A pair of plasmids pGBKT7-53 and pGADT7 was used as a positive control. A pair of plasmids pGBKT7-Lam and pGADT7 was used as a negative control. (B) Domain architecture of the BcRim15 identified by Pfam (http://pfam.xfam.org/). (C) Phosphorylation of BcRim15PK (the protein kinase domain of BcRim15) in ΔBcMkk1 was dramatically higher than that in the wild type, and was reduced in the double mutant ΔBcMkk1-BcSch9. (D) Acid production and protease activity of each strain. (E) Both BcMkk1 and BcSch9 bind directly to BcRim15PK. Proteins associated with the GST beads or in the total proteins before GST pull-down (input) were probed by western blot analysis using the monoclonal mouse anti-His or anti-GST antibodies. (F) BcMkk1 inhibits the interaction of BcSch9 with BcRim15PK. BcRim15PK bound to Ni sepharose beads was incubated with 5 μg of BcSch9-GST and different amounts of BcMkk1-GST as indicated. Proteins associated with the beads or in the total proteins before His pull-down (input) were probed by western blot analysis using the monoclonal mouse anti-His or anti-GST antibodies. (G) Quantification of the binding data in (F). The relative binding activity of BcSch9 with BcRim15PK is calculated as the BcSch9 band intensity at a given concentration of BcMkk1 divided by the BcSch9 band intensity in the absence of BcMkk1. Values on the bars followed by the same letter are not significantly different at P = 0.05. ER -