Fig. 4: Overexpression of AtMKK7 in the active form (AtMKK7ac) activates MAPK signaling in apple protoplast cells.
From: Development of potent promoters that drive the efficient expression of genes in apple protoplasts
a Comparison of AtMKK7ac expression driven by CaMV 35S and Pro-BIUTNT. b Comparison of AtMKK7ac and MdMAPK6 expression driven by CaMV 35S. c AtMKK7ac overexpression induced hypersensitive responses in tobacco leaves. The white arrows denote the phenotype of hypersensitive reactions. Agrobacterium tumefaciens LBA 4404 carrying GFP, MdMAPK6-GFP, or AtMKK7ac-GFP was infiltrated into the leaves of 30-day-old tobacco seedlings, and the phenotypes were characterized 2 days later. d AtMKK7ac overexpression activates MAPK cascades in apple protoplast cells. AtMKK7ac was expressed in apple protoplast cells, and MAPK phosphorylation was detected by an anti-pERK antibody. CT untransformed protoplast cells. MER represents the control sample (a, b, d). e AtMKK7ac overexpression activates MdWRKY33 posttranslational modification, potentially phosphorylation, in apple protoplast cells. MdWRKY33-HA was expressed or coexpressed with AtMKK7ac-FLAG in protoplast cells generated from wild-type apple callus cells or from MdMAPK6-FLAG-overexpressing apple callus cells. MdWRKY33-HA was detected by western blot using an anti-HA antibody. f AtMKK7ac phosphorylates MdMAPK6 or activates MdMAPK6 autophosphorylation, which is mediated by Thr-231. An in vitro kinase assay was conducted as described in the Materials and methods section. The mutation of T231A/D but not T236A in MdMAPK6 blocked MdMAPK6 phosphorylation. Upper: phosphorylation statuses of MBP-AtMKK7ac and MdMAPK6 as determined by autoradiography; middle: expression of MdMAPK6 and its mutants in apple protoplast cells; lower: Coomassie blue staining of MBP-AtMKK7ac in the reaction samples. g AtMKKac overexpression activates MdMAPK3/6/4 phosphorylation in apple callus cells. Apple calli transformed with Agrobacterium carrying AtMKK7ac were placed on callus maintenance medium for 2 days. Apple calli transformed with Agrobacterium carrying an empty vector were used as the control (CT). MAPK phosphorylation was detected as described in d
