Fig. 4: ZmBLK1 is a cytoplasmic kinase downstream of the ZmWAKL/ZmWIK complex.
From: The ZmWAKL–ZmWIK–ZmBLK1–ZmRBOH4 module provides quantitative resistance to gray leaf spot in maize
a, Interaction between ZmBLK1 and ZmWIK in an SLC assay. b–d, In Co-IP assay, ZmBLK1 interacts with ZmWIK (b), ZmWIKICD (c) and ZmWAKLY/ZmWAKLQ (d), but not with ZmWIKECD,TM (c). e, Interaction among ZmWIK, ZmBLK1 and ZmWAKLY (or ZmWAKLQ) in Co-IP assay. f, Autophosphorylation and kinase activity of ZmBLK1. g, Reciprocal phosphorylation between ZmBLK1 and ZmWIK. His-ZmWIKICD and ZmBLK1-GST and their kinase-inactive variants His-ZmWIKICD,K339E and ZmBLK1K114E-GST were used for in vitro phosphorylation assays. h,i, Reciprocal phosphorylation between ZmBLK1 and ZmWAKL. The in vitro phosphorylation assays were performed using two ZmWAKLICD-MBP isoforms with the kinase-inactive ZmBLK1K114E-GST (h) and two kinase-inactive ZmWAKLICD-MBP variants with ZmBLK1-GST (i). j, Mutual phosphorylation assays among ZmWAKL, ZmWIK and ZmBLK1. Phosphorylation levels of His-ZmWIKICD,K339E and ZmBLK1K114E-GST (I; n = 2 independent experiments), His-ZmWIKICD and ZmBLK1K114E-GST (II; n = 3 independent experiments), His-ZmWIKICD,K339E and ZmBLK1-GST (III; n = 3 independent experiments) and His-ZmWIKICD and ZmBLK1-GST (IV; n = 4 independent experiments) in the presence of ZmWAKLY or ZmWAKLQ or their kinase-inactive variants. In f–j, autoradiograph (upper) and CBB staining (lower) show phosphorylation and loading, respectively. In j, the data were shown as means ± s.d., and the statistical significance was determined by a paired t test. The Co-IP, SLC and phosphorylation assays were repeated at least two times.
