INDETERMINATE-DOMAIN 4 (IDD4) coordinates immune responses with plant-growth in Arabidopsis thaliana
Fig 5
Interaction analysis and kinase assay of IDD4 with MPK6.
(A) In-vitro pull-down assays of MBP-tagged IDD4 showed prevalent interaction with MPK6 and minor association to MPK3 and MPK4. The negative control GST and MBP did not show interaction with MPK3,-4,-6. (B) Nuclear interaction study by Bimolecular Fluorescence Complementation of IDD4 with MPK6 in Nicotiana benthamiana epidermal cells. SCL3 served as positive control, UBIQUITIN10 and empty vector (EV) acted as negative control. Scale bar = 50 μm. (C-D) in-vitro kinase assays were performed by using recombinant IDD4 and constitutively active MPK6 and analyzed by LC/MS-MS. Depicted are the spectra of the obtained phosphorylated IDD4 peptides containing SERINE-73 (Ser-73) and THREONINE-130 (Thr-130). (E) Domain map of IDD4. IDD4 contains a nuclear-localisation signal (NLS) at the very N-terminus and a highly conserved ID domain that comprises 4 zinc finger (ZF). MPK6-targeted phospho-peptides reside in front of ZF1 (S73) and inside of ZF2 (T130).