Fig. 4: TaPP2C-7A directly dephosphorylates TaNHLP1-A phosphorylated by TaSnRK2-1A in vitro, thereby mitigating its degradation.

a Split-ubiquitin yeast two-hybrid assays confirmed the interaction between TaNHLP1-A and TaSnRK2-1A and TaPP2C-7A. Yeast cells were first selected on synthetic dropout (SD) medium lacking Leu and Trp (SD-LW) and then transferred to SD-Leu/-Trp/-His/-Ade (SD-LWHA) selective medium with 25 mM 3-AT for protein interaction analysis. b TaNHLP1-A interacts with TaSnRK2-1A and TaPP2C-7A, respectively, in Luciferase complementation imaging assays conducted in N. benthamiana leaves. c The interactions between MYC-TaNHLP1-A, HA-TaSnRK2-1A, and HA-TaPP2C-7A were revealed through co-immunoprecipitation assays. Following immunoprecipitation with anti-HA magnetic beads, the precipitated proteins were probed using anti-HA and anti-MYC antibodies, respectively. d TaSnRK2-1A phosphorylates TaNHLP1-A and is negatively regulated by TaPP2C-7A in vitro. Kinase assays were performed for 90 min at 30 °C. Prior to the addition of TaSnRK2-1A, TaNHLP1-A, and ATP, TaPP2C-7A was incubated with TaPYL-1D and ABA on ice for 20 min. The phosphorylation status of MBP-^△TaNHLP1-A was analyzed using a Phos-tag gel assay (top, anti-MBP). The phosphorylated (MBP-p^△TaNHLP1-A) and unphosphorylated (MBP-^△TaNHLP1-A) forms of TaNHLP1-A are indicated by the red and black arrows, respectively. Red stars indicate non-specific bands. e TaPP2C-7A directly dephosphorylates TaNHLP1-A in vitro. MBP-^△TaNHLP1-A and Flag-TaSnRK2-1A plasmids were co-expressed in Escherichia coli strain BL21, then the cell lysates were incubated with GST-TaPP2C-7A in a protein phosphatase reaction buffer for 90 min. The phosphorylated MBP-^△TaNHLP1-A, indicated by the red arrow, were detected by Phos-tag gel (top, anti-MBP). ^△TaNHLP1-A refers to TaNHLP1-A with the signal peptide (1-27 aa) and transmembrane domain (241–263 aa) removed. f Subcellular localization of TaNHLP1-A-GFP with or without HA-TaSnRK2-1A in N. benthamiana leaves. GmMan1-mCherry was used as a cis-Golgi marker protein. Scale bars, 50 μm. g TaSnRK2-1A triggers TaNHLP1-A degradation in N. benthamiana, possibly by influencing its phosphorylation. Red star indicates non-specific bands. The experiments shown in (c–e) and (f, g) were performed independently two and three times, respectively, with similar results. Source data are provided as a Source Data file.