Phosphorylation of Novel Interactors of Wild Soybean GsSnRK1 Protein Kinase

Plant SnRK1 kinases have been reported to participate in regulating many aspects of plant biology including resistance to biotic and abiotic stresses. Wild soybean ( Glycine soja ) has strong adaptation abilities to extreme environmental conditions and has four GsSnRK1 genes in its genome. However, the functions of GsSnRK1 are still largely unknown. In this work, we employed yeast two-hybrid and co-immunoprecipitation to determine the physical interactions of GsSnRK1 and three unknown proteins (GsKIP1, GsKIP2, and GsKIP3). To further characterize these proteins, we expressed and purified them in E. coli system. Biochemical analyses indicated that Thr176 of GsSnRK1 could be directly phosphorylated by its upstream kinase GsGRIK1 and the activated GsSnRK1 could phosphorylate its interactors GsKIP1, GsKIP2, GsKIP3, and ABF2 revealed by PhosTag-based approaches and specific phospho-antibodies. Intriguingly, a constituted ABA receptor complex consisting of GsPYL, GsPP2C, could perceive exogenous ABA, and subsequently, the ABA-bound receptor could activate GsSnRK1 to phosphorylate ABF2, suggesting that GsSnRK1 is an essential and bona fide component of ABA signaling pathway. This study is the first time to comprehensively characterize the in vitro functions of wild soybean GsSnRK1 kinase, providing the clues to unveil the novel molecular roles of SnRK1 kinase in cell signaling.