The mitogen-activated protein kinase kinase kinase, ILK5, regulates plant purinergic receptor-mediated, innate immunity

Mitogen-activated protein (MAP) kinase signaling cascades play important roles in the regulation of eukaryotic defense against various pathogens. Activation of the extracellular ATP (eATP) receptor P2K1 triggers MAP kinase 3 and 6 (MPK3/6) phosphorylation, which leads to elevated defense responses in Arabidopsis. However, the mechanism by which P2K1 activates the MAPK cascade is unclear. In this study, we identified Raf-like MAPKKK ILK5 as a downstream substrate of the P2K1 kinase. P2K1 phosphorylates ILK5 on serine 192. The interaction between P2K1 and ILK5 was confirmed both in vitro and in planta and their interaction was enhanced by ATP treatment. Similar to P2K1 expression, ILK5 expression levels were highly induced by treatment with ATP, flg22, Pseudomonas syringae pv. tomato DC3000, and various abiotic stresses, such as wounding. ILK5 interacts with both MKK4 and MKK5, but only MKK5 is phosphorylated by ILK5. Moreover, phosphorylation of MPK3/6 was significantly reduced upon ATP treatment in ilk5 mutant plants, relative to wild-type. The ilk5 mutant plants showed higher susceptibility to P. syringae pathogen infection relative to wild-type plants. Plants expressing only the mutant ILK5S192A protein, lacking kinase activity, did not activate the MAPK cascade upon ATP addition. Taken together, the results suggest that eATP activation of P2K1 results in transphosphorylation of the Raf-like MAPKKK ILK5, which subsequently triggers the MAPK cascade, culminating in activation of MAPK3 and 6 associated with an elevated innate immunity response. Significance statementPathogens invasion and subsequent wound stress activates extracellular ATP-mediated purinergic signaling cascades, a danger associated molecular pattern (DAMP) signal, which induces phosphorylation of MAPKs. Previous studies revealed that the P2K1 purinergic receptor increases MPK3/6 phosphorylation in response to eATP signaling cascades in Arabidopsis. However, the specific mechanism by which this occurs remains unknown. Here, we describe the isolation and characterization of Raf-like MAPKKK ILK5 (Integrin-linked Kinase 5) as a downstream substrate of P2K1 kinase activity. Initiation of an eATP-dependent signaling pathway by phosphorylation of ILK5 with subsequent activation of MKK5, leading to activation of MPK3/6 and downstream events is crucial to the plant innate immunity response.