The Wheat Intrinsically Disordered Protein Td RL1 Negatively Regulates the Type One Protein Phosphatase Td PP1

Type 1 protein phosphatases (PP1s) are crucial in various plant cellular processes. Their function is controlled by regulators known as PP1-interacting proteins (PIPs), often intrinsically disordered, such as Inhibitor 2 (I2), conserved across kingdoms. The durum wheat Td RL1 acts as a positive regulator of plant stress tolerance, presumably by inhibiting PP1 activity. In this work, co-immunoprecipitation and bimolecular fluorescence complementation (BiFC) assays demonstrate that the durum wheat Td PP1 interacts with both Td RL1 and At -I2 in vivo. YFP fluorescence restored after Td RL1- Td PP1 interaction decorated specifically the microtubular network of the tobacco co-infiltrated cells. In vitro phosphatase assays revealed that Td RL1 inhibited the activity of wild-type Td PP1 and two mutant forms (T243M and H135A) in a concentration-dependent manner, showing a novel and potent inhibition mechanism. Structural modeling of the Td PP1-inhibitor complexes suggested that both At -I2 and Td RL1 bind to Td PP1 by wrapping their flexible C-terminal tails around it, blocking access to the active site. Remarkably, the model showed that Td RL1 differs from At -I2 in its interaction with Td PP1 by trapping the phosphatase with its N-terminal tail. These findings provide important insights into the regulatory mechanisms governing the activity of PP1s in plants and highlight the potential for targeted inhibition to modulate plant stress responses.