B4-subgroup RAF kinases function as direct cellular osmosensors that detect hyperosmolarity through reversible condensation of their intrinsically disordered regions, recruiting subclass-I SnRK2 kinases into co-condensates that spatially exclude inhibitory PP2C phosphatases to enable rapid SnRK2 activation—a mechanism that can be fully reconstituted with three minimal components and represents an evolutionarily conserved osmosensing strategy across kingdoms.

Hyperosmolarity caused by drought, high salinity, or cold stress inhibits plant growth and crop productivity. A conserved protein-kinase cascade of cytosolic B-RAFs and SnRK2s is rapidly activated upon osmotic stresses to initiate downstream adaptive responses, which represents one of the fastest known responses to osmotic stress in plants. How the kinase cascade is activated by osmotic stress is unknown. Here, we show that Arabidopsis B4 subgroup RAFs have intrinsically disordered regions and directly sense both ionic and non-ionic hyperosmolarity by reversible condensation. B4-RAFs recruit and co-condense with subclass-I SnRK2s to phosphorylate and turn on SnRK2s, evading the non-condensable inhibitory A-clade PP2C phosphatases. This straightforward osmosensing and relaying module can be fully reconstituted in E. coli by co-expressing three components or in solution in a test tube using recombinant proteins. Our findings identify B-RAFs as the chief cellular osmosensors that detect low water potential by co-condensation, forming a signal hub with SnRK2s to orchestrate adaptive responses in plants, and represent an evolutionarily conserved osmosensing mechanism across kingdoms.