A tonoplast cytokinin riboside transporter gates intracellular hormone availability at the plant-microbe interface

Cytokinin ribosides are major mobile and precursor forms of cytokinins, but the mechanisms that control their intracellular availability remain poorly understood. Here, we identify Arabidopsis thaliana EQUILIBRATIVE NUCLEOSIDE TRANSPORTER1 (ENT1) as a tonoplast-localized cytokinin riboside transporter that gates intracellular cytokinin riboside homeostasis. ENT1 transported trans-zeatin riboside and isopentenyladenosine, but not the corresponding free bases, and mutational analysis combined with molecular docking identified residues required for riboside recognition and discrimination from nucleobases. A functional internally tagged reporter resolved previous ambiguity in ENT1 localization, showing predominant tonoplast association under native-like conditions and enrichment in epidermal and other outer root tissues. Increasing ENT1 abundance enhanced plant sensitivity specifically to cytokinin ribosides and elevated cytokinin signalling output, while radiotracer assays supported a role for ENT1 in cytokinin riboside accumulation in roots. Conversely, loss of ENT1 did not cause a pronounced developmental phenotype under standard conditions, but reconfigured tissue-specific profiles of adenosine and cytokinin riboside-related metabolites, with the strongest effects on cis-zeatin-type and riboside O-glucosylated cytokinin pools. ENT1 function became physiologically apparent during plant-microbe interactions, where ent1 mutants showed reduced beneficial microbe-associated protection against Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000. These findings reveal a vacuolar gatekeeping mechanism that controls intracellular cytokinin riboside availability and links cytokinin riboside homeostasis to beneficial microbe-associated defence outputs.