Cytokinin arabinosides show increased efficacy in priming tomato immunity and inhibiting the fungal phytopathogen B. cinerea

• Cytokinin (CK) has been reported to prime plant immunity, however, previous research on CK hormopriming has largely focused on classical synthetic cytokinins like kinetin or 6-benzylaminopurine (BAP). Here, we asked whether synthetic aromatic CK derivatives could offer enhanced efficacy in priming immunity and inhibiting pathogens due to their unique chemical structure. • Our work investigated how CK arabinosides compare to classical CKs in terms of immunity priming efficacy and undesirable effects on plant growth, uncovering the underlying molecular mechanisms and signalling pathways involved in CK arabinoside-mediated immunity in plants and their inhibitory effects on fungi. • Our findings show that CK arabinosides are substantially more effective at promoting immunity and reducing disease caused by the necrotrophic fungal pathogen Botrytis cinerea and the hemibiotrophic bacterium Xanthomonas euvesicatoria in tomato compared to the classical CK 6-BAP. • Crucially, unlike classical CKs, CK arabinosides achieve these benefits without strongly activating canonical cytokinin signalling pathways in plants. Thus, they possess immunity-promoting and fungal inhibitory activities with minimal unwanted effects on plant growth and development. • On the mechanistic level, our work reveals that CK arabinoside-mediated immunity in tomato primarily operates via a salicylic acid (SA)-dependent mechanism and is generally jasmonic acid (JA)-independent; and that CK arabinosides alter sugar metabolism and virulence pathways in B. cinerea . • Importantly, unlike classical CKs, CK arabinosides do not reverse growth inhibition under sugar-limited conditions in fungi, contributing to their stronger antifungal effects and distinguishing their mechanism of action from classical CKs. Cytokinins (CKs) regulate numerous biological processes in plants, and are involved in plant growth and development as well as responses to biotic and abiotic stresses. Although CKs have been reported to prime plant immunity, thus far, research on CK hormopriming has focused on classical synthetic CKs such as kinetin or 6-benzylaminopurine (BAP). Here, we show that synthetic aromatic CK derivatives—specifically, 3-methoxy-6-benzylaminopurine-9-arabinosides—effectively induce immune responses and promote resistance to Botrytis cinerea in tomato ( Solanum lycopersicum ) primarily via a salicylic acid (SA)-dependent mechanism. Additionally, like 6-BAP, these compounds directly inhibit B. cinerea . Transcriptomic analyses of B. cinerea treated with CK arabinosides revealed significant changes in expressed gene profiles, notably in pathways associated with sugar metabolism. Unlike classical CKs, CK arabinosides do not alleviate growth inhibition under sugar-limited conditions, contributing to their stronger antifungal effects. Furthermore, CK arabinosides induce tomato immune responses more robustly than classical CKs, yet without activating canonical cytokinin signaling pathways, likely owing to their exceptional chemical stability which prevents release of the free active CK base, and due to their inability to directly bind CK receptors. Altogether, our findings suggest that CK arabinosides act via a dual mechanism—immune priming and direct pathogen inhibition—and hold promise as next-generation hormopriming agents for sustainable crop protection.