The CsGL3–CsERF22–CsTBH–CsMYS1 pathway governs multicellular trichome development from initiation to maturation in cucumber

Multicellular trichomes serve as protective barriers by providing physical protection (non-glandular trichomes) and chemical defenses (glandular trichomes). Although key transcription factors (TFs) regulate developmental transitions during organogenesis, few have been linked to the early stages of multicellular trichome development. Here, we identify a core regulatory pathway involving four TFs-CsGL3, CsERF22, CsTBH, and CsMYS1-that coordinates the stepwise development of multicellular trichomes in cucumber (Cucumis sativus). Loss of CsGL3 results in a completely glabrous phenotype, with no trichome bulge formation. Loss of CsERF22 does not prevent trichome bulge initiation but blocks subsequent cell proliferation and the formation of multicellular structures. Disruption of CsTBH causes trichome development to arrest at the stacked multicellular stage, preventing further maturation into functional glandular or pointed trichomes. By contrast, loss of CsMYS1 does not affect trichome maturation but disrupts type specification, with some non-glandular trichomes developing swollen glandular heads instead of pointed tips. Genetic and molecular analyses indicate that these TFs operate in a sequential cascade, each directly activating the next to drive trichome morphogenesis from initiation to functional specialization. This study identifies key TFs that govern the formation of specific micro-organs, the multicellular trichomes, providing mechanistic insight into organogenesis and suggesting potential strategies to enhance plant resistance.