Cycloartenol-derived triterpenoid pathway genes alter the root metabolome and microbiome in tomato

Plant triterpenoids derived from cycloartenol are central to sterol homeostasis and specialized metabolite production, yet their roles in shaping rhizosphere interactions remain poorly understood. Here, we investigated the function of key cycloartenol-derived triterpenoid biosynthetic genes in tomato (Solanum lycopersicum) by transiently silencing CYCLOARTENOL SYNTHASE 1 (SlCAS1), STEROL METHYLTRANSFERASE 1 (SlSMT1), STEROL SIDE CHAIN REDUCTASE 2 (SlSSR2), and PHYTOENE DESATURASE (SlPDS). SlCAS1 suppression caused severe growth inhibition, confirming the essential role of cycloartenol for plant development. Silencing of SlSMT1 and SlSSR2 altered root sterol composition, with SlSMT1 reducing β-sitosterol and stigmasterol, and SlSSR2 causing decreases in cholesterol as well as significant reductions in steroidal glycoalkaloids (SGAs) and steroidal saponins (SAs). By contrast, SlPDS silencing unexpectedly led to elevated sterol levels and broad metabolome shifts. Untargeted metabolomics revealed gene-specific alterations in root and exudate profiles, while molecular networking highlighted the rapid loss of SGAs in exudates, suggesting microbial degradation. Integration of metabolomic and 16S rRNA sequencing data showed that changes in sterols, SGAs, and saponins were associated with distinct bacterial families, including Comamonadaceae and Sphingomonadaceae. Together, these findings demonstrate that cycloartenol-derived triterpenoid pathway genes strongly influence root metabolite composition and shape the assembly of tomato root-associated microbial communities.