Probing the genomic and proteomic basis of encystment in Oxytricha granulifera

Protozoan encystment constitutes a pivotal survival strategy against environmental stressors; however, the molecular architecture governing this transition remains enigmatic, owing to limited genomic resources and a scarcity of integrated multi-omics investigations. Here, we elucidate the mechanisms underlying encystment in Oxytricha granulifera by reporting the first macronuclear genome assembly and conducting a comprehensive integration of transcriptomic, proteomic, and morphological analyses across vegetative and cyst stages. Morphological restructuring, typified by ciliary dedifferentiation and cyst wall formation, is molecularly supported by the downregulation of microtubule dynamics-associated genes and the concurrent upregulation of vesicle transport machinery. Furthermore, expanded gene families linked to carbohydrate metabolism and cellular acidification coincide with observed autophagic clearance and mucocyst activity, highlighting a coordinated metabolic shift essential for cyst formation. Elevated expression of the ubiquitin-proteasome system and autophagy pathways, which mediate protein turnover, along with upregulation of antioxidant enzyme genes, contributes to alleviating oxidative damage. Notably, we identified rewired post-transcriptional regulation that increases spliceosome activity and alternative splicing frequency, with each trend validated at the protein level. Concurrently, we observed a distinct epigenetic signature characterized by the significant downregulation of DNA N⁶-adenine methylation (6mA) methyltransferases (homologs of AMT1 and AMT6/7), suggesting a potential repressive role of methylation during the cyst stage. Collectively, these findings provide a multidimensional atlas of the encystment process, revealing that O. granulifera accomplishes cellular structural remodeling through a multilayered regulatory network spanning morphological, genetic, transcriptomic, and proteomic levels. IMPORTANCE Oxytricha species are widely distributed in freshwater and terrestrial ecosystems, playing significant ecological roles in microbial communities. Their ability to undergo encystment provides a powerful model for studying cellular differentiation and stress adaptation in microbial eukaryotes. This study presents the first multi-omics analysis of encystment in Oxytricha granulifera, revealing microbial survival strategies through enhanced protein turnover, autophagy, alternative splicing, and DNA methylation reprogramming. These findings offer fundamental insights into dormancy mechanisms and environmental adaptation in protists, advancing our understanding of microbial resilience, evolutionary innovation, and ecological success in fluctuating environments.