Probing the genomic and proteomic basis of encystment in Oxytricha granulifera

Abstract

Protozoan encystment is a crucial survival strategy for resisting environmental stresses, yet its molecular mechanisms remain poorly understood due to limited genomic resources and lack of integrated multi-omics analyses in model ciliates. In this study, we aimed to elucidate the molecular mechanisms underlying the encystment process in Oxytricha granulifera by sequencing and assembled its genome, and by conducting comprehensive transcriptome, proteome and morphology analyses to reveal gene expression and morphologic changes between vegetative and cyst stages. Morphological observation revealed ciliary dedifferentiation and cyst-wall formation during encystment, events respectively supported by the downregulation of microtubule dynamics related genes and the upregulation of vesicle transport related genes in cyst stage. Expanded gene families for carbohydrate metabolism, autophagy and cellular acidification align with the species mucocyst signature and their observed autophagic clearance, hinting at previously unrecognized mechanisms underlying cyst formation. Elevated expression of the ubiquitin-proteasome system and autophagy pathways facilitates protein turnover, and upregulation of antioxidant enzymes genes helps mitigate oxidative damage, while a rewired post-transcriptional regulation that increases spliceosome activity and alternative splicing frequency--each trend validated at the protein level. Concurrently, the methyltransferases responsible for DNA N6-adenine methylation (6mA), with homologous genes of AMT1 and AMT6/7 significantly downregulated. These results suggesting a multilayered regulation of ciliate encystment and the first integrated evidence that alternative splicing modulates dormancy. Our findings establish the first multi-omics framework for O. granulifera encystment, offering baseline data and tentative clues to the dormancy mechanism that will inform future inquiries into how single-celled eukaryotes endure adverse environments. IMPORTANCEOxytricha 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.