Novel insights into molecular mechanisms of Pseudourostyla cristata encystment using comparative transcriptomics

Abstract

The encystment of many ciliates is an advanced survival strategy against adversity and the most important reason for ciliates existence worldwide. However, the molecular mechanism for the encystment of free-living ciliates is poorly understood. Here, we performed comparative transcriptomic analysis of dormant cysts and trophonts from Pseudourostyla cristata using transcriptomics, qRT-PCR and bioinformatic techniques. We identified 2565 differentially expressed unigenes between the dormant cysts and the trophonts. The total number of differentially expressed genes in GO database was 1752. The differential unigenes noted to the GO terms were 1993. These differential categories were mainly related to polyamine transport, pectin decomposition, cytoplasmic translation, ribosome, respiratory chain, ribosome structure, ion channel activity, and RNA ligation. A total of 224 different pathways were mapped. Among them, 184 pathways were upregulated, while 162 were downregulated. Further investigation showed that the calcium and AMPK signaling pathway had important induction effects on the encystment. In addition, FOXO and ubiquitin-mediated proteolysis signaling pathway jointly regulated the encystment. Based on these findings, we propose a hypothetical signaling network that regulates Pseudourostyla cristata encystment. Overall, these results provide deeper insights into the molecular mechanisms of ciliates encystment and adaptation to adverse environments.of Strepkiella histriomuscorum has been studied in the encystment and trophont stages through biochip technology, which suggested that cyst formation is associated with Ribosomal L7, Ribosomal acidic P2, Cathepsin B, Cathepsin H, Ubiquitin, Ca 2+ -ATPase and Actin1 7 . Other studies investigated the differential proteins of the cysts and the trophonts from Pseudourostyla cristata (P. cristata) using shotgun LC-MS/MS, finding the association of fibrillarin-like rRNA methylase, methylmalonyl-coenzyme mutase, ADP ribosylation factor, Rab12, MAPK-related kinase and KR multi-domain proteins with cyst formation 8 . However, the reports of systematically studying the genes and signaling pathways involved in the formation of ciliate cysts at the molecular level are rare 8 . In this study, we have investigated the molecular mechanism underlying the cyst formation in ciliates using comparative transcriptomic analysis, quantitative real-time PCR (qRT-PCR) and bioinformatic techniques. Furthermore, we have focused on the analysis of several important signaling pathways related to the encystment. Therefore, we have proposed a hypothetical network that regulates Pseudourostyla cristata encystment. Our study generates novel insights into the molecular mechanisms of the cyst formation in free-living ciliates.www.nature.com/scientificreports www.nature.com/scientificreports/ dormant cysts of P. cristata (Fig. 4). Validation of results by transcriptome sequencing showed that mRNA outcomes were consistent with the transcriptomic data of trophonts and dormant cysts in P. cristata (Fig. 4).

The Encystment-Related MicroRNAs and Its Regulation Molecular Mechanism in Pseudourostyla cristata Revealed by High Throughput Small RNA Sequencing

“…These results suggested that downregulation of miR-23b-3p not only enhanced autophagy in the encystment of P. cristata but also improved the ability of the cysts to resist stress with increased GPX. These findings are consistent with our previous results [3].…”

“…The upregulated miR-106b-3p was participated in the downregulation of the target gene cytochrome c oxidase subunit 5b (COX5B) in the oxidative phosphorylation (ko00190) signaling pathway. Above all, differentially expressed genes such as GPX, UBE1, SGK, CPT1, and F-Actin were consistent with our previously reported findings [3]. According to the current investigation, we listed a miRNA-mRNA interaction relationship related to the encystment (Table 4).…”

“…Jiang et al found AMPK, CaMK, ABCC4, ERK1/2, and PP1 were associated with the cyst formation of Colpoda aspera through transcriptomic techniques [10]. Our previous report found calcium, AMPK, FOXO, and a ubiquitin-mediated proteolysis signaling pathway jointly regulated the encystment of P. cristata [3]. However, to the best of our knowledge, there is no attention that has been paid to the function of miRNAs involved in the regulatory formation of ciliate cysts.…”

“…Many ciliates adopt encystment as a survival strategy, i.e., the vegetative cells of ciliates can transform into dormant cysts in responding to harsh environmental conditions e.g., starvation, crowding, sudden changes in temperature, and lack of food [2]. Studies on the dormant cyst formation contribute to the understanding of the mechanism of eukaryotic cell morphogenesis [3]. Several early studies have reported the induction factors, physiological and biochemical changes in the process of ciliate encystment [4,5], and there were also reports that documented the changes in macronuclear DNA demethylation during encystment and the protein post-transcriptional modification of cyst dormancy [6].…”

Analysis of Water-Soluble Proteins by Two-Dimensional Electrophoresis in the Encystment Process of Colpoda cucullus Nag-1 and Cytoskeletal Dynamics

“…Thereafter, the signaling pathways for encystment were extensively elucidated by Colpoda aspera transcriptome analysis performed by Jiang et al (2019); in the signaling pathways activated by Ca 2+ /calmodulin, the expression of genes for AMP-activated protein kinase (AMPK), eukaryotic elongation factor-2 kinase (eEF2K), AKT and several genes for autophagy are upregulated. Transcriptomic analysis of encysting Pseudourostyla also proposed the outline of signaling pathways for encystment including Ca 2+ /calmodulin signaling pathways (Pan et al 2019).…”