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MRNA-seq and miRNA-seq profiling analyses reveal molecular mechanisms regulating induction of fruiting body in Ophiocordyceps sinensis.

ABSTRACT: Ophiocordyceps sinensis has been a source of valuable materials in traditional Asian medicine for over two thousand years. With recent global warming and overharvest, however, the availability of these wild fungi has decreased dramatically. While fruiting body of O. sinensis has been artificially cultivated, the molecular mechanisms that govern the induction of fruiting body at the transcriptional and post-transcriptional levels are unclear. In this study, we carried out both mRNA and small RNA sequencing to identify crucial genes and miRNA-like RNAs (milRNAs) involved in the development of fruiting body. A total of 2875 differentially expressed genes (DEGs), and 71 differentially expressed milRNAs (DEMs) were identified among the mycoparasite complex, the sclerotium (ST) and the fruiting body stage. Functional enrichment and Gene Set Enrichment Analysis indicated that the ST had increased oxidative stress and energy metabolism and that mitogen-activated protein kinase signaling might induce the formation of fruiting body. Integrated analysis of DEGs and DEMs revealed that n_os_milR16, n_os_milR21, n_os_milR34, and n_os_milR90 could be candidate milRNAs that regulate the induction of fruiting body. This study provides transcriptome-wide insight into the molecular basis of fruiting body formation in O. Sinensis and identifies potential candidate genes for improving induction rate.

SUBMITTER: Zhang H

PROVIDER: S-EPMC8217512 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundOphiocordyceps sinensis, which is only naturally found in the high-elevation extreme environment of the Tibetan Plateau, has been used in traditional Chinese medicine. Information concerning the evolutionary and geologic context of O. sinensis remains limited, however.MethodsWe constructed the high-quality genome of O. sinensis and provided insight into the evolution and ecology of O. sinensis using comparative genomics.ResultsWe mapped the whole genome of the anamorph/asexual form Hirsutella of O. sinensis using Illumina and PacBio sequencing technologies and obtained a well assembled genome of 119.2 Mbp size. Long-read Single Molecule Real Time (SMRT) sequencing technology generated an assembly with more accurate representation of repeat sequence abundances and placement. Evolutionary analyses indicated that O. sinensis diverged from other fungi 65.9 Mya in the Upper Cretaceous, during the uplift of the Tibetan Plateau. Gene family expansions and contractions in addition to genome inflation via long terminal repeat (LTR) retrotransposon insertions were implicated as an important driver of O. sinensis divergence. The insertion rate of LTR sequences into the O. sinensis genome peaked ~ 30-40 Mya, when the Tibetan Plateau rose rapidly. Gene Ontology (GO) enrichment analysis suggested that O. sinensis contained more genes related to ice binding compared to other closely related fungi, which may aid in their adaptability to the cold Tibetan Plateau. Further, heavy metal resistance genes were in low abundance in the O. sinensis genome, which may help to explain previous observations that O. sinensis tissues contain high levels of heavy metals.ConclusionsOur results reveal the evolutionary, geological, and ecological context for the evolution of the O. sinensis genome and the factors that have contributed to the environmental adaptability of this valuable fungus. These findings suggest that genome inflation via LTR retrotransposon insertions in O. sinensis coincided with the uplift of the Tibetan Plateau. LTRs and the specific genetic mechanisms of O. sinensis contributed to its adaptation to the environment on the plateau.

Project description:Ophiocordyceps sinensis is a highly valued fungus that has been used as traditional Asian medicine. This fungus is one of the most important sources of income for the nomadic populations of the Tibetan Plateau. With global warming and excessive collection, the wild O. sinensis resources declined dramatically. The cultivation of O. sinensis hasn't been fully operational due to the unclear genetic basis of the fruiting body development. Here, our study conducted pairwise comparisons between transcriptomes acquired from different growth stages of O. sinensis including asexual mycelium (CM), developing fruiting body (DF) and mature fruiting body (FB). All RNA-Seq reads were aligned to the genome of O. sinensis CO18 prior to comparative analyses. Cluster analysis showed that the expression profiles of FB and DF were highly similar compared to CM. Alternative splicing analysis (AS) revealed that the stage-specific splicing genes may have important functions in the development of fruiting body. Functional enrichment analyses showed that differentially expressed genes (DEGs) were enriched in protein synthesis and baseline metabolism during fruiting body development, indicating that more protein and energy might be required for fruiting body development. In addition, some fruiting body development-associated genes impacted by ecological factors were up-regulated in FB samples, such as the nucleoside diphosphate kinase gene (ndk), β subunit of the fatty acid synthase gene (cel-2) and the superoxide dismutase gene (sod). Moreover, the expression levels of several cytoskeletons genes were significantly altered during all these growth stages, suggesting that these genes play crucial roles in both vegetative growth and the fruiting body development. Quantitative PCR (qPCR) was used to validate the gene expression profile and the results supported the accuracy of the RNA-Seq and DEGs analysis. Our study offers a novel perspective to understand the underlying growth stage-specific molecular differences and the biology of O. sinensis fruiting body development.

Project description:In this study, using an isobaric tags for relative and absolute quantitation (iTRAQ ) approach coupled with LC-MS / MS and bioinformatics, the proteomes were analyzed for the crucial three stages covering the fruiting body development of Ophiocordyceps sinensis, including sclerotium (ST), primordium (PR) and mature fruiting body (MF), with a focus on fruiting body development-related proteins and the potential mechanisms of the development. A total of 1,875 proteins were identified. Principal Component Analysis (PCA) demonstrated that the protein patterns between PR and MF were more similar than ST. Differentially accumulated proteins (DAPs) analysis showed that there were 510, 173 and 514 DAPs in the comparisons of ST vs. PR, PR vs. MF and ST vs. MF, respectively. A total of 62 shared DAPs were identified and primarily enriched in proteins related to 'carbon transport and mechanism', 'the response to oxidative stress', 'antioxidative activity' and 'translation'. KEGG and GO databases showed that the DAPs were enriched in terms of 'primary metabolisms (amino acid/fatty acid/energy metabolism)', 'the response to oxidative stress' and 'peroxidase'. Furthermore, 34 DAPs involved in reactive oxygen species (ROS) metabolism were identified and clustered across the three stages using hierarchical clustering implemented in hCluster R package . It was suggested that their roles and the underlying mechanisms may be stage-specific. ROS may play a role in fungal pathogenicity in ST, the fruit-body initiation in PR, sexual reproduction and highland adaptation in MF. Crucial ROS-related proteins were identified, such as superoxide dismutase (SOD, T5A6F1), Nor-1 (T5AFX3), electron transport protein (T5AHD1), histidine phosphotransferase (HPt, T5A9Z5) and Glutathione peroxidase (T5A9V1). Besides, the accumulation of ROS at the three stages were assayed using 2,7-dichlorofuorescin diacetate (DCFH-DA) stanning. A much stronger ROS accumulation was detected at the stage MF, compared to the stages of PR and ST. Sections of ST and fruit-body part of MF were stained by DCFH-DA and observed under the fluorescencemicroscope, showing ROS was distributed within the conidiospore and ascus. Besides, SOD activity increased across the three stages, while CAT activity has a strong increasement in MF compared to the stages of ST and PR. It was suggested that ROS may act in gradient-dependent manner to regulate the fruiting body development. The coding region sequences of six DAPs were analyzed at mRNA level by quantitative real-time PCR (qRT-PCR). The results support the result of DAPs analysis and the proteome sequencing data. Our findings offer the perspective of proteome to understand the biology of fruiting body development and highland adaptation in O. sinensis, which would inform the big industry of this valuable fungus.

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MRNA-seq and miRNA-seq profiling analyses reveal molecular mechanisms regulating induction of fruiting body in Ophiocordyceps sinensis.

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