Identification of cordycepin biosynthesis-related genes through de novo transcriptome assembly and analysis in Cordyceps cicadae

Cordyceps cicadae (Chanhua) is a parasitic fungus that grows on Cicada flammata larvae and is used to relieve exhaustion and treat numerous diseases, in part through its active constituent, cordycepin. We used de novo Illumina HiSeq 4000 sequencing to obtain transcriptomes of C. cicadae mycelium, fruiting body, and sclerotium, and identify differentially expressed genes. In the mycelium versus sclerotium libraries, 1576 upregulated and 2300 downregulated genes were identified. In the mycelium versus fruiting body and fruiting body versus sclerotium body libraries, 1604 and 1474 upregulated and 1365 and 1320 downregulated genes, respectively, were identified. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses identified 19 genes differentially expressed in mycelium versus fruiting body as related to the purine pathway, along with 28 and 16 genes differentially expressed in the mycelium versus sclerotium and fruiting body versus sclerotium groups, respectively. Gene expression of six key enzymes was validated by quantitative polymerase chain reaction. Specifically, 5′-nucleotidase (c62060g1) and adenosine deaminase (c35629g1) in purine nucleotide metabolism, which are involved in cordycepin biosynthesis, were significantly upregulated in the sclerotium group. These findings improved our understanding of genes involved in the biosynthesis of cordycepin and other characteristic secondary metabolites in C. cicadae.


Recommendation?
Major revision is needed (please make suggestions in comments)

Comments to the Author(s)
This manuscript (MS) RSOS-181247 reported the "Identification of Cordycepin Biosynthesisrelated genes through de novo transcriptome assembly and analysis of Cordyceps cicadae". The MS is relatively well written, scientifically sound and the outcomes are relatively well explained, and it is of interest to the readership of Royal Society Open Science. Therefore, I recommend its acceptance for publication after major revision. The MS needs an extensive proofreading. There are extra full points and missing spaces between words and references. I strong suggest reviewing all the citations, there are two changed references. I also recommend looking after the Royal Society Open Science rules for authors, example: Fig The editors assigned to your paper ("Identification of Cordycepin Biosynthesis-related Genes Through de novo Transcriptome Assembly and Analysis of Cordyceps cicadae") have now received comments from reviewers. While some of the reviewers are very positive about publication, the reviewers also raise some substantive points which merit careful consideration. We would like you to revise your paper in accordance with the referee and Associate Editor suggestions which can be found below (not including confidential reports to the Editor). Please note this decision does not guarantee eventual acceptance.
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. Comments to the Author(s) Major comments: I suggest the authors organize their manuscript in a logical way, and present their data in a simple and direct way. It's better if they provide a hypothesis for their study. The language should also be polished by an English-speaking expert. L15-24 What is the difference between "gene expression analysis" and "GO and KEGG analyses"? Each analysis produced different numbers of differential genes between libraries. Why? L24 Why were the six genes chosen? L66 Ophiocordyceps sinensis and Hirsutella sinensis are representing the same species. The fungus is known to produce undetectable cordycepin. L113-117 Primers ITS1 and ITS4 are actually amplifying the ITS region instead of 18S rDNA. PCR extension time of 90 s at 72℃ is too long for the 500-bp ITS region. L155-156 Are "cultured in canned bottles" and "buried in soil" describing the same thing? L238-250 The paragraph can be simplified to avoid duplicating methods. L253 What's the difference between a unigene and a transcript? In later parts of the manuscript, it seems that only unigenes were analyzed, and transcripts were not mentioned again. L311, L314, and other parts. It's not a conventional way to show and cite data by providing the file name directly in the text. The subsection 3.5 is partially duplicating subsection 3.2.
Minor comments: L27 are responsible L34 Cordycipitaceae is a family name and Cordyceps is a genus name.
L47 O. sinensis L105-106 What's the difference between fruiting body and stromata? L126 "strain buc" should not be italic. Figure 5B The venn diagram is unusual. Each big circle should be a library instead of a comparison as shown.
Reviewer: 2 Comments to the Author(s) In the manuscript, the authors provide the first comparative transcriptomic data of Cordyceps cicadae, one of the most famous fungi as traditional Chinese medicinal material. In addition, the authors were also trying to identify genes related to cordycepin biosynthesis. This study improved our understanding of secondary metabolism in C. cicadae. The study is solid and the results reported are interesting. The manuscript is also nicely written. Therefore, this manuscript is worth publication. Following please a few examples to improve this manuscript. 1) Line 51 The second and subsequent generic name Cordyceps in the text should be abbreviated. Comments to the Author(s) This manuscript (MS) RSOS-181247 reported the "Identification of Cordycepin Biosynthesisrelated genes through de novo transcriptome assembly and analysis of Cordyceps cicadae". The MS is relatively well written, scientifically sound and the outcomes are relatively well explained, and it is of interest to the readership of Royal Society Open Science. Therefore, I recommend its acceptance for publication after major revision. The MS needs an extensive proofreading. There are extra full points and missing spaces between words and references. I strong suggest reviewing all the citations, there are two changed references. I also recommend looking after the Royal Society Open Science rules for authors, example: Fig

RSOS-181247.R1 (Revision)
Review form: Reviewer 1 Is the manuscript scientifically sound in its present form? Yes

Are the interpretations and conclusions justified by the results? Yes
Is the language acceptable? Yes

Recommendation?
Accept with minor revision (please list in comments)

Comments to the Author(s)
Title in Cordyceps cicadae Introduction Cordyceps cicadae can parasitize many hosts, not just Cicada flammata. 3.2 The last sentence is unclear. Why? 3.3 Why did you use fruiting bodies cultured in canned bottles while sclerotium samples buried in soil? 4.1 Mycelium-1-3 References Not all authors were listed consistent with papers for some literatures (e.g., 8) while only one author was listed for some followed by et al (e.g., 19).

Are the interpretations and conclusions justified by the results? Yes
Is the language acceptable? Yes

Recommendation?
Accept as is

Comments to the Author(s)
The authors answered the questions appropriately, reviewed the text formatting and the English language. I recommend its acceptance for publication.

14-Nov-2018
Dear Dr Liu: On behalf of the Editors, I am pleased to inform you that your Manuscript RSOS-181247.R1 entitled "Identification of Cordycepin Biosynthesis-related Genes Through de novo Transcriptome Assembly and Analysis of Cordyceps cicadae" has been accepted for publication in Royal Society Open Science subject to minor revision in accordance with the referee suggestions. Please find the referees' comments at the end of this email.
The reviewers and Subject Editor have recommended publication, but also suggest some minor revisions to your manuscript. Therefore, I invite you to respond to the comments and revise your manuscript.
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• Data accessibility It is a condition of publication that all supporting data are made available either as supplementary information or preferably in a suitable permanent repository. The data accessibility section should state where the article's supporting data can be accessed. This section should also include details, where possible of where to access other relevant research materials such as statistical tools, protocols, software etc can be accessed. If the data has been deposited in an external repository this section should list the database, accession number and link to the DOI for all data from the article that has been made publicly available. Data sets that have been deposited in an external repository and have a DOI should also be appropriately cited in the manuscript and included in the reference list.
If you wish to submit your supporting data or code to Dryad (http://datadryad.org/), or modify your current submission to dryad, please use the following link: http://datadryad.org/submit?journalID=RSOS&manu=RSOS-181247.R1 • Competing interests Please declare any financial or non-financial competing interests, or state that you have no competing interests.
• Authors' contributions All submissions, other than those with a single author, must include an Authors' Contributions section which individually lists the specific contribution of each author. The list of Authors should meet all of the following criteria; 1) substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; 2) drafting the article or revising it critically for important intellectual content; and 3) final approval of the version to be published.
All contributors who do not meet all of these criteria should be included in the acknowledgements.
We suggest the following format: AB carried out the molecular lab work, participated in data analysis, carried out sequence alignments, participated in the design of the study and drafted the manuscript; CD carried out the statistical analyses; EF collected field data; GH conceived of the study, designed the study, coordinated the study and helped draft the manuscript. All authors gave final approval for publication.
• Acknowledgements Please acknowledge anyone who contributed to the study but did not meet the authorship criteria.
• Funding statement Please list the source of funding for each author.
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. Decision letter (RSOS-181247.R2)

20-Nov-2018
Dear Dr Liu, I am pleased to inform you that your manuscript entitled "Identification of Cordycepin Biosynthesis-related Genes Through de novo Transcriptome Assembly and Analysis of Cordyceps cicadae" is now accepted for publication in Royal Society Open Science.
You can expect to receive a proof of your article in the near future. Please contact the editorial office (openscience_proofs@royalsociety.org and openscience@royalsociety.org) to let us know if you are likely to be away from e-mail contact. Due to rapid publication and an extremely tight schedule, if comments are not received, your paper may experience a delay in publication.
Royal Society Open Science operates under a continuous publication model (http://bit.ly/cpFAQ). Your article will be published straight into the next open issue and this will be the final version of the paper. As such, it can be cited immediately by other researchers. As the issue version of your paper will be the only version to be published I would advise you to check your proofs thoroughly as changes cannot be made once the paper is published. Response to L311, L314, and other parts.
All supplementary material section have been replaced by "electronic supplementary material,name". We are sorry for our negligence of this. We deleted the section 3.5 and insert into section 3.2.

Response to reviewer 1 minor comments:
Line 27 has been revised to " are responsible".
Line 34 has added genus before Cordyceps.
Line 47 was also revised to O. sinensis.
Line 105-106 Stromata was the mat-like structure which formed by mycelium combines with host substrate. And fruiting body was a multicellular structure on which spore-producing structures.
L126 "strain buc" has been changed normal. Figure 5B has corrected as MF, FS and MS.

Response to reviewer 2
Thank you very much for your kindly comments on our manuscript . Based on your suggestions, we carefully revised the manuscript.

1) Cordyceps in the text has been abbreviated;
2) Ophiocordyceps sinensis were more used than Cordyceps sinensis and we unifed in O. sinensis in the whole manscuript.
3) I think the reviewer means Line131 was line 141,and

Response to reviewer 3
We are truly grateful to yours critical comments and thoughtful suggestions. 1ine 125 ITS region was used for amplified and conduct phylogenetic analysis.
Line 190, The reference of BLAST2GO was added.

Lines 199 to 200
The abbreviation of FPKM( fragments per kilobase of exon per million mapped reads) was added.

Line 201
The reference has been checked.

Line 213-215
The references of the bioinformatics tools used for bioinformatics analysis were cited.
Line 225 References 21-24 was cited in the previous research.

Line 388-389
The top 20 metabolic pathways was replaced. And Figure S9 ABC was cited in the text and a red box was marked in the figure.
Lines 419 to 425 Validation was replaced. The validation of DEGs by qRT-PCR and cited the Figure 6 (lines 424 to 425). Figure 6 was revised as your suggesstions.The name of c35629 in Figure 6D was ADA2 and the gene names in the Figure 6 below the graphic were added.

Legend of
Bsed on the comments we received, careful modifications have been made to the R1 manuscript. All changes were marked in red text. We hope that these revisions are satisfactory and that the revised version will be acceptable for publication in Royal Society Open Science.
Thank you very much for your work concerning my paper.
Wish you all the best! Sincerely yours