Chitin Synthases in Cordyceps militaris: Genome-Wide Gene Identification, Evolutionary Insights, and Life Cycle Transcript Profiling

Round 1

Reviewer 1 Report

In the manuscript ‘Chitin Synthases in Cordyceps militaris: Genome-wide Gene Identification, Evolutionary Insights, and Life Cycle Transcript Profiling’, the authors performed thorough genome mining of known Cordyceps militaris genomes, establishing in silico chitin synthases analysis and their distribution. Moreover, these results have been  supplemented with the set of the transcriptional experiments, proving that in C. militaris various chitin synthases are crucial for different steps of mycelium development, including the fruiting body.

In my opinion, presented work is interesting and it is important to the other fungal researchers, working on Cordyceps sp. as well as on other fruiting body-formed fungi. The carefully thought-out experiments gave the interesting results, and it was pleasure to read. I have few small points to the presented data:

Row 19: I would not exaggerate with the commercial cultivation of C. militaris. C. sinensis, of course, has a long tradition in China, albeit C. miliaris is a rarely found in forest, wild fungus without any significant value for a broad medical uses.

Row 28-9: please, rewrite this sentence. May be general number of families among Dikarya has no significant differences. The sentence in the present form suggests that each fungus has the same number, which is not true, as the authors indicate in rows 57-60.

Row 44: nutrition tonics wordwide? If it is true, pleae, provide the wordwide statistics and citation; the same rows 45-6.

Row 49: Chitin is a minor constituent of the fungal cell wall. The main component in Dikarya is the 1-6 beta glucan.

Row 60: Ustilago is a type of smut fungi, far away from filamentous fungi, such as Metarhizium

Row 87: Phytophthora is not a fungus. It has its own type of chitin synthase, albeit this organism is not comparable with fungi, and cannot be presented in this context

Materials and methods – lack of information what was the source of silkworm pupae

Row 401: the duplication event should have result from the phylogenetic analysis. If so, then You may show that CmChs3, 4, 5 form one family of duplicated chitin synthases. If not, better omit this speculation

The language is fine, I have no remarks

Author Response

In the manuscript ‘Chitin Synthases in Cordyceps militaris: Genome-wide Gene Identification, Evolutionary Insights, and Life Cycle Transcript Profiling’, the authors performed thorough genome mining of known Cordyceps militaris genomes, establishing in silico chitin synthases analysis and their distribution. Moreover, these results have been supplemented with the set of the transcriptional experiments, proving that in C. militaris various chitin synthases are crucial for different steps of mycelium development, including the fruiting body.

In my opinion, presented work is interesting and it is important to the other fungal researchers, working on Cordyceps sp. as well as on other fruiting body-formed fungi. The carefully thought-out experiments gave the interesting results, and it was pleasure to read. I have few small points to the presented data:

Response: Thanks for the positive comments. We have revised the Ms. carefully according to the suggestions one by one.

Row 19: I would not exaggerate with the commercial cultivation of C. militaris. C. sinensis, of course, has a long tradition in China, albeit C. miliaris is a rarely found in forest, wild fungus without any significant value for abroad medical uses.

Response: We think C. miliaris is valued for its medicinal and nutritional benefits. However, the sentence of “These properties have led to their widespread use as food and nutrition tonics world-wide” has been deleted.

Row 28-9: please, rewrite this sentence. May be general number of families among Dikarya has no significant differences. The sentence in the present form suggests that each fungus has the same number, which is not true, as the authors indicate in rows 57-60.

Response: Thanks for your suggestion. “In general” and P values have been added (Rows 28-29).

Row 44: nutrition tonics word wide? If it is true, please, provide the word wide statistics and citation; the same rows 45-6.

Response: Agreed. We have deleted that sentence and added citation at Rows 43-44.

Row 49: Chitin is a minor constituent of the fungal cell wall. The main component in Dikarya is the 1-6 beta glucan.

Response: Agreed. We have changed the key component to important component (Row 49).

Row 60: Ustilago is a type of smut fungi, far away from filamentous fungi, such as Metarhizium

Response: Thanks for the suggestion. Ustilago maydis has been changed to Trichoderma reesei (Row 60).

Row 87: Phytophthora is not a fungus. It has its own type of chitin synthase, albeit this organism is not comparable with fungi, and cannot be presented in this context. Materials and methods – lack of information what was the source of silkworm pupae

Response: Agree and have deleted. The source of silkworm pupae has been added at Rows 170-171.

Row 401: the duplication event should have result from the phylogenetic analysis. If so, then You may show that CmChs3, 4, 5 form one family of duplicated chitin synthases. If not, better omit this speculation

Response: Agreed. We have omitted that speculation (Rows 398-401).

Reviewer 2 Report

In this research, eight chitin synthase genes from in Cordyceps militaris were identified and analyzed. Moreover, transcript patterns of the chitin synthase genes during key developmental stages such as conidia germination, infection of silkworm, and fruiting body development were characterized to infer their potential roles. This research fits into the latest research on the use of chitin and chitosan, a natural cationic copolymer of chitin, in the pharmaceutical and biomedical fields as well as in animal nutrition and wastewater treatment. The results are described in a concise manner. The discussion section clearly interprets the significance of the findings, and the conclusion summarizes the main findings of the study.

I have only one suggestion for the manuscript:

1. Line 153: The full name of the abbreviation should be given the first time it is mentioned, NCBI- The National Center for Biotechnology Information.

Also, the reference of the open genome from the NCBI database should be written in the text.

Author Response

In this research, eight chitin synthase genes from in Cordyceps militaris were identified and analyzed. Moreover, transcript patterns of the chitin synthase genes during key developmental stages such as conidia germination, infection of silkworm, and fruiting body development were characterized to infer their potential roles. This research fits into the latest research on the use of chitin and chitosan, a natural cationic copolymer of chitin, in the pharmaceutical and biomedical fields as well as in animal nutrition and wastewater treatment. The results are described in a concise manner. The discussion section clearly interprets the significance of the findings, and the conclusion summarizes the main findings of the study.

Response: Thanks for the positive comments. We have revised the Ms. carefully according to the suggestions one by one.

I have only one suggestion for the manuscript:

1. Line 153: The full name of the abbreviation should be given the first time it is mentioned, NCBI-The National Center for Biotechnology Information.

Also, the reference of the open genome from the NCBI database should be written in the text.

Reponse: Agreed. We have added the full name of NCBI at Lines 147-148. The reference of the open genome from the NCBI database has been listed at Table S4 and S5.

Reviewer 3 Report

In this paper, the authors identified chitin synthases (CHSs) encoding genes from the Cordyceps militaris genome. They analysed the chromosomal localization of this gene and the relationship of the number of CHSs in the genomes of Ascomycota and Basidiomycota fungi, in relation to their ecological strategies, was revealed. Transcription patterns of CHS protein genes during key developmental stages such as conidia germination, silkworm infection and fruiting body development were characterized to infer their potential roles. These results may provide useful information for further functional studies of the CHS protein gene. The subject was presented in a rather interesting and innovative way. I believe that some materials included in the supplement (S1, S2, S3) would enrich the work (manuscript) from a visual and aesthetic perspective. This manuscript requires minor revisions before it is ready for publication. Further detailed comments for consideration are provided below.

Comment 1#

Please provide additional information about the strain (for example, natural isolate or comes from a collection?)

Comment 2#

Line 50: Add EC number

Comment 3#

Line 507: correct to the italic

Author Response

In this paper, the authors identified chitin synthases (CHSs) encoding genes from the Cordyceps militaris genome. They analysed the chromosomal localization of this gene and the relationship of the number of CHSs in the genomes of Ascomycota and Basidiomycota fungi, in relation to their ecological strategies, was revealed. Transcription patterns of CHS protein genes during key developmental stages such as conidia germination, silkworm infection and fruiting body development were characterized to infer their potential roles. These results may provide useful information for further functional studies of the CHS protein gene. The subject was presented in a rather interesting and innovative way. I believe that some materials included in the supplement (S1, S2, S3) would enrich the work (manuscript) from a visual and aesthetic perspective. This manuscript requires minor revisions before it is ready for publication.

Response: Thanks for the positive comments. We have revised the Ms. carefully according to the suggestions one by one.

Further detailed comments for consideration are provided below.

Comment 1#

Please provide additional information about the strain (for example, natural isolate or comes from a collection?)

Response: Thanks. We have added the additional information about the strain at Lines 112-113.

Comment 2#

Line 50: Add EC number

Response: Agreed. We have added at Line 51.

Comment 3#

Line 507: correct to the italic

Response: The CmCHS at this sentence represented protein not gene, so we used bold.