Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript title "Altered stereostructures of the DNA-binding domains in the mutant mating proteins of Ophiocordyceps sinensis and the wild insect–fungal complex" is very well drafted and scientifically sound. The manuscript is truly constructed based on various bioinformatics tools. All comments, suggestions, and minor corrections are flagged in the manuscript and incorporated into the final draft. 

General Comments

1 All figure descriptions are very lengthy and may be condensed for clarity.

2 In the title, mention "the wild insect–fungal complex", what is the wild in this context, is it different from "the natural insect–fungal complex"

 

Comments for author File: Comments.pdf

Author Response

Responses1 to the comments from Reviewer #1:

Thank you very much for your positive comments.

To respond to the general comment #1 from Reviewer #1: “All figure descriptions are very lengthy and may be condensed for clarity”, we have shortened the figure descriptions as much as possible.

 

To respond to the general comment #2 from Reviewer #1: “In the title, mention "the wild insect–fungal complex", what is the wild in this context, is it different from "the natural insect–fungal complex.”

“The wild insect–fungal complex” is indeed a natural insect–fungal complex. The word “wild” is used to distinguish between wild insect–fungal complexes and artificially cultivated insect–fungal complexes. To date, no mating protein sequence information is available from cultivated insect–fungal complexes in public depository databases.

 

We are greatly grateful for all the comments, suggestions, and minor corrections flagged in the revised manuscript attached to your review comments. We have made changes accordingly and forwarded it to a professional English editing team at American Journal Experts. We have worked closely with the team to finalize the manuscript.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The aim of this study was to characterize the primary structures of MAT1-1-1 and MAT1-2-1 protein variants obtained from diverse wild-type Cordyceps sinensis insect–fungal complex isolates, comprising the Ophiocordyceps sinensis fruiting body and the remains of a Hepialidae moth larva. The protein sequences revealed various amino acid substitutions in the functional domains and clustered into several Bayesian clades correlated with the secondary and tertiary structures of the high significance function domains. Intriguingly, the findings of this research in combination with other data refute the hypothesis that O. sinensis can self-fertilize via homothallic mating and instead suggest that O. sinensis is self-sterile and requires a compatible partner for sexual reproduction under heterothallism or hybridization during the sexual phase of natural C. sinensis insect-fungal complex.

In general, the manuscript is well-written, the presented data are many and the findings expand the current knowledge on this topic.

However, in my view, some modifications should be made prior to the consideration of this manuscript for publication by the Journal.

To begin with, the goal and the originality of this study must be presented in a clearer way in the Introduction of this manuscript.

Furthermore, the authors should consider divide the first paragraph of the Introduction section into smaller ones so as to be easier for the reader to comprehend its content.

Another problem is that in many parts of the Discussion section the text feels more like representation of results instead of commentary on them and comparison with the existing relative literature. Therefore, proper rephrasing should be done where needed throughout this section of the manuscript.

In addition, further analysis is needed in other aspects of Discussion, e.g. lines 1054-1056, 1142-1146.

Lines 1146-1149: according to the literature, if any relative information is available, what would be the most suitable culture conditions for the C. sinensis. Comment on this is also needed.

Lastly, the authors should pay attention to misstypes, e.g. animo acids instead of amino acids.

Author Response

Responses2 to the comments from Reviewer #2:

Thank you very much for your positive comments: “In general, the manuscript is well-written, the presented data are many and the findings expand the current knowledge on this topic.”

 

Reviewer #2 commended: “the goal and the originality of this study must be presented in a clearer way in the Introduction of this manuscript …… the authors should consider divide the first paragraph of the Introduction section into smaller ones so as to be easier for the reader to comprehend its content”

The last paragraph of the Introduction describes the goal of this study. Following the reviewer’s comment, we have revised this portion in the Introduction section:

“Our previous study [50] showed variations in the 3D structures of complete mating proteins. This study continues our previous study, focusing on amino acid substitutions within the MATα_HMGbox and HMG-box_ROX1-like domains of the full-length MAT1-1-1 and MAT1-2-1 proteins, respectively, and the impact of the variable primary structures of the DNA-binding domains on the changes in the hydrophobic properties and the secondary and tertiary structures of the functional domains in wild-type C. sinensis isolates. Correlations between changes in hydrophobicity and the primary and secondary structures of the DNA-binding domains of mating proteins encoded by the genome, transcriptome and metatranscriptome assemblies of H. sinensis and C. sinensis insect–fungal complexes were also analyzed.” (Lines 120−129)

 

Reviewer #2 commended: “Another problem is that in many parts of the Discussion section the text feels more like representation of results instead of commentary on them and comparison with the existing relative literature. Therefore, proper rephrasing should be done where needed throughout this section of the manuscript.”

Following this comment, we have rephrased many sentences in the discussion section. Our manuscript has been edited by an editing team of the professional language editing company, American Journal Experts, for re-editing.

 

Reviewer #2 commended: “further analysis is needed in other aspects of Discussion, e.g. lines 1054−1056, 1142−1146: according to the literature, if any relative information is available, what would be the most suitable culture conditions for the C. sinensis. Comment on this is also needed ……”

Lines 1054−1056 of the original submission, we wrote “The analysis of mutant full-length mating proteins derived from wild-type C. sinensis isolates (Figures 3−8 and 10−13) revealed their heterogeneous fungal sources (Table S3) [5,34,36,52,56−57].”

Following the comment from Reviewer #2, we revised this sentence as follows:

“The analysis of wild-type C. sinensis isolates revealed their heterogeneous fungal components, which belong to genomically independent GC-biased Genotypes #1 and #3 of O. sinensis fungi (Table S4), suggesting that the mutant mating proteins derived from the wild-type C. sinensis isolates (Figures 3-8 and 10-13) may have different fungal sources [5,7,10,34, 36,52,56−57]. In addition to the impure wild-type C. sinensis isolates, Li et al. [23] reported 8 heterogeneous O. sinensis strains, which are cultures of C. sinensis monoascospores that contain both genome-independent GC-biased Genotype #1 and AT-biased Genotype #5 of O. sinensis fungi. These O. sinensis strains produced MAT1-1-1 and MAT1-2-1 proteins with different truncation mutations and various amino acid substitutions at various mutation sites (the detailed analytical data will be published elsewhere).” (Lines 1104−1113)

 

Lines 1142−1146 of the original submission, we wrote “In addition to the formal reports of unsuccessful attempts at artificial cultivation of the fruiting bodies of C. sinensis [49,120-121], Zhang et al. [36] summarized the 40-year history of cultivation failures of insect–fungal complexes via a “pure” mycological strategy in academic research-oriented settings, and Qin et al. [122] summarized the obstacles to the cultivation of O. sinensis fruiting bodies and ascospores.”

Following the comment from Reviewer #2, we revised the sentences in the revised manuscript as follows: “Hu et al. [49], Holliday & Cleaver [120], and Stone [121] reported unsuccessful attempts at artificial cultivation of the fruiting bodies and ascospores of C. sinensis in academic settings. Faced with the unsuccessful experiment reported by Hu et al. [49], their coauthors Zhang et al. [36] summarized the 40-year history of cultivation failures of insect–fungal complexes using a “pure” mycology strategy in academic research-oriented settings. Qin et al. [122] further analyzed the unsuccessful situation and summarized the obstacles to the cultivation of O. sinensis fruiting bodies and ascospores.” (Lines 1196−1202)

 

Reviewer #2 commended: “Lines 1146-1149: according to the literature, if any relative information is available, what would be the most suitable culture conditions for the C. sinensis. Comment on this is also needed.”

Lines 1146−1149 of the original submission, we wrote “In contrast, Wei et al. [32] reported success in such a cultivation effort in industrial product-oriented settings. This industrial success might be attributed to the application of a “mycologically impure” cultivation strategy on the basis of at least two facts:”

Previous studies (Wei et al. 2016) [32] reported a successful cultivation project in industrial product-oriented settings, which was designed and conducted by a commercial company. However, Wei et al. [32] reported contradictory information regarding the anamorphic fungal inoculant (GC-biased Genotype #1) and the sole teleomorph (AT-biased Genotype #4) in the fruiting body of the cultivated C. sinensis insect−fungal complex. We have briefly described and commented on the species contradiction presented by Wei et al. [32] and the modified information published by Li et al. (2020, 2023) [118−119]. (Lines 70−74, 1158−1162, 1174−1195, and 1202−1212). Apparently, these authors have hidden secrets, probably because of some commercial interests, which is understandable. Because of the contradiction surrounding artificial cultivation success, we provided our analysis and explanation of the species contradiction in Lines 1146−1166 of the original submission.

To respond to the reviewer’s comment “the most suitable culture condition”, we can only assume that the key cultivation condition is “mycological impurity”, leading to synergistic symbiosis among O. sinensis fungi, based on the limited publicly available information that Wei et al. [32] and other scientists provided. (Lines 1202−1212)

 

Another comment from Reviewer #2: “the authors should pay attention to misstypes, e.g. animo acids instead of amino acids. (Line 19)” Thank you for identifying the typo, which has been corrected.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This article systematically studied the sequence variation and structural changes of the DNA binding domains (MAT α - HMGBox and HMG-box-ROX1 like) of mating proteins MAT1-1-1 and MAT1-2-1 in Ophiocordyceps sinensis in wild-type isolates, and their effects on mating patterns. The study used AlphaFold to predict three-dimensional structures, Bayesian clustering analysis, and other methods to reveal structural heterogeneity caused by multiple amino acid substitutions, further supporting the view that Cordyceps sinensis is propagated through allopatric coordination or hybridization, and denying the possibility of self pollination. The research design is relatively systematic and the data analysis is detailed, which has important scientific value for understanding the sexual reproduction mechanism of Cordyceps sinensis.

1. The author should summarize more clearly in the abstract how the structural changes identified directly affect DNA binding ability, in order to highlight the mechanistic contribution of the research.
2. The biological significance of the low confidence regions predicted by AlphaFold in the structure was not fully discussed in the article. It is suggested to explain their potential impact in the discussion.
3.Although the sample size is large, does it cover all known genotypes?
4. The article mentions the possibility of hybridization or allogenetic cooperation, but does not delve into how to identify and contact mating partners in natural environments.
5.The use of H. sinensis, O. sinensis, and C. sinensis in the text is relatively complex and can easily cause confusion. Suggest briefly summarizing the naming history in the introduction or discussion, and maintaining consistency throughout the entire text.
6. Some sentences are long and structurally complex, which affects readability. For example: Section 3.5.
7.The sequence alignment in Panel A is incomplete in the paper. It is recommended to ensure clear charts and complete labeling.

Comments on the Quality of English Language

Some sentences are long and structurally complex, which affects readability. For example: Section 3.5.

Author Response

Responses3 to the comments from Reviewer #3:

Thank you very much for your positive comment: “The research design is relatively systematic and the data analysis is detailed, which has important scientific value for understanding the sexual reproduction mechanism of Cordyceps sinensis.”

 

Reviewer #3 commended: “1. The author should summarize more clearly in the abstract how the structural changes identified directly affect DNA binding ability, in order to highlight the mechanistic contribution of the research.”

Following the reviewer’s comment, we revised the abstract as follows:

“Correlation analysis revealed that 1-3 amino acid substitutions in the DNA-binding domains of the mating proteins resulted in altered hydrophobicity and secondary and tertiary structures of the DNA-binding domains of the proteins, particularly the stereostructures of the hydrophobic cores of the 3 critical α-helices within the functional domains of the proteins. Fungal origin analysis revealed possible heterospecific fungal sources of mating proteins with stereostructure variations in wild-type C. sinensis isolates, suggesting that alterations in DNA-binding function and the subsequent regulation of mating-related gene transcription are involved in ensuring the accuracy and genetic diversity of heterothallic and hybrid reproduction of O. sinensis during the lifecycle of the C. sinensis insect–fungal complex.” (Lines 40−50)

 

Reviewer #3 commended: “2. The biological significance of the low confidence regions predicted by AlphaFold in the structure was not fully discussed in the article. It is suggested to explain their potential impact in the discussion.”

We provide information in the Materials and Methods section of the original submission: “The AI-based AlphaFold database provides per-residue model confidence, predicts scores between 0 and 100 on the local distance difference test (pLDDT) and provides a per-residue score that is assigned to each individual residue [73−76,78−79]. The model confidence bands are used to color-code the residues in the 3D structures: residues with very high confidence (pLDDT>90) are shown in dark blue, those with high confidence (90>pLDDT>70) appear in light blue, residues with low confidence (70>pLDDT>50) are shown in yellow, and residues with very low confidence (pLDDT<50) are shown in orange [50,80,83]. The AlphaFold database provides an average pLDDT score for each of the AI-predicted 3D structural models.” (Lines 202−208 in the original submission)

Following the reviewer’s comment, we have added a paragraph to explain the low-confidence predicted regions:

The main reasons for low-confidence region AlphaFold predictions include the following: (1) There are insufficient supporting data because the AlphaFold-based prediction relies on the quality of multiple sequence alignment (MSA). If few homologous sequences are available in a certain region of the MSA, the model will lack sufficient evolutionary information to infer the structure, resulting in low confidence. (2) Some protein regions are naturally flexible under physiological conditions, such as the activation domains of transcription factors, which do not have a fixed 3D structure, leading to low prediction confidence assigned by AlphaFold. (3) In terms of special structural regions, such as small-molecule binding sites and artificial linkers in fused proteins, the AlphaFold system may have limited prediction power, leading to low confidence in the prediction of complex structures. Thus, the low-confidence regions in AlphaFold predictions are informative flags for highlighting prediction challenges and should not be interpreted too confidently.

(Lines 219−230)

 

Reviewer #3 commended: “3. Although the sample size is large, does it cover all known genotypes?”

Unfortunately, the answer is “No”.

On the basis of the study findings for C. sinensis isolates CH1 and CH2 (Lines 1118-1134) [86]; in general, wild-type C. sinensis isolates likely contain multiple fungal components, which were detected with use of a strict and rigorous PCR strategy, involving the use of multiple primer pairs, a Touch-Down PCR protocol, and a PCR amplicon cloning technique with selecting and sequencing >30 white colonies, as well as the use of SNP MALDI−TOF mass spectrometry with the use of multiple extension primers. Thus, the original studies [5,34,36,52,56−57] on wild-type C. sinensis isolates published by Prof. Zhang Y-J’s group did not thoroughly profile the entire fungal community in the study samples, leading to the limited number of ITS sequences uploaded to the GenBank database for phylogenetic and genotypic analysis of these wild-type isolates. On the basis of the limited number of ITS sequence information, our analysis revealed only 2 GC-biased genotypes, unfortunately, not able to cover all naturally cooccurring genotypes (cf. Sections 3.9 and 4.3).

 

Reviewer #3 commended: “4. The article mentions the possibility of hybridization or allogenetic cooperation, but does not delve into how to identify and contact mating partners in natural environments.”

This is a great question, which may inspire scientists to design and conduct follow-up reproductive physiology studies.

The scientific studies to date revealed the differential occurrence, differential transcription, and alternative splicing of the MAT1-1-1 and MAT1-2-1 genes, as well as pheromone receptor genes. Tertiary protein structure studies revealed the heteromorphic stereostructures of the MAT1-1-1 and MAT1-2-1 proteins, especially and much critically the altered stereostructures of the DNA-binding domains of the proteins. These findings at the genetic, transcriptional, and protein structural levels refute the possibility of self-fertilization for O. sinensis under homothallism. Thus, O. sinensis must be self-sterile, using heterothallism or hybridization, even parasexuality, to accomplish sexual reproduction during the lifecycle of natural C. sinensis insect−fungal complexes.

To date, there has not been direct evidence of reproductive physiology in terms of identifying natural mating partners for sexual reproduction. However, some indirect evidence has been reported.

(1) Wei et al. (2016) [32] reported a successful artificial cultivation study conducted in an industrial product-oriented setting with the use of a mycologically impure cultivation strategy (Lines 1202−1212) and reported that the genomically independent AT-biased Genotype #4 of O. sinensis was the sole teleomorph in the fruiting body of cultivated C. sinensis. (Lines 70−74, 1158−1162, 1174−1195, and 1202−1212) Thus, the genomically independent AT-biased Genotype #4 of O. sinensis fungus may be considered a candidate of the mating partners, in addition to GC-biased Genotype #1 H. sinensis.

(2) Zhu et al. (2010) [111] reported the co-occurrence of GC-biased Genotypes #1 and #2 in the immature, maturing, and mature stromata of natural C. sinensis insect−fungal complexes.

(3) Chen et al. (2011) [109] reported the detection of a GC-biased Genotype #7 in the stroma of a single sample of a natural C. sinensis insect-fungal complex. Simultaneously, the authors reported the detection of GC-biased Genotype #1 in the caterpillar body of the same sample.

(4) Li et al. (2023; DOI: 10.1371/journal.pone.0270776) [10] reported the detection of GC-biased Genotypes #13 and #14 in the semi-ejected and fully ejected ascospores, respectively, of natural C. sinensis insect−fungal complexes, respectively. Genetically, the ITS sequences of Genotypes #13 and #14 of O. sinensis featured large DNA segment reciprocal substitutions and genetic material recombination between the genomes of the 2 parental fungi, i.e., Group-A Genotype #1 H. sinensis (GenBank accession # AB067721) and the AB067719-type Group-E fungus, as illustrated in the figure below. The taxonomic position of the AB067719-type Group-E fungus has not been determined, but it is a heterospecific fungus for sure. This phenomenon of genetic material recombination indicates the possibility of fungal hybridization or parasexuality between the 2 parental fungi.

 

Reviewer #3 commended: “5. The use of H. sinensis, O. sinensis, and C. sinensis in the text is relatively complex and can easily cause confusion. Suggest briefly summarizing the naming history in the introduction or discussion and maintaining consistency throughout the entire text.”

The Latin name issue is indeed complex. We have elucidated this in detail in the literature [7,9−10]. To avoid confusion while reading the current paper, we briefly summarized the taxonomic/nomenclatural issues in the first paragraph of the Introduction of the original submission (Lines 72−93) and now in the third paragraph of the revised Introduction (Lines 76−97).

Both Reviewers #3 and #4 have commented on Latin name usage and suggested including a summary section in the Discussion section. We realize that the confusion of Latin name usage is a very serious historical problem in the O. sinensis research field, and the brief summary in the Introduction section may not be sufficiently knowledgeable for our readers to understand and differentiate between different study materials. We then followed the Reviewers’ suggestion and added a subsection to the Discussion.

4.1. Historical and current usage of Latin names regarding Cordyceps sinensis, Hirsutella sinensis, and Ophiocordyceps sinensis to refer to fungus/fungi and the insect−fungal complex

Natural C. sinensis was introduced to Western countries by the French missionary Dominicus Parennin in 1723. Its intrinsic fungus was identified by Jonathan Pereira in 1843 as belonging to the Sphaeria genus, and the insect portion was identified by Edward Doubleday as belonging to Agrotis in 1842 [87−89]. Historical taxonomic examinations of natural C. sinensis clearly revealed that it is an insect−fungal complex. Miles Joseph Berkeley described the fungus as Sphaeria Sinensis Berkeley in 1843 and renamed it Cordyceps Sinensis in 1857 [90−91]. Pier Andrea Saccardo renamed it Cordyceps sinensis (Berkeley) Saccardo in 1883 [92−93]. Unfortunately, since then, the Latin name C. sinensis, which was originally given to the intrinsic fungus, has been indiscriminately used in academia and the mass market to refer to both the fungus C. sinensis and the wild insect–fungal complex.

The fungus was renamed O. sinensis in 2007 by Sung et al. [33] with the use of the H. sinensis strain EFCC 7287 as the nomenclatural reference [4,7,33−35]. Since 2001, >600 ITS1-5.8S-ITS2 sequences of O. sinensis have been uploaded to the GenBank database under the GenBank Taxid 72228, representing 17 genome-independent genotypes of O. sinensis with numerous, scattered transition, transversion, or insertion/deletion mutant alleles or hereditary variations with reciprocal substitutions of large DNA segments and genetic material recombination [7,9−10,86]. Li et al. [7,10,86] have shown that 17 genotypes are genomically independent and belong to different O. sinensis fungi, which have been postulated to share a common hereditary ancestor [17].

To date, only Genotype #1 of O. sinensis (i.e., H. sinensis) has been purified and taxonomically characterized [7,94]. The genome-independent Genotypes #2-17 have not been purified, and their taxonomic positions have not been determined [7], although these genotypes are assigned the same Latin name, O. sinensis, under the same GenBank taxonomic ID 72228. Controversies, hypotheses, and scientific arguments surrounding genotypic mutations of O. sinensis pervade the entire field of O. sinensis research.

This historical situation prompted us to embrace the currently available information and use different names, i.e., H. sinensis, O. sinensis, C. sinensis insect−fungal complex, and wild-type C. sinensis isolates, to refer to different study materials. This practice might not be perfect because of historical uncertainties in academia. However, we hope that the taxonomic/nomenclatural uncertainties will encourage taxonomists in mycology, botany, and traditional Chinese medicine to design and conduct future studies to solve these historical academic problems.

(Lines 1015−1048)

 

Reviewer #3 commended: “6. Some sentences are long and structurally complex, which affects readability. For example: Section 3.5 …… Comments on the Quality of English Language: Some sentences are long and structurally complex, which affects readability. For example: Section 3.5.”

Following these the comments, we have sent our manuscript to a professional English editing team at American Journal Experts for language re-editing and have worked closely with the team to finalize our manuscript revision.

 

Reviewer #3 commended: “7. The sequence alignment in Panel A is incomplete in the paper. It is recommended to ensure clear charts and complete labeling.”

The first panels (Panel A) of Figures 3–16 and S3 in the manuscript and supplementary files show alignments of the sequences of DNA-binding domains, plus 9 amino acid residues both upstream and downstream of the domains because of ExPASy ProtScale plotting for Panels B-F. We have described the alignment method in Section 2.5. It is true that “The sequence alignment in Panel A is incomplete” because this study focuses on the DNA-binding domain sequences. However, we have included Figures S1−S2 in the Supplementary files, showing alignments of full-length sequences of the MAT1-1-1 and MAT1-2-1 proteins that represent the proteins under all AlphaFold 3D structure models. Owing to the size (52 pages in total) of our paper, which contains 16 figures, we then reluctantly ground the full-length sequence alignments in the Supplementary files.

 

 

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

General comments to the Authors

Summary

The manuscript extends your previous structural work on mating-type proteins in O. sinensis by concentrating on the DNA-binding domains (MATα_HMGbox and HMG-box_ROX1-like) of MAT1-1-1 and MAT1-2-1. You compile AlphaFold-predicted structures for 118 full-length MAT1-1-1 and 69 full-length MAT1-2-1 proteins, identify variants in wild C. sinensis insect–fungal complexes and H. sinensis strains, and analyze how 1–4 amino-acid substitutions correlate with changes in hydrophobicity, secondary/tertiary structure, and Bayesian clustering patterns.

Strengths

• Large and carefully curated set of mating-type protein sequences from genomes, transcriptomes, and metatranscriptomes.
• Systematic use of AlphaFold, ProtScale, and Bayesian phylogenetic tools to connect sequence variants with predicted domain structures.
• Clear biological motivation: understanding sexual reproduction of O. sinensis and the implications for artificial cultivation of Chinese cordyceps.

Major points

1. Clarify novelty versus the J. Fungi 2025 article.

The introduction and discussion repeatedly revisit material already presented in your prior paper on 3D heteromorphs of full-length mating proteins (https://doi.org/10.3390/jof11040244). It would help the reader if you explicitly and succinctly state, early in the introduction and again in the discussion, what is new here (e.g., exclusive focus on DNA-binding domains, integration with hydrophobicity/secondary structure plots, specific new inferences) and what is confirmatory or elaborative.

1. Moderate the strength of the main conclusions.

Statements that the findings “demonstrated that O. sinensis experiences self-sterility and uses a heterothallic or hybrid strategy, even parasexuality” and that this constitutes a “fundamental paradigm shift” are very strong given that the study is wholly based on sequence and structure predictions without direct mating or functional assays. I suggest rephrasing throughout to “our data are consistent with…” or “support the hypothesis that…,” and adding a short “Limitations” paragraph explicitly acknowledging the inferential nature of the conclusions and the lack of experimental validation.

1. Clarify datasets and definitions.
• Please make more explicit how many sequences come from each source (H. sinensis genomes, C. sinensis metatranscriptomes, other genotypes), perhaps in a concise summary table.
• Define clearly what you call “mutant” proteins (any deviation from AGW27560/AEH27625?) and what is meant by “heterospecific fungal sources of mating proteins” in the context of the insect–fungal complex.
2. Improve structure and concision.

The introduction includes a lengthy taxonomic/nomenclatural digression (e.g., on “One Fungus=One Name”) that could be shortened or moved in part to the discussion or supplement.

Similarly, the discussion contains an extended narrative on 40 years of cultivation attempts and species contradictions, which, while interesting, somewhat distracts from the core structural findings. Consider focusing the discussion more tightly on how your structural data refines hypotheses about mating behavior and cultivation.

 

Line-by-line comments – selected examples

1. Line 11: “Correspondence: zhujoah@163.com)” – remove the final parenthesis after the email address.
2. Lines 18–19 (Simple Summary): “The protein sequences exhibited various animo acid substitutions…” – correct “animo” to “amino”; avoid line-break artefacts (“se-quences”).
3. Line 21 (Simple Summary): “critical function domains” – consider “critical functional domains” for more natural phrasing.
4. Line 26 (Simple Summary): “ sinensis insect-fungal complex” – use the same form as elsewhere (“insect–fungal complex”) and standardize the hyphen/en dash.
5. Line 55 (Introduction): “Natural Cordyceps sinensis is one of most expensive therapeutic agents…” – add “the”: “one of the most expensive therapeutic agents…”
6. Lines 76–86 (Introduction): Very long sentence beginning “Although Zhang et al. [36] proposed improper implementation of the ‘One Fungus=One Name’…”. Consider splitting into two or three sentences to improve clarity and reduce repetition of “genomically independent genotypes…”.
7. Lines 116–122 (Introduction): Sentence starting “Continuing our previous study [50]…” is long and dense; consider breaking it and standardizing “DNA binding domains” vs “DNA-binding domains.”
8. Lines 128–141 (Materials and Methods 2.1): The list of genome and protein accessions is very detailed; these could be summarized in a table or reserved for Supplementary material to streamline the main text.
9. Lines 180–192 (Section 2.5): “The MATα_HMGbox domain of MAT1-1 proteins…” – elsewhere you refer to “MAT1-1-1 proteins”; please check whether “MAT1-1 proteins” is a typo and ensure consistent nomenclature.
10. Line 193 (heading): “II-6 AlphaFold-based prediction of the 3D structures…” – likely meant to be “6.” Correct the section numbering format.
11. Lines 232–233 (Results 3.1): “The MATα_HMGbox domains of 10 (34.5%) of the 29 mutant proteins are 100% identical…” – by definition “mutant” suggests a difference; perhaps reserve “mutant” for the subset with substitutions and use “variant” or similar for the whole set.
12. Lines 267–268 (Results 3.2): “The HMG-box_ROX1-like domain sequences of 3 of the 30 variable AMT1-2-1 proteins…” – “AMT1-2-1” appears to be a typo; it should be “MAT1-2-1.”
13. Line 283 (Results 3.2): “metatranscriptome assembly GAGW00000000 of the C. sinensis insect–fungi complex…” – consider standardizing to “insect–fungal complex” throughout.
14. Lines 370-406 (Figure 3 legend and associated text): Several references to changes “ultimately changed the DNA binding affinities and the functionalities of gene transcriptional regulation…” are quite assertive, given that no binding assays were performed; consider softening to “are expected to influence” or “may affect.”
15. Line 1084 (Section 4.3): “self-fertilization via a homothallic or psuedohomothallic strategy” – correct “psuedohomothallic” to “pseudohomothallic.”
16. Lines 1088-1089 (Section 4.3): “natural C. sinensis insect–fungal complex specimens” – consider standardizing to “insect–fungal complex” throughout.
17. Lines 1096–1097 (Section 4.3): “These findings demonstrated that O. sinensis experiences self-sterility and uses a heterothallic or hybrid strategy, even parasexuality…” – This is a very strong claim; I recommend changing “demonstrated” to “suggest” or “are consistent with,” and reconsider mentioning “parasexuality” unless directly supported by data.
18. Lines 1167–1181 (Discussion): Long sentence starting “Regardless of whether the MAT1-1-1 and MAT1-2-1 proteins form heterodimers…” becomes difficult to follow; consider splitting and clarifying the logical steps linking occurrence/transcription patterns to self-sterility under heterothallic or hybrid reproduction.
19. Lines 1182–1199 (Conclusions): The conclusions section repeats much of the results at length. You might condense this to a shorter paragraph focusing on the key take-home messages, while moving detailed numbers and cluster counts to a table or leaving them in the Results.

Concise suggestions for improving the work

• Clarify novelty relative to your previous J. Fungi article and more clearly state what new biological insights the domain-focused analysis adds.
• Streamline the manuscript by shortening repeated background sections (taxonomy, nomenclature, cultivation history) and condensing the Conclusions to the essential messages.
• Temper causal language about self-sterility, heterothallism, and parasexuality; reframe these as hypotheses supported by structural and occurrence data, and explicitly acknowledge the lack of direct functional assays.
• Improve presentation of results by adding one or two summary figures or schematic models that synthesize the many individual examples into an overarching conceptual picture.
• Standardize terminology and style (names of complexes, genotypes, domains; hyphenation; section numbering) and correct the specific typos listed in the line-by-line section.
• Undertake thorough English language editing to simplify sentence structure and remove remaining typographical and formatting artefacts.

 

Overall recommendation

The work addresses a biologically relevant question and assembles an interesting set of structural predictions, but in its current form, the manuscript is too long, somewhat repetitive, linguistically difficult to read, and contains interpretative claims that are stronger than the evidence presented. With substantial restructuring, clearer delimitation of novelty, more cautious conclusions, and comprehensive language editing, it could become suitable for publication.

Comments on the Quality of English Language

Language and style.

The manuscript contains numerous long sentences with multiple embedded clauses, frequent hyphenation artefacts from line breaks, and several typographical issues (e.g., “animo acid,” “psuedohomothallic,” inconsistent use of “insect–fungal” vs “insect–fungi complex”). I strongly recommend a thorough edit by a fluent English speaker or professional editing service, with particular attention to simplifying sentences and standardizing terminology.

Overall, I think the work has potential but would benefit from being shorter, more focused, and more cautious in its interpretations.

 

Assessment of the English language

Overall assessment: the English needs to be improved.

Although the manuscript is generally understandable, there are:

• Numerous typographical errors (“animo acid,” “psuedohomothallic,”).
• Very long and complex sentences with multiple subclauses, which hinder readability, especially in the Introduction and Discussion.
• Inconsistent terminology and hyphenation (e.g., “insect–fungal complex” vs “insect–fungi complex;” “DNA binding” vs “DNA-binding”).

A careful, line-by-line language edit by a native or very fluent English speaker is strongly recommended.

Author Response

Responses4 to the comments from Reviewer #4:

Thank you very much for your positive comments by listing the 3 strengths of our paper.

 

Reviewer #4 commended: “1. Clarify novelty versus the J. Fungi 2025 article: The introduction and discussion repeatedly revisit material already presented in your prior paper on 3D heteromorphs of full-length mating proteins (https://doi.org/10.3390/jof11040244). It would help the reader if you explicitly and succinctly state, early in the introduction and again in the discussion, what is new here (e.g., exclusive focus on DNA-binding domains, integration with hydrophobicity/secondary structure plots, specific new inferences) and what is confirmatory or elaborative.”  And  “Clarify novelty relative to your previous J. Fungi article and more clearly state what new biological insights the domain-focused analysis adds.”

Our previous study presented in “the J. Fungi 2025 article” [50] is the first to demonstrate variations in the 3D structures of 138 MAT1-1-1 and 74 MAT1-2-1 proteins derived from numerous wild-type C. sinensis isolates and O. sinensis strains. The previous paper [50] is quite long (41 pages in total) and analyzed entire length sequences of mating proteins by use of the Bayesian clustering technique, ExPASy ProtScale plotting for analysis of hydrophobicity and secondary structure of proteins, and AlphaFold 3D structure predictions. However, because of the size (41 pages in total) of the paper, we have to leave behind the very lengthy, detailed analysis of the critical functional DNA-binding domains to the subsequent papers (the current paper and a following paper).

The current paper is a continuation study of “the J. Fungi 2025 article” [50] and focuses on a detailed analysis of the primary structures of the MATα_HMGbox and the HMG-box_ROX1-like domains of the MAT1-1-1 and MAT1-2-1 proteins, respectively, derived from numerous wild-type C. sinensis isolates, and the impact of the primary structural changes in the hydrophobic properties and the secondary and tertiary structures of the critical functional DNA-binding domains within the mating proteins.

However, the analytical results in this paper are already quite lengthy (52 pages in total and 16 figures, many of which contain multiple panels), exceeding the length typically considered for a single paper. Thus, the current paper involves the results for the 118 full-length MAT1-1-1 proteins and 69 MAT1-2-1 full-length proteins that were derived from wild-type C. sinensis isolates of insect−fungal complex specimens collected from nearly all C. sinensis production regions on the Qinghai−Tibet Plateau by the research team led by Prof. Zhang Y-J.

Unfortunately, we have to leave behind second part of the detailed analytic results of the MATα_HMGbox and the HMG-box_ROX1-like domains of the truncated and mutant MAT1-1-1 and MAT1-2-1 proteins derived from numerous O. sinensis strains to the next paper. We have completed all analysis and drafted a manuscript, which probably has 50+ pages. These O. sinensis strains were initially obtained through the collaborative research efforts of the research teams led by Profs. Yao Y-J, Bushley KE, and Spatafora JW [23,48].

 

Reviewer #4 commended: “2. Moderate the strength of the main conclusions: Statements that the findings “demonstrated that O. sinensis experiences self-sterility and uses a heterothallic or hybrid strategy, even parasexuality” and that this constitutes a “fundamental paradigm shift” are very strong given that the study is wholly based on sequence and structure predictions without direct mating or functional assays. I suggest rephrasing throughout to “our data are consistent with…” or “support the hypothesis that…,” and adding a short “Limitations” paragraph explicitly acknowledging the inferential nature of the conclusions and the lack of experimental validation.”

We have followed the suggestions and revised the simple summary and conclusion sections by adjusting the language intensity/strength. Again, our manuscript has been sent to a professional English editing team at American Journal Experts for re-editing and we have worked with the editing team to finalize the paper.

 

Reviewer #4 commended: “3. Clarify datasets and definitions:

• Please make more explicit how many sequences come from each source ( sinensis genomes, C. sinensis metatranscriptomes, other genotypes), perhaps in a concise summary table.

Following Reviewer #4’s comment, we have added a new Table S1 listing how many sequences of MAT1-1-1 and MAT1-2-1 proteins come from each of the sample sources: O. sinensis strains of different genotypes (including Genotype #1 H. sinensis strains), wild-type C. sinensis isolates, and C. sinensis insect‒fungal complexes (metatranscriptome).

Based on the available information in literature and the GenBank database, numerous wild-type C. sinensis isolates are impure, coexisting with several or multiple endophytic/symbiotic fungi. Thus, the numerous mutant MAT1-1-1 and MAT1 -2-1 proteins, regardless of their co-occurrence or differential occurrence, detected from the impure samples by several research groups indicate possible heterogeneous fungal sources of the mating proteins. Without the additional efforts and help from the original owners (the research group of Prof. Zhang Y-J) of the impure study samples, the actual fungal origins of the detected mating proteins derived from the impure samples remain scientifically unclear.

 

• Define clearly what you call “mutant” proteins (any deviation from AGW27560/AEH27625?) and what is meant by “heterospecific fungal sources of mating proteins” in the context of the insect–fungal complex.”

The MAT1-1-1 protein AGW27560 was derived from pure H. sinensis strain CS68-2-1229 [48]. This authentic protein and 82 other MAT1-1-1 proteins have identical primary structures and the same predicted 3D structures under the AlphaFold 3D structure code U3N942.

The MAT1-2-1 protein AEH27625 was derived from pure H. sinensis strain CS2 [56]. This authentic protein and 31 other MAT1-2-1 proteins have identical primary structures and the same predicted 3D structures under the AlphaFold 3D structure code D7F2E9.

Other mating proteins with various primary structures and different 3D structures other than the AlphaFold 3D structure codes U3N942 and D7F2E9 are considered to have altered conformations. Mating proteins with amino acid substitutions or peptide chain truncations are considered mutants.

The fungal portion of the natural C. sinensis insect–fungal complex has been systematically examined and proven to be a combination of multiple fungi, including >90 species of at least 37 fungal genera [7−23,26,32,56,86]. The phylogenetic and genotypic complexity is even more complex than that of impure O. sinensis strains and wild-type C. sinensis isolates. Thus, the differentially mutated mating proteins derived from natural C. sinensis insect–fungal complexes may be produced by different cooccurring fungi.

 

Reviewer #4 commended: “4. Improve structure and concision: The introduction includes a lengthy taxonomic/nomenclatural digression (e.g., on “One Fungus=One Name”) that could be shortened or moved in part to the discussion or supplement …... Similarly, the discussion contains an extended narrative on 40 years of cultivation attempts and species contradictions, which, while interesting, somewhat distracts from the core structural findings. Consider focusing the discussion more tightly on how your structural data refines hypotheses about mating behavior and cultivation.” …… “Streamline the manuscript by shortening repeated background sections (taxonomy, nomenclature, cultivation history) …...”

It appears to be that “The introduction includes a lengthy taxonomic/nomenclatural digression (e.g., on “One Fungus=One Name”)”, although this portion of Introduction has been largely shortened and concentrated. The Latin name issue is quite complex. We have elucidated this in detail in the literature [7,9−10]. To avoid confusion while reading the current paper, we briefly summarized the taxonomic/nomenclatural problem in the first paragraph of the Introduction of the original submission (Lines 72−93) and now in the third paragraph of the revised Introduction (Lines 76−97).

Both Reviewers #3 and #4 have commented on Latin name usage and suggested including a summary in the Discussion section. We realize that the confusion of Latin name usage is a very serious historical problem in the O. sinensis research field, and the brief summary in the Introduction section may not be sufficiently knowledgeable for our readers to understand and differentiate between different study materials. We then followed the Reviewers’ suggestion and added a subsection to the Discussion.

4.1. Historical and current usage of Latin names regarding Cordyceps sinensis, Hirsutella sinensis, and Ophiocordyceps sinensis to refer to fungus/fungi and the insect−fungal complex

Natural C. sinensis was introduced to Western countries by the French missionary Dominicus Parennin in 1723. Its intrinsic fungus was identified by Jonathan Pereira in 1843 as belonging to the Sphaeria genus, and the insect portion was identified by Edward Doubleday as belonging to Agrotis in 1842 [87−89]. Historical taxonomic examinations of natural C. sinensis clearly revealed that it is an insect−fungal complex. Miles Joseph Berkeley described the fungus as Sphaeria Sinensis Berkeley in 1843 and renamed it Cordyceps Sinensis in 1857 [90−91]. Pier Andrea Saccardo renamed it Cordyceps sinensis (Berkeley) Saccardo in 1883 [92−93]. Unfortunately, since then, the Latin name C. sinensis, which was originally given to the intrinsic fungus, has been indiscriminately used in academia and the mass market to refer to both the fungus C. sinensis and the wild insect–fungal complex.

The fungus was renamed O. sinensis in 2007 by Sung et al. [33] with the use of the H. sinensis strain EFCC 7287 as the nomenclatural reference [4,7,33−35]. Since 2001, >600 ITS1-5.8S-ITS2 sequences of O. sinensis have been uploaded to the GenBank database under the GenBank Taxid 72228, representing 17 genome-independent genotypes of O. sinensis with numerous, scattered transition, transversion, or insertion/deletion mutant alleles or hereditary variations with reciprocal substitutions of large DNA segments and genetic material recombination [7,9−10,86]. Li et al. [7,10,86] have shown that 17 genotypes are genomically independent and belong to different O. sinensis fungi, which have been postulated to share a common hereditary ancestor [17].

To date, only Genotype #1 of O. sinensis (i.e., H. sinensis) has been purified and taxonomically characterized [7,94]. The genome-independent Genotypes #2-17 have not been purified, and their taxonomic positions have not been determined [7], although these genotypes are assigned the same Latin name, O. sinensis, under the same GenBank taxonomic ID 72228. Controversies, hypotheses, and scientific arguments surrounding genotypic mutations of O. sinensis pervade the entire field of O. sinensis research.

This historical situation prompted us to embrace the currently available information and use different names, i.e., H. sinensis, O. sinensis, C. sinensis insect−fungal complex, and wild-type C. sinensis isolates, to refer to different study materials. This practice might not be perfect because of historical uncertainties in academia. However, we hope that the taxonomic/nomenclatural uncertainties will encourage taxonomists in mycology, botany, and traditional Chinese medicine to design and conduct future studies to solve these historical academic problems.

(Lines 1015−1048)

 

Reviewer #4 provided “Line-by-line comments”

1. Line 11: “Correspondence: zhujoah@163.com)” – remove the final parenthesis after the email address. Following this comment, we have deleted it.

 

1. Lines 18–19 (Simple Summary): “The protein sequences exhibited various animo acid substitutions…” – correct “animo” to “amino”; avoid line-break artifacts (“se-quences”). Following this comment, we have corrected it. For the “line-break artifacts”, this is the automatic formatting style requested by the Journal.

 

1. Line 21 (Simple Summary): “critical function domains” – consider “critical functional domains” for more natural phrasing. Following this comment, we have revised it.

 

1. Line 26 (Simple Summary): “sinensis insect-fungal complex” – use the same form as elsewhere (“insect–fungal complex”) and standardize the hyphen/en dash. Following this comment, we have standardized all the hyphens/endashes.

 

1. Line 55 (Introduction): “Natural Cordyceps sinensis is one of most expensive therapeutic agents…” – add “the”: “one of the most expensive therapeutic agents…” Following this comment, we have revised the text.

 

1. Lines 76–86 (Introduction): Very long sentence beginning “Although Zhang et al. [36] proposed improper implementation of the ‘One Fungus=One Name’…”. Consider splitting into two or three sentences to improve clarity and reduce repetition of “genomically independent genotypes…”. Following this comment, we have revised it.

 

1. Lines 116–122 (Introduction): Sentence starting “Continuing our previous study [50]…” is long and dense; consider breaking it and standardizing “DNA binding domains” vs “DNA-binding domains.” Following this comment, we have standardized “DNA-binding domains”.

 

1. Lines 128–141 (Materials and Methods 2.1): The list of genome and protein accessions is very detailed; these could be summarized in a table or reserved for Supplementary material to streamline the main text. The GenBank accession numbers listed in Section 2.1 are listed in Tables S2−S4 of the Supplementary files.

 

1. Lines 180–192 (Section 2.5): “The MATα_HMGbox domain of MAT1-1 proteins…” – elsewhere you refer to “MAT1-1-1 proteins”; please check whether “MAT1-1 proteins” is a typo and ensure consistent nomenclature. Following this comment, we have revised it. It should be “MAT1-1-1 protein” rather than “MAT1-1 protein” in Line 183 of original submission.

 

1. Line 193 (heading): “II-6 AlphaFold-based prediction of the 3D structures…” – likely meant to be “6.” Correct the section numbering format. Following this comment, we have revised it. It should be 2.6, rather than II-6.

 

1. Lines 232–233 (Results 3.1): “The MATα_HMGbox domains of 10 (34.5%) of the 29 mutant proteins are 100% identical…” – by definition “mutant” suggests a difference; perhaps reserve “mutant” for the subset with substitutions and use “variant” or similar for the whole set.

The “29 mutant proteins” contain amino acid substitutions at different mutation sites. Ten of them have identical “the MATα_HMGbox domain” sequences but have amino acid substitutions outside the functional domains, while the other 19 proteins have amino acid substitutions within the functional domains. To avoid potential confusion, we revised this sentence to the following:

The MATα_HMGbox domains of 10 (34.5%) of the 29 mutant proteins are 100% identical to the domain sequence of the query protein AGW27560 when the amino acid substitutions outside the functional domains are not considered. (Lines 254−256)

 

1. Lines 267–268 (Results 3.2): “The HMG-box_ROX1-like domain sequences of 3 of the 30 variable AMT1-2-1 proteins…” – “AMT1-2-1” appears to be a typo; it should be “MAT1-2-1.” Yes. It is a typo. We have corrected it.

 

1. Line 283 (Results 3.2): “metatranscriptome assembly GAGW00000000 of the C. sinensis insect–fungi complex…” – consider standardizing to “insect–fungal complex” throughout. Following this comment, we have standardized the term.

 

1. Lines 370-406 (Figure 3 legend and associated text): Several references to changes “ultimately changed the DNA binding affinities and the functionalities of gene transcriptional regulation…” are quite assertive, given that no binding assays were performed; consider softening to “are expected to influence” or “may affect.” In accordance with the comment, we have revised it with the use of “may ultimately affect”. (Line 639)

 

1. Line 1084 (Section 4.3): “self-fertilization via a homothallic or psuedohomothallic strategy” – correct “psuedohomothallic” to “pseudohomothallic.” Following this comment, we have revised the typo.

 

1. Lines 1088-1089 (Section 4.3): “natural C. sinensis insect–fungal complex specimens” – consider standardizing to “insect–fungal complex” throughout. Following this comment, we have standardized the term.

 

1. Lines 1096–1097 (Section 4.3): “These findings demonstrated that O. sinensis experiences self-sterility and uses a heterothallic or hybrid strategy, even parasexuality…” – This is a very strong claim; I recommend changing “demonstrated” to “suggest” or “are consistent with,” and reconsider mentioning “parasexuality” unless directly supported by data.

Following this comment, we have revised it with the use of “suggested”.

Li et al. (2023; DOI: 10.1371/journal.pone.0270776) [10] reported the detection of GC-biased Genotypes #13 and #14 in the semi-ejected and fully ejected ascospores, respectively, of natural C. sinensis insect−fungal complexes. Genetically, the ITS sequences of Genotypes #13 and #14 of O. sinensis featured large DNA segment reciprocal substitutions and genetic material recombination between the genomes of the 2 parental fungi, i.e., Group-A Genotype #1 H. sinensis (GenBank accession # AB067721) and the AB067719-type Group-E fungus, as illustrated in the figure below. The taxonomic position of the AB067719-type Group-E fungus has not been determined, but it is a heterospecific fungus. This phenomenon of genetic material recombination indicates the possibility of fungal hybridization or parasexuality between the 2 parental fungi.

 

 

1. Lines 1167–1181 (Discussion): Long sentence starting “Regardless of whether the MAT1-1-1 and MAT1-2-1 proteins form heterodimers…” becomes difficult to follow; consider splitting and clarifying the logical steps linking occurrence/transcription patterns to self-sterility under heterothallic or hybrid reproduction.

Thank you for the comment. To keep the sentence compact and more understandable, we have deleted “Regardless of whether the MAT1-1-1 and MAT1-2-1 proteins form heterodimers or interact via complementary electrostatic surfaces and thereby enable cooperative DNA binding,” and leave the main sentence unchanged. This revision does not change the meaning of the sentence. (Lines 1224–1226)

 

• Lines 1182–1199 (Conclusions): The conclusions section repeats much of the results at length. You might condense this to a shorter paragraph focusing on the key take-home messages, while moving detailed numbers and cluster counts to a table or leaving them in the Results. And Streamline the manuscript by …… condensing the Conclusions to the essential messages.

Following the comments, we have revised the conclusion section accordingly:

This study demonstrates the variations in the tertiary structures of the mating proteins in numerous wild-type C. sinensis isolates, H. sinensis strains, and C. sinensis insect–fungal complex specimens, focusing on the DNA-binding domains, i.e., the MATα_HMGbox domain of the MAT1-1-1 protein and the HMG-box_ROX1-like domain of the MAT1-2-1 protein. These DNA-binding domains are critical for specifically controlling the expression of genes related to the sexual reproduction of O. sinensis. The sequences of the functional DNA-binding domains with various amino acid substitutions at different mutation sites alter the tertiary protein structures based on heteromorphic AlphaFold-predicted stereostructures. These findings suggest self-sterility of O. sinensis under heterothallic or hybrid mating at the protein structural level, further complementing the genetic and transcriptional evidence to refute the self-fertilization hypothesis for O. sinensis during homothallic or pseudohomothallic reproduction. Thus, the identification of an appropriate mating partner is the only mechanism by which self-sterile O. sinensis can accomplish sexual reproduction during the lifecycle of the C. sinensis insect−fungal complex on the Qinghai–Tibet Plateau. This conceptual shift in sexual reproduction mode will aid in the design of future reproductive physiology studies for experimental validation.

(Lines 1238−1254)

 

Reviewer #4 also commended:

• Temper causal language about self-sterility, heterothallism, and parasexuality; reframe these as hypotheses supported by structural and occurrence data and explicitly acknowledge the lack of direct functional assays.

We understand Reviewer #4’s concern of “the lack of direct functional assays” with respect to “self-sterility, heterothallism, and parasexuality”. We would like to state that the studies from our group and the research groups of Profs. Zhang Y-J and Yao Y-J [48−50,52,54−55, as well as this study] demonstrated differential occurrence, differential transcription, and alternative splicing of the MAT1-1-1 and MAT1-2-1 and pheromone receptor genes and altered the tertiary structures of the functional DNA-binding domains of the MAT1-1-1 and MAT1-2-1 proteins. These findings systematically refute the hypothesis of self-fertilization at the genetic, transcriptional, and protein structure levels . Thus, “self-sterility and heterothallism” is definitely the case for O. sinensis.

However, with respect to hybridization or “parasexuality”, Li et al. [10] detected GC-biased Genotypes #13−14, which featured large DNA segment reciprocal substitutions and genetic material recombination between the genomes of the 2 parental fungi, i.e., Group-A Genotype #1 H. sinensis (GenBank accession # AB067721) and the AB067719-type Group-E fungus. Please see our response to the comment “Lines 1096–1097 (Section 4.3): “These findings demonstrated that O. sinensis experiences ……” above. These discoveries, with their reliability in terms of genetics, gene expression, and protein structure, encourage us to use an affirmative language to describe “hybridization” or “parasexuality”: “These findings suggest that O. sinensis experiences self-sterility and uses a heterothallic or hybrid strategy, even parasexuality, to accomplish sexual reproduction during the lifecycle of the C. sinensis insect–fungal complex [10,50,54−55,108−115].” (Lines 1151–1154)

 

• Improve presentation of results by adding one or two summary figures or schematic models that synthesize the many individual examples into an overarching conceptual picture.

The results section of our paper contains 16 figures, among which Figures 3−16 contain 6 to 15 panels. Because of the number of the figures in our paper, we unwillingly put 4 summary tables (Tables S6-S9) in the Supplementary files, to summarize the MAT1-1-1 proteins in various wild-type C. sinensis isolates (Table S6), the MAT1-1-1 proteins in the genome assemblies of H. sinensis strains and the metatranscriptome assemblies of natural C. sinensis (Table S7), the MAT1-2-1 proteins in various wild-type C. sinensis isolates (Table S8), and the MAT1-2-1 proteins in the genome and transcriptome assemblies of H. sinensis strains and the metatranscriptome assembly of natural C. sinensis (Table S9).

 

• Standardize the terminology and style (names of complexes, genotypes, domains; hyphenation; section numbering) and correct the specific typos listed in the line-by-line section. Thank you for the comments, and we have carefully checked and revised them.

 

• Undertake thorough English language editing to simplify sentence structure and remove remaining typographical and formatting artefacts.

Thank you for the comment; we have sent our manuscript to a professional language editing company, American Journal Experts, for language re-editing.

 

Overall recommendation

The work addresses a biologically relevant question and assembles an interesting set of structural predictions, but in its current form, the manuscript is too long, somewhat repetitive, linguistically difficult to read, and contains interpretative claims that are stronger than the evidence presented. With substantial restructuring, clearer delimitation of novelty, more cautious conclusions, and comprehensive language editing, it could become suitable for publication.

 

Comments on the Quality of English Language

Language and style.

The manuscript contains numerous long sentences with multiple embedded clauses, frequent hyphenation artefacts from line breaks, and several typographical issues (e.g., “animo acid,” “psuedohomothallic,” inconsistent use of “insect–fungal” vs “insect–fungi complex”). I strongly recommend a thorough edit by a fluent English speaker or professional editing service, with particular attention to simplifying sentences and standardizing terminology.

Thank you for the comment; we have sent our manuscript to a professional language editing team at American Journal Experts for language re-editing and worked closely with the team to finalize the paper.

 

Reviewer #4 also commended:

Overall, I think the work has potential but would benefit from being shorter, more focused, and more cautious in its interpretations.

 

Assessment of the English language

Overall assessment: the English needs to be improved. Thank you for the comment; we have sent our manuscript to a professional language editing ream at American Journal Experts for language re-editing and worked closely with the team to finalize the paper.

 

Although the manuscript is generally understandable, there are:

• Numerous typographical errors (“animo acid,” “psuedohomothallic,”). Thank you for the comment; we have corrected the typos.

 

• Very long and complex sentences with multiple subclauses, which hinder readability, especially in the Introduction and Discussion. Thank you for the comment; we have tried to shorten the sentences in the Introduction and Discussion sections. We have sent our manuscript to a professional language editing team at American Journal Experts for language re-editing and worked closely with the team to finalize the paper.

 

• Inconsistent terminology and hyphenation (e.g., “insect–fungal complex” vs “insect–fungi complex;” “DNA binding” vs “DNA-binding”). Thank you for the comment; we have revised the terms.

 

A careful, line-by-line language edit by a native or very fluent English speaker is strongly recommended.

Thank you for the comment; we have sent our manuscript to a professional language editing team at American Journal Experts for language re-editing and worked closely with the team to finalize the paper.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

In my view, this manuscript can be published in its current form.

Author Response

Thank you very much

Reviewer 4 Report

Comments and Suggestions for Authors

Dear Authors,
Thank you for submitting the revised version. The revision successfully fixes multiple typographical and formatting problems (e.g., email punctuation, “amino acid,” corrected typos such as MAT1-2-1, section numbering), and it is helpful that you added explicit caution about interpreting low-confidence AlphaFold regions.

To make the manuscript publishable within a short revision window, I suggest focusing on the following:

1. Clarify novelty vs. your prior J. Fungi paper in 2–3 sentences early in the Introduction and again in the Discussion (what is new here vs. confirmatory).

2. Define and standardize terminology: replace blanket “mutant proteins” with “variants” (or define “mutant” precisely), especially where sequences are stated to be 100% identical.

3. Further moderate causal language in Discussion/Conclusions (replace remaining “demonstrated/refute/invalidates” where appropriate with “consistent with/supports/suggests”).

4. Reconsider “parasexuality” unless you can directly support it with evidence; otherwise remove or clearly bracket it as speculation.

5. Tighten Introduction/Discussion by shortening the nomenclature/taxonomy digression and simplifying long sentences; ensure consistent hyphenation (e.g., “DNA-binding”).

Overall, the revision is moving in the right direction, but these remaining points are important for clarity and evidentiary balance.

Author Response

Responses to the Round-2 review comments from Reviewer #4

Reviewer #4 commended: “Clarify novelty vs. your prior J. Fungi paper in 2–3 sentences early in the Introduction and again in the Discussion (what is new here vs. confirmatory).”

Following the comment, we have added a new paragraph in the Discussion section, right before subsection 4.1. (Lines 1014−1036)

Previous studies [54−55] reported the differential occurrence, differential translation, and alternative splicing of MAT1-1-1 and MAT1-2-1 and pheromone receptor genes of H. sinensis, wild-type C. sinensis isolates, and C. sinensis insect-fungal complex. The evidence from the genetic and transcriptional level studies indicates that O. sinensis experiences self-sterility and uses a heterothallic or hybrid (even parasexuality) strategy to accomplish sexual reproduction during the lifecycle of the C. sinensis insect–fungal complex [10,108−115]. Li et al. [50] reported the 3D structural changes of the complete mating proteins, further validating the self-sterility hypothesis for O. sinensis at the protein structure level. They demonstrated that the entire MAT1-1-1 and MAT1-2-1 proteins contribute to 15 and 17 stereostructure heteromorphs, belonging to 5 and 5 Bayesian clusters, respectively. The research presented in this paper continues our previous studies, with a focus on various amino acid substitutions within the MATα_HMGbox and HMG-box_ROX1-like domains of the full-length MAT1-1-1 and MAT1-2-1 proteins, respectively, which are derived from wild-type C. sinensis isolates. The MATα_HMGbox domains of MAT1-1-1 proteins with different amino acid substitutions are clustered to 5 Bayesian clusters, with or without branches, as shown in Figure 1 and summarized in Tables S6-S7. The HMG-box_ROX1-like domains of the MAT1-2-1 proteins are clustered to 2 branched Bayesian clusters, as shown in Figure 2 and summarized in Tables S8-S9. The amino acid substitutions within the DNA-binding domains significantly affect the hydrophobicity of functional domains and alter the secondary and tertiary structures of mating proteins, especially the tertiary structures of hydrophobic core formed by 3 α-helices within the DNA-binding domains, ultimately affecting the synergistic functionality of the key functional domains of mating proteins in the sexual reproduction of O. sinensis.

 

Reviewer #4 commended: “Define and standardize terminology: replace blanket “mutant proteins” with “variants” (or define “mutant” precisely), especially where sequences are stated to be 100% identical.”

We have replaced all “mutant” with “variant”.

 

Reviewer #4 commended: “Further moderate causal language in Discussion/Conclusions (replace remaining “demonstrated/refute/invalidates” where appropriate with “consistent with/supports/suggests.”

We have moderated the language with replacing “demonstrated” with “revealed” and replacing “refute/invalidates” with “disprove” in Discussion/Conclusions.

 

Reviewer #4 commended: “Reconsider “parasexuality” unless you can directly support it with evidence; otherwise remove or clearly bracket it as speculation.”

Following your suggestion, we have bracketed the word “parasexuality” in order to avoid controversy.

However, as we have responded to the comment in the Round-1 review, we do have solid evidence depicting 2 cases of reciprocal substitutions and genetic material recombination between the genomes of the 2 parental fungi, i.e., Group-A Genotype #1 H. sinensis (GenBank accession #AB067721) and the AB067719-type Group-E fungus. Please see our previous response #17 (Lines 1096–1097 (Section 4.3): ……) to your comments last time.

 

Reviewer #4 commended: “Tighten Introduction/Discussion by shortening the nomenclature/taxonomy digression and simplifying long sentences; ensure consistent hyphenation (e.g., “DNA-binding”).”

We understand that the “nomenclature/taxonomy” issue is quite complex and has led and will continuously lead to difficulties in reading our papers and understanding the differential usage of relevant Latin names. For this complex issue, we have published several papers and books [7,9−10,86] [and other papers and books].

In the original submission of this paper, we provided a short description in the Introduction regarding the complex Latin name issue. However, you and Reviewer #3 requested us to provide more information regarding the historical and current usage of Latin names in the Discussion section.

We have shortened some of the long sentences in this portion according to your request.

We have discussed the possibility of shortening this subsection in the Discussion section. In response to the requests and comments from you and Reviewer #3 during the first round of review and in order to enable our readers to fully understand the complexity and rationality of the usage of Latin scientific names in our paper, we summarize the full historical story regarding the Latin name usage. We feel that it is difficult to omit any content from this section.

As for the “hyphenation (e.g., “DNA-binding”)”, we have checked throughout the paper. For the domains, we consistently use hyphenated “DNA-binding” domain(s) but for the biological functions, we use non-hyphenated “DNA binding”.