Chemical Signaling and Metabolomic Crosstalk in Endophytic Fungi–Medicinal Plant Symbioses for Natural Product Discovery and Sustainable Bioproduction
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsMajor revision
Comments for author File: 
Comments.pdf
Author Response
Comments 1: The manuscript title Chemical Dialogue and Metabolomic Crosstalk in Endophytic Fungi-Medicinal Plant Symbioses for Natural Product Discovery and Sustainable Bioproduction needs major revision with correction of typo error. The title of the study needs to be revised. The word “Chemical Dialogue” seems to more metaphorical or exaggerated. Could have been used Chemical signalling.
Response 1: Thank you for pointing this out. We completely agree with this comment. The term “Chemical Dialogue” was indeed too metaphorical for a rigorous scientific review. Therefore, we have changed the title to be more scientifically precise by using “Chemical Signalling”. Furthermore, we have systematically replaced the phrase “chemical dialogue” with “chemical signalling” throughout the entire manuscript, including the abstract, keywords, section headings, and figure captions, and thoroughly corrected typo errors. This change can be found on [Page 1, Title] and throughout the revised manuscript.
Comments 2: Manuscript is scientifically stronger having conceptual strength linking all the domain metabolomic, Multi-omic, BGC and ecology. Detail and comparative description of holobiont and spatial metabolomic (MALDI-MSI) gives it reader clear understanding for natural product discovery from symbiotic plants.
Response 2: Thank you very much for your positive and encouraging feedback. We are greatly motivated to know that our effort to integrate these diverse domains into a cohesive framework was well received.
Comments 3: However, review is too long and can be concise in several section where there is an overlap. BCG regulation, CRISPR, Metabolomic section overlap can be concise.
Response 3: We agree with this comment. We realized that these sections were previously too lengthy and contained overlapping information. Therefore, we have substantially streamlined the text. Specifically, we merged and condensed the redundant descriptions of BGC regulation and CRISPR-based activation into a single, highly focused paragraph. This revision eliminates the overlap while retaining the core scientific mechanisms. This change can be found in the revised manuscript in Section 3.3 [Page 7, Lines 350-361].
Comments 4: Authors have used several examples which can be concise but work out to generalise all the examples findings together in few lines.
Response 4: Agree. We have, accordingly, generalized all the specific findings into a concise paragraph to reduce the length and improve readability. We deleted the lengthy subsections that previously detailed individual metabolites and specific fungal strains (former Sections 4.1 to 4.4). Instead, we synthesized these findings into a few lines summarizing the structural diversity and target-specific bioactivities, while moving the extensive specific examples to Supplementary Table S1. This change can be found in the revised manuscript in Section 4.1 [Pages 9-10, Lines 527-541].
Comments 5: There are limitation and challenges in the natural product from endophytes that needs to be discussed.
Response 5: Thank you for pointing this out. We agree with this comment. Addressing the current bottlenecks is crucial for a comprehensive review. Therefore, we have added a dedicated new subsection right before the conclusions to explicitly discuss the major hurdles in the field, including the “unculturable” barrier, yield attenuation during axenic fermentation, and bioprocessing scale-up difficulties. This new addition can be found in the revised manuscript in Section 6.6 [Page 18, Lines 1063-1075].
Reviewer 2 Report
Comments and Suggestions for AuthorsThis manuscript presents a comprehensive, timely, and technically sophisticated review on metabolomic crosstalk in endophytic fungi–medicinal plant symbioses. It integrates metabolomics, BGC regulation, spatial MSI, and synthetic biology in a way that aligns well with the scope of Metabolites. However, the manuscript would benefit from streamlining, clearer conceptual signposting, and reduced redundancy, particularly in later sections.
Abstract:
- The “Methods” subsection reads more like a narrative justification than a methods summary; consider tightening to focus on what was surveyed rather than why.
- The Results section could be more specific.
Introduction:
- Consider adding a brief paragraph at the end of the Introduction outlining the review structure, which would improve navigation for readers.
Metabolomic Strategies for Deciphering Interactions
- Untargeted Metabolomics: Consider a short schematic table summarizing axenic vs. co-culture vs. in planta metabolomics designs.
- Spatial Metabolomics (MALDI-MSI): Clarify whether cited MSI studies are primarily plant–pathogen or plant–endophyte, to avoid conceptual overgeneralization.
- 4–2.5 Integrative Multi-Omics & Bioinformatics: Slightly reduce tool listing density; focus more on decision logic (when to use what).
- Consider a brief concluding paragraph synthesizing Sections 2.1–2.5.
BGCs and Regulatory Networks
- Add a summary figure or table mapping regulatory layers → activation strategies.
- Consider merging Sections 3.3 and 3.6 conceptually (regulation + CRISPR activation).
Pharmacologically Active Natural Products
- Reduce repetition in concluding sentences across 4.1–4.4.
- Explicitly link each compound class back to metabolomic discovery strategies discussed earlier.
Chemical Dialogue and Symbiotic Interplay
- Add short “Key Concepts” or “Mechanistic Summary” paragraphs at the end of Sections 5.2–5.5.
- Explicitly distinguish plant-driven vs. endophyte-driven metabolic changes.
- There are some repetition with later Sections 2 and 5 regarding “chemical dialogue” and silent BGC activation. These themes are introduced multiple times without clear conceptual progression, leading to redundancy rather than cumulative insight and weakening the overall narrative coherence of the manuscript.
Author Response
Comments 1 & 2: Abstract: The “Methods” subsection reads more like a narrative justification than a methods summary; consider tightening to focus on what was surveyed rather than why. The Results section could be more specific.
Response 1 & 2: Thank you for pointing this out. We agree with this comment. Therefore, we have rewritten the “Methods” and “Results” sections in the Abstract. The Methods now explicitly state what was surveyed, and the Results are far more specific regarding the types of metabolic cues and compound classes discussed. These changes can be found in the Abstract [Page 1, Lines 16-28].
Comments 3: Consider adding a brief paragraph at the end of the Introduction outlining the review structure, which would improve navigation for readers.
Response 3: Agree. We have, accordingly, added a brief paragraph at the very end of the Introduction to outline the structure of the entire review, providing a clear roadmap for the reader. This change can be found in Section 1 [Page 2, Lines 96-102].
Comments 4: Untargeted Metabolomics: Consider a short schematic table summarizing axenic vs. co-culture vs. in planta metabolomics designs.
Response 4: Thank you for the excellent suggestion. We agree and have created a new schematic table that summarizes and compares the applications and limitations of axenic, co-culture, and in planta metabolomics designs. This change can be found in Section 2.1 [Page 3, Table 1].
Comments 5: Spatial Metabolomics (MALDI-MSI): Clarify whether cited MSI studies are primarily plant–pathogen or plant–endophyte, to avoid conceptual overgeneralization.
Response 5: We agree with this comment. Therefore, we have added clarifying text explicitly noting that while MSI is well-validated in plant-pathogen models, its application in mutualistic endophyte symbioses is emerging and requires careful differentiation. This change can be found in Section 2.3 [Page 4, Lines 178-182].
Comments 6: 4–2.5 Integrative Multi-Omics & Bioinformatics: Slightly reduce tool listing density; focus more on decision logic (when to use what).
Response 6: Agree. We have, accordingly, reduced the mere listing of tools and added a sentence focusing on the “decision logic”—specifically when to use GNPS for rapid dereplication versus targeted tools for novel discovery. This change can be found in Section 2.5 [Page 6, Lines 268-272].
Comments 7: Consider a brief concluding paragraph synthesizing Sections 2.1–2.5. Response 7: Thank you for this suggestion. We agree and have added a concluding paragraph at the end of Section 2, synthesizing how these tools form a progressive discovery workflow. This change can be found at the end of Section 2 [Page 6, Lines 277-281].
Comments 8: Add a summary figure or table mapping regulatory layers → activation strategies.
Response 8: Agree. We have, accordingly, added a new table mapping the different regulatory layers directly to their corresponding activation strategies to provide clear signposting. This change can be found in Section 3.3 [Pages 7-8, Table 2].
Comments 9, 10 & 11: Consider merging Sections 3.3 and 3.6 conceptually (regulation + CRISPR activation). Reduce repetition in concluding sentences across 4.1–4.4. Explicitly link each compound class back to metabolomic discovery strategies discussed earlier.
Response 9, 10 & 11: We completely agree with these observations (Note: Reviewer 1 raised a similar point). Therefore, we have conceptually merged the BGC regulation and CRISPR sections into a single revised Section 3.3 to eliminate overlap. Furthermore, we drastically reduced the repetition across Sections 4.1–4.4 by synthesizing them into a single, concise section and moving the exhaustive examples to Supplementary Table S1. We also explicitly linked structural plasticity back to hybrid modular systems (PKS-NRPS) driven by integrative multi-omics. These major changes can be found in Sections 3.3[Page 7, Lines 350-361] and 4.1[Pages 9-10, Lines 527-541].
Comments 12 & 13: Add short “Key Concepts” or “Mechanistic Summary” paragraphs at the end of Sections 5.2–5.5. Explicitly distinguish plant-driven vs. endophyte-driven metabolic changes.
Response 12 & 13: Thank you for pointing this out. We agree with this comment. To clarify the conceptual progression and reduce redundancy, we have added a dedicated summary paragraph at the end of Section 5. Crucially, this paragraph explicitly distinguishes between plant-driven metabolic cues and endophyte-driven changes. This change can be found at the end of Section 5.6 [Page 15, Lines 901-910].
Comments 14: There are some repetition with later Sections 2 and 5 regarding “chemical dialogue” and silent BGC activation. These themes are introduced multiple times without clear conceptual progression, leading to redundancy rather than cumulative insight and weakening the overall narrative coherence of the manuscript.
Response 14: Thank you for pointing this out. We completely agree with this comment. Therefore, we have systematically restructured these sections to eliminate redundancy and establish a clear conceptual progression.
First, as suggested by both reviewers, we removed the repetitive and metaphorical term “chemical dialogue” replacing it with the precise term “chemical signalling” throughout the manuscript. Second, we strictly delineated the distinct themes of these sections to build cumulative insight: Section 2 is now rigorously focused on the methodological and analytical tools (i.e., how to detect and analyze BGC activation via multi-omics and MSI), while Section 5 is dedicated exclusively to the biological mechanisms and directionality of these interactions (i.e., why and how plant-driven vs. endophyte-driven signalling occurs).
Finally, to explicitly guide the reader through this progression, we added synthesizing “Mechanistic Summary” paragraphs at the end of both Section 2 and Section 5. This bridges the technical discovery workflow with the ecological reality, thereby significantly strengthening the narrative coherence of the manuscript. These structural adjustments can be found throughout Sections 2 and 5, particularly with the newly added summaries at [Page 6, Lines 277-281] and [Page 15, Lines 901-910].
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors have revised the title suitable for this review paper. Authors have conscize the several section and generalize the examples to give the direction to the review paper. the addition of new section limitation and challenges gives overall clear understanding to the research gap in this field.
Reviewer 2 Report
Comments and Suggestions for AuthorsAll the comments have been satisfactorily addressed.
