Review Reports

Round 1

Reviewer 1 Report

            In this work, Seo et al. investigate the molecular basis underlying morphological divergence between two L. edodes strains exhibiting contrasting pileus development. By integrating transcriptomic profiling, protein–protein interaction network analysis, ortholog comparison across multiple strains, and genome-wide variant detection, the authors attempt to identify candidate genes and genomic features associated with the pileus-deficient phenotype. Overall, the study addresses a relevant question in fungal developmental biology and mushroom biotechnology, and the multi-layered analytical framework represents a potentially valuable approach for linking gene regulation and structural genomic variation to phenotypic traits in basidiomycetes.

            This manuscript presents a well-designed and carefully executed study investigating the genetic basis of the pileus-deficient phenotype in Lentinula edodes. The experimental approach is robust, and the data are thoroughly analyzed using a comprehensive set of bioinformatic tools. Additionally, the results are clearly presented and the discussion is appropriate. Importantly, although no single gene explains the phenotype, the study convincingly shows that it arises from a complex interplay of transcriptional regulation and structural genomic variation. This is a significant contribution, as it highlights the multifactorial nature of fungal developmental processes. The identification of distinct transcriptional programs and the involvement of transporter-related genes are particularly relevant findings. A brief addition of a conceptual model or further discussion on biological implications would strengthen the manuscript, but this is not essential. Overall, this is a solid and meaningful contribution.

 

1. The manuscript would benefit from a slightly stronger emphasis on the biological implications of the findings, particularly how altered transporter activity could mechanistically influence pileus development.

2. While the bioinformatic analyses are extensive, the inclusion of even limited experimental validation (e.g., qPCR of key hub genes or functional assays) would further strengthen the conclusions. However, I recognize this may be beyond the current scope.

3. The figures are clear and informative, but given the applied relevance of the study, the manuscript could benefit from adding a schematic model or graphical summary integrating the main findings into a conceptual framework.

4. It may be useful to briefly discuss how these findings could be leveraged in strain improvement or industrial cultivation, given the importance of L. edodes.

 

Author Response

We sincerely thank the reviewer for the positive and insightful evaluation of our work.

Comment 1. Biological implications (transporter mechanism)

Response: We appreciate this important suggestion. We have expanded the Discussion to provide a clearer biological interpretation of transporter-associated genes. Specifically, we now discuss how MFS and sugar transporters may influence pileus development through regulation of nutrient uptake, metabolite distribution, osmotic balance, and developmental coordination during fruiting body differentiation. 

Comment 2. Experimental validation (qPCR, functional assay).

Response: We agree that experimental validation would further strengthen the study. However, such analyses are beyond the scope of the present work. We have now explicitly acknowledged this limitation and highlighted future directions for validation (e.g., qPCR, gene functional studies) in the revised Discussion and Conclusion.

Comment 3. Conceptual model / graphical summary

Response: We appreciate this insightful suggestion. Rather than introducing an additional figure within the main text, we have incorporated a graphical abstract that presents the conceptual model of this study. This graphical summary integrates the major findings, including (i) the transporter- and regulator-centered transcriptional program associated with normal pileus development in Le_L, (ii) the ribosome-centered transcriptional state observed in Le_S, and (iii) the potential contribution of structural variation affecting transporter-related genes to the pileus-deficient phenotype. We believe that providing this model as a graphical abstract enhances clarity and accessibility while maintaining the logical flow of the manuscript.

Comment 4. Industrial/application relevance

Response: We have entirely revised the Conclusion to include the applied significance of our findings, particularly their potential use in strain selection, breeding strategies, and improving cultivation stability in L. edodes.

Reviewer 2 Report

The molecular basis of pileus morphogenesis in the mushroom Lentinula edodes was for the first time studied using a set of experimental and bioinformatic procedures. As a result, candidate factors and genes responsible for abnormal pileus development were identified.

Figure 2A: I see significant differences in RNA expression patterns between the three replicates, in contrast to the "high reproducibility of the RNA-seq data" stated in lines 239-241. Large differences between biological replicates are also visible in Figure 4A.

Figure 2E is puzzling, apparently not related to this study.

Author Response

We thank the reviewer for the careful reading and helpful comments.

Comment 1. Replicate variability (Figure 2A, 4A)

Response: We agree that the original wording overstated reproducibility. We have revised the text to more accurately describe the results as showing clear group-level separation with some within-group variation, rather than “tight clustering” or “high reproducibility.”

Comment 2. Figure 2E interpretation

Response: We appreciate this important point. We agree that the original KEGG pathway presentation could be misleading due to the inclusion of broad or non-specific pathway categories. We have revised Figure 2E to emphasize biologically relevant functional categories (e.g., ribosome, proteostasis), and we clarified the interpretation in the Results section.

Reviewer 3 Report

This study explores the molecular mechanisms responsible for differences in shiitake mushroom cap morphology. A bioinformatics analysis of the transcriptome of a normal, developed mushroom and a mushroom with abnormal morphology was conducted. The data were processed using a number of bioinformatics programs. It was shown that regulatory genes and proteins are largely responsible for morphological changes, rather than changes in the structure of the mushroom genome. The study is interesting, and the manuscript is well written. However, beyond minor technical comments, the question remains: what is the practical significance of this research beyond its obvious scientific interest? What should readers understand about transposing new knowledge for their own research, beyond the methodology of consistent application of a range of bioinformatics methods? Please discuss the applied aspect of the research in the Conclusions section.

I have just minor comments:

- Figure 2 needs to be made clearer.
- Part of Figure 4 is not visible.
- If publicly accessible websites were used, the website itself and the date it was accessed must be indicated. If commercially available software packages were used, the company must be identified.
- I believe a section on statistical data processing (Materials and Methods) is needed.

Author Response

We thank the reviewer for the constructive feedback.

Comment 1. Practical significance (Conclusion)

Response: We have revised the Conclusion to explicitly discuss the applied implications of our findings, including their relevance for strain improvement, breeding, and cultivation stability.

Comment 2. Figure clarity (Figure 2)

Response: We improved figure resolution, labeling, and layout for better readability.

Comment 3. Figure 4 visibility issue

Response: We corrected the layout and replaced the figure with a higher-resolution version.

Comment 4. Software / website / access date

Response: We revised the Methods and References sections to provide complete information on software tools, databases, and access dates where applicable.

Comment 5. Statistical analysis section

Response: We added a new Statistical Analysis subsection in Materials and Methods describing all statistical procedures used.

Round 2

Reviewer 2 Report

The molecular basis of pileus morphogenesis in the mushroom Lentinula edodes was for the first time studied using a set of experimental and bioinformatic procedures. As a result, candidate factors and genes responsible for abnormal pileus development were identified.

The text was corrected.