Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study presents valuable insights into optimizing Bacillus subtilis for recombinant peptide production and seed delivery applications. The experimental design is rigorous and addresses important agricultural biotechnology challenges. While the findings on spore-based seed treatments are particularly impactful, several aspects of the presentation and interpretation require strengthening to maximize the manuscript's contribution.

Major concerns.

1. The introduction section should be refinement. The rationale for focusing on spore-based seed treatments requires deeper justification. While spore survival advantages are mentioned, the argument would benefit from specific references to literature on spore resilience during commercial seed storage conditions. The practical advantages of seed treatments over root drenches (e.g., reduced labor, scalability) should be explicitly highlighted to strengthen the applied context. Strengthen the spore-treatment rationale with targeted references and explicitly contrast seed treatments with alternative delivery methods.
2. The central finding regarding stationary phase yield superiority (Fig 1C) deserves more prominent emphasis in the narrative. While higher cell numbers compensate for lower per-cell expression, the manuscript doesn't sufficiently explore why this trade-off occurs at the molecular level.
3. Regarding bioactivity assessments (Fig 4), testing spore treatments only immediately after application overlooks their key practical advantage: longevity. The absence of bioactivity data after storage (e.g., 30-90 days) represents a significant gap, as it doesn't demonstrate whether the growth-promoting effects persist through realistic storage periods.
4. The SEM images (Fig 3) provide qualitative support but lack quantitative analysis. Including cell density measurements per seed surface area would significantly strengthen the adhesion claims.

5. The details of endospore preparation is not clear enough. Please specify the detail.

6. How to separate vegetative cells and spores？When added treatment, how many vegetative cells and spores?

Minor concerns. 

1. Only one PGPR used, it is not necessary to define.
2. Fig. 3: How about after 3 months? 
3. Fig. 4: Clarify whether "B.+GmPEP3" includes both spore and vegetative cell treatments.
4. Add bioactivity data for spores after storage periods matching the CFU experiments.
5. Include quantitative analysis of SEM images.
6. Provide mechanistic interpretation of the temperature/media interactions.

Author Response

Dear Reviewer 1,

Thank you for your helpful feedback and suggestions for revision.  We have carefully revised the manuscript to address your comments.  Please see the attached Word document responding in detail and listing the edits we have made, which are also in marked in "track changes" mode in the revised manuscript.

Thanks again for your consideration and assistance in making this paper the best it can be!

Best, 

Fiona Goggin

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

In this study, the authors investigated the effect of temperature, medium composition, and growth phase on the ectopic expression of a Plant Elicitor Peptide from soybean (GmPEP3) in Bacillus subtilis. Additionally, soybean seeds were treated with spores and vegetative cells, both with and without carboxymethyl cellulose, to enhance the viability of B. subtilis during seed application. The study is well-designed, with experiments that logically follow one another. However, several issues regarding the structure and organization of the manuscript need to be addressed before publication.

Introduction:

The introduction is currently divided into subsections with subheadings, which is unnecessary. It is recommended to remove all subheadings and present the introduction as a continuous narrative.

The objectives of the study are stated multiple times throughout the introduction. Delete Lines 44–46 and 85–88, as the aims are already clearly summarized at the end of the introduction.

The results should not be introduced in the Introduction section. Delete Lines 121–125 and 130–135, which describe findings that belong in the Results section.

Results:

Remove subheading "2.1.1. Influence of Growth Phase and Growth Conditions on the Number of Viable Cells and Transcripts Per Cell" (Lines 139–140)

The sentence in Lines 164–165 “This is consistent with previous reports that 2xYT [18] and growth at 30 °C [30] can promote high ectopic expression in bacteria.” should be either removed or moved to the Discussion section, as it represents interpretation rather than results.

The figure legends for Figures 1 and 2 are too long and include experimental details and result interpretation. These should be shortened to include only a brief description of what is shown in the figure, an explanation of all symbols and labels and the statistical tests used

In Figure 1, the numbers on the Y-axis should be formatted in exponential notation (e.g., 1×10⁶ instead of 1000000) to improve readability

Discussion:

The Discussion would benefit from a more in-depth comparison with similar studies, particularly those involving heterologous peptide expression in bacterial systems and seed treatment efficacy using B. subtilis.

 

Author Response

Dear Reviewer 2,

Thank you for your helpful feedback and suggestions for revision.  We have carefully revised the manuscript to address your comments.  Please see the attached Word document responding in detail and listing the edits we have made, which are also in marked in "track changes" mode in the revised manuscript.

Thanks again for your consideration and assistance in making this paper the best it can be!

Best, 

Fiona Goggin

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Improved. The Figures need further beautification to meet the publication standards.

Author Response

Comments 1: The English is fine and does not require any improvement.  Selected "Yes" for all six questions about the whether the manuscript's quality is sufficient.

Reply 1: We appreciate Reviewer 1's positive feedback.

Comments 2: "Improved. The Figures need further beautification to meet the publication standards."

Reply 2: We appreciate the reviewer's assessment that the manuscript revisions were beneficial.  To address the comment of beautification, we have 1) adjusted the colors of Figure 1 to make them bolder, which we believe makes the figure easier to read; and 2) we have adjusted the font sizes and data point sizes across the figures for consistency, attractiveness, and readability. 

 

Reviewer 2 Report

Comments and Suggestions for Authors

No comments

Author Response

We thank Reviewer 2 very much for their assessment that the English of this paper is fine, and that the manuscript meets all six criteria outlined in the journal's questions.