Dear Dr Akimoto-Tomiyama,

Thank you very much for submitting your manuscript "BglaTNB6, a tailocin produced by a plant-associated nonpathogenic bacterium, prevents rice seed-borne bacterial diseases" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Three reviewers have evaluated the manuscript and all find the experimental data and results clear and compelling. Each reviewer has suggested a variety of editorial changes that will enhance the clarity and readability of the manuscript, but I don't think that new sets of data or experiments are necessary for this submission.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

David A Baltrus

Guest Editor

PLOS Pathogens

Bart Thomma

Section Editor

PLOS Pathogens

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

Three reviewers have evaluated the manuscript and all find the experimental data and results clear and compelling. Each reviewer has suggested a variety of editorial changes that will enhance the clarity and readability of the manuscript, but I don't think that new sets of data or experiments are necessary for this submission.

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: The authors have demonstrated convincingly that B. gladioli strain NB6 releases a tailocin able to inhibit the growth of strains of B glumae and B plantarii.

Reviewer #2: This manuscript reports that a nonpathogenic stain of Burkholderia gladioli, NB6, shows a strong disease suppression activity against seedling blight caused by B. glumae and B. plantarii, and that a new tailocin (a phage-tail-like bacteriocin) produced by B. gladioli NB6, BglaTNB6 (B6 hereafter), is a key antibacterial element of this antagonistic strain.

Overall, this study was performed very well from the characterization of disease suppression activity by the cell suspension and the cell-free filtrate of B. gladioli NB6 to the identification of the tailocin B6 and demonstration of its direct antibacterial activity against strains of Burkholderia spp. including B. glumae and B. plantarii. Manuscript writing and data presentation were also nicely done, describing each step of molecular genetics and biochemistry experiments very well. I think this study brings some useful information about an important mechanism of biological control against bacterial pathogens.

Reviewer #3: This manuscript describes the isolation and characterization of a B. gladioli strain (NB6), which was isolated from a genetically resistant line of rice (Nona Bokra) and is able to suppress multiple Burkholderia pathogens of rice. In general this paper does a thorough job of establishing the link between the tailocin of NB6 and the disease suppressing capability of this strain. This paper makes an important contribution to the field, however, I have a couple of concerns about the manuscript in its current form.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: Although not the main focus of the paper they have also alluded to the host genetic control and recruitment of appropriate Burkholderia biocontrol strains as mediating Nona Bokra’s tolerance to these rice pathogens. On this point the evidence is both circumstantial and unconvincing. The variation in bacterial diversity in different cultivars is interesting although there are more changes than just Burkholderia (which counters claims on Line 396) and this also ignores that these are japonica vs indica cultivars. It would be helpful to also look at the Bleiyo microbiome. Based on the authors hypothesis, Bleiyo should lack enrichment for Burkholderia. Also, while testing in rice seedlings is interesting, the authors should conduct in vitro tests for Bg and Bp viability after being mixed with NB6, CF and tailocin using the same conditions for prepping inoculum. What is the amount of time between mixing and seed vacuum infiltration? I'm curious about the M17 transposon mutant. Were you actively seeking mutants with reduced protein secretion? EM analysis should also be conducted of the DFII and DFII complement lines for loss and restoration of tailocin-like structures. Also while BglaTNB6 makes strong contributions to restricting B. plantarii growth it is clearly not “essential”.

Reviewer #2: No major issues found

Reviewer #3: -In general, I found the manuscript reasonably easy to read, however there were a number of sentences in the discussion that I found hard to interpret (I’ve indicated each in minor comments). In my opinion, these sentences need to be addressed.

-Although the authors acknowledge the data showing that there are at least two systems operating to suppress disease (one contact-independent tailocin and another, likely contact dependent system), this is not really addressed at all in the discussion. I think this is a more important point to discuss its implications for disease suppression and if there are other examples in plants (or elsewhere), where a biocontrol strain has multiple systems that contribute to biocontrol.

-There should be more explicit descriptions of how genetic data is confirmed. It looks as though multiple deletion mutants were used in Figs 2, 3, and 5 as a way to confirm that the phenotypes weren’t related to auxiliary mutations? However, I don’t believe this is stated clearly in the text (either in methods or results section). Additionally, the methods section contains language indicating that a complement strain of the FII mutant was generated. I don’t recall seeing this strain in the results (but I might have missed it)?

-L236 – This statement indicates that the capsid proteins are not present in the predicted prophage regions, however, table S3 indicates that this progaphe region was scored as intact, which I would think would be odd if the capsid genes are missing. This discrepancy should be addressed.

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: L63: Using a mutant that secretes proteins in low abundance

L111: why would high specificity limit emergence of tolerance in the targeted organism? There are clear examples of mutations that allow evasion of tailocins

L114: citation 19 "The sensitivity of phages to environmental stressors such as UV irradiation and soil (type, pH, moisture, organic matter content) is still an obstacle to phage therapy on plants" Tailocins lack nucleic acid and so should be less sensitive to UV

L128: I see no mention of this pathogen resistance correlating with biocontrol strains in Truyens et al The Upreti and Thomas seems very limited and circumstantial.

L134: Upload the 16S sequences to a public repository

Fig 1D: Relative bacterial population data should be log transformed. Which strains of Bg and Bp were used in inoculation tests?

L205: inhibitory effect on seedling growth

L227: encoded by a prophage-like region

L347: There’s a noticeable shift in writing quality in the first paragraph of the discussion (for instance, distinct feather). Also, why is it unexpected to have a tailocin region in the genome. Where else would it be?

L478: Which strains of Bg and Bp were used in inoculation tests?

Reviewer #2: 1. To distinguish from B. plantarii, ‘Bp’ indicated for base pairs should be ‘bp’ (e.g. lines 185 and 539)

2. Line 269-315: The authors presented a TEM image of the tailocin from the CF fraction of B. gladioli NB6. To claim that the phage tail-like structure is the tailocin B6, the authors should mention or present data that no such structure was observed in the CF of the FII or region1-deleted mutant.

3. It is not clear why the authors mentioned ‘as expected’ in the sentence on line 322. How did they expect that the cell suspension of the FII-deficient mutant would also exhibit reduced protective activity unlike Bg, with the observation that B6 was effective to both Bg and Bp?

4. Line 334: The sentence is for Bp, not for Bg because B6 is not required for the activity of the NB6 cells. The sentence should be modified, reflecting this fact.

5. Please refine the sentence on lines 350 – 353.

6. Line 396: Please provide more information of the ‘M gene’ with one or two sentences.

7. Line 482: Please describe more detail about the vacuum inoculation method, such as duration of vacuum, psi, drying process before planting, etc.

8. Lines 541 and 547: Please describe how the bacterial cells were transformed. Heat shock, electroporation, or conjugation?

Reviewer #3: L43-44 - This sentence should be reworded since species (not strains) are listed.

L49-50 - I don’t think this sentence logically flows form the preceeding sentences, since it is not clear/established that disease suppression is connected to the competitive fitness of NB6.

L60 – Nona Bokra was described as resistant in the abstract, but as tolerant in this line. These (and all other designations in the manuscript) should be harmonized.

L91-94 – This sentence seems to go in a direction not relevant to this manuscript, I think this can be removed.

L135 – Was this strain only tested agains Nipponbare? Or was it tested for pathogenicity toward other rice cultivars?

L135 – 137 - This statement seems to require the reader to compare the number of isolates from table S1 to ref. 12, which makes it unnecessarily complicated to review this statement. This should be more supported directly in this text.

L181 – 182 – How does the 60% saturation fraction confirm proteinaceous identity? Can the authors cite something to support this statement?

L226 – For this line and all similar lines related to the other figures, are the results of one representative experiment shown or are the graphs representative of all of the combined data?

L234 – Please specify what is meant by “homologies” – nt or aa identity/similarity?

L241 – What genes specifically were deleted? Do these numbers refer to the locus tags? Or some other gene identifier?

L250 – Why compare region 1 and 2 in this manner? Do the authors think these two regions are evolutionarily related?

L293-294 – This sentence seems unwarranted given the data. It might be true, but I don’t think there is sufficient data to conclude this.

L322 – itals deltaFII

L336 – This figure heading should be revised, since the data shows that the BglaTNB6 contributes to biocontrol activity (but is not essential, given the other contact dependent system)

L352 – I don’t understand this sentence

L353 – I don’t understand what is meant by “distinct feather”.

L357-358 – This sentence doesn’t make sense to me since tailocins are by definition phage genomes that have inserted into the bacterial genome. What makes this “unexpectedly”?

L358-359 – I don’t get the logic of this clause.

L361-363 – What is “it” in this sentence?

L383 – What tailocin loci don’t originate from prophages that would necessitate “some” for this sentence.

L386-388 – This sentence seems out of place for what is being reported, I suggest removing.

L414 – How does this figure confirm this statement?

L435 – It should be indicated that Rhizoviticin is a tailocin.

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Jong Hyun Ham

Reviewer #3: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

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Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Dear Dr Akimoto-Tomiyama,

Thank you very much for submitting your manuscript "BglaTNB6, a tailocin produced by a plant-associated nonpathogenic bacterium, prevents rice seed-borne bacterial diseases" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Thank you for your resubmission. All reviewers now agree that the revised manuscript is nearly acceptable for publication. I would just like to see a bit more clear and direct language concerning construction of multiple mutants incorporated into the manuscript as the reviewer suggests.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

David A Baltrus

Guest Editor

PLOS Pathogens

Bart Thomma

Section Editor

PLOS Pathogens

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

Thank you for your resubmission. All reviewers now agree that the revised manuscript is nearly acceptable for publication. I would just like to see a bit more clearl and direct language concerning construction of multiple mutants incorporated into the manuscript as the reviewer suggests.

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: My concerns have been addressed

Reviewer #3: This paper makes an important contribution to the field of biological controls, particularly tailocin-mediated biological control of an important bacterial rice pathogen. The authors have done a reasonably good job of addressing my previous comments.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: (No Response)

Reviewer #3: None

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: (No Response)

Reviewer #3: It would still be good for the authors to state explicitly in the text (either figure legends, results section, or methods section) that the multiple mutant isolates shown in figures 2, 3, and 5 are all included to confirm that the phenotype of the mutants is related to the deletion that was introduced, and not a spurious mutation that was picked up during the mutant generation process. I think this is why multiple mutant isolates are included in the figures, but it isn't actually confirmed in any text.

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #3: Yes: Kevin L. Hockett

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Dear Dr Akimoto-Tomiyama,

We are pleased to inform you that your manuscript 'BglaTNB6, a tailocin produced by a plant-associated nonpathogenic bacterium, prevents rice seed-borne bacterial diseases' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

David A Baltrus

Guest Editor

PLOS Pathogens

Bart Thomma

Section Editor

PLOS Pathogens

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

Thank you for your resubmission. I think that all of the reviewer's comments have now been adequately addressed.

Reviewer Comments (if any, and for reference):