Dear Dr. Gao,

Thank you very much for submitting your manuscript "The protein segregase VCP/p97 promotes host antifungal defense via regulation of SYK activation" 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.

The authors addressed most of previous reviewers' editorial and experiment comments in this revised manuscript. The reviewers also agreed that the revised manuscript is much improved. Having said that, they still raised some concerns that need to be addressed in the second round of revision, as can be seen in the reviewers' comments. These include more quantitative measurement of VCP phosphorylation (reviewer 1), providing raw blotting data for Ig pulldown (reviewer 3), and several comments from reviewer 2. I think that the authors could reasonably address these questions.

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).

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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,

Yong-Sun Bahn, Ph.D.

Guest Editor

PLOS Pathogens

Michal Olszewski

Section Editor

PLOS Pathogens

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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The authors addressed most of previous reviewers' editorial and experiment comments in this revised manuscript. The reviewers also agreed that the revised manuscript is much improved. Having said that, they still raised some concerns that need to be addressed in the second round of revision, as can be seen in the reviewers' comments. These include more quantitative measurement of VCP phosphorylation (reviewer 1), providing raw blotting data for Ig pulldown (reviewer 3), and several comments from reviewer 2. I think that the authors could reasonably address these questions.

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: This is a revised manuscript and the authors responded to reviewers' point by providing additional experiments and adding more robust statistic analysis. Overall responses are acceptable. However, the phosphorylation of VCP upon dectin-1 signaling is not really convincing based on western blot experiment in fig 1. What's difference between no activation (0 zyme) vs activation (Zymo treatment)?

They need to provide clear, convincing evidence to make the case.

Reviewer #2: The revised submission has addressed most of my concerns and the quality has been improved. Impressive amount of work has been included. I have a few additional comments.

1. It is nice to have the ATPase activity-dead VCP mutant. Not sure whether such mouse mutant strain is available. To confirm the ATPase activity of VCP is required for fungal infectivity, in addition to using the NMS-872 inhibitor, testing the VCP (K542A) ATPase-dead mutant mouse strain would be more convincing. What is the rationale to use NMS-872 at 2 mg/Kg dosage?

2. Line 401, please cite a reference on the role of VCP as an ATPase in targeting and transporting ubiquitinated substrate for degradation.

3. Figure 2B. The image used for the ikBa signals is partially cut out and should use a better image with complete bands. Also comparing with the original blot (have two strong bands), the up band was cut out. Please explain what is the up band and why was it cut out?

4. Figure 3E. please add the label to the right panel.

5. Figure 6. In the model, the letters on the dectin-1 phosphorylation site are hard to see. May change the color of the letters.

Reviewer #3: The authors’ revisions, including toning down their language regarding the role of VCP in regulation of Dectin-1 signaling, quantification of experimental replicates, and new data have strongly improved the manuscript. This is an important study identifying a new regulator of CLR signaling. The authors have addressed our concerns well and their data now fit their conclusions.

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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: Further verification of phosphorylaion of VCP upon dectin-1 activation is needed.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

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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: (No Response)

Reviewer #2: (No Response)

Reviewer #3: (No Response)

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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 #2: No

Reviewer #3: No

Figure Files:

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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. Gao,

We are pleased to inform you that your manuscript ' The protein segregase VCP/p97 promotes host antifungal defense via regulation of SYK activation ' 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,

Yong-Sun Bahn, Ph.D.

Guest Editor

PLOS Pathogens

Michal Olszewski

Section Editor

PLOS Pathogens

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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

The two original reviewers appreciated that the authors address their concerns and comments nicely.

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 addressed the issues raised by the reviewers well.

Reviewer #2: The authors have addressed my concerns. This is a nice study with impressive amount of data.

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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: (No Response)

Reviewer #2: (No Response)

**********

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 #2: (No Response)

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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 #2: No