Dear Dr Soldati-Favre,

Thank you for submitting your Portable Peer Review manuscript entitled "RNG2 tethers the conoid to the apical polar ring in Toxoplasma gondii: a key mechanism controlling parasite motility and invasion" for consideration as a Research Article by PLOS Biology.

Your manuscript (and the PLOS Pathogens reviews and your responses) has now been evaluated by the PLOS Biology editorial staff, as well as by an academic editor with relevant expertise, and I'm writing to let you know that we would like to send your submission out for further review.

However, before we can send your manuscript to reviewers, we need you to complete your submission by providing the metadata that is required for full assessment. To this end, please login to Editorial Manager where you will find the paper in the 'Submissions Needing Revisions' folder on your homepage. Please click 'Revise Submission' from the Action Links and complete all additional questions in the submission questionnaire.

Once your full submission is complete, your paper will undergo a series of checks in preparation for peer review. After your manuscript has passed the checks it will be sent out for re-review. To provide the metadata for your submission, please Login to Editorial Manager (https://www.editorialmanager.com/pbiology) within two working days, i.e. by Sep 22 2025 11:59PM.

Feel free to email us at plosbiology@plos.org if you have any queries relating to your submission.

Kind regards,

Roli Roberts

Roland G Roberts PhD

Senior Editor

PLOS Biology

rroberts@plos.org

on behalf of

Melissa Vazquez Hernandez, Ph.D.

Associate Editor

PLOS Biology

mvazquezhernandez@plos.org

Dear Dr Soldati-Favre,

Thank you for your patience while we considered your revised manuscript "RNG2 tethers the conoid to the apical polar ring in Toxoplasma gondii: a key mechanism controlling parasite motility and invasion" for publication as a Research Article at PLOS Biology. I'm handling your paper temporarily while my colleague Dr Vazquez-Hernandez is out of the office. This revised version of your manuscript has been evaluated by the PLOS Biology editors, the Academic Editor, and one of the original reviewers.

Based on the review (see the foot of this email) and on our Academic Editor's assessment of your revision, we are likely to accept this manuscript for publication, provided you satisfactorily address the following data and other policy-related requests.

a) We like to avoid punctuation in Titles. Please change it to: "RNG2 tethers the conoid to the apical polar ring in Toxoplasma gondii to enable parasite motility and invasion"

b) Please supply the original uncropped blots or gels for the appropriate Figure panels (Figs 2BCE, 3BC, 5C, 6BI, 7BJ, S1B, S2A, S3B, S4B).

c) Please address my Data Policy requests below; specifically, we need you to supply the numerical values underlying Figs 2D, 3E, 4DF, 5E, 6DEFJKL, 7DEFHL, S1AD, S3CD, S4ACE, either as a supplementary data file or as a permanent DOI’d deposition.

d) Please cite the location of the data clearly in all relevant main and supplementary Figure legends, e.g. “The data underlying this Figure can be found in S1 Data” or “The data underlying this Figure can be found in https://zenodo.org/records/XXXXXXXX

e) Please include the URLs of your funders in the Financial Disclosure statement.

As you address these items, please take this last chance to 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 cover letter that accompanies your revised manuscript.

In addition to these revisions, you may 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 shortly. If you do not receive a separate email within a few days, please assume that checks have been completed, and no additional changes are required.

We expect to receive your revised manuscript within two weeks.

To submit your revision, please go to https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' to find your submission record. Your revised submission must include the following:

- a cover letter that should detail your responses to any editorial requests, if applicable, and whether changes have been made to the reference list

- a Response to Reviewers file that provides a detailed response to the reviewers' comments (if applicable, if not applicable please do not delete your existing 'Response to Reviewers' file.)

- a track-changes file indicating any changes that you have made to the manuscript.

NOTE: If Supporting Information files are included with your article, note that these are not copyedited and will be published as they are submitted. Please ensure that these files are legible and of high quality (at least 300 dpi) in an easily accessible file format. For this reason, please be aware that any references listed in an SI file will not be indexed. For more information, see our Supporting Information guidelines:

https://journals.plos.org/plosbiology/s/supporting-information

*Published Peer Review History*

Please note that 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. Please see here for more details:

https://plos.org/published-peer-review-history/

*Press*

Should you, your institution's press office or the journal office choose to press release your paper, please ensure you have opted out of Early Article Posting on the submission form. We ask that you notify us as soon as possible if you or your institution is planning to press release the article.

*Protocols deposition*

To enhance the reproducibility of your results, we recommend that if applicable 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 for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Please do not hesitate to contact me should you have any questions.

Sincerely,

Roli Roberts

Roland G Roberts PhD

Senior Editor

rroberts@plos.org

on behalf of

Melissa Vazquez Hernandez, Ph.D.

Associate Editor

mvazquezhernandez@plos.org

PLOS Biology

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DATA POLICY:

You may be aware of the PLOS Data Policy, which requires that all data be made available without restriction: http://journals.plos.org/plosbiology/s/data-availability. For more information, please also see this editorial: http://dx.doi.org/10.1371/journal.pbio.1001797

Note that we do not require all raw data. Rather, we ask that all individual quantitative observations that underlie the data summarized in the figures and results of your paper be made available in one of the following forms:

1) Supplementary files (e.g., excel). Please ensure that all data files are uploaded as 'Supporting Information' and are invariably referred to (in the manuscript, figure legends, and the Description field when uploading your files) using the following format verbatim: S1 Data, S2 Data, etc. Multiple panels of a single or even several figures can be included as multiple sheets in one excel file that is saved using exactly the following convention: S1_Data.xlsx (using an underscore).

2) Deposition in a publicly available repository. Please also provide the accession code or a reviewer link so that we may view your data before publication.

Regardless of the method selected, please ensure that you provide the individual numerical values that underlie the summary data displayed in the following figure panels as they are essential for readers to assess your analysis and to reproduce it: Figs 2D, 3E, 4DF, 5E, 6DEFJKL, 7DEFHL, S1AD, S3CD, S4ACE. NOTE: the numerical data provided should include all replicates AND the way in which the plotted mean and errors were derived (it should not present only the mean/average values).

IMPORTANT: Please also ensure that figure legends in your manuscript include information on where the underlying data can be found, and ensure your supplemental data file/s has a legend.

Please ensure that your Data Statement in the submission system accurately describes where your data can be found.

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CODE POLICY

Per journal policy, if you have generated any custom code during the course of this investigation, please make it available without restrictions. Please ensure that the code is sufficiently well documented and reusable, and that your Data Statement in the Editorial Manager submission system accurately describes where your code can be found.

Please note that we cannot accept sole deposition of code in GitHub, as this could be changed after publication. However, you can archive this version of your publicly available GitHub code to Zenodo. Once you do this, it will generate a DOI number, which you will need to provide in the Data Accessibility Statement (you are welcome to also provide the GitHub access information). See the process for doing this here: https://docs.github.com/en/repositories/archiving-a-github-repository/referencing-and-citing-content

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BLOT AND GEL REPORTING REQUIREMENTS:

We require the original, uncropped and minimally adjusted images supporting all blot and gel results (Figs 2BCE, 3BC, 5C, 6BI, 7BJ, S1B, S2A, S3B, S4B) reported in an article's figures or Supporting Information files. We will require these files before a manuscript can be accepted so please prepare and upload them now. Please carefully read our guidelines for how to prepare and upload this data: https://journals.plos.org/plosbiology/s/figures#loc-blot-and-gel-reporting-requirements

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DATA NOT SHOWN?

- Please note that per journal policy, we do not allow the mention of "data not shown", "personal communication", "manuscript in preparation" or other references to data that is not publicly available or contained within this manuscript. Please either remove mention of these data or provide figures presenting the results and the data underlying the figure(s).

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REVIEWER'S COMMENTS:

Reviewer #2:

The revision of the article by Hasse et al. represents a significantly improved version of the manuscript, now presenting conclusions that are rigorously supported by two sets of new experiments. In particular, the authors have abandoned their initial model, which proposed that proteolytic processing and oligomerization of RNG2 could provide the flexibility required to span the distance from the APR to the conoid. The revised conclusion—that RNG2 is sufficiently long to bridge this intervening space—does not diminish the scope or impact of the study. My second major criticism, regarding microneme secretion, has also been carefully addressed, and the authors now clarify the primary phenotype associated with RNG2. Furthermore, the inclusion of cryo-ET analysis of the RNG2 mutant adds valuable structural information on RNG2 defect in apical complex. Finally, the authors provide a well-reasoned explanation for the apparent discrepancy between the 50% defect in rhoptry secretion and the detachment of rhoptries from the apex observed in nearly all mutants.

Dear Dominique,

Thank you for the submission of your revised Research Article "RNG2 tethers the conoid to the apical polar ring in Toxoplasma gondii to enable parasite motility and invasion" for publication in PLOS Biology. On behalf of my colleagues and the Academic Editor, Kami Kim, I am pleased to say that we can in principle accept your manuscript for publication, provided you address any remaining formatting and reporting issues. These will be detailed in an email you should receive within 2-3 business days from our colleagues in the journal operations team; no action is required from you until then. Please note that we will not be able to formally accept your manuscript and schedule it for publication until you have completed any requested changes.

*Just a quick note. I took a quick look through the manuscript and I noticed that in some figures the legends seemed to have the description letter wrong. For example on Figure 6. Just letting you know for when you have to do the checks.

Please take a minute to log into Editorial Manager at http://www.editorialmanager.com/pbiology/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production process.

PRESS

We frequently collaborate with press offices. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximise its impact. If the press office is planning to promote your findings, we would be grateful if they could coordinate with biologypress@plos.org. If you have previously opted in to the early version process, we ask that you notify us immediately of any press plans so that we may opt out on your behalf.

We also ask that you take this opportunity to read our Embargo Policy regarding the discussion, promotion and media coverage of work that is yet to be published by PLOS. As your manuscript is not yet published, it is bound by the conditions of our Embargo Policy. Please be aware that this policy is in place both to ensure that any press coverage of your article is fully substantiated and to provide a direct link between such coverage and the published work. For full details of our Embargo Policy, please visit http://www.plos.org/about/media-inquiries/embargo-policy/.

Thank you again for choosing PLOS Biology for publication and supporting Open Access publishing. We look forward to publishing your study. 

Sincerely, 

Melissa

Melissa Vazquez Hernandez, Ph.D., Ph.D.

Associate Editor

PLOS Biology

mvazquezhernandez@plos.org