Dear Ping,

Thank you for submitting your manuscript entitled "Cuticular collagens mediate cross-kingdom predator-prey interactions between nematodes and trapping fungi" for consideration as a Research Article by PLOS Biology.

Your manuscript 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 external peer review.

IMPORTANT: As previously mentioned, we think this would be best considered as an Update Article, and the Academic Editor agreed. No re-formatting is required, but please select "Update Article" as the article type when you upload your additional metadata (see next paragraph).

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 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 Apr 25 2025 11:59PM.

If your manuscript has been previously peer-reviewed at another journal, PLOS Biology is willing to work with those reviews in order to avoid re-starting the process. Submission of the previous reviews is entirely optional and our ability to use them effectively will depend on the willingness of the previous journal to confirm the content of the reports and share the reviewer identities. Please note that we reserve the right to invite additional reviewers if we consider that additional/independent reviewers are needed, although we aim to avoid this as far as possible. In our experience, working with previous reviews does save time.

If you would like us to consider previous reviewer reports, please edit your cover letter to let us know and include the name of the journal where the work was previously considered and the manuscript ID it was given. In addition, please upload a response to the reviews as a 'Prior Peer Review' file type, which should include the reports in full and a point-by-point reply detailing how you have or plan to address the reviewers' concerns.

During the process of completing your manuscript submission, you will be invited to opt-in to posting your pre-review manuscript as a bioRxiv preprint. Visit http://journals.plos.org/plosbiology/s/preprints for full details. If you consent to posting your current manuscript as a preprint, please upload a single Preprint PDF.

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

Kind regards,

Roli

Roland Roberts, PhD

Senior Editor

PLOS Biology

rroberts@plos.org

Dear Ping,

Thank you for your patience while your manuscript "Cuticular collagens mediate cross-kingdom predator-prey interactions between nematodes and trapping fungi" was peer-reviewed at PLOS Biology. It has now been evaluated by the PLOS Biology editors, an Academic Editor with relevant expertise, and by three independent reviewers.

You'll see that reviewer #1 is very positive, but presents a list of questions, some of which require textual or presentational changes, but some of which imply additional analyses. Reviewer #2 is also positive, but suggests a number of experiments (using a reporter line, attempting rescue with a different collagen gene). Reviewer #3 also praises the study; most of his/her requests are presentational, but points 4 and 5 suggest additional analysis and experiment, respectively.

Based on the reviews and on our Academic Editor's assessment, we are likely to accept this manuscript for publication, provided you satisfactorily address the points raised by the reviewers (by textual and/or presentational changes only) and the following data and other policy-related requests.

IMPORTANT - please attend to the following:

a) I discussed the reviewers' comments with the Academic Editor. While they recognise that some of the experimental and analytical requests would indeed improve the paper, they said that these are not required to support the current claims, and so we would only require textual and/or presentational changes to address the concerns raised.

b) Please address my Data Policy requests below; specifically, we need you to supply the numerical values underlying Figs 1BC, 2ABCD, 3ABCDFG, 4, 5BCD, S2, S3, S4, S5, either as a supplementary data file or as a permanent DOI’d deposition.

c) 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

d) Please make any custom code available, either as a supplementary file or as part of your data deposition.

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 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 shortly.

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

Roland Roberts, PhD

Senior Editor

rroberts@plos.org

PLOS Biology

------------------------------------------------------------------------

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 1BC, 2ABCD, 3ABCDFG, 4, 5BCD, S2, S3, S4, S5. 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.

------------------------------------------------------------------------

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

------------------------------------------------------------------------

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).

------------------------------------------------------------------------

REVIEWERS' COMMENTS:

Reviewer #1:

Review of PLoS Biology

Organisms do not exist in isolation but in a complex ecological web with other organisms that involves predation, mutualism, and symbioses. Despite many examples of these highly nuanced and evolutionarily important relationships, little is known about the molecular basis underlying these interactions. One reason for this is because model systems - which have the genetic power to uncover molecular mechanisms - are often studied in lab settings outside their natural niches. One relationship that has captured much interest are worm-trapping fungi that capture comparatively large metazoans in predator-prey relationships. The present study is important because it exploits the powerful genetics of C. elegans to understand their vulnerabilities in this specific predator-prey relationship. A genetic screen was performed to look for predation-resistant mutants. This effort uncovered a transcription factor of the hormone receptor family. Further analysis revealed a new role of target adhesion proteins in predator escape, but not without a trade off in other physiologically relevant phenotypes. In addition to the uniqueness of the genetic screen, several other novel aspects of the paper include the ultrastructural analysis of the worm cuticle. In these ways, the study breaks new ground in an important and interesting area. Below follow some questions for the authors to consider:

1. Why does the hormone receptor normally regulate collagen gene expression? Do worms have the option of altering their surface properties?

2. "Furthermore, sequence analysis of natural C. elegans populations revealed no obvious loss-of-function variants in nhr-66, suggesting selective pressures exist that balance adhesion-mediated predation risk with physiological robustness." Were changes observed in the collagen genes?

3. It is clear the authors are striving to make the study clear and relevant. Nevertheless, going from the results to Figure 1B and 1C is difficult for the general reader to understand. What's a fosmid? What is the purpose of the red rectangle? What are all the gene names? The figure legend does not clear this up.

4. For clarification, how many total collagen genes are there in worms?

5. Figure 2B, the wild type and mutant do not look the same - there is a difference in granularity. What do the authors think underlies this difference?

6. Do the mutant worms have any motility defects? Do collagen overexpressing strains have altered motility?

7. Does purified collagen bind to the fungus?

8. Is cuticle left behind on the fungus after escape?

Reviewer #2:

This manuscript show very interesting results. To pick a few exciting points, they show that A. oligospora targets and kills the cuticle/collagen of the nematode; that nhr-66, which regulates the cuticle genes, was identified through forward genetics.

They also show that nhr-66 is an essential gene selected for survival in nature by showing that there is no significant natural variations defective in nhr-66 functions.

Overall, the manuscript will be appropriate for publication in PLOS Biology after a minor revision as suggested below. In addition, I felt that it would be interesting to examine natural variations of collagen genes controlled by nhr-66 to see if there is any selection during evolution.

Major points

- Figure 2: They describe that macroscopic cuticle structure has not been changed. Would it be possible to provide evidence for that, for example, by showing cuticle structures using translational reporter lines such as col-19::gfp in nhr-66 mutants?

- line 268: It would be nicer if they can show some other collagen genes that can rescue mutant phenotypes besides col-14.

- Other studies (for example, Nasrallah MA et al., 2023) seem to show that nhr-66 is a transcriptional repressor. It would be necessary to elaborate / explain their notion that nhr-66 is an activator.

Minor points

- line 148: Adding information on the insertion site STOP-IN cassette in Fig 1D would be helpful.

- line 163: Please revise the phrase 'the tissues key for cuticle synthesis' to 'the key tissues for cuticle synthesis'.

- line 194: It appears that only a single allele (yph413) was analyzed. Therefore, it would be better to revise the title from 'Mutations' to 'Mutation' to reflect this.

- Figure 2: Placing the Greek letters with the title of corresponding graphs would be easier for readers to comprehend.

- line 377: It is mentioned in discussion that nhr-49 mutant does not show trap resistance. However, relevant data are not present in the main text and figures. Please rephrase the sentence or show evidence.

Reviewer #3:

In this manuscript, the authors follow up on their prior study that identified a new family of fungal protein that mediate the trapping of prey nematodes. Using the fungus Arthrobotrys oligospora, they previously showed that the TEP genes encode proteins that facilitate the capture of C. elegans worms via a process that seemingly involved carbohydrate-mediated adhesion. Here, they employ a nifty genetic screen to identify C. elegans mutants that are capable of evading capture. Through detailed phenotypic characterization of the resulting nhr-66 mutants, the screen ultimately resulted in the demonstration that nematode collagens are likely adhesive targets that mediate capture by A. oligosperma. Although removing these targets confers resistance to capture, it comes with the associated cost of increased sensitivity to osmotic stress. Accordingly, this resistance mechanism is not observed in natural isolates. However, results from the study shed light on the initial adhesive interactions that mediate prey capture by A. oligosperma and identify strategies that could be used to engineer nematode-trapping fungi (NTF) as biocontrol agents in the field.

Overall, this is a well-conceived and performed study that provides significant new insight into interactions between worms and NTF. The genetic, phenotypic, and molecular analysis are all first rate, such that the author's conclusions are sound and well-justified. Overall, I only have the following minor suggestions or concerns;

1. Fig. 1A would be much easier to interpret if the authors used a darker colour scheme to represent nematodes and the traps.

2. The plots presented in Figs. 2B and 2C are very difficult to read given the light colouring..

3. Line 288 and Fig 4A. In addition to A. musiformis, the protective affect of the nhr-66 mutation on A. amerospora is also reduced, although this occurs in a high background of general resistance to this species in wild type. This might be worth commenting on as well.

4. Fig. 1B and Line 221. Was GO enrichment performed on the 168 up-regulated genes? Were any patterns observed?

5. Fig. 5B and Line 312. Were the natural variants within nhr-66 also tested for their effects on hypo-osmotoic stress? One would expect there to be a much lower frequency of ruptured worms given the negligible impact on escape.

Dear Ping,

Thank you for the submission of your revised Update Article "Cuticular collagens mediate cross-kingdom predator-prey interactions between trapping fungi and nematodes" for publication in PLOS Biology. On behalf of my colleagues and the Academic Editor, Aaron Mitchell, I'm 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.

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, 

Roli

Roland G Roberts, PhD, PhD

Senior Editor

PLOS Biology

rroberts@plos.org