Transfer Alert

This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present.

Dear Dr Tang,

Thank you very much for submitting your Research Article entitled 'Phosphatidylcholine mediates the crosstalk between ER and cytosolic stress response pathways' to PLOS Genetics.

The manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the current manuscript. Based on the reviews, we will not be able to accept this version of the manuscript, but we would be willing to review a much-revised version. We cannot, of course, promise publication at that time.

Should you decide to revise the manuscript for further consideration here, your revisions should address the specific points made by each reviewer. We will also require a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

If you decide to revise the manuscript for further consideration at PLOS Genetics, please aim to resubmit within the next 60 days, unless it will take extra time to address the concerns of the reviewers, in which case we would appreciate an expected resubmission date by email to plosgenetics@plos.org.

If present, accompanying reviewer attachments are included with this email; please notify the journal office if any appear to be missing. They will also be available for download from the link below. You can use this link to log into the system when you are ready to submit a revised version, having first consulted our Submission Checklist.

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. For instructions see our guidelines.

Please be aware that our data availability policy requires that all numerical data underlying graphs or summary statistics are included with the submission, and you will need to provide this upon resubmission if not already present. In addition, we do not permit the inclusion of phrases such as "data not shown" or "unpublished results" in manuscripts. All points should be backed up by data provided with the submission.

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool.  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.

PLOS has incorporated Similarity Check, powered by iThenticate, into its journal-wide submission system in order to screen submitted content for originality before publication. Each PLOS journal undertakes screening on a proportion of submitted articles. You will be contacted if needed following the screening process.

To resubmit, use the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.

[LINK]

We are sorry that we cannot be more positive about your manuscript at this stage. Please do not hesitate to contact us if you have any concerns or questions.

Yours sincerely,

Coleen T. Murphy

Associate Editor

PLOS Genetics

Gregory P. Copenhaver

Editor-in-Chief

PLOS Genetics

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: In this study, He et al. identify that depletion of the let-607 transcription factor leads to activation of the stress-related transcription factor DAF-16, resulting in enhanced cytotoxic protection and lifespan extension. They term this induction of DAF-16 activity as the ER-to-cytosol stress pathways communication (ECSPC), based on the assumption that LET-607 is the C. elegans CREBH homolog, embedded in the ER membrane. They further show that let-607 depletion alters fatty acid metabolism, and specifically reduced the levels of unsaturated PC, which turned out to be important for DAF-16 activation under these conditions. The authors identified additional genes required for DAF-16 activation by let-607 depletion including the IP3 receptor itr-1, which releases Calcium stores from the ER to the cytoplasm and the calcium-dependent kinase PKC-2.

I find that the beneficial effects of let-607 inactivation on lifespan and stress resistance, the associated activation of DAF-16, and the alterations in lipid metabolism are all well established. Specifically, the regulation of DAF-16 by unsaturated PC is novel and of wide-interest. Based on these, I find this work interesting and novel.

That being said, the main take-home message conveyed by the authors is that this is a pathway that links between the cytosolic and the ER stress responses. Nevertheless, the connection to the ER is mainly based on the finding that its transcript levels rise upon ER stress and the annotation of let-607 as the C. elegans ortholog of CREBH (whose connection to the ER is well established). However, this statement is based on homology spanning only 19% of the protein, corresponding to the b-leucine zipper domain. I could not find any indication for the presence of a putative transmembrane domain in the LET-607 protein. Such a transmembrane domain would be essential for anchoring the protein to the ER. I understand that let-607 has been referred to as the C. elegans ortholog of CREBH in previous publications, but this still needs to be established, especially since this is the “major player” in this work. Thus, the authors should investigate the intercellular localization of LET-607. Is it indeed embedded in the ER or is it cytosolic/nuclear? If this is done by tagging the protein, be careful not to perturb any putative signal peptide. If this experiment does not demonstrate ER localization – the paper should be re-written accordingly. To clarify – this experiment is needed and should be included in the final manuscript whatever the result may be – to confirm the ER association of this protein or to prevent further confusion.

Additional major comments:

1) Epistatic analysis demonstrated that let-607 and germline depletion might act in the same or similar pathway as the lifespan effects were not additive. Likewise, both treatments lead to nuclear localization of DAF-16 specifically in the intestine. The question is – are these similar pathways that converge downstream, or does let-607 activate the germline pathway? This should be addressed in 2 ways: 1) What is the state of the germline in let-607 RNAi treated animals? 2) The longevity of glp-1 animals depends on unique genes such as daf-9, daf-12, kri-1, tcer-1 etc. Are these genes required for lifespan extension by let-607? Please do these experiments using mutants and let-607 RNAi (not double RNAis).

2) Does sms-5 RNAi/PC(18:1N9) supplementation interfere with other daf-16 activating pathways such as daf-2,glp-1, oxidative stress, heat shock?

3) Some of the experiments are done with multiple RNAis. This is especially problematic for epistasis analysis. These should be repeated using mutants when possible, or to the very least with a comparison of RNAi efficiencies.

Additional comments:

Page 8 – “LET-607, like its mammalian ortholog, could also be activated upon ER stress18” – activation has not been shown here, only increased transcript levels have been demonstrated.

Page 8 – “Gene functional classification analysis revealed that the LET-607-upregulated genes were enriched in stress responses” - I am not sure if stress responses are the best definition here. The explicit stress response GO categories did not show up in this analysis. Please word this more carefully.

Page 10 – “as RNAis of none of the ER UPR genes (ire-1, pek-1 and atf-6) had any effects on MTL-1::GFP expression” – negative result. Better done with mutants rather than RNAi.

Page 10 – “We compared the let-607 RNAi-induced genes with the DAF-16 class I targets, and found that 74 genes upregulated by let-607 RNAi (13.6%) belonged to the DAF-16 class I targets, which was significantly greater than that would be expected by chance7 (Figure S2E), further supporting that let-607 RNAi regulates the activity of DAF-16. ” Please comment whether these genes also contain LET-607 binding sites.

Figure 2 – number of animals scored is not the number of biological repeats.

A Summary model would be helpful

Reviewer #2: This manuscript by He et al. describes heretofore unappreciated genetic pathway connecting suppression of the ER stress factor CREBH/LET-607 to activation of a FoxO-dependent stress resistance pathway mediated by reduction in phosphatidylcholine containing fatty acids. The authors term this pathway the ER-to-cytosol stress pathways communication (ECSPC). The survival data are generally robustly gathered and I appreciate the inclusion of ~4 biological replicates for each of the conditions tested. Some of the mechanistic work based upon simultaneous knockdown of multiple genes at once with RNAi require more rigorous proof. Additionally, stronger genetic evidence for the involvement of LET-607 and factors previously reported to act in a pathway with LET-607 should be provided. I am generally positive on the novelty of the findings, but the work requires more rigor to substantiate a solid enough mechanism to garner publication in PLoS Genetics.

Although intelligible, the manuscript is written in broken English and absolutely requires proofreading by a native English speaker. There are too many errors to enumerate here.

There is no mention of significance for the calculated enrichment score and what the comparator group is for the analysis conducted in Figure 1B. Also, I assume that what the authors are referring to in Fig 1B when they say “LET-607-upregulated genes” is the set of genes upregulated when LET-607 is knocked down by RNAi. The manuscript would be well served if this was made more clear.

Are there other genes that activate the ECSPC in concert with CREBH, including previously reported partners/activators in CREBH/LET-607 activity BMAL1/AHA-1, GSK3b/GSK-3, or PPARalpha/NHR-49? Does genetic inactivation of the S1P/S2P also activate the ECSPC?

Does the nuclear form of CREBH/LET-607 act as a dominant suppressor of the ECSPC?

In addition to “n”, the tests used to calculate significance should be indicated in the main figure legends (for example but not limited to Figs. 2B, 2C), as well as significance for survival data (even if they are indicated with repeats in Table S2).

It is possible that the doses of PC(16:0) and PC(18:0) were insufficient to raise C. elegans levels of these metabolites and that is the reason they “fail” to suppress daf-16 responses in figure 3E. The authors should measure abundances of these species in treated animals along with the successful supplementation of PC(18:1n9) to substantiate the claim that it is unsaturated PC that is mediating the signaling pathway. Certainly because more than just 18:1n9 fatty acids are changing in the PC fraction of let-607 RNAi treated animals in figure 4D, even though, as the authors point out, the majority of changes are seen in PC unsaturated fatty acid abundances.

While I believe that the authors are likely correct on the conclusion of the involvement of calcium in the ECSPC activation, more evidence is needed to solidify some of the lines of evidence. Specifically, for the double RNAi experiments, the authors are already diluting let-607 RNAi to prevent lethality, how do they know, for example in Figure 5A/D/E/F that this is because they are no longer effectively knocking down let-607? This is not proven by the nicely done qPCR in Figure S1A. Further, I don’t know what to think when three RNAi are combined—this is generally not accepted as standard practice in the field without proof of equally good knockdown (i.e.figure 5D).

The demonstration of pkc-2 and sgk-1 dependency of the ECSPC are interesting and compelling, but the interpretation of these data are complicated. First, Sgk-1 mutants are viable, so consideration should be given to repeating the experiment in figure 5F with sgk-1 mutants, and not double RNAi. Second, while pkc-2 and sgk-1 are reported to be in a genetic pathway with each other with regard to cold survival in the worm, more canonical descending input into sgk-1 is through mTORC2. The authors should determine whether disruption of mTORC2 also recapitulates the sgk-1 result. Third, the best described input into daf-16 are the akt kinases. The authors should disrupt akt-1 and akt-2 by genetic mutation or RNAi and determine whether this mitigates activation of daf-16 in the ECSPC (e.g. in response to let-607 RNAi).

Minor comments

Please describe the pathogen stress used in the figure legend (Fig. 1C) or the text, as well as more detail about the stresses (i.e. duration, dose) used in the legend for figures 1C-1G.

Sphingomyelin on P.8 is misspelled.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: None

Reviewer #2: Yes

**********

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

Dear Dr Tang,

Thank you very much for submitting your Research Article entitled 'Phosphatidylcholine mediates the crosstalk between LET-607 and DAF-16 stress response pathways' to PLOS Genetics.

The manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important topic but identified some concerns that we ask you address in a revised manuscript

We therefore ask you to modify the manuscript according to the review recommendations. Your revisions should address the specific points made by each reviewer.

In addition we ask that you:

1) Provide a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

2) Upload a Striking Image with a corresponding caption to accompany your manuscript if one is available (either a new image or an existing one from within your manuscript). If this image is judged to be suitable, it may be featured on our website. Images should ideally be high resolution, eye-catching, single panel square images. For examples, please browse our archive. If your image is from someone other than yourself, please ensure that the artist has read and agreed to the terms and conditions of the Creative Commons Attribution License. Note: we cannot publish copyrighted images.

We hope to receive your revised manuscript within the next 30 days. If you anticipate any delay in its return, we would ask you to let us know the expected resubmission date by email to plosgenetics@plos.org.

If present, accompanying reviewer attachments should be included with this email; please notify the journal office if any appear to be missing. They will also be available for download from the link below. You can use this link to log into the system when you are ready to submit a revised version, having first consulted our Submission Checklist.

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool. 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.

Please be aware that our data availability policy requires that all numerical data underlying graphs or summary statistics are included with the submission, and you will need to provide this upon resubmission if not already present. In addition, we do not permit the inclusion of phrases such as "data not shown" or "unpublished results" in manuscripts. All points should be backed up by data provided with the submission.

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

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.

PLOS has incorporated Similarity Check, powered by iThenticate, into its journal-wide submission system in order to screen submitted content for originality before publication. Each PLOS journal undertakes screening on a proportion of submitted articles. You will be contacted if needed following the screening process.

To resubmit, you will need to go to the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.

[LINK]

Please let us know if you have any questions while making these revisions.

Yours sincerely,

Coleen T. Murphy

Associate Editor

PLOS Genetics

Gregory P. Copenhaver

Editor-in-Chief

PLOS Genetics

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: I am overall pleased with the revised version of the manuscript, but still must insist on one follow-up experiment to clearly demonstrate the intra-cellular localization of LET-607.

I appreciate the attempts of the authors to determine let-607 localisation by generating a translational fusion. However, the provided images in Fig 1B are of poor quality, of low resolution and lack markers. From the little I can see, I think the localization may be ER after all, rather than nuclear. Better imaging is required. Higher resolution, confocal imaging, contrast labelling of the nuclei and an ER marker to examine possible colocalization

On the same note, DAF-16::gfp images are also of poor quality (Fig 2C. Fig 4C). Please provide better images.

Page 11, line 221 - since PC reduction alone did not activate DAF-16 - this sentence should be toned down.

Page 12, libe 250 - the suppression of mlt-1 expression in glp-1 mutants was only partial - tone down

Page 10, line 191 - Replace reproductivity with a better word

Page 14, line 271 - in defective in

Reviewer #2: This revised manuscript by He et al. for the most part addresses my concerns. I appreciate the addition of the data demonstrating interaction with the daf-2 and germlineless pathway, as well as the epistasis analyses with daf-9, daf-12, tcer-1 and kri-1. The PC-calcium pathway analyses are also compelling and well presented, though quantification of some of the reporter data would go a long way in strengthening the conclusions. It is an interesting story that is relevant to the broad readership of PLoS Genetics.

I have a few lingering concerns, mostly statistical with some minor points.

t-test is not appropriate for establishing significance when more than 1 test is being done, e.g. fig 2B where daf-16 and sod-3 expression are both tested—correction for multiple hypothesis testing is needed.

Paired student t-tests are not appropriate without multiple hypothesis testing for figures 5B, C, and F. B and C should be corrected as A and D for multiple hypothesis testing, and F should be compared by 2-way ANOVA with post-hoc tests for significance.

Minor points:

The authors use dilutional RNAi. Did they consider post-developmental RNAi to see if the lifespan extension and developmental lethality represent antagonistic pleiotropies?

Line 60: based upon their findings, the authors may want to say “In mammals, CREBH is an ER-bound transcription factor…”

Line 68 LET-607 “is located in the nucleus” or “localizes to the nucleus”

In figure S1B, membrane is spelled “membrance” in 2 places.

It may be useful, when making decisions about additivity of factors in stress pathway activation, to quantify data—e.g. fig S2f-H. Then again, is it really valuable to be making comments about additivity of hypomorphic effects by RNAi? A hypomorphic effect can be enhanced almost by definition. As such the analyses are not terribly informative as presented.

The authors may consider quantifying expression of mtl-1 to make conclusions about synthetic interactions in figs. 4 and S4.

Please state the statistical test used in Fig 2E.

The data in figure 6 are very interesting and while S6 is quantified, Fig 6 is not, especially in glp1 6A where a claim of dependence on PC is made. These data should be quantified.

Sgk-1 RNAi has been reported to be long lived (Baumeister lab, 2004 Dev Cell). Can the authors explain why their RNAi is not long lived in fig. 7G?

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Dear Dr Tang,

We are pleased to inform you that your manuscript entitled "Phosphatidylcholine mediates the crosstalk between LET-607 and DAF-16 stress response pathways" has been editorially accepted for publication in PLOS Genetics. Congratulations!

Before your submission can be formally accepted and sent to production you will need to complete our formatting changes, which you will receive in a follow up email. Please be aware that it may take several days for you to receive this email; during this time no action is required by you. Please note: the accept date on your published article will reflect the date of this provisional acceptance, but your manuscript will not be scheduled for publication until the required changes have been made.

Once your paper is formally accepted, an uncorrected proof of your manuscript will be published online ahead of the final version, unless you’ve already opted out via the online submission form. If, for any reason, you do not want an earlier version of your manuscript published online or are unsure if you have already indicated as such, please let the journal staff know immediately at plosgenetics@plos.org.

In the meantime, please log into Editorial Manager at https://www.editorialmanager.com/pgenetics/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production and billing process. Note that PLOS requires an ORCID iD for all corresponding authors. Therefore, please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field.  This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager.

If you have a press-related query, or would like to know about making your underlying data available (as you will be aware, this is required for publication), please see the end of this email. If your institution or institutions have a press office, please notify them about your upcoming article at this point, to enable them to help maximise its impact. Inform journal staff as soon as possible if you are preparing a press release for your article and need a publication date.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Genetics!

Yours sincerely,

Coleen T. Murphy

Associate Editor

PLOS Genetics

Gregory P. Copenhaver

Editor-in-Chief

PLOS Genetics

www.plosgenetics.org

Twitter: @PLOSGenetics

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

Comments from the reviewers (if applicable):

The authors have addressed the reviewers' remaining concerns.

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

Data Deposition

If you have submitted a Research Article or Front Matter that has associated data that are not suitable for deposition in a subject-specific public repository (such as GenBank or ArrayExpress), one way to make that data available is to deposit it in the Dryad Digital Repository. As you may recall, we ask all authors to agree to make data available; this is one way to achieve that. A full list of recommended repositories can be found on our website.

The following link will take you to the Dryad record for your article, so you won't have to re‐enter its bibliographic information, and can upload your files directly: 

http://datadryad.org/submit?journalID=pgenetics&manu=PGENETICS-D-20-01382R2

More information about depositing data in Dryad is available at http://www.datadryad.org/depositing. If you experience any difficulties in submitting your data, please contact help@datadryad.org for support.

Additionally, please be aware that our data availability policy requires that all numerical data underlying display items are included with the submission, and you will need to provide this before we can formally accept your manuscript, if not already present.

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

Press Queries

If you or your institution will be preparing press materials for this manuscript, or if you need to know your paper's publication date for media purposes, please inform the journal staff as soon as possible so that your submission can be scheduled accordingly. Your manuscript will remain under a strict press embargo until the publication date and time. This means an early version of your manuscript will not be published ahead of your final version. PLOS Genetics may also choose to issue a press release for your article. If there's anything the journal should know or you'd like more information, please get in touch via plosgenetics@plos.org.