Dear Dr Ma,

Thank you very much for submitting your Research Article entitled 'Drosophila Myc restores immune homeostasis of Imd pathway via activating miR-277 to inhibit imd/Tab2' to PLOS Genetics. Your manuscript was fully evaluated at the editorial level and by three 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.

The reviewers concerns were quite significant and a revised manuscript will need to include additional experimental work including, inclusion of proper controls, adding loss-of-function analysis to bolster claims that primarily rely on over-expression analysis, and substantial improvement of English grammar (this will likely require a professional editing service).

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,

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 manuscript, Li et al study the link between IMD pathway, Myc and miR-277

They propose that Myc represses the IMD pathway and that this is mediated by the miR-277.

There are many problems in this manuscript that are listed here.

- All the presented experiments analyzed the effects of Myc over expression using the Gal4 system. So, they do not study the role of Myc in the immune homeostasis but the consequences of Myc overexpression. This is therefore non-physiological and might have nothing to do with the endogenous role of Myc. Loss-of-function mutants should be use to study the role of a gene in a given process.

- Most of the experiments presented here use the diptericin as a read out of the IMD pathway. Hundreds of papers have shown that, as most Antimicrobial peptides, diptericin transcripts can increase us to 10.000 times upon infection. Even in the absence of infection, their levels vary quite a bit. The fold changes reported in this manuscript are around 1,5 or even sometimes less. There is something wrong here. I do not what. When a gene can have its transcript produced 10.000 times more in control conditions, what does a 1,3 fold difference mean? My guess is that there is a technical problem.

- The experiments always miss important controls. Just one example but this is true for all of them. Fig 8, the authors test the resistance of Myc overexpressing flies to E. Cloacae infection. The survival rate of these flies in the absence of infection and after sterile wounding should be tested.

- Immune genes can be activated by stress. Overexpressing the oncogene Myc in a cell is probably stressful for the flies. I am not sure this work studies immune response but rather stress response.

- The text is very difficult to follow. Just one example from the first sentence of the results “as we know that the diptericin expression is an important readout of Drosophila Imd pathway activation”. We expect the phrase to continue but it does not.

Reviewer #2: The manuscript “Drosophila Myc restores immune homeostasis of Imd pathway via activating miR-277 to inhibit imd/Tab2”(PGENETICS-D-19-02099) has revealed that Drosophila transcription factor dMyc could serve as a novel negative regulator of Imd pathway immune response using the loss- and gain-of-function screening. Especially, their works illuminate a feasible mechanism that dMyc as a transcription factor may activate miR-277 transcription to inhibit imd and/or Tab2 expression to restore immune homeostasis of Drosophila Imd pathway and improve the survival of flies. Overall, this manuscript has provided new insights into further studying the maintenance mechanism of innate immune homeostasis. The content of this manuscript is interesting. Thus I think that this manuscript is suitable to be published in PLOS Genetics. However, there are several shortcomings as following:

Major comments

Comment 1: In response to Gram-negative bacteria, the Imd pathway produces not only Dpt but also other antimicrobial peptides (AMPs). Thus, what are the expression levels of other AMPs in response to E.coli?

Comment 2: Although the expression level of dMyc in the dMyc and dMyc-RNAi co-highexpressed flies could be recovered to some extent, whether the expression level of miR-277 could also be restores?

Comment 3: Myc may recognize some binding motifs so-called E-boxes (e.g. CACGTG, CATGTG and alternative sequences) in the promoter region of target genes to activate their expression. Whether there is a similar E-box in the promoter region of miR-277? Especially, when the E-box of miR-277 is deleted or mutated, whether Myc could still activate the transcription of miR-277？

Comment 4: Why choose these five time points to collect and test samples after infection?

Minor comments

Comment 1: Some abbreviations, such as E.coli and LPS, should write the full name at the first appearance.

Comment 2: In Fig 3, the font of the title is inconsistent.

Comment 3: In Fig 7, line 326: “E.col” should be “E.coli”.

Comment 4: Line 430: “togther” should be “together”.

Comment 5: “(D)”should be “(B)” in Fig 1 legend (line 140).

Comment 6: “Drosophila” should be italic (line 116 and 148).

Comment 7: The legend in Fig4A is not consistent with other figures. It should be “Gal80ts” not “Gal80”.

Comment 8: Two legends in Fig 3B and 3C could look better when they are adjusted up and down.

Reviewer #3: Li et al. have studied the effect of Drosophila Myc on innate immune signaling. They have discovered that it acts as a negative regulator of the IMD pathway and they further show that it acts by inducing expression of miR-277 transcription which in turn down-regulates the expression of imd and Tab2-Ra/b (but not Tab2-Rc). Notably, dMyc was shown to bind to the promoter region of miR-277.

Major comments:

Most of all, the level of the English grammar – and the way of scientific writing – is not at the required level. As I see potential in this manuscript I hope that there would be careful editing for the paper for scientific English.

Figure 1 A, Dipt expression should be shown also in uninfected (0h) flies

Figure 3B: S2 cells respond poorly to LPS as in Drosophila, gram-negative bacteria are recognized by Peptidoglycan recognistion proteins, predominantly via PGRP-LC (use Choe et al., 2002 Nature, Ramet et al., 2002 Nature, Gottar et al., 2002 Science for references). I suggest repeating this experiments using heat-killed E. coli instead of LPS.

Minor comments:

Lines 75-77 ‘Furthermore, the Imd-mediated immune response can also be negatively regulated by some immune suppressors, such as WntD, Die, PGRP-LF, pirk, dUSP36, CYLD, Dnr1, dRYBP and Caspar [15-24]’ lacks the notion and reference to the most important negative regulator of this pathway, namely Pirk (references Kallio et al., 2005 Microbes and Infection and Kleino et al., J Immunol 2008 needs to be cited).

Line 117: ‘As we all know…’ Rephrase

Lines 124 Avoid phrases such as ‘remarkably’ – the results will speak to themselves

Lines 128-129: ‘Especially, the expression level of Diptericin in the dMyc and dMyc-RNAi co-highexpressed flies’ – I see no point – is I understand this correctly – to both overexpress dMyc and to suppress its expression by RNAi at the same time.

Fig 1C: I see no reason to cut the y-axis

Line 189: ‘This dMyc can activate…’ Remove word ‘This’

Figure 3 B and C – statistical significance

Chapter ‘miR-277 inhibits the expression of imd and Tab2-Ra/b’ – Authors should –shortly introduce why they are interested in Tab2, i.e. that it has been shown to be a key component of the Drosophila Imd pathway (Kleino et al., 2005 EMBO J)

Fig 7C – there is a mistake in the y-axis (spelling)

Line 326 - E.col infection

Figure 8 – there is no reason to cut the x-axis

Line 451: ‘And that dMyc…’ Rephrase

**********

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: None

Reviewer #3: 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

Reviewer #3: No

Dear Dr Ma,

We are pleased to inform you that your manuscript entitled "Drosophila Myc restores immune homeostasis of Imd pathway via activating miR-277 to inhibit imd/Tab2" 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 accept, 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 one way to make 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,

Gregory P. Copenhaver

Editor-in-Chief

PLOS Genetics

www.plosgenetics.org

Twitter: @PLOSGenetics

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

Comments from the reviewers (if applicable):

Reviewer's Responses to Questions

Comments to the Authors:

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

Reviewer #2: All questions are well answered or explained in the revision. I recommend accpetance and publication of this manuscript.

Reviewer #3: May concerns are sufficiently addressed and I would favour accepting this manuscript

**********

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 #2: None

Reviewer #3: 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 #2: No

Reviewer #3: No

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

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-19-02099R1

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.