Dear Dr. Kim,

Thank you very much for submitting your manuscript "Immune mediation of HMG-like DSP1 via Toll-Spätzle pathway and its specific inhibition by salicylic acid analogs" 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.

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).

Important additional instructions are given below your reviewer comments.

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,

Francis Michael Jiggins

Associate Editor

PLOS Pathogens

Karla Satchell

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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

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 manuscript characterizes the mechanisms by which X. hominikii suppresses the immune response in the insect S. exigua. It identified the genes involved and uses both ectopic expression and dsRNA knockdown and/or CRISPR mutants to define the roles of DSP1, Toll receptors, and Spatzle (a Toll ligand) in the immune response in S. exigua. It goes on to define the secondary metabolite from X. hominikii that suppresses this immune response. These findings provide significant insight into how x. hominikii causes immunosuppression. The manuscript is well-written, and the data presented are generally clear and convincing. However, there are a few areas that could be modified to improve the clarity.

Reviewer #2: This study investigated the immune signaling pathway of a damage-associated molecular pattern, DSP1. It reports that DSP1 activates Toll/Spz pathway for defending Gram-positive bacterial infection. Especially, Toll9 among 10 Toll receptors was a main signal component. This is further supported by a deletion mutant of Toll9, which lost immune activation. Furthermore this study identified an immune blocker, EMP, which exhibited a tight binding to DSP1 not to be released. Thus the entomopathogenic bacterium, X. hominickii induces a fatal pathogenicity by releasing EMP to suppress host immune defense. Overall, this reports a new immune signaling pathway in insects using a DAMP signal, DSP1 and a novel immunosuppressive agent, EMP. Thus, this study fits to the journal scope and standard of PLoS Pathogens. However, following issues need to be addressed.

1. This study showed the release of DSP1 from nucleus to plasma. The released DSP1 appears to be transported in plasma using exosome cargo. Here are questions. What does the intracellular signal stimulate DSP1 release from nucleus? Why does the DSP1 take exosome in the plasma?

2. In addition to Toll9, DSP1 may use Toll6, Toll7, or Toll8 to induce the AMP or PO activation from Fig. 4. Why did you determine Toll9 as DSP1 signal component?

3. I am not sure that DSP1 directly activated Spz to trigger Toll sigaling pathway?

4. What is the relationship of Bt pathogenicity and immunosuppression? Is it required for the Bt to exhibit its insecticidal activity?

5. How can you explain the inhibitory activity of EMP on SP1 release from the nucleus?

Reviewer #3: Precis

Some insect pathogenic microbes overcome host immune responses to infection by secreting compounds that inhibit insect immune reactions to infection. Park and Kim (2000; doi: 10.1016/s0022-1910(00)00071-8) first reported that the bacterial lethality of the insect pathogen Xenorhabdus nematophilus was attenuated after injecting arachidonic acid (AA), a direct precursor to biosynthesis of prostaglandins and other eicosanoids, into the abdomens of infected lepidopterans, Spodoptera exigua. They put forth the idea the bacterium suppresses insect immunity by inhibiting biosynthesis of eicosanoids. Now, after 20 years and many publications on eicosanoid signaling in insect immunity, they turned attention to a related insect pathogen, X. hominickii. This pathogen also secretes metabolites that inhibit the first step in eicosanoid biosynthesis, phospholipase A2 (PLA2), which effectively suppresses eicosanoid signaling to suppress host immunity. They reported that a damage-associated molecular pattern called dorsal switch protein 1 (DSP1), which activates PLA2 early in infection in S. exigua. They used immunofluorescence to record increased hemocyte spreading and Se-DSP1 in fat body. They used western blots to show Se-DSP2 in hemolymph of infected, but not naïve larvae at 6 h post-infection. Their western blot analysis also indicated the Se-DSP1 was transported out the cells as via exosomes. The authors created a recombinant Se-DSP1 (rSe-DSP1) and used it to demonstrate that bacterial infection with another insect pathogen, E. mundtii, leads to increased PO activity, as does rDSP1 treatments, but not after denaturing the protein. Treating larvae with a dsRNA construct, dsDSP1 + E. mundtii blocked infection-triggered PO activity. They used enzyme activity assays to show rDSP1 treatments led to significant increases in secretory PLA2 and cellular PLA2 activities. Translating to the influence of rDSP1 on humoral immunity, the authors show that rDSP1 injections led to significant increases in mRNAs encoding a range of antimicrobial peptides and proteins, including apolipophoren-III, cecropin and lysozyme. They report ten Toll receptors and show accumulations of mRNAs encoding them. Expression of each of the ten genes was substantially reduced at 24 or 48 h after injecting dsRNA constructs specific to each gene. Co-injections with each dsRNA + rDSP1 led to substantial PO activity, sPLA2 activity and cPLA2 activity. Similar co-injections led to substantial expression of genes encoding three anti-microbial proteins. Digging deeper into humoral immunity, the authors report two genes encoding Spätzels, showing gene structures and a phylogenetic tree placing them withing Diptera-Lepidoptera. They show the genes are expressed in hemocytes and fat body. rDSP1 treatments led to increased gene expression in hemocytes, but not in fat body. Bacterial injections led to increased expression of both genes in fat body. They show dsRNA treatments led to decreased expression of both genes encoding Spätzes from 24 – 72 h post-injections. Bacterial injection and DSP1 injections led to increased PO activity, but not to increased PLA2 activity. Co-inections with dsSpätze1 + DSP1 did not lead to increased expression of three anti-microbial peptides, although dsSpätze2 +DSP1 did.

The authors continued drilling into humoral immune signaling by creating CRISPR/Cas9 deletion mutants in SeToll9. Although rDSP1 injections into wild-type larvae led to increased sPLA2 and cPLA2 activities and to increased expression of the three anti-microbial proteins, similar injections into the deletion mutants did not. The changes in immune parameters just mentioned translated into increased mortality following co-injections with most dsRNAs designed to each of the ten toll receptors + the insect pathogen, Bacillus turingiensis Mortality did not increases following dsTOLL1, 8 or 10. Similarly, dsSpätze1 and -2 treatments also led to increased mortality. The authors identified X. hominickii metabolites that bind to rSeDSP1, and thus, block its translocation. They used GC-MS analysis to identify several metabolites with binding affinities in the low �M range, of which 3-eethoxy-4-methoxyphenol (EMP) had the highest binding affinity. In a direct test of DSP1 translocation, the authors presented a western blot showing the presence of Se-DSP1 in plasma from larvae injected with the pathogen E. mundtii, but not in larvae injected with the pathogen + EMP. EMP + rDSP1 treatments led to sharp reductions in PO, sPLA2 and cPLA2 activities and virtually eliminated accumulations of mRNAs encoding gallerimycin, gloverin and lyzozyme. Injections of pathogen B. thuringiensis led to increased mortality in control larvae. The bacterial injections into larvae treated with dsDSP1 led to higher mortality and still higher mortality followed in larvae treated with EMP.

The authors complete their story with a meaningful model of a hypothesized mechanism of DSP1 action. In their view, the bacterial product EMP damages hemocytes and DSP1 is translocated from cells into hemolymph, where it activates one or more serine protease cascades that leads to active phenol oxidase and melanization reactions. It also activates two Spätzels, with interacts with toll receptors that lead to biosynthesis of eicosanoids that mediate cellular immune reactions and lead to expression of genes encoding lysozyme and other anti-microbial protains.

They present three supplementary figures. Fig. S1 reports accumulations a mRNAs encoding the 10 toll receptors. Fig. S2 shows their extraction scheme for analysis of the X. hominickii metabolites and Fig. S3 shows the chemical structures of selected metabolites.

Critique

The authors present a well-reasoned and thorough series of experiments to test their hypothesis.

**********

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: n/a

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: 1. Abstract: The abstract is incredibly long and it seems like much of this information belongs in the Introduction. Further, from the abstract, the reader expected eicosanoid biosynthesis to be studied in the manuscript and it was not.

2. Line 104 – explain to the reader what Pelle kinase is.

3. Paragraph starting at line 107 – it is not clear what the authors think the order of events is.

4. Figure 1 – The change in Se-DSP1 immunofluorescence isn’t convincing. It seems like it is relocalizes, perhaps to the nucleolus, but the intensity does not appear reduced. Some quantification is needed to clarify this.

5. The terminology in the results and in the figures is not always consistent. For example, Line 134-135, Se-DSP1 (rSe-DSP1) is not how it is written in the Figure 2. Another example is in relation to Figure 7

6. The authors need use PLA2 activity to indicate eicosanoid biosynthesis, but PLA2 has many other roles. The authors need to discuss this limitation. Further, in the results it is not discussed what samples are used for the analysis and why they are the correct samples.

7. It seems it is worth discussing why AMP genes are activated by heat inactivated rSe-DSP1

8. The description of the data in Figure 4 is difficult to follow. Sometimes the authors say which Toll receptors have no effects and for other data which Toll receptors play a role. It would be clearer to the reader to talk about the Toll receptors that are involved in the process. Also a discussion of the difference in the Toll receptor role in PLA activity vs AMP gene expression is warranted. It is unclear what the reader should conclude about who is involved.

9. Line 183 – the data in figure 5c would say that neither Spz gene is induced in the hemocytes in response to EM, and Spz1 may be inhibited. This is not what is stated in the results and it seems worth talking about. Lines 184-185 seems to contradict the prior sentence

10. In the section starting at 209, it would help the reader for the authors to explain their method of assessing pathogenicity.

11. Figure 1 legend talks about Tubulin western blots that are not shown in the figure

12. The RNAi knockdown data in Figure 4a and Figure6a are shown as connected lines. Such a graph implies the expression was assessed repeatedly in the same cells, when in reality it was done on different cells. The data should be presented as individual data points or bar graphs.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: David Stanley

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. 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.

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Reproducibility:

To enhance the reproducibility of your results, PLOS recommends that you deposit 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 http://journals.plos.org/plospathogens/s/submission-guidelines#loc-materials-and-methods

Dear Dr. Kim,

Thank you very much for submitting your manuscript "Immune mediation of HMG-like DSP1 via Toll-Spätzle pathway and its specific inhibition by salicylic acid analogs" for consideration at PLOS Pathogens. Thank you for thoroughly revising the manuscript, it is a great paper. I am happy with all the revisions in the main text, but would like you to look again at the first comment about the abstract. At the moment the abstract is hard to read unless you have technical knowledge of this area. This is because it includes a large amount of technical detail and goes through many different experiments. In contrast, it does not make clear the wider significance and importance of the work. This contrasts with the author summary, which did this job very well. I would ask that the abstract is simplified and makes clearer the wider significance of your work. I apologise for returning the manuscript a second time, but I hope this will be a quick change to make that will help your work reach a wider audience.

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).

Important additional instructions are given below your reviewer comments.

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,

Francis Michael Jiggins

Associate Editor

PLOS Pathogens

Karla Satchell

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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

Thank you for thoroughly revising the manuscript which is a very interesting body of work. I am happy with all the revisions in the main text, but would like you to look again at the first comment about the abstract. At the moment the abstract is not suitable for PlOS Pathogens as it is very hard to read unless you have a technical knowledge of this area. This is because it includes a large amount of technical detail and goes through many different experiments. However, it does not make clear the wider significance and importance of the work. This contrasts with the author summary, which did this job very well. I would ask that the abstract is simplified and makes clearer the wider significance of your work.

Reviewer Comments (if any, and for reference):

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. 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.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, PLOS recommends that you deposit 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 http://journals.plos.org/plospathogens/s/submission-guidelines#loc-materials-and-methods

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.

Dear Dr. Kim,

We are pleased to inform you that your manuscript 'Immune mediation of HMG-like DSP1 via Toll-Spätzle pathway and its specific inhibition by salicylic acid analogs' 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.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Francis Michael Jiggins

Associate Editor

PLOS Pathogens

Karla Satchell

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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

Reviewer Comments (if any, and for reference):