Dear Dr Gassmann,

Thank you very much for submitting your Research Article entitled 'Opposing functions of the plant TOPLESS gene family during SNC1-mediated autoimmunity' to PLOS Genetics. Your 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 again a 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. In particular, please focus your efforts on infection assays and the role of TPR2 and TPR3 with respect to SNC1. These points were made by all reviewers, and I agree that they should be addressed. 

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[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,

Gitta Coaker, PhD

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: Garner et al. describe the negative role of TOPLESS family members in SRFR1/SNC1-dependent immunity. Using plant dwarf morphology as a proxy to defense autoactivation, they performed epistasis analysis between srfr1 and tpr mutants, looking for enhancement or reversion of srfr1 phenotype. They demonstrated that trp2 and tpr3 loss-of-function mutant both enhance srfr1/snc1 stunting growth and defense gene expression. Whereas, TPR2 overexpression suppressed srfr1 and srfr1 tpr2 growth. They further showed that srfr1 tpr2 tpr3 phenotypes depend on SNC1. Coupled with gene expression analysis, the authors claimed that SRFR1 functions upstream of SNC1 transcription, while TPR2, on the other hand, may act downstream.

The manuscript is overall convincing and easy to follow. The genetic data is solid and clear; however, infection assays and mechanisms are lacking. To justify publication in PLOS Genetics, more characterization of the mutants and some mechanism are needed:

Major comments:

- Infection assays: why the authors did not perform a single infection assay for the mutants? Plant size and marker gene expression are helpful. But without bona fide pathogen challenges, it is insufficient to draw immune-related conclusions. It is reasonable bacterial infections are difficult to be done on dwarf mutants, but not wild-type like plants.

- The role of TPR2/3 on SNC1 is unclear. At least some molecular mechanism should be provided.

Minor comments:

- It would be helpful to place parental lines in the same images:

Fig3A, srfr1 tpr3 double mutant

Fig4A, tpr2, tpr3 single mutant

- L140: to claim tpr1-2 is not a true knockout need to show T-DNA genotyping and RNA level.

- L166, it is insufficient to claim tpr2 tpr3 act redundantly to enhance srfr1. Under autoimmune mutant background, this could also due to additive effects of independent pathways.

- L168, may worthwhile performing infection assays on tpr2 tpr3 double mutants. Certain mutant with mild EDR phenotype may not exhibit obvious growth defects.

- L232: should it be “decrease” in SNC1 expression?

- L240: no evidence supports TPR2 compete with TPR1

- Fig8C, in vitro assays are fine for protein interactions. I would like to see at least an additional in vivo IP to support the claim. Transient expression in tobacco would be sufficient.

- Fig9: Without additional data the model depicting competition between TPR2/SNC1 and TPR1/SNC1 cannot be made. These can also be tested via transient expression in tobacco.

Reviewer #2: The work is beautiful and the genetic data to reveal the interaction between SRFR1 and TPR2 and TPR3 is clearly present. However, about the function of TPR2 and TPR3 in negatively regulating plant autoimmunity, the conclusions are not that convincing, and the proposed mechanisms are a little speculative. Additive experiments are recommended to confirm the conclusions and strengthen the hypothesis.

Major

1.The authors claim a novel role of TPR2 and TPR3 in suppressing plant autoimmunity, pathogen assays are recommended to proof such a statement. PstDC3000 (virulent) or Pst DC3000 AvrRps4 (avirulent) strains could be used to test the srfr1-4 tpr2-2 and srfr1-4 tpr2-2 tpr3-1 mutants to provide further information on the effect of TPR2 and TPR3 on pathogen resistance.

2. The authors propose that TPR2 and to some degree TPR3 act downstream of SNC1 transcription by repressing expression of a positive regulator of SNC1 or activating a negative regulator. RNA-seq of srfr1-4, srfr1-4 tpr2-2, and srfr1-4 tpr2-2 tpr3-1 mutants are recommended, which could better explain the diverse function of these two TOPLESS members.

Minor

1. In figure 1, the letters that denote the significant differences are placed on top of the figure, which is difference from the letter placements in other figures. It is better to be consistent.

2. In page 5, line 85 “PR genes” should be “PR genes”, “PR” should be in italics.

3. In page 9, line 171 “Fig 4C and 4D” should be “Fig 4B and 4C”.

Reviewer #3: please see attachment

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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

Reviewer #3: Yes

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Attachments

Attachment

Submitted filename: PG-20-Gassmann.docx

* 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. *

Dear Dr Gassmann,

Thank you very much for submitting your Research Article entitled 'Opposing functions of the plant TOPLESS gene family during SNC1-mediated autoimmunity' 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. All reviewer comments can be textually addressed and do not require new experimentation. In particular, reviewer 1 suggests addressing redundancy in TPL/TPR loci as well as consolidating some of the main and supplemental figures. I agree with the comment about TPL/TPR loci and leave the decision of figure consolidation to your discretion. 

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.

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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.

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,

Gitta Coaker, PhD

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: The authors have addressed most of my concerns. This reviewer has a couple of final suggestions:

1. As a dominant-negative mutant of topless tpl-1 is lethal, it is predicted that knocking out all five TPL/TPR related genes would yield a lethal phenotype. Therefor the whole TPL family must be redundant in many aspects. This may explain the weak phenotypes of the mutants the authors describe. The new interaction data between TPR1 and TPR3 can also support such redundancy since they are exchangeable in the complex. Some discussion should be added regarding this, so the readers would bear in mind the diverse, and sometimes even opposite roles of these repressor proteins in regulating plant biology. An alternative explanation for the story can be that the TPL family proteins all serve both positive and negative roles, the previous 3 more positive, while 2/3 more negative. I would also suggest the authors to include such alternative model in their model figure.

2. Some main figures are quite thin, and there are too many figures/sup figures. I would suggest the authors to reduce the number of figures below 7, combining some simples ones but include some of the important supplementary figures in main.

Reviewer #2: In the revised manuscript, the authors added data on planta bacterial growth assays. Their conclusions about the role of TPR2 and TPR3 in suppressing plant autoimmunity are more solid and convincing now with the addition of the new generated data. Most of my previous concerns have been well addressed.

One of my previous concerns was not entirely addressed: although the authors showed that TPR2 directly interacts with SNC1, the mechanism on how this interaction represses negative regulators of immunity is still not quite clear.

Since the main focus of this manuscript is to establish the functional relationship between SRFR1 and TPR, it is acceptable that the authors did not look into the detailed differences in gene regulation by RNA-Seq in this manuscript.

Reviewer #3: My concerns have been sufficiently addressed.

**********

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

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 Gassmann,

We are pleased to inform you that your manuscript entitled "Opposing functions of the plant TOPLESS gene family during SNC1-mediated autoimmunity" has been editorially accepted for publication in PLOS Genetics. Congratulations!

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Yours sincerely,

Gitta Coaker, PhD

Associate Editor

PLOS Genetics

Gregory P. Copenhaver

Editor-in-Chief

PLOS Genetics

www.plosgenetics.org

Twitter: @PLOSGenetics

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Comments from the reviewers (if applicable):

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