Dear Dr Lundberg,

Thank you for submitting your manuscript entitled "Field conditions greatly modify a major growth-defense tradeoff in Arabidopsis thaliana" for consideration as a Research Article by PLOS Biology.

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Kind regards,

Roli Roberts

Roland Roberts, PhD

Senior Editor

PLOS Biology

rroberts@plos.org

Dear Dr Lundberg,

Thank you for your patience while your manuscript "Field conditions greatly modify a major growth-defense tradeoff in Arabidopsis thaliana" went through peer-review at PLOS Biology. Your manuscript has now been evaluated by the PLOS Biology editors, an Academic Editor with relevant expertise, and by two independent reviewers. Please accept my apologies for the delay incurred over the holiday period.

You'll see that reviewer #1 is very positive and has no requests. Reviewer #2 is also largely positive, but wants much clearer description of the methods, especially the stats, and thinks that the framing of the Intro and Discussion needs to be improved in order to bring out the advance for a broader audience (their point 3).

IMPORTANT: I discussed these comments with the Academic Editor, and we both agree fully with the points raised by reviewer #2, and especially the need to bring out the advance that it most relevant to the broader readership, namely the striking and reproducible disparity between field and greenhouse experiments.

In light of the reviews, which you will find at the end of this email, we are pleased to offer you the opportunity to address the [comments/remaining points] from the reviewers in a revision that we anticipate should not take you very long. We will then assess your revised manuscript and your response to the reviewers' comments with our Academic Editor aiming to avoid further rounds of peer-review, although might need to consult with the reviewers, depending on the nature of the revisions.

We expect to receive your revised manuscript within 1 month. Please email us (plosbiology@plos.org) if you have any questions or concerns, or would like to request an extension.

At this stage, your manuscript remains formally under active consideration at our journal; please notify us by email if you do not intend to submit a revision so that we withdraw the manuscript.

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Thank you again for your submission to our journal. We hope that our editorial process has been constructive thus far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Roli Roberts

Roland Roberts, PhD

Senior Editor

PLOS Biology

rroberts@plos.org

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REVIEWERS' COMMENTS:

Reviewer #1:

In this paper, Lundberg et al. investigate effects of ACD6 alleles, using parallel approaches with near-isogenic lines and gene editing. ACD6 is associated with defense responses, which typically come at the cost of reduced growth and fecundity. The authors studied these growth-defense tradeoffs in a series of field and greenhouse experiments, in great detail focusing on visual phenotypic traits, gene expression, hormone levels, and leaf microbiome composition.

The key finding, which really is surprising and extremely important, is that the expected growth-defense tradeoff only manifested under greenhouse conditions. There, defense-associated gene expression was high in the corresponding genotypes, and associated costs such as reduced growth and leaf necrosis were observed. In the field, these effects were not observed, neither in near-isogenic lines derived from Col-0 x Est-1 crosses, nor in comparisons of lines in which the Est-1 allele was knocked out by editing with CRISPR/Cas9.

Overall, these strong G x E interaction demonstrate that findings from controlled-condition studies may not be transferable to the field, which has far-reaching consequences. One would have to understand the specific environmental drivers of this GxE interaction, which the authors have tried. They investigated and discuss different possibilities, including differences in temperature between field and greenhouse and differences in foliar microbiome, but did not find an explanation. I would like to stress that the authors are not to blame for this lack of explanation. On the contrary, I highly appreciate the level of detail in which this was done, and how transparently this field-greenhouse discrepancy is presented.

This fascinating paper was a pleasure to read, is well written, the methods clearly described, and in my opinion publishable as is.

Reviewer #2:

Overall this is a high quality manuscript describing an interesting partial-null result, in which naturally occurring allelic variants of the ACD6 gene in Arabidopsis thaliana have major effects on growth and disease resistance in greenhouse experiments, but not in the outdoors. It is well written and (except for the statistical analysis, see detailed comments below) the work appears to be well executed. The authors demonstrate this using both heterogeneous inbred families to compare the two alleles directly to each other, and using a mutant line to compare one of the two alleles to a knockout. The HIF approach is more useful for understanding the functional consequences of naturally occurring genetic variation, but it is nice that the results from the two methods made for a consistent story. I appreciated that the alleles' effects (or lack thereof) were assessed both at the phenotype level and the molecular (transcription) level, and also that the authors tested for evidence of selection in the invisible fraction by bulk-genotyping multiple generations of plants in each environment. These elements make the paper a fairly thorough exploration of the fitness consequences of ACD6 in greenhouse and field environments, with a particular focus on its effect on plant-microbe interactions.

Here are my major comments.

1. Unfortunately, I am unable to evaluate the data analysis because many key details were missing from the manuscript. Basic information such as the sample sizes for each experiment, replication of each genotype, was not provided. For the DESeq2 analysis, the model being tested was not mentioned, making it impossible to interpret the results. For example, did the model simultaneously account for genotype and environment or was it done separately for each environment? Details about the statistical analysis of rosette size data were not provided - the figure captions mention Mann-Whitney U tests, which implies that many such tests were conducted to compare pairs of genotypes in various experiments and environments. This is a classic case where P-values must be adjusted to correct for the inflated Type I error rate, but there is no mention of this being done.

2. Following my first comment: Anyway, repeated pairwise tests cannot provide sufficient statistical support for the main conclusion of this paper. As aptly put by Gelman and Stern in 2012 (https://doi.org/10.1198/000313006X152649), the difference between P<0.05 in one environment and P>0.05 in a second environment is not necessarily statistically significant. Fundamentally this paper is about a genotype-by-environment interaction, which corresponds to a hypothesis that can only be tested with an interaction term followed by post-hoc testing. This applies to the rosette size data as well as the gene expression and microbiome analyses.

3. In my opinion the potential impact is unclear and the paper is missing a broader context. Will these results be interesting to readers who are not interested in Arabidopsis genetics, plant immunology, or microbiomes? I do see how it is useful as a cautionary tale for inferring ecological and evolutionary phenomena from greenhouse studies. The authors do mention balancing selection but it is not really clear how this story advances our understanding of balancing selection. Possibly this just requires some re-framing in the introduction and discussion.

4. I am confused about how and when the HIF individuals were genotyped - page 4 states that only the HIF:Est-1 and HIF:Col-0 genotypes (along with Est-1 wildtype) were planted in the greenhouse and outdoors, but the method for genotyping required destructive harvesting. Presumably half of the HIF seed would have been heterozygous, were these also included in the experiment but their phenotypic data not analyzed? Or was there some way of discerning ACD6 genotype prior to planting in the field?

5. Figure 2c, it was not clear whether this analysis used data from all environments, or one environment. The methods for this analysis seem to be missing as well.

6. More information on the "secure open-air room" field setting would be helpful to put in context how comparable the results should be to a "normal" field setting. For example, how is it enclosed? Is it accessible to insects? Are the plants exposed to wind?

7. A few unexplained acronyms - page 12, Pv-ATUE5, I assume these are different species of Pseudomonas? Page 3, EMS

8. Figure 4, panel c.i, the "NS" arrows seem to be pointing to nothing

Dear Dr Lundberg,

Thank you for your patience while we considered your revised manuscript "Field conditions greatly modify a major growth-defense tradeoff in Arabidopsis thaliana" for publication as a Short Report at PLOS Biology. This revised version of your manuscript has been evaluated by the PLOS Biology editors, the Academic Editor and one of the original reviewers.

Based on the review, we are likely to accept this manuscript for publication, provided you satisfactorily address the following data and other policy-related requests.

IMPORTANT - please attend to the following:

a) Please could you make your Title more explicit and accessible to a broader readership? We suggest: "A major tradeoff between growth and pathogen defense in hyperactive ACD6 Arabidopsis thaliana is absent in field conditions"

b) After some discussion, we've decided that your paper would be more appropriate for a Short Report format. As your manuscript is already succinct, no re-formatting is needed, and I have simply flipped the article type on your behalf. No further action is required.

c) Please address my Data Policy requests below; specifically, we need you to supply the numerical values underlying Figs 1B, 2ABDEFG, 3C, 4ABCD, S2, S3, S5, S6AB, S7ABCDE, either as a supplementary data file or as a permanent DOI’d deposition.

d) 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

e) 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 Roberts

Roland Roberts, PhD

Senior Editor

rroberts@plos.org

PLOS Biology

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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 1B, 2ABDEFG, 3C, 4ABCD, S2, S3, S5, S6AB, S7ABCDE. 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.

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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

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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).

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REVIEWER'S COMMENTS:

Reviewer #2:

The authors have done a good job of addressing my concerns with the original manuscript, particularly by presenting their results focused on effect sizes, and by adding statistical detail. I also thought the introduction was much improved, and the paper is an interesting story of a polymorphism that appears to be maintained by balancing selection, but which resists characterization under natural field conditions.

Dear Dr Lundberg,

Thank you for the submission of your revised Short Report "A major tradeoff between growth and defense in Arabidopsis thaliana can vanish in field conditions" for publication in PLOS Biology. On behalf of my colleagues and the Academic Editor, Pamela Ronald, 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 Roberts

Roland G Roberts, PhD, PhD

Senior Editor

PLOS Biology

rroberts@plos.org