Dear Dr. Stougaard,

Thank you for submitting your manuscript entitled "A Lotus japonicus glycan receptor kinase facilitates intracellular accommodation of arbuscular mycorrhiza and symbiotic rhizobia." for consideration as a Research Article by PLOS Biology.

Your manuscript has now been evaluated by the PLOS Biology editorial staff, as well as by an academic editor with relevant expertise, and I am writing to let you know that we would like to send your submission out for external peer review.

However, before we can send your manuscript to reviewers, we need you to complete your submission by providing the metadata that is required for full assessment. To this end, please login to Editorial Manager where you will find the paper in the 'Submissions Needing Revisions' folder on your homepage. Please click 'Revise Submission' from the Action Links and complete all additional questions in the submission questionnaire.

Once your full submission is complete, your paper will undergo a series of checks in preparation for peer review. After your manuscript has passed the checks it will be sent out for review. To provide the metadata for your submission, please Login to Editorial Manager (https://www.editorialmanager.com/pbiology) within two working days, i.e. by Jun 22 2022 11:59PM.

If your manuscript has been previously peer-reviewed at another journal, PLOS Biology is willing to work with those reviews in order to avoid re-starting the process. Submission of the previous reviews is entirely optional and our ability to use them effectively will depend on the willingness of the previous journal to confirm the content of the reports and share the reviewer identities. Please note that we reserve the right to invite additional reviewers if we consider that additional/independent reviewers are needed, although we aim to avoid this as far as possible. In our experience, working with previous reviews does save time.

If you would like us to consider previous reviewer reports, please edit your cover letter to let us know and include the name of the journal where the work was previously considered and the manuscript ID it was given. In addition, please upload a response to the reviews as a 'Prior Peer Review' file type, which should include the reports in full and a point-by-point reply detailing how you have or plan to address the reviewers' concerns.

During the process of completing your manuscript submission, you will be invited to opt-in to posting your pre-review manuscript as a bioRxiv preprint. Visit http://journals.plos.org/plosbiology/s/preprints for full details. If you consent to posting your current manuscript as a preprint, please upload a single Preprint PDF.

Feel free to email us at plosbiology@plos.org if you have any queries relating to your submission.

Kind regards,

Paula

Paula Jauregui, PhD,

Senior Editor

PLOS Biology

pjaureguionieva@plos.org

Dear Dr. Stougaard,

Thank you for your patience while your manuscript "A Lotus japonicus glycan receptor kinase facilitates intracellular accommodation of arbuscular mycorrhiza and symbiotic rhizobia." was peer-reviewed at PLOS Biology. Your manuscript has been evaluated by the PLOS Biology editors, an Academic Editor with relevant expertise, and by several independent reviewers.

As you will see in the reviewer reports, which can be found at the end of this email, although the reviewers find the work potentially interesting, they have also raised a substantial number of important concerns. Based on their specific comments and following discussion with the Academic Editor, it is clear that a substantial amount of work would be required to meet the criteria for publication in PLOS Biology. However, given our and the reviewer interest in your study, we would be open to inviting a comprehensive revision of the study that thoroughly addresses all the reviewers' comments. Given the extent of revision that would be needed, we cannot make a decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript would need to be seen by the reviewers again, but please note that we would not engage them unless their main concerns have been addressed.

You will see that the reviewers agree that the manuscript is an interesting article and a potentially important contribution to the field, particularly because there are just a few ECD from RLKs that have been described to bind glycans, but consider that the work would need a substantial revision, additional experiments, and probably a relevant reorientation of the conclusion and models, to provide the strength of advance that PLOS Biology strives to publish. At the end of this letter you will find the comments from the academic editor where the most important points to address are highlighted.

We appreciate that these requests represent a great deal of extra work, and we are willing to relax our standard revision time to allow you 6 months to revise your study. Please email us (plosbiology@plos.org) if you have any questions or concerns, or envision needing a (short) 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 may withdraw it.

**IMPORTANT - SUBMITTING YOUR REVISION**

Your revisions should address the specific points made by each reviewer. Please submit the following files along with your revised manuscript:

1. A 'Response to Reviewers' file - this should detail your responses to the editorial requests, present a point-by-point response to all of the reviewers' comments, and indicate the changes made to the manuscript.

*NOTE: In your point by point response to the reviewers, please provide the full context of each review. Do not selectively quote paragraphs or sentences to reply to. The entire set of reviewer comments should be present in full and each specific point should be responded to individually, point by point.

You should also cite any additional relevant literature that has been published since the original submission and mention any additional citations in your response.

2. In addition to a clean copy of the manuscript, please also upload a 'track-changes' version of your manuscript that specifies the edits made. This should be uploaded as a "Revised Article with Changes Highlighted " file type.

*Resubmission Checklist*

When you are ready to resubmit your revised manuscript, please refer to this resubmission checklist: https://plos.io/Biology_Checklist

To submit a revised version of your manuscript, please go to https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' where you will find your submission record.

Please make sure to read the following important policies and guidelines while preparing your revision:

*Published Peer Review*

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. Please see here for more details:

https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/

*PLOS Data Policy*

Please note that as a condition of publication PLOS' data policy (http://journals.plos.org/plosbiology/s/data-availability) requires that you make available all data used to draw the conclusions arrived at in your manuscript. If you have not already done so, you must include any data used in your manuscript either in appropriate repositories, within the body of the manuscript, or as supporting information (N.B. this includes any 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

*Blot and Gel Data Policy*

We require the original, uncropped and minimally adjusted images supporting all blot and gel results reported in an article's figures or Supporting Information files. We will require these files before a manuscript can be accepted so please prepare them now, if you have not already uploaded them. Please carefully read our guidelines for how to prepare and upload this data: https://journals.plos.org/plosbiology/s/figures#loc-blot-and-gel-reporting-requirements

*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

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,

Paula

---

Paula Jauregui, PhD,

Senior Editor

PLOS Biology

pjaureguionieva@plos.org

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

REVIEWS:

Reviewer #1: Rhizobia and Arbuscular Mycorrhizal Symbiosis including receptor signaling

Reviewer #2: Glycan sensing in plants

Reviewer #1: In the manuscript entitled "A Lotus japonicus glycan receptor kinase facilitates intracellular accommodation of arbuscular mycorrhiza and symbiotic rhizobia" Kelly et al. identified a novel LysM-RLK protein EPR3a from L. japonicus. They performed biochemical, molecular, and functional characterization of EPR3a in arbuscular and legume-rhizobia symbioses. In this manuscript, the authors investigated the binding of EPR3a to branched glycans and the perception of rhizobial exopolysaccharide, which is one of the determinants of the specificity of plant-microbe interaction. The authors used LORE1 mutant lines of EPR3a for functional characterization of this protein in arbuscular mycorrhiza association. Finally, the authors used kinase assays to validate in vitro kinase activity of EPR3 and EPR3a. In my opinion, this manuscript suffers from significant flaws.

1- Many essential controls and critical experiments are missing. This story is highly biased towards symbiosis while it seems likely that EPR3 is a general fungal MAMP receptor:

* The authors performed binding assays between EPR3a ectodomain and glycans obtained from algae (E. bicyclis and L. digitata)... and a fungus irrelevant to the biology of L. japonicus. Since they try to make a case for the role of EPR3a protein in AM symbiosis, the authors should have used fungal extracts from AM fungi and possibly pathogenic fungi relevant to L. japonicus. Using biologically relevant fungal cell wall extracts, the authors would have addressed whether the EPR3a activity is conserved across fungi, involving symbiotic and pathogenic interactions and whether symbiotic or pathogenic interaction dominates in virtue of EPR3a activity.

* The authors checked the nodule number and arbuscular formation, the mature stages of root nodule, and AM symbiosis. Since the protein acts downstream of Nod factor perception, they should have checked the change in expression of genes involved in the SYM pathway, such as NIN and nuclear calcium spiking. Do glycans activate calcium spiking in L. japonicus? Do glycans activate or repress defense signaling in L. japonicus?

* Since the binding seems conserved with algal glycans, it seems likely that glycans from pathogenic fungi will bind EPR3a too. The authors should not frame their story so narrowly around symbiosis and test phenotypes of L. japonicus epr3a mutants with pathogenic fungi too.

* Since the authors used LORE1 mutants in this study which possess several insertions in the genome, it is not sure if the phenotype obtained is due to non-functional EPR3 and EPR3a only. It is necessary to perform some complementation assays to determine that the observed phenotypes are due to mutations in EPR3a or EPRw, not mutations in other genes.

* The authors performed an RT-qPCR in the root, nodules, and arbuscular mycorrhizal symbiosis. However, they have not shown any data on the expression of EPR3a in other plant tissues or response to fungal pathogens. While missing the expression analysis in other tissues, the authors neglected its role in different plant tissues or biotic interactions.

2- Inconsistencies between the data presented in figures and text.

* Line 102: "In this study, we report on the identification and characterization of a novel glycan receptor kinase in Lotus that we have designated EPR3a." The authors identified a novel receptor kinase; however, Line 125 says, "A wider search in the genome databases shows that this class of LysM-RLKs is widely conserved in the plant kingdom (22), with either EPR3a or EPR3 or both present in genomes of most plants forming endosymbiosis with mycorrhiza and/or rhizobia (26)." The authors have not presented any data to show the conservation of EPR3a in other plant genomes, and the same is not present in the references cited.

* In the glycans binding assays in Fig. 4, the authors have not used the EPR3 binding ligand. EPR3 binding ligand would have ensured that the EPR3 protein used in the assay was functionally active. Additionally, there are two bands in the EPR3 lanes; can the authors give a plausible reason behind the two bands in the case of EPR3.

* In Supplementary Fig. 6, the authors showed that EPR3 could bind with a low affinity to E. bicyclis glycan; however, the gel in Fig. 4 does not correlate with this statement.

* EPR3a binds with a stronger affinity to S. indica decasaccharide (Kd:~51.2 μM) compared to E. bicyclis glycan (Kd:~247.7 μM), which again indicates that more assays are required to determine the binding affinity of EPR3a with pathogenic or symbiotic signals.

* As per Fig. 5, EPR3a shows higher binding affinity with R. leguminosarum EPS (Kd: ~12.5 μM) compared to M. loti EPS (Kd: ~44.4 μM). This result is contradictory as M. loti is a host for L. japonicus.

3- Some statements are inaccurate:

* Line 135: "In contrast, Epr3 expression did not differ between mock and AM treatments." however, the expression of EPR3 differed between mock and AM treatments at 2 DPI.

* In Line 171 authors state, "Arbuscule development in epr3a mutants appears indistinguishable from arbuscule development in wild-type plants, suggesting a role for EPR3a in fungal entry into cortical cells rather than in arbuscule accommodation." however, in line 182 authors make a statement that, "Localisation of Epr3a expression in arbusculated cells and the reduced frequency of arbuscule development in epr3a-1 and epr3a-2 mutants." These two statements are contradictory; maybe the authors wanted to make a statement about arbuscular structure.

Reviewer #2: The article by Kelly et al., describes the identification and characterization of a glycan receptor kinase, EPR3a, closely related to the exopolysaccharide (EPS) receptor EPR3 previously described. EPR3a seems to bind β-1,3 glucans with different β-1,6 branching patterns, which have been described to be characteristic of surface-exposed fungal glucans. EPR3a plays a function in the colonization of Arbuscular Mycorrhiza (AM), since epr3a mutants are defective in different steps of the colonization process. Since cell walls/surface of AM has been suggested to contain β-1,3/β-1,6 glucan, the authors conclude that EPR3a is involved in the perception of this type of glucans from AM. However, purification of these β-1,3/β-1,6 glucans from AM cell walls has not been either performed previously nor in the current article. The demonstration of β-1,3/β-1,6 glucans binding by the extracellular domains (EC), with LysM structures, of EPR2a and EPR has been demonstrated using two approaches: i) affinity gel electrophoresis assays, that is not a very accurate approach, and ii) MST, that is a more precise technology, though clearly has some limitations in EC-glucan binding assays due to precipitation of glucans at high concentration. Based on MST data, the authors conclude that EPR3a binds different types of β-1,3/β-1,6 glucans, but also rhizobial exopolysaccharide (EPS) that is mainly enriched in β-1,6 glucans. Notably, the affinities of EPR3a for the complex mixtures of glucans tested are comparable to those previously described for EPS-EC-EPR3. Though the article lacks the characterization of the specific β-1,3/β-1,6 glucan structure/s recognized by EPR3a and EPR3, the authors show that their ECs bind β-GD, a well-defined β-1,3/β-1,6 glucan present in different fungi. Based on these findings they propose that the activity of EPR3-type receptors in plants is to monitor glycans produced by microbes during their colonization and to regulate intracellular accommodation of microbes and the adaptative response of AM. The results of the article suggest contrasting expression patterns and the authors suggest divergent ligand affinities of EPR3a and EPR3 that explain their distinct functions in AM colonization and rhizobial infection in Lotus japonicus, though these differences in specificity for some ligands tested are quite low and accordingly some conclusion should be weakened. The article contains very interesting new data, and clearly contributes to increase the number of plants RLKs that have been shown to bind glycans, expanding our knowledge in this field of RLK-ligan (glycan) recognition.

Major Points:

1. Purified glucans from AM fungal cell walls are not available, therefore, the binding capacity of EPR3a ectodomains was tested in native affinity gel electrophoresis and MST using β-glucans that might or not be present in AM cell walls. The laminarins used in the experiments, that are β-1,3 glucans with β-1,6 branched found in fungal cell wall, are quite complexes in their composition. Though the results of MST seem to be clear, the fact that these very complex mixtures of glucans were used for the binding assays do not contribute to clarify the minimal structure that is perceived by these ECs. Though the article shows that β-GD, a well-defined β-1,3/β-1,6 glucan is bound by both EC receptors, it is not clear if this minimal structure is present in AM cell walls. It would be very interesting to test the binding of short structures (Degree of Polymerizayion of 6 to 12) of pure linear β-1,3 and linear β-1,6 glucan in MST assays or ITC . Some of these pure oligosaccharides are commercially available. These analyses will clarify if β-1,6 branching is really required or not for ECs binding of β-glucans. It is hard to understand at the structural level that ECs of EPR3a and EPR could bind laminarin and EPS.

2. The use of glucan preparations (e.g. scleroglucan, pustulan) enriched in some β-glucans as negative controls might not be appropriate since, as the authors indicate, to work with these polymeric preparations is kind of risky because they contained a diversity of unknown structures and some binding/not binding results could be due to changes in solution viscosity (gelation) and precipitation, which is a well-known property of β-glucans at high concentrations. This is clearly a limitation of working with glucans, but some additional experiments (see 1) to further support authors conclusion could be performed.

3. Interestingly, EPR3a ectodomains can also bind rhizobial EPS from M. loti, S. meliloti, and R. leguminosarum with μM affinity, and the affinity for M. loti EPS is comparable to that of S. indica decasaccharide β-glucan. As indicated in 1, this is quite surprising and interesting, but also indicates that the binding specificity for some glucans is low or the glucan structures present in EPS and laminarin which are bound by ECs are more similar than anticipated.

4. Surprisingly, epr3a epr3 double mutants were less severely impaired than epr3a single mutants, forming IT numbers comparable to epr3-11, This result suggests direct interaction between the EPR3a and EPR3 receptors or convergence of downstream signal transduction pathway. Based on these results, the authors suggests that EPR3 in the absence of EPR3a is a negative regulator and EPR3a normally acts to counter this negative regulation. However, this hypothesis has not been validated testing the regulation by EPR3a and EPR3 of downstream components of AM and rhizobia colonization pathways. The characterization of downstream events is in general poor in this article.

Minor Points

1. Legends of Figures with MST data are incomplete. Some concepts like n, different time points, etc. are not well explained in the legend to figures. The values in the Y axe is not explained either.

2. The authors used to ecotypes of Lotus japonica Gifu and Lotus Base (mutants background). Are those genotypes the same? This must be clarified.

3. Reference 27 is incomplete

4. Perception of β-GD, a well-defined β-1,3/β-1,6 glucan present in different fungi was found to be perceived by EPR3 and EPR3a receptors, but it has been described to be released by fungi for ROS scavenging beta-glucan to subvert immune responses. How this function of β-GD match with the binding data and functions of EPR3a and EPR3? Is Ros production altered in EPR3a and EPR3 mutants treated with β-GD?

5. The Figure of the expression of EC proteins is not clear explained in Legends to Figures.

COMMENTS FROM THE ACADEMIC EDITOR:

In particular, I would like to see the following major points addressed:

1) the limitations of using of crude preps from algae rather than relevant fungi or purified glucans was brought up by both reviewers and calls into question the specificity of the receptor. This should be addressed with either commercially available purified molecules or crude preps from AM fungi with relevant controls.

2) I also agree that testing how EPR3a and EPR3 affect downstream regulation of symbiosis signaling is important to put these receptors into the context of the extensive literature.

3) I do agree that the findings are biased towards AM fungi/symbiosis and it is entirely possible that the proteins also play a role in general immunity against fungi. I think addressing this point from reviewer 1 would strengthen the paper. However, I don’t think their fundings or conclusions preclude this idea and so I think this could also be addressed by modifying the conclusions.

4) a point-by-point response to all other concerns brought up by reviewers.

Dear Dr. Stougaard,

Thank you for your patience while we considered your revised manuscript "A Lotus japonicus glycan receptor kinase facilitates intracellular accommodation of arbuscular mycorrhiza and symbiotic rhizobia." for publication as a Research Article 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 reviews and our Academic Editor's assessment of your revision, we are likely to accept this manuscript for publication, provided you satisfactorily address the remaining points raised by the reviewers. You should respond to the reviewer's concerns and we consider that the issues can be addressed by text modifications. Regarding the issue about "pure structures should be tested”, we think you should clarify earlier in the manuscript that “laminarin, which has the beta-1,6 branched beta-1,3 glucans also found in fungal cell walls” is a mix of different beta-1,6 branched beta 1,3 glucans as you resolve later by NMR. Regarding EPR3 having similar binding affinity as EPR3a (page 27, line 238-240), you show that EPR3a uniquely binds to beta-1,6 branched beta-1,3 glucans while both bind to beta-1,3 glucans with similar affinity, and we think that this does not undermine the central conclusions of the manuscript. Regarding the ROS data, we consider that pathogen assays are beyond the scope of the manuscript.

Please also make sure to address the following data and other policy-related requests.

1. 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:

A) 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).

B) 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: Figures 1A, 2A, 3ABC, 5BCD, 6ABCD, 7A, 8ABC, and Supplementary Figures S4A, S6ABCD, S7BC, S8AC, S9BE, S10ABCDEFG, S11ABCD, S13ABCD.

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).

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.

2. We suggest a change in the title to include that Lotus japonicus is a legume: "A glycan receptor kinase facilitates intracellular accommodation of arbuscular mycorrhiza and symbiotic rhizobia in the legume Lotus japonicus".

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.

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)

- 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://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/

*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,

Paula

---

Paula Jauregui, PhD,

Senior Editor,

pjaureguionieva@plos.org,

PLOS Biology

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

Reviewer remarks:

Reviewer #2: This is a clear improved version of the previous submitted article. Authors have clarified the majority of questions addressed, and have completed the majority of the information that was lacking in the previous article. The articles now shows that EPR3a binds β-1,3 glucans (Lam5 and Lam6) though with lower affinity than β-glucans with β-1,6-branches. However, this analysis is incomplete (additional pure structures should be tested) but based on the β-glucans available for comparison we might accept the experiments performed as those that were feasible. Surprisingly, the difference in specificity of EPR and EPR3a for the decasaccharide β-glucan is very low (it is almost identical).

The RNaseq data clarify some of the questions regarding the transcriptional responses activated. It is a shame that the authors did not include in the RNaseq analyses the beta-glucan that they used for binding experiments for comparison since this had been provided cleaner information about the responses triggered by the ligand and not just by the whole microorganism.

Last, the ROS response observed upon treatment with the β-glucan provides important information. However, since the decasaccharide is not triggering ROS, as described previously, it seems that ROS production observed upon L. digitata laminarin treatment, that is not reduced in EPR and EPR3a mutants, should be triggered by other β-glucan structures distinct from the decasaccharide. The interpretation of the authors might not be correct. I agree with reviewer 2 that the infection of mutants with a Lotus pathogen will contribute to clarify some key aspects of the function of the EPR3a receptor, but these experiments could be the matter of a different history.

Dear Dr. Stougaard,

Thank you for the submission of your revised Research Article "A glycan receptor kinase facilitates intracellular accommodation of arbuscular mycorrhiza and symbiotic rhizobia in the legume Lotus japonicus" for publication in PLOS Biology. On behalf of my colleagues and the Academic Editor, Cara Haney, I am 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, 

Paula

---

Paula Jauregui, PhD,

Senior Editor

PLOS Biology

pjaureguionieva@plos.org