Arabidopsis assemble distinct root-associated microbiomes through the synthesis of an array of defense metabolites

Enoch Narh Kudjordjie; Rumakanta Sapkota; Mogens Nicolaisen

doi:10.1371/journal.pone.0259171

Peer Review History

Original SubmissionSeptember 5, 2021
25 Sep 2021 Decision Letter - Zonghua Wang, Editor PONE-D-21-28774Arabidopsis assemble distinct root-associated microbiomes through the synthesis of an array of defense metabolitesPLOS ONE Dear Dr. Nicolaisen, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Nov 09 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: No Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors carried out a microbiota survey around the rhizosphere of the selected Arabidopsis mutants that are defects in metabolites such as plant hormone, Glucosinolates, and found that there is a relationship between specific mutations and certain bacterial or fungal groups that are differentially enriched or depleted. Although there is no mechanistic relationship investigated, and it is hard to conclude if the target metabolites are causative of the microbiota phenotype, this report provide valuable information to generate hypothesis regarding plant metabolites influencing rhizosphere microbiota. Some suggestions to further improve this manuscript: 1. Please show the identification of the mutants used in this study and if corresponding metoblites are indeed altered in the rhizosphere. 2. For certain metabolites, exogenous application and microbiota survey have been reported. More comparisons between this study and corresponding previous work will provide stronger evidence that the metabolites studied contribute to the phenotype. 3. According to the methods described, the seeds are directly germinated in soil without sterilization. How to make sure if pre-existing microbes in seeds didn’t significantly affects the analysis. Another issue is about sampling. How to control amount of fine soil attached to roots that are sampled? This will affect how to define rhizosphere in this study. 4. The figures and charts presented are not clear and sharp and should be in higher resolution. 5. Please double-check the references. There was duplicated references (for example, Ref#19, #22). 6. There are some writting errors to be corrected: Line 71: “single and few mutants” should be “signle and high-order mutants” Line 83: “Clean” could be deleted Line 87. “aba3-2” should be italic. Please check other mutant name. Line196: the nahG mutant expresses a foreign gene of which the product degrades SA. Reviewer #2: The study entitled “Arabidopsis assemble distinct root-associated microbiomes through the synthesis of an array of defense metabolites” by Kudjordjie et al. took advantage of various Arabidopsis mutant lines defective in the synthesis of glucosinolates (GLS), flavonoids (FLV) and defense hormones (DSMs) to assess their effects on root microbiome assembly. This study revealed dramatic yet complex effects the disruptions of different genes in these pathways have on both bacterial and fungal communities. Overall, this study presents an interesting link between plant genes and root-associated microbiota, the information of which could be further used for a more thorough multi-omics analysis in the future. Following are my comments: 1. Line 82: 21 mutant lines in total were used in this study. However, in Figure 1, only results for 17 mutant lines were presented. How about the remaining lines? Related to this question, in Figure S2 (A and B), sample npr1_2 showed extremely low numbers of reads and OTUs. What is the reason for that? 2. As the authors have pointed out in the paper, some resident root microbes could potentially degrade or transform plant metabolites. In addition, many root-associated microbes possess the ability to promote plant growth by producing plant hormones or antibiotics to ward off other microbes. Therefore, these possibilities could perhaps interfere with the assembly of root microbiota. In the future experiments, quantitative measurements of root exudates of these mutant lines are warranted to establish the link between plant metabolite and microbiota. Perhaps, a better experimental design is desired in the future to tease out the impacts from plant-derived metabolites and microbe-derived metabolites. Also some minor corrections need to be made in the text of manuscript: -Line 158, “Table ST2”: revised to “Table S2” -Lines 167-168, 170-171: Statements should be accompanied with results of statistical tests. -Line 186: tt3, tt3_1tt5_1 and tt5 should be compared to their parental line Ler-0, not Col-0. -Line 203, “p>0.01”: Did the authors actually mean “p<0.01”? -Line 223, “Bacteroidia”: revised to “Bacteroidetes” -Lines 238-239: Cite Table S11 in the text. -Line 259, “iGLS”: Provide the full name. -Line 266, “…although significant, effects…” -Line 324, “Holtermanniella takashimae”: This is a species name, not a genus name. -Line 372: Add “on” before “its host associated microbial communities”. ****** 6. 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 [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment 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. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. 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Revision 1
11 Oct 2021 Author Response Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Yes ________________________________________ 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ________________________________________ 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: No Response We have provided all data used in this study as stated in the availability of data and materials section “The metadata and sequencing data from MiSeq paired end reads for bacterial 16 s rRNA gene (V3-V4) and fungal ITS2 regions have been deposited in the NCBI Sequence Read Archive (SRA) database under the accession number PRJNA579829” in lines 424-426. Reviewer #2: Yes ________________________________________ 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ________________________________________ 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors carried out a microbiota survey around the rhizosphere of the selected Arabidopsis mutants that are defects in metabolites such as plant hormone, Glucosinolates, and found that there is a relationship between specific mutations and certain bacterial or fungal groups that are differentially enriched or depleted. Although there is no mechanistic relationship investigated, and it is hard to conclude if the target metabolites are causative of the microbiota phenotype, this report provide valuable information to generate hypothesis regarding plant metabolites influencing rhizosphere microbiota. Some suggestions to further improve this manuscript: 1. Please show the identification of the mutants used in this study and if corresponding metoblites are indeed altered in the rhizosphere. Response We have provided a detailed description of mutant lines, as well as essential references in Table S1. These lines are well characterized in other studies and constituted the basis for their selection for this study. ¬ 2. For certain metabolites, exogenous application and microbiota survey have been reported. More comparisons between this study and corresponding previous work will provide stronger evidence that the metabolites studied contribute to the phenotype. Response We acknowledged the suggestion by the reviewer for a more detailed comparisons of our results with previous in-vitro studies. We provided further comparisons as suggested by the reviewer in the revised manuscript. Please, see lines 290-294 and 331-334. 3. According to the methods described, the seeds are directly germinated in soil without sterilization. How to make sure if pre-existing microbes in seeds didn’t significantly affects the analysis. Response All the seeds were multiplied in arasystem using the same soil. (https://www.arasystem.com). The aim of the present work was to study microbiomes in a natural setting, and we therefore used non-sterilized seeds (both mutants and their parental lines) that were produced and stored in clean tubes. We assume that both mutants and their parental lines would assemble similar microbiomes and in effect would not differentially affect mutant and wildtype Arabidopsis root microbiomes. Moreover, sampling was done 4 weeks after planting, a time period we considered relatively long to enable the manifestation of soil/root-mediated (caused by metabolites) microbiome assembly. Another issue is about sampling. How to control amount of fine soil attached to roots that are sampled? This will affect how to define rhizosphere in this study. Response To our knowledge, no method for sampling roots/rhizosphere would be completely unbiased. We shook roots until all loose soils were removed. The remaining fine soil and root were then collected for analysis. Although this approach may result in some biases, we were meticulous in ensuring uniformity during shaking. Moreover, our aim was to profile microbiotas associated with the plant compartment (i.e roots and rhizosphere) that are strongly influenced by the metabolites, and thus make this sampling approach more appropriate. 4. The figures and charts presented are not clear and sharp and should be in higher resolution. Response We have regenerated most of the figures (Figs 1-4) and modified Figs-5 and 6 to meet the publication criteria. 5. Please double-check the references. There was duplicated references (for example, Ref#19, #22). Response We have cross-checked the references and edited accordingly. 6. There are some writting errors to be corrected: Line 71: “single and few mutants” should be “signle and high-order mutants” Response We have rephrased to clarify. See line 72 Line 83: “Clean” could be deleted Response This was corrected. See line 87 Line 87. “aba3-2” should be italic. Please check other mutant name. Response We corrected and italized all mutants in the text. See line 89. Line196: the nahG mutant expresses a foreign gene of which the product degrades SA. Response For simplicity, we stated that SA is degraded as well in the pen3_1_NahG mutant. See lines 201-205. Reviewer #2: The study entitled “Arabidopsis assemble distinct root-associated microbiomes through the synthesis of an array of defense metabolites” by Kudjordjie et al. took advantage of various Arabidopsis mutant lines defective in the synthesis of glucosinolates (GLS), flavonoids (FLV) and defense hormones (DSMs) to assess their effects on root microbiome assembly. This study revealed dramatic yet complex effects the disruptions of different genes in these pathways have on both bacterial and fungal communities. Overall, this study presents an interesting link between plant genes and root-associated microbiota, the information of which could be further used for a more thorough multi-omics analysis in the future. Following are my comments: 1. Line 82: 21 mutant lines in total were used in this study. However, in Figure 1, only results for 17 mutant lines were presented. How about the remaining lines? Related to this question, in Figure S2 (A and B), sample npr1_2 showed extremely low numbers of reads and OTUs. What is the reason for that? Response That is correct. 21 mutant lines were used in the study. However, few of the mutant lines including for example, the cyp79B2, pad2-1, fad3_2 npr1_2 in the bacterial dataset had less than 3 replicates after quality filtering (Figure S2) and were thus removed in downstream analysis. We stated this in lines 131-134. 2. As the authors have pointed out in the paper, some resident root microbes could potentially degrade or transform plant metabolites. In addition, many root-associated microbes possess the ability to promote plant growth by producing plant hormones or antibiotics to ward off other microbes. Therefore, these possibilities could perhaps interfere with the assembly of root microbiota. In the future experiments, quantitative measurements of root exudates of these mutant lines are warranted to establish the link between plant metabolite and microbiota. Perhaps, a better experimental design is desired in the future to tease out the impacts from plant-derived metabolites and microbe-derived metabolites. Response We agree with this assertion and we strongly anticipated that future studies will be guided by the findings presented herein, in designing inter-omics experiments that will increase our understanding of mechanisms involved in plant-microbiome assembly. Also some minor corrections need to be made in the text of manuscript: -Line 158, “Table ST2”: revised to “Table S2” Response Corrected. See line 163. -Lines 167-168, 170-171: Statements should be accompanied with results of statistical tests. Response We provided detailed statistics with asterisks in Figure 1, and text description of Figure 1; “Test of statistical significance was performed by comparing each mutant to their respective background parental lines, and the significantly affected taxa are indicated as * (P<0.001), (P<0.01) and * (P<0.05). ANOVA tests were followed by the Tukey–Kramer post hoc test using the Benjamini and Hochberg (BH) FDR for multiple comparisons”. -Line 186: tt3, tt3_1tt5_1 and tt5 should be compared to their parental line Ler-0, not Col-0. Response Thank you. This was a typo. We corrected it in the revised manuscript. See line 191. -Line 203, “p>0.01”: Did the authors actually mean “p<0.01”? Response Thank you. This was corrected. See line 208. -Line 223, “Bacteroidia”: revised to “Bacteroidetes” Response Thank you. This has been corrected. See line 228 -Lines 238-239: Cite Table S11 in the text. Response Thank you. This has been corrected. See line 249 -Line 259, “iGLS”: Provide the full name. Response Thank you. This has been edited. See line 264 -Line 266, “…although significant, effects…” Response This statement was corrected to … “but significant effect on the microbiome”. See line 272 -Line 324, “Holtermanniella takashimae”: This is a species name, not a genus name. Response Thank you. This has been edited. See line 338 -Line 372: Add “on” before “its host associated microbial communities”. Response Thank you. This has been edited. See line 387. Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0259171.r002
14 Oct 2021 Decision Letter - Zonghua Wang, Editor Arabidopsis assemble distinct root-associated microbiomes through the synthesis of an array of defense metabolites PONE-D-21-28774R1 Dear Dr. Mogens Nicolaisen, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Zonghua Wang, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0259171.r003
Formally Accepted
18 Oct 2021 Acceptance Letter - Zonghua Wang, Editor PONE-D-21-28774R1 Arabidopsis assemble distinct root-associated microbiomes through the synthesis of an array of defense metabolites Dear Dr. Nicolaisen: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Prof. Zonghua Wang Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0259171.r004

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