Linnemannia elongata (Mortierellaceae) stimulates Arabidopsis thaliana aerial growth and responses to auxin, ethylene, and reactive oxygen species

Natalie Vandepol; Julian Liber; Alan Yocca; Jason Matlock; Patrick Edger; Gregory Bonito

doi:10.1371/journal.pone.0261908

Peer Review History

Original SubmissionDecember 8, 2021
11 Feb 2022 Decision Letter - Sabrina Sarrocco, Editor PONE-D-21-37581Linnemannia elongata (Mortierellaceae) stimulates Arabidopsis thaliana aerial growth and responses to auxin, ethylene, and reactive oxygen speciesPLOS ONE Dear Dr. Bonito, 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. The paper has been submitted to te revision of two experts who both generally positively evaluated it. However both suggested some minor revisions that should be included in the paper in order to improve it and render acceptable for publication. Authors are invited to carefully follow suggestions from reviewers and modify the text according it. Please submit your revised manuscript by Mar 28 2022 11:59PM. 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Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.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: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. 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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: This is an interesting study to examine the role of Linnemannia elongata (Mortierellaceae) and its endosymbionts in Arabidopsis thaliana aerial growth, although endosymbionts had no significant effects on Arabidopsis performance and the expression of genes in Arabidopsis in this study. Overall, the study is solid, and the manuscript is well written. However, there are a few areas of the manuscript that could be strengthened a little. Abstract: it may be necessary to state the significance of the study at the end of abstract. Introduction: L47: Please add the refs. M&M: Please provide some information about two isolates of L. elongata, NVP64 and NVP80. For example, where did you isolate? L143. Please correct the format of the ref. L193. Please show the ingredients for the ratio. L234. How many replicates/plates for each treatment in the agar-based experiment. L324. What do you mean of “2x”? L352. Superscript L373. Please correct the format of ref. L.376. You randomly selected five plants for extracting root RNA. Why are there only three biological replicates? Results: L408. Somehow, fungal treatment can offset the negative effect of millet on plant growth? L414. Fig. 3a L416-417. From Fig. 3b, it seems that uninoculated control also had slightly higher average seed mass than NVP64wt and NVP80cu. L428. Plants? L433. Please correct the format of ref. L443-451. It is not clear where are these data from. Please cite figures or tables here. L485. How did you get this? Try to use statistical analysis (e.g., PERMANOVA) to reveal it. L508-509. Please show the specific GOs here and site Table 2. Discussion: L529. It is surprising that bacterial endosymbionts negatively impact the growth of Linnemannia. According to the previous study (Büttner et al., 2021), bacterial endosymbionts (Ca. Mycoavidus necroximicus) can protect Mortierella/ Linnemannia from the attack of micro-predators. L540-544. This sentence is too long. Try to break it down into two sentences. L555. This sentence is less connected to previous ones. Is Serendipita (Piriformospora) indica phylogenetically close to L. elongate? L637. Refs? L649. Although K, P, and Fe are available in PNM medium, there is a great demand for plant growth in the fungal treatment. This is probably the main reason for so many down-regulated genes related to nutrients. L651. Correct the format of ref. Reviewer #2: The manuscript by Vandepol and colleagues presents the results of several experiments aiming to demonstrate Linnemania elongate as effective plant growth promoting organism and verifying the role of endobacteria in this process. Apart of mRNA sequencing Authors performed a series of classical agar and pot experiments aiming to measure plant biomass and seed production. I appreciate presentation of results, showing no significant influence of EHB on the PGP. These are rarely demonstrated and highly valuable for the community. However, it is not clearly explained why Authors assume EHB may have such an effect on PGP processes. The study is a valuable contribution proving existence and describing mechanisms of PGP in Mortierellomycotina. Below I present some minor comments. Lines 49-51: This sentence seems to be a far-reaching simplification. Especially, speaking about Mucoromycota it would be valuable to discuss at least mycorrhiza-like or paramycorrhiza concepts. Line 60: I would remove “some of which are EM fungi” – see previous comment Line 88: Arabidopsis once is italicized once without – please unify throughout the text Line 486: “DESeq2 provided p-values for each comparison” – DESeq2 is software package you used to perform specific type of analysis, please reformulate here and further eg. line 492. Line 540: I miss information on number of these replicates in methodological part. Please complete. The used strains should be deposited in public culture collection. ****** 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. 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Attachments Attachment Submitted filename: Recommendation.docx https://doi.org/10.1371/journal.pone.0261908.r001
Revision 1
26 Feb 2022 Author Response We appreciate the reviewers for their time and comments. Below we provide a point-by-point response to the peer-reviews. Reviewer 1 This is an interesting study to examine the role of Linnemannia elongata (Mortierellaceae) and its endosymbionts in Arabidopsis thaliana aerial growth, although endosymbionts had no significant effects on Arabidopsis performance and the expression of genes in Arabidopsis in this study. Overall, the study is solid, and the manuscript is well written. However, there are a few areas of the manuscript that could be strengthened a little. 1. Abstract: it may be necessary to state the significance of the study at the end of abstract. We’ve added a concluding significance statement: “Together, these results indicate that beneficial plant growth promotion and seed mass impacts of L. elongata on Arabidopsis are likely driven by plant hormone and defense transcription responses after plant-fungal contact, and that plant phenotypic and transcriptional responses are independent of whether the fungal symbiont is colonized by Mollicutes or Burkholderia-related endohyphal bacteria.” 2. Introduction : L47: Please add the refs. New references have been included. 3. M&M: Please provide some information about two isolates of L. elongata, NVP64 and NVP80. For example, where did you isolate? This has been included 4. L143. Please correct the format of the ref. This has been done. 5. L193. Please show the ingredients for the ratio. This has been added: “transplanted into 4 in3 pots with SureMix mixed with the appropriate millet mix treatment at 3% by volume.” 6. L234. How many replicates/plates for each treatment in the agar-based experiment. This has been added “The number of biological replicates per treatment varied by experiment as follows: 27 for the bolting assay, 12 for the media panel, and 27 for the endobacteria panel.” 7. L324. What do you mean of “2x”? We replaced with ‘twice’ 8. L352. Superscript Fixed 9. L373. Please correct the format of ref. Fixed 10. L.376. You randomly selected five plants for extracting root RNA. Why are there only three biological replicates? This was added to at LN311 to clarify: ‘three of which were selected as triplicate biological replicates for RNA sequencing based on having aerial dry weights closest to the mean for that treatment’ 11. Results:L408. Somehow, fungal treatment can offset the negative effect of millet on plant growth? This is correct, and is elaborated on further in the paragraph. 12. L414. Fig. 3a fixed 13. L416-417. From Fig. 3b, it seems that uninoculated control also had slightly higher average seed mass than NVP64wt and NVP80cu. This is reported on Ln 423: “Unlike total seed mass, the average seed mass of the Uninoculated control was slightly higher than NVP80wt and NVP64cu, but not significantly different from the NoMillet control (Fig. 3b)”. This pertains to the average mass of each seed (total seed mass/number of seeds). 14. L428. Plants? We removed ‘fungal’ so it now reads ‘treatments’, which refers to the inoculum used (NVP64+, NVP64-, NVP80+, NVP80-, control). 15. L433. Please correct the format of ref. fixed 16. L443-451. It is not clear where are these data from. Please cite figures or tables here. Analysis of variance analyses were used to determine which factors were significant for inclusion in the linear models. Following standard convention, non-significant factors based on ANOVAs are reported only in the text directly. 17. L485. How did you get this? Try to use statistical analysis (e.g., PERMANOVA) to reveal it. Thank you for suggesting we test this. Initially, this was an observation that there was no clustering based on the first two principal components in 2-dimensional space. Applying a PERMANOVA test in R (code shown at the bottom of the rnaseq.R file) revealed there was no significant clustering based on either isogenic strain or cured/wild-type. 18. L508-509. Please show the specific GOs here and site Table 2. We have included specific GO numbers here and have cited table 2. 19. Discussion: L529. It is surprising that bacterial endosymbionts negatively impact the growth of Linnemannia. According to the previous study (Büttner et al., 2021), bacterial endosymbionts (Ca. Mycoavidus necroximicus) can protect Mortierella/ Linnemannia from the attack of micro-predators. The negative impact of endosymbionts on Linnemannia growth in pure culture was previously published by Uehling et al 2017 and Desiro et al. 2018. It was not assessed in the Büttner et al., 2021 paper. 20. L540-544. This sentence is too long. Try to break it down into two sentences. This sentence has been rewritten. “While it is difficult to draw strong conclusions with so few replicates, fitness trade-offs in seed size and seed mass of plants growing in stressful environmental conditions compared to non-stressful conditions is an interesting topic for future work [47,48].” 21. L555. This sentence is less connected to previous ones. Is Serendipita (Piriformospora) indica phylogenetically close to L. elongate? Thank you. The sentence has been better connected by adding this transition. “However, although Serendipita and Linnemannia may both be found in plant roots, these fungi are phylogenetically distant, as Serendipita are Basidiomycetes while Linnemannia belong to Mucoromycota. Without further experiments to characterize nutrient exchange through these intracellular structures, it is not possible to ascribe structure with function [55].” 22. L637. Refs? Refs added 23. L649. Although K, P, and Fe are available in PNM medium, there is a great demand for plant growth in the fungal treatment. This is probably the main reason for so many down-regulated genes related to nutrients. We have revised this sentence Ln643: “Considering the availability of potassium, phosphorus, and iron in the PNM growth medium, it is striking that so many genes related to deficiencies of these nutrients were down-regulated compared to the control plants. The fungal mycelial network may have increased the availability of these nutrients to plant roots, by serving as an extension of the root system.” 24. L651. Correct the format of ref. This has been fixed Attachments Attachment Submitted filename: ResponseToReviewers.docx https://doi.org/10.1371/journal.pone.0261908.r002
1 Mar 2022 Decision Letter - Sabrina Sarrocco, Editor Linnemannia elongata (Mortierellaceae) stimulates Arabidopsis thaliana aerial growth and responses to auxin, ethylene, and reactive oxygen species PONE-D-21-37581R1 Dear Dr. Bonito, 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, Sabrina Sarrocco Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0261908.r003
Formally Accepted
10 Mar 2022 Acceptance Letter - Sabrina Sarrocco, Editor PONE-D-21-37581R1 Linnemannia elongata (Mortierellaceae) stimulates Arabidopsis thaliana aerial growth and responses to auxin, ethylene, and reactive oxygen species Dear Dr. Bonito: 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 Dr Sabrina Sarrocco Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0261908.r004

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