PONE-D-23-14214Comparative transcriptome analysis reveal the Sclerotinia sclerotiorum  resistance mechanism in sunflowerPLOS ONE

Dear Dr. Liu,

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.

Upon careful evaluation of the Reviewer(s)' comments and my own assessment, it is evident that significant revisions are necessary for your manuscript before it can be further considered for publication. I would like to inform you that I reserve the right to send the revised manuscript to the original Reviewer(s) or seek input from new Reviewer(s) to ensure a thorough evaluation before making a final decision on its suitability for publication.The comments of the Reviewer(s) are included at the bottom of this letter.

Main concerns

Unavailability of raw RNA-seq data

General findings

Lack of comprehensive analysis

Insufficient detail in data processing methods

Missing critical genome assembly information

Please submit your revised manuscript by Sep 16 2023 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 would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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We look forward to receiving your revised manuscript.

Kind regards,

Asela J. Wijeratne, Ph.D.

Academic Editor

PLOS ONE

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The study was supported by China Agriculture Research System of MOF and MARA (CARS-14), and Shenyang Seed Industry Innovation Project (22-318-2-19). 

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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: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

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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: Yes

Reviewer #2: No

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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: No

Reviewer #2: Yes

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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 paper provides good insights into transcriptional changes occurring in susceptible and resistant sun flower following Sclerotinia sclerotiorum infection. There is a good attempt to measure physiological and correlate it with infection. The authors also provide a good model and graphical abstract as figure 8. For this, I commend the authors.

Having said that, my biggest sticking point with the paper is the poor writing. It was difficult to understand some parts of the paper because of the way poor English and grammar. I pointed out a few and suggested revisions, but the whole paper will need to go through a thorough editing process.

I was also less impressed by the quality of figures 4 and 5.

Below are my specific comments:

Line 3

S. sclerotiorum

Page 4 line 2

Delete “thus far”

Page 4 line 18-19

Rephrase as follows:

“Both DR genotype C6 and DS genotype B728 are oilseed sunflower inbred lines which were bred by Liaoning Academy of Agricultural Sciences”

Line 23

Italicize “S. Sclerotiorum”

Line 5 Page 5

Replaced “were washing” with “washed”

Page 7 line 2 and throughout the paper,

Italicize “S. sclerotiorum”

Page 8 lines 3-12

Consider rephrasing as follows:

“The physiological response of leaves to S. sclerotiorum inoculation was further evaluated based on the activity of antioxidant enzymes, such as superoxide dismutase activity (SOD), peroxidase (POD), catalase (CAT), glutathione-S-transferase (GST), ascorbate peroxidase (APX) and monodehydroascorbate reductase (MDAR). These data are shown in Table 1. Following infection, the activity of POD, CAT and GST increased significantly in both genotypes, while the SOD activity increased significantly in DR genotype rather than DS genotype. APX and MDAR activity in DS genotype were decreased significantly by S. sclerotiorum infection, while DR genotype showed no significant change. S. sclerotiorum infection, caused higher activity of the enzymes relative to the DS genotype.”

Page 12 line 7

Replace “validate” with “validating”

Page 12 lines 18-24

The sentence in the beginning of the discussion section is poorly written and confusing. Please revise the sentence or consider removing it.

Reviewer #2: The presented study performed pairwise DEG analyses within susceptible and resistant genotypes, resulting in lists of genes and general pathways that may be involved in the sunflower defense response against the pathogen Sclerotinia sclerotiorum. Some main concerns are also listed below:

(1) There are new RNA-seq data generated, but the raw reads are unavailable.

(2) The findings are too general, with little information to conduct further studies or cross validations.

(3) There are expression profiles measured for susceptible and resistant genotypes, but functional/enrichment analyses are mainly focused on DEGs within genotypes. This will mainly lead to functions or pathways that are common between the two different genotypes. Related analyses for uniquely up/down-regulated genes in the resistant genotypes is recommended to identify candidate resistance genes that contribute to the resistance in the DR genotype compared with DS genotype. The analysis described in line 9-16 of page 10 is not comprehensive.

(4) The data processing parts in the method section needs to be revised to add detail information, including software and parameters used. For example, using "BMKCloud platform" does not provide sufficient information, and MapMan is a software instead of an analysis. A lot of information is missing.

(5) The genome assembly information is missing, which is critical for further use and validation of this study. Besides, the gene ID does not sound correct or published ones. For example, "Helianthus_annuus_newGene_2831" in table S2 and "NewGene_11665" in Figure 5B.

Other minor concerns:

(1) There should be a figure for mock-inoculated sample in Figure 1, which only presents inoculated leaves.

(2) Nearly a half of expressed genes are differentially expressed, which seems too many. The author may want to double check their data. For example, compare their result to previously published studies.

(3) The reads mapping ratio ranges from 74.8% to 91.8%. This imbalanced mapping ratio may contribute to the super-high number of DEGs.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-23-14214R1Comparative Transcriptome Analysis in Two Contrasting Genotypes for Sclerotinia Sclerotiorum  Resistance in SunflowerPLOS ONE

Dear Dr. Liu,

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.

 I've read your current version of the manuscript and your responses to the reviewers. Together with the two external reviewers, this manuscript still needs major changes. The analyses need to be fully described for reproducibility (this was a main issue last round as well). The clarity of writing should be improved for readability.

Please submit your revised manuscript by May 01 2024 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.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. 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.

We look forward to receiving your revised manuscript.

Kind regards,

Asela J. Wijeratne, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

**********

2. 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 #2: Partly

Reviewer #3: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: No

Reviewer #3: No

**********

4. 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 #2: No

Reviewer #3: Yes

**********

5. 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 #2: Yes

Reviewer #3: No

**********

6. 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 #2: Please add the NCBI BioProject ID ‘PRJNA908908’ in the manuscript.

According to figure 4 B-E (figure 6 in previous version), nearly most genes are differentially expressed in both DS and DR genotypes (|log2(FC)| > 1), although the fold change varied. The expression of these genes was not statistically compared between DS and DR genotypes to narrow down the candidates, which could have been inferred from this data.

The read cleaning method is unclear and irreproducible. For example, a read should not be ‘annotated based on the reference genome’. This revised method section still contains many errors, making the results unreliable.

The reference genome information is still not provided, making it impossible to assess the results. Not only the ‘new genes’, but also the ‘reference genes’ have only an ‘ID’ available, which largely limits the contribution to the community.

The numbers of expressed genes were previously 22,077 and 22,579, but in the present version, they increased a lot. Again, without the information of the reference genome, it is impossible to know what happened.

Without sufficient data, I cannot comment on the numbers listed.

Reviewer #3: Authors have conducted transcriptome analysis of a resistant and a susceptible genotype of sunflower against Sclerotinia sclerotiorum. I have some suggestions and comments, which are given below. Page numbers are based on the revised, track-change version.

# Major comments:

1) Figure-4, S1, and S2 are not legible. Hence, I couldn’t check the information associated with these figures. These figures need to be updated.

2) One major criticism is that all the follow-up analyses were done with the DEGs (Differentially Expressed Genes) upon infection in both the resistant and the susceptible genotypes. This was pointed out by one of the reviewers. However, the author didn’t do a good job of resolving the issue. Ideally, it would be better to focus on DEGs that are unique to either the resistant or the susceptible genotype. Alternatively, performing clustering of DEGs could have been better.

3) English is very poor in some instances. For example, on page 17, lines 2-3, sentence construction is not correct. The same is true for line-8-9. There are multiple examples like this throughout the manuscript. Correcting those will improve the readability of the manuscript.

4) Did you use any filtering to get rid of the low-expressed genes, otherwise there will be DEGs that were expressed barely.

# Minor comments:

1) Usage of abbreviations should be kept at a minimum level. CWDE for ‘cell wall degrading enzyme’ (page 3, line-11) is unacceptable.

2) Page-5, line-6, why 24 hpi was used for the experiment?

3) Page 6, line 20, how the raw reads were processed?

4) What kind of analysis was performed using MapMan? MapMan is a software, not an analysis.

5) Page 8, line 27, what are the criteria for being expressed?

6) GO analysis of all DEGs is meaningless, authors should have focused on unique DEGs.

7) Page-9, line-23, “In the CC category, “cell”, “cell part”……were most frequent”. Is this kind of information meaningful?

8) Common GOs for both genotypes represented in Figure-3, are not very informative.

9) Page 10, lines 11-18, describe GO terms associated with the unique DEGs for each genotype. This section is useful. Authors should expand this section.

10) The result section for the analysis using MapMan is very rudimentary.

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Reviewer #2: Yes: Honghe Sun

Reviewer #3: Yes: Manohar Chakrabarti

[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. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-23-14214R2Comparative Transcriptome Analysis in Two Contrasting Genotypes for Sclerotinia Sclerotiorum  Resistance in SunflowerPLOS ONE

Dear Dr. Liu,

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.

 I've read your current version of the manuscript and your responses to the reviewers. As the external reviewer noted, the manscript still require editing. Please go through reviewer's suggestion carefully.

Please submit your revised manuscript by Oct 24 2024 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.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. 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.

We look forward to receiving your revised manuscript.

Kind regards,

Asela J. Wijeratne, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Please 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 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.

Additional Editor Comments:

I've read your current version of the manuscript and your responses to the reviewers. As the external reviewer noted the manscript still require editing. Please go through reviewers suggestion carefully.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

**********

2. 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: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

**********

4. 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)

**********

5. 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: (No Response)

**********

6. 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 ms has improved but still require editorial attention. Please see attached document with comments.

Figures are also low resolution - especially the supplementary figures that show enrichment. Labels on the X-axis have small fonts that are pixelated.

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7. 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: Yes: Steven Runo

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[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. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Attachments

Attachment

Submitted filename: PONE-D-23-14214_R2_reviewer_R1_19_6_24.pdf

Comparative Transcriptome Analysis in Two Contrasting Genotypes for Sclerotinia Sclerotiorum  Resistance in Sunflower

PONE-D-23-14214R3

Dear Dr. Liu,

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.

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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,

Asela J. Wijeratne, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The authors have addressed all comments and issues raised by the reviewers, and the manuscript has been updated accordingly. However, there are still some editorial and grammatical mistakes that need to be corrected for the final submission. I kindly request you to carefully review the manuscript, identify any remaining issues, and make the necessary corrections.

Reviewers' comments: