PONE-D-24-52977NPR1-Like Genes in Theobroma cacao: Evolutionary Insights and Potential in Enhancing Resistance to Phytophthora megakarya

Dear Dr. Ali,

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.

You will find the comments from the reviewers below which will help you improve your manuscript.

Please submit your revised manuscript by Feb 03 2025 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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We look forward to receiving your revised manuscript.

Kind regards,

Valentine Otang Ntui, Ph.D

Academic Editor

PLOS ONE

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Additional Editor Comments:

Having gone through the manuscript, I see a major flaw with the gene expression analysis presented in Fig. 8. The authors should inoculate cacao with Phytophthora megakarya and check the expression of the mentioned genes rather than relying on information deposited on NCBI. It should be noted that several parameters would have changed from the time the information was deposited on NCBI to the time the analysis was done.

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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: Yes

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

Reviewer #1: N/A

Reviewer #2: N/A

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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: Yes

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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: No

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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: 1. The manuscript needs major English language revision. A number of typo and grammatical mistakes have been seen in your manuscript, Please try to improve the readability of your manuscript as possible by consulting with an English language expert.

2. Abstract should be more precise. In Abstract, briefly explain how study was conducted and what measured.

3. Figure 1, The values of relationship and similarity index is missing. Try to improve the figure quality and please indicate the species names in the figure legend.

4. One limitation of this study is the reliance on the previous published data for gene expression profiling of Cacao varieties responses to Phytophthora Megakarya inoculation and the absence of experimental validation.

Reviewer #2: GENERAL

The authors used in silico approach to identify NPR-like genes in Cocoa and analyzed the implications especially in relation to their possible role in enhancing resistance to Phytophthora megakarya. No laboratory experiment was carried out.

I found that the given the nature of the study, the appropriate softwares were used for analysis and the results presented were largely reflective of the software output. There was no "big" need for the traditional statistical analysis except in Fig. 8 where I have commented below that the authors should state exactly what analysis was done since they derived raw data from a previously published paper.

Other section-by-section comments are compiled below:

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INTRODUCTION

1. Overall, the manuscript is REPLETE with poor grammatical constructions that have substantially impeded logical flow and understanding of the work presented. A few examples are found in lines 49, 53, 68-69, 88, 90-91, 107-110, etc. The paper MUST NOT be accepted in the present form without a careful and extensive English language check and thorough revision. Also, attention should be given to standard scientific formats such as italicizing scientific names, capital letters beginning genus names and small letters beginning species name, writing the full form of an abbreviation at first mention (lines 70, 88, etc).

2. There are too many old references in the introduction and some of them do not seem to connect with the statements preceding them. For example, in lines 59-61, 78-80, the authors made mention of expressions like "recent studies", "until now", but cited references as old as "2004" and "2009". It would suggest that adequate literature review was not done to build in new references and connect them smoothly with the older references. As it stands currently, the older references in the manuscript seem to have been merely imported from a previous similar paper.

3. Lines 78-87: It is quite confusing whether the authors were reviewing a past literature or talking about the current study under consideration since they kept using "we/our". The entire construction needs review.

MATERIALS AND METHODS

1. Section title should be: "Materials and Methods", not "Material and Methods"

2. Be consistent with T. cacao, not T. cocoa or T. Cacao

3. TcNPR-like sequences and not TcNPR like sequences (line 123-124).

4. BTB domain means what?...line 124

5. Line 130-131: Any special reason for the choice of species whose NPR amino acid sequences were used in the phylogenetic analysis with TcNPRs?

6. Line 135: Do you mean Physico-chemical?

7. Lines 139-140: ...locations of chromosomal what???

8. Line 141: "...was used to assess..." does not sound right. Recast.

9. Line 143: CERs or CREs? At first mention, state full meaning.

10. Lines 150-152: Statement does not make a lot of sense. Review and re-present.

11. Lines 163-164: I think the sentence can be re-presented like this: "To investigate the expression levels of T. cacao NPR genes in response to Phytophthora megakarya inoculation, 2 variants of cocoa plants (SCA6 and NA32) were utilized".

RESULTS

1. Lines 221-223: Poor construction. Recast.

2. Lines 223-224: what does "thrice" mean in the context of its use?

3. Lines 278-279: The statement: "In T. cacao no gene happened to be a singleton as no duplication was found via synteny analysis" is somewhat confusing. If there was no duplication, does it not suggest that the genes exist as singletons?? Please clarify!

4. Line 290: It is unclear what analysis the authors exactly did under expression profiling. From the presentation, the transcriptomics data was obtained from a previously published report. So, what exactly did the authors do with this data to generate Fig. 8?

DISCUSSION

1. There are too many poor constructions, bad punctuations, and illogical flow. Please review extensively.

FURTHER CONSIDERATIONS

1. The authors should relook at this section. It would appear they are merely stating what they should have done instead of leaving it up to the readers to supply those information. In fact, as it stands currently, it seems as though the points raised under this section were for the authors to fill in before sending out the manuscript but somehow forgot to attend to them. This section should be re-done.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-24-52977R1

NPR1 -Like Genes in Theobroma cacao: Evolutionary Insights and Potential in Enhancing Resistance to Phytophthora megakarya

Dear Dr. Ali,

Thank you for submitting your revised manuscript. 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 Mar 01 2025 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,

Valentine Otang Ntui, 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.

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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: All comments have been addressed

Reviewer #2: (No Response)

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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: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: N/A

Reviewer #2: N/A

**********

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: Yes

Reviewer #2: 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 #1: Yes

Reviewer #2: Yes

**********

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: I appreciate the efforts made by authors to improve the readability of the manuscript in the revised version. Now the manuscript can be accepted for publication after a minor revision.

1. In abstract, state full name of the gene NPR1 at first mention.

2. Lines 25-27, Statement does not make a lot of sense, review and re-present.

3. Lines 52-53, Poor construction, rewrite the statement.

4. Lines 64-65, Are pathogens susceptible or cocoa plants are susceptible to pathogens? Please correct the statement.

5. Conclusion section should be as concise as possible and should not be more than one or two paragraphs.

Reviewer #2: There has been a major English language revision in the manuscript and it is currently much better to understand. Also, most of the issues raised in the first review seem to have been addressed.

However, a few observations are noted below;

1. Lines 41-44: Authors referred to "Recent investigations" yet cited references as old as 1999 and 2004. This should be addressed.

2. Line 257: The last sentence: "However, only one duplication is found in..." is incomplete.

3. Physiochemical or physicochemical? Be consistent. See line 294

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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: 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. 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.

NPR1 -Like Genes in Theobroma cacao: Evolutionary Insights and Potential in Enhancing Resistance to Phytophthora megakarya

PONE-D-24-52977R2

Dear Dr. Ali,

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.

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Kind regards,

Valentine Otang Ntui, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

None

Reviewers' comments:

None