PONE-D-19-28852

Genome-wide Transcriptomic Analysis of the Response of Botrytis cinerea to Wuyiencin

PLOS ONE

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Additional Editor Comments (if provided):

You are requested to undertake a major revision of the paper addressing all the points raised by the reviewers. Please provide your reply for each of the comments made by the reviewers

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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

Reviewer #3: Partly

Reviewer #4: Partly

Reviewer #5: No

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

Reviewer #5: 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: No

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: 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: Yes

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: No

Reviewer #5: 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: This is a decent study of genome-wide transcriptomic analysis of B. cinerea treated with wuyiencin. The authors identified 2067 differentially expressed genes, and performed and discussed pathway analysis, which revealed the molecular mechanisms of wuyiencin in treating B. cinerea.

Here are some minor questions.

1, Table S1 and Table S2 may be mis-labeled or uploaded in the opposite order. Can you double check?

2, Figure 2 looks nice. However, for the inner circle, I can barely read their colors and match them to the legend. Is there anyway to improve? Also for the third circle, what does the length of each bar represent?

Reviewer #2: The authors describe the inhibitory effects on Botrytis cinerea of the antifungal metabolite wuyiencin from the biological control bacterium Streptomyces ahygroscopicus var. wuyiensis, and present transcriptome data showing changes in gene expression in wuyiencin-treated B. cinerea compared to untreated controls.

The morphological effects of wuyiencin treatment are quite superficially, from the pictures shown in Fig. 1, it is difficult to draw any deeper conclusion about the molecular processes that are affected. The main part of the manuscript deals with the analyis of differential gene expression based on RNA sequencing. As expected from a potent growth inhibitor, many genes encoding metabolic enzymes are up- or downregulated, however, it is difficult to draw more specific conclusions about the molecular target(s) of wuyiencin. This applies also to the differential upregulation by wuyiencin of major plant cell degrading enzymes encoding genes (Bcxyn11A, Bcpg1/2, Bcpme1/2), which are interesting observations but don’t provide clear evidences towards a certain mode of action. In general, the interpretation of differential transcriptome studies taken at only one time point is very difficult because they are unable to describe the dynamics of gene expression changes induced by inhibitor treatment, and therefore yield only limited informations.

Part of the text is not written very well and carefully. Line numbers are missing. Some recent papers (e.g. about the latest B. cinerea genome sequence, van Kan et al., Mol Plant Pathol. 2017 18:75-89) have not been cited.

Reviewer #3: In this manuscript, Shi and colleagues present the transcriptome-wide response of Botrytis cinerea to exposure to the anti-fungal agent, wuyiencin. The manuscript is well written and provides an in-depth characterization of the genes which are differentially expressed in response to the drug. I had no major criticisms with the science, although I did find some of the results/conclusions a bit perplexing (discussed below) and felt that inclusion of p-values at certain points in the text would be informative to the reader (also discussed below). Aside from these two points, which can be easily fixed by some minor additions to the text, I feel that this manuscript is appropriate for publication in PLOS One.

Comments:

1. When first using the abbreviation for control (I assume this is what ck means) please define it (first paragraph of results).

2. When using phrases such as "almost significantly" please include p-values. My interpretation of "almost" may different than another reader's, particularly since the adjusted p-value cutoff was 0.05.

3. I found the upregulation of certain genes involved in pathogenesis, such as the CWDEs and the CAZymes, in response to the antifungal agent to be counter-intuitive. Perhaps a little more discussion on why this might be occurring would be helpful.

4. It is interesting that no genes involved in cell death are upregulated. In addition, the genes associated with stress responses that were identified in this study were all down-regulated.

5. Out of the pathways shown to be differentially expressed, could the authors point to a particular one which they believe is most likely the target of wuyiencin? The authors mention in the discussion that this work highlights potential mechanisms but I think I missed the ones that they predict to be most likely.

Reviewer #4: In order to elucidate the effects of wuyiencin on the transcriptional regulation in B. cinerea, the authors used genome-wide transcriptomic analysis of B. cinerea treated with wuyiencin.

The found that putative genes involved in amino acid metabolism were influenced in response to wuyiencin treatment. Moreover, the suggest expression of genes involved in protein synthesis and energy metabolism and of those encoding ATP-binding cassette transporters was markedly upregulated, whereas that of genes participating in DNA replication, cell cycle, and stress response was downregulated. Furthermore, wuyiencin resulted in mycelial malformation and negatively influenced cell growth rate and conidial yield in B. cinerea. Even though the subject is very important and the transcriptional analysis can give hint on wuyencin mode of actions this paper is mainly descriptive and not supported by functional analysis.

Major concerns:

1. Is there any correlation between the phenotypic influence by SEM and TEM to the transcriptional changes- this need to be addresses and discussed.

2. Lack of functional analysis of the effect demonstrated by transcriptional changes (e.g. are AA levels or metabolism really changed? ; are CWDE active?; Do cell cycle/DNA replication impaired?; how can athoures conclude that wuyiencin active efflux is occurs?)

3. Not enough details on the RNA-seq experiment and analysis such as PCA , reads number etc.

4. Missing statistic on qRT-PCR analysis

5. Figure 2 is unreadable! - must be replaced with better way of demonstration the transcriptional changes by GO- annotation diagrams or pies., KEGG analysis is missing and need to be added

6. The authors fail to discuss how come some of the pathway they suggested to be involved is upregulate but other genes in the same pathway are down regulated- in AA metabolism or CWDE (Figures 3 and 4)?

7. Is ROS effected by wuyiencin, may check similar effect demonstrated by Vogel et al., 2011

8. Authors fail to discuss their results and explain properly how specific the changes wuyiencin are and not part of general stress response??

9. Reference 11 is very important backbone for this paper and it is not available in pubmed

Minor concerns:

1. Figures legends are not sufficient

2. Statistic is missing Figures 3-4

3. Fig1. no size bars, arrows are not defined, pigment is not shown?

4. wuyiencin is not biological control agent??

5. Is there any correlation between RNAseq and qRT-PCR?

6. Sometime there is no correlation between genes marked on Fig 3 to the text in result section

7. In results section may parts belong to discussion

8. Conidial germination S1 figure should be in main text, also demonstrate other results on conidia

9. Why control called ck?? It is very confusing

10. Some earlier studies references are missing (e.g. in results second part line 3)

11. In discussion part paragraph 4 “ In the past…” is not connected and need to be removed

12. The manuscript is not written fluently and very hard to read

13. The MS must go through English professional editing

Reviewer #5: The manuscript titled "Genome-wide Transcriptomic Analysis of the Response of Botrytis cinerea to Wuyiencin" by Shi et al is well written and provides a solid base for the future investigation of the mode of action of Wuyiencin however, the authors did not provide access to their data nor did they provide citations for how their analyses were carried out. Just stating that a given software package was used does provide sufficient information to asses the statistical analyses carried out. A further point is that the quality of the images in the figure in the version of the manuscript I dowloaded was insufficient to illustrate what the authors indicated in the text especially for figure 1 and 2. A final major point is that there are instances in where the RT-qPCR data does not fully support the RNA-seq data and as such the athors need to moderate statments that suggest it is fully consistant with the RNA-seq data

Apart from these major issues that need to be addressed the authors also need to improve the language associated with their analyse of the data.

For example In the abstract the sentence beginning "We could identify 2067 differentially .." should be edited to improve clarity

Change the phrase "remarkably influence" to improve precision of the statement give specifics, up regulated ? down regulated? or even change the proceeding structure to something like " transcript levels of genes involved in amino acid metabolism and those encoding putative secreted proteins were altered..."

IN this regard it is more precise that the authors refer to altered transcript levels since they are not specifically measuring gene expression.

Also the last sentence of the abstract is too general the data presented does not suppot this statement so it should be edited to clarify what the data does support.

In the first paragraph of the introduction edit the phrase "relatively suitable condition" to more precisely indicate which conditions are required for conidiation.

Introduction second paragraph the evidence is not conclusive enough to make the statment that differential expression of enzymes is the only basis for B. cinerea infection of multiple hosts.

QRT_PCR should be RT-qPCR since it is the PCR that is quantitative and RT-qPCR does not validate RNA-seq data it provides data that support that obtained by RNA-seq.

IN the results, Figure 2 does not illustrate clearly the number of genes with altered transcript levels

KEGG does not simply classify genes it assess the representation of specific functional groups of genes edit the text to clarify this indicating that the DEGs were enriched in the following functional categories ...

The phrase almost significantly down regulated is meaningless indicate specifically what the data shows.

Statements like "Protein synthesis, although an exceedingly complicated process ..." are overly obvious and add nothing to the text add such statements to indicate more precisely what the authors are trying to convey

The sentence "These findings suggest that B. cinerea uses different protein synthesis metabolism when exposed to phytoalexin from plants and antibiotic in microorganisms." is not clear do the authors mean " ... and when exposed to antibiotics from microorganisms"?

The phrase "most significant" is not meaningful, edit to clarify precisely what the authors intend to convey

The statement "The downregulation of the expression of these genes is directly related to cell growth

rate impairment in B. cinerea upon wuyiencin treatmen." is poorly worded and not support. The authors need to cite data that supports the contention that altering the transcript levels of these genes alters growth rate

To further validate was not the reason RT-qPCR was carried out it was carried out to assess if RNA_seq data was supportable by a different method of analysis

The authors state "significantly downregulated" but never define this nor do they provide sufficient information on their statistical analyses such that this can be independently assessed. this must be edited for clarification.

Sun et al is referenced but no date is provided for this reference

The statement "We strongly believe" has no place in a scientific publication. This is not religion, evidence must be presented to support conclusions.

The authors state "...in this study, we identified candidate pathways and target genes that may offer insights into the protective and antagonistic mechanisms underlying the action of biological control agents." but do not eleborate on what information they can gain from the data about the potential mode of action of the antifungal used and how that relates to other antifungal of the same class

In the conclusion the phrase "actual situations" is meaningless the authors must edit this phrase and the sentence it is in to provide clarity of the point they are trying to convey

The authors must define how they denatured the RNA during agarose gel electrophoresis

The authors must provide more information on how they used DESeq R and provide a citation for this analysis

Eliminate any reference of RT-qPCR being carried out to confirm RN-seq data this suggests that you had expectations of what the data would show which would have influenced interpretation of he reults

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Reviewer #1: No

Reviewer #2: Yes: Matthias Hahn

Reviewer #3: Yes: Andrew D. L. Nelson

Reviewer #4: No

Reviewer #5: No

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Genome-wide Transcriptomic Analysis of the Response of Botrytis cinerea to Wuyiencin

PONE-D-19-28852R1

Dear Dr. Zhang,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Kandasamy Ulaganathan

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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 #3: (No Response)

Reviewer #5: All comments have been addressed

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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 #3: Yes

Reviewer #5: Yes

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

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #5: Yes

**********

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 #3: Yes

Reviewer #5: Yes

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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 #3: Yes

Reviewer #5: Yes

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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 author has addressed all my concerns. I have no further questions. I would suggest to accept this paper.

Reviewer #3: I thank the authors for addressing my concerns. I still do not think that they really address in the discussion that they observe downregulation of stress response genes (lines 420-423) and upregulation of genes that a naive reviewer would assume to help the pathogen be more virulent (lines 277-283) or better degrade the cell wall of their host. Finally, the authors mention on line 323-324 that downregulation of the expression of these genes (Bcin09g01360, etc) are directly related to cell growth impairment in B. cinerea... This sentence is speculation and as such should be moved to the discussion or changed to "may be related".

Overall though, I think the data will be useful to the community. Aside from the above points and a few areas where grammar needs to be addressed, I think this manuscript is suitable for publication.

Reviewer #5: I feel that the authors have addressed the concerns I raised adequately. The improved images make a big difference. There is still some minor issues with wording but it is acceptable.

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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 #3: Yes: Andrew Nelson

Reviewer #5: No