PONE-D-19-16123

Transcriptome analysis of Xanthomonas oryzae pv. oryzicola exposed to H2O2 reveals horizontal gene transfer contributes to its oxidative stress response

PLOS ONE

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PLOS ONE

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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: Partly

Reviewer #2: Partly

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

Reviewer #1: No

Reviewer #2: Yes

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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

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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: This manuscript described a time-resolved RNA-seq result that reveals Xoc transcriptome change related to oxidative stress response during exposed to hydrogen peroxide. Following the differentially expressed gene analysis, further investigations were carried out to determine 4 DEGs’ role in terms of antioxidation and virulence. This study provided useful information to related professional researcher. However, according to this manuscript, it is regretful to learn that part of result could not sufficient support the conclusion made by the authors.

Major issues

1. The virulence assay result could not support the conclusion made by the authors.

According to Fig 4, I assumed the authors using leaf piercing method to inoculate Xoc on rice leaf. But the authors did not clearly describe this in manuscript, the inoculate process was missing. By using leaf piercing method, the virulence should be evaluated by either calculating the means of lesion length (n >= 10), or establishing bacterial growth curves, or both. But in Fig 4, authors only showed a typical lesion caused by leaf piercing inoculation for each Xoc strain, which is not enough to represent their virulence.

2. To demonstrate whether a gene is required for full virulence of Xoc to rice, the virulence mutant should be assayed along with its complementation strain, which have not been showed in this manuscript.

3. The GEO data is not accessible. The authors declared all raw RNA-seq data is available on GEO at line 315-318. But the given accession numbers are invalid.

Minor issues

1. The organization of Introduction part is poor. And I suggest authors describe some known oxidative stress response process in microbe as part of background.

2. Some bioinformatic methods mentioned in Materials and Methods need proper citation. e.g. in line 135, the TMM method, and in line 155, the 2(-ΔΔC(T)) method, both need a proper citation.

3. At line 101, author mentioned “Cultures were treated with 0.1mM H2O2 in a 28 ℃ shaking incubator.” without describing how to conduct H2O2 treatment. I assumed the authors were closely following the method in Upadhya at.el work. I recommended the authors to provide more details in this part to make it more readable.

4. At line 146, the total RNA extraction process is missing.

5. At line 182, more details for how to interduce H2O2 into NB agar plates is required.

6. At line 190 to 193. author mentioned “Plant phenotypes were scored 24 h post-inoculation for the HR in tobacco, 3 days post-inoculation (dpi) for water-soaked symptoms, and 14 dpi for lesion length.” But there is not any hypersensitive reaction or water-soaked symptom result in this manuscript. Please make sure only the involved methods were included in this manuscript.

7. At line 203 to 206, the result indicated there were about 75% clear reads mapped on ribosomal RNA. Is this a typical rRNA/mRNA ratio for RiboMinus treated sample?

8. At line 235, authors should provide the full form of GO and KEGG.

9. At line 266, what’s the mean of “encompassed a range of expression levels at 7, 15, and 45 minutes”?

10. At line 267-272, author mentioned a microarray dataset but never described it. I assume author intend to refer the RNA-seq data in this study.

11. In Fig 3, the authors did not describe the meaning of captions “1x”, “3x”, and “9x”.

12. At line 315-318, the authors should not put the accession numbers in the main part of manuscript. The authors should list all accession numbers instead of an accession number range.

13. At line 347-358, the phylogenetic analysis result should be moved to the Result section.

14. The order of supporting information label incorrect.

15. The S1 Table, titled as “Differential expressed genes in the early, middle and late stage.”, missing heading at column one.

16. The S4 Table missing its heading.

Overall, the writing of this manuscript is very poor. The authors should proof the writing of the manuscript seriously, or consider hiring a professional copyeditor to handle it.

Reviewer #2: In the manuscript ‘Transcriptome analysis of Xanthomonas oryzae pv. oryzicola exposed to H2O2 reveals horizontal gene transfer contributes to its oxidative stress response’ the authors study the gene expressions of XoC strain BLS256 in response to time series H2O2 treatment, following RNA-Seq analysis. They claim in the Abstract, that ‘soxR triggers and regulates XoC oxidative stress response in early infection, gene expression kinetics among the time-series samples namely for TonB-dependent receptors and suf and pst operons. However, there is no clear data in the Results to corroborate these claims.

The finding that a MntR-like hypothetical protein is highly upregulated at a later time-point and that it is likely to be a horizontally transferred gene is interesting.

However, the English and the overall presentation of the manuscript needs to be improved considerably.

The descriptions of the Supporting data do not match the actual Supporting Tables. Hence,

In lines 249, 261 and 340: S3 Table needs to be replaced by S1 Table.

The authors have validated the role of 4 selected genes for their role in H2O2 sensitivity and pathogenicity by mutant generation and their complementation. It might be better to perform the experiments with at least two-independently generated mutants for each gene.

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Reviewer #1: No

Reviewer #2: No

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Transcriptome analysis of Xanthomonas oryzae pv. oryzicola exposed to H2O2 reveals horizontal gene transfer contributes to its oxidative stress response

PONE-D-19-16123R1

Dear Dr. Rao,

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.

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With kind regards,

Kandasamy Ulaganathan

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Please carry out the minor corrections pointed by the Reviewer

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: 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 #2: Yes

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

Reviewer #2: Yes

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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: 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 #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 #2: The manuscript looks considerably better and improved.

Following are some minor corrections:

Line 70: limited instead of limite

line 71: space between elucidate and the

line 105: delete Cell

line 106: delete was

line 133: space between having and the

line 276: remove additional space between different and levels

line 362: organic instead of organis

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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 #2: No