PONE-D-19-15638

Intergrating long noncoding RNAs and mRNAs expression profiles of response to Plasmodiophora brassicae infection in Pakchoi (Brassica campestris ssp. chinensis Makino)

PLOS ONE

Dear Dr. Yuying Zhu,

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.

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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: No

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

Reviewer #1: I Don't Know

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: This manuscript describes the changes in the expression of protein coding genes (mRNA) as well as those encoding long non-coding (lnc)RNAs in Brassica rapa in response to infection with the clubroot causing pathogen, Plasmodiophora brassicae. It attempts to integrate the findings with respect to lncRNA expression and mRNA expression.

Experiments have been designed and conducted appropriately although there are some significant issues. Please see major comments below.

The manuscript has been generally written well but there are still many issues--see minor comment for a non-exhaustive list of errors. It is impossible for me to list all the errors and the authors should ensure that the submission is free from these types of mistakes.

Major comments:

1. The samples were collected 6 weeks after sowing when full-blown root galls had developed. I do not understand the logic of investigating molecular responses after the development of galls. It would have been more revealing at earlier stages of pathogenesis. In my opinion, earlier stages in the disease should be sampled and mRNA and lncRNA profiles investigated.

2.Were there any pathogen mRNA and lncRNA detected? This is such a late stage that this may have been possible. IF so, it should be reported and if not, a statement should be made to that effect.

3. Line 376, are you talking about QTLs associated with resistance? However, in this case you used susceptible lines. This part of the discussion is confusing and should be clarified.

4. The Discussion section is extremely weak, or non-existent. It has be significantly strengthened to discuss their findings and integrating them with the information that is available in the literature. Currently this section is a sum total of 31 lines or just about one page. The most important part of these types of descriptive articles is their discussion about possible biological relevance of their findings. This should be rewritten in its entirety.

5. Why does the title of the manuscript say Brassica campestris? I thought that Pakchoi is Brassica rapa? Please be accurate and consistent.

Minor comments:

1. Some typographical and grammatical errors in the abstract include, line 11 “researches”; line 12 “works”; line 13 “….profiles of response to…”.

2. Line 28, “…a kind of soil-borne disease…”. It is a soil-borne disease, NOT “a kind-of soil-borne disease”.

3. Line 36, not “clubroot resistance crops” but “clubroot resistant crops”.

4. Line 32, Brassica rapa????

5. Lines 53 and 60, specify the Brassica species! In general, please specify the Brassica (or any plant) species when you are talking about the results from another study.

6. Line 69—do not begin a sentence with “And”.

7. Line 79, wrong reference format.

8. Line 122, NOT “reversely” transcribed.

9. Line 181, should be “Functional” enrichment.

10. Line 281, should be “Functional” annotation.

11. Line 345, “Pearson” correlation coefficient NOT “person”.

Reviewer #2: I have carefully read the manuscript PONE-D-19-15638 “Integrating long noncoding RNAs and mRNAs expression profiles of response to Plasmodiophora brassicae infection in Pakchoi (Brassica campestris ssp. Chinensis Makino)” by Zhu et al. The topic of the manuscript is interesting and the integration of lncRNAs to increase our understanding of clubroot disease is timely and novel. However, there are some shortcomings of the manuscript in its present form most notably the absence of an acceptable discussion of the results. More detailed comments can be found below.

The English of the manuscript is understandable, but I would strongly advice the authors to consult someone proficient in scientific English for language editing as there are several grammatical errors in the MS. There are many sloppy errors (inconsistent spelling, spelling mistakes, etc) throughout the text. I have pointed out some issues in the detailed comments below, but this list is not exhaustive.

Overall the manuscript would add important findings to our understanding of clubroot disease, but I strongly recommend that the authors considerably revise the manuscript to highlight and describe the very interesting findings they collected.

Detailed comments:

Abstract:

L 11: delete “Although lots of researches have been conducted during past decades” – grammer errors and IMHO this is not important in the abstract.

L17: specify which type of enrichment analyses

L19: define which type of interaction relationship, list the most important groups of interactions to provide more info for the reader on the biological relevance of those rather than just providing numbers.

L21: change “15 clubroot disease related” into “15 P. brassicae mRNAs”, maybe provide more detail on these RNAs.

Introduction

The introduction informative about the aims and guides the reader to the topic of the manuscript. Some phrases are difficult to understand, especially if the reader is not familiar with the original literature cited, therefore I would advise the authors to carefully edit the introduction.

L29: no need to write P. brassicae in brackets.

L60: “between CR and clubroot-resistant (CS) lines” this sentence is not clear, should this be clubroot susceptible (CS)?

L60: The sentence starting with “It was also found….” is unclear, please rephrase and explain what you mean with “updated SA function”.

Material and Methods

L102ff: Sample collection: the authors state that they use race 7 of P. brassice (also check for consistent spelling!). Was this a single spore isolate or a field population showing the characteristics of race 7 using the Williams differential? For all further experiments it will make a huge difference if the experiments were conducted using a single spore isolate or a field population.

L105ff: when were the plants inoculated and with approximately which amount of P. brassicae spores?

L107: please change “6 weeks after sowing” by the days post inoculation as this will provide more information on the disease progression

L129ff: what does “clean data” and “dirty reads” refer to? Please rephrase this and avoid laboratory jargon. Having a flow chart of the procedure including versions of the software in the Supplement is usually very helpful for such methods sections (also for LncRNA).

L172: state the type of mastermix used

L181ff: This section is not fully clear, please provide more information on the statistical analyses (R packages, settings, assumptions etc used in the analyses)

L188: The sentence “the p value…” is incomplete, please rephrase

L193ff: this section is not clear.

L193: lncRNAs - please make sure the correct spelling throughout the MS

Results

L217: use control or C instead of CK, this abbreviation is not intuitive.

L223: use “the number of reads after qality filtering (or quality control) were….” Instead of “clean reads”

L225: the sentence startin with Q30 is incomplete

L293: “these findings indicate that mRNAs were participating in the defence of clubroot” – This sentence is not correct. There is not evidence that the mRNAs themselves are involved in clubroot defence, rather it can be assumed that the genes/proteins the mRNA codes for are involved in the reaction. Please rephrase.

L301: The phrase “previous researches” is odd, rephrase to “previous studies” or similar.

L307: why were the GO terms restricted to “biological process” and why were molecular function and cellular component omitted? Is there a reason for this choice?

L346: is it really “person correlation coefficient” or should it be Pearson’s

Tables and Figures:

Overall the figure and table capitations could be a bit more informative and descriptive. Not every image can be easily understood, therefore some comments on specific issues:

Tables: Please describe what the different values stand for. I suspect that “annotated” means the number of transcripts that were assigned this GO term in the full dataset – or does this refer to the number of transcripts that were upregulated in clubroot tissue and assigned a certain GO term? Significant – is the number of these GO terms that were significant in which respect – significantly up/downregulated? What does “expected” mean and where does this come from? Classic Fisher refers to what – the significance of the GO-term, the up/downregulated GO-term, the transcripts?

Fig 2, 5, 6 Heatmap figures: can you provide any biological information to the genes other than the BRA accession? Maybe adding some sort of functional annotation (GO term, gene name, function of the gene,….). Which values are displayed? FPKM values, and if yes were these normalised? DEGs – but then which values were compared to give the values?

Fig3A: what do the numbers on the x-axis refer to? Please describe in the figure capitation or in the image.

Fig7: This figure is not very informative in the present form and also its not a network but a series of correlated genes. Please add information and annotations (which genes are we looking at?) to the figure or convert it into a table (which would provide more information on the individual correlations)

Fig S1: please provide a description of the figure. It is nearly impossible to understand this figure the way it is currently presented.

Discussion:

Unfortunately, the discussion feels very incomplete especially as the authors present a number of fascinating results. The authors fail to discuss what the findings of the correlation of lncRNAs and mRNAs presented mean for the biology of clubroot disease. The concept of lncRNAs is employed to clubroot for the first time, so there are plenty of factors that can be discussed and described here. Also there is no comparison to other transcriptomics studies of which there are plenty on a multitude of clubroot hots, resistant and susceptible interactions, on the intraplant variation etc. Please use this pool of references to discuss the results in a broader context.

L357ff: the first paragraph of the discussion is mostly results. Please move the description of the KEGG analyses into the results section, where only GO terms are described currently. Many of these processes have been identified in previous transcriptomic studies of clubroot, please cite those studies and compare their results to the ones generated in this study.

L376ff: This information is interesting, but most of the data are not yet available. Therefore the validity of more than half of the second paragraph of the discussion cannot be assessed.

Data availability

Data are available.

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Reviewer #1: No

Reviewer #2: No

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Integrating long noncoding RNAs and mRNAs expression profiles of response to Plasmodiophorabrassicaeinfection in Pakchoi (Brassica campestris ssp. chinensisMakino)

PONE-D-19-15638R1

Dear Dr. Yuying Zhu,

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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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

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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

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

Reviewer #1: I Don't Know

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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 #1: 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

**********

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 authors have addressed my comments.

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Reviewer #1: No