How Does the Sample Preparation of Phytophthora infestans Mycelium Affect the Quality of Isolated RNA?

Round 1

Reviewer 1 Report

The manuscript presents a comparison between different approaches for RNA extraction from P. infestans and pointed the most efficient combination of mycelial grinding manner and RNA isolation protocol. The subject of the work fit the journal scope.

The aim of the work is to compare different protocols for the isolation of RNA from P. infestans. The authors determine the most efficient approach that would be useful for researchers that have a problem with extraction of the high-quality RNA from the mycelium.

The topic of the manuscript is relevant for the field and is presented. The study explores and proposes an approach for a fast and efficient method for isolating quality RNA of P. infestans. An improved and reproducible protocol for the extraction of high quality fungal RNA from a specific oomycetes species is presented in a well-structured manner.

The methodology is presented in detail and consistently. The experimental design is appropriate and the manuscript is well-structured. All methods are described in detail and they could be repeated by other researchers.

The conclusions correspond to the experiments and the results obtained.

The cited references are suitable and recently published.

Figures and tables are clear, informative and present the results in an appropriate manner.

I have a small remark related to the Acknowledgments that have to be specified, if any.

Author Response

Response letter

We are grateful to the Reviewer for the time and effort in providing the feedback and appreciate all the comments offering us valuable insights for refining our manuscript.

Comments and Suggestions for Authors

The manuscript presents a comparison between different approaches for RNA extraction from P. infestans and pointed the most efficient combination of mycelial grinding manner and RNA isolation protocol. The subject of the work fit the journal scope.

The aim of the work is to compare different protocols for the isolation of RNA from P. infestans. The authors determine the most efficient approach that would be useful for researchers that have a problem with extraction of the high-quality RNA from the mycelium.

The topic of the manuscript is relevant for the field and is presented. The study explores and proposes an approach for a fast and efficient method for isolating quality RNA of P. infestans. An improved and reproducible protocol for the extraction of high quality fungal RNA from a specific oomycetes species is presented in a well-structured manner.

The methodology is presented in detail and consistently. The experimental design is appropriate and the manuscript is well-structured. All methods are described in detail and they could be repeated by other researchers.

The conclusions correspond to the experiments and the results obtained.

The cited references are suitable and recently published.

Figures and tables are clear, informative and present the results in an appropriate manner.

I have a small remark related to the Acknowledgments that have to be specified, if any.

Authors’ response to the comments:

Dear Reviewer, we appreciate your time and effort devoted to examining our manuscript. We are glad that our work left a pleasant impression. We thank you for your remark on the Acknowledgments: we have excluded the unnecessary section.

With kind regards, the Authors.

Reviewer 2 Report

Dear Author,

The article in general is well written and the information presented in a logical manner but some information in sections Materials and Methods and Results must be completed.

I have made several queries throughout the manuscript. For details, see the manuscript

Therefore, the present draft needs revision before further process.

Comments for author File: Comments.pdf

Author Response

Response letter

We are grateful for the Reviewer’s time and effort dedicated to providing the feedback. We appreciate all the comments that allow us to refine our manuscript. In the following, we provide our point-by-point answers.

Comments to the Author

Dear Author,

The article in general is well written and the information presented in a logical manner but some

information in sections Materials and Methods and Results must be completed.

I have made several queries throughout the manuscript. For details, see the manuscript

Therefore, the present draft needs revision before further process.

Authors’ response to the comments:

Dear Reviewer, we are deeply grateful for your patience and helpful comments aimed at eliminating any discrepancies in the manuscript. Here we provide our corrections for each mistake you have pointed out in the pdf file:

Line 28, 30, and further: We apologize for that mistake and have corrected all the references in the manuscript.

Line 55: Thank you for pointing out this glaring inaccuracy. We have checked the log and specified the mycelium growth time for all samples.

Line 70: The missing information has been added.

Line 79: We gratefully acknowledge this remark. The explanation of the sample quantity has been provided in Materials and Methods to eliminate any potential misunderstanding.

Lines 120 and 165: We are gravely sorry for that draft-carried x. We have revised and corrected the descriptions of all the figures and tables to ensure that they are no longer confusing. The P-values for the ANOVA, the absence of which you have rightfully noted, are now presented in the heading of Table 3.

Line 127: We appreciate your suggestion and have added a figure with the best sample of RNA analyzer data.

Line 147: We apologize that our incomplete explanation was misleading. We have added a passage to clarify the lack of methods in Figure 3.

With kind regards, the Authors.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Dear Authors,

Your explanations are sufficient but showed deficiencies in the methodology.

The number of repetitions is insufficient.

You wrote ‘For the NB_T-15 and B_T-15 methods, the number of samples was reduced due to the insufficient amount of mycelium material in the relevant Petri dishes.

It's not a problem to inoculate more Petri dishes to make the material homogeneous and with the same number of repetitions of at least five and the whole experiment should be carried out twice.

Author Response

Response letter

We are grateful for the Reviewer’s time and effort dedicated to providing the feedback. We appreciate all the comments that allow us to refine our manuscript.

Comments to the Author

Dear Authors,

Your explanations are sufficient but showed deficiencies in the methodology.

The number of repetitions is insufficient.

You wrote ‘For the NB_T-15 and B_T-15 methods, the number of samples was reduced due to the insufficient amount of mycelium material in the relevant Petri dishes.

It's not a problem to inoculate more Petri dishes to make the material homogeneous and with the same number of repetitions of at least five and the whole experiment should be carried out twice.

Authors’ response to the comments:

Dear Reviewer,

We are deeply grateful for your patience and helpful comments aimed at eliminating any discrepancies in the manuscript. We hope that our honest answers to your comments on staging the experiment will clear up several concerns about our work.

You’re absolutely right, it's not a problem to inoculate more Petri dishes. Unfortunately, the Major Revision only gives us 10 days to correct your comment, and we are not able to complete all the additional experimental work during this period. We regret, but “mission impossible”, as the saying goes …

In the existing realities, we can only say in defense of our manuscript that the addition of repetitions in the samples you indicated will not radically change the meaning of our manuscript, nor will it affect our proposed optimal method for isolating RNA from Phytophthora.

In conclusion, we hope that our arguments and corrections, as well as the opinion of reviewer 1 about the high scientific value of our work, will be taken into account by you when making a decision. We also note that no detailed protocol for RNA isolation from Phytophthora has been described so far, our manuscript may be the first, which will undoubtedly be useful to other researchers who can improve it.

With kind regards, the Authors

Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

Dear Authors,

I accept the explanation.

When planning subsequent experiments, please pay attention to the number of repetitions.