PONE-D-19-32656

Rapid Alkalinization Factor (RALF) gene family genomic structure and transcriptional regulation during host-pathogen crosstalk in Fragaria vescaand Fragariax ananassastrawberry

PLOS ONE

Dear Prof. Baraldi,

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.

I agree with both reviewers that this was an interesting study on an important topic. However, a number of concerns were raised. While all of the reviewer's comments should be addressed, most can likely be done so by editing or expanding the text. However, Reviewer 2 noted that more detailed searches for new RALFs are warranted, and this might require additional experimentation. In addition, Reviewer 1 requested more details about Figure 2 and noted difficulties with the quality of this figure.  Reviewer 1 also asked about the 5' upstream sequence of FaRALF6, and I wonder whether some functional analysis of this promoter is possible.

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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

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

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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: Yes

Reviewer #2: 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: Title: “Rapid Alkalinization Factor (RALF) gene family genomic structure and transcriptional regulation during host-pathogen crosstalk in Fragaria vesca and Fragaria x ananassa strawberry”

Authors: Francesca Negrini, Kevin O’Grady, Marko Hyvönen, Kevin M. Folta and Elena Baraldi.

General Comments:

The paper consists in the identification, characterization and genomic organization of the RALF gene family of F. x ananassa and F. vesca strawberry species based on the information available of Arabidopsis ortholog genes. Results revealed that whereas F. vesca contains 13 RALF genes, F. x ananassa contains 50, and they do not show conserved localization among the genotypes analyzed. Evaluation of the effect of infection with the fungal pathogens C. acutatum or B. cinerea on the expression of 7 selected FaRAFL genes in strawberry fruits showed that only FaRALF3 was upregulated, and FaRALF6 downregulated after fungal infection. The paper also includes the characterization of the 5’ upstream sequence of the gene FaRALF3, corresponding to the putative promoter region, and the evaluation of its functionality when fruits were infected with the pathogens C. acutatum or B. cinerea. Experiments were carried out by agroinfiltrating strawberry fruit with FaRALF3 truncated 5’ upstream sequences and evaluating the level of expression of GFP and GUS genes in fruits infected with C. acutatum or B. cinerea.

The paper is interesting and provides important information about the structure, genomic arrangement and transcriptomic of the RALF genes of strawberry species.

Overall, my opinion is that the paper deserves to be published after minor revision and answering the comments pointed out below.

Specific Comments

1) Line 63. Revise what “Buddah’s Paper Seal 1, 2” means.

2) Line 117. Revise, “..to gaining...” . Should be “..to gain...” or “…of gaining…”.

3) Lines 153-159. When fruits were inoculated with the pathogens, they were evaluated 24 hpi. However, whereas for B. cinerea that time may be adequate for assessing the effect on RALF expression, that time may not be adequate for C. acutatum. Normally the latter requires more time; likewise can be discussed about the optimal temperatures required for each pathogen. These experimental details may have consequences when evaluating transcriptional levels of RALF genes. I suggest that authors should add some comments about this issue.

4) In Figure 2, authors show a complex figure in which they present a transcriptional analysis of RALD genes in F. vesca. The figure I have received is not quite clear, therefore I could not see clearly plants stages. Anyhow authors should explain better in the text or in the figure legend the stages indicated at the botton of this figure. Since the transcription level of all genes evaluated may change with time, authors should be more precise about the physiological state of plants used, as they did with fruits. They should more details about how were the plants grown (i.e. photosynthetic photon flux density used), and how old were the plants, the leaves, and roots at the moment of taking the samples? What roots did they use for the analysis (e.g. whole root, principal, lateral)? Likewise with the leaves. Did they take the three leaflets, or only the central one for the analysis?

5) Line 413. Revise. There is a “t” that should be deleted.

6) Line 419. Revise. “...since the F.ve progenitor...”. What is F.ve? F. vesca?

7) It is interesting the study carried out by the authors of the 5’ upstream sequence of the gene FaRALF3, and understand why did they focused on that gene, however I wonder why did not pay equal attention to the 5’ upstream sequence of the gene FaRALF6. These genes exhibit an opposite behavior when infected with C. acutatum or B. cinerea, mostly in red fruits. Do they share similar cis-regulatory sequences, or cis-acting elements? It would be desirable if the authors add some comment about it.

Reviewer #2: As a qualification to my reply to the prior question (about standard English), the manuscript could benefit by careful editing by a native English speaker. While the writing is clear and unambiguous, there are some systematic departures from standard English in the use, or lack of use, of articles such as "the", and also distinction between singular/plural forms.

That minor detail aside, the manuscript content is generally interesting and useful, but in my view a number of issues would need to be addressed to warrant publication. These issues are described, and other minor editorial suggestions are provided, on a line-by-line basis below.

Line 21: Replace “Factor” with “Factors”

Line 21: Why are RALFs referred to here and throughout the manuscript as peptides rather than polypeptides or proteins? What criteria distinguish these three terms/classes and on what basis is the term “peptide” utilized in this manuscript? This needn’t be explained in the Abstract, but it should be addressed in the Introduction. Once the distinction is explained, consistent terminology should be used throughout the manuscript.

Line 23: insert “,” after “development”

Line 23: replace “regulator” with “regulators”, and check throughout manuscript for proper usage of singular/plural.

Line 25: are incomplete citations such as this permitted in the Abstract in PLOS-ONE?

Line 28: delete “type of” (in what sense are these two varieties “types”?)

Line 29: insert comma after “expression”

Line 29: specific F. vesca cultivar/accession: it may not be typical.

Line 30: specific F. x ananassa cultivar: it may not be typical.

Line 31: it has not yet been established that F. x ananassa has four definable subgenomes, or that the subgenome composition of variety “Camaroso” is typical or representative of F. x ananassa.

Lne 31: the statement about transposable elements should be deleted: the manuscript provides no data on TE distributions in the studied species.

Line 32: replaces “genes” with “gene”

Line 33: state pathogen genus names on first usage.

Lines 36 and 38: should the gene name end in -1?

Line 47: replace “to respond” with “that respond”.

Line 48: replace “organs” with “organ”

Line 50: replace “in plant” with “in the plant”

Line 63: spelling error in protein name

Line 65: What is “pollen the tube”?

Lines 92-95: cite information sources.

Line 115: it has not yet been established that F. x ananassa cultivars have four, consistently definable subgenomes, or that “Camarosa” is typical. No other studies have defined octoploid subgenome composition in the same way. When referring to the subgenome model for Camarosa published by Edger et al., 2019, it would be advisable to qualify your statements to make it clear that their model is based on one study of one cultivar, and that it is still best regarded as “tentative” or “putative”.

Similarly, statements made about F. vesca Hawaii 4 (FvH4) should be qualified to limit conclusions specifically to this highly atypical representative of F. vesca, which in its wild type form has red fruit and short-day photoperiodic response as opposed to the yellow fruit and day neutrality of H4.

Line 130: Compared to what, and integrated into what? Not clear.

Line 140: Run a spell-check here and throughout manuscript.

Lines 258-260. It does not appear that the authors used a Blast search of the F. vesca genome, but relied only of the effectiveness of one search term “RALF”. They found 13 genes that had been previously identified as RLAF genes in the process of annotation by prior investigations. Thus, it seems unjustified for the authors to state that they have “identified” these 13 genes. They simply used a word search for find those RALF genes previously identified by others. Also, this word search approach is in my view insufficient for the intended purpose, which was to discover new RALF genes,, especially given that the investigators later used the 13 F. vesca RALF genes as queries in Blast searches of the octoploid Fxa genome. Why not also search back through to vesca genome using the 13 genes as queries to see if anything had been missed by the published genome annotation? This would be easy to do, and I would consider it a requirement in the revision.

Line 294: define “Fxa” upon first use.

Lines 351-352. Some additional justification is needed for the choice of “available RNA-seq datasets” in the performance of comparative expression analyses in F. vesca. Seemingly, the original purpose of these datasets was to assist in gene annotation of the F. vesca genome by other investigators. But what makes this set of sequence suitable for comparative expression analysis? Please explain.

Line 494: the examined upstream region was only 400 bp in length, which seems very small to warrant the term “putative promoter”. It is quite possible that interesting upstream polymorphisms would have been discovered by sequencing further upstream, on which basis it is not justifiable to state, as the authors have done, that “susceptibility to anthracnose disease cannot be associated with allelic polymorphisms”. It would appear accurate to say that in the limited sequence region examined, no association was detected.

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Reviewer #1: No

Reviewer #2: No

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Genomic structure and transcript analysis of the Rapid Alkalinization Factor (RALF) gene family during host-pathogen crosstalk in Fragaria vesca and Fragaria x ananassa strawberry

PONE-D-19-32656R1

Dear Dr. Baraldi,

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.

Please note that Reviewer 1 has recommended a minor revision, but this can likely be addressed during the proofing stage.

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,

Richard A Wilson

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 #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 #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: N/A

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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

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 #1: (No Response)

Reviewer #2: 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: Authors have answered all the queries and comments I have formulated, and completed satisfactorily those aspects that were pointed out in the previous version. They have also incorporated a new figure (Fig. 6) and a new Table (Table S3) to analyze the 5’ upstream, as suggested.

However, there is still a minor comment I would suggest the authors to incorporate.

In Figure 3 there is no indication that the gene expression levels shown are relative to the reference gene Elongation Factor 1. The latter can be done by adding at the figure caption a line saying that the numbers correspond to the “relative expression level”, or at the ordinate.

As far as I am concerned, my opinion is that the paper can be published after incorporating the above mentioned comment.

Reviewer #2: (No Response)

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Reviewer #1: No

Reviewer #2: No