PONE-D-19-24276

Comparative transcriptomics of Diuraphis noxia and Schizaphis graminum fed wheat plants containing different aphid-resistance genes

PLOS ONE

Dear Dr. Smith,

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.  In particular:

• Both reviewers have indicated that more information is required about the wheat lines that were tested, in particular how genetically similar (or different) they are aside from the presence/absence of the resistance alleles
• Reviewer 2 also requested more information about genetic diversity in the aphid populations tested, in particular as this relates to the phenotypic variation observed on wheat lines.
• Both reviewers made cautionary statements related to drawing conclusions based on annotations that are not functionally validated.
• Consider Reviewer 2's suggestion to provide a hypothesis in the Introduction as a context for your Discussion and Conclusions, which may help non-specialist readers follow the logic of the paper.

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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: Yes

Reviewer #2: No

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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: No

Reviewer #2: Yes

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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: The authors investigated plant phenotypic responses and aphid gene expression responses using three wheat cultivars(Yuma, Yumar, and 94M370) and two aphid species (Schizaphis graminum and Diuraphis noxia). Most of the results section consists of a listing of gene expression changes, along with speculation regarding what the identified genes might be good for. There is no confirmation of candidate gene function.

Specific comments:

1. “The Russian wheat aphid, Diuraphis noxia, (Kurdjumov) has invaded all wheat producing regions in the world since being identified in 1900 and causes major losses in yield of bread wheat, Triticum aestivum L.,(Quisenberry and Peairs 1998, Baker et al. 2016). Based on recent CLIMEX projections, D. noxia distribution will soon expand into Brazil, China, eastern North America, Northern Europe and New Zealand (Avila et al. 2018).” These sentences seem contradictory. On the one hand, D. noxia has invaded all wheat-growing areas and, on the other hand, there are wheat-growing areas to which it will expand soon.

2. Some of the information under “Insect and Plant Material” is better placed in the Results than in the Methods section. How the authors identified aphid species is the method. The outcome of the identification process should be in the results section.

3. The DNA sequencing results that confirm the aphid species should be deposited in GenBank (or some other public database), and the corresponding GenBank ID number should be included in the manuscript. This is particularly important given that the authors have previously had issues with misidentification of aphid species.

4. There should be better description of the relatedness of the three wheat cultivars (Yuma,

Yumar, and 94M370). How near-isogenic are they? It is mentioned that Dn7 is a translocation from rye. How similar is the rest of the genome between the three lines? In addition to the Dn4 and Dn7 regions, do other parts of the wheat genome also influence plant symptom formation and aphid gene expression responses in these experiments?

5. Lines 317-318: “which showed 95% and 100% nucleotide identity to Buchnera aphidicola strains associated with S. graminum, respectively.” I think that this sentence is missing “D. noxia”.

6. Lines 409-410. “All four GH family 1 unigenes have highest scoring blastp matches to myrosinases, which are involved in degrading toxic glucosinolates produced by plants in the order Brassicales.” Myrosinases do not degrade toxic glucosinolates. Instead, they are involved in cleaving glucose off glucosinolates to produce toxic breakdown products. It should also be noted that the myrosinase homology that the authors found is not to plant enzymes, but to the myrosinase of Brevicoryne brassicae (cabbage aphid), which sequesters glucosinolates and produces its own myrosinase for activation as a defense against predators. Given that very few aphid glucosidases have been functionally investigated, it is not surprising that the closest identified match is to B. brassicae myrosinase. However, I would not read any big meaning into this.

7. Figure 5 legend “clusters of highly expressed (2 - 8 fold) unigenes” This should be “highly differentially expressed”, as the selection is based on fold-induction rather than absolute expression level.

8. It would be helpful if the first line of each Excel supplemental table included some sort of header stating what is shown in the table.

9. “Quality filtered reads from all nine S. graminum samples were pooled and a de novo transcriptome

assembly was performed” and “All transcripts containing protein coding genes have been submitted to NCBI’s Transcriptome Shotgun Assembly database (accession number pending).” Why limit this to protein-coding assemblies? There is also information content in the assemblies that were deemed to be non-protein-coding. I recommend uploading all of the assemblies, as they might be useful in future analyses by the authors and others.

Reviewer #2: The authors planned to conduct the comparative transcription analysis of D. noxia biotype 1 and 2 feeding on three wheat cultivars, each contains a different D. noxia resistance gene. However, they found later that “D. noxia biotype 2” was S. graminum biotype I and reorganised the manuscript as submitted here.

Major problems

I had a hard time to read the manuscript because the manuscript does not contain any hypothesis but contains a long description of the differentially expressed (DE) genes and their GO categories. The authors tried to make a story out of the list of DE genes, but to me, it looks like many things were happening in those aphids. In case of D. noxia, it is not clear to me if they were simply dying or actively responding to the plants. It is pity that the authors had to pool the samples from different time points and missed the opportunity to dissect the DE genes. More information of aphid performance (developmental time, survival rate, growth curve) may help to understand the transcription data.

In the manuscript, there is not enough description of the wheat genotypes (it’s not nice to let readers dig up references for such info), and no data was presented regarding the interaction between D. noxia and the wheat genotypes: therefore, based on this manuscript, readers cannot make their own hypothesis and examine the data presented. I think that more information on the wheat genotypes (How were they created? How genetically close are they?) and their interactions with D. noxia biotype 1 (like Table 1 and also need to show survival rate and developmental time etc) should be presented. The authors presented three wheat lines as “Dn0”, “Dn4” and “Dn7”, but I think it is misleading the readers because these lines contain many different genes in addition to the Dn genes. Especially, the Dn7 line was created by transferring rye genomic segments (chromosome arms) into wheat cultivars. Both tested aphids responded quite differently on Dn7 compared to Dn0 and Dn4, but the differences might be caused by the rye genes in Dn7 wheat line and may not be related to the Dn7 gene itself. I think this point should be clearly explained in the manuscript. The subtitles like “Impacts of Dn7 on global gene expression in S. graminum” is not correct. Especially, in case of Dn4, there was no impact of Dn4 on wheat responses to S. graminum or on S. graminum fecundity, so such a subtitle is simply confusing.

I also worry about their aphid stocks. Authors wrote “D. noxia biotype 1 aphids were collected… (L110)” Those multiple aphids may have the same COI sequence, but they may have many genetic differences… It is also not clear whether S. graminium biotype I stock is a mixture of many S. graminum genotypes or not. The mixture of various aphid lines (genotypes) may be a cause of the huge variation of aphid numbers presented in Table 1.

Minor points

L42-44

Dn7 carrying wheat line had no effect on the fecundity of S. graminum, so, I don’t agree with this sentence. What they saw in wheat (less chlorosis) should be considered as a tolerance of the wheat and not a resistance reaction. Same for L604.

L73-75: Although experimental support is lacking, “stealth” feeding behaviors is not thought to mask aphid effectors from plant perception (at least, the cited manuscripts don’t say that). Although effectors recognised by plant resistance genes are not identified yet, infestation of specific aphids are recognised by these resistance genes.

L79: In my knowledge, no one showed the variation in sensitivity of olfactory, gustatory and salivary?! receptors. IAGC did not compare biotypes, and Smadja showed high level of sequence differences in chemosensory genes in different A. pisum biotypes. The sentence is incorrect.

Fig.4 legend, last sentence: cluster 2 instead cluster 1?

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Reviewer #1: No

Reviewer #2: No

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PONE-D-19-24276R1

Comparative transcriptomics of Diuraphis noxia and Schizaphis graminum fed wheat plants containing different aphid-resistance genes

PLOS ONE

Dear Dr. Smith,

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.

Both reviewers have responded similarly (and consistently to my own response) to the additional information you have provided in this revision on the genetic relatedness of the wheat lines tested.  You should address in your revision the two related questions raised:

• Is Yuma an appropriate control plant for the 94M370 resistant line containing Dn7?
• Can the DEGs observed between 94M370 and Yuma reasonably be attributed to the Dn7 resistance gene?

Neither reviewer believe this issue precludes publication of this research, but both have indicated that the limitations of your design need to be made more explicit.  You should also address the reviewers' other comments.

We would appreciate receiving your revised manuscript by May 04 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Owain Rhys Edwards, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

The information provided by the authors on the genetic relationship among the wheat genotypes tested provides some concerns about the interpretation of the results. The first concern is that the best control plant has not been included for the interpretation of the 94M370 line ("Dn7"). Previous studies have compared the resistance phenotype (and its transcriptome) in 94M370 to Gamtoos as a near-isogenic susceptible (Zaayman et al. 2009), the results of which are very relevant to the submitted work and really should be referenced and discussed. The authors need to defend why Gamtoos was not included in their experiments to provide a better control for the 94M370 results.

The second (related) issue is that all the clustering results provided (visible in Figs. 1, 3, 6, 7) shows quite clearly that there is more variability in transcriptomic response associated with genetic background than with the resistance phenotype. The authors have attributed this variation to the resistance provided by Dn7, which is a legitimate hypothesis, but it is equally legitimate to attribute a large part of this variation to the genetic background differences between 94M370 and the other two near-isogenic lines. It is not unreasonable for the authors to propose their preferred hypothesis, but it needs to be presented with an appropriate level of uncertainty and the alternative hypothesis should be given (especially considering that there is a translocation containing additional genes, including R genes, present in 94M370 that is absent from the other two lines).

The authors may need to reconsider referring to the lines as "Dn0", "Dn4", and "Dn7" - as this may suggest to a reader who has not read the methods in detail that the hosts are all near-isogenic, differing only in the presence/absence of these genes. While this is the case for "Dn0" and "Dn4", it is definitely not the case for "Dn7".

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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: (No Response)

Reviewer #2: (No Response)

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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: Partly

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

Reviewer #1: Yes

Reviewer #2: 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

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: Yes

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: My comments and those of the other reviewer have been mostly addressed.

Thank you for providing more information about the genetic background of the wheat cultivars that were used. How genetically similar is Gamtoos, into which Dn7 was crossed, to Yuma wheat. Is Yuma really the appropriate control for Gamtoos with a 1RS translocation from rye?

Lines 537-538. “All four GH family 1 unigenes have highest scoring blastp matches to

538 myrosinases.” Please note at this point that these are myrosinases from cabbage aphids, so that there is no confusion with the more commonly studied cruciferous plant myrosinases. A reference describing cabbage aphid myrosinases would also be appropriate here.

Reviewer 2 commented that this manuscript is difficult to read with a long listing of differentially expressed genes, no hypothesis being tested, and no experimental validation of the speculated function of differentially expressed genes. I agree. However, this manuscript is being submitted to PLoS One, which does not require results to be a significant advance over what was previously known.

Reviewer 2 questioned how genetically homogeneous the selected aphid populations are. The authors’ response that they are all biotype 1 does not mean that they are genetically homogeneous. Unless they started their colony with a single parthenogenetic aphid, there is no easy way to determine that they all have the same genotype. Or, if the different biotypes are known to be single parthenogenetic clones, is there a reference for this?

Response to reviewers: “It is highly unlikely that other rye genes on the 1RS translocation function in aphid resistance, as Dn7 has been mapped specifically to D. noxia resistance” Based on information provided in the references, the Dn7 gene has not been identified. Given the size of the current genetic mapping interval (2.4 cM and likely a few million bp of DNA), it is not yet possible to say that a single gene affects aphid resistance.

Reviewer #2: I appreciate the information added by the authors, but I have the same concerns as before. The three wheat genotypes (especially the one containing Dn7) seem quite different from each other. The DE of the aphid genes feeding on those three genotypes may reflect various physiological differences of the plants but may not reflect the presence of Dn4 or Dn7. The manuscript concludes that “Overall, the variation in transcriptional responses of D. noxia and S. graminum to the Dn7 and Dn4 resistance genes suggests that the mechanisms underlying the evolution of virulent biotypes of these aphids are likely to be species specific… (Line 777-)”, but it should assume and discuss the limitation of their work.

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Reviewer #1: No

Reviewer #2: No

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Comparative transcriptomics of Diuraphis noxia and Schizaphis graminum fed wheat plants containing different aphid-resistance genes

PONE-D-19-24276R2

Dear Dr. Smith,

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Owain Rhys Edwards, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: