Ecotype-specific blockage of tasiARF production by two different RNA viruses in Arabidopsis

Péter Gyula; Tamás Tóth; Teréz Gorcsa; Tünde Nyikó; Anita Sós-Hegedűs; György Szittya

doi:10.1371/journal.pone.0275588

Peer Review History

Original SubmissionMarch 29, 2022
25 Jul 2022 Decision Letter - Rajarshi Gaur, Editor PONE-D-22-09203Ecotype-specific blockage of tasiARF production by two different RNA viruses in ArabidopsisPLOS ONE Dear Dr. Gyula, 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. Please submit your revised manuscript by Sep 08 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're 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. 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The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf. [Note: HTML markup is below. Please do not edit.] 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: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 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 ****** 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 ****** 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: Comments to authors Aims and methoda Dr Gyula co-authors characterised the sRNA and RNA profiles of two Arabidopsis ecotypes in terms of interaction with two viruses belonging to different taxonomic groups. The aim of the study was to identify patterns/responses that could explain the common phenotype induced by the two viruses in the 'bur' ecotype alone and not in the 'col' ecotype. Relevance of the study The scientific questions posed by the authors are relevant, all the more so since plant ecotypes differenziate in precise geographic area and stabilise genetic traits whose study is a) in continuity with the establishment of platforms for the conservation and characterisation of plant genetic resources; b) fundamental for investigating and characterising traits of fast-changing environmental adaptation with regard to biotic and abiotic stresses. The main results The main answer in the study lies in the functionality of the long non-coding RNA called TAS3 and the abnormal production (abolished in the 'bur' ecotype) of tasiARFs. The results are consistent with the known and well-studied function of ARFs in leaf development (also well illustrated in the introduction and discussions). In itself, this finding might be sufficient to support the main conclusion. In this frame, however, the authors discuss that AGOs involved in the function of tasiARFs could have differences in the two ecotypes, but they do not show it (different amino acid composition SNPs/indels at functional points) : the authors are invited to evaluate and show (if present) such differences available in the 1001 genomes project at https://1001genomes.org/. Main criticisms There are, however, some notable criticisms. The authors adopt a very interesting 'omic' approach to identifying genes involved in the symptomatological expression of the disease without definitely and fully considering (even only in the introduction) what has been discovered so far in terms of viral-associated siRNAs derived from coding genes. In particular, I refer to the work of Cao et al, 2012 (mentioned but not well discussed, being source of valuable information in the supplementary data concerning infection with TuMV, the same virus analysed in the present study), Leonetti et al, 2020 and Pitzalis et al, 2020. In particular, a cursory analysis of the Data_S1 table reveals that a large proportion of the unique sRNAs are 22-nt long (6,863, as opposed to 16,336 of 21-nt). This is why the authors, should take the opportunity of the knowledge they have gained (see leonetti et al., 2020 on the functionality of 22-nt vasiRNAs) and possibly extend it. The authors are therefore invited to consider the issue of vasiRNAs as being responsible, together with tasiARFs. Alternatively they should eliminate the analysis of vasiRNAs from this study. Moreover, with regard to vasiRNAs we note that there are no sRNAs in DATA_S1 that are derived from FIL and this finding is inconsistent with two of the panels in Figure 10 (i.e. vasiRNAs-FIL). It is also unclear whether in the histograms of the figures, when referring to sRNAs from specific loci, only those of 21-nt are considered or also those of other lengths: to be clarified. The authors are therefore invited to consider the issue of vasiRNAs as being responsible of the specific diseased phenotype, together with tasiARFs. Alternatively they should eliminate the analysis of vasiRNAs from this study. Other criticisms In the discussions an important concept is reported . The first part of the sentence is related to what is requested above (see “The main resuls” of comments to authors) but is not fully supported in the part concerning the evolution of the VSRs. In this regard, it is well known that VSRs of different viral taxonomic groups target different steps in the silencing pathways with different mechanisms, implying convergent evolution, i.e., having analogous features but non-homologous motifs. Strong VSR evolution/diversification is attributable to episodic selection rather than to pervasive positive selection (Murray et al., 2013). In other words, the diversification of plant virus VSRs is strongly boosted by frequent jumps between host species/genotypes rather than a one-to-one co-evolution. If what shown by Murray et al (2013) is in line with your discussion, please clarify. Minor: GWAS is only mentioned once in the discussions: do not use (unnecessary) acronym, which is instead required when the term is recurrent in the text more than 2-3 times. Reviewer #2: Although this manuscript did not conclude with genetic examinations, this reviewer thinks it provides helpful suggestions for future studies to identify host factors involved in the symptom expressions the authors focused on, including leaf deformation caused by two distinct virus infections in Arabidopsis. The authors explored the host factors by comparison analysis with RNA-seq and small RNA-seq between virus-infected Col and Bur ecotypes. Bur showed leaf deformation with these virus infections, but Col did not. Their RNA-seq showed that two genes, ARF4 and FIL, were specifically induced and reduced in Bur. Then, sRNA-seq consistently revealed that tasiARF that regulates the ARF4 expression was specifically reduced among other tasiRNAs. QTL analysis with RILs between Bur and Col supported their transcriptome analyses; that is, one of the QTL detected was around the locus of ARF4, though the QTL analysis is perhaps considered preliminary level. Minor points: P2, L16, and L17, These ARF4 mean protein and thus should be roman. P3, L17-22, These descriptions are of the case when plants have appropriate R genes that recognize invaded viruses. Rewrite them. P25, L17, Fig 10 should be Fig 12 ****** 6. 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 #1: No Reviewer #2: Yes: Yes ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0275588.r001
Revision 1
6 Sep 2022 Author Response First of all, we appreciate the time and effort the reviewers invested to provide us with their valuable criticism and recommendations. We rewrote the introduction, results, and discussion part according to the suggestions which we believe significantly improved the manuscript. Please find our detailed reactions below. Reviewer #1: Aims and methods Dr Gyula co-authors characterised the sRNA and RNA profiles of two Arabidopsis ecotypes in terms of interaction with two viruses belonging to different taxonomic groups. The aim of the study was to identify patterns/responses that could explain the common phenotype induced by the two viruses in the 'bur' ecotype alone and not in the 'col' ecotype. Relevance of the study The scientific questions posed by the authors are relevant, all the more so since plant ecotypes differenziate in precise geographic area and stabilise genetic traits whose study is a) in continuity with the establishment of platforms for the conservation and characterisation of plant genetic resources; b) fundamental for investigating and characterising traits of fast-changing environmental adaptation with regard to biotic and abiotic stresses. The main results The main answer in the study lies in the functionality of the long non-coding RNA called TAS3 and the abnormal production (abolished in the 'bur' ecotype) of tasiARFs. The results are consistent with the known and well-studied function of ARFs in leaf development (also well illustrated in the introduction and discussions). In itself, this finding might be sufficient to support the main conclusion. In this frame, however, the authors discuss that AGOs involved in the function of tasiARFs could have differences in the two ecotypes, but they do not show it (different amino acid composition SNPs/indels at functional points): the authors are invited to evaluate and show (if present) such differences available in the 1001 genomes project at https://1001genomes.org/. Answer: Thank you for your suggestion. We aligned the amino acid sequences of AGO1, AGO2, AGO7, ARF4, and FIL from 19 different ecotypes, including Col and Bur (Supplementary Fig S2). The analysis revealed that there were 5 amino acid changes in the Bur version of AGO7 compared to the Col version (all in the N-terminal part), while there were 0, 1, 2, and 0 amino acid changes in AGO1, AGO2, ARF4, and FIL, respectively (Fig S2). The Bur version of AGO7 was more different than the versions in the rest of the ecotypes. The 2 amino acid changes in ARF4 were not conserved (E/K and T/I in Col and Bur, respectively), and again, the Bur variant was rare among the investigated ecotypes. The FIL and AGO1 sequences were remarkably conserved among the investigated 19 ecotypes suggesting a strong selection pressure on them. There was some degree of variability in the AGO2 sequence but the Bur was not the most divergent ecotype in this respect. Main criticisms There are, however, some notable criticisms. The authors adopt a very interesting 'omic' approach to identifying genes involved in the symptomatological expression of the disease without definitely and fully considering (even only in the introduction) what has been discovered so far in terms of viral-associated siRNAs derived from coding genes. In particular, I refer to the work of Cao et al, 2012 (mentioned but not well discussed, being source of valuable information in the supplementary data concerning infection with TuMV, the same virus analysed in the present study), Leonetti et al, 2020 and Pitzalis et al, 2020. In particular, a cursory analysis of the Data_S1 table reveals that a large proportion of the unique sRNAs are 22-nt long (6,863, as opposed to 16,336 of 21-nt). This is why the authors, should take the opportunity of the knowledge they have gained (see leonetti et al., 2020 on the functionality of 22-nt vasiRNAs) and possibly extend it. The authors are therefore invited to consider the issue of vasiRNAs as being responsible, together with tasiARFs. Alternatively they should eliminate the analysis of vasiRNAs from this study. Answer: We admit that the discussion part could have been more thorough, especially regarding the research on the function vasiRNAs in plants (we fixed this in the Introduction and Discussion part, see the yellow text). It was because we focused on the differences rather than the similarities between the two ecotypes regarding their antiviral responses. We wanted to avoid discussing the commonly regulated sRNAs that were possibly not responsible for the differential symptoms observed in the two ecotypes. Additionally, we focused on the genes/sRNAs that were similarly differentially expressed only in the Bur ecotype infected by two different viruses and not discussed those genes/sRNAs that were unique for the two viruses or that were changed in the opposite direction. However, we observed that the vast majority of the commonly downregulated genes are potentially regulated by vasiRNAs that are commonly upregulated only in the Bur ecotype infected with both viruses. This is surprising, and therefore, we decided to discuss them in a little more detail, especially because these genes have functions that could be related to leaf development (i.e. auxin metabolism, cell wall expansion, etc). Regarding the 22-nt-long vasiRNAs, the number of 22-nt sRNAs (6863) is the number of unique sequences in all the samples, including mock samples, therefore, it does not tell us how virus infection affects this size class. Actually, we found no such markedly increased population of this size class as in the case of CaMV infection investigated by Leonetti et al (see our corrected Fig 7). Maybe this is a CaMV-specific feature. Moreover, with regard to vasiRNAs we note that there are no sRNAs in DATA_S1 that are derived from FIL and this finding is inconsistent with two of the panels in Figure 10 (i.e. vasiRNAs-FIL). Answer: We are sorry for being confusing here: the vasiRNA-FIL is derived from a transposon (not from FIL) and potentially targets FIL in trans. It is analogous to tasi-ARF, when the functional siRNA is derived from the TAS3 non-coding RNA and targets ARF3- and ARF4-coding transcripts. We are aware that vasiRNAs could target the gene they derived from (in this case, a transposon). To predict potential vasiRNA/target pairs, we only considered those vasiRNAs and target genes that were differentially expressed only in the Bur virus samples in the opposite direction, i.e. upregulated vasiRNAs vs downregulated target genes. We did not predict all the possible targets of all the vasiRNAs, because we considered it irrelevant to our goal to find the possible cause of the differential leaf deformation. It is also unclear whether in the histograms of the figures, when referring to sRNAs from specific loci, only those of 21-nt are considered or also those of other lengths: to be clarified. Answer: When we show the expression of certain sRNA specimens (i.e. miR390a,b-5p, tasiARF a.k.a. TAS3 5’D7(+), TAS1abc-siR255, etc), we considered only the conserved, mature sequence; iso-miRs or other sequence variants (or other, non-functional siRNAs from the same TAS locus) were not included in the analysis. Other criticisms In the discussions an important concept is reported <>. The first part of the sentence is related to what is requested above (see “The main resuls” of comments to authors) but is not fully supported in the part concerning the evolution of the VSRs. In this regard, it is well known that VSRs of different viral taxonomic groups target different steps in the silencing pathways with different mechanisms, implying convergent evolution, i.e., having analogous features but non-homologous motifs. Strong VSR evolution/diversification is attributable to episodic selection rather than to pervasive positive selection (Murray et al., 2013). In other words, the diversification of plant virus VSRs is strongly boosted by frequent jumps between host species/genotypes rather than a one-to-one co-evolution. If what shown by Murray et al (2013) is in line with your discussion, please clarify. Answer: We guess that the incriminated sentence was about the parallel evolution of the VSRs of different viruses targeting rice ARF17. We suppose that in our case the VSRs of the two different viruses interfere with the biosynthesis of the tasi-ARF in different ways. For example, one VSR might bind miR390 while the other might block AGO7 activity. As the reviewer pointed out, this would require non-homologous protein motifs. Both VSRs were reported to bind double-stranded sRNAs and HCPro was reported to bind AGO1. We analyzed the sequence of the miR390 duplex in the two ecotypes because we theorized that the different duplex structure (i.e. because of a possibly different star sequence) could result in a differential binding of miR390 duplex by the VSR(s). However, we found no evidence for such a difference. AGO1 amino acid sequences of the two ecotypes are also identical. We think that the best target of the VSRs is either the AGO7 or AGO2 proteins, or some unknown component of the tasi-ARF biosynthesis pathway. Murray et al argue that plant VSRs are subject to episodic positive selection due to frequent jumps between different hosts rather than a one-to-one co-evolution with a single host. This is consistent with our findings since both viruses we worked with have a broad host range (i.e. the turnip vein-clearing virus isolate we used, TVCV-ApH, was isolated from Alliaria petiolata, a.k.a. garlic mustard). Minor: GWAS is only mentioned once in the discussions: do not use (unnecessary) acronym, which is instead required when the term is recurrent in the text more than 2-3 times. Answer: The Reviewer is right, we replaced the acronym with the full name. Reviewer #2: Although this manuscript did not conclude with genetic examinations, this reviewer thinks it provides helpful suggestions for future studies to identify host factors involved in the symptom expressions the authors focused on, including leaf deformation caused by two distinct virus infections in Arabidopsis. The authors explored the host factors by comparison analysis with RNA-seq and small RNA-seq between virus-infected Col and Bur ecotypes. Bur showed leaf deformation with these virus infections, but Col did not. Their RNA-seq showed that two genes, ARF4 and FIL, were specifically induced and reduced in Bur. Then, sRNA-seq consistently revealed that tasiARF that regulates the ARF4 expression was specifically reduced among other tasiRNAs. QTL analysis with RILs between Bur and Col supported their transcriptome analyses; that is, one of the QTL detected was around the locus of ARF4, though the QTL analysis is perhaps considered preliminary level. Minor points: P2, L16, and L17, These ARF4 mean protein and thus should be roman. Answer: We changed the format to refer to protein products. P3, L17-22, These descriptions are of the case when plants have appropriate R genes that recognize invaded viruses. Rewrite them. Answer: Thank you for noticing this, we rephrased the sentence as suggested. P25, L17, Fig 10 should be Fig 12 Answer: We fixed it. https://doi.org/10.1371/journal.pone.0275588.r002
20 Sep 2022 Decision Letter - Rajarshi Gaur, Editor Ecotype-specific blockage of tasiARF production by two different RNA viruses in Arabidopsis PONE-D-22-09203R1 Dear Dr. Gyula, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Rajarshi Gaur Academic Editor PLOS ONE 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 ******** 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: N/A ****** 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 ****** 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 ****** 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: Thank you for accepting addressing all the and criticisms were raised during the first reviewing round. Reviewer #2: (No Response) ****** 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 #1: No Reviewer #2: No ******** https://doi.org/10.1371/journal.pone.0275588.r003
Formally Accepted
26 Sep 2022 Acceptance Letter - Rajarshi Gaur, Editor PONE-D-22-09203R1 Ecotype-specific blockage of tasiARF production by two different RNA viruses in Arabidopsis Dear Dr. Gyula: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Professor Rajarshi Gaur Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0275588.r004

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