Characterization of an unusual carlavirus-like RNA from papaya (Carica papaya) lacking essential genes

Milenka Vera; Raúl Cifuentes; Ronan Keener; Neil Olszewski; Robert A. Alvarez-Quinto; Diego F. Quito-Avila

doi:10.1371/journal.pone.0329708

Peer Review History

Original SubmissionFebruary 14, 2025
23 May 2025 Decision Letter - Abozar Ghorbani, Editor Dear Dr. Quito-Avila, 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 Jul 07 2025 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. A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. 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Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript. [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? 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 Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes ****** Reviewer #1: The manuscript entitled 'Characterization of an unusual carlavirus-like RNA from papaya (Carica papaya) lacking essential genes' represents the presence of a new carlavirus-like RNA elements in papaya latex. The authors have meticulously performed the experiments to justify the manuscript; but few points have to be addressed before publishing this manuscript: 1. Authors sampled only papaya latex, what about different plant parts or tissues? 2. The authors should check/confirmed the presence of CPMMV in the papaya plants harbouring the unusual carlavirus-like RNA as the helper virus; as both having close similarity with indentical terminal seqiences as written in Figure 2. 3. It is suggested to conduct a complementaion study by co-inoculating CPMMV or other carlavirues (PaMV, PaMMV) infecting papaya along with agroinoculation of pLX-AS_D to prove the infectivity of carlavirus-like RNA (PapDfV1) in papaya. 4. The quality of figures in the manuscript must improved. 5. Some minor typo-graphical errors (in attached files) must be corrected. Reviewer #2: 1. In line 1, the title of the article is clear, attractive, and descriptive, and reflects the overall structure and content of the article 2. This article is written at a good level and the different sections for example introduction, abstract, discussion is readability and having sufficient organization. 3. In the introduction, the importance and position of the research are explained in an acceptable format. 4. In the results section, the data related to the phylogenetic analysis are explained in a suitable manner. 5. In line 38, add a short section on the importance of the discovery of PapDfV1 or other defective viruses in the Abstract and Introduction sections for understanding the evolution of plant viruses 6. In line 66, in the introduction section, provide a reference on possible mechanisms of compensation for deleted genes in carlaviruses or other viruses. 7. In line 226, in the results section, it is better to present a schematic diagram of the genomic structure of PapDfV1 compared to typical carlaviruses. To better understand their differences in terms of genomic structure, based on the figure 8. In line 373, in the conclusion section, further emphasis is placed on the importance of the findings of this research for the direction of future studies. 9. There are a number of sentences in the long text. Please write them in shorter sentences. 10. In line 373, the conclusion title should be written 11. The main headings, including introduction, materials and methods, etc., should be numbered respectively. 12. Please write references to format of journal 13. In total, this article contains a correct writing structure and very desirable content, and with some minor revisions and corrections, it is acceptable. ****** 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: Farideh Farahbakhsh Plant Protection Department, Agricultural Research, Education and Extension Organization (AREEO), Darab, Fars, Iran ******** [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. Attachments Attachment Submitted filename: PONE-D-25-08143 AC.pdf Attachment Submitted filename: Comment.docx Attachment Submitted filename: Papaya Carlavirus Final_02-13-25.docx https://doi.org/10.1371/journal.pone.0329708.r001
Revision 1
2 Jul 2025 Author Response Response to Reviewer’s Comments We sincerely acknowledge the time and effort that both reviewers and the editor have dedicated to evaluating our manuscript. We are confident that each comment and suggestion was intended to enhance the quality and readability of our work, and we greatly appreciate the constructive feedback provided. Below, please find our detailed responses to each of the reviewers' comments and suggestions. As part of our rebuttal, we have included a tracked-changes version of the manuscript for verification. Please note that we have carefully considered and addressed all comments, both those included in the email body and those provided in the attached documents. ____________________________________________________________________ Reviewer #1: The manuscript entitled 'Characterization of an unusual carlavirus-like RNA from papaya (Carica papaya) lacking essential genes' represents the presence of a new carlavirus-like RNA elements in papaya latex. The authors have meticulously performed the experiments to justify the manuscript; but few points have to be addressed before publishing this manuscript: 1. Authors sampled only papaya latex, what about different plant parts or tissues? Response: We appreciate this observation. The viral sequence was detected from total RNA extracted from both leaf and latex tissues, and additionally from dsRNA extracted from leaf tissue. We acknowledge that this detail was not explicitly stated in the Results section. However, Figure 1C (in its current version) shows detection using two distinct primer sets from total RNA (from latex and leaf) and from dsRNA (from leaves). Furthermore, the Discussion does address this point: “Despite the lack of experimental evidence for systemic infection and movement of PapDfV1, its infection under natural conditions was supported by its detection in total RNA and dsRNA, a replicative intermediate, both extracted from leaf tissue. Notably, RT-PCR amplification of the fragment spanning the deleted region was prominent from dsRNA, indicating active replication of viral RNA not only in latex but also in leaf tissues.” Nevertheless, to directly address the reviewer’s observation, we have added a sentence in the Results section referring explicitly to the detection of the virus in the tested tissues. We have also slightly modified the subheading from “Virus genome assembly” to “Virus detection and genome assembly.” In this revised section, we now begin by describing the detection of the virus in both latex and leaf tissues. To align with this change in narrative, we have also swapped the order of panels B and C in Figure 1. We believe these adjustments, though minor, significantly enhance the clarity and readability of the manuscript. 2. The authors should check/confirmed the presence of CPMMV in the papaya plants harbouring the unusual carlavirus-like RNA as the helper virus; as both having close similarity with indentical terminal seqiences as written in Figure 2. Response: We appreciate the reviewer’s suggestion. We did test for the presence of CPMMV using CPMMV-specific primers; however, no amplification was obtained. Accordingly, we have added this information to the Methods section (see "Search for missing genes and helper viruses"), including the primer sequences and relevant citation. This has also been noted in the Results section (see the last paragraph of "Virus detection and genome assembly"). 3. It is suggested to conduct a complementaion study by co-inoculating CPMMV or other carlavirues (PaMV, PaMMV) infecting papaya along with agroinoculation of pLX-AS_D to prove the infectivity of carlavirus-like RNA (PapDfV1) in papaya. Response: We acknowledge the validity and relevance of this suggestion.We have addressed this possibility as a direction for future studies in the Discussion section. 4. The quality of figures in the manuscript must improved. Response: We appreciate the reviewer’s comment. The reduced quality of figures in the current manuscript is due to the PDF conversion process. We assure that the final submitted figures will meet the high-quality standards required by the journal. 5. Some minor typo-graphical errors (in attached files) must be corrected. Response: Thank you. We have accepted all those typos diligently caught by the reviewer. _________________________________________________________________ Reviewer #2: In line 1, the title of the article is clear, attractive, and descriptive, and reflects the overall structure and content of the article Response: Thank you. 2. This article is written at a good level and the different sections for example introduction, abstract, discussion is readability and having sufficient organization. Response: Thank you. 3. In the introduction, the importance and position of the research are explained in an acceptable format. Response: Thank you. 4. In the results section, the data related to the phylogenetic analysis are explained in a suitable manner. Response: Thank you. 5. In line 38, add a short section on the importance of the discovery of PapDfV1 or other defective viruses in the Abstract and Introduction sections for understanding the evolution of plant viruses Response: Thank you. We have added the information suggested both at the end of the abstract and the introduction section. 6. In line 66, in the introduction section, provide a reference on possible mechanisms of compensation for deleted genes in carlaviruses or other viruses. Response: We respectfully disagree with this suggestion. The sentence referenced by the reviewer does not mention defective carlaviruses or the loss of viral genes, and thus, it would not be appropriate to cite compensation mechanisms in that specific context. However, we do address the concept of defective carlaviruses in the Discussion section, where we reference relevant literature and explain how previously reported events relate to our findings. We appreciate the reviewer’s attention to detail and thank them for their understanding. 7. In line 226, in the results section, it is better to present a schematic diagram of the genomic structure of PapDfV1 compared to typical carlaviruses. To better understand their differences in terms of genomic structure, based on the figure Response: We appreciate the reviewer’s suggestion. However, the genome organization of ZhBV2, the closest known relative of PapDfV1, is already presented in Figure 1 and represents the typical carlavirus genomic structure. As indicated in the figure legend, “The canonical carlavirus genome structure of ZhBV2 is illustrated.” Therefore, we believe that including an additional, separate schematic of a typical carlavirus genome would be redundant. 8. In line 373, in the conclusion section, further emphasis is placed on the importance of the findings of this research for the direction of future studies. Response: We appreciate the reviewer’s comment. We believe that the concluding paragraph already emphasizes the significance of our findings and their implications for future research. This emphasis is consistent with the context provided in both the Introduction and the Abstract, where we also highlight the relevance and potential of this study to guide further investigations. 9. There are a number of sentences in the long text. Please write them in shorter sentences. Response: We have shortened a few long sentences in the discussion section. We appreciate the detailed attention to this matter. 10. In line 373, the conclusion title should be written Response: We thank the reviewer for this observation. According to the PLOS ONE submission guidelines, including a titled "Conclusions" section is optional. As such, we have chosen to retain the current structure of the manuscript. Thank you for your understanding. (see guidelines here: https://journals.plos.org/plosone/s/submission-guidelines). 11. The main headings, including introduction, materials and methods, etc., should be numbered respectively. Response: We thank the reviewer for the suggestion. However, PLOS ONE does not require numbering of main headings such as Introduction, Materials and Methods, etc. This is confirmed in the journal's formatting guidelines and the official manuscript template, which can be found here: https://journals.plos.org/plosone/s/submission-guidelines#loc-style-and-format:~:text=Download%20sample%20manuscript%20body%20(PDF) 12. Please write references to format of journal Response: Thank you. Yes. We have changed to the “Vancuver Style” required by the journal. 13. In total, this article contains a correct writing structure and very desirable content, and with some minor revisions and corrections, it is acceptable. Response: Thank you! Attachments Attachment Submitted filename: Response to Reviewer_Carla-Defective_MV.docx https://doi.org/10.1371/journal.pone.0329708.r002
21 Jul 2025 Decision Letter - Abozar Ghorbani, Editor Characterization of an unusual carlavirus-like RNA from papaya (Carica papaya) lacking essential genes PONE-D-25-08143R1 Dear Dr. Quito-Avila, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Abozar Ghorbani, Ph.D Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 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??> Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes ****** Reviewer #1: The authors have addressed all the question asked; this can be accepted for publication in the modified form. Reviewer #2: Dear Editor, All comments have been answered carefully and clearly. In my opinion, the article is eligible for acceptance in this journal. All the best. ****** 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: Farideh Farahbakhsh, Assistance Professor, Plant Pathology Department, Fars agricultural and natural resources, AREEO, Darab, Iran. ******** https://doi.org/10.1371/journal.pone.0329708.r003
Formally Accepted
Acceptance Letter - Abozar Ghorbani, Editor PONE-D-25-08143R1 PLOS ONE Dear Dr. Quito-Avila, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps. Lastly, 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. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. If we can help with anything else, please email us at customercare@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 Dr. Abozar Ghorbani Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0329708.r004

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