Unraveling the genomic complexity of secretion systems in the most virulent Xanthomonas arboricola pathovars

Sara Cuesta-Morrondo; Jerson Garita-Cambronero; Jaime Cubero

doi:10.1371/journal.pone.0332834

Peer Review History

Original SubmissionMay 9, 2025
30 Jun 2025 Decision Letter - Luis D. Alcaraz, Editor PONE-D-25-24971Unraveling the Genomic Complexity of Secretion Systems in the Most Virulent Xanthomonas arboricola pathovarsPLOS ONE Dear Dr. Cubero, 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. Your manuscript presents an engaging comparative genomics study focused on Xanthomonas and the diversity of its secretion systems. Following peer review, both reviewers concurred that the manuscript would benefit from further refinement, particularly regarding the definition of homologous genes and the application of more robust sequence alignment tools beyond BLAST to enhance validation. I do agree with them. I would be inclined to consider the manuscript for publication, provided that the authors address the reviewers’ comments. Doing so will significantly strengthen both the clarity of the findings and the overall impact of the manuscript. Please submit your revised manuscript by Aug 14 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. 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). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. 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Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols . We look forward to receiving your revised manuscript. Kind regards, Luis D. Alcaraz, Ph.D. Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. 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 2. Thank you for stating the following financial disclosure: [This study was supported by PID2021-123600OR-C41 funded MICIU / AEI /10.13039/501100011033/, by “ERDF A way of making Europe” and PRE2019-090846 “ESF Investing in your future”.]. 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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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: N/A ****** 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: This is a well-conceived and methodologically solid comparative genomics study that explores the diversity of secretion systems (T1SS–T5SS) across six closed genomes representing the most virulent Xanthomonas arboricola pathovars: pruni, juglandis, and corylina. The work contributes meaningfully to the field of plant pathology by offering insights into the genomic architecture underlying host specificity and virulence potential. The study uses complete (closed) genome assemblies, enabling high-resolution structural comparisons. Multiple secretion system clusters are characterized comprehensively across multiple strains. The visual presentation of gene clusters and secretion system comparisons (Figures 1–6) is clear, well-integrated, and supports the narrative effectively. The manuscript includes an appropriate discussion of potential horizontal gene transfer (HGT) events contributing to genomic diversity and cluster acquisition. The discovery and discussion of a putative non-canonical PctAB-like T1SS are particularly valuable and appropriately discussed. Recomendations: The Methods section states that protein hits were considered homologs when they satisfied three criteria: E-value < 0.001, query coverage >80%, and sequence identity >40%. The authors cite Pearson (2013) [DOI: 10.1002/0471250953.bi0301s42] in support of this threshold, which is appropriate given that the referenced work emphasizes the importance of statistical significance (e.g., E-values and bit scores) over raw percent identity when inferring homology. However, I recommend that the authors explicitly elaborate on this rationale in the manuscript, especially given the potential concern that a 40% identity threshold may allow the inclusion of distant homologs with uncertain functional conservation, even when high query coverage is used. This is particularly important for secretion system effectors, where minor sequence divergence may translate into major functional differences. To improve clarity and strengthen the reliability of the results, I suggest the authors: Briefly summarize Pearson’s argument (e.g., that statistical measures are more reliable than identity percentages alone in homology inference). Clarify whether domain conservation (e.g., InterProScan) or other functional annotations were used to validate low-identity hits. Minor Changes -Typographical edits: Line 52: "Thes pathovars" → "These pathovars" Lines 69–70: Consider rephrasing slightly for clarity: “Secretion systems are complex molecular machineries…” Line 156: “appeared to have been interrupted…” to “were likely disrupted…” Optional addition: -A brief sentence in the conclusions and/or discussing how the findings could guide future experimental design (e.g., validation of T1SS function or virulence assays) would help bridge the gap to applied research. Reviewer #2: In this manuscript, Cuesta-Morrondo and colleagues present a comparative genomic analysis of six previously sequenced Xanthomonas arboricola genomes. Given the well established role of secretion systems in bacterial pathogenicity, the authors focus their analysis on the occurrence and genomic organization of type I, II, III, and V secretion systems across Xa pathovars of agricultural relevance.The authors provide a systematic description of the presence/absnece patterns and genomic arrangement of the aforementioned secretion systems. While primarily descriptive in nature, with the authors limiting their analysis to the identification of gene clusters encoding these secretion systems, the study offers insights that will be of interest to researchers investigating Xanthomonas pathogenicity. Taking advantage of available high quality genomes, the authors report some interesting findings, including the identification of a TALE effector, which is often overlooked in draft genome analyses due to its repetitive structure. The authors also provide a detailed description of the distribution of distinct type III effectors across the analyzed genomes which may, at least partially, help explain differences in pathogenic profiles and host-pathogen interactions among X. arboricola pathovars. However, before recommending this manuscript for publication, I have a significant concern regarding the methodological aspect of secretion systems identification. MAjor comment: The key methodological limitation of the manuscript is the approach taken to detect secretion systems, as the authors rely only on BLAST searches, mainly against known components of secretion systems from Xanthomonas genomes. While effective for detecting conserved systems, this method may fail to identify divergent or lineage-specific secretion systems, therefore limiting the depth and completeness of the analysis. To address this limitation, I strongly recommend the use of TXSScan (https://github.com/macsy-models/TXSScan), a dedicated tool for the comprehensive identification of bacterial secretion systems. TXSScan integrates HMM profile searche and synteny criteria, offering enhanced sensitivity compared to BLAST based methods alone. Since the primary claim of the study concerns the diversity and organization of secretion systems, reanalysis using TXSScan would strengthen the conclusions and, potentially, could reveal additional systems not detected with the current approach. Given that TXSScan is relatively simple and straightforward to implement, this reviewer recommend reanalyzing the genomes using TXSScan to cross-validate, and potentially expand,the identification of secretion systems in the studied Xa genomes. This would ensure the completeness and robustness of the findings prior to publication. Minor comments: Line 34: "variation was" instead of "variation were." line 113: Review and correct citation format (Cuesta-Morrondo et al., 2022). line 189: The authors mention that xcsK is frameshifted…consider claryfing here whether xcsK is essential for T2SS function, especially for readers unfamiliar with the system nomenclature in Xanthomonas. line 190: Redaction needs to be double checked...consider rephraseing “Moreover, orthologs of eight Xanthomonas citri pv. citri experimentally validated T2SS substrates [42] were” to “Moreover, orthologs of eight experimentally validated T2SS substrates from Xanthomonas citri pv. citri were” Line 396: Rephrase: "Xac strains harbored two XAC2853 orthologs" to "...harbored two homologs of XAC2853", as the presence of two related genes within the same genome indicates paralogs, not orthologs. Fig. 1A: Labels are too small to read. Fig. 3 and 4: These both relate to T3SS and could be merged into a single figure for clarity and consistency Fig. 5: Again…gene labels are too small, especially in panel 5C Fig. 6A: Clarify the meaning of the lighter blue boxes in the figure legend. I guess they represent predicted non-functional clusters (due to frameshifts) but this should be explicitly stated at least in the figure legend. Fig. 6B: This panel conveys essentially the same information as Figure 4A. Consider replacing 4A with 6B to avoid redundancy ****** 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: Yes: Israel Muñoz-Velasco Reviewer #2: No ******** [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.0332834.r001
Revision 1
17 Jul 2025 Author Response Rebuttal Letter We are highly grateful to the reviewers for their thorough and valuable suggestions for improving the manuscript, all of them adressed Editor: “Your manuscript presents an engaging comparative genomics study focused on Xanthomonas and the diversity of its secretion systems. Following peer review, both reviewers concurred that the manuscript would benefit from further refinement, particularly regarding the definition of homologous genes and the application of more robust sequence alignment tools beyond BLAST to enhance validation. I do agree with them.” We appreciate the positive comments regarding the article and the work carried out. Concerning the definition of homologous genes, as suggested by one of the reviewers, we have added information in the text about the parameters used to determine homology and emphasized that the identification was based not only on sequence similarity criteria. Reviewers' comments: Reviewer #1: This is a well-conceived and methodologically solid comparative genomics study that explores the diversity of secretion systems (T1SS–T5SS) across six closed genomes representing the most virulent Xanthomonas arboricola pathovars: pruni, juglandis, and corylina. The work contributes meaningfully to the field of plant pathology by offering insights into the genomic architecture underlying host specificity and virulence potential. Thanks for your positive comments. The Methods section states that protein hits were considered homologs when they satisfied three criteria: E-value < 0.001, query coverage >80%, and sequence identity >40%. The authors cite Pearson (2013) [DOI: 10.1002/0471250953.bi0301s42] in support of this threshold, which is appropriate given that the referenced work emphasizes the importance of statistical significance (e.g., E-values and bit scores) over raw percent identity when inferring homology. However, I recommend that the authors explicitly elaborate on this rationale in the manuscript, especially given the potential concern that a 40% identity threshold may allow the inclusion of distant homologs with uncertain functional conservation, even when high query coverage is used. This is particularly important for secretion system effectors, where minor sequence divergence may translate into major functional differences. To improve clarity and strengthen the reliability of the results, I suggest the authors: Briefly summarize Pearson’s argument (e.g., that statistical measures are more reliable than identity percentages alone in homology inference). Clarify whether domain conservation (e.g., InterProScan) or other functional annotations were used to validate low-identity hits. Thank you for the comment that helps clarify the focus of our analysis. We have modified the text according to your suggestion and included the argument used by the authors of the cited manuscript. Indeed, we have encompassed in the text the parameters employed to determine homology and have clarified that the identification relied on more than just sequence similarity criteria. Minor Changes -Typographical edits: Line 52: "Thes pathovars" → "These pathovars" Ok, corrected. Lines 69–70: Consider rephrasing slightly for clarity: “Secretion systems are complex molecular machineries…” Ok, we’ve rephrased it. However, because Type IV and VI secretion systems target the cytoplasm of other bacteria rather than the host or extracellular space, we decided to retain that part of the sentence to reflect this. Line 156: “appeared to have been interrupted…” to “were likely disrupted…” Ok, changed. Optional addition: -A brief sentence in the conclusions and/or discussing how the findings could guide future experimental design (e.g., validation of T1SS function or virulence assays) would help bridge the gap to applied research. Thanks for the comment, we have added a sentence regarding. We have added a sentence to this effect at the end of the conclusions, reinforcing the idea that the results may serve as a basis for future functional studies and contribute to the control of phytopathogenic bacteria. Reviewer #2: In this manuscript, Cuesta-Morrondo and colleagues present a comparative genomic analysis of six previously sequenced Xanthomonas arboricola genomes. Given the well established role of secretion systems in bacterial pathogenicity, the authors focus their analysis on the occurrence and genomic organization of type I, II, III, and V secretion systems across Xa pathovars of agricultural relevance.The authors provide a systematic description of the presence/absnece patterns and genomic arrangement of the aforementioned secretion systems. While primarily descriptive in nature, with the authors limiting their analysis to the identification of gene clusters encoding these secretion systems, the study offers insights that will be of interest to researchers investigating Xanthomonas pathogenicity. Taking advantage of available high quality genomes, the authors report some interesting findings, including the identification of a TALE effector, which is often overlooked in draft genome analyses due to its repetitive structure. The authors also provide a detailed description of the distribution of distinct type III effectors across the analyzed genomes which may, at least partially, help explain differences in pathogenic profiles and host-pathogen interactions among X. arboricola pathovars. We sincerely thank you for your positive feedback and for recognizing the significance and relevance of the work presented. Major comment: The key methodological limitation of the manuscript is the approach taken to detect secretion systems, as the authors rely only on BLAST searches, mainly against known components of secretion systems from Xanthomonas genomes. While effective for detecting conserved systems, this method may fail to identify divergent or lineage-specific secretion systems, therefore limiting the depth and completeness of the analysis. To address this limitation, I strongly recommend the use of TXSScan (https://github.com/macsy-models/TXSScan), a dedicated tool for the comprehensive identification of bacterial secretion systems. TXSScan integrates HMM profile searche and synteny criteria, offering enhanced sensitivity compared to BLAST based methods alone. Since the primary claim of the study concerns the diversity and organization of secretion systems, reanalysis using TXSScan would strengthen the conclusions and, potentially, could reveal additional systems not detected with the current approach. Given that TXSScan is relatively simple and straightforward to implement, this reviewer recommend reanalyzing the genomes using TXSScan to cross-validate, and potentially expand,the identification of secretion systems in the studied Xa genomes. This would ensure the completeness and robustness of the findings prior to publication. Thank you for the suggestion. We have reanalyzed our data using the proposed tool and confirmed the results in most cases. The outcomes obtained with this tool have been cited throughout the text for each of the gene groups studied, thereby completing the work and following the reviewer's recommendation. Moreover, we have included the reference for the tool and an additional supplementary material to compare results between BLAST and TXSScan. Minor comments: Line 34: "variation was" instead of "variation were." Ok, changed line 113: Review and correct citation format (Cuesta-Morrondo et al., 2022). Ok, changed line 189: The authors mention that xcsK is frameshifted…consider claryfing here whether xcsK is essential for T2SS function, especially for readers unfamiliar with the system nomenclature in Xanthomonas. Thanks for your comment. While there are some studies suggesting possible functions for the Xcs T2SS, its role remains unclear. Therefore, it is not possible to assess if xcsK is essential. We have included some information in the main text regarding this issue line 190: Redaction needs to be double checked...consider rephraseing “Moreover, orthologs of eight Xanthomonas citri pv. citri experimentally validated T2SS substrates [42] were” to “Moreover, orthologs of eight experimentally validated T2SS substrates from Xanthomonas citri pv. citri were” Ok, changed. We also revised the whole text. Line 396: Rephrase: "Xac strains harbored two XAC2853 orthologs" to "...harbored two homologs of XAC2853", as the presence of two related genes within the same genome indicates paralogs, not orthologs. Ok, changed Fig. 1A: Labels are too small to read. The figure has been modified to increase the size of the labels. Fig. 3 and 4: These both relate to T3SS and could be merged into a single figure for clarity and consistency By merging 4 with 6b, we preferred to keep 3 and 4 separate for greater clarity for the reader. Fig. 5: Again…gene labels are too small, especially in panel 5C Figure was modified to increase label size. Fig. 6A: Clarify the meaning of the lighter blue boxes in the figure legend. I guess they represent predicted non-functional clusters (due to frameshifts) but this should be explicitly stated at least in the figure legend. The legend was modified to clarify that they are incomplete secretion systems, containing only some of the components. Fig. 6B: This panel conveys essentially the same information as Figure 4A. Consider replacing 4A with 6B to avoid redundancy Part of Figure 6 was merged with Figure 4, but we believe that the summary presented in the new Figure 6 is quite informative for the reader and it should be preserved Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0332834.r002
28 Aug 2025 Decision Letter - Luis D. Alcaraz, Editor PONE-D-25-24971R1Unraveling the Genomic Complexity of Secretion Systems in the Most Virulent Xanthomonas arboricola pathovarsPLOS ONE Dear Dr. Cubero, 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. The requested revisions have been satisfactorily addressed, as confirmed by both reviewers. Nonetheless, one reviewer and I concur that the manuscript’s clarity would benefit from refining certain statements—particularly within the figure captions—to make them more self-explanatory and comprehensive. You are encouraged to implement these improvements, after which I will be able to accept the article without requiring further revision. Please submit your revised manuscript by Oct 12 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. 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). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols . We look forward to receiving your revised manuscript. Kind regards, Luis D. Alcaraz, Ph.D. Academic Editor PLOS ONE Journal Requirements: 1. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. 2. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Additional Editor Comments: Reviewer #1: Reviewer #2: 1) The figure legends are, in general, too brief and would benefit from being more descriptive. The legend of Figure 4 is particularly vague: “B. T3Es Venn diagram.” … a Venn diagram of what? Please elaborate (e.g. “Venn diagram showing the shared and unique repertoires of…”). 2) The sentence “TXSScan also supported the T3SS in all the strains under study”....should read "TXSScan also supported the PRESENCE OF the T3SS in all…" ? [Note: HTML markup is below. Please do not edit.] 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 ******** 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: Yes ****** 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: The authors have addressed the major points raised during the first round of review. In particular, they have clarified the rationale behind the homology criteria, expanded their methodological framework with additional validation (including TXSScan analyses and domain-based approaches), and refined the manuscript’s language and presentation. The revisions substantially strengthen the methodological rigor and interpretative clarity of the study. The manuscript now provides a clearer explanation of the thresholds used for homology inference, appropriately contextualized, and integrates complementary validation strategies. These additions address the concern regarding the reliability of homology assignments. Minor textual and stylistic issues noted in the earlier review have been corrected, and the discussion now better situates the findings within the broader context of bacterial pathogenesis and applied plant pathology. Overall, the revised version of the manuscript is clear and scientifically sound. Reviewer #2: Cuesta-Morrondo et al. satisfactorily addressed my main concerns, and I have no further major comments on the revised version. I do, however, have a couple of minor observations for authors consideration: 1) The figure legends are, in general, too brief and would benefit from being more descriptive. The legend of Figure 4 is particularly vague: “B. T3Es Venn diagram.” … a Venn diagram of what? Please elaborate (e.g. “Venn diagram showing the shared and unique repertoires of…”). 2) The sentence “TXSScan also supported the T3SS in all the strains under study”....should read "TXSScan also supported the PRESENCE OF the T3SS in all…" ?????? Aside from these minor points, I believe the manuscript meets the publication criteria of PLOS One, and I recommend it for acceptance. ****** 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: Yes: Israel Muñoz-Velasco Reviewer #2: No ******** [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.0332834.r003
Revision 2
2 Sep 2025 Author Response Rebuttal Letter Once again we thanks to the reviewers for their interest on the manuscript and their final suggestions for improving it. All of them were addressed. Editor: “The requested revisions have been satisfactorily addressed, as confirmed by both reviewers. Nonetheless, one reviewer and I concur that the manuscript’s clarity would benefit from refining certain statements—particularly within the figure captions—to make them more self-explanatory and comprehensive. You are encouraged to implement these improvements, after which I will be able to accept the article without requiring further revision.” We appreciate again the positive comments regarding the article and the work carried out and we have addressed the final suggestions and corrections from one of the reviewers. Reviewers' comments: Reviewer #2: The figure legends are, in general, too brief and would benefit from being more descriptive. The legend of Figure 4 is particularly vague: “B. T3Es Venn diagram.” … a Venn diagram of what? Please elaborate (e.g. “Venn diagram showing the shared and unique repertoires of…”). Indeed the reviewer is right. We have modified all the legends according to such comment in this new version The sentence “TXSScan also supported the T3SS in all the strains under study”....should read "TXSScan also supported the PRESENCE OF the T3SS in all…" ? Thank you for the comment. The sentence was modified according to reviewer´s comment. Attachments Attachment Submitted filename: Response to Reviewers 2.docx https://doi.org/10.1371/journal.pone.0332834.r004
5 Sep 2025 Decision Letter - Luis D. Alcaraz, Editor Unraveling the Genomic Complexity of Secretion Systems in the Most Virulent Xanthomonas arboricola pathovars PONE-D-25-24971R2 Dear Dr. Cubero, 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. For questions related to billing, please contact billing support . 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, Luis D. Alcaraz, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0332834.r005
Formally Accepted
Acceptance Letter - Luis D. Alcaraz, Editor PONE-D-25-24971R2 PLOS ONE Dear Dr. Cubero, 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 Prof. Luis D. Alcaraz Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0332834.r006

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