Production of the light-activated elsinochrome phytotoxin in the soybean pathogen Coniothyrium glycines hints at virulence factor

Nicholas Greatens; Harun M. Murithi; Danny Coyne; Steven J. Clough; Michael Sulyok; Wahab Oluwanisola Okunowo; Hamed K. Abbas; W. Thomas Shier; Rachel A. Koch Bach

doi:10.1371/journal.pone.0321896

Peer Review History

Original SubmissionSeptember 27, 2024
27 Sep 2024 Author Response https://doi.org/10.1371/journal.pone.0321896.r001
15 Nov 2024 Decision Letter - Katherine Borkovich, Editor PONE-D-24-43331Presence of a gene cluster for a light-activated phytotoxin in the soybean pathogen Coniothyrium glycines hints at possible virulence mechanismPLOS ONE Dear Dr. Koch Bach, 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 address the suggestions of the reviewers in your revised manuscripts. I do not believe that additional experimentation is warranted. Please submit your revised manuscript by Dec 30 2024 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 . 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Thank you for stating the following in the Acknowledgments Section of your manuscript: “Financial support for this project was provided by USDA-ARS project 8044-22000-051-00D (RAKB and NG), USDA-ARS project 5012-22000-023-00D (SJC, and HMM) and the USDA-ARS National Plant Disease Recovery System (HKA and WTS). The authors would like to thank Glen Hartman, Doug Luster, Sprine Misiani and Aaron Sechler for project support, as well as express our appreciation to all who assisted with the RLB field and lab work including Clint Slocum, Godfree Chigeza, Florence Kamwana, Mathe Lukanda, Learnmore Mwadzingeni, Christabell Nachilima, Irodino Saraivia, Yechalew Sileshi, Munashe Sithole, Phinehas Tukamuhabwa, and Maserasha Yirga. This research used resources provided by the SCINet project and was also supported in part by a postdoctoral fellowship funded by the USDA Agricultural Research Service's SCINet Program and AI Center of Excellence, ARS project numbers 0201-88888-003-000D and 0201-88888-002-000D, and administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the U.S. Department of Energy (DOE) and the U.S. Department of Agriculture (USDA). ORISE is managed by ORAU under DOE contract number DE-SC0014664. All opinions expressed in this paper are the authors’ and do not necessarily reflect the policies and views of USDA, DOE, or ORAU/ORISE. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture (USDA). The USDA is an equal opportunity provider and employer.” We note that you have provided funding information that is currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: “Financial support for this project was provided by USDA-ARS project 8044-22000-051-00D (RAKB and NG) and the USDA-ARS National Plant Disease Recovery System (HKA and WTS).” Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 5. When completing the data availability statement of the submission form, you indicated that you will make your data available on acceptance. We strongly recommend all authors decide on a data sharing plan before acceptance, as the process can be lengthy and hold up publication timelines. Please note that, though access restrictions are acceptable now, your entire data will need to be made freely accessible if your manuscript is accepted for publication. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor's input on an exemption. Please be assured that, once you have provided your new statement, the assessment of your exemption will not hold up the peer review process. 5. 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. [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: Yes Reviewer #2: Partly ******** 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: The authors carried out a study to investigate the presence of a secondary metabolite gene cluster in the genome of C. glycines, and how exposure to light/darkness affected pigment production in axenic cultures and symptom development in detached leaves. The study is timely and the manuscript is well written. Minor correction: Line 289: Indicate that the counts of genes g6532-g6539 are from Coniothyrium glycines. Reviewer #2: This manuscript describes potential perylenequinone production from Coniothyrium glycines, an important soybean pathogen. The authors showed via whole genome sequencing that this species has a secondary metabolite cluster very closely related to those that produce cercosporin; a well-studied secondary metabolite effector produced by Cercospora species and other Dothidiomycetes. Isolates of C. glycines were grown in light/dark conditions, which showed a reddish phenotype in the mycelium potentially characteristic of cercosporin in the light-grown cultures, which was absent in the dark-grown cultures. The authors showed that several of the genes in this cluster were up-regulated in the light, also typical of cercosporin. The authors used a detached leaf assay to show that there was less disease when inoculated leaves were in the dark versus the light, suggesting that a light-activated effector is at play with this fungus. I found the manuscript to be well-written and a pretty compelling argument for perylenequinone production by this fungus, and likely cercosoporin. This manuscript, however, relies heavily on association (especially to phenotypes) without development of mutants, etc to really show that this fungus is producing cercosporin. The detached leaf assay is somewhat compelling, but there could be a host of other reasons why there was less disease in the dark. However, with a little extra lab work, cercosporin production can be shown very easily (see below) which will help add impact to this work. Points to consider: Figure 1 cultures show light/day-related color and morphology, but is not diagnostic for cercosporin. In fact, cercosporin should be secreted into the medium in advance of the mycelium… typically. The “eight gene cluster” of cercosporin is mentioned in several cases; it is not produced via only eight genes (see de Jonge et al PNAS 2018). The bottom C. beticola genome should really be removed in figure 2 as it doesn’t add anything to the figure. Figure captions (and Tables) within the main text itself makes reading the manuscript difficult. Table 2: C. beticola has at least 13 CTB genes, including MFS transporter… Why aren’t the others included here? Finally, consider the standard “KOH Assay” for cercosporin detection: 1. Grow isolates on 9 cm Petri plates containing 15 ml of PDA a. 7d, 25C, Natural light 2. Using a #2 cork borer, remove three plugs from each isolate from the edge, middle and center of each colony and placed in small screw cap glass vials. (scrape off excess PDA) 3. Add 5mls of 5N KOH to each vial to cover the surface of the plugs and shake at room temperature for 4 hours (this can be shortened more like 15-30min) 4. Supernatants were examined for cercosporin spectrophotometrically. 5. Spectrophotometer set to broad spectrum using 472 nm as the target wavelength. Cercosporin (Sigma) can be used as a positive control in this assay (dissolve in acetone to 100 mM) ****** 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: Emma W. Gachomo 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.0321896.r002
Revision 1
11 Mar 2025 Author Response Editor comments: 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. In your Methods section, please provide additional information regarding the permits you obtained for the work. Please ensure you have included the full name of the authority that approved the field site access and, if no permits were required, a brief statement explaining why. 3. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. 4. Thank you for stating the following in the Acknowledgments Section of your manuscript: “Financial support for this project was provided by USDA-ARS project 8044-22000-051-00D (RAKB and NG), USDA-ARS project 5012-22000-023-00D (SJC, and HMM) and the USDA-ARS National Plant Disease Recovery System (HKA and WTS). The authors would like to thank Glen Hartman, Doug Luster, Sprine Misiani and Aaron Sechler for project support, as well as express our appreciation to all who assisted with the RLB field and lab work including Clint Slocum, Godfree Chigeza, Florence Kamwana, Mathe Lukanda, Learnmore Mwadzingeni, Christabell Nachilima, Irodino Saraivia, Yechalew Sileshi, Munashe Sithole, Phinehas Tukamuhabwa, and Maserasha Yirga. This research used resources provided by the SCINet project and was also supported in part by a postdoctoral fellowship funded by the USDA Agricultural Research Service's SCINet Program and AI Center of Excellence, ARS project numbers 0201-88888-003-000D and 0201-88888-002-000D, and administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the U.S. Department of Energy (DOE) and the U.S. Department of Agriculture (USDA). ORISE is managed by ORAU under DOE contract number DE-SC0014664. All opinions expressed in this paper are the authors’ and do not necessarily reflect the policies and views of USDA, DOE, or ORAU/ORISE. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture (USDA). The USDA is an equal opportunity provider and employer.” We note that you have provided funding information that is currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: “Financial support for this project was provided by USDA-ARS project 8044-22000-051-00D (RAKB and NG) and the USDA-ARS National Plant Disease Recovery System (HKA and WTS).” Please include your amended statements within your cover letter; we will change the online submission form on your behalf. We have included the amended statement in the cover letter. It reads as the following: Our funding sources are as follows: USDA-ARS projects 8044-22000-051-00D, 5012-22000-023-00D, 6066-42000-007-000D, 0201-88888-003-000D, and 0201-88888-002-000D, as well as the USDA-ARS National Plant Disease Recovery System. 5. When completing the data availability statement of the submission form, you indicated that you will make your data available on acceptance. We strongly recommend all authors decide on a data sharing plan before acceptance, as the process can be lengthy and hold up publication timelines. Please note that, though access restrictions are acceptable now, your entire data will need to be made freely accessible if your manuscript is accepted for publication. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor's input on an exemption. Please be assured that, once you have provided your new statement, the assessment of your exemption will not hold up the peer review process. 5. 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. Reviewer #1: The authors carried out a study to investigate the presence of a secondary metabolite gene cluster in the genome of C. glycines, and how exposure to light/darkness affected pigment production in axenic cultures and symptom development in detached leaves. The study is timely and the manuscript is well written. Minor correction: Line 289: Indicate that the counts of genes g6532-g6539 are from Coniothyrium glycines. Accepted Reviewer #2: This manuscript describes potential perylenequinone production from Coniothyrium glycines, an important soybean pathogen. The authors showed via whole genome sequencing that this species has a secondary metabolite cluster very closely related to those that produce cercosporin; a well-studied secondary metabolite effector produced by Cercospora species and other Dothidiomycetes. Isolates of C. glycines were grown in light/dark conditions, which showed a reddish phenotype in the mycelium potentially characteristic of cercosporin in the light-grown cultures, which was absent in the dark-grown cultures. The authors showed that several of the genes in this cluster were up-regulated in the light, also typical of cercosporin. The authors used a detached leaf assay to show that there was less disease when inoculated leaves were in the dark versus the light, suggesting that a light-activated effector is at play with this fungus. I found the manuscript to be well-written and a pretty compelling argument for perylenequinone production by this fungus, and likely cercosoporin. This manuscript, however, relies heavily on association (especially to phenotypes) without development of mutants, etc to really show that this fungus is producing cercosporin. The detached leaf assay is somewhat compelling, but there could be a host of other reasons why there was less disease in the dark. However, with a little extra lab work, cercosporin production can be shown very easily (see below) which will help add impact to this work. Points to consider: Figure 1 cultures show light/day-related color and morphology, but is not diagnostic for cercosporin. In fact, cercosporin should be secreted into the medium in advance of the mycelium… typically. With our additional LC-MS/MS data, we were able to test this out and we found that not much of the perylenequinone elsinochrome is secreted into the agar, but is instead concentrated in the tissue. Perhaps the different chemical properties of cercosporin and elsinochrome may explain the difference. The “eight gene cluster” of cercosporin is mentioned in several cases; it is not produced via only eight genes (see de Jonge et al PNAS 2018). The eight gene cluster refers to the eight genes present in C. glycines that form prehypocrellin, a precursor to perylenequinones, such as elsinochromes. Hu et al. 2019 show these genes, when expressed in Aspergilllis nidulans in absence of other enzymes that modify prehypocrellin, form hypocrellins. We acknowledge the other genes involved in cercosporin biosynthesis in Fig 2, and, following recommended edits below, in Table 2. The bottom C. beticola genome should really be removed in figure 2 as it doesn’t add anything to the figure. Accepted. The gene visualization has been removed, but the PKS gene has been retained for use as an outgroup in the tree. Figure captions (and Tables) within the main text itself makes reading the manuscript difficult. Captions are now reformatted to comply with PLOSONE requirements. Hopefully, this makes reading the manuscript easier. Table 2: C. beticola has at least 13 CTB genes, including MFS transporter… Why aren’t the others included here? Accepted. Other CTB genes have been added for comparison. The MFS transporter in the C glycines cluster is not predicted as orthologous to the CTB MFS transporter. Both are now included in the table. Finally, consider the standard “KOH Assay” for cercosporin detection: 1. Grow isolates on 9 cm Petri plates containing 15 ml of PDA a. 7d, 25C, Natural light 2. Using a #2 cork borer, remove three plugs from each isolate from the edge, middle and center of each colony and placed in small screw cap glass vials. (scrape off excess PDA) 3. Add 5mls of 5N KOH to each vial to cover the surface of the plugs and shake at room temperature for 4 hours (this can be shortened more like 15-30min) 4. Supernatants were examined for cercosporin spectrophotometrically. 5. Spectrophotometer set to broad spectrum using 472 nm as the target wavelength. Cercosporin (Sigma) can be used as a positive control in this assay (dissolve in acetone to 100 mM) In response to reviews, we used a comparable technique with lc-ms to analyze the toxin produced by the cluster and found the primary toxin to be elsinochrome, rather than cercosporin or hypocrellin, although the latter is produced to a lesser extent. Cercosporin is not produced. 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: Emma W. Gachomo Reviewer #2: No Attachments Attachment Submitted filename: Response_to_reviewers.docx https://doi.org/10.1371/journal.pone.0321896.r003
14 Mar 2025 Decision Letter - Katherine Borkovich, Editor Production of the light-activated elsinochrome phytotoxin in the soybean pathogen Coniothyrium glycines hints at virulence factor PONE-D-24-43331R1 Dear Dr. Koch Bach, 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, Katherine A. Borkovich, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0321896.r004
Formally Accepted
Acceptance Letter - Katherine Borkovich, Editor PONE-D-24-43331R1 PLOS ONE Dear Dr. Koch Bach, 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. 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. Katherine A. Borkovich Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0321896.r005

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