Castanea crenata Ginkbilobin-2-like Recombinant Protein Reveals Potential as an Antimicrobial against Phytophthora cinnamomi, the Causal Agent of Ink Disease in European Chestnut

Round 1

Reviewer 1 Report

The authors have conducted an interesting study building on earlier work investigating the use of an antimicrobial protein found in Asian chestnuts but absent in the European species.  The results look quite promising.  There are parts of the manuscript that read very well, but others where more thorough English edits are needed.  The authors must be more diligent in citing earlier work by others.  There were numerous statements made that need references.  I have made numerous suggestions for specific revisions in the attached pdf.

Comments for author File: Comments.pdf

Author Response

We thank the Reviewers for their valuable corrections and suggestions. We also thank the editor in charge of the present article.

Reviewer 1:

• The suggestions in the Abstract were added according to the Reviewer’s recommendations.
• We modified the keywords in alphabetic order (modification not asked by the Reviewer).
• The suggestions in the Introduction were added and modified according to the reviewer’s recommendations. For example, the word wood was changed to “forest.”
• Answer to a question in the Introduction: Castanea sativa is considered to be the only native species in Europe.
• Answer to another question in the Introduction: The economy of the mountainous Mediterranean regions of southwest Europe is important for the income derived from chestnut culture.

We deleted the paragraph: “Achieving good pathogen management according to sustainable agriculture principles also requires parallel actions to prevent the spread of phytopathogens,” as we considered it evident in the work context (modification not asked by the Reviewer).

• The suggestions in the M&M section were added and modified according to the reviewer’s recommendations.
• The suggestions in the Results section were added and modified according to the reviewer’s recommendations.
• The sentence “In pictures we can see the plugs from the control treatment much white/cream colored, this plugs aspect represents a higher amount of mycelia dried; however, in the treatment with added protein, the plugs seem to more transparent with considerable less amount of cinnamomi mycelia,” was deleted.
• We decided to modify Fig. 6 to clarify the image (modification not asked by the Reviewer).
• The suggestions in the Discussion section were added and modified according to the reviewer’s recommendations.
• More references were added.
• The paragraph “In a previous study where we used a histological approach to observe the responses exhibited by susceptible and resistant chestnut genotypes by characterizing the early stages of cinnamomi infection and the cellular responses it induces in roots, we found that the early accumulation of phenolic-like compounds in cell walls was observed from 0.5 hours after inoculation in C. crenata root tissues which may prevent the spread of some hyphae”, was modified according to the reviewer suggestion.
• The word “wild type” in the following sentence “In an in vitro tolerance assay with the pathogen cinnamomi, Serrazina et al. (2022) [20], observed that transgenic plants of holm oak (Quercus ilex) with the Cast_Gnk2-like were able to survive longer than wild type [20]” was not removed because it refers to the control holm oak.
• The following paragraph: “We believe this work is exploratory research, and more studies and tests need to be done. However, we are developing studies and tests for the in-silico protein structure prediction, in vitro 7-day antagonist assays with cinnamomi, and metabolomics analysis”, was modified according to the reviewer’s suggestion for a better understanding.
• We decided to delete the following paragraph as we considered it unnecessary: “Though, we consider this preliminary work important for referencing in further publications, as we can here find the basis and justification of the present and future research considering Cast_Gnk2-like (pure protein) against cinnamomi pathogen.” (modification not asked by the Reviewer).
• The suggestions in the Conclusion section were added and modified according to the reviewer’s recommendations.
• The recommended English changes were all added to the manuscript. We deeply acknowledge the effort of the reviewer.

Author Response File: Author Response.docx

Reviewer 2 Report

The present work is part of an ongoing research program that aims to find solutions to this important problem regarding Chestnut/P. cinnamomi pathosystem in Europe. It was reported that Gnk2 inhibits the growth of pathogenic fungi such as Fusarium oxysporum, F. culmorum, and Candida albicans; and activates actin-dependent by inducing hypersensitive response (HR-related) plant cell death. The title and subject of the manuscript are very interesting from the methodological and practical point of view, suitable and adequate. The manuscript is well designed and written, and therefore could be acceptable after minor revision for possible publication in forests journal. However, some corrections should be considered as listed below:

The abstract should illuminate the main findings of the paper that can serve as a stand-alone document.However, authors have represented abstract in more generalized form. Authors should emphasize the levels of increment/reduction of different parameters assessed in % age values. I suggest revising abstract and incorporate significant results (wherever missing) in the abstract for comparative framework.

Add latest references in the introduction. Line 70-72, please add the below reference at the end of sentence: Over time, several approaches have been proposed to deal with soil diseases (which affect the xylem of trees), such as the intensive application of pesticides and the selection and production of improved plant material with some disease resistance (Niu et al., 2022).

Niu, F., Xing, Y., Jia, N. et al. Effectiveness of entomopathogenic fungal strains against poplar/willow weevil (Cryptorhynchus lapathi L.) larvae. J. For. Res. 33, 1691–1702 (2022). https://0-doi-org.brum.beds.ac.uk/10.1007/s11676-021-01428-3.

Experimental design: it is poorly written. It is critical part of the research and worth’s to describe more details providing a good image of your experiments for readers. The authors should clarify about experimental design used?

Materials and methods are standard and well-drafted.

From where authors provided Bacterial strains?

The English needs to be revised in all sections so that the content is written more succinctly, coherently and logically, avoiding unnecessary repetition; the Introduction, Results and Discussion sections in particular need revision in this respect.

Please use the below reference in discussion:

Miyakawa T, Miyazono K, Sawano Y, Hatano K, Tanokura M. Crystal structure of ginkbilobin-2 with homology to the extracellular domain of plant cysteine-rich receptor-like kinases. Proteins. 2009 Oct;77(1):247-51. doi: 10.1002/prot.22494.

Conclusion is well written. Remove all the errors including spacing and revise the entire manuscript by removing typographical errors.

Author Response

Reviewer 2:

• The suggestions in the Abstract were added according to the Reviewer’s suggestions, and the abstract now has significant results for a comparative framework.
• The suggestions in the Introduction were added and modified according to the reviewer’s recommendations. We added the proposed reference of Niu et al., 2022, in the sentence: “Over time, several approaches have been proposed to deal with soil diseases (which affect the xylem of trees), such as the intensive application of pesticides and the selection and production of improved plant material with some disease resistance.”
• The reviewer’s suggestions for the M&M section were considered. The experimental design was specified in section: 2.7. Cast_Gnk2-like in vitro Antagonist Activity Against cinnamomi. We improved and gave more details in the M&M section for a better understanding. We added the information regarding the question: “From where authors provided Bacterial strains?”. The competent cells bacteria TOP10 and BL21 (DE3) pLysS were taken from Thermo Fisher Scientific Inc. This was specified in the manuscript.
• The English was revised throughout the manuscript by a native speaker and by another reviewer.

Author Response File: Author Response.docx

Reviewer 3 Report

Dear Editor, 

The manuscript by Colavolpe et al., entitled 'Castanea crenata Ginkbilobin-2-like recombinant protein reveals potential as an antimicrobial against Phytophthoracin namomi, the causal agent of ink disease in European chestnut' is a well-written and well-performed research work. However, there are several things that need to be considered.

Most of my comments have been included in the attached manuscript. Please go via the comments made directly in the pdf file (attached). Several typos and different scientific styles were found in the manuscript, and please make them uniform. Usage of the English language is poor, so the help of native English speakers should prove fruitful.

Comments for author File: Comments.pdf

Author Response

Reviewer 3:

• The references in the Introduction were added.
• The information in the M&M section regarding the Escherichia coli bacteria strains used was added. The PCR was performed using Phusion™ High-Fidelity DNA Polymerase (2 U/µL). The manufacturer’s guide has the details of the master mix and PCR conditions, which we consider unnecessary to explain. The size of the Cast_GNK2-like coding sequence is represented in Fig. 1 (726 bp).
• In the Results section, the Reviewer asks for Figures with a black background. Unfortunately, all the pictures we took had a white background, but we marked the Oomycete growth in black at the bottom of the Petri plate to represent the growth area. The Reviewer also mentioned that the western blot is unclear. The Western was performed with the total cell extract after sonication, using the insoluble fraction, pellet resulting from centrifugation of the total extract, and the supernatant, corresponding to the soluble fraction. It is predictable that other bands will appear. We deleted the pink picture. We simplified the image and added an SDS-Page of the protein purification procedure. We hope the editor understands that sometimes some imperfections are present.
• The English was revised throughout the manuscript by a native speaker and by another reviewer.

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

Dear Editor,

The manuscript entitled' Castanea crenata Ginkbilobin-2-like recombinant protein re- 2 veals potential as an antimicrobial against Phytophthora cin- 3 namomi, the causal agent of ink disease in European chestnut' by Colavolpe et al. has now been modified as per my comments. The authors have addressed all my comments in a very succinct way. Please try to see the typos once again. Thus, I approve the acceptance of the manuscript for publishing in the Forests (MDPI) journal. Thank you.