Functional Study of the Chitinase CaChi93 Gene from the Mycoparasitic Cladosporium sp. SYC23

Round 1

Reviewer 1 Report

Respect to the manuscript entitled "Functional Study of the Chitinase CaChi93 Gene from the Mycoparasitic Cladosporium sp. SYC23":

1. The manuscript meets the quality standards required for publication in your prestigious journal.
2. The methodology employed is appropriate and represents a state-of-the-art approach within the field.
3. The bioinformatic and molecular analyses are properly conducted and are supported by current and well-established references.
4. Although the manuscript demonstrates generally good English grammar, several grammatical and/or technical errors were identified during the review process and should be corrected prior to publication. All specific comments and annotations are provided in the attached PDF document

Respect to the manuscript entitled "Functional Study of the Chitinase CaChi93 Gene from the Mycoparasitic Cladosporium sp. SYC23":

1. The manuscript meets the quality standards required for publication in your prestigious journal.
2. The methodology employed is appropriate and represents a state-of-the-art approach within the field.
3. The bioinformatic and molecular analyses are properly conducted and are supported by current and well-established references.
4. Although the manuscript demonstrates generally good English grammar, several grammatical and/or technical errors were identified during the review process and should be corrected prior to publication. All specific comments and annotations are provided in the attached PDF document

Comments for author File: Comments.pdf

Author Response

Dear Editor and Reviewers,
      We sincerely appreciate the thorough review and constructive comments on our manuscript. These suggestions are highly valuable for improving the quality and clarity of our work. We have carefully addressed each comment and made corresponding revisions to the manuscript. Below is our detailed response to each point raised by the reviewers.
Reviewer comment 1: Do not leaf spaces on sheet 7, it is wasted space.
Response 1:The layout of Table 2 has been adjusted, and there is no longer excessive blank space on page 7.
Reviewer comment 2: “ the 2^-△△Ct method”I suggest, indicate the method reference.
Response 2: References have been inserted according to the reviewers’ comments.
Reviewer comment 3: In Figure 1, Add sections a), b), and c)
Response 3: We have added labels (a), (b), and (c) to Figure 1, and explained what they represent in the figure legend.

Reviewer comment 4: In figure 7, the function of the blue box must be indicated.
Response 4: Figure 7c; The blue box indicates the expression of protein Cachi93. They have been labeled in both the figure legend and the main text.
Reviewer comment 5: Figure 9, Indicate with arrows
Response 5:In Figure 9, intact spores and enzyme-treated broken spores have been labeled with arrows.

Reviewer comment 6:Are there significant differences? ANOVA?
Response 6:Figures 6 and 8 were generated based on ANOVA analysis. Since the significance differences were not distinguishable by color, we have revised the colors of these figures.
      All changes introduced in the revised version of the manuscript are marked with highlight or underline. We believe these revisions have significantly improved the clarity, rigor, and presentation of our manuscript. We are grateful for the reviewers' time and expertise, and we hope the revised manuscript now meets the standards for publication in this journal.
Sincerely,
Chen Chen
Southwest Forestry University

Author Response File: Author Response.pdf

Reviewer 2 Report

The logic of the paper needs careful work.  You need to make his tagging clear, where the rust spores come from, why one gene was used for recombinant expression...  It needs to be a story that follows clearly and logically.

This is informationally a very simple paper.  A set of chitinase genes are identified to be regulated in the mycoparasitic fungus Cladosporium sp. strain SYC23 during treatment with rust spores.  It must be made clear at the beginning of the manuscript the source of these rust spores and the species of rust being used.  As it is written this is very unclear, and as this is the central point of the paper, this needs to be carefully laid out. Eight genes are identified that have sequence similarity to chitinase genes from other fungi.  The description of the encoded proteins is provided (molecular weights, isoelectric points) and the pattern of gene expression at various time during the presence of the rust spores for these chitinase encoding genes are established.  One of the genes (CaChi93) was expressed in E. coli to generate chitinase activity that could degrade rust spore walls and inactivate the spores, with the idea that this could be exploited as an anti-rust treatment.

Line 62 of the introduction is presenting the idea that a chitinase from the strain under investigation (SYC23) can act as a bioactive agent. Unfortunately, as written, it is apparently acting on the plant Photinia prionophylla, rather than the rust pathogen infecting the plant.  This needs clarity.

Ultimately the active part of the chitinase CaChi93 is cloned and expressed successfully in E.coli.  Purification is done on nickel affinity columns, but why nickel affinity is useful is not explained.  The presence of a his-tag is not explained.

Figure 1 describes the structures of the chitinase genes identified in the study, highlighting motifs like transmembrane domains and signal sequences, and showing the extensive differences among the proteins in size and domain structure.  Figure 2 positions the identified chitinases in terms of their phylogenetic relationships.  During the description of Figure 2 the gene names switch from CaChi… to CaCHi… – not sure why the H gets capitalized?  Figure 3 lays out the structures of the promoters of the various genes; this can make some predictions of the possible regulatory controls.  The role of elements like light-response and pathogen response sequences are not investigated.

There is an issue with describing the family of proteins together.  I think the term CaCHis means members of the set, but this needs to be defined, and if this is the case the term has to be plural; not CaCHis protein but rather proteins, not CaCHis sequence, but sequences.  Figure 4 shows tertiary structure predictions using SWISS-MODEL; was this compared with the predictions of alpha-fold?  The expression pattern of the genes was determined in the presence of rust spores over time, this is figure 5.  In figure 6 the production of extracellular chitinase is measured over time – induction of activity in the presence of spores does not impressively increase over time, which seems surprising.

The next section describes the protein purification from the recombinant strain.  At the start of the results section 3.7 we need to be told what this strain is.  Why was the 93 protein chosen for the recombinant expression?  Is the protein his-tagged?  It must be based on doing westerns with an anti-his-tag antibody and using nickel columns for purification, but this is never really made clear.  This purified protein is used to attack rust spores, and images are shown of spore disruption.  Because the overall idea is that this activity could serve as an antirust treatment measurement of spore viability would be a better assay than loss of content measured by staining.

 

English style

There are a lot of issues with “idiomatic” English - the text is relatively comprehensible, but frequently the terms used are confusing or the sentence structure is awkward.  I will note a small number of points, but the paper must be edited by a native English speaker to improve the whole manuscript, because in some cases the linguistic problems confuse the point the authors are trying to make.

Examples

Rewriting text starting at line 13  

In order to select chitinase genes from the mycoparasitic Cladosporium sp. Strain SYC23, real-time fluorescence quantitative PCR was used to identify sequences induced by treatment with rust spores at 12, 24, 48 and 72 hours.  A total of 8 regulated chitinase genes were identified through bioinformatic analysis.  I think this is what the authors are hoping to say;  “excavate” is certainly not the English word to be used here.

Line 19 …molecular weights range…

Line 23  SYC23 is not related to chitinase sequences, it is a fungal strain

Line 25 …CaChi45 was most…  …CaChi93 was continuously…

Line 26 reorganization CaChi93 protein   reorganization is not a term to describe a protein, so this phrase is confusing. Are the authors trying to say recombinant or heterologously expressed protein?

As you can see there are issues with many lines just in the abstract, and as this continues throughout the paper, careful editing will be essential.

Make sure E. coli is in italics everywhere it is written.

Make sure that genes are not confused with proteins.  For example, line 194 talks about genes being localized extracellularly – it is the gene products that are extracellular.

Referencing is inconsistent, in some places just a number, in others the reference number and the first and last names of the authors.  Be consistent, follow the journal style.

The heading “Patents” is used for the author contributions (line 433)

 

 

Author Response

Dear Editor and Reviewers,
    We sincerely appreciate the thorough review and constructive comments on our manuscript. These suggestions are highly valuable for improving the quality and clarity of our work. We have carefully addressed each comment and made corresponding revisions to the manuscript. Below is our detailed response to each point raised by the reviewers.
1. Key Scientific Issues and Experimental Design
1.1 Source of Rust Spores and Target Pathogen
Reviewer comment: It must be made clear at the beginning of the manuscript the source of these rust spores and the species of rust being used. As it is written this is very unclear, and as this is the central point of the paper, this needs to be carefully laid out.
Response: We apologize for the ambiguity. We have added a detailed description in the Materials and Methods section (Section 2.1, line 79). The rust spores employed in the present experiment were harvested from rust-diseased Photinia specimens growing at Southwest Forestry University.
1.2 Ambiguity in Bioactive Agent Target
Reviewer comment: Line 62 of the introduction is presenting the idea that a chitinase from the strain under investigation (SYC23) can act as a bioactive agent. Unfortunately, as written, it is apparently acting on the plant Photinia prionophylla, rather than the rust pathogen infecting the plant. This needs clarity.
Response: We have revised the relevant paragraph in the introduction (line 64) to explicitly state that the bioactive agent is the chitinase from Cladosporium sp. strain SYC23, which targets the cell wall of the rust pathogen Puccinia photiniae, thereby inhibiting spore germination and pathogenicity, rather than acting directly on the host plant Photinia prionophylla.
1.3 Protein Purification Details
Reviewer comment: Ultimately the active part of the chitinase CaChi93 is cloned and expressed successfully in E. coli. Purification is done on nickel affinity columns, but why nickel affinity is useful is not explained. The presence of a his-tag is not explained.
Response: We have added a detailed explanation in the Materials and Methods section (Section 2.6, line 166):The CaChi93 gene was cloned into the pET-28a(+) vector, which introduces a 6×His-tag at the N-terminus of the recombinant protein. Nickel affinity chromatography (Ni-NTA resin) was used for purification because the 6×His-tag specifically binds to Ni²⁺ ions, allowing for one-step purification of the recombinant protein with high purity and yield.
1.4 Choice of Recombinant Strain
Reviewer comment: At the start of the results section 3.7 we need to be told what this strain is. Why was the 93 protein chosen for the recombinant expression?
Response: We have added a clear statement in section 3.7 (line 317):The E. coli strain used for recombinant expression is Rosetta(DE3). CaChi93 was selected for recombinant expression because: It showed the highest and most sustained upregulation during rust spore induction (Figure 5). Phylogenetic analysis (Figure 2) placed it in a clade with well-characterized antifungal chitinases, suggesting strong potential for rust spore degradation.
1.5 Gene Nomenclature Consistency
Reviewer comment: During the description of Figure 2 the gene names switch from CaChi… to CaCHi… – not sure why the H gets capitalized?
Response: This was an inadvertent typographical error. We have standardized all gene names to CaChi (e.g., CaChi45, CaChi93) throughout the manuscript, including in Figure 2 and its legend, to ensure consistency.
1.6 Definition of CaChis Family
Reviewer comment: There is an issue with describing the family of proteins together. I think the term CaChis means members of the set, but this needs to be defined, and if this is the case the term has to be plural; not CaChis protein but rather proteins, not CaChis sequence, but sequences.
Response: We apologize for the confusion. We have: Corrected all instances of incorrect grammar, changing "CaChis protein" to "CaChis proteins" and "CaChis sequence" to "CaChis sequences" throughout the manuscript.
1.7 Interpretation of Extracellular Chitinase Activity
Reviewer comment: In figure 6 the production of extracellular chitinase is measured over time – induction of activity in the presence of spores does not impressively increase over time, which seems surprising.
Response: Changes in reducing sugars in fermentation broth of strain SYC23 under different culture conditions. a: DNS color reaction; b: Changes in N-acetylglucosamine content. 1 is Blank control; 2 is 1% colloidal chitosan treatment; 3 is Pyronium rust spore treatment. Figure 6b shows that the N-acetylglucosamine content in the fermentation broth of the strain induced by Photinia prionophylla rust spore walls was overall higher than that in the blank control group and the 1% colloidal chitin treatment group.
2. English Style and Formatting
2.1 Specific Language Corrections
Reviewer comment: Rewriting text starting at line 13: "excavate" is certainly not the English word to be used here. Line 26: "reorganization CaChi93 protein" is confusing. Are the authors trying to say recombinant or heterologously expressed protein?
Response: Line 13: We have replaced "excavate" with "identified" to accurately describe the process of finding chitinase genes through bioinformatic analysis. Line 28: We have corrected "reorganization CaChi93 protein" to "recombinant CaChi93 protein" to clearly indicate that it is a heterologously expressed protein in E. coli.
2.3 Formatting and Terminology Standards
Reviewer comment: Make sure E. coli is in italics everywhere it is written. Make sure that genes are not confused with proteins. For example, line 194 talks about genes being localized extracellularly – it is the gene products that are extracellular. Referencing is inconsistent, in some places just a number, in others the reference number and the first and last names of the authors. Be consistent, follow the journal style. The heading "Patents" is used for the author contributions (line 433).
Response: We have ensured that E. coli is consistently italicized throughout the manuscript. We have carefully reviewed all instances where genes and proteins are mentioned. For example, line 219 has been corrected to state that the four genes products (proteins) were predicted to be extracellular. All references have been reformatted to be consistent with MDPI's specified citation style (numbered in order of appearance). The incorrect heading "Patents" at line 433 has been corrected to "author contributions".
    All changes introduced in the revised version of the manuscript are marked with highlight or underline. We believe these revisions have significantly improved the clarity, rigor, and presentation of our manuscript. We are grateful for the reviewers' time and expertise, and we hope the revised manuscript now meets the standards for publication in this journal.
Sincerely,
Chen Chen
Southwest Forestry University

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

 

 

The relationship between the chitinase producing organism, the host plant, and the plant-invading rust is still not particularly clear.  When I read the abstract, I see the source of the chitinase genes as Cladosporium strain SYC23.  These genes are identified because they look like chitinase genes based on their sequence, and they are responding to the presence of rust spores by changing their expression.  The rust spore producer reads as Photinia prionophylla, but this is not a fungus but rather a plant.  So I assume there is some rust fungus that is growing on this plant that serves as the inducing element?  What is this rust?  At this point, my understanding would be that the plant that is infected by the rust is basically unimportant to the logic of the paper.  What is important is the source of the rust spores, the actual rust fungus.  So if I am correct in my understanding here, you want to be saying something like “spores from the rust fungus Aecidium pourthiaea, growing on the plant Photinia prionophylla, were used to induce gene expression…

But you really need to lay out the logic and details of the paper much better, so the reviewer is not actually unclear on such fundamental details.  This paper still really needs lots of work.

,.

When I say the paper needs to be carefully proof-read by a native English speaker I am serious. It is not just the sections I have highlighted below, it is the whole paper.  These corrections noted represent a somewhat serious analysis of just the abstract, but the whole paper needs to be corrected at this level.  I cannot proof the entire paper, but someone must do so before publication.

In order To identify chitinase genes from the genome…bioinformatics analysis methods and real-time … mycoparasitism-associated genes of SYC23 at 12,24, … growth during Photinia…

A total of 8 chitinase genes were detected from SYC23 using bioinformatics analysis methods and were named CaChi34…

…revealed that their molecular weights ranged from… and their theoretical isoelectric points ranged

… that the SYC23 sequences …

CaChi45 was the most…CaChi93 was continuously …CaChi83 was continuously…

The analysis shows that enzymatic …

The rust spores were treated with recombinant chitinase CaChi93, and the results show that CaChi93 is able to dissolve rust spore cell walls.

In conclusion, this study confirms that by secreting chitinase, mycoparasitic Cladosporium sp strain SYC23 could dissolve the rust spore walls and kill the spores, while providing new gene resources and a theoretical basis for …

Author Response

Response to Reviewers' Comments on Manuscript ID:4152270

Dear Editor and Reviewers,

We are deeply grateful for the time, effort, and constructive feedback you have dedicated to our manuscript. Your critical insights have been invaluable in helping us identify key areas for improvement, and we have thoroughly addressed each of your concerns in the revised version of the paper. Below, we provide a detailed, point-by-point response to your comments.

1. Clarity of the relationship between the chitinase-producing organism, host plant, and rust fungus

Comment: The relationship between the chitinase producing organism, the host plant, and the plant-invading rust is still not particularly clear. The rust spore producer reads as Photinia prionophylla, but this is not a fungus but rather a plant.

Response: We sincerely apologize for this critical ambiguity. In the revised manuscript, we have completely restructured the Introduction and Results sections to explicitly clarify the tripartite interaction: We have clearly stated that Cladosporium sp. strain SYC23 is the mycoparasite producing chitinases. We have specified that Photinia prionophylla is the host plant, and the rust fungus Aecidium pourthieae is the pathogen growing on this plant. The revised text now clearly states: "Spores from the rust fungus Aecidium pourthieae, growing on the plant Photinia prionophylla, were used to induce gene expression in Cladosporium sp. strain SYC23."

2. Comprehensiveness and conciseness of the knowledge overview

Comment: The introduction does not provide a comprehensive yet concise overview. It confuses "rust diseases" with "plant pathogens" and fails to justify the economic importance of P. prionophylla as a target.

Response: We have completely rewritten the Introduction section to address these points: We have corrected the conceptual confusion by clearly defining "rust fungi" as the causal agents of "rust diseases." We have added a new paragraph explicitly stating the economic and ecological importance of Photinia prionophylla and the rust fungi that infect it, justifying why this pathosystem is a critical target for biocontrol research.

3. Clarity of the central aim

Comment: The statement "Currently, the role of chitinase... remains unclear" leads to confusion about the target of SYC23 and the paper's central aim.

Response: We have rephrased this critical sentence to eliminate ambiguity. The revised text clearly states: "Currently, the specific role of chitinases in the mycoparasitic process of Cladosporium sp. strain SYC23 against rust fungi remains unclear. This study aims to characterize these chitinases and determine their function in dissolving rust spore cell walls." This directly states the paper's central objective.

4. Adequacy of the methods description

Comment: Key details are missing: How is SYC23 isolated? What is "PSKA activating"? Which rust species is used? How are spores inactivated?

Response: We have thoroughly revised and expanded the Methods section to include all missing details: Isolation of SYC23: We have added a detailed protocol for the isolation of strain SYC23 from rust spore masses, including the specific growth medium and incubation conditions. "PSKA activation": We have defined this term and provided the exact protocol for the activation of strain SYC23 using potato sucrose agar (PSA) medium. Rust species and spore preparation: We have explicitly identified the rust species as Aecidium pourthieae, described the method of spore collection from infected Photinia prionophylla, and detailed the heat-inactivation protocol.

5. Appropriateness of the phylogenetic tree

Comment: The term "beautified" for the phylogenetic tree is unprofessional and its standard is questionable.

Response: We agree that the term "beautified" was unprofessional and has been removed. The phylogenetic tree was reconstructed using the maximum-likelihood method in MEGA X with 1000 bootstrap replicates.

6. English language and proofreading

Comment: The paper needs careful proofreading by a native English speaker.

Response: We take your professional comments on the English expression very seriously. Due to objective constraints, we were unable to engage a native English-speaking professional editor. However, the entire manuscript has been comprehensively and meticulously revised and proofread by our supervising instructor, who has long-term experience in writing and publishing English scientific papers. The instructor focused on refining the grammatical structure, technical terminology, and logical expression throughout the text, with particular attention to correcting the capitalization errors and awkward phrasing you highlighted. We believe that after this round of proofreading, the clarity and academic rigor of the paper's English presentation have been significantly improved, and it now meets the publication standards of the journal. Furthermore, we are willing to use MDPI’s professional foreign language editing and proofreading service to further improve the language quality of the manuscript.

All changes introduced in the revised version of the manuscript are marked with highlight and underline. We believe these revisions have significantly improved the clarity, rigor, and presentation of our manuscript. We are grateful for the reviewers' time and expertise, and we hope the revised manuscript now meets the standards for publication in this journal.

Sincerely,

Chen Chen

Southwest Forestry University

Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

I think whenever you are talking about the spores you should say “Aecidium pourthieae spores” and not P. prionophylla spores – the rust is growing on the plant, but they are not “Photinia prionophylla rust spores”

Jof F revision 3

We are getting there.

Line 14  bioinformatical analyses

Line 15 using rust spores cultured on the plant Photinia prionophylla.

Line 17 and designated CaChi34…

Line 27 His-tagged recombinant CaChi93 protein was purified from E. coli and characterized.  The results demonstrate that the …

Line 31 …dissolve rust spore walls.

Line 33   …for the biological control of Aecidium…

Line 56 …[11, 12] are extensive.

Line 72  Fungal chitinases mainly belong…

Line 83  We examined chitinase activity at different stages during rust spore induction, investigated the expression… and analysed the physiological ….by expression in E. coli.  The research findings provide insight into the role of chitinase genes… and into the biosafety control…

Line 102  On the third day of incubation a small aliquot…

If you absolutely have to have the table of the bioinformatics tools, put it in an appendix – not as Table one of the paper.  But really, if you say “here is a tool” you should also be saying “and I used it to perform this job and here is the result”.  A tool with no context provides the reader with no way of using the tool to repeat your results, and fundamentally providing the framework for repeating results is the role of the methods section.

Line 180 the protein sequence was not ligated into the vector – the DNA sequence encoding the protein sequence of interest was ligated.  Tell us at this point that the vector adds a His6 tag, don’t wait until line 207.

Line 460 …in E. coli cells (no need to say prokaryotic)

Line 506 destroy rust spores from infected P. prionophylla plants.

Line 509  Furthermore, recombinant CaChi93 chitinase was successfully expressed in E. coli and shown to effectively inactivate Aecidium pourthieae spores.

 

 

Author Response

Response to Reviewers' Comments
Dear Reviewers,
We greatly appreciate your valuable and constructive comments on our manuscript. These suggestions have significantly helped us improve the clarity, accuracy, and scientific rigor of our work. We have carefully addressed each point raised, and the manuscript has been revised accordingly. Below is our detailed response to each comment.
Major Comments
1. Comment: I think whenever you are talking about the spores you should say "Aecidium pourthieae spores" and not P. prionophylla spores – the rust is growing on the plant, but they are not "Photinia prionophylla rust spores".
Response:We sincerely apologize for the inaccuracy in the nomenclature of the rust spores. We fully agree with the reviewer that the spores belong to the rust fungus Aecidium pourthieae, not the host plant Photinia prionophylla. We have systematically revised the entire manuscript to replace all instances of "P. prionophylla spores", or "Photinia prionophylla rust spores" with the correct and precise term "Aecidium pourthieae spores".
Detailed Comments
1. Comment (Line 14): bioinformational analyses
Response: Following the reviewer's suggestion, we have updated the manuscript to use the term "bioinformatical analyses" in place of "bioinformatics analysis,".
2. Comment (Line 15): using rust spores cultured on the plant Photinia prionophylla.
Response: Following the major comment, we have revised this line to read: "using Aecidium pourthieae spores cultured on the plant Photinia prionophylla." This explicitly identifies the source of the spores and adheres to the correct nomenclature.
3. Comment (Line 17): and designated CaChi34…
Response: Following the reviewer's suggestion, we have updated the manuscript to use the term "and designated as CaChi34" in place of "and designated CaChi34…".
4. Comment (Line 27): His-tagged recombinant CaChi93 protein was purified from E. coli and characterized. The results demonstrate that the …
Response: Following the reviewer's suggestion, we have updated the manuscript to use the term "The recombinant CaChi93 protein was obtained by prokaryotic expression," in place of "His-tagged recombinant CaChi93 protein was purified from E. coli and characterized. The results demonstrate that the …".
5. Comment (Line 31): …dissolve rust spore walls.
Response: Following the reviewer's suggestion, we have updated the manuscript to use the term "dissolve rust spores"in place of "…dissolve rust spore walls. ".
6. Comment (Line 33): …for the biological control of Aecidium…
Response: Following the reviewer's suggestion, we have updated the manuscript to use the term "for the green prevention and control of Aecidium pourthiaea." in place of "for the biological control of Aecidium pourthiaea.  ".
7. Comment (Line 56): …[11, 12] are extensive.
Response: We have updated the manuscript to use the term " [11,12] is incalculable." in place of "[11, 12] are extensive.".
8. Comment (Line 72): Fungal chitinases mainly belong…
Response: We have updated the manuscript to use the term "Fungal chitinase genes mainly belong to " in place of "Fungal chitinases mainly belong…".
9. Comment (Line 83): We examined chitinase activity at different stages during rust spore induction, investigated the expression… and analysed the physiological ….by expression in E. coli. The research findings provide insight into the role of chitinase genes… and into the biosafety control…
Response: We have updated the manuscript to use the term "It were examined in chitinase activity at different stage induced by rust spores, investigate the expression patterns of chitinase-related genes associated with mycoparasitism using real-time fluorescence quantitative PCR (RT-qPCR), and finally analyze the physiological activity of mycoparasitism-related chitinases by prokaryotic expression in Escherichia coli. The research findings will lay the groundwork for revealing the role of chitinase genes in the mycoparasitism process of Cladosporium sp., while also providing insights into the biosafety control mechanisms for Photinia prionophylla rust disease." in place of "We examined chitinase activity at different stages during rust spore induction, investigated the expression patterns of chitinase-related genes associated with mycoparasitism using real-time fluorescence quantitative PCR (RT-qPCR), and analysed the physiological activity of mycoparasitism-related chitinases by expression in Escherichia coli. The research findings  provide insight into the role of chitinase genes in the mycoparasitism process of Cladosporium sp., and into the biosafety control mechanisms for Photinia prionophylla rust disease.".
10. Comment (Line 102): On the third day of incubation a small aliquot…
Response: We have updated the manuscript to use the term "On the 3rd day of incubation," in place of "On the third day of incubation a small aliquot…".
11. Comment: If you absolutely have to have the table of the bioinformatics tools, put it in an appendix – not as Table one of the paper. But really, if you say "here is a tool" you should also be saying "and I used it to perform this job and here is the result". A tool with no context provides the reader with no way of using the tool to repeat your results, and fundamentally providing the framework for repeating results is the role of the methods section.
Response: We have taken this important methodological advice seriously. The table listing the bioinformatics tools has been moved from the main text to an Appendix at the end of the manuscript.
12. Comment (Line 180): the protein sequence was not ligated into the vector – the DNA sequence encoding the protein sequence of interest was ligated. Tell us at this point that the vector adds a His6 tag, don't wait until line 207.
Response: We have moved the information about the vector adding a His6 tag to this location in the text, so that the experimental design is fully explained at the earliest appropriate point, rather than later in the manuscript.
13. Comment (Line 460): …in E. coli cells (no need to say prokaryotic)
Response: We have removed the redundant term "prokaryotic" from this line.
14. Comment (Line 506): destroy rust spores from infected P. prionophylla plants.
Response: This line has been revised to: "destroy Aecidium pourthieae spores from infected Photinia prionophyllaplants," 
15. Comment (Line 509): Furthermore, recombinant CaChi93 chitinase was successfully expressed in E. coli and shown to effectively inactivate Aecidium pourthieae spores.
Response: It has been added to line 516.
All changes introduced in the revised version of the manuscript are marked with highlight. We believe these revisions have significantly improved the clarity, rigor, and presentation of our manuscript. We are grateful for the reviewers' time and expertise, and we hope the revised manuscript now meets the standards for publication in this journal.
Sincerely,
Chen Chen
Southwest Forestry University

Author Response File: Author Response.pdf