Proteomics-Based Approaches to Decipher the Molecular Strategies of Botrytis cinerea: A Review

Round 1

Reviewer 1 Report

The authors present a review about proteomics-based approaches to decipher the molecular strategies of Botrytis cinerea‘. They present and discuss technological advances and several recent papers involving proteomics related to Botrytis cinerea, and emphasize the importance of this technology to improve our understanding of the biology, infection mechanisms and our ability to  develop new strategies for grey mould control.

Compared to a previous version submitted to Genes, the manuscript has significantly improved. Because of the outstanding role of the secretome, the chapter ‘Secretome: A Key Virulence Compartment’ which contains just two (!) is underrepresented. Several important papers with secretome data have not been cited, for example Müller et al. (2018), New Phytol. 2018 Aug;219(3):1062-1074. doi: 10.1111/nph.15221, showing a relationship between transcripts and proteome of secreted proteins, and Li et al., 2020, mSystems 5(1):e00732-19. doi: 10.1128/mSystems.00732-19, showing the effects of actin mutants on the fungal secretome.

Sometimes, the authors present a biased and even naive view on the role of the proteome, implicating that simply the knowledge of a (sub)proteome and its changes will unravel mechanisms of pathogenicity and mode of action of fungicide (e.g. line 24: ‘Moreover, proteomic approaches have deepened our understanding of host–pathogen interactions, shedding light on the plant’s defensive responses and the molecular mechanisms triggered by infection’; line 77: ‘Additional factors, such as protein stability over time, activation and deactivation cycles, and the reversible binding of side groups, play critical roles in fully comprehending the organism’s biological dynamics. By studying the proteome, we gain a deeper understanding of the true molecular components responsible for a given pheno type.’ Line544: ‘The proteome is a dynamic entity that evolves depending on developmental stages and environmental context. Understanding these modulations is key to comprehending the behavior of this organism.’ Line 606: ‘The proteome is a dynamic entity that evolves depending on developmental stages and environmental context. Understanding these modulations is key to comprehending the behavior of this organism’)... rather than accepting that a combined genetic, transcriptomic, proteomic, metabolomic, cytological and molecular biology approach is required for a true understanding of such complex biological phenomena. The authors should therefore tone down their statements regarding the unique role of proteomics.

 

I think chapter 2.2.  (Proteomic Insights into B. cinerea–Host Interactions) might not be of great interest for the reader, because it focuses on plant proteomes which are far away from the B. cinerea proteome, even when they are analysed in the context of B. cinerea infection. If maintained, the chapter should clearly state in the beginning of the chapter which doesn’t deal with the Botrytis proteome. More interesting are mixed proteomes of both partners, there are  examples of such publications, which could be included. 

Overall, there is a lot of text with general statements of little scientific value, which could be shortened. Furthermore, there are many statements in the text which are unclear or poor, I have mentioned in the detailed comments just a few examples:

lane 94: ‘The publication of its genome in 2011 [2] was a crucial step forward...’ The first genome sequence still contained many errors, only the final sequence published in 2017 (‘A gapless genome sequence of the fungus Botrytis cinerea’) by Jan van Kan, allowed a markedly improved annotation and predictability of proteins.

Table 1: On what basis were these papers listed selected? One of the papers (‘PAMP Activity of Cerato-Platanin during Plant Interaction: An -Omic Approach’) does not describe the fungal but the plant proteome and should be deleted.

line 123: ‘B. cinerea secretome has been one of the fungal subproteomes with a relevant number...’ should better read: ‘B. cinerea secretome has been the fungal subproteomes with most contributions’

line 135: ‘This study confirmed that the secretome of B.cinerea contains proteins that have an exclusive role in pathogenicity on the host, specifically on the mycelial growth.’ Rephrase this sentence, its orthography is wrong and it doesn’t make sense.

Line 137:  ‘This approach allows us to understand the infection strategy of the fungus in situ’ Sentence should read: ‘This approach helps us to understand ...’

Line 146: ‘An explanation provided by the author is that BcPacC is not a direct virulence regulator but might be related to the incapacity of the fungus...’ Rephrase this sentence, e.g.: ... is that the lack of BcPacC might lead to the impaired capacity of the fungus.....

Line 156: ‘Given the crucial role of B. cinerea virulence factors in host colonization, a deep description of its secreted proteins is essential.’ This sentence makes no sense.

Line 229: ‘In this study 1010 proteins were found, which 229 increased the percentage of proteins detected in proteomic studies from 10% to 54% coverage of the genome-predicted proteins.’ I don’t understand this calculation; the B. cinerea genome encodes 11700 predicted proteins (excluding variants)!

Author Response

The authors present a review about proteomics-based approaches to decipher the molecular strategies of Botrytis cinerea. They present and discuss technological advances and several recent papers involving proteomics related to Botrytis cinerea, and emphasize the importance of this technology to improve our understanding of the biology, infection mechanisms and our ability to  develop new strategies for grey mould control.

Compared to a previous version submitted to Genes, the manuscript has significantly improved. Because of the outstanding role of the secretome, the chapter ‘Secretome: A Key Virulence Compartment’ which contains just two (!) is underrepresented. Several important papers with secretome data have not been cited, for example Müller et al. (2018), New Phytol. 2018 Aug;219(3):1062-1074. doi: 10.1111/nph.15221, showing a relationship between transcripts and proteome of secreted proteins, and Li et al., 2020, mSystems 5(1):e00732-19. doi: 10.1128/mSystems.00732-19, showing the effects of actin mutants on the fungal secretome.

Following the reviewer suggestion, “secretome” section has been revised accordantly, with the inclusion of the selected references. Both articles have been carefully reviewed, and the corresponding findings have now been incorporated into the revised version of the manuscript. These additions help to expand the discussion on the functional relevance of the secretome, particularly regarding transcript-protein correlations and the impact of actin dynamics.

Sometimes, the authors present a biased and even naive view on the role of the proteome, implicating that simply the knowledge of a (sub)proteome and its changes will unravel mechanisms of pathogenicity and mode of action of fungicide (e.g. line 24: ‘Moreover, proteomic approaches have deepened our understanding of host–pathogen interactions, shedding light on the plant’s defensive responses and the molecular mechanisms triggered by infection’; line 77: ‘Additional factors, such as protein stability over time, activation and deactivation cycles, and the reversible binding of side groups, play critical roles in fully comprehending the organism’s biological dynamics. By studying the proteome, we gain a deeper understanding of the true molecular components responsible for a given pheno type.’ Line544: ‘The proteome is a dynamic entity that evolves depending on developmental stages and environmental context. Understanding these modulations is key to comprehending the behavior of this organism.’ Line 606: ‘The proteome is a dynamic entity that evolves depending on developmental stages and environmental context. Understanding these modulations is key to comprehending the behavior of this organism’)... rather than accepting that a combined genetic, transcriptomic, proteomic, metabolomic, cytological and molecular biology approach is required for a true understanding of such complex biological phenomena. The authors should therefore tone down their statements regarding the unique role of proteomics.

We thank the reviewer for their comment. In response, we have revised the tone of several sentences to make them more neutral and less assertive. This includes, for example, the sections referring to the contribution of proteomics, which are now framed more clearly as part of a broader multi-omics approach.

 I think chapter 2.2.  (Proteomic Insights into B. cinerea–Host Interactions) might not be of great interest for the reader, because it focuses on plant proteomes which are far away from the B. cinerea proteome, even when they are analysed in the context of B. cinerea infection. If maintained, the chapter should clearly state in the beginning of the chapter which doesn’t deal with the Botrytis proteome. More interesting are mixed proteomes of both partners, there are  examples of such publications, which could be included. Table 1: On what basis were these papers listed selected? […] One of the papers (‘PAMP Activity of Cerato-Platanin during Plant Interaction: An -Omic Approach’) does not describe the fungal but the plant proteome and should be deleted.

Thank you for the helpful suggestion. We agree that including studies analyzing mixed proteomes from both partners would strengthen the manuscript. However, identifying recent publications that comprehensively analyze both the B. cinerea and host proteomes simultaneously remains challenging. For example, the study by Müller et al. mentioned by the reviewer focuses primarily on the tomato proteome in quantitative terms, with limited discussion on defense responses. Nevertheless, following the suggestion, we have incorporated this reference and clarified the scope of the current proteomic studies within the revised text.

Overall, there is a lot of text with general statements of little scientific value, which could be shortened. Furthermore, there are many statements in the text which are unclear or poor, I have mentioned in the detailed comments just a few examples:

Detailed comments

lane 94: ‘The publication of its genome in 2011 [2] was a crucial step forward...’ The first genome sequence still contained many errors, only the final sequence published in 2017 (‘A gapless genome sequence of the fungus Botrytis cinerea’) by Jan van Kan, allowed a markedly improved annotation and predictability of proteins.

Thank you for the comment. We have added the reference to the 2017 gapless genome sequence by Jan van Kan and updated the text accordingly to reflect the improved annotation and protein predictability achieved with this final genome assembly.

line 123: ‘B. cinerea secretome has been one of the fungal subproteomes with a relevant number...’ should better read: ‘B. cinerea secretome has been the fungal subproteomes with most contributions’

Thank you for the suggestion. The sentence has been modified accordingly in the revised version.

 

line 135: ‘This study confirmed that the secretome of B.cinerea contains proteins that have an exclusive role in pathogenicity on the host, specifically on the mycelial growth.’ Rephrase this sentence, its orthography is wrong and it doesn’t make sense.

We agree with the reviewer that the original phrasing was unclear. We have rewritten this section to improve clarity and better convey the differences observed between in vitro and in vivo secretome expression, as well as the functional relevance of the proteins identified.

Line 137:  ‘This approach allows us to understand the infection strategy of the fungus in situ’ Sentence should read: ‘This approach helps us to understand ...’

Thank you for the suggestion. The sentence has been modified accordingly in the revised version.

Line 146: ‘An explanation provided by the author is that BcPacC is not a direct virulence regulator but might be related to the incapacity of the fungus...’ Rephrase this sentence, e.g.: ... is that the lack of BcPacC might lead to the impaired capacity of the fungus.....

We thank the reviewer for the suggestion. As part of the revision, we have incorporated the reference the reviewer recommended regarding pH regulation, and this led to a restructuring of the corresponding section. As a result, the sentence in question no longer appears in the revised version.

Line 156: ‘Given the crucial role of B. cinerea virulence factors in host colonization, a deep description of its secreted proteins is essential.’ This sentence makes no sense.

We agree with the reviewer that the sentence was unclear. It has been removed from the revised version of the manuscript.

Line 229: ‘In this study 1010 proteins were found, which 229 increased the percentage of proteins detected in proteomic studies from 10% to 54% coverage of the genome-predicted proteins.’ I don’t understand this calculation; the B. cinerea genome encodes 11700 predicted proteins (excluding variants)!

We thank the reviewer for pointing this out. We agree that the calculation was not clearly explained. A clarifying sentence has been added to the manuscript to specify that the 54% figure results from combining newly identified proteins in this study with all non-redundant proteins reported in previous proteomic studies, using the updated prediction of 11,707 proteins in the current B. cinerea genome annotation (excluding isoforms). This provides a more accurate estimate of the proteome coverage achieved to date

Reviewer 2 Report

Please refer to the detailed comments.

Comments to the author (JoF-3772389)

The review article, “Proteomics-Based Approaches to Decipher the Molecular Strategies of Botrytis cinerea: A Review” by Olivier B.N. Coste and team is mainly focusing on the most common fungal pathogen having wide host range. The manuscript is well-structured and covers a comprehensive range of proteomics strategies applied to this pathogen. However, some grammatical issues, inconsistent tense usage, article placement, and punctuation errors appear throughout while reading the article. Below are some corrections and suggestions for the improvement that can be considered (where applicable).

Line 10: “postharvest pathogens in the agricultural industry”. please consider “postharvest pathogens affecting global horticulture” for better scope clarity.

Line 12: “fungicides, resistance development” revise it as: “Despite the use of fungicides, the development of resistance”

Line 18: “revealing dynamic, condition-dependent mechanisms of pathogenicity” replace rephrasing as: “revealing condition-specific pathogenic mechanisms”

Line 23: “including pH-sensing factors” can be replaced as “such as pH-responsive elements” that would be clearer.

My personnel suggestions: add 1-2 quantitative examples or numerical advances (e.g., number of proteins identified via secretome studies) to strengthen the impact of abstract and to improve the flow by combining shorter clauses for readability.

In line 40: instead of using “species of the vegetal kingdom” using “plant kingdom” would be more appropriate.

Similarly, in line 44: “fungus’s ability” replaces “fungus’ ability” for the consistency of style.

Line 51: replace “50% or more within one month in cold storage” with “up to 50% incidence within a month under cold storage”.

Line 56: Revise “flowers and vegetative parts are the main commercial products” as “where both flowers and foliage represent key market components”.

I would suggest the subsection titles like “1.1” and “1.2” could be renamed for better attraction. e.g., “1.1 “Economic and ecological threat” and “1.2” as unveiling virulence through proteomics. Also, consider linking the introduction more explicitly with the proteomics focus e.g., a brief timeline or trends.

Please include a small timeline figure or bullet-point history of technical advancements (e.g., 2DE LC-MS/MS iTRAQ).

In 2.1 Subproteomes, please clarify how the deletion mutants affected specific virulence phenotypes with suitable previously published examples and a comparative table of proteins identified via different secretome techniques would be more helpful.

In the part Surface Proteins (Surfactome) and PTMs, the surfactome study could benefit from elaboration on cross-kingdom RNAi, please provide suitable citations particularly on fungal proteins or plant transcripts affected by separating PTMs into a standalone subsection.

As this is review article, so in the host interaction section, adding a schematic figure showing host-pathogen molecular interplay via proteomic markers would be more attractive rather than discussing theoretically. Further, please discuss the limitations more explicitly, such as cross-contamination in mixed samples.

In section 2.3 (functional profiling of botryticides), adding a table on comparing the proteomic responses to different antifungal treatments would be nice and also provide overview if any of the identified proteins overlap with those in drug resistance studies.

Section 4 is an important section, I would suggest emphasize emerging AI-guided proteomics pipelines and cite real-world tools (e.g., AlphaFold for structural prediction). Further, provide a brief overview summarizing underexplored tools and their potential applications in B. cinerea proteomics. Shift/merge this section after/within the conclusion section.

Author Response

 

Reviewer 2:
The review article, “Proteomics-Based Approaches to Decipher the Molecular Strategies of Botrytis cinerea: A Review” by Olivier B.N. Coste and team is mainly focusing on the most common fungal pathogen having wide host range. The manuscript is well-structured and covers a comprehensive range of proteomics strategies applied to this pathogen. However, some grammatical issues, inconsistent tense usage, article placement, and punctuation errors appear throughout while reading the article. Below are some corrections and suggestions for the improvement that can be considered (where applicable).

 

Line 10: “postharvest pathogens in the agricultural industry”. please consider “postharvest pathogens affecting global horticulture” for better scope clarity.

Thank you, we have updated the text to “postharvest pathogens affecting global horticulture” as suggested.

Line 12: “fungicides, resistance development” revise it as: “Despite the use of fungicides, the development of resistance”

Thank you, the sentence has been revised to: “Despite the use of fungicides, the development of resistance.”

Line 18: “revealing dynamic, condition-dependent mechanisms of pathogenicity” replace rephrasing as: “revealing condition-specific pathogenic mechanisms”

Thank you, we have rephrased it as: “revealing condition-specific pathogenic mechanisms.”

Line 23: “including pH-sensing factors” can be replaced as “such as pH-responsive elements” that would be clearer.

Thank you, we have replaced the phrase with “such as pH-responsive elements” for clarity.

 

My personnel suggestions: add 1-2 quantitative examples or numerical advances (e.g., number of proteins identified via secretome studies) to strengthen the impact of abstract and to improve the flow by combining shorter clauses for readability.

Thank you for the comment. We agree the abstract could be improved, so we have restructured and rewritten parts of it to make it clearer and more engaging. We also added some quantitative examples to strengthen the impact and improve the flow.

 

In line 40: instead of using “species of the vegetal kingdom” using “plant kingdom” would be more appropriate.

Thank you, we have replaced “species of the vegetal kingdom” with “plant kingdom” as suggested.

Similarly, in line 44: “fungus’s ability” replaces “fungus’ ability” for the consistency of style.

Thank you, we have updated “fungus’ ability” to “fungus’s ability” for consistency in style.

Line 51: replace “50% or more within one month in cold storage” with “up to 50% incidence within a month under cold storage”.

Thank you, we have revised the phrase to “up to 50% incidence within a month under cold storage.”

Line 56: Revise “flowers and vegetative parts are the main commercial products” as “where both flowers and foliage represent key market components”.

Thank you, we have revised the phrase to “where both flowers and foliage represent key market components.”

I would suggest the subsection titles like “1.1” and “1.2” could be renamed for better attraction. e.g., “1.1 “Economic and ecological threat” and “1.2” as unveiling virulence through proteomics. Also, consider linking the introduction more explicitly with the proteomics focus e.g., a brief timeline or trends.

Thank you for the helpful suggestion. We have renamed subsections 1.1 and 1.2 to “Economic and ecological threat” and “Unveiling virulence through proteomics,” respectively, to improve clarity and engagement.

Please include a small timeline figure or bullet-point history of technical advancements (e.g., 2DE LC-MS/MS iTRAQ).

We appreciate the reviewer’s comment. However, this information was deleted during previous rounds of review due to technological advancements in proteomics falls outside the scope and focus of this review.

In 2.1 Subproteomes, please clarify how the deletion mutants affected specific virulence phenotypes with suitable previously published examples and a comparative table of proteins identified via different secretome techniques would be more helpful.

We appreciate the suggestion and have revised the section to clarify how deletion mutants affect specific virulence phenotypes. In fact, the inclusion of a list of thousands of identified proteins will make the paper illegible. References and previously obtained conclusions has been included as suggested.

 

In the part Surface Proteins (Surfactome) and PTMs, the surfactome study could benefit from elaboration on cross-kingdom RNAi, please provide suitable citations particularly on fungal proteins or plant transcripts affected by separating PTMs into a standalone subsection.

Thank you for the suggestion. Although we find the topic of cross-kingdom RNAi very interesting, elaborating on it would go beyond the focus of this review. Regarding the separation of the PTMs section, following reviewer suggestion is presented as a standalone subsection.

As this is review article, so in the host interaction section, adding a schematic figure showing host-pathogen molecular interplay via proteomic markers would be more attractive rather than discussing theoretically. Further, please discuss the limitations more explicitly, such as cross-contamination in mixed samples.

Following the review suggestions, We have added a paragraph to clarify this distinction and highlighted the interest in future studies addressing the proteomes of both host and pathogen for a more comprehensive interaction view.

 

In section 2.3 (functional profiling of botryticides), adding a table on comparing the proteomic responses to different antifungal treatments would be nice and also provide overview if any of the identified proteins overlap with those in drug resistance studies.

Following reviewer suggestion, we have added modifications in the text to highlight the overlap between the pathways affected by the novel antifungal agents and those targeted by traditional chemical fungicides.  The addition of more table or comments about fungicides against botrytis was limited in previous rounds of reviews.

Section 4 is an important section, I would suggest emphasize emerging AI-guided proteomics pipelines and cite real-world tools (e.g., AlphaFold for structural prediction). Further, provide a brief overview summarizing underexplored tools and their potential applications in B. cinerea proteomics. Shift/merge this section after/within the conclusion section.
We appreciate the reviewer’s suggestion. We have emphasized emerging AI-guided proteomics pipelines by citing relevant tools such as AlphaFold, DIA-NN, and Prosit, and briefly discussing their potential applications in B. cinerea research. However, according with the valorization of others reviewers, we chose to keep Section 4 separate from the conclusion to preserve structural clarity, while ensuring a smooth transition between future perspectives with the present state of the art.