Review Reports - The Velvet Complex Is Essential for Sclerotia Formation and Virulence in <i>Sclerotinia sclerotiorum</i>

Round 1

Reviewer 1 Report

The study aimed to elucidate the molecular mechanisms underlying sclerotia formation in Sclerotinia sclerotiorum. The authors generated mutants and compared their virulence, sclerotia formation, and appressoria development with the wild type. Through forward and reverse genetic analyses, SsLae1 and SsVel1, core components of the conserved fungal velvet complex,were identified as essential regulators of these processes. Mutants P20D12, MK63, and CZ-1 exhibited defects in sclerotia formation, appressoria development, and both pre- and post-penetration virulence. The findings demonstrate that disruption of either SsLae1 or SsVel1 abolishes sclerotia formation, impairs appressorium development, and significantly reduces virulence, highlighting the crucial role of the velvet complex in pathogenicity and sclerotia formation in S. sclerotiorum.

This is a valuable and well-conducted study, and I recommend it for publication after the authors have addressed the comments in the attached document.

Comments regarding general concepts

This is a valuable and well-conducted study, and I recommend it for publication after the authors have addressed the following comments.

Specific comments

Line 100: “Thess” — please clarify the meaning of this term.

Lines 100–106: Consider moving this section under subheading 2.2 to align the text more closely with Figure 4, improving clarity and flow.

Figure 1, Line 108: Please spell out the abbreviation UBC in full upon first mention.

Figure 4c, Line 182: Add a brief explanation in the results section to clarify what the phylogenetic tree (Fig. 4c) indicates. Also, consider placing the text closer to the figure for better readability.

Page 4, Figure 2, Lines 124 and 127: The phrases “Quantification evaluation of lesion area” should be revised to “Quantitative evaluation”. Additionally, the reference to panel (D) appears inconsistent; please verify whether it should be (E).

Page 5, Figure 3, Lines 134–135: Revise “Quantification evaluation” to “Quantitative evaluation”. Also, the reference to panel (D) should likely be (E)—please confirm.

Line 152: Remove the period after “appressorial development (Figure 5C and 5D)”

Lines 190–195: This section contains interpretation and discussion. Consider moving it to the Discussion section.

Lines 209–213: The statement “The velvet complex has been reported…” is more appropriate for the Introduction or Discussion section. Please remove it from the Results.

Lines 213–216: The sentence “To gain deeper insights into…” describes methodology and should be relocated to the Materials and Methods section.

Lines 229–237: This passage presents interpretation and should be moved to the Discussion section.

Discussion

Lines 252–266: Consider beginning the discussion section by interpreting your most significant findings, followed by references to supporting or contrasting studies. This will strengthen the contextual relevance of your results.

Lines 260–261: The term “developmental processes” in S. sclerotiorum requires clarification. Are you referring to infection-related processes such as cushion and appressoria formation, or sclerotia development? Please specify.

Lines 311–312: The statement “sclerotia development and pathogenicity of S. sclerotiorum does not appear to be light dependent” should be supported by a reference. While it may be assumed that light is not a major factor due to the pathogen’s soil-borne nature, citing relevant literature would strengthen the claim.

Lines 319–320: The suggestion that “host-induced gene silencing or RNA interference may offer a viable strategy to control Sclerotinia stem rot in crops” would benefit from further elaboration and supporting evidence. It is important to discuss how your findings could be translated into practical disease management strategies. While gene knockout experiments demonstrate the potential to disrupt pathogenicity and sclerotia formation in laboratory conditions, please address how this approach could be applied in field settings, where large sclerotia banks serve as inoculum sources.

M&M

Lines 332, 339, and 358: The abbreviation PDB is unclear. Please clarify what the “B” stands for. Did you mean PDA (Potato Dextrose Agar)?

Line 382: Avoid starting a paragraph with an abbreviation. Please write WT as “Wild type” in full.

NB: Please note that I have not checked the reference list or the in-text citations.

Comments for author File: Comments.pdf

Author Response

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

The response in /Word is attached for your reference.

2. Point-by-point response to Comments and Suggestions for Authors

Comments 1:

Line 100: “Thess” — please clarify the meaning of this term.

Response 1:

Thank you for pointing this out. The sentence has been revised to: “These results suggest that mutants P20D12, MK63, and M73 are allelic to each other, each likely carrying different mutations in the same gene.” In addition, to improve the flow, we have moved the paragraph describing the allelism test to Section 2.2.

Comments 2:

Lines 100–106: Consider moving this section under subheading 2.2 to align the text more closely with Figure 4, improving clarity and flow.

Response 2:

Agree. we have moved the paragraph describing the allelism test to Section 2.2.

Comments 3:

Figure 1, Line 108: Please spell out the abbreviation UBC in full upon first mention.

Response 3:

Thank you for pointing this out. We’ve made changes to the main text in Section 2.1, to spell out the full name of both UBC and SCU, “As shown in Figure 1A and 2A, mutant P20D12 and MK63 identified at the University of British Columbia (UBC) in Canada, and CZ-1 found from Sichuan University (SCU) in China, could not produce sclerotia.”.

Comments 4:

Figure 4c, Line 182: Add a brief explanation in the results section to clarify what the phylogenetic tree (Fig. 4c) indicates.

Response 4:

Agree. It has been improved as “In the NGS data of CZ-1 mutant, a frameshift mutation in sscle_11g084630 was identified, which, based on our phylogenetic analysis and protein sequence alignment, corresponds to the VeA orthologue in S. sclerotiorum”.

Comments 5:

Response 5:

Thank you for pointing this out. We have revised the text accordingly to improve clarity.

Comments 6:

Page 5, Figure 3, Lines 134–135: Revise “Quantification evaluation” to “Quantitative evaluation”. Also, the reference to panel (D) should likely be (E)—please confirm.

Response 6:

Thank you for pointing this out. We have revised the text accordingly to improve clarity.

Comments 7:

Line 152: Remove the period after “appressorial development (Figure 5C and 5D)”

Response 7:

Thank you for pointing this out. We have revised the text accordingly.

Comments 8:

Lines 190–195: This section contains interpretation and discussion. Consider moving it to the Discussion section.

Response 8:

This section provides a brief summary of the characterization results of the SsLae1 and SsVel1 mutants. Retaining it here helps maintain logical flow, as the subsequent section introduces the RNA-seq analysis of the SsVel1 mutant.

Comments 9:

Lines 209–213: The statement “The velvet complex has been reported…” is more appropriate for the Introduction or Discussion section. Please remove it from the Results.

Response 9:

Agree. We have revised the text accordingly.

Comments 10:

Lines 213–216: The sentence “To gain deeper insights into…” describes methodology and should be relocated to the Materials and Methods section.

Response 10:

We chose to keep it unchanged, as the description in this sentence is concise but necessary to understand the RNA-seq data presented in Figure 7, which includes both 3-day and 5-day samples. In addition, detailed information about the RNA-seq analysis is provided in the Materials and Methods section.

Comments 11:

Lines 229–237: This passage presents interpretation and should be moved to the Discussion section.

Response 11:

We prefer to keep it where it is, as it serves as a short summary of the results.

Comments 12:

Response 12:

Thank you for your suggestions. We have summarized our main findings in the first paragraph of the Discussion, highlighting that the velvet complex in S. sclerotiorum is essential for both virulence and sclerotia formation. We then further elaborate on these findings in the following two paragraphs, focusing separately on virulence and sclerotia formation.

Comments 13:

Lines 260–261: The term “developmental processes” in S. sclerotiorum requires clarification. Are you referring to infection-related processes such as cushion and appressoria formation, or sclerotia development? Please specify.

Response 13:

Thank you for pointing this out. We have revised the text accordingly. “In this study, our forward genetic screens revealed that SsLae1 and SsVel1 encoded part of the velvet complex that seems to be essential for compound appressorial development, virulence, and sclerotia formation in S. sclerotiorum.”

Comments 14:

Lines 311–312: The statement “sclerotia development and pathogenicity of S. sclerotiorum does not appear to be light dependent” should be supported by a reference. While it may be assumed that light is not a major factor due to the pathogen’s soil-borne nature, citing relevant literature would strengthen the claim.

Response 14:

Agree. We have revised the text accordingly and provided a reference. “However, based on our observations and a previous report [28], sclerotia development and pathogenicity of S. sclerotiorum do not appear to be light dependent.”

Comments 15:

Response 15:

Thank you for your suggestion. We have provided more information and references on this. “Moreover, the velvet complex itself represents a promising target for disease management. Recent studies have provided evidence that host-induced gene silencing (HIGS) of several virulence-related genes effectively reduces lesion size on N. benthamiana leaves infected by S. sclerotiorum [6,10,12]. Targeting SsLae1 and SsVel1 through similar approaches may offer a viable strategy to control Sclerotinia stem rot in crops. Future studies will be needed to evaluate the feasibility and effectiveness of such approaches in planta.”

Comments 16:

Lines 332, 339, and 358: The abbreviation PDB is unclear. Please clarify what the “B” stands for. Did you mean PDA (Potato Dextrose Agar)?

Response 16:

Thank you for pointing this out. We have revised the text accordingly. PDB is short for potato dextrose broth.

Comments 17:

Line 382: Avoid starting a paragraph with an abbreviation. Please write WT as “Wild type” in full.

Response 17:

Thank you for pointing this out. We have revised the text accordingly.

Reviewer 2 Report

Dear authors, I have reviewed your manuscript, which contains elements of interest for elucidating the mechanisms of fungal phytopathogenicity. In particular, those related to sclerotium formation and the velvet complex. The discussion focuses on molecular aspects with documented support. In this regard, I recommend its publication with minor improvements addressing the comments described in the following section of detailed comments. Additionally, I recommend including quality parameters, such as gels and DNA and RNA absorbance ratios.

The introduction is adequate and sufficient. However, the hypothesis is not identified

In the Materials and Methods section.

Line 325: The authors mention that the strain was provided by Dr. Jeffery. However, the authors should provide more information about the strain’s background: where was it isolated from? How is it identified?

Line 333: the authors should indicate how they counted the spores.

Line 332: they must indicate what PDB means

Line 334: How many Petri dishes were incubated? What was the diameter of the plates?

Line 336: the authors must indicate how they carried out the sclerotia observations.

Line 339: the authors should provide the reference for the CTAB protocol or, if applicable, describe it, since it may vary among authors.

Line 352: the authors should provide more information about the pCH-EF-1 plasmid.

Line 356: indicate the concentration of hygromycin used.

Line 361: How did they select the colonies to be transferred? Did they have to be a particular diameter? What were the selection criteria? It should be indicated.

Line 374: the authors must indicate which symptoms they evaluated.

Line 380: At what magnification were the observations made?

In the results section, the authors discuss when this should be in a separate section.

Regarding the velvet complex, the authors should complement the discussion on its impact on virulence and sclerotium formation by mentioning the main biochemical aspects involved in addition to the molecular ones.

Author Response

1. Summary

2. Point-by-point response to Comments and Suggestions for Authors

Comments 1:

Additionally, I recommend including quality parameters, such as gels and DNA and RNA absorbance ratios.

Response 1:

Thank you for pointing this out. We have revised the manuscript accordingly. A DNA gel image of the Sslae1 and Ssvel1 mutants is included in Supplemental Figure 2.

Comments 2:

The introduction is adequate and sufficient. However, the hypothesis is not identified.

Response 2:

Thank you for your suggestion. The objective of this study is to identify new regulators of sclerotia formation in S. sclerotiorum, and this has been stated in the last paragraph of the introduction section.

Comments 3:

Response 3:

Thank you for pointing this out. We have provided a reference accordingly.

Comments 4:

Line 333: the authors should indicate how they counted the spores.

Response 4:

Thank you for pointing this out. We have revised the manuscript accordingly. “In short, ascospores of S. sclerotiorum strain 1980 WT and Ssoah1 were obtained from apothecia [50], resuspended in sterilized potato dextrose broth (PDB) (Shanghai Bio-Way Technology), and diluted to 10⁴ spores/ml after counting with a hemocytometer.”

Comments 5:

Line 332: they must indicate what PDB means

Response 5:

Thank you for pointing this out. We have revised the manuscript accordingly, as “potato dextrose broth (PDB)”.

Comments 6:

Line 334: How many Petri dishes were incubated? What was the diameter of the plates?

Response 6:

Thank you for pointing this out. We have revised the manuscript, and cited the original paper establishing the pipeline of UV mutagenesis and screening.

Comments 7:

Line 336: the authors must indicate how they carried out the sclerotia observations.

Response 7:

Thank you for pointing this out. After grown in 96-well plates for one to two weeks, the sclerotia, if there are any, can be observed with the naked eye.

Comments 8:

Line 339: the authors should provide the reference for the CTAB protocol or, if applicable, describe it, since it may vary among authors.

Response 8:

Thank you for pointing this out. We have revised the manuscript accordingly, and added a supporting reference.

Comments 9:

Line 352: the authors should provide more information about the pCH-EF-1 plasmid.

Response 9:

Thank you for pointing this out. The pCH-EF-1 plasmid carries the Hygromycin B resistance gene hph with a PtrpC promoter and terminator. As stated in section 3.4, we amplified the hph gene from this plasmid.

Comments 10:

Line 356: indicate the concentration of hygromycin used.

Response 10:

Agree. We have revised the manuscript accordingly. “Transformants were obtained on PDA plates supplemented with hygromycin B at a final concentration of 50 µg/mL”

Comments 11:

Line 361: How did they select the colonies to be transferred? Did they have to be a particular diameter? What were the selection criteria? It should be indicated.

Response 11:

Thank you for your suggestion. Positive colonies were selected based on their growth on PDA plates containing hygromycin B, where normal mycelial growth was observed spreading from the germination site. We transferred only single, isolated colonies, ensuring that each transfer was made before any colonies came into contact or fused with one another.

Comments 12:

Line 374: the authors must indicate which symptoms they evaluated.

Response 12:

Thank you for pointing this out. We have revised the manuscript accordingly. “Disease symptoms, represented by lesion area, were recorded at 24-36 hpi. Each assay was repeated two times with consistent results.”

Comments 13:

Line 380: At what magnification were the observations made?

Response 13:

Agree. We’ve added more information on this. “Compound appressoria formed on glass slides were subsequently observed and photographed by a ZEISS AXIO Imager M2 microscope with a 63x objective lens. Compound appressoria formed on parafilm were photographed using a smartphone digital camera.”

Comments 14:

In the results section, the authors discuss when this should be in a separate section.

Response 14:

Thank you for pointing this out. We have revised the manuscript accordingly.

Comments 15:

Response 15:

Thank you for your suggestion. The biochemical function of the velvet complex remains unclear. We have included a discussion in the fifth paragraph of the Discussion section about potential future experiments to further investigate the roles of the SsLae1 and SsVel1 proteins.