COMPASS subunit Bre2 regulates chromatin remodeler Arp9 to control Aspergillus flavus aflatoxin synthesis and virulence

Domain: chemistry
Published: 2026-02-20
Editorial outcome: major_revision

Abstract
Aspergillus flavus, along with its notorious secondary metabolite aflatoxin B1 (AFB1), seriously endangers human health. Histone methyltransferase complex COMPASS (complex of proteins associated with Set1) plays a crucial role in regulating aflatoxin biosynthesis and virulence of A. flavus, but the underlying mechanism is unclear. Here, we find that Bre2, the key subunit of COMPASS, regulates AFB1 biosynthesis, fungal morphogenesis, and virulence through modulation of H3K4 methylation. ChIP-seq and biochemical analyses reveal that chromatin remodeling factor (CRF) Arp9 is directly targeted by Bre2, and Arp9 exerts bio-functions through interacting with the other CRFs such as RSC8, Arp7, and Sth1. ATAC-seq results indicate that Arp9 contributes to fungal pathogenicity by modulating chromatin conformation of genes that are involved in secondary metabolism, morphogenesis, and virulence. The study reveals an epigenetic signaling pathway mediated by chromatin remodeler Arp9 and provides a potential strategy for the control of pathogenic fungi and mycotoxins.

Introduction
Aspergillus flavus is a notorious fungus that produces toxic aflatoxins (AFs). It poses a serious threat to human health by food contamination1–4. Characterized by hepatotoxicity, teratogenicity, and immunotoxicity, AFs can be taken in through the respiratory, mucous, or even skin pathways. AFB1 is the most toxic and productive aflatoxin. It acts synergistically with hepatitis B virus (HBV), leading to significantly elevated risk of chronic liver disease (CLD) and hepatocellular carcinoma (HCC)5,6. Therefore, it is crucial to reveal the intricate regulatory mechanisms governing A. flavus development and secondary metabolite biosynthesis to control contamination caused by this pathogenic fungus.

Various factors affecting secondary metabolism and pathogenicity of A. flavus have been revealed in recent years, including environmental factors, transcriptional regulators, and histone post-translational modifications (HPTM)7–9. Nucleosome is the fundamental structural component of chromatin. Histones are involved in nucleosome assembly and thus influence chromatin packing10. Amino acid residues at the N-terminal-end of the histones are susceptible to post-translational modifications (PTMs), including acetylation, methylation, phosphorylation, and ubiquitination11. As a hotspot in epigenetic modification, research on HPTM has revealed its important role in A. flavus morphogenesis, secondary metabolite biosynthesis, and pathogenicity12–14. Methylation of H3K4, H3K36, and H3K79 is linked to transcriptional activation, whereas methylation of H3K9, H3K20, and H3K27 is linked to transcriptional repression and gene silencing15. In fungi, chromatin conformation is regulated by the methylation status of histone-associated sites, which in turn affects development and biosynthesis of secondary metabolites16,17. Methylation of lysine residues in histones is catalyzed by lysine (K) methyltransferases (KMTs)18. Set1, which contains a highly conserved SET (suppressor of variegation, enhancer of zeste, trithorax) domain, is the first and only H3K4 methyltransferase identified in yeast. The Set1-containing complex was named COMPASS (complex of proteins associated with Set1)19–21. The following studies have uncovered the catalytic activity of COMPASS toward H3K4 methylation22–24. In yeast, structure integrity of the COMPASS and methyltransferase activity of Set1 are maintained by Swd3, Swd1, Bre2, and Sdc125. In yeast, Bre2 is the homolog of Drosophila Ash2 (absent, small, or homeotic 2-like protein) and functions as scaffold protein of COMPASS22. Available reports show that COMPASS is closely associated with meiotic DSBs (double-strand breaks)26,27, and Bre2 is indispensable for H3K4me3 modification during yeast meiosis28,29. Bre2 and Sdc1 interact directly at the C-terminus of Bre2 and the Dpy-30 domain of Sdc1 in yeast. This interaction is required for appropriate H3K4 methylation and subsequent gene expression regulation30. In addition, CclA, the Bre2 ortholog in A. nidulans, was found to be related to the activation of cryptic secondary metabolites31. Thus, it is important to elucidate whether Bre2 plays a role in determining A. flavus virulence and AFB1 biosynthesis.

Arps (actin-related proteins) are essential functional subunits in the SWI/SNF, SWR1, and INO80 remodeling complexes. They play a critical role in regulating the ATPase activity of chromatin remodeler by binding to the helicase/SANT associated (HAS) domain of the remodeler at the N-terminal-end32. Among Arps, Arp9 binds to Sth1 and Snf2 of the SWI/SNF ATPases32. In Saccharomyces cerevisiae, the arp9 mutant has impaired growth, a phenotype similar to that of the Swi-/Snf- mutant. Therefore, it was predicted that Arp9 functions in chromatin remodeling as a component of the remodeling complex rather than via enzymatic activity33. In Penicillium oxalicum, ARP9 plays an essential developmental role and is involved in the regulation of the expression of cellulase and amylase34. In A. flavus, Set1 mediates dimethylation and trimethylation of H3K4 and regulates the secondary metabolite and pathogenicity of A. flavus, which indicates that COMPASS plays a crucial role in regulating various important biological processes35. But the relationship between Bre2-mediated gene expression and Arp-mediated chromatin remodeling (especially Arp9) in secondary metabolite biosynthesis warrants further exploration.

In this work, Bre2 is chosen to explore its biological functions in fungal virulence and production of secondary metabolites, mainly AFB1. The role of Arp9 in the Bre2-mediated epigenetic signaling pathway is explored by ATAC-seq analysis. We elucidate the regulatory mechanism of Bre2-mediated H3K4 methylation and its potential role in fungal morphogenesis and secondary metabolite biosynthesis, thereby providing a theoretical basis for early prevention and control of contamination by A. flavus and its notorious secondary metabolites AFs.

Results
Bre2 plays a critical role in fungal morphogenesis and aflatoxin B1 synthesis
With the aim of exploring the role of Bre2 in fungal virulence from an evolutionary perspective

Methods

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this study, the authors first explore the consequences of inactivating the COMPASS subunit Bre2 in different biological processes of A. flavus. They show that Bre2 deletion (∆bre2) reduces the expression of key transcription factors and thereby affects fungal morphogenesis and the level of the secondary metabolite AFB1. In agreement with these results, they show that Bre2 is required for virulence A. flavus to silkworm. They further show that A. flavus is localized in the nucleus and that deletion or mutations in the SPRY phenocopy the ∆bre2 deletion mutant. Mapping the peaks of H3K4me3 in WT reveals that this mark is mainly located in promoters and exons of genes notably involved in metabolite synthesis and in several genes of the SNF2 family. Interestingly, they show that Bre2 and Arp9 are each required for the expression of the one by the other. In the second part of the manuscript, the authors show that the deletion of Arp9 produces effects similar to the deletion of Bre2 indicating that Arp9 collaborates with Bre2 to modulate fungal morphogensis and metabolite production. ATAC-seq experiments reveal that Arp9 acts by remodeling the promoter of target genes, including genes involved secondary metabolites related process. In the paper, it is improper to write (lane 298):\" H3K4me3 modification catalyzed by Bre2\"; H3K4me3 is catalyzed by the Set1 complex\nThe experiments are solid and support the conclusions of this manuscript. My major problem is with the title, the summary, and the role is assigned to Bre2. Actually, the observed effects are not specifically due to Bre2, but rather to the absence of H3K4me3 and the reduction of H3K4me2. It is likely that the deletion of Sdc1 would produce the same effects as that of Bre2. The more appropriate title would be \"H3K4me3 promotes CRF Arp9 expression to regulate the bio synthesis of secondary metabolite AFB1 and virulence in Aspergillus flavus”. In this sense, part of the novelty of the article has already been revealed in their previous publication: “Methyltransferase AflSet1 Is Involved in Fungal Morphogenesis, AFB1 Biosynthesis, and Virulence of Aspergillus flavus\".\nThe results on Arp9 and the link between H3K4me3 and the RSC complex are more novel and echo the MS of Schlichter and Cairns BR (PMID: 15775977).\nSpecifically, on the results, I have few comments:\n- In figure 1K, what are the neighboring peaks of the ABF1 peak which also disappear in the Δbre2 mutant? Specify the abbreviation TLC\n- H3K4me3 ChIP-seq reveal 2871 enriched genes and 723 depleted genes ( WT compared to the Δbre2 strain). What are these 723 depleted genes ?\n- Please provide the 63 proteins pulled down by Arp9.", "reviewer_2": "The manuscript describes regulation of a target to control the biosynthesis of secondary metabolites and virulence in Aspergillus. I appreciate the authors’ robust process used for genetic confirmations. The study is well presented and organized.\nGrammatical clarity could be improved.\nDefine COMPASS and ATAC\nAdd reference to line 88 (i.e., Our group previously reported…)\nLine 125: italics to ‘bre2’\nLine 130: CM defined already?\nLine 136: TLC and HPLC defined? Check throughout if terms from methods are defined within the Results for first use\nFigure 1 – suggest increasing the size of the plots A-L (excluding K) to improve readability and decrease empty white space on the figures.\nMost figures appeal too small within the panels – sizes could be adjusted to optimize the figure size and reduce white space.", "reviewer_3": "In the manuscript entitled \"The COMPASS subunit Bre2 targets CRF Arp9 to regulate the bio synthesis of secondary 1 metabolite AFB1 and virulence in Aspergillus flavus\", the authors focused on the gene of Bre2 subunit of the histone methyltransferase complex and showed that bre2 is involved in H3K4 trimethylation in A. flavus. Through ChIP-seq analysis, they found that the gene of Arp9, which is involved in chromatin remodeling, is located downstream of bre2. The analysis of bre2 and arp9 knockout strains demonstrated that these genes are essential for the morphological development and aflatoxin production of A. flavus. Additionally, for bre2, they revealed functionally important protein domains and amino acid residues. Using epigenetic analysis of arp9 mutants, they investigated downstream genes affected by Arp9-mediated chromatin remodeling, constructed knockout strains for some of these genes, and examined their phenotypes.\nThe authors utilized a variety of approaches including seq analysis on the generated mutant strains to elucidate the relationship between epigenetic modifications—initiated by histone H3K4 trimethylation—and aflatoxin production of A. flavus, the notorious mycotoxigenic fungus. The experimental results clearly establish that arp9 functions downstream of bre2 and suggest that Arp9 exerts a distal effect on the aflatoxin biosynthetic pathway and morphological development pathway through SMAP and other factors.\nHowever, the manuscript does not fully elucidate the mechanism by which Bre2 and Arp9 regulates aflatoxin production and morphological development. Although the ATAC-seq analysis identified several genes as potential components of the Arp9 regulatory pathway, only SMAP knockout strains exhibited significant effects on aflatoxin production and morphology, leaving the relationship between SMAP and these factors unclear. Simply to say, in Figure S6, the arrow which leads to the red boxes enclosing the gene clusters is unidentified. Due to this limitation, the overall framework linking histone H3K4 trimethylation initiated by Bre2 to aflatoxin production has not been fully clarified. Further analyses are needed to bridge this missing link.\nThe authors concluded that Bre2 is related to the colonization of peanuts and maize based on experiments using the poorly growing deletion strain ⊿bre2. However, since poor growth is associated with colonization, it is not possible to discuss a direct relationship between Bre2 and colonization ability. Similarly, in the infection experiment with silkworms, the strong relationship between the poor growth of the deletion strain and its virulence makes it difficult to discuss the relationship between Bre2 and virulence in silkworms. Additionally, it should be noticed that the relationship between fungal AF productivity and pathogenicity to silkworms is unclear. In fact, the ⊿bre2 strain still maintains lethality, with more than half of the silkworms dying within 6 days. As the same methods were used in experiments using other poorly growing deletion strains, no conclusions can be drawn from this paper regarding the relationship between Bre2 and virulence in A. flavus.\nSpecific comments:\nThe structure and explanations of the figures are inadequate, making it difficult for readers to understand the intended information. This issue is particularly pronounced from Figure 5 onward. Specifically:\n• Figure 4A: Appears to be a screenshot from IGV, but lacks sufficient explanation.\n• Figure 5B: The meaning of the mass spectrum is unclear.\n• Figure 5C-E: The differences between lanes are not explained, and it is difficult to read what the detected bands represent.\n• Figure 5F-H: Additional explanations for the bands are needed.\n• Figure 6A: The descriptions of the lanes and labels lack.\n• Figure 7: The text is too small to read. The labeling may be inappropriate (e.g., \"arp9\" should be \"Δarp9\", isn’t it?). The same issue applies to Figure S3.\n• Figure 8A: The method for calculating the CAI index is unclear. Is there an easier metric to understand?\nIn terms of overall structure of the manuscript, the Results section includes elements that belong in the Discussion, while the Discussion section reads more like a Results and Discussion section, mixing result descriptions with interpretations. A thorough revision is needed to clearly separate these sections.\nLine 40-42: As relationship of AF productivity of A. flavus with its pathogenicity in humans has not been proved, this sentence should be separated to avoid misunderstandings.\nFigure 1I: Spots are overlapped. It necessary to show the AFB1 spot clearly.\nFigure 2H: It’s necessary to show the data of a week.\nFigure 2K: Rf values are different among the spots.\nLine 215: The website should be included in the Methods section.\nFigure 3A: The intensity of the H3 bands varies greatly between lanes, making it unsuitable as a control.\nLine 313-314: The statement \"the enrichment level in WT 313 was significantly higher (more than twice times)\" lacks numerical data, making it unclear.\nLine 363: The sentence \"Immunoprecipitation was used to enrich ~\" is unclear because Figure 5A presents a Western blot result, and it is not evident what was enriched.\nLine 364: Information about the outsourced company should be placed in the Methods section.\nLines 365-367: There is an inconsistency between Line 365, which states \"totally 63 proteins,\" and Line 367, which states \"64 proteins.\" Additionally, it would be helpful to provide a table listing these proteins including the five screened-out proteins.\nLine 370: What is the reasoning for stating that \"~was found to tightly interact with Arp9\"?\nLines 374-375: It is unclear how the dual-tagged A. flavus was constructed and verified. In addition, the company name should be included in the Methods section.\nLine 408: Does this statement align with the observation that bre2 gene expression was reduced in the arp9 knockout strain?\nLine 451-452: Explanation is need for the criteria for selecting target genes in ATAC-seq analysis. Were they screened based on GO terms?\nLine 456: As mentioned above, the chromatin accessibility index is not well defined. The Methods section only mentions that it was calculated using the 2-ΔΔCT method, which is typically used for qPCR quantification.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Point-by-point response to the reviewer comments\nReviewer #1 (Remarks to the Author):\nIn this study, the authors first explore the consequences of inactivating the COMPASS subunit\nBre2 in different biological processes of A. flavus. They show that Bre2 deletion (∆bre2) reduces\nthe expression of key transcription factors and thereby affects fungal morphogenesis and the level\nof the secondary metabolite AFB1. In agreement with these results, they show that Bre2 is\nrequired for virulence A. flavus to silkworm. They further show that A. flavus is localized in the\nnucleus and that deletion or mutations in the SPRY phenocopy the ∆bre2 deletion mutant.\nMapping the peaks of H3K4me3 in WT reveals that this mark is mainly located in promoters and\nexons of genes notably involved in metabolite synthesis and in several genes of the SNF2 family.\nInterestingly, they show that Bre2 and Arp9 are each required for the expression of the one by the\nother. In the second part of the manuscript, the authors show that the deletion of Arp9 produces\neffects similar to the deletion of Bre2 indicating that Arp9 collaborates with Bre2 to modulate\nfungal morphogensis and metabolite production. ATAC-seq experiments reveal that Arp9 acts by\nremodeling the promoter of target genes, including genes involved secondary metabolites related\nprocess.\n1. In the paper, it is improper to write (lane 298):\" H3K4me3 modification catalyzed by Bre2\";\nH3K4me3 is catalyzed by the Set1 complex\nAnswer: Thanks, we have revised the sentence according the professional advice.\nIt has been revised into “The above results indicated that H3K4me3 modification catalyzed by\nCOMPASS with Bre2 as the key subunit plays an indispensable role in the fungal growth and\ndevelopment, response to stress, and secondary metabolites production.”\nThe revision position was marked with purple background at Line 318-319 (Page 13).\n2. My major problem is with the title, the summary, and the role is assigned to Bre2. Actually, the\nobserved effects are not specifically due to Bre2, but rather to the absence of H3K4me3 and the\nreduction of H3K4me2. It is likely that the deletion of Sdc1 would produce the same effects as\nthat of Bre2. The more appropriate title would be \"H3K4me3 promotes CRF Arp9 expression to\nregulate the bio synthesis of secondary metabolite AFB1 and virulence in Aspergillus flavus”.\nAnswer: Thanks for the thoughtful advice.\nIn fact, according to our pre-experiment, the phenotype of sdc1 deletion strain is not the same as\nthose of Bre2. It reflected that each subunit in the complex has its own characteristics. And in\nview of the bre2 deletion strain was used as control strain in the ChIP-seq analysis, the title has\nbeen revised according to your professional advice to highlight the role of H3K4me3, and\nprovided below:\n“The COMPASS subunit Bre2 targets CRF Arp9 to regulate the bio-synthesis of secondary\nmetabolite AFB1 and virulence by H3K4me3 in Aspergillus flavus”\nIt was marked with purple background at Line 1 and 2 (Page 1).\n3. In figure 1K, what are the neighboring peaks of the ABF1 peak which also disappear in the\nΔbre2 mutant? Specify the abbreviation TLC\nAnswer: The neighboring peaks of the ABF1 peak are AFG2, AFG1, and the AFB2 next to the\nAFB1. According to your kind hint, the abbreviation of TLC have been specified in Line 142-143\n(Page 5) with purple background.\n4. H3K4me3 ChIP-seq reveal 2871 enriched genes and 723 depleted genes (WT compared to the\nΔbre2 strain). What are these 723 depleted genes?\nAnswer: Thanks, it is 723 down-regulated DAPs (differently accumulated peaks) in the WT\nsamples compared to Δbre2 samples. And we have check and revise it in Line 307 (Page 13)\naccording to kind reminder with purple background.\n5. Please provide the 63 proteins pulled down by Arp9.\nAnswer: Thank, we have provided the 63 proteins in an Attached Excel File titled with “63\nproteins pulled down by Arp9”. As shown in Line 387 (Page 16)\nReviewer #2 (Remarks to the Author):\nThe manuscript describes regulation of a target to control the biosynthesis of secondary\nmetabolites and virulence in Aspergillus. I appreciate the authors’ robust process used for genetic\nconfirmations. The study is well presented and organized.\n1. Grammatical clarity could be improved\nAnswer: Thanks, the grammars have been carefully improved according to your kind advice.\nIncluding Line 54, Line 66, Line 81, Line 92, Line 98, Line 99, Line 101, Line 112, Line 118,\nLine 131, Line 133, Line 153, Line 172, Line 173, Line 206, Line 207, Line 212, Line 217, Line\n240, Line 245, Line 248, Line 251, Line 252, Line 259, Line 269-273, Line 294, Line 305, Line\n306, Line 309, Line 313-315, Line 325, Line 330, Line 336, Line 342, Line 344, Line 345, Line\n361, Line 364, Line 365, Line 372, Line 376, Line 380, Line 393, Line 397, Line 416-417, Line\n430, Line 434-435, Line 488-489, Line 631, Line 640. They are all marked with purple\nbackground.\n2. Define COMPASS and ATAC\nAnswer: Thanks, COMPASS is defined as “Complex of Proteins Associated with Set1” in Line\n70-71 (Page 3); and ATAC is defined as “Assay for Transposase-Accessible Chromatin Using\nSequencing” in Line 456 (Page 21).\n3. Add reference to line 88 (i.e., Our group previously reported…)\nAnswer: Thanks, we have added related reference (Refernce No. 35) to the original line 88\nwhich is Line 96 (Page 4) in the revision version with purple underground.\nAs shown in the Reference Section (Line 1015-1016, Page 40):\n35. Liu, Y., Zhang, M., Xie, R., Zhang, F.&Zhuang, Z. J. F. i. M. The Methyltransferase AflSet1 Is\nInvolved in Fungal Morphogenesis, AFB1 Biosynthesis, and Virulence of Aspergillus flavus. 11,\n(2020).\n4. Line 125: italics to ‘bre2’\nAnswer: Thanks, we have revised it in Line 131 (Page 5) (purple background) according to your\nkind advise.\n5. Line 130: CM defined already?\nAnswer: Thanks, CM is defined as “6 g/L tryptone, 6 g/L yeast extract, 10 g/L glucose” in the\nLine 738-739 (Page 32) in the part of “Materials and methods”, and it is defined as “complete\nmedium” in Line 136 (Page 5) according to your kind advise. And they are marked with purple\nbackground.\n6. Line 136: TLC and HPLC defined? Check throughout if terms from methods are defined within\nthe Results for first use\nAnswer: Thanks, TLC and HPLC were defined as “thin layer chromatography” and “high\nperformance liquid chromatography” according to your kind advice in Line 142-143 (Page 5)\nwith purple background.\nAnd others are listed as below:\nCOMPASS (Complex of Proteins Associated with Set1) in Line 70-71 (Page 3)\nCM (complete medium) in Line 136 (Page 5)\nATAC-seq (Assay for Transposase-Accessible Chromatin Using Sequencing) in Line 456 (Page\n21)\n7. Figure 1 – suggest increasing the size of the plots A-L (excluding K) to improve readability and\ndecrease empty white space on the figures.\nMost figures appeal too small within the panels – sizes could be adjusted to optimize the figure\nsize and reduce white space.\nAnswer: Thanks, we have adjusted the figures according to your kind advice. As shown in Figure\n1 in Page 6; Figure 2 in Page 9; Figure 4 in Page 15; Figure 5 in Page 18;Figure 6 in Page 20;\nFigure 7 in Page 22; Figure 8 in Page 24.\nReviewer #3 (Remarks to the Author):\nIn the manuscript entitled \"The COMPASS subunit Bre2 targets CRF Arp9 to regulate the bio\nsynthesis of secondary 1 metabolite AFB1 and virulence in Aspergillus flavus\", the authors\nfocused on the gene of Bre2 subunit of the histone methyltransferase complex and showed that\nbre2 is involved in H3K4 trimethylation in A. flavus. Through ChIP-seq analysis, they found that\nthe gene of Arp9, which is involved in chromatin remodeling, is located downstream of bre2. The\nanalysis of bre2 and arp9 knockout strains demonstrated that these genes are essential for the\nmorphological development and aflatoxin production of A. flavus. Additionally, for bre2, they\nrevealed functionally important protein domains and amino acid residues. Using epigenetic\nanalysis of arp9 mutants, they investigated downstream genes affected by Arp9-mediated\nchromatin remodeling, constructed knockout strains for some of these genes, and examined their\nphenotypes.\nThe authors utilized a variety of approaches including seq analysis on the generated mutant\nstrains to elucidate the relationship between epigenetic modifications—initiated by histone H3K4\ntrimethylation—and aflatoxin production of A. flavus, the notorious mycotoxigenic fungus. The\nexperimental results clearly establish that arp9 functions downstream of bre2 and suggest that\nArp9 exerts a distal effect on the aflatoxin biosynthetic pathway and morphological development\npathway through SMAP and other factors.\n1. The manuscript does not fully elucidate the mechanism by which Bre2 and Arp9 regulates\naflatoxin production and morphological development. Although the ATAC-seq analysis identified\nseveral genes as potential components of the Arp9 regulatory pathway, only SMAP knockout\nstrains exhibited significant effects on aflatoxin production and morphology, leaving the\nrelationship between SMAP and these factors unclear. Simply to say, in Figure S6, the arrow\nwhich leads to the red boxes enclosing the gene clusters is unidentified. Due to this limitation, the\noverall framework linking histone H3K4 trimethylation initiated by Bre2 to aflatoxin production\nhas not been fully clarified. Further analyses are needed to bridge this missing link.\nAnswer: Thanks for your professional advice. To fully clarify the overall framework linking\nhistone H3K4 trimethylation initiated by Bre2 to aflatoxin production and fungal morphological\ndevelopment, four more targets of Arp9 (including G4B84_002788, G4B84_009741,\nG4B84_009537 and G4B84_008657) were deeply analyzed in Line 493-511 (Page 23) with\npurple background, and Fig. 8 (Page 24).\n2. The authors concluded that Bre2 is related to the colonization of peanuts and maize based on\nexperiments using the poorly growing deletion strain ⊿bre2. However, since poor growth is\nassociated with colonization, it is not possible to discuss a direct relationship between Bre2 and\ncolonization ability.\nThanks, to discuss a direct relationship between Bre2 and fungal colonization ability, the fungal\nvirulence related factors, including cp1, fbg1, lysM, mhp1, mpg1 and tox1, were monitored with\nqRT-PCR. The results showed that the absence of Bre2 significantly depressed the expression of\nthese virulence factors, as shown in Line 176-180 (Page 7) and Fig. S2A.\n3. Similarly, in the infection experiment with silkworms, the strong relationship between the poor\ngrowth of the deletion strain and its virulence makes it difficult to discuss the relationship between\nBre2 and virulence in silkworms.\nAnswer: Thanks, to deeper explore the virulence of Bre2 to animals, we constructed a Galleria\nmellonella larvae model. In the model, we compared the virulence of ⊿bre2 and WT stain to the\nimmune system of Galleria mellonella larvae, and the results showed the absence of Bre2 serious\naffects the response of cellular-immunity and humoral-immunity of the larvae compared with the\nWT group, which reflected that Bre2 play an important role in fungal virulence to animal hosts.\nThe revision part is in Line 181-201 (Page 7-8) with purple back ground and in Figure 2G-2N\n(Page 9).\n4. Additionally, it should be noticed that the relationship between fungal AF productivity and\npathogenicity to silkworms is unclear. In fact, the ⊿bre2 strain still maintains lethality, with more\nthan half of the silkworms dying within 6 days. As the same methods were used in experiments\nusing other poorly growing deletion strains, no conclusions can be drawn from this paper\nregarding the relationship between Bre2 and virulence in A. flavus.\nAnswer: Thanks for your professional advice. To explore the role of AFs in the virulence of A.\nflavus to hosts, the silkworms were injected with equal volume of 10% DMSO dissolved AFs\nextracted from Δbre2, Com-bre2 and WT strain each day. And as shown in Fig. S2, the results\nshowed that the survival rate of Δbre2 group was significantly highly than that of Com-bre2 and\nWT group, especially at 96 h and 120 h, which reflected that the concentration of AFs is one of\nthe critical factors in fungal virulence against hosts regulated by Bre2. The revision is in Line\n195-201 (Page 8) with purple background, and in Fig. S2B and S2C.\n5. Figure 4A: Appears to be a screenshot from IGV, but lacks sufficient explanation.\nAnswer: Thanks, more detail explanation was added as “reflecting the trimethylation modification\nlevel of H3K4 on this chromatin fragment” to help readers understand the intended information\naccording to your kind advice. And the revision is in the Line 331-332 (Page 14) with purple\nbackground.\n6. Figure 5B: The meaning of the mass spectrum is unclear.\nAnswer: Thanks, more explanation- “the samples were sent for mass spectrometry identification\nin Applied Protein Technology Biotechnology Co., Ltd., and the interaction protein peaks of Arp9\nwere obtained as shown in Fig.5B and in the Attached Excel File, with WT sample as control.” in\nLine 385-387 (Page 16) was added to make the mass spectrum clear according to your kind\nadvice.\n7. Figure 5C-E: The differences between lanes are not explained, and it is difficult to read what\nthe detected bands represent.\nAnswer: Thanks, the lanes have been clearly explained in Line 399-402 (Page 17) with purple\nbackground according to your kind advice.\nIt is “Western-blotting confirmed that in the Arp9-3HA strain, Strep was fused with RSC8\n(Fig. 5C, two transformants of Arp9-3HA/RSC8-Strep strain), Sth1 (Fig. 5D, four transformants\nof Arp9-3HA/Sth1-Strep strain), Arp7 (Fig. 5E, five transformants of Arp9-3HA-Arp7-Strep\nstrain) protein, respectively.”\n8. Figure 5F-H: Additional explanations for the bands are needed.\nAnswer: Thanks, explanations for the bands have been added in Line 402-414 (Page 17) with\npurple background according to your kind advice.\nIt is shown as “Co-immunoprecipitation experiments showed that, in the\nArp9-3HA/RSC8-Strep strain, Arp9-3HA could be pull-down, enriched and detected with anti-HA\nantibody, and anti-Strep antibody detection showed that RSC8 was interacted with Arp9 and\npull-down together with Arp9 by anti-HA antibody (Fig. 5F above). The lower panel of Fig. 5F\nshowed that, in the same fungal strain, RSC8-Strep could be pull-down, enriched and detected\nwith anti-RSC8 antibody, while anti-HA antibody detection showed Arp9 was interacted with\nRSC8 and pull-down together with RSC8 by anti-Strep antibody (Fig. 5F below). Using the same\nmethod, it was found that Arp9 also interacts directly with Arp7 of the SWI/SNF complex, as\nshown in Figure 5G. Interestingly, after Arp9 was immunoprecipitated by anti-HA magnetic\nbeads from Arp9-3HA/Sth1-Strep strain, in the final Co-IP analysis, Sth1-Strep could not be\ndetected in the experimental group (Fig. 5H, the above two panels). However, when\nimmunoprecipitation of Sth1 was carried out firstly with anti-strep magnetic beads, Arp9 could be\ndetected with HA antibody in the experimental group (Fig. 5H, the lower two panels). ”\n9. Figure 6A: The descriptions of the lanes and labels lack.\nAnswer: Thanks, the descriptions of the lanes and labels are provided in the legend of Figure 6A\nin Line 446-449 (Page 20) with purple background as below: “Compared to WT strain, the bre2\nORF and arp9 ORF could not be amplified from the Δbre2Δarp9 strain, while AP and BP\nfragments could amplified from Δbre2Δarp9 strain, but could not detected from WT. The DNA\nfragments amplified from WT is bigger than that form the Δbre2Δarp9 strain with nested primers\nBre2-pyrG-NF and Bre2-pyrG-NR.”, which confirmed that the Δbre2Δarp9 strain had been\nsuccessfully constructed.\n10. Figure 7: The text is too small to read. The labeling may be inappropriate (e.g., \"arp9\" should\nbe \"Δarp9\", isn’t it?). The same issue applies to Figure S3.\nAnswer: Thanks, the text of Figure 7 has been enlarged according to your nice advice.\nYes, arp9 in this figure should be \"Δarp9\", and we have added the corresponding\nexplanation/descriptions in the end of the legend in Line 485 (Page 22) with purple background.\n11. Figure 8A: The method for calculating the CAI index is unclear. Is there an easier metric to\nunderstand?\nAnswer: Thanks, we have introduced the method for calculating the CAI index according to the\noriginal reference (Gonzalez, R, and C Scazzocchio. “A rapid method for chromatin structure\nanalysis in the filamentous fungus Aspergillus nidulans.” Nucleic acids research vol. 25,19 (1997):\n3955-6. doi:10.1093/nar/25.19.3955) in the Methods section in Line 864-869 (Page 36) with\npurple background according to your professional advice.\nIt is shown as “Due to this characteristic, the region where DNaseⅠ is prone to digestion is the\narea where chromatin is open. qPCR analysis of the DNaseI-treated samples was performed to\nmeasure the relative abundance (accessibility) of target regions. The results were calculated by\n2-ΔΔCt and normalized with the housekeeping gene - tubulin, then the final result is the relative\nchromatin accessibility index (CAI). Primer sequences are listed in Table S4.”\n12. Line 40-42: As relationship of AF productivity of A. flavus with its pathogenicity in humans\nhas not been proved, this sentence should be separated to avoid misunderstandings.\nAnswer: We agree, and we have revised the sentence in Line 46-48 (Page 2) with purple\nbackground according to your professional advice.\nIt is revised into “Due to producing the most toxic secondary metabolite-aflatoxins (AFs), the\nnotorious Aspergillus flavus poses a serious threat to human health by food contamination”\n13. Figure 1I: Spots are overlapped. It necessary to show the AFB1 spot clearly.\nAnswer: Thanks, we have repeated the TLC in Figure 1I (Page 6) according to your nice advice.\n14. Figure 2H: It’s necessary to show the data of a week.\nAnswer: Thanks, to deeper explore the virulence of Bre2 to animals, we constructed a Galleria\nmellonella larvae model in Line 181-195 (Page 7-8) with purple background and the Figure\n2G-2N in Page 9. And the data of the experiment was collected for a week following your kind\nadvice.\n15. Figure 2K: Rf values are different among the spots.\nAnswer: Thanks, to deeper explore the virulence of Bre2 to animals, we constructed a Galleria\nmellonella larvae model. And the quality of Figure 2K, Figure 2N (Page 9) in the revision version\nwas improved according to your professional advice.\n16. Line 215: The website should be included in the Methods section.\nAnswer: Thanks, the website \"MEME (https://meme-suite.org/meme/tools/meme)” has been\nincluded in the Methods section in Line 751 (Page 32) according to your nice advice.\n17. Figure 3A: The intensity of the H3 bands varies greatly between lanes, making it unsuitable as\na control.\nAnswer: Thanks, the WB has been repeated according to your kind advice, and the intensity of\nthe H3 bands is stable in the new WB analysis in the Figure 3A (Page 12).\n18. Line 313-314: The statement \"the enrichment level in WT was significantly higher (more than\ntwice times)\" lacks numerical data, making it unclear.\nAnswer: Thanks, we have changed it into “the enrichment level of this chromatin fragment from\nWT is 21.1 times (P<0.05) of that from Δbre2 strain (Fig. 4A)” in Line 333-334 (Page 14) with\npurple background.\n19. Line 363: The sentence \"Immunoprecipitation was used to enrich ~\" is unclear because Figure\n5A presents a Western blot result, and it is not evident what was enriched.\nAnswer: Thanks, we have made our description more precisely in Line 382-384 (Page 16)\naccording to your kind advice.\nIt is revised into “Immunoprecipitation was used to enrich the proteins interacting with Arp9, and\nthe outcome of the enrichment (more HA tagged Arp9 was enriched in Arp9-3HA strain of IP\ngroup than the fungal strain in the Input group) was confirmed by western blotting as showed in\nFig. 5A”.\n20. Line 364: Information about the outsourced company should be placed in the Methods section.\nAnswer: Thanks, the detail information about the outsourced company “Applied Protein\nTechnology Biotechnology Co., Ltd. (Shanghai, China)” has been placed in the Methods section\nin Line 878-879 (Page 37) with purple background.\n21. Lines 365-367: There is an inconsistency between Line 365, which states \"totally 63 proteins,\"\nand Line 367, which states \"64 proteins.\" Additionally, it would be helpful to provide a table\nlisting these proteins including the five screened-out proteins.\nAnswer: Thanks, it should be 63 proteins, and after add Arp9 itself, it become 64, we have\nrevised it in Line 387 and 389 (Page 16) in purple background.\nAnd we have provided the list of these proteins in an Excel file titled “63 proteins pulled down\nby Arp9” as one of the attachment materials according to your professional advice.\n22. Line 370: What is the reasoning for stating that \"~was found to tightly interact with Arp9\"?\nAnswer：Thanks, we have revised the sentence into “RSC8 is a subunit in the SWI/SNF complex,\nhowever, it has not been reported whether there is a direct interaction between Arp9 and RSC8” in\nLine 390-392 (Page 16) in purple background.\n23. Lines 374-375: It is unclear how the dual-tagged A. flavus was constructed and verified.\nIn addition, the company name should be included in the Methods section.\nAnswer: Thanks, in this experiment, we constructed dual-tagged strains by homologous\nrecombination. Firstly, the 3HA-pyrG gene fragment was ligated behind the Arp9 gene through\nfusion PCR. Then, to obtain Apr9-3HA single-tag strain by transforming protoplasts of A.\nflavus-CA14 PTS with amplified Apr9-3HA-pyrG fragment. After the pyrG gene was deleted\nfrom Apr9-3HA single-tagged strain under the stress of 5-FOA, RSC8-Strep-pyrG fragment was\nintroduced into the protoplasts of the Apr9-3HA single-tagged strain by homologous\nrecombination, and the Apr9-3HA/RSC8-Strep dual-tagged strain was screened with the\nresuscitation medium without uracil and uridine. Apr9-3HA/Sth1-Strep dual-tagged strain and\nArp9-3HA/Arp7-Strep dual-tagged strain were prepared with the same method. Then, the\ndual-tagged A. flavus strains were verified by sequencing in Beijing Tsingke Biotech Co., Ltd.,\nand western-blotting. And the company name has been included in the Methods section in Line\n783 according to your kind advice. All related information was provided in Line 774-784 (Page\n33) in purple background.\n24. Line 408: Does this statement align with the observation that bre2 gene expression was\nreduced in the arp9 knockout strain?\nAnswer: Thanks, we have made corresponding revision in Line 441-442 (Page 19) according to\nyour nice reminder.\nIt has been revised into “All the above results suggested that Bre2 and Arp9 are deeply\ninvolved in hyphal growth and conidiation, indispensable in sclerotia formation and AFB1\nbio-synthesis, and Arp9 might be downstream of Bre2 in the Bre2 triggered epigenetic signaling\npathway, additionally, they might establish a positive-feedback in view of that Arp9 dramatically\npromotes the expression of bre2 (Fig 4C).”\n25. Lines 451-452: Explanation is need for the criteria for selecting target genes in ATAC-seq\nanalysis. Were they screened based on GO terms?\nAnswer: Yes, the criteria for selecting target genes in ATAC-seq analysis is the possibility of\nbeing a virulence factor in A. flavus according to GO terms, and we have added the explanation in\nLine 488-489 (Page 23).\nIt is revised into “To further reveal the signaling pathway mediated by Arp9, the targets\npotentially regulating fungal development, secondary metabolites bio-synthesis and pathogenicity\naccording to GO terms are screened out from the results of ATAC-seq analysis”\n26. Line 456: As mentioned above, the chromatin accessibility index is not well defined. The\nMethods section only mentions that it was calculated using the 2-ΔΔCT method, which is\ntypically used for qPCR quantification.\nAnswer: Thanks, we have defined the chromatin accessibility index according to the original\nreference (Gonzalez, R, and C Scazzocchio. “A rapid method for chromatin structure analysis in\nthe filamentous fungus Aspergillus nidulans.” Nucleic acids research vol. 25,19 (1997): 3955-6.\ndoi:10.1093/nar/25.19.3955) in the Methods section in Line 864-869 (Page 36) according to your\nprofessional advice.\nIt is changed into “Due to this characteristic, the region where DNaseⅠ is prone to digestion is the\narea where chromatin is open. qPCR analysis of the DNaseI-treated samples was performed to\nmeasure the relative abundance (accessibility) of target regions. The results were calculated by\n2-ΔΔCt and normalized with the housekeeping gene - tubulin, then the final result is the relative\nchromatin accessibility index (CAI). Primer sequences are listed in Table S4.”\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nAnswer: Thanks, we have carefully and cautiously revised our manuscript according to the\nprofessional advice provided by all reviewers."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors did not respond to my major criticism regarding the specific role that Bre2 could play in relation to another subunit of the Se1 complex, such as Scd1. To validate a number of the article's conclusions, it is essential to compare and show in the article the differences in phenotypes between bre2∆ and sdc1∆.\nThe new title is incomprehensible.", "reviewer_2": "The authors have addressed my points; I appreciate the grammatical clarity and figure revisions.", "reviewer_3": "The manuscript has been substantially improved by the addition of new experiments, revisions of the text, and significant improvements to the figures (including enlargement of labels). These efforts have strengthened the work overall. However, several major issues remain that should be addressed.\nMajor comments:\n1. Four additional genes were proposed as putative targets of Arp9, and corresponding knockout mutants were generated and analyzed. However, aside from Stromal membrane-associated protein (SMAP, G4B84_002937), none of the knockouts appear to have a substantial impact on aflatoxin production.\nThe role of SMAP in aflatoxin biosynthesis, fungal growth, and conidiation has not been previously reported and remains unclear. The mechanism underlying the observed effect of SMAP deletion is entirely unknown. In the Discussion, the authors only mention its involvement in stroma-supported erythropoiesis and erythropoietic activity in mice, which has no apparent relevance to Aspergillus flavus. The authors should therefore provide at minimum a plausible mechanistic hypothesis, and ideally, supporting validation experiments.\n2. In the Materials and Methods, the Data Analysis section initially states that “All data were expressed as mean ± standard deviation,” but later specifies that “Error bars represented the standard error of at least three repetitions.” Which is correct? Furthermore, the section indicates that statistical analysis was performed using Tukey’s test. However, Tukey’s test is not appropriate in all cases (e.g., for comparisons between two groups). It is also unclear whether proper attention was paid to the issue of multiple comparisons. Statistical methods should be described not only in Materials and Methods but also clarified in each relevant figure legend where statistical data are presented.\n3. It is not appropriate to directly compare the virulence of the WT strain and the Δbre2 strain when there is a significant difference in their growth. The authors argue that the Δbre2 strain exhibits reduced virulence because it has a weaker impact on the immune system of the larvae compared to the WT strain. However, this reduced impact may simply be due to the impaired growth of the Δbre2 strain, rather than a direct consequence of the Bre2 deletion. Additionally, the authors suggest that the survival rate of larvae injected with extracts from the WT and Δbre2 strains is related to the amount of AF contained in those extracts. To support this claim, it is necessary to conduct experiments using authentic AF at varying concentrations, and to quantify the AF content in the extracts before repeating the injection experiments.\n4. There remain numerous grammatical errors, especially in the newly added texts with purple highlight (e.g., incorrect verb tenses). The manuscript should undergo professional English editing.\nMinor comments:\n1. Title: The expression “by H3K4me3” is unclear. In addition, abbreviations such as “me3” should be avoided in the title.\n2. It would be appropriate to mention SMAP in the summary.\n3. Line 47: The primary cause of aspergillosis is A. fumigatus rather than A. flavus. The relevance of the present study to aspergillosis is uncertain, and therefore this description should be deleted.\n4. Line 334: In “the enrichment level of this chromatin fragment from WT is 21.1 times (P < 0.05) of that from Δbre2 strain,” it is unclear how the enrichment level was calculated, and thus the meaning of “21.1 times” is ambiguous. Moreover, without information on sample size and the specific statistical test applied, the interpretation of reported P value is uncertain.\n5. Line 350 (Figure 4): The label “H3K36me3” appears to be a mistake; it should likely read “H3K4me3.”\n6. Lines 385–386 and Figure 5B: The name of the company should be reported in the Methods section, while the description here should focus on clarifying what experiments were performed and how the data were analyzed. In particular, Fig. 5B raises questions: based on the MS peaks of Arp9-3HA and WT, it seems unlikely that Arp9-3HA contains peaks corresponding to 63 additional proteins compared with WT. Were these peaks derived from peptides following tryptic digestion, or from another protease?\n7. Line 390: The criteria by which the five proteins were “screened out” should be clearly defined.\n8. Lines 397–414: This section contains many grammatical errors and requires thorough editing. A concise explanation of the “Strep” tag is also needed.\n9. Figure 5 (F–H): Each panel requires a more detailed explanation. What detection methods were used—silver staining or Western blotting (and if Western blotting, with which antibody)? What proteins do the detected bands represent? In panel H, the band sizes are not indicated.\n10. Figure 6A: The meaning of “AP” and “BP” should be explained.\n11. Lines 473–476: From Fig. 7F, it does not appear that “metabolism, oxidative phosphorylation, and fatty acid degradation” are particularly heavily enriched. Rather, a variety of KEGG categories seem to be represented. What is the basis for concluding that these specific categories are heavily enriched?\n12. Line 478: The statement “especially oxidative and secondary metabolites related process” does not seem well supported by the information in Fig. 7. A more detailed explanation of results of the KEGG and GO term enrichment is required.\n13. Line 490: The basis for narrowing down 214 downregulated genes from the ATAC-seq results to nine candidate genes should be described. From which GO terms were they selected? Were there other genes belonging to the same GO terms that were excluded?\n14. Line 511: “Fig. 8M” likely refers to “Fig. 8O.” Additionally, the phrase “significantly decreased” should be reserved for statistically significant differences. Although the AFB1 spot appears weaker, the criteria for significance are not provided.\n15. Figures 8J and 8L: Each panel shows two plate images (top and bottom), but this is not described in the figure legends. Furthermore, in panel L, there should be no “empty position,” yet the legend mentions.\n16. Discussion: I suggest that the Discussion be made more concise, focusing on the insights derived from the findings rather than merely summarizing the results.\n17. Figure S7: This figure is important for understanding the manuscript, and I recommend that it be moved from the Supplementary section to the main figures (e.g., Figure 9). However, several points require clarification. Currently, it appears that all nine genes affected by Arp9, including SMAP, influence the aflatoxin gene cluster and other genes (red boxes). In reality, their effects are likely to differ. For example, gene G4B84_008657 (DAK) clearly does not affect aflatoxin production. It would be preferable to draw arrows only from the strongly influential genes, such as SMAP. In addition, the term “other biosynthetic gene clusters” is vague and may be better omitted."}, "author_response": "Point-by-point response to the reviewer comments\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors did not respond to my major criticism regarding the specific role that Bre2 could play\nin relation to another subunit of the Se1 complex, such as Scd1. To validate a number of the\narticle's conclusions, it is essential to compare and show in the article the differences in\nphenotypes between bre2∆ and sdc1∆.\nAnswer: Thanks for your professional advice. The ∆sdc1 strain is constructed and its phenotype is\ncompared with that of ∆bre2 according to your kind advice. The results showed that, different\nfrom Bre2, Sdc1 participates in fungal morphogenesis, but not involved in the regulation of AFB1\nbiosynthesis, and the related contents are shown in L633-639 (P 30) with blue background and in\nFig. S9.\nThe new title is incomprehensible.\nAnswer: Thanks, the title has been revised according to your kind advice. It is as below now：\n“The COMPASS subunit Bre2 targets CRF Arp9 to regulate the biosynthesis of secondary\nmetabolite AFB1 and virulence through H3K4 trimethylation in Aspergillus flavus”\nReviewer #2 (Remarks to the Author):\nThe authors have addressed my points; I appreciate the grammatical clarity and figure revisions.\nAnswer: We sincerely appreciate your professional advice and valuable reminders, which will\ngreatly facilitate the quality improvement of our manuscript.\nReviewer #3 (Remarks to the Author):\nThe manuscript has been substantially improved by the addition of new experiments, revisions of\nthe text, and significant improvements to the figures (including enlargement of labels). These\nefforts have strengthened the work overall. However, several major issues remain that should be\naddressed.\nAnswer: Thanks very much for your professional reminders and suggestions for the improvement\nof the quality of our manuscript.\nMajor comments:\n1. Four additional genes were proposed as putative targets of Arp9, and corresponding knockout\nmutants were generated and analyzed. However, aside from Stromal membrane-associated protein\n(SMAP, G4B84_002937), none of the knockouts appear to have a substantial impact on aflatoxin\nproduction.\nThe role of SMAP in aflatoxin biosynthesis, fungal growth, and conidiation has not been\npreviously reported and remains unclear. The mechanism underlying the observed effect of SMAP\ndeletion is entirely unknown. In the Discussion, the authors only mention its involvement in\nstroma-supported erythropoiesis and erythropoietic activity in mice, which has no apparent\nrelevance to Aspergillus flavus. The authors should therefore provide at minimum a plausible\nmechanistic hypothesis, and ideally, supporting validation experiments.\nAnswer: Thanks for your professional advice. We agree and have performed further RT-qPCR to\nexplore the regulatory mechanism of SMAP, with relevant details presented in L763-765 (P35) of\nDiscussion section with blue background and in Fig. S8.\n2. In the Materials and Methods, the Data Analysis section initially states that “All data were\nexpressed as mean ± standard deviation,” but later specifies that “Error bars represented the\nstandard error of at least three repetitions.” Which is correct?\nFurthermore, the section indicates that statistical analysis was performed using Tukey’s test.\nHowever, Tukey’s test is not appropriate in all cases (e.g., for comparisons between two groups).\nIt is also unclear whether proper attention was paid to the issue of multiple comparisons.\nStatistical methods should be described not only in Materials and Methods but also clarified in\neach relevant figure legend where statistical data are presented.\nAnswer: Thanks for this professional reminder. We have revised related contents and figures\naccording to your advice. As the revision in the Section of “Materials and methods” in L919-926\n(P40): All the results in this study were expressed as the mean value of triplicates, and the error\nbars represented the standard deviation of at least three repetitions. The statistical analysis was\nperformed using the software GraphPadPrism 10 (La Jolla, CA), and one-way ANOVA (one\nindependent variable involved) or two-way ANOVA (more than one independent variable\ninvolved) was used to compare the statistical significance by Tukey’s multiple comparisons test.\nIn the manuscript, three groups (WT strain, deletion strain and complementary strain) were\ncompared together, so Tukey’s test is applicable. The unpaired two-tailed t-test was used to\ncompare the statistical significance between two groups. The log-rank test was used in the\ncomparison of the difference in survival rates between animal injection groups in median survival\ntime. And statistical significance was determined as * (P <0.05), **(P<0.01) and ***(P<0.001).\nStatistical methods were briefly introduced in each relevant figure legend in L166-168 (P7),\nL246-248 (P11), L302-303 (P13), L370-371 (P16), L480-481 (P22), L565-567 (P27), and the\nlegends of Fig. S1, Fig. S2, Fig. S3, Fig. S6, Fig. S7, Fig. S8. and Fig. S9 with blue background.\n3. It is not appropriate to directly compare the virulence of the WT strain and the Δbre2 strain\nwhen there is a significant difference in their growth. The authors argue that the Δbre2 strain\nexhibits reduced virulence because it has a weaker impact on the immune system of the larvae\ncompared to the WT strain. However, this reduced impact may simply be due to the impaired\ngrowth of the Δbre2 strain, rather than a direct consequence of the Bre2 deletion. Additionally, the\nauthors suggest that the survival rate of larvae injected with extracts from the WT and Δbre2\nstrains is related to the amount of AF contained in those extracts. To support this claim, it is\nnecessary to conduct experiments using authentic AF at varying concentrations, and to quantify\nthe AF content in the extracts before repeating the injection experiments.\nAnswer: Thanks for this professional reminder, and we have performed fungal spore injection\nexperiment to explore if the reduced virulence of Δbre2 strain just due to the impaired growth of\nthe Δbre2 strain in Fig. S3, and the results demonstrated that fungal mycelium loading (reflecting\nthe growth of fungal hyphae) is not the key factor in the reduced virulence of Δbre2 strain as\nshowed in L205-213 (P8).\nThe pre-experiments for AF content quantification had been performed prior to the presented\nAF-injected larvae results, and these pre-experiment data for AF quantification are presented in\nFig. S2B and S2C and in L194-202 (P8) according to your professional advice.\n4. There remain numerous grammatical errors, especially in the newly added texts with purple\nhighlight (e.g., incorrect verb tenses). The manuscript should undergo professional English editing.\nAnswer: Thanks, the grammatical errors have been carefully corrected according to your kind\nreminder, and they are marked with blue background in Line 31, 35, 53, 80-82, 91, 101, 132, 139,\n164, 189, 204, 223, 226, 227, 237, 241, 244, 256，257，265, 274, 283, 288, 317, 323, 341, 365, 368,\n376, 387，394, 395, 449, 457, 465, 471, 525, 557, 571-573, 580, 593, 597, 600, 611，617, 625, 678,\n727, 729, 761, 763, 766, 871, 893, and 906.\nMinor comments:\n1. Title: The expression “by H3K4me3” is unclear. In addition, abbreviations such as “me3”\nshould be avoided in the title.\nAnswer: Thanks for your advice, “me3” has been fully expressed as “trimethylation”, and the title\nhas been revised into “The COMPASS subunit Bre2 targets CRF Arp9 to regulate the biosynthesis\nof secondary metabolite AFB1 and virulence through H3K4 trimethylation in Aspergillus\nflavus”\n2. It would be appropriate to mention SMAP in the summary.\nAnswer: Thanks for your professional advice, SMAP has been appropriately mentioned in the\nsummary in Line 38 (P2)with blue background, as “ATAC as “ATAC results revealed that mainly through\nSMAP, Arp9 deeply participates in fungal pathogenicity through regulating a serial secondary\nmetabolism, morphogenesis and virulence associated regulators by modulating the chromatin\nconformation.”\n3. Line 47: The primary cause of aspergillosis is A. fumigatus rather than A. flavus. The relevance\nof the present study to aspergillosis is uncertain, and therefore this description should be deleted.\nAnswer: Thanks, we have deleted this description, and the revised sentence is marked with blue\nbackground in Line 46-47 (P2).\n4. Line 334: In “the enrichment level of this chromatin fragment from WT is 21.1 times (P < 0.05)\nof that from Δbre2 strain,” it is unclear how the enrichment level was calculated, and thus the\nmeaning of “21.1 times” is ambiguous. Moreover, without information on sample size and the\nspecific statistical test applied, the interpretation of reported P value is uncertain.\nAnswer: Thanks for the professional reminder. It has been checked and corrected and revised into\n“the enrichment level of this chromatin fragment from WT is 21.1 times (FDR< 0.05) of that from\nΔbre2 strain.” And the enrichment levels were calculated with peak areas. It is showing in\nL347-349 (P15).\n5. Line 350 (Figure 4): The label “H3K36me3” appears to be a mistake; it should likely read\n“H3K4me3.”\nAnswer: Thanks, we have corrected it into “H3K4me3” in L365 (P16) according to your nice\nreminder.\n6. Lines 385–386 and Figure 5B: The name of the company should be reported in the Methods\nsection, while the description here should focus on clarifying what experiments were performed\nand how the data were analyzed. In particular, Fig. 5B raises questions: based on the MS peaks of\nArp9-3HA and WT, it seems unlikely that Arp9-3HA contains peaks corresponding to 63\nadditional proteins compared with WT. Were these peaks derived from peptides following tryptic\ndigestion, or from another protease?\nAnswer: Thanks for the kind advice. The name of the company has been given in L916-917 (P40)\nin the Methods section. And related experiments are further clarified in L398-402 (P17) according\nto your professional advice.\nTo the peaks, we have not shown this clear in the original manuscript. In fact, the MS peaks in\nfigure 5B do not indicate the number of proteins or peptides, but they reflect the separation\nresolution of the sample and the signal intensity of the peptides. The result of Figure 5B reflects\nthat the tested samples are characterized with high separation resolution and signal intensity. And\nthe protein samples in this study were digested by trypsin. We have revised and improved these in\nthis version (L399-401, P17).\n7. Line 390: The criteria by which the five proteins were “screened out” should be clearly\ndefined.\nAnswer: Through joint analysis with the STRING database (a tool for predicting functional\nprotein association networks; string-db.org), the Arp9 interacting protein, RSC8, was screened out\nfrom these 63 candidates based on the criterion that the enrichment levels in the Arp9-HA strain\nwere fourfold higher than those in the WT strain. And in the chromatin remodeling complex\nSWISNF, Arp9 is highly conserved and needs to form a heterodimer with another subunit of the\ncomplex, Arp7, to function. Hence, it is speculated that there is an interaction between Arp7 and\nArp9. Similarly, the core subunit of the chromatin remodeling complex SWISNF, Sth1, is central\nto the function of the complex, and a heterodimer of Arp9 and Arp7 can bind to the HAS domain\nof Sth1. Thus, we speculate that there is an interaction between Arp9 and core subunit Sth1, which\nplays a chromatin remodeling role. Therefore, Co-IP analysis was carried out to further verify\nwhether arp9 interacted with RSC8, Arp7 and Sth1. The related introduction is presented in\nL402-411 (P17).\n8. Lines 397–414: This section contains many grammatical errors and requires thorough editing. A\nconcise explanation of the “Strep” tag is also needed.\nAnswer: Thanks, the grammatical errors from L394 to L432 (P17-18) have been thoroughly\nrevised according to your professional advice. And the“Strep”is a tag composed of 8 amino acids\nthat can specifically bind to Streptavidin or its derivative StrepTactin. It is widely used in protein\npurification, immunoprecipitation, and protein-protein interaction research. The “Strep” tag is\nconcisely explained in L412-413 (P17-18) as: “Arp7 further labeled with eight-amino-acid Strep\ntag that can specifically bind to Streptavidin or its derivative StrepTactin”.\n9. Figure 5 (F–H): Each panel requires a more detailed explanation. What detection methods were\nused—silver staining or Western blotting (and if Western blotting, with which antibody)? What\nproteins do the detected bands represent? In panel H, the band sizes are not indicated.\nAnswer: Thanks for your kind advice. The detection methods were Western blotting, and each\npanel was given a detailed explanation. The band sizes in Panel H have been indicated, and it has\nbeen revised as follows in L438-447 (P19) according to your professional advice:\n(F) The upper panel western-blotting results were obtained from the Arp9-3HA/RSC8-Strep strain:\nafter IP with anti-HA magnetic beads, Arp9 (100 kDa) was detected using an anti-HA antibody,\nand RSC8 (100 kDa) using an anti-Strep antibody; the lower panel shows IP with anti-Strep beads,\nfollowed by western-blotting detection of RSC8 using an anti-Strep antibody and Arp9 using an\nanti-HA antibody. (G) The upper panel shows western-blotting results from the\nArp9-3HA/Arp7-Strep strain: after IP with anti-HA magnetic beads, Arp9 was detected using an\nanti-HA antibody, and Arp7 (70 kDa) using an anti-Strep antibody. The lower panel was IP with\nanti-Strep beads, followed by western-blotting detection of Arp7 via an anti-Strep antibody, and\nArp9 via an anti-HA antibody. (H) The upper panel shows western-blotting results from the\nArp9-3HA/Sth1-Strep strain: After IP with anti-HA magnetic beads, Arp9 was detected using an\nanti-HA antibody and Sth1 (180 kDa) using an anti-Strep antibody. The lower panel presents IP\nwith anti-Strep beads, followed by western-blotting detection of Sth1 via an anti-Strep antibody,\nand Arp9 via an anti-HA antibody. The band sizes in panel H are indicated according to your kind\nreminder.\n10. Figure 6A: The meaning of “AP” and “BP” should be explained.\nAnswer: Thanks, we have revised it according to your kind advice, as following in L473-475\n(P21-22): The \"AP\" fragment, which overlaps the 5’ flanking sequence and pyrG, was amplified\nwith the validation primers Arp9-AP-F and Arp9-AP-R. The \"BP\" fragment, overlapping pyrG\nand the 3’ flanking sequence, was amplified by primers Arp9-BP-F and Arp9-BP-R.\n11. Lines 473–476: From Fig. 7F, it does not appear that “metabolism, oxidative phosphorylation,\nand fatty acid degradation” are particularly heavily enriched. Rather, a variety of KEGG\ncategories seem to be represented. What is the basis for concluding that these specific categories\nare heavily enriched?\nAnswer: Thanks to this professional advice. From the Fig.7F, according to the “percent of genes”,\nthe genes in metabolism related KEGG categories (the purple color ones) accounts for 56%,\nso they are heavily involved in metabolism process. Related revision, according to your kind advice,\nis given in Line L501 (P22).\n12. Line 478: The statement “especially oxidative and secondary metabolites related process” does\nnot seem well supported by the information in Fig. 7. A more detailed explanation of results of the\nKEGG and GO term enrichment is required.\nAnswer: Thanks for the professional advice. The related content is revised as “Further KEGG\npathways analysis showed that these down-regulated DAPs genes of ΔArp9 were heavily involved\n(accounts for 56% genes in KEGG, and 73% GO annotated genes) in the process of metabolism\n(including Lipid metabolism, Carbohydrate metabolism, Amino acid metabolism, Nucleotide\nmetabolism, Metabolism of terpenoids and polyketides, and Biosynthesis of other secondary\nmetabolites.)”. It is shown in L500-504 (P22-23) according to your kind advice.\n13. Line 490: The basis for narrowing down 214 downregulated genes from the ATAC-seq results\nto nine candidate genes should be described. From which GO terms were they selected? Were\nthere other genes belonging to the same GO terms that were excluded?\nAnswer: 214 downregulated genes were first filtered by the molecular-function of the GO\nenrichment, by which those related to fundamental molecular-level activities such as catalysis or\nbinding, as well as those involved in the ordered biochemical or physiological processes at the cell,\ntissue or organism level listed under biological process were screened out (P < 0.05). Finally, the\nnine candidate genes were figured out in the second round of selection by KEGG pathway\nannotation for genes implicated in growth/development, reproduction, virulence, autophagy,\nproliferation, cell wall integrity, drug resistance, and immunity which are the key areas concerned\nby our lab.\n14. Line 511: “Fig. 8M” likely refers to “Fig. 8O.” Additionally, the phrase “significantly\ndecreased” should be reserved for statistically significant differences. Although the AFB1 spot\nappears weaker, the criteria for significance are not provided.\nAnswer: Thanks for your kind reminder, it has been corrected into “Fig. 8O” in L539 (P25). And\nsignificant analysis among the AFB1 spots has been provided in Fig. 8P (P26) according to your\nprofessional advice.\n15. Figures 8J and 8L: Each panel shows two plate images (top and bottom), but this is not\ndescribed in the figure legends. Furthermore, in panel L, there should be no “empty position,” yet\nthe legend mentions.\nAnswer: The top, middle and bottom plates are described in the figure legends according to your\nkind reminder as following from L555-557 and L559-560 (P27): (J) The spores of\nΔG4B84_010461, ΔG4B84_002937 and ΔG4B84_005528 were incubated onto CM media under\n37℃ for 7 d (above panels); Middle panels were sprayed with 75% ethanol, and the lower panels\nwere the enlarged pictures of the middle panels under a dissecting microscope. (L) The spores of\nΔG4B84_002788, ΔG4B84_009741, ΔG4B84_009537 and ΔG4B84_008657 were incubated onto\nCM media under 37℃ for 7 d (above panels); Middle panels were sprayed with 75% ethanol, and\nthe lower panels were the enlarged pictures of the middle panels.\nAnd thanks, we have removed the unnecessary content from the legend of panel L according\nto your kind remainder as shown in L559 (P27).\n16. Discussion: I suggest that the Discussion be made more concise, focusing on the insights\nderived from the findings rather than merely summarizing the results.\nAnswer: Thanks for the professional advice, we have made the Discussion more concise and\nfocusing on the insights in L595-596 (P29), L633-639 (P30), L644-646 (P30-31), L658-659\n(P31), L663-665 (P31), L686-688 (P32), L714-716 (P33), L763-765 (P35).\n17. Figure S7: This figure is important for understanding the manuscript, and I recommend that it\nbe moved from the Supplementary section to the main figures (e.g., Figure 9). However, several\npoints require clarification. Currently, it appears that all nine genes affected by Arp9, including\nSMAP, influence the aflatoxin gene cluster and other genes (red boxes). In reality, their effects are\nlikely to differ. For example, gene G4B84_008657 (DAK) clearly does not affect aflatoxin\nproduction. It would be preferable to draw arrows only from the strongly influential genes, such as\nSMAP. In addition, the term “other biosynthetic gene clusters” is vague and may be better omitted.\nAnswer: We agree with your opinion. Figure S7 has been moved from the Supplementary section\nto the main figures, as the Fig. 9 in Page 28. Arrow is drawn from the strongly influential gene,\nSMAP. The term “other biosynthetic gene clusters” has been deleted according to your kind\nadvice."}, {"round": 3, "reviewer_comments": {"reviewer_3": "The authors’ revisions have addressed most of the previous concerns, and the manuscript has been further improved. Only two minor points still require attention.\n1. Statistical Analysis\nAs one-way ANOVA has been applied to panels G and H in Fig. 1, it appears that no analyses using two-way ANOVA remain in the manuscript. If this is the case, the description of two-way ANOVA should be removed from the Methods section.\n2. Clarity of Expression\nOn page 25, line 530, the phrase “which are the key areas concerned by our lab” is unclear. This phrase should either be removed or replaced with a more objective expression."}, "author_response": "Point-by-point response to the reviewer comments\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors have now answered my main concern.\nFor the title, I suggest to remove \"Through H3K4 trimethylation\". The title would be:\nThe COMPASS subunit Bre2 targets CRF Arp9 to regulate the biosynthesis of secondary\nmetabolite AFB1 and virulence in Aspergillus flavus.\nAnswer: We agree, and we have revised the title (The COMPASS subunit Bre2 targets CRF Arp9\nto regulate the biosynthesis of secondary metabolite AFB1 and virulence in Aspergillus flavus)\naccordingly to your kind advice with purple background (See Line 1-2, Page 1).\nReviewer #3 (Remarks to the Author):\nThe revised manuscript has been substantially improved through the authors’ careful\nconsideration of the review comments and the addition of new experiments. In particular,\nthe RT-qPCR analyses conducted on the SMAP (G4B84_002937) deletion strains have\nhelped to clarify the pathway linking Bre2 to aflatoxin production.\nHowever, several issues still require further attention.\nMinor comments\n1. Statistical analysis\nIn response to the question regarding statistical analysis (Reviewer #3, Major Comment 2),\nthe authors answered:\n“All the results in this study were expressed as the mean value of triplicates, and the error\nbars represented the standard deviation of at least three repetitions. The statistical\nanalysis was performed using the software GraphPadPrism 10 (La Jolla, CA), and\none-way ANOVA (one independent variable involved) or two-way ANOVA (more than one\nindependent variable involved) was used to compare the statistical significance by\nTukey’s multiple comparisons test. In the manuscript, three groups (WT strain, deletion\nstrain and complementary strain) were compared together, so Tukey’s test is applicable.”\nTwo-way ANOVA is a method specifically designed for analyses involving two\nindependent variables, not “more than one independent variable.” This description should\ntherefore be corrected. In addition, ANOVA itself is not a method to “compare statistical\nsignificance”; rather, ANOVA assesses overall differences among groups, followed by\npost hoc tests such as Tukey’s test to determine which specific groups differ.\nAnswer: Thanks for the professional reminder, we have revised “more than one independent\nvariable” into “two independent variables” in Line 934 Page 41. And the relationship between\nANOVA and Tukey’s test was made clearer in Line 934-937 Page 41 according to your nice\nadvice, as below shown in purple background:\n“The statistical analysis was performed using the software GraphPadPrism 10 (La Jolla, CA), and\none-way ANOVA for one independent variable involved or two-way ANOVA for two independent\nvariables involved was used to assess overall differences among groups (Determining whether\nsignificant differences exist among all intergroup differences), and the statistical significance\nbetween specific groups was further compared by Tukey’s multiple comparisons test.”\nFurthermore, with respect to the statistical analyses, the legend of Fig. 1 states:\n“One-way ANOVA was used to compare the statistical significance by Tukey’s multiple\ncomparisons test for Panels B–D, F, J, and L. Two-way ANOVA was used to compare the\nstatistical significance by Tukey’s multiple comparisons test for Panels G and H.”\nIt is unclear how two-way ANOVA was applied to the results shown in Panels G and H.\nDid the authors treat time points (48 h and 72 h) as independent variables? If so, this\napproach may not be appropriate in this context. Instead, it would seem more suitable to\nperform one-way ANOVA separately at each time point (48 h and 72 h), using strain (WT,\ndeletion strain, complementary strain) as the independent variable, followed by Tukey’s\npost hoc test.\nAnswer: Thanks, the treat-time-points (48 h and 72 h) are not the independent variables, they\nwere analyzed with one-way ANOVA separately at each time point (48 h and 72 h). The related\ncontent has been revised as “One-way ANOVA was used to assess the overall statistical\nsignificances among groups, and Tukey’s multiple comparisons test was further performed for\npost-hoc analysis for Panel B-D, F-H, J and L.” (See Line 166-168, Page 7) in purple ground\naccording to your professional reminder.\nFor all figures, the results of Tukey’s test would be more appropriately presented using a\ncompact letter display (alphabetical notation) rather than asterisks, since the use of\nasterisks can give the impression that multiple pairwise comparisons were performed\nrepeatedly.\nAnswer: Thanks, the asterisks in the results of Tukey’s test in all figures have been replaced with\nlowercase letters according to your kind advice. (See Figure 1, Figure 2, Figure 3, Figure 4,\nFigure 6, Figure 8, Figure S1, Figure S2, Figure S3, Figure S6, Figure S7, Figure S8, Figure\nS9).\n2. Figure 5B\nIn response to the question regarding Figure 5B (Reviewer #3, Minor Comment 6), the\nauthors answered:\n“To the peaks, we have not shown this clear in the original manuscript. In fact, the MS\npeaks in figure 5B do not indicate the number of proteins or peptides, but they reflect the\nseparation resolution of the sample and the signal intensity of the peptides. The result of\nFigure 5B reflects that the tested samples are characterized with high separation\nresolution and signal intensity. And the protein samples in this study were digested by\ntrypsin.”\nThe explanation referring to “separation resolution and signal intensity” does not appear to\nbe sufficiently informative. Based on the presentation of Figure 5B—where the x-axis and\ny-axis appear to represent retention time and relative abundance, respectively, and the\nlabel “Base Peak FTMS + p NSI Full MS” is shown—the figure appears to represent a ion\nchromatogram obtained from LC–MS analysis using a full MS scan in positive ion mode\nover an m/z range of 300–1800.\nTaken together, it seems that Figure 5B shows extracted ion chromatograms derived from\nLC/MS analysis of peptides obtained by trypsin digestion following immunoprecipitation of\nthe target proteins. Is this interpretation correct? Please provide a precise and careful\ndescription of the experimental procedure in the figure legend.\nAnswer: Thanks for this professional advice. We have revised it into “The target proteins\nobtained from immunoprecipitation were digested with trypsin, and the peptide segments were\nanalyzed using LC-MS/MS (nanoLC QE) after enzymatic hydrolysis. The data was collected in\npositive ion mode, and the ion chromatogram (the peak chromatogram) was obtained by full MS\nscanning (m/z range 300-1800).” (See Line 439-441, Page 19) according to your kind advice.\n3. Figure 7F\nRegarding Figure 7F (Reviewer #3, Minor Comment 11), the authors answered:\n“From the Fig.7F, according to the ‘percent of genes’, the genes in metabolism related\nKEGG categories (the purple color ones) accounts for 56%, so they are heavily involved\nin metabolism process. Related revision, according to your kind advice, is given in Line\nL501 (P22).”\nIt is unclear how the value of 56% was calculated. This number appears to represent the\nsimple sum of genes classified under metabolism-related KEGG categories (assuming\nthat the numbers shown on the bars represent gene counts). If so, expressing this value\nas a percentage may be misleading. Please clearly explain how the value of 56% was\nderived and describe the basis for this percentage calculation in the main text.\nAnswer：Thanks, we recalculated the percentage according to your nice advice. The number of\ngenes in the metabolism related KEGG pathway divided by the total number of KEGG annotated\ngenes is 60%, so the number of genes in the metabolic related KEGG pathway accounts for 60%\nof the total KEGG annotated genes. It is described as “(accounts for 60% genes in KEGG (the\nnumber of genes in the metabolism related KEGG pathway divided by the total\nnumber of KEGG annotated genes)” (See Line 506-508, Page 22).\n4. Selection of nine candidate genes\nIn response to the question regarding the criteria used to narrow down 214 downregulated\ngenes to nine candidate genes (Reviewer #3, Minor Comment 13), the authors answered:\n“214 downregulated genes were first filtered by the molecular-function of the GO\nenrichment, by which those related to fundamental molecular-level activities such as\ncatalysis or binding, as well as those involved in the ordered biochemical or physiological\nprocesses at the cell, tissue or organism level listed under biological process were\nscreened out (P < 0.05). Finally, the nine candidate genes were figured out in the second\nround of selection by KEGG pathway annotation for genes implicated in\ngrowth/development, reproduction, virulence, autophagy, proliferation, cell wall integrity,\ndrug resistance, and immunity which are the key areas concerned by our lab.”\nIt is recommended that this explanation be incorporated into the main text (for example,\naround line 521) to ensure transparency and clarity regarding the gene selection process.\nAnswer: Thanks, we have incorporated the related content into the main text in Line 523-530\nPage 25 according to your kind advice as below in purple background:\n“To further reveal the signaling pathway mediated by Arp9, 214 downregulated genes were first\nfiltered according to the molecular-function of the GO enrichment, by which those related to\nfundamental molecular-level activities such as catalysis or binding, as well as those involved in\nthe ordered biochemical or physiological processes at the cell, tissue or organism level listed\nunder biological process were screened out (P < 0.05). Finally, nine candidate genes were figured\nout in the second round of selection by KEGG pathway annotation for genes implicated in\ngrowth/development, reproduction, virulence, autophagy, proliferation, cell wall integrity, drug\nresistance, and immunity which are the key areas concerned by our lab”"}]

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