SntB triggers the antioxidant pathways to regulate development and aflatoxin biosynthesis in Aspergillus flavus

Reviewer #1 (Public Review):

The study identifies the epigenetic reader SntB as a crucial transcriptional regulator of growth, development, and secondary metabolite synthesis in Aspergillus flavus, although the precise molecular mechanisms remain elusive. Using homologous recombination, researchers constructed sntB gene deletion (ΔsntB), complementary (Com-sntB), and HA tag-fused sntB (sntB-HA) strains. Results indicated that deletion of the sntB gene impaired mycelial growth, conidial production, sclerotia formation, aflatoxin synthesis, and host colonization compared to the wild type (WT). The defects in the ΔsntB strain were reversible in the Com-sntB strain.

Further experiments involving ChIP-seq and RNA-seq analyses of sntB-HA and WT, as well as ΔsntB and WT strains, highlighted SntB's significant role in the oxidative stress response. Analysis of the catalase-encoding catC gene, which was upregulated in the ΔsntB strain, and a secretory lipase gene, which was downregulated, underpinned the functional disruptions observed. Under oxidative stress induced by menadione sodium bisulfite (MSB), the deletion of sntB reduced catC expression significantly. Additionally, deleting the catC gene curtailed mycelial growth, conidial production, and sclerotia formation, but elevated reactive oxygen species (ROS) levels and aflatoxin production. The ΔcatC strain also showed reduced susceptibility to MSB and decreased aflatoxin production compared to the WT.

This study outlines a pathway by which SntB regulates fungal morphogenesis, mycotoxin synthesis, and virulence through a sequence of H3K36me3 modification to peroxisomes and lipid hydrolysis, impacting fungal virulence and mycotoxin biosynthesis.

The authors have achieved majority of their aims at the beginning of the study, finding target genes, which led to catC mediated regulation of development, growth and aflatoxin metabolism. Overall most parts of the study is solid and clear.

Reviewer #2 (Public Review):

Summary:

This work is of great significance in revealing the regulatory mechanisms of pathogenic fungi in toxin production, pathogenicity, and in its prevention and pollution control. Overall, this is generally an excellent manuscript.

Strengths:

The data in this manuscript is robust and the experiments conducted are appropriate.

Weaknesses:

(1) The authors found that SntB played key roles in oxidative stress response of A. flavus by ChIP-seq and RNA sequencing. To confirm the role of SntB in oxidative stress, authors have better to measure the ROS levels in the ΔsntB and WT strains, besides the ΔcatC strain.
(2) Why the authors only studied the function of catC among the 7 genes related to oxidative response listed in Table S14.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

The manuscript by Wu et al. explores the role of the histone reader protein SntB in Aspergillus flavus, claiming it to be a key regulator of development and aflatoxin biosynthesis. While the study incorporates various techniques, including gene deletion, ChIP-seq, and RNA-seq, several concerns and omissions in the paper raise questions about the validity and completeness of the presented findings.

(1) Omissions of Prior Work:

The authors fail to acknowledge and integrate prior research by Pfannenstiel et al. (2018) on the sntB gene in A. flavus, which covered phenotypic changes, RNA-seq data, and histone modifications. This omission raises concerns about the transparency and completeness of the current study.

The absence of reference to studies by Karahoda et al. (2022, 2023) revealing SntB's involvement in the KERS complex in A. flavus and A. nidulans is a major oversight. This raises questions about the specificity of SntB's regulatory functions, as it may be part of a larger complex. The authors should clarify why these studies were omitted and how they ensure that SntB alone, and not the entire KERS complex, is responsible for the observed effects.

We very appreciate reviewer’s professional question. As reviewer mentioned, Pfannenstiel et al. (2018) reported the functions of sntB gene covered secondary metabolism, development and global histone modifications in A. flavus and we also cited this paper (please see reference 20). In their study, the functions of sntB gene were analyzed by both ΔsntB and overexpression sntB genetic mutants. SntB deletion impaired several developmental processes, such as sclerotia formation and heterokaryon compatibility, secondary metabolite synthesis, and the ability to colonize host seeds, which were consistent with our results (Figure 1 and 2). Unlike, a complementation strain was constructed in our study which further clarified and confirmed the function of sntB gene. What’s more, our main purpose is to find the downstream regulatory mechanism of SNTB, which was reported to be a transcription factor, not only as an important epigenetic reader. Please see lane 452-457 and lane 486-500.

For the function of KERS complex in A. nidulans (Karahoda et al., 2022), we had cited the papers, please see reference 29. For the report about the function of KERS complex in A. flavus (Karahoda et al., 2023), this paper was published recently. We are sorry for the omissions of this work. In our revised manuscript, we have cited this paper and compared with our work. Please see lane 97-98 and reference 30. Based solely on our experiments, we cannot confirm whether it is acting alone or in conjunction with others, what we can confirm is that SntB plays a key role in the process. And we will conduct related research in the future.

(2) Transparency and Accessibility of Data:

The lack of accessibility and visualization tools for ChIP-seq and RNA-seq data poses a challenge for independent verification and in-depth analysis. The authors should address this issue by providing more accessible data or explaining the limitations of data availability. A critical component missing from the paper is a detailed presentation of ChIP-seq data, specifically demonstrating SntB binding patterns on key promoters. This omission weakens the link between SntB and the mentioned regulatory genes. The authors should include these crucial data visualizations to strengthen their claims.

To review GEO accession GSE247683, you can go to https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE247683, and enter the token “ipilouscnruprsl” into the box. And after our paper being published, the data will be released. For the SntB binding patterns on key promoters, we have added in the Figure 4, please see Figure 4D, 4E, 5F, 5G, and table S9.

(3) SntB Binding Sites and Consensus Sequence:

The study mentions several genes upregulated in the sntB mutant without demonstrating SntB binding sites on their promoters. A detailed analysis of SntB binding maps is necessary to establish a direct link between SntB and these regulatory genes.

Thanks for your suggestion. We have added the binding maps of SntB, please see Figure 5F, 5G; lane 362-364.

(4) Mechanistic Insight into Peroxisome Biogenesis:

If SntB indeed regulates peroxisome biogenesis, the absence of markers for peroxisomes and the localization of peroxisomes in the sntB mutant vs. WT strains is a significant gap. Providing evidence for peroxisome regulation is crucial for understanding the proposed mechanism and validating the study's claims.

Thanks for your suggestion. Catalase is ubiquitously present in aerobic organisms and plays a crucial role in mitigating oxidative stress through the scavenging of reactive oxygen species (ROS). So, we detected the ROS level in sntB mutant and WT strain, as well as ∆catC strain (Figure 6H).

In summary, while the manuscript presents intriguing findings regarding SntB's role in A. flavus, the omissions of prior work, lack of transparency in data accessibility, and insufficient mechanistic insights call for revisions and additional experimental evidence to strengthen the validity and impact of the study. Addressing these concerns will enhance the manuscript's contribution to the field.

Thanks. We have revised our manuscript depending on the valuable comments provided above.

Additionally, the way the English language is used could be improved.

Thanks. We have asked a native English-writing assistant to proof read the paper and revised the grammar errors and typos and improve the readability and quality of the manuscript.

Reviewer #2 (Public Review):

Summary:

This work is of great significance in revealing the regulatory mechanisms of pathogenic fungi in toxin production, pathogenicity, and in its prevention and pollution control. Overall, this is generally an excellent manuscript.

Strengths:

The data in this manuscript is robust and the experiments conducted are appropriate.

Weaknesses:

(1) The authors found that SntB played key roles in the oxidative stress response of A. flavus by ChIP-seq and RNA sequencing. To confirm the role of SntB in oxidative stress, the authors have to better measure the ROS levels in the ΔsntB and WT strains, besides the ΔcatC strain.

Thanks for your suggestion. We have supplemented the relevant experiments and the results were shown in Figure 6G and lane 185-192 and 395-398.

(2) Why did the authors only study the function of catC among the 7 genes related to an oxidative response listed in Table S14?

The function of some genes in Table S15 (Table S14 in old version of our manuscript) had been studied, such as cat1 [1]. In this study, we just choose catC for further validation, which was the most up-regulated gene in ΔsntB strain. The others may also have important roles in SntB triggered antioxidant pathways to regulate development and aflatoxin biosynthesis in A. flavus. We will focus on this in the following work.

(1) Zhu Z., Yang M., Bai Y., Ge F., Wang S. Antioxidant-related catalase CTA1 regulates development, aflatoxin biosynthesis, and virulence in pathogenic fungus Aspergillus flavus [J]. Environ Microbiol, 2020, 22(7): 2792-2810.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

Line 52: Change "shad light" to "shed light"

Thanks. We have revised. Please see lane 50.

Line 62: Change "has" to "have" to match the plural noun "aflatoxins."

Original: "Aflatoxins produced by A. flavus has strong toxicity..."

Suggested: "Aflatoxins produced by A. flavus have strong toxicity..."

Thanks. We have revised it. Please see lane 62.

Line 79: Consider rephrasing for clarity.

Original: "...which may result in the modulation of the expression of genes involved in toxin production [15-17]."

Thanks. We have revised. Please see lane 77-80.

Line 105: Add a comma after "host strain."

Original: "A. flavus Δku70 ΔpyrG was used as a host strain for genetic manipulations."

Suggested: "A. flavus Δku70 ΔpyrG was used as a host strain, for genetic manipulations."

Thanks. We have revised it. Please see lane 107.

Line 113, Table 1: Remove the extra "r" in "from" in the Source column.

Original: "Kindly presented form Prof. Chang[1]"

Suggested: "Kindly presented from Prof. Chang[1]"

Thanks. We have revised it. Please see Table 1.

Line 140: Typo - Change "reaches" to "reach."

Original: "when silkworm larva reaches about 1 g in weight."

Suggested: "when silkworm larvae reach about 1 g in weight."

Thanks. We have revised it. Please see lane 141.

Line 158: Typo - Change "pervious" to "previous."

Original: "Data processing was according pervious study [39]."

Suggested: "Data processing was according to a previous study [39]."

Thanks. We have revised it. Please see lane 150.

Line 138 The animal invasion assay using silkworms was conducted according to a previous study.

Change "according" to "conducted according to" for clarity.

Thanks. We have revised it. Please see lane 139.

Line 148 Was carried out by APPLIED PROTEIN TECHNOLOGY, Shanghai (www. aptbiotech.com).

Change "TECHNOLOY" corrected to "TECHNOLOGY."

Thanks. We have revised it. Please see lane 149.

Line 148 Data processing was conducted according to a previous study [39].

Change "according to" to "conducted according to" for clarity.

Thanks. We have revised it. Please see lane 139.

Line 429 Schizzosaccharomyces pombe, Correct the spelling to "Schizosaccharomyces pombe [55]."

Thanks. We have revised it. Please see lane 448.

Reviewer #2 (Recommendations For The Authors):

(1) The resolution of the words written in Figures 3 and 4 is not clear (or high) enough.

Thanks. We have revised them. Please see Figures 3 and 4.

(2) Which kind of protein marker (protein ladder) was used in Figure 4A, you should mark out the size of the related protein.

Thanks. We have revised. Please see Figure 4A and lane 332-333.

(3) Latin names do not necessarily need to be written in full when they are not the first time used in the text.

Thanks. We have revised them throughout the manuscript.

(4) The complementary strain of sntB was labeled as sntB-C in Figure 2B, while in other figures was Com-sntB. You should correct all related problems.

Thanks. We have revised it. Please see Figure 2B.

(5) What is the meaning of "1" in Table 1?

Thanks. The meaning of "1" in Table 1 was a citation. We have revised. Please see Table 1.