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 the 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 are solid and clear.

Reviewer #2 (Public Review):

Summary:

Wu et al. explores the role of the histone reader protein SntB in Aspergillus flavus. They not only studied its function related to the growth, development, and secondary metabolite through gene knockout and complement, but also explored the underlying potential mechanisms by RNA-seq and ChIP-seq. The response of oxidative stress in ΔsntB strain and ΔcatC strain were further analyzed. Their study revealed a potential machinery that SntB regulated fungal morphogenesis, mycotoxin anabolism, and fungal virulence through the axle of from epigenetic modification to fungal virulence and mycotoxin bio-synthesis via SntB, i.e. H3K36me3 modification-SntB-Peroxisomes-Lipid hydrolysis-fungal virulence and mycotoxin bio-synthesis. 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.

Strengths:

One of the main advantages of this study is that the author constructed HA fused strains for ChIP seq analysis, rather than using antibodies related to epigenetic modifications. Nancy et al. reported the functions of sntB as a histone methylation regulator, but in addition to being an epigenetic regulator, there are also reports that it has transcriptional regulatory activity. Through integration analysis with RNA-seq data, it was found that SntB played key roles in oxidative stress response of A. flavus. This study can increase our understanding of more functions of the SntB in A. flavus.

Weaknesses:

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 previous reviews.

Reviewer #1 (Recommendations For The Authors):

Inclusion of other catalase, peroxidase or superoxide dismutase gene promoters (with ChiP-seq screen shots) and whether they contain sntB binding sites is important to provide other potential downstream pathways controlling oxidative stress mediated regulation of development and aflatoxin metabolism. This can be presented as supplementary material.

or

Some more examples of ChiP-seq peaks in the promoters of nsdC, nsdD, sclR, steA, wetA, veA, fluG, sod2, catA, catC would strengthen the paper for the reliability of the ChiP-seq data. Currently, visualisation of the ChIP-seq data is only limited to catC gene promoter, where background ChIP-seq signals are very high (Figure 5F).

The binding region and motif of SntB on the catA, catB, sod1, and sod2 genes were shown in Figure S7 and described in lane 531-536 and 881-884. The background of ChIP-seq signals is high, but the enrich level in the ip-sntB-HA samples is significant compared to IP-WT.

Figure 5F, letters are too small, and difficult to read. The same is true for Figure 4. Letters should be enlarged for the readers to read it without problem.

Thanks. We have revised the Figure 5F and Figure 4. Please see these Figures.

Reviewer #2 (Recommendations For The Authors):

The authors fully addressed my concerns and made appropriate changes in the manuscript. The quality of the manuscript is now improved.

Thanks. We would like to express our sincere gratitude for your affirmation and thoughtful feedback. Your positive comments have been extremely encouraging and have strengthened my confidence in my work. Your time and effort in reviewing my submission are greatly appreciated.