Set2 family regulates mycotoxin metabolism and virulence via H3K36 methylation in pathogenic fungus Aspergillus flavus

Abstract

Aspergillus flavus infects various crops with aflatoxins, and leads to aspergillosis opportunistically. Though H3K36 methylation plays an important role in fungal toxin metabolism and virulence, no data about the biological function of H3K36 methylation in A. flavus virulence has been reported. Our study showed that the Set2 histone methyltransferase family, AshA and SetB, involves in morphogenesis and mycotoxin anabolism by regulating related transcriptional factors, and they are important for fungal virulence to crops and animals. Western-blotting and double deletion analysis revealed that AshA mainly regulates H3K36me2, whereas SetB is mainly responsible for H3K36me3 in the nucleus. By construction of domain deletion A. flavus strain and point mutation strains by homologous recombination, the study revealed that SET domain is indispensable in mycotoxin anabolism and virulence of A. flavus, and N455 and V457 in it are the key amino acid residues. ChIP analysis inferred that the methyltransferase family controls fungal reproduction and regulates the production of AFB1 by directly regulating the production of the transcriptional factor genes, including wetA, steA, aflR and amylase, through H3K36 trimethylation in their chromatin fragments, based on which this study proposed that, by H3K36 trimethylation, this methyltransferase family controls AFB1 anabolism through transcriptional level and substrate utilization level. This study illuminates the epigenetic mechanism of the Set2 family in regulating fungal virulence and mycotoxin production, and provides new targets for controlling the virulence of the fungus A. flavus.AUTHOR SUMMARYThe methylation of H3K36 plays an important role in the fungal secondary metabolism and virulence, but no data about the regulatory mechanism of H3K36 methylation in the virulence of A. flavus have been reported. Our study revealed that, in the histone methyltransferase Set2 family, AshA mainly catalyzes H3K36me2, and involves in the methylation of H3K36me1, and SetB mainly catalyzes H3K36me3 and H3K36me1. Through domain deletion and point mutation analysis, this study also revealed that the SET domain was critical for the normal biological function of the Set2 family and that N455 and V457 in the domain were critical for AshA. By ChIP-seq and ChIP-qPCR analysis, H3K36 was found to be trimethylation modified in the promotors and ORF positions of wetA, steA, aflR and the amylase gene (AFLA_084340), and further qRT-PCR results showed that these methylation modifications regulate the expression levels of these genes. According to the results of ChIP-seq analysis, we proposed that, by H3K36 trimethylation, this methyltransferase family controls the metabolism of mycotoxin through transcriptional level and substrate utilization level. All the results from this study showed that Set2 family is essential for fungal secondary metabolism and virulence, which lays a theoretical groundwork in the early prevention and treatment of A. flavus pollution, and also provides an effective strategy to fight against other pathogenic fungi.

Keywords: Aspergillus flavus; Set2 family; histone methyltransferase; mycotoxin; virulence.

Figure 1.

The role of AshA in the development and aflatoxin biosynthesis. (a) Conidial quantification of cultures of WT, ΔashA and Com-ashA strains on YES agar and PDA at 37°C in the dark for 4 d. (b) Relative expression levels of abaA gene in the above strains in YES at 37°C at 48 h and 72 h. (c) Relative expression levels of brlA gene in the above strains in YES at 37°C at 48 h and 72 h. (d) Sclerotia quantification of WT, ΔashA and Com-ashA strains on CM media at 37°C in the dark for 6 d. (e) Relative expression level of nsdC gene in the above strains in CM media at 37°C at 48 and 72 h. (f) Relative expression level of nsdD gene in the above strains in CM media at 37°C at 48 and 72 h. (g) TLC analysis of aflatoxins production from WT, ΔashA and Com-ashA strains after 6 d of incubation in dark in liquid YES media. (h) Relative quantification of AFB1 production of WT, ΔashA and Com-ashA strains according to the result of panel G. (i) HPLC analysis of aflatoxins from WT, ΔashA and Com-ashA strains cultivated in liquid YES media for 6 d. (j) Relative expression levels of aflatoxin bio-synthesis and regulation genes monitored by qRT-PCR at 48 h. All A. flavus strains were cultivated in liquid YES media. (k) Relative expression levels of aflatoxin bio-synthesis and regulation genes at 72 h. All fungal strains were cultivated in liquid YES media.

Figure 2.

The role of AshA in the A. flavus virulence against silkworm and crop kernels, and its subcellular location and the methylation site and levels catalyzed by it. (a) Photographs of the silkworms infected with the WT (WT group), ΔashA (ΔashA group) and Com-ashA (Com group) of A. flavus strains after 1 week incubation. (b) the survival rate of silkworms 1 week after injection of above A. flavus strains, respectively. (c) Photographs of the dead silkworms infected by A. flavus after 6 d incubation in dark under 28°C. (d) TLC analysis of AFB1 levels produced in infected dead silkworms that are shown in C. (e) the histogram showing the relative amount of AFB1 in silkworms according to panel D. (f) Photographs of the peanut seeds and corn kernels infected with the A. flavus WT, ΔashA and Com-ashA strains after 7 d incubation in the dark. (g) Quantification of conidia from the surface of A. flavus strains colonized peanut and corn grains in panel a and B. (h) TLC analysis of AFB1 levels of A. flavus strains colonized peanut and corn grains shown in panel F. (i) Relative amount of AFB1 in peanut and corn grains, according to the result of H. (j) Point inoculated cultures of A. flavus WT, ΔashA, and Com-ashA strains in YES and YES+80 µg/ml CFW. (k) Relative inhibition rate of CFW to A. flavus strains on the 4th d. (l) Point inoculated cultures of A. flavus WT, ΔashA and Com-ashA strains in YES and YES+5 mM H₂O₂ in the dark for 4 d. (m) the histogram showing relative inhibition rate of H₂O₂ to above A. flavus strains on the 4th d. (n) Maps of the germinating spore and hyphae shown by DIC imaging, the location of the nucleus with DAPI staining (Exciting wavelength: 405 nm UV light. Emission wavelength: 420–460 nm), the location of mCherry-AshA (Exciting wavelength: 552 nm. Emission wavelength: 600–630 nm), and merged photo of nucleus and mCherry-AshA. The mCherry-AshA was expressed under the promotor of gpdA(p). (o) Maps of hyphae under the stress of H₂O₂ with DIC imaging, DAPI staining, mCherry-AshA fluorescence imaging, and merged photo of DAPI staining and fluorescence imaging. (p) Western-blotting analysis on the bio-function of AshA in methylation level of H3K36me (1–3). (q) the histogram showing the relative 1–3 methylation level of H3K36 according to the results of western-blotting analysis in panel P according to the size and density of bands of H3K36me (1–3), respectively.

Figure 3.

The bio-function of SetB in the methylation of H3K36, and the role of SetB in the development, AFB1 biosynthesis and virulence of A. flavus. (a) Western-blotting analysis on the bio-function of setB gene, SET domain, and the interaction of setB and ashA genes in the methylation of H3K36. (b) the histogram showing the relative 1–3 methylation levels of H3K36 according to the results of western-blotting analysis. (c) the fungal strains, including ΔsetB, WT, Com-setB, setB^ΔSET,ΔsetB/ashA, and H3K36A, were inoculated on PDA media under 37°C in the dark for 4 d. (d) Colony diameter comparison among ΔsetB, WT, δsetb-Com, setB^ΔSET,ΔsetB/ashA, and H3K36A fungal strains. (e) the comparison of conidia numbers produced by above fungal strains on PDA media under 37°C for 4 d. (f) the sclerotia were formed by WT, ΔsetB and Com-setB strains on CM media under 37°C in the dark for 7 d. (g) Histogram showing the statistical analysis results on the role of SetB, SET domain, and the interaction of SetB and AshA in sclerotia formation. (h) Expression levels of abaA and brlA genes among ΔsetB, WT, Com-setB strains at 48 h. The fungal strains were inoculated in PDB media under 37°C in the dark. (i) Relative expression level of nsdC and nsdD genes among ΔsetB, WT, Com-setB strains at 48 h. The fungal strains were inoculated in liquid CM media under 37°C in the dark. (j) the productions of AFB1 were monitored by TLC after above strains cultured in liquid YES media under 28°C in the dark for 6 d. (k) the relative mycotoxin productions were shown with histogram according to the results from panel J. (l) qRT-PCR analysis was carried out on orthodox AFB1 biological synthesis pathway genes after above fungal strains were cultured in liquid YES media under 28°C for 48 h. (m) the colonization of setB, WT, δsetb-Com, setB^ΔSET,ΔsetB/ashA, and H3K36A fungal strains on the surface of maize kernels after 7 d incubation under 28°C. (n) the histogram showing the amount of conidia produced on the surface of maize kernels. (o) TLC analysis of AFB1 produced on these maize kernels colonized by above fungal strains. (p) the histogram showing the production of AFB1 in these A. flavus infected host kernels, according to the result from panel O. (q) Point inoculated cultures of A. flavus WT, ΔsetB, and Com-setB strains in PDA+0.02% SDS under 37°C in the dark for 4 d. (r) Relative inhibition rate of SDS to A. flavus WT, ΔsetB, and Com-setB strains on the 4th d. Relative inhibition rate = (diameter of the colony without inhibitor - diameter of the colony with inhibitor)/diameter of the colony without inhibitor.

Figure 4.

H3k36me3 catalyzed by SetB and AshA regulates many important biological processes. (a) Distributions of log2 FC (WT/ΔsetB/ashA), and 1995 DAPs for all 5592 peaks are significantly accumulated in WT sample (Red color; -log2 FC ≥1, p < 0.01), 23 DAPs are accumulated in the sample from δsetb/asha strain (green color; log2 FC ≤-1, p < 0.01), and 3574 peaks are not differentially regulated (black color; log2 FC < 1 or > −1, p>0.01 if log2 FC > 1 or < −1). (b) the annotation of biological processes, cellular components and molecular function for the genes which are up-regulated at WT strain compared to δsetb/asha strain. (c) the bubble graph of KEGG pathways of up-regulated gene fragments in the WT strains compared to δsetb/asha strain. (d) Comparation of the enriching levels of the H3K36me3 modified promotor and coding sequence of wetA gene between WT and δsetb/asha strain through ChIP-seq analysis. (e) qRT-PCR analysis on the expression level of the sporulation regulating gene wetA. (f) ChIP-qPCR analysis on the H3K36me3 modified promotor of wetA (−529 to −427 bp upstream of the TSS, IgG was used as negative control). (g) ChIP-qPCR analysis on second H3K36me3 modified promotor position for wetA (−373 to −207 bp upstream of the TSS). (h) ChIP-qPCR analysis on the H3K36me3 modified gene coding sequence (open read fragment, ORF) of wetA (167 to 308 bp downstream of the TSS). (i) Comparation of the enriching levels of the H3K36me3-modified chromatin fragment of steA gene between WT and δsetb/asha strains. (j) the expression level of the sclerotia regulating gene SteA. (k) ChIP-qPCR analysis on H3K36me3 modified ORF region of steA gene (1909 to 2023 bp downstream of the TSS).

Figure 5.

SetB and AshA regulate the biological synthesis of AFB1 through catalyzing the trimethylation of H3K36. (a) Comparation of the enrichment of H3K36me3-modified aflR chromatin fragments between WT and δsetb/asha strains (Red and blue peaks are the repetition for WT strain; green and purple are repetition for δsetb/asha strain). (b) qRT-PCR analysis monitoring the expression level of AFB1 biological synthesis regulating gene aflR. (c) ChIP-qPCR analysis on the promotor of regulating gene aflR (at the position of −627 to −491 bp upstream of the TSS, IgG was used as negative control). (d) ChIP-qPCR analysis on the second position of aflR promotor (−197 to −93 bp upstream of the TSS). (e) ChIP-qPCR analysis on the ORF region of aflR (337 to 520 bp downstream of the TSS). (f) HPLC analysis on the AFB1 production of WT and δsetb/asha strain (The black curve is for the WT strain, the red curve is for the δsetb/asha strain). (g) Comparation of the enrichment of H3K36me3-modified chromatin fragments of the amylase gene (AFLA_084340) between WT and δsetb/asha strains (Red and green peaks are the repetition for WT strain; blue and yellow are repetition for δsetb/asha strain). (h) qRT-PCR analysis monitoring the expression of the amylase gene. (i) TLC analysis of AFB1 produced by WT strain and the amylase gene knock-out strain (ΔAFLA_084340). (j) the histogram showing the relative production of AFB1 in panel I. (k) ChIP-qPCR analysis on the ORF of AFLA_084340 (at the position of 1609 to 1740 bp downstream of the TSS, IgG was used as negative control).

Figure 6.

The regulatory model of AshA and SetB in the virulence of A. flavus.