BcEaf2 cofactor links histone methylation to fungal growth and virulence

A laboratory study of the plant-pathogenic fungus Botrytis cinerea identifies BcEaf2 as a chromatin-associated cofactor that supports hyphal growth, spore production, stress tolerance and infection by promoting the activity of the histone methyltransferase BcSET2. The researchers silenced BcEaf2 in three independent strains and separately deleted BcSET2, then found that both perturbations produced closely matching defects. These included slow colony expansion, sensitivity to oxidative, osmotic and DNA-damage stress, reduced asexual spore formation, weak spore germination and markedly attenuated disease on detached grape berries.

BcEaf2 depletion selectively lowered the global level of trimethylation at histone H3 lysine 36, or H3K36me3, without measurably changing several other histone acetylation and methylation marks examined. The research focused on BcEaf2, a 568-amino-acid protein containing a conserved SANT domain that had been characterized in other eukaryotes as a component of chromatin-regulatory machinery. A genome-wide search identified the corresponding gene in B. cinerea, and phylogenetic analysis placed fungal and other animal homologs within a broader Eaf2/DMAP1 group.

Transcript measurements showed that BcEaf2 expression was developmentally regulated: its messenger RNA abundance was higher in conidia and during germination than in the mycelial baseline and rose further during host infection, with the highest levels reported at later invasion stages. Investigators screened more than 300 transformants in an attempt to create a homozygous BcEaf2 null mutant but did not recover one. Overexpression of BcEaf2 in one silenced strain largely restored wild-type growth, conidiation and stress tolerance, supporting a causal role for the targeted gene.

The absence of a specific antibody against endogenous BcEaf2 meant that the corresponding change in protein abundance could not be measured directly, an important limitation of the transcript-silencing approach. The silenced strains grew more slowly throughout measurements made one, three and six days after inoculation, rather than showing only a temporary developmental delay. By day six, wild-type, empty-vector and complemented colonies had covered the culture plates, whereas the silenced colonies had not.

Microscopy also revealed sparse networks with fewer branches and wider gaps between hyphae. On media containing hydrogen peroxide, potassium chloride or sorbitol, the silenced strains showed greater growth inhibition than the controls, indicating reduced tolerance to reactive oxygen species and osmotic pressure. A further assay with methyl methanesulfonate showed that all three silenced strains were more sensitive to DNA-damaging stress, while complementation restored tolerance.

Asexual reproduction was similarly impaired. Wild-type and empty-vector cultures produced abundant conidia, whereas each BcEaf2-silenced strain produced markedly fewer spores. Germ tubes emerged from more than 60% of spores in the control strains but fewer than 20% in the silenced strains.

Counts at seven and 11 days showed that the reduction persisted throughout the observation period, and reintroducing BcEaf2 largely restored conidial production. In the infection assay, the silenced strains caused substantially less tissue damage on detached Shine Muscat grapes. Confocal microscopy after wheat-germ agglutinin staining showed that their invasive hyphae rarely extended beyond the inoculation site, while fungal DNA measurements confirmed lower pathogen accumulation in infected tissue than after inoculation with wild-type or empty-vector controls.

Analysis of histone modifications revealed that BcEaf2 did not have a broad effect on every chromatin mark tested. Global levels of H3K18ac, H3K27ac and H4K5ac, as well as H3K4me3 and H3K27me3, were not significantly changed in the silenced strains under the in vitro conditions used. H3K36me3, however, was consistently and markedly reduced in all three mutants.

The selective biochemical phenotype connected BcEaf2 to the deposition of a specific histone mark rather than to a general increase or decrease in all forms of histone modification. The experiments measured global abundance in cultured mycelia; they did not map H3K36me3 to particular genes or establish the transcriptional targets responsible for the observed phenotypes. A yeast two-hybrid screen identified BcSET2, a protein containing the conserved SET catalytic domain associated with H3K36 methylation, as a BcEaf2 partner.

Targeted two-hybrid assays and co-immunoprecipitation confirmed the interaction, while deletion-mapping experiments showed that the SANT domain of BcEaf2 was required for binding in the two-hybrid system. Removal of the N-terminal or C-terminal regions did not significantly disrupt the association. The researchers could not recover a stable BcEaf2 mutant lacking the SANT domain, however, so the domain’s direct contribution to H3K36me3 regulation could not be tested in a deletion strain.

BcSET2 messenger RNA abundance did not differ significantly between the BcEaf2-silenced strains and the wild type under the tested conditions. That measurement showed no detectable change in BcSET2 transcription, but it did not identify the molecular basis of the protein interaction. Purified recombinant proteins provided a biochemical test of the proposed mechanism.

BcSET2 exhibited intrinsic H3K36 methylation activity in vitro, and adding BcEaf2 markedly increased the amount of H3K36me3 deposited. BcEaf2 stimulated wild-type BcSET2 in a dose-dependent manner. By contrast, a catalytic-deficient BcSET2 variant with the conserved NHXCXPN motif removed showed markedly reduced activity and was not detectably stimulated by BcEaf2.

Comparable protein abundance in the wild-type and catalytic-deficient preparations argued against the activity difference simply reflecting unequal BcSET2 abundance. The results therefore support BcEaf2 as a positive regulator of BcSET2-dependent H3K36me3 formation, although the assays do not establish whether the cofactor stabilizes the enzyme, improves substrate recognition, promotes an active conformation or influences recruitment to chromatin. Deleting BcSET2 produced the same broad phenotype previously associated with BcEaf2 silencing.

Three independent knockout strains had reduced radial growth, sparse hyphae, greater sensitivity to hydrogen peroxide, potassium chloride, sorbitol and methyl methanesulfonate, and sharply reduced conidiation. Fewer than 20% of their conidia germinated, compared with more than 60% in wild-type, empty-vector and complemented strains. The knockouts also caused weaker lesions on grape berries, showed limited invasive hyphal expansion and accumulated less fungal biomass in infected tissue.

Genetic complementation restored these traits toward wild-type levels. The parallel results support functional coupling between BcEaf2 and BcSET2, but they do not by themselves prove that every phenotype is caused exclusively by reduced H3K36me3 rather than by additional molecular effects of these proteins. Overall, the study presents BcEaf2 and BcSET2 as a regulatory module that connects histone H3 lysine 36 trimethylation with fungal fitness and pathogenicity in B. cinerea.

The findings extend the known repertoire of Eaf2/DMAP1 proteins beyond their better-characterized roles in DNA methylation maintenance or histone acetylation in mammals and yeast, showing that the fungal homolog can instead enhance a histone methyltransferase. The work also illustrates why null mutants are informative: repeated failures to obtain BcEaf2 knockouts and SANT-domain deletions suggest severe consequences for viability or transformant recovery, although that interpretation remains inferential. Because the virulence tests used wounded detached grapes under controlled laboratory conditions and the methyltransferase evidence came from purified proteins, the results do not establish field disease control, clinical benefits or an effective antifungal treatment.

They instead identify a mechanistic target for future research that would require chromatin mapping, structural analysis, proteomics and testing across additional fungal species.

Subject of Research: Molecular regulation of fungal growth and virulence by the BcEaf2–BcSET2 histone-methylation module in Botrytis cinerea

Article Title: BcEaf2 plays a critical role in hyphal growth, asexual reproduction, pathogenic infection, and histone methylation in Botrytis cinerea

Article References: Wu, J., Wu, W., Wu, Y., Hao, J., Chen, W., Wang, H., Yang, C., & Shen, F. (2026). BcEaf2 plays a critical role in hyphal growth, asexual reproduction, pathogenic infection, and histone methylation in Botrytis cinerea. Frontiers in Microbiology, 17, Article 1978891. https://doi.org/10.3389/fmicb.2026.1978891

Image Credits: AI Generated

DOI: 10.3389/fmicb.2026.1978891

Keywords: Botrytis cinerea, BcEaf2, BcSET2, H3K36me3, histone methylation, fungal virulence, plant pathology, chromatin regulation, fungal stress response, asexual reproduction, plays, critical