Catalytic Activities And Biological Functions Of Four Histone Lysine Methyltransferases In Beauveria Bassiana

Fungal insect pathogens including Beaurveria bassiana serve as main sources of fungal insecticides and acaricides that are environment friendly and safe for honeybees in the field.Biological control potential of such fungi relies upon their virulence,stress tolerance and asexual developmental traits that favour large-scale production and quality control of aerial conidia serving as active ingredients of fungal pesticides.Therefore,the fungal biocontrol potential is an overall output of various cellular processes and events controlled by complicated signalling and epigenetic networks.However,pleiotropic effects of histone H3 lysine-specific methylation(H3K_me)on the fungal potential against arthropod pests remains understood insufficiently.It is a main task in the post-genomic era to explore the H3K_me-mediated gene expression and phenotypes that are of special merits for the pest control potential of fungal insect pathogens.This study seeks to characterize catalytic activities and biological functions of four histone lysine methyltransferases(KMTs),namely Dim5/KMT1,Setl/KMT2,and Set2/KMT3 and its partner Ash1.The main results are summarized below.Dim5/KMT1 controls fungal insect pathogenicity and genome stability by methylation of histone H3K4,H3K9 and H3K36 in B.bassiana.Mono-,di-and tri-methylation of histone H3 Lys 9,Lys 4,and Lys 36(H3K_mel/me2/me3)required for mediation of DNA-based cellular events in eukaryotes usually rely upon the activities of KMTs classified to the KMT1,KMT2,and KMT3 families,respectively.In B.bassiana,H3K9me-specific Dim5/KMT1 orthologue lacking a C-terminal post-SET domain,was proven to localize mainly in nucleus and have both conserved and noncanonical roles in methylating core H3 lysines.Disruption of dim5 led to not only abolished H3K9me3 but also marked attenuation of H3K4me1/me2,H3K9me1/me2 and H3K36me2.Consequently,the Δdim5 mutant lost the whole insect pathogenicity through normal cuticle infection,and was compromised severely in virulence through cuticle-bypassing infection(hemocoel injection)and also in a series of cellular events critical for the fungal virulence and lifecycle in vivo and in vitro.The compromised cell events included reduced hyphal growth,blocked conidiation,impeded proliferation in vivo,altered carbohydrate epitopes,disturbed cell cycle,reduced biosynthesis and secretion of cuticle-degrading enzymes,and increased sensitivities to various stresses.Among 1,201 dysregulated genes(up/down ratio:712:489)associated with those phenotypic changes,92(up/down ratio:59:33)encode transcription factors and proteins and enzymes involved in posttranslational modifications,implying that the Dim5-methylated core H3 lysines could act as preferential marks of those transcription-active genes crucial for global gene regulation.These findings uncover a novel scenario of Dim5 and its indispensability for insect-pathogenic lifestyle and genome stability of B.bassiana.Setl/KMT2-governed H3K4me coordinates the lifecycle in vivo and in vitro of B.bassiana.Biological control potential of insect-pathogenic fungi against pests is an overall output of various cellular processes regulated by signaling and epigenetic networks.In B.bassiana,H3K4me1,H3K4me2 and H3K4me3 were abolished by inactivation of Setl(KMT2),leading to marked virulence loss,impeded proliferation in insect haemocoel,severe growth and conidiation defects,reduced hydrophobicity,hypersensitivity to cell wall perturbing or lysing,and increased sensitivities to H2O2 and heat shock.Such compromised phenotypes correlated well with transcriptional abolishment or repression of those genes encoding carbon catabolite-repressing transcription factor Crel,classes I and II hydrophobins Hydl and Hyd2 required for cell hydrophobicity,key conidiation regulators,and stress-responsive enzymes/proteins.Particularly,crel,a direct target activated by KMT2-mediated H3K4me3 for upregulation of hyd4 in Metarhizium robertsii,was nearly abolished at transcription level in the Δset1 mutant,leading to abolished expression of hyd1 and hyd2 as homologs of hyd4 in M.robertsii.These data implicate that a Setl-CrelHydl/2 pathway works in B.bassiana like the KMT2-Crel-Hyd4 pathway previously reported to mediate infection-related morphogenesis and pathogenicity in M.robertsii.Our findings unveil that Setl/KMT2-governed H3K4me3 coordinates not only the host infection and virulence but also the asexual cycle in vitro of B.bassiana.Conserved and noncanonical activities of H3K36me-specific Set2 and Ash1 required for fungal insect-pathogenic lifestyle.Both Set2 and Ashl are KMT3 family members to catalyze H3K36me but remain unexplored in fungal insect pathogens.In B.bassiana,Set2 and Ash1 were proven to have broader/greater roles in catalyzing mono-diand tri-methylation of H3K4 than of H3K36 and function like Setl/KMT2,which catalyzes specifically H3K4me3 as an epigenetic mark of cre1 to upregulate the classes I and II hydrophobin genes hydl and hyd2 required for conidial hydrophobicity and adherence to insect cuticle.Particularly,H3K4me3 was more attenuated than H3K36me3 in the absence of set2(72%versus 67%)or ash1(92%versus 12%),leading to sharply repressed or nearly abolished expression of cre1,hyd1 and hyd2 and reduced hydrophobicity.Consequently,theΔset2 and Δash1 mutants were differentially compromised in radial growth on various media or under different stresses,aerial conidiation under normal culture conditions,virulence,and cellular events crucial for normal cuticle infection and haemocoel colonization,accompanied by transcriptional repression of clustered genes involved in or required for asexual development and multiple stress responses.These findings unravel novel roles of Set2 and Ash1 in the co-catalysis of usually Setl-reliant H3K4me3 required for fungal insect-pathogenic lifestyle.