The Epigenetic Mechanism Analysis Of A Novel Xylanase Gene BCG1 In Fusarium Graminearum

Fusarium head blight(FHB)mainly caused by Fusarium graminearum is a devastating disease that causes extensive yield and quality losses to wheat.Contaminate grain produces various mycotoxins,in particular deoxynivalenol(DON),Zearalenone(ZEN),which are detrimental to human and animal health.Application of chemical fungicides is still one main approach for controlling FHB because of the lack of effective disease-resistant cultivars.However,the problems with pesticide residues and fungicide resistance are increasing.Controlling F.graminearum-caused diseases will benefit from an in-depth understanding of how F.graminearum spreads inside the host.Histone methylation has been implicated as a critical epigenetic switch.H3 lysine 4trimethylation(H3K4me3)is generally a chromatin marker for transcriptional activation,while genes marked with H3 lysine 27 trimethylation(H3K27me3)are associated with transcriptional silencing.Evidence has accumulated that transcriptional induction or repression through H3K4me3 and H3K27me3 contributes to the virulence and secondary metabolism in fungus-host interactions.However,how H3K4me3 and H3K27me3 coordinately regulate fungal pathogenesis remains unknown.“Bivalent chromatin” is a novel chromatin modification pattern that marked with both active and repressive histone modifications on the same nucleosomes,which can flexibly regulate gene expression.To date,bivalent H3K4me3-H3K27me3 histone modifications were reported only in animals and plants,the presence and function of bivalent histone modifications in fungi remain to be elucidated.In this study,we found notable increased bivalent H3K4me3-H3K27me3 histone modifications during F.graminearum infection via Chromatin immunoprecipitation sequencing(Ch IP-seq)and Sequential Ch IP-seq(Re Ch IP-seq),which has supported the crucial roles of bivalent modifications in F.graminearum virulence.Then we found that BCG1 is significantly marked by bivalent chromatin domains during infection.The biological functions and elucidate the epigenetic regulatory mechanism analysis of BCG1 were investigated during F.graminearum infection.It will provide a scientific basis for the epigenetic regulatory mechanism in plant-pathogen interactions.The results are summarized as follows:1.Identification and function study of a novel xylanase BCG1 in F.graminearumWe obtained 45 genes marked with both H3K4me3 and H3K27me3 modifications,and by combined analysis of Ch IP-seq and Re Ch IP-seq data,we found that 21 of the 45 comarked genes showed significantly increased bivalent signals during infection.Among these21 genes with significantly increased bivalent signals,BCG1 encoding for a novel xylanase showed the highest increase in bivalent signals in planta(120 hpi)as compared to those in vitro(0 hpi)condition.RNA-seq analyses revealed that BCG1 was highly expressed at the early stage of infection(24 hpi)and significantly decreased at 120 hpi,supporting the crucial roles of BCG1 in F.graminearum virulence.In this study,BCG1 was required for F.graminearum virulence.There was other seven xylanases in F.graminearum,sequence comparisons showed that BCG1 evolves a previously unrecognized G/Q-rich motif,however,this novel motif was absent in other seven xylanases.The results indicated that this G/Q-rich motif contribute to the high xylanase activity of BCG1,which BCG1 is the major xylanase required for F.graminearum virulence.2.BCG1 is an apoplastic effector and functions as a PAMP in plantsPlants and pathogens are engaged in a continuous coevolutionary struggle for survival.Increasing evidence indicates that plant innate immune system can sense the presence of pathogen-secreted virulence factors and initiate plant defense responses to block pathogens infection.BCG1 triggers plant immune responses in plants.Co-IP assays showed that BCG1 could interact with both Nb BAK1 and Nb SOBIR1,however,loss of G/Q-rich domain blocked these associations and triggered significantly decreased plant immune responses.Interestingly,the G/Q-rich motif is highly conserved and widely present in Fusarium species,but no proteins containing this domain were found in other organisms.In the evolutionary struggle between plants and pathogens,evolving protein with stronger xylanase activity in F.graminearum degraded plant cell wall,however,the plants,in turn,recognized the BCG1 to initiate plant defense against F.graminearum infection.3.Bivalent H3K4me3-H3K27me3 histone modification determines the transcriptional reprogramming of BCG1 gene,conferring host immunity evasion during F.graminearum infection.The result showed that the expression of BCG1 was highly induced by ～23-fold at early infection stage(24 hpi),and then rapidly declined to low levels at 48 hpi-120 hpi.Ch IPq PCR assays showed markedly elevated H3K4me3 level at BCG1 locus at 24 hpi,as compared to that at 0 hpi;however,little H3K27me3 signals were observed at 0 and 24 hpi.Therefore,we proposed that F.graminearum activates BCG1 expression via H3K4me3 modification during early infection stage,thus effectively overcoming the physical barriers of plant cell wall.Re Ch IP-q PCR assays were performed and the results showed that bivalent H3K4me3-H3K27me3 modification was significantly enriched on BCG1 gene at 48-120 hpi,but not at 0-24 hpi.Bivalent H3K4me3-H3K27me3 histone modification maintains a repressive chromatin environment that facilitates the epigenetic silencing of BCG1 at 48-120 hpi,thus evading host immunity.Thus,our study indicated that F.graminearum deploys an elegant epigenetic strategy that fine-tunes BCG1 expression in a temporal manner at different phase of infection,thereby facilitating infection as well as evading host immunity.This epigenetic strategy may be widely used in fungal pathogens,which provide new insight into fungus-host interactions.