Biological Function Of Fusarium Graminearum FgNrp1

Fusarium graminearum is the main pathogen of wheat scab,one of the the most devastating diseases in wheat.Wheat scab not only seriously reduces the yield of wheat,but also contaminate the grains with mycotoxins,endangering human and animal health.Therefore,understanding the growth and development regulation mechanism of F.graminearum is important to control wheat scab.DNA methylation,histone modification and RNA interference are important gene regulation mechanism in fungi.The 3’→5’exoribonuclease ERI-1 negatively regulates RNAi,and also participates in endogenous si RNA production in Caenorhabditis elegans,and is related to heterochromatin formation in Schizosaccharomyces pombe.Phylogenetic analysis showed that ERI-1 was lost in filamentous ascomyces.And based on its exonuclease domain we found NRP1 gene in filamentous fungi.NRP1 in Neurospora Crassa is an important regulator of co-transcriptional gene silencing and post-transcriptional RNA metabolism,linking the RNAi mechanism with heterochromatin formation.We previously identified a class of 24-nt s RNAs in the later stage of sexual development of F.graminearum.The production sites of these s RNAs highly overlaps with the heterochromatin marked with H3K27 methylation.Based on the function of NRP1 in Neurospora Crassa,we hypothesized that these s RNAs are associated with histone methylation through NRP1.Therefore,the functions of FgNRP1 in F.graminearum growth and pathogenicity,gene expression,H3K27 methylation were investigated by reverse genetics.Gene knockout analysis showed that the grwoth rate of Fgnrp1 mutant decreased significantly,indicating its important roles in growth.Crossing with the MAT1-1 mutant suggested that the Fgnrp1 mutant is female sterile.In determine the relationship between FgNRP1 and histone methylation,we examined the levels of H3K27me3 and H3K36me in Fgnrp1 mutant by Western blot.The results showed that FgNRP1 negatively regulated the heterochromatin related H3K27me3 and positively regulated euchromatin related H3K36me.In this study,the differentially expressed genes of each mutant in 24h hypha stage were analyzed by RNA-seq.The expression of the FgDCR1 and FgARB1 related to si RNA production pathway were reduced in the Fgnrp1 mutant,suggesting that FgNRP1 may participate in si RNA production.The up-regulation of FgMLH1 and FgRAD50 in Fgnrp1 suggests that FgNRP1may be related to DNA damage repair pathway.FgNRP1 may also be involved in inhibiting the expression of methyltransferase FgRID1 in asexual stage,thus indirectly participating epigenetic modification.When mapping the differential expressed genes onto the euchromatin or heterochromatin,more genes were mapped on heterochromatin than on euchromatin.These results of RNA-seq suggest that FgNRP1 may be involved in DNA damage repair,histone modification and heterochromatin formation.To futher study the effect of FgNRP1 on DNA damage repair,the effects of DNA damage repair inhibition reagents on the growth of all mutants were assayed.The results showed that the growth rate of FgNRP1^△EXOand FgNRP1^△RRMmutants had increased sensitivity to H₂0₂,indicating ERI-1＿3’h Exo＿like and RRM＿ARP＿like domain may regulate the expression of DNA damage repair pathway genes by participating in the process of reactive oxygen species damage repair.In this study,the proteins interacting with FgNrp1 at 14-16 h stage of hypha were also found by Pull down-MS.We found some genes related to histone acetylation and DNA damage repair and selected three genes related histone acetylation pathway,FgASF1,FgHAT1 and FgHAT2,for yeast two-hybrid verification.The results showed that there was no direct interaction between FgNrp1 and FgHat1 and FgAsf1,suggesting that they may be form a complex.In conclusion,this study started from FgNRP1 gene,clarify its effects on asexual reproduction,pathogenicity and sexual reproduction of Fusarium graminearum,and its possible correlation with chromatin modification.It provides a theoretical basis for further research on the function and regulatory mechanism of FgNRP1 gene.