The Mechanism Of Intron And Small RNA In The Regulation Of Fungicide Sensitivity In Fusarium Graminearum

Wheat,an important grain crop,is second only to rice in area and yield and the second largest grain crop in China.However,Fusarium head blight(FHB)has been seriously affecting the safety production of triticeae crops in the middle and lower reaches of Yangtze river in China.The disease can not only cause serious yield loss,but also produce trichothecene mycotoxins which is toxic to human and animal.Due to the lack of resistance gene resources,there are no wheat varieties that are immune to or highly resistant to F.graminearum.At present,the most effective strategy to control FHB is chemical control,which includes benzimidazoles,triazoles,methoxyacrylates,cyanoacrylates and so on.With the long-term use of fungicides in large quantities alone,the problem of fungicide resistance has become increasingly prominent.It has been proved that point mutations in the ?2-tubulin gene lead to resistance to benzimidazoles and point mutations in myosin-5 gene lead to resistance to Phenamacril in F.graminearum.In addition to the main cause that gene point mutations lead to changes in fungicide sensitivity,there are other related elements and pathways regulating sensitivity in vivo.For example,in cancer research,many studies have shown that non-coding RNA is involved in the regulation of anti-cancer drug resistance,and the development of new drugs targeting non-coding RNA may become a new strategy for cancer treatment.However,the role of non-coding RNA in the regulation of fungicide sensitivity has not been reported.With the progress and application of sequencing technology,it has been found that the genome not only encodes a huge proteome,but also transcribes some even larger amounts of non-coding RNA.Non-coding RNAs in the eukaryotic transcriptome include introns spliced from protein-coding transcripts and non-coding RNAs that function independently and closely related to the development regulation process,including small non-coding RNAs(microRNA,siRNA,piRNA)and long non-coding RNAs(lncRNAs).In addition,the sequences of intron RNAs are highly complex and contain important conserved patterns,suggesting that these RNAs contain important regulatory information that can be expressed simultaneously with protein-coding sequences.Genome-wide bioinformatics analysis of introns in F.graminearum indicated that the genome of F.graminearum includes 11 600 genes.Of which,75.57%of genes contain intron(s)and each gene on average contains 1.91 introns.At the same time,sRNA-sequencing showed that a total of 108 micro-like RNAs were identified in F.graminearum.However,until now,the functions of these endogenous non-coding RNAs in F.graminearum have not been revealed in detail.On this basis,this study will explore the effects of non-coding RNAs,especially the introns in fungicide target gene,on the regulation of host gene transcription and translation and regulation of fungicide sensitivity in F.graminearum.It is well known that Fg-?2tub,cyp51A and myosin-5 are important target genes of benzimidazoles,triazoles and cyanoacrylates respectively in F.graminearum.Sequence analysis showed that Fg-?2tub,cyp51A and myosin-5 genes of F.graminearum contain six,one and two introns respectively.However,the underlying functions of these introns are unknown.To explore the sensitivity regulation functions of introns in these target genes,several detailed deletion studies were completed on the intronic regions of Fg-?2tub,cyp51A and myosin-5 using a homology recombination strategy.Phenotypic analyses showed that deletion of the fourth intron from Fg-?2tub gene(designated ?2?i4),the sole intron from cyp51A gene(cyp51A-?i)and the second intron from myosin-5 gene(myo5-?i2)exhibited an increased sensitivity to corresponding fungicides.In contrast,deletion of the first or second intron from gene exhibited a decreased sensitivity to carbendazim.qRT-PCR showed that the mRNA transcript levels of target genes were significantly down-regulated in ?2?i4,cyp51A-?i and myo5-?i2 respectively.Meanwhile,Western blot assays revealed that the protein expression levels of Fg-?2tub were also dramatically reduced in ?2?i4,but accumulated in and ?2?i2.This not only indicates that introns i1 and i2 have negative regulatory effects on host gene translation,but also reveals that the transcription level is not always completely consistent with the translation level.Overall,our results indicate that introns in target genes significantly regulate the fungicide-sensitivity by influencing expression of the corresponding resident genes in F.graminearum.F.graminearum is known to have two ?-tubulin genes(named Fg-?1tub and Fg-?2tub).Among them,more than 95%of the amino acid sequences of Fg-?1tub are homologous with ?-tubulin isotypes related to benzimidazole resistance in other fungi,while only 77%of the amino acid sequences of Fg-?2tub are homologous with?-tubulin isotypes related to benzimidazole resistance in other fungi.However,mutations in Fg-?2tub rather than in Fg-?1tub have been shown to confer resistance to benzimidazoles in F.graminearum.Sequence alignment of ?-tubulin isotypes related to benzimidazole resistance showed that the number and position of introns in Fg-?2tub are more consistent than Fg-?1tub to those in other ?-tubulin genes.In detail,Fg-?1tub lacks three introns i.e.intron i3,i4 and i6 corresponding to positions in Fg-?2tub of F.graminearium.To investigate the effects of the divergence introns on the function of ?-tubulins in F.graminearum,a strategy of intron deletion and insertion was used.Our results showed that inserting introns Fgi3 and Fgi6 from Fg-?2tub into the corresponding positions in Fg-?1tub can increase Fg-?1tub expression leading to increased sensitivity to carbendazim(MBC)in F.graminearum.Inserting introns Fgi4 from Fg-?2tub into the corresponding positions in Fg-?1tub can increase both Fg-?1tub and Fg-?2tub expression.Meanwhile,the sensitivity to MBC remained unchanged.Deletion experiments of three introns in Fg-?1tub gene showed that deletion of the 2nd intron from Fg-?1tub gene increased Fg-?1tub expression levels leading to increased sensitivity to MBC.Besides,we also found that the transcription expression of Fg-?1tub was also significantly up-regulated by inserting exogenous introns into the Fg-?1tub gene,suggesting that intron-mediated Fg-?1tub gene expression is not species and sequence-specific in F.graminearum.Besides,the insertion and deletion of introns in Fg-?1tub gene has no significant effect on hyphal growth,conidiation and virulence in F.graminearum.In order to study the regulatory roles of small RNAs in MBC sensitivity of Fusarium graminearum,we applied high-throughput sequencing technology to elucidate small RNA(18-40 nucleotides)(sRNA)transcriptome in MBC sensitive strain 2021,MBC medium resistance nt167,and MBC high resistance nt198,and found that a total of one hundred and eight micro-like-RNA(milRNA)candidates were identifed.Through bioinformatics analysis,we found that there were significant differences in milRNA expression between the sensitive and resistant strains.The KEGG pathway was used to analyze the candidate target genes of differentially expressed milRNAs,and it was found that the ABC transporter pathway was significantly up-regulated in the resistant strains.In addition,two Dicer genes,FgDicer1 and FgDicer2,were identified in F.graminearum.Single and double deletion of FgDicer genes were obtained in 2021 and nt198 by homologous recombination strategy.Phenotypic analysis showed that FgDicer1 and FgDicer2 deletion mutants had no effect on mycelial growth,asexual reproduction and pathogenicity.However,the MBC sensitivity test found that in nt198,the EC90 value of FgDicer1 and FgDicer2 deletion mutants were significantly increased compared with the original strain nt198.Overall,our results show that small RNAs are significantly involved in the regulation of MBC sensitivity in Fusarium graminearum.