Genome-wide exonic small interference RNA-mediated gene silencing regulates sexual reproduction in the homothallic fungus Fusarium graminearum
[['The ascomycete fungus Fusarium graminearum is a major causative agent of Fusarium head blight ( FHB ) in small-grain cereals worldwide [1] .', 'The fungus reproduces using sexual spores ( ascospores ) and asexual spores ( conidia ) .', 'Although both spore types contribute to disease initiation and propagation , ascospores serve as the primary inocula for FHB outbreaks because these spores are forcibly discharged into the air and can move long distances [2] .', 'Additionally , the sexual development process ensures the production of survival structures required for overwintering [3] and the genetic diversity of the population [4]; therefore , understanding the molecular mechanisms underlying F . graminearum sexual reproduction is important for developing novel FHB disease-control strategies .', 'Perithecia ( fruiting bodies ) produced via sexual reproduction have complex multicellular structures with three distinguishable layers [5] .', 'The perithecial outer layer composed of black and hard tissues/cells protects the inner components of the perithecia .', 'Asci are biseriately arranged within perithecia and generate turgor pressure by accumulating osmolytes for forcible ascospore discharge [6] .', 'Inside an ascus , a diploid zygote nucleus undergoes normal meiosis and mitosis to produce eight haploid nuclei , which are then sequestered into eight ascospores [7] .', 'Various genetic and metabolic processes are intricately involved in sexual reproduction; these processes are likely fine-tuned temporally and spatially depending on each step of sexual development [5 , 8] .', 'Many upstream signal transduction pathways and hundreds of signaling mediators , such as transcription factors and kinases , orchestrate sexual reproduction in F . graminearum [9–11] .', 'RNA interference ( RNAi ) is a conserved gene-silencing mechanism that occurs at the post-transcriptional or post-translational level in eukaryotes , including fungi [12–14] .', 'RNAi is triggered by small noncoding RNAs ( sRNAs ) of approximately 20–30 nucleotides , which are generally categorized as short interfering RNAs ( siRNAs ) , microRNAs ( miRNAs ) , and piwi-interacting RNAs ( piRNAs ) ; siRNAs and miRNAs have been extensively studied in fungi , although some piRNA-like sRNAs have been identified in Neurospora crassa [13 , 15] .', 'The siRNA and miRNA duplexes are produced from double-stranded RNA ( dsRNA ) precursors that are cleaved by RNase III-family nuclease Dicers .', 'The resulting siRNA or miRNA duplexes are loaded into an Argonaute protein of the RNA-induced silencing complex ( RISC ) .', 'Subsequent removal of the passenger strands of sRNA duplexes leads to the activation of the RISC , and this RISC-incorporated guide-strand sRNA is then used to identify complementary mRNA for silencing by either mRNA degradation or translational repression .', 'Elucidating the involvement of RNAi in quelling [16] and meiotic silencing by unpaired DNA ( MSUD ) [17 , 18] in N . crassa has been a major recent breakthrough in the genetics of filamentous fungi .', 'Most fungal species possess functional RNAi components except for some single-cell yeasts [19] .', 'Moreover , dsRNA-mediated gene silencing has been widely reported in fungi [13 , 20] , which is promising for the development of an efficient gene-silencing method or the utilization of host-induced gene silencing for disease control [21 , 22] .', 'Advanced next-generation sequencing technologies and bioinformatics tools have enabled researchers of non-model filamentous fungi to identify sRNAs .', 'However , the biological roles of sRNAs in fungal development have rarely been identified , while RNAi pathways have crucial regulatory functions in cellular processes such as development , host defenses , mRNA processing , transcription , and translation in animals and plants [23–25] .', 'Few sRNAs with important biological roles have been characterized in fungi , and sRNAs have been hypothesized to function under specific developmental or environmental conditions [26] .', 'RNAi generally functions in genome defense from viruses and transposable elements in eukaryotes , including fungi [14 , 22] .', 'The possible involvement of sRNAs in fungal development or responses to environmental stimuli has been proposed in Aspergillus flavus ( responses to water activity and temperature ) [27] , Magnaporthe oryzae ( fungal development ) [28] , and Penicillium marneffei ( transition between mycelia and yeast phases ) [29] .', 'Previous studies have proposed biological roles for RNAi based on phenotypic defects of RNAi-deficient mutants .', 'In the ascomycete fungus Trichoderma atroviride , different components of the RNAi machinery are involved in light-dependent asexual reproduction and light-independent hyphal growth [30] .', 'Sexually induced RNAi machinery is required for sexual reproduction but not for virulence in the basidiomycete human pathogen Cryptococcus neoformans [31] .', 'Components of the RNAi machinery involved in MSUD are also important for ascospore formation in N . crassa [18 , 32 , 33] .', 'Null mutants of RNAi components showed various developmental defects , including dysfunction during sexual and asexual reproduction , in Mucor circinelloides [34] .', 'In plant pathogenic fungi , important roles of pathogen-derived sRNAs in host-microbe interactions have been recently reported [35] .', 'Botrytis cinerea microRNA-like RNAs ( milRNAs ) silence genes involved in host defense by inhibiting the host RNAi pathway [36] .', 'However , there is little evidence from genome-wide approaches to characterize transcriptional reprograming by sRNAs during fungal development .', 'Our previous study revealed that the homothallic fungus F . graminearum possesses a functional MSUD mechanism , as is the case with the heterothallic fungus N . crassa [7] .', 'The presence of MSUD led us to investigate the roles of sRNA-mediated RNAi in F . graminearum sexual development .', 'A recent study revealed that hairpin RNA silenced target genes , and specific RNAi components were involved in this silencing process in F . graminearum [37] .', 'Moreover , one of the Argonaute genes ( FgSMS2/FgAGO2 ) was shown to be under the control of a mating-type gene and was required for sexual development in this fungus [9] .', 'Thus , the objectives of the present study were to provide an in-depth characterization of the sexual phenotypes of RNAi component mutants and to determine the genome-wide correlation of sRNAs with the transcriptome and degradome during the sexual development of F . graminearum .', 'This is the first genome-wide characterization of sRNAs involved in balancing transcript levels during fungal sexual development , and it provides insights into the role of sRNAs in fungi .'], ['F . graminearum possesses two Dicers and two Argonautes , as previously reported [37] .', 'Both Dicers ( FgDicer1 and FgDicer2 ) contain domain architectures similar to those of other representative orthologs; however , FgDicer1 and Dcl1 of Schizosaccharomyces pombe do not carry the double-stranded RNA-binding ( DSRM ) domain ( Fig 1A ) .', 'Each Argonaute has a conserved catalytic triad ( Asp-Asp-His/Asp , DDH/D ) ; FgAgo2 contains the third histidine residue but is replaced by an aspartate in FgAgo1 .', 'FgAGO2 was also annotated as FgSMS2 in a previous study [9] .', 'We measured the transcript levels of these genes during vegetative growth and sexual development ( Fig 1B ) .', 'The expression levels of FgDICER1 and FgAGO2 were significantly increased after sexual induction , suggesting that they have active roles during perithecia development in F . graminearum .', 'The transcript levels of FgDICER2 were similarly maintained throughout the developmental stages , while FgAGO1 transcripts accumulated to high levels as mycelia aged ( V5 , 5 days after inoculation ) and as perithecia matured ( S3–7 , 3–7 days after sexual induction ) .', 'To characterize the roles of these genes in fungal development , we generated each gene deletion mutant using the homologous recombination method ( S1 Fig and S1 Table ) .', 'Although all of the tested F . graminearum strains produced mature perithecia with similar numbers , the Fgdicer1 and Fgago2 mutants were severely defective in forcible ascospore discharge , while the Fgdicer2 and Fgago1 mutants showed indistinguishable phenotypes compared to those of the wild type ( Fig 1C and 1D ) .', 'Except for defective sexual development , we could not find any defect in vegetative growth , virulence , or stress responses in the mutants , in accordance with a previous report [37] .', 'Double-deletion mutants were generated to verify the redundant roles of Dicers or Argonautes .', 'Fgdicer2 and Fgago1 mutants were first outcrossed with the Δmat2 strain to produce the HK338 ( Δmat2 ΔFgdicer2 ) and HK339 ( Δmat2 ΔFgago1 ) strains , respectively ( S1 Table ) .', 'Then , the double-deletion mutants HK340 ( ΔFgdicer1 ΔFgdicer2 ) and HK341 ( ΔFgago1 ΔFgago2 ) were generated from the outcrosses HK338 × Fgdicer1 and HK339 × Fgago2 , respectively .', 'Deletion of both FgDICER1 and FgDICER2 almost completely abolished ascospore discharge , whereas Fgdicer1 mutants discharged reduced amounts of ascospores compared to those of the wild type , suggesting a synergistic effect of mutant phenotypes between single- and double-deletion mutants ( Fig 1C and 1D ) .', 'Double-deletion Fgago1 Fgago2 mutants showed similar phenotypes to those of Fgago2 mutants with respect to forcible ascospore discharge .', 'Mutant phenotypes and synergisms were confirmed using several complementation assays ( S2 Fig ) .', 'First , reintroduction of the intact FgDICER1 and FgAGO2 genes into each corresponding single deletion mutant restored defective sexual development ( Fig 1C and 1D ) .', 'Next , the roles of FgDICER2 and FgAGO2 were further verified by introducing each gene into the double-deletion mutants using the outcrosses Δmat2 × HK340 ( ΔFgdicer1 ΔFgdicer2 ) and Δmat2 × HK341 ( ΔFgago1 ΔFgago2 ) .', 'The resulting strains ( ΔFgdicer1 FgDICER2 and ΔFgago1 FgAGO2 in Fig 1C and 1D ) showed similar phenotypes to those of the Fgdicer1 and Fgago1 mutants .', 'To determine the mechanism underlying defective ascospore discharge , we dissected mature perithecia to observe rosette asci ( Fig 1E ) .', 'The F . graminearum wild-type strain produced eight normal spindle-shaped ascospores per ascus .', 'However , some asci of the mutants that were defective in ascospore discharge ( ΔFgdicer1 , ΔFgago2 , ΔFgdicer1 ΔFgdicer2 , and ΔFgago1 ΔFgago2 ) contained abnormally shaped ascospores and fewer than eight per ascus .', 'The morphologies of abnormal-shaped ascospores vary , including larger ones , smaller ones , and broken ones .', 'Moreover , small , ghost-like ascospores with incomplete spore delimitation were observed in these mutants ( Fig 1E ) .', 'Thus , we concluded that defective forcible ascospore discharge of Dicer or Argonaute mutants was due to abnormal ascospore production .', 'To investigate the molecular mechanisms underlying defective ascosporogenesis of the RNAi component mutants , we analyzed the transcriptomes of the F . graminearum strains using RNA-seq .', 'Based on a threshold of reads per kilobase of exon per hundred million mapped reads ( RPKHM ) values ( ≥ 10 ) under all tested conditions , 11 , 908 of 13 , 820 genes were selected for further analyses ( S2 Table ) .', 'Differentially expressed genes ( DEGs ) were identified as genes displaying a greater than 3-fold change in transcript levels compared to those of the wild type .', 'Deletion of both FgDICER1 and FgDICER2 induced the differential expression of 380 genes; 241 genes were upregulated , and 139 genes were downregulated ( Fig 2A and 2B ) .', 'While most of the upregulated genes in the Fgdicer2 mutant overlapped with those of the Fgdicer1 Fgdicer2 mutant ( 15/18 ) , 70% of the genes ( 58/84 ) were specifically upregulated in the Fgdicer1 mutant ( Fig 2A ) .', 'Similarly , 80% ( 297/378 ) and 61% of the genes ( 73/119 ) were increased or decreased , respectively , in their expression only in the Fgago2 mutant but not in the Fgago1 Fgago2 mutant ( Fig 2A and 2B ) .', 'In contrast , most of the downregulated genes in each Dicer single mutant were also reduced in transcript levels in the Fgdicer1 Fgdicer2 mutant ( Fig 2B ) .', 'Transcriptomes between the double-deletion mutants , Fgdicer1 Fgdicer2 and Fgago1 Fgago2 , were highly positively correlated ( R2 = 0 . 91; Fig 2C ) , and most DEGs overlapped in these mutants ( Fig 2D ) , suggesting that these two mutants were impaired in the same regulatory pathway .', 'However , each transcriptome of Fgdicer1 and Fgdicer2 showed a mild positive correlation with that of Fgdicer1 Fgdicer2 , supporting the functional synergism of FgDicer1- and FgDicer2-mediated transcriptional regulation during ascospore formation in F . graminearum ( Fig 2E ) .', 'While the transcriptome of the Fgago1 mutant showed a relatively lower positive correlation with that of the Fgago1 Fgago2 mutant , transcriptomes between the Fgago2 and Fgago1 Fgago2 mutants had a much higher correlation ( slope = 0 . 72 and R2 = 0 . 36 ) .', 'Taken together , these results showed that the two Dicers and two Argonautes have redundant roles in the same biological pathway affecting ascospore formation in F . graminearum .', 'To dissect the characteristics of DEGs via disruption of the RNAi pathway , we examined the expression profiles of the upregulated ( 200 ) and downregulated ( 112 ) genes in both the Fgdicer1 Fgdicer2 and Fgago1 Fgago2 mutants compared to those of the wild type .', 'Raw RNA-seq data from F . graminearum sexual developmental stages were obtained from a previous study [8] , and the time-course expression patterns during sexual development were clustered into 10 groups using the R package Mfuzz [39] with the default setting , which performs fuzzy c-means clustering ( S3 Fig and S3 Table ) .', 'Mating-type genes ( MATs ) are important sex-specific upstream regulators that orchestrate sexual reproduction processes in F . graminearum , and the RNAi pathway was proposed to be a downstream regulatory mechanism of MATs [9] .', 'MAT1-1-2 and MAT1-1-3 were included in group 2 , and MAT1-1-1 and MAT1-2-1 were members of group 9 ( Fig 3A and S3 Table ) .', 'More than half of the upregulated genes were included in groups 2 or 9 , in which the transcript levels were decreased at 4 days after sexual induction ( Fig 3A and S4 Table ) .', 'Group 5 possessed approximately 40% of the downregulated genes , and the transcript levels of group 5 genes were generally increased 4 days after sexual induction ( Fig 3A ) .', 'In short , the RNAi pathway affects the transcript abundance of genes closely related to the MAT-mediated regulatory mechanism during the late stages of sexual development in F . graminearum .', 'Our transcriptome analysis revealed that the RNAi pathway affects several important sexual reproduction-related molecular processes in F . graminearum ( S2 Table ) .', 'Pheromone precursor gene PPG1 ( Gene ID: FGSG_05061 ) , two putative transcription factors ( Gene ID: FGSG_01366 and FGSG_16753 ) , and transaldolase ( Gene ID: FGSG_13162 ) , which are known to be important for sexual development , were significantly downregulated in RNAi-deficient mutants [9 , 10 , 42] .', 'Moreover , three of these genes ( FGSG_01366 , FGSG_13162 , and FGSG_16753 ) are under the control of MAT genes in F . graminearum [9] .', 'In addition , 11 fungal-specific transcription factors with a Zn ( II ) 2Cys6 fungal-type DNA-binding domain ( FGSG_01760 , FGSG_03892 , FGSG_03912 , FGSG_04782 , FGSG_04786 , FGSG_09111 , FGSG_11186 , FGSG_11358 , FGSG_11672 , FGSG_12597 , and FGSG_16075 ) were downregulated in RNAi component mutants; null mutants of these factors did not show any mutant phenotype [10] .', 'Gene Ontology ( GO ) enrichment analysis was additionally applied to classify the functions of the predicted DEGs , and the GO terms were statistically analyzed using GOstats [40] and subsequently visualized using REVIGO [41] .', 'REVIGO provides a network structure of non-redundant GO terms ( Fig 3B and 3C ) .', 'Although we did not find any functionally characterized genes from the upregulated DEGs , they were assigned to 62 categories; 27 were included in biological processes , 26 in molecular functions , and 9 in cellular components .', 'Accordingly , the most significant GO terms were related to the transport of organic compounds such as “amino acid transport” and “carbohydrate transport” of biological processes and "transmembrane transporter activity" of molecular functions ( Fig 3B ) .', 'Downregulated genes were assigned to 76 categories ( 36 in biological processes , 36 in molecular functions , and 4 in cellular components ) .', 'Most GO terms corresponding to negatively regulated genes were involved in carbohydrate catabolism , such as “carbohydrate catabolism” and “polysaccharide catabolism” ( Fig 3C ) .', 'Based on our GO enrichment analysis , we demonstrated that one of the important molecular processes regulated by the RNAi pathway is carbon metabolism .', 'Production of triacylglycerol is important for perithecia development , and therefore , dynamic changes in carbon metabolism should occur during sexual reproduction processes in F . graminearum [43] .', 'Accordingly , acetyl coenzyme A production and translocation between cellular organelles are closely involved in various steps of sexual development in F . graminearum [44–47] .', 'In our transcriptome data , transaldolase ( Gene ID: FGSG_13162 ) is involved in the pentose-phosphate pathway , and ascospore formation [9] was markedly downregulated in RNAi-deficient mutants ( S2 Table ) .', 'To determine the relationship between sRNA production and transcriptome alteration , we isolated total low-molecular-weight RNAs from the wild-type and RNAi component mutant strains 5 days after sexual induction and used them for sRNA sequencing ( S5 Table ) .', 'After the adaptor sequences were trimmed , raw reads were normalized using DESeq to adjust for differences in library sizes [48] .', 'Total reads with 18–32 nt perfect matches were used for alignment to the genome of F . graminearum [4 , 49] .', 'The size distributions and 5′ end frequencies of sRNAs were analyzed .', 'Identified sRNAs were 18–32 nt long , with a clear peak at approximately 24 nt in the F . graminearum wild-type strain ( Fig 4A ) .', 'The sRNAs with a 5′ end with U ( 5′-U ) accounted for 70% of the total sRNAs in the wild-type strain and were mostly 21–26 nt long ( Fig 4A and 4B ) .', 'Deletion of either FgDICER1 , FgDICER2 , or FgAGO2 attenuated the biases of sRNAs toward specific sizes ( 21–26 nt ) and 5′-U; these changes were milder in Fgdicer1 than those in Fgdicer2 and Fgago2 .', 'Deletion of FgAGO1 did not result in distinct changes in sRNA characteristics , but results for the Fgago2 mutant were similar to those of Fgdicer2 ( Fig 4B ) .', 'Fgdicer1 Fgdicer2 and Fgago1 Fgago2 mutants typically abolished these biases .', 'Taken together , these findings showed that F . graminearum has distinct FgDicer1- , FgDicer2- , or FgAgo2-dependent sRNA production/accumulation mechanisms and that these sRNAs have strong tendencies toward being 24 nt long and 5′-U .', 'Moreover , both members of Dicers and Argonautes have functional redundancies .', 'To investigate the origin of sRNAs , we mapped sRNA sequences to the F . graminearum genome [4] with a recent annotation [49] to identify their various genomic features , such as coding sequences of rRNA , tRNA , and predicted proteins as well as intergenic regions ( S6 Table ) .', 'In the wild-type strain , sRNAs were in the rRNA region composed of approximately 40% of the total sRNAs ( Fig 4C ) .', 'Approximately 30% and 27% of the sRNAs mapped to protein-coding genes and intergenic regions , respectively .', 'sRNA-producing loci frequently covered both protein-coding sequences and adjacent regions .', 'Because there is little information about the untranslated region ( UTR ) in a recent annotation of F . graminearum [49] , 500 bp upstream and downstream regions were arbitrarily denoted as 5′-UTR and 3′-UTR , respectively .', 'Thereafter , 27% of the intergenic sequences were again separated into 16% of the UTRs and 11% of the intergenic sequences; predicted sRNAs corresponding to putative transcript sequences ( protein-coding regions and UTRs ) accounted for 46% ( Fig 4C ) .', 'Henceforth , we will call these sRNAs exonic siRNAs ( ex-siRNAs ) , as previously reported [50] .', 'When Dicer or Argonaute genes were deleted , the ratios of most genomic features aligned with sRNAs , except that the rRNA , protein-coding gene ( sense strand ) , and UTR ( sense strand ) , were decreased with analogous tendencies shown for size distributions and 5′-U in the corresponding deletion mutants ( Fig 4C ) .', 'In particular , sRNAs mapped to antisense sequences of putative transcripts were markedly reduced in the Fgdicer1 Fgdicer2 and Fgago1 Fgago2 mutants .', 'Various genomic features of sRNA-producing sequences were further assessed in detail with total normalized reads .', 'The sRNAs produced from protein-coding genes converged on exons rather than introns ( Fig 4D ) .', 'The total amounts of sRNAs were decreased in the Fgdicer1 , Fgdicer2 , Fgdicer1 Fgdicer2 , Fgago2 , and Fgago1 Fgago2 mutants , principally because of the reduced number of introns , exons , and UTRs with an antisense direction .', 'Dicer- and Argonaute-dependent sRNAs with 5′-U ( 22–25 nt ) were particularly enriched in antisense sequences of putative transcripts ( Fig 4D and S6 Table ) .', 'Moreover , the results confirmed that Dicers and Argonautes have redundant roles .', 'Interestingly , total counts of sRNAs with antisense sequences of tRNA and rRNA were also markedly reduced in the Fgdicer1 Fgdicer2 and Fgago1 Fgago2 mutants , primarily because of a decreased number of sRNAs with 5′-U .', 'The results suggest that Dicer- and Argonaute-dependent biogenesis/accumulation of sRNAs occurs at a genome-wide level .', 'As most sRNAs were produced from gene regions , except for rRNA-mediated sRNAs , we focused on the characteristics of sRNA-producing genes ( S7 Table ) .', 'The F . graminearum wild-type strain produces sRNAs from 5180 genes , and 80% ( 4155/5180 ) of the genes produced only sense-specific sRNAs , suggesting that sense sRNAs were byproducts of mRNA degradation ( Fig 5A and S8 Table ) .', 'Accordingly , the number of genes producing only sense sRNAs was similar among the wild-type and RNAi component mutant strains .', 'We investigated 5′-U sRNA-producing genes from F . graminearum strains ( Fig 5B and S8 Table ) .', 'The genes were selected when the amount of 5′-U sRNA was more than 70% of the total sRNAs ( S7 and S8 Tables ) .', 'Approximately 1000 genes preferentially produced 5′-U sRNAs in the F . graminearum wild-type strain .', 'Among them , more than half of the genes generated sRNAs with both orientations , and 10% of the genes produced only sense-specific sRNAs .', 'A markedly reduced number of genes produced 5′-U sRNAs in Fgdicer2 and Fgago2 compared to those of the wild type , whereas the Fgdicer1 mutant showed a slight reduction in the number of 5′-U sRNA-producing genes .', 'Double deletion of FgDICER1 and FgDICER2 largely abolished 5′-U sRNA production .', 'Genes producing antisense-specific or sense-specific sRNAs were counted in the Fgago1 Fgago2 mutant .', 'Taken together , these findings show that most sRNAs originating from the gene region with a 5′-U or antisense direction were distinctly produced in a Dicer-dependent manner .', 'We investigated whether sRNAs affected the transcript levels of corresponding genes in F . graminearum .', 'First , all genes were categorized depending on the levels of sense or antisense sRNA counts , and they were applied in correlation analyses of transcript abundance between Fgdicer1 Fgdicer2 and Fgago1 Fgago2 versus the wild-type strain ( Fig 5C ) .', 'As the genes produced more sense or antisense sRNAs , a greater number of genes tended to accumulate more transcripts in Fgdicer1 Fgdicer2 and Fgago1 Fgago2 than those in the wild type .', 'In particular , most genes producing either sense or antisense sRNAs at more than 1000 counts per kilobase ( red dots in Fig 5C ) were upregulated in the RNAi-deficient mutants .', 'To analyze the negative correlation between sRNA production and transcript amounts of genes in detail , we assessed the gene numbers depending on transcript abundance in Fgdicer1 Fgdicer2 or Fgago1 Fgago2 versus the wild-type strain ( Fig 5D ) .', 'Whereas both sense and antisense sRNAs produced below 100 counts per kilobase did not show substantial changes in transcript levels of the corresponding genes , most genes producing antisense sRNAs at more than 1000 counts per kilobase ( red graphs ) were positively regulated in Fgdicer1 Fgdicer2 and Fgago1 Fgago2 compared to those in the wild type ( Fig 5D ) .', 'Both sense and antisense 5′-U sRNA ( 22–25 nt ) -producing genes showed similar results to those obtained from antisense sRNAs ( S4 Fig ) .', 'Taken together , these findings show that a sRNA-mediated gene regulatory mechanism is not a critical factor that determines final transcript levels of most genes , but highly expressed sRNAs with antisense orientations participate in the negative transcriptional gene regulation of corresponding genes at a genome-wide level .', 'Although we found that F . graminearum possesses a global negative transcriptional regulatory mechanism involving antisense sRNAs during sexual development , only 57 genes produced antisense sRNAs among 200 upregulated DEGs ( S2 and S7 Tables ) .', 'Instead , a defective phenotype in ascospore production of RNAi-deficient mutants appears to be derived by the downregulation of sexual reproduction-related genes , such as PPG1 ( Gene ID: FGSG_05061 ) , transcription factors ( Gene ID: FGSG_01366 and FGSG_16753 ) , and transaldolase ( Gene ID: FGSG_13162 ) , as noted above [9 , 10 , 42] .', 'Thus , genes with substantial changes in transcript levels were not directly affected by RNAi-mediated gene regulation .', 'Thirty-two transcription factor genes are possibly modulated by antisense sRNAs , and three of them ( FGSG_01022 , FGSG_09654 , and MAT1-2-1 ) are closely related to perithecia development in F . graminearum [10] .', 'In particular , RNAi participates in the regulation of one of the MATs , MAT1-2-1 , during sexual development .', 'In the case of kinases , another important mediator for signal transduction pathways , four genes ( FGSG_00469 , FGSG_16383 , FGSG_05418 , and FGSG_16493 ) produced antisense sRNAs , and one of them ( FGSG_05418 ) was shown to be important for ascospore formation in F . graminearum [11] .', 'Although most of the mentioned genes involved in sexual development are known to be related to the perithecium development or the early stages of sexual development , our additional transcript analysis during sexual development demonstrated that they also have important functions during the late stages of sexual development ( S5 Fig ) .', 'In particular , most of tested genes were dynamically changed in expression approximately 5 days after sexual induction , and these patterns of several genes were altered in RNAi-deficient mutants compared to those of the wild type .', 'Thus , the RNAi mechanism fine-tunes the transcriptome to modulate various molecular processes , including signal transduction networks , during ascospore production in F . graminearum .', 'We visualized patterns of sequence alignments at the ex-siRNA-producing genes using Integrative Genomics Viewer ( IGV ) [51] .', 'The IGV image of the gene ( FGSG_11711 ) showed that ex-siRNAs are produced over the entire ranges of the gene ( S6 Fig ) , demonstrating that total amount of siRNAs of the gene does not always reflect abundances of specific ex-siRNAs .', 'Therefore , we identified ex-siRNAs with over 500 raw counts ( S9 Table ) .', 'Since repeated trials of the standard Northern blot analysis of six highly expressed ex-siRNAs ( S7 Fig ) were unsuccessful in detecting signals , we quantified the absolute amount of the ex-siRNA with the highest expression ( siRNA of FGSG_09213 ) using stem-loop RT-PCR ( S8 Fig ) .', 'Whereas Northern blot analysis of our system could detect 1–10 fmoles synthetic siRNA of FGSG_09213 , approximately 0 . 05 fmoles of siRNAs were detected in 100 ng of small RNA-enriched RNA samples from the F . graminearum wild type .', 'Thereafter , we analyzed siRNAs using stem-loop RT-PCR ( Fig 6 ) .', 'Abundances of siRNAs in the wild-type and the other mutant strains determined by RT-PCR were mostly consistent with our sRNA-seq results ( S7 Fig and Fig 7A ) .', 'We also verified that transcript levels of FGSG_10502 and FGSG_03222 were negatively correlated with corresponding ex-siRNA production ( Fig 7B ) .', 'We analyzed the degradome of the F . graminearum wild-type strain to elucidate the mechanism for genome-wide sRNA-mediated gene silencing .', 'Total RNAs were isolated from the wild-type strain 5 days after sexual induction , and RNA fragments with a 5′ monophosphate and polyA tail , which were cleaved products of transcripts , were subjected to the degradome library construction and deep sequencing .', 'In our degradome sequencing method , we expected 16 nt reads after trimming the adapter sequences , and enriched reads of 16 nt and 17 nt were further used for alignment ( S10 Table ) .', 'Approximately 95% of the trimmed sequences from two independent degradome libraries perfectly matched one or more positions in the F . graminearum genome ( S11 Table ) .', 'To monitor transcript degradation , we used degradome tags with a sense direction for further analyses .', 'The libraries of degradome tags were markedly biased toward the 3′ ends of protein coding sequences ( CDS ) and 3′-UTR regions ( Fig 8A ) .', 'Specifically , most degradome tags were enriched in the exon and 3′-UTR regions ( Fig 8B and S11 Table ) .', 'The average of the total abundances of degradome tags for the CDS and 3′-UTR regions was calculated as reads per kilobase of sequence per two billion mapped sequence reads with a cutoff value of 10 ( S12 Table ) .', 'Comparison of the degradome abundance of the wild-type strain to the transcript values of the wild-type and RNAi component mutant strains similarly showed a distinct positive correlation ( Fig 8C ) .', 'Accordingly , genes with higher degradome counts tended to accumulate more transcripts in Fgdicer1 Fgdicer2 and Fgago1 Fgago2 than those in the wild type ( Fig 8D ) .', 'Taken together , these findings revealed that degradome tag abundance is primarily determined by the level of the corresponding gene transcript in F . graminearum .', 'The only known mechanism for siRNA-mediated gene silencing in filamentous fungi is siRNA-guided mRNA degradation [34] .', 'As siRNA-guided endonucleolytic cleavage events should lead to the rapid decay of transcripts [52] , we investigated the correlation between the abundance of degradome tags and antisense sRNA-mediated gene regulation .', 'As genes produced more sRNAs with an antisense direction , the absolute number of degraded transcripts increased in the F . graminearum wild-type strain ( Fig 8E ) .', 'As the degradome generally reflects transcript levels , the abundances of degradome tags were normalized with transcript counts , and total genes were listed in the order of their given degradome tags:transcript counts ratios .', 'When a higher portion of transcripts was degraded in the wild-type strain ( higher percentile rank classes ) , corresponding genes tended to be upregulated in Fgdicer1 Fgdicer2 mutants ( Fig 8F ) , suggesting that Dicer-dependent gene regulation globally triggers transcript degradation in this fungus .', 'When sRNA-producing genes were evenly distributed among the percentile rank classes , markedly reduced and increased portions of genes produced sRNAs in the down- ( Fgdicer1 Fgdicer2/Z-3639 < 1 ) and upregulated ( Fgdicer1 Fgdicer2/Z-3639 > 1 ) gene groups , respectively ( Fig 8G ) .', 'More than 70% of the genes were concentrated in the top 20% of the percentile rank class in the genes that were upregulated more than 2-fold in Fgdicer1 Fgdicer2 compared to that of the wild type ( Fgdicer1 Fgdicer2/Z-3639 > 2; Fig 8G ) .', 'Taken together , these results show that RNAi-mediated negative gene regulation occurs post-transcriptionally by degrading corresponding gene transcripts during sexual reproduction in F . graminearum .'], ['Fungi have evolved several un
['Introduction', 'Results', 'Discussion', 'Methods']
['Various ascomycete fungi possess sex-specific molecular mechanisms , such as repeat-induced point mutations , meiotic silencing by unpaired DNA , and unusual adenosine-to-inosine RNA editing , for genome defense or gene regulation .', 'Using a combined analysis of functional genetics and deep sequencing of small noncoding RNA ( sRNA ) , mRNA , and the degradome , we found that the sex-specifically induced exonic small interference RNA ( ex-siRNA ) -mediated RNA interference ( RNAi ) mechanism has an important role in fine-tuning the transcriptome during ascospore formation in the head blight fungus Fusarium graminearum .', 'Approximately one-third of the total sRNAs were produced from the gene region , and sRNAs with an antisense direction or 5′-U were involved in post-transcriptional gene regulation by reducing the stability of the corresponding gene transcripts .', 'Although both Dicers and Argonautes partially share their functions , the sex-specific RNAi pathway is primarily mediated by FgDicer1 and FgAgo2 , while the constitutively expressed RNAi components FgDicer2 and FgAgo1 are responsible for hairpin-induced RNAi .', 'Based on our results , we concluded that F . graminearum primarily utilizes ex-siRNA-mediated RNAi for ascosporogenesis but not for genome defenses and other developmental stages .', 'Each fungal species appears to have evolved RNAi-based gene regulation for specific developmental stages or stress responses .', 'This study provides new insights into the regulatory role of sRNAs in fungi and other lower eukaryotes .']
['Control of gene expression by small noncoding RNA ( sRNA ) has recently been highlighted as a significant post-transcriptional regulatory mechanism .', 'To date , researchers have predominantly focused on the identification of microRNA-like RNAs ( milRNAs ) in fungi because microRNAs ( miRNAs ) are key regulators in animals and plants .', 'In this study , we discovered that the sex-induced RNA interference ( RNAi ) mechanism had important roles in sexual reproduction in the head blight fungus Fusarium graminearum .', 'In the late stages of sexual reproduction , small interference RNAs that were produced from gene regions ( ex-siRNAs ) were involved in post-transcriptional gene regulation at a genome-wide level .', 'Based on our results , we concluded that F . graminearum specifically utilizes ex-siRNA-mediated RNAi for sexual development but not for other biological processes .', 'This is the first genome-wide characterization of the sRNAs involved in fungal development .']
['rna interference', 'fungal genetics', 'gene regulation', 'developmental biology', 'fungi', 'genome analysis', 'epigenetics', 'morphogenesis', 'small interfering rnas', 'mycology', 'genetic interference', 'gene expression', 'biochemistry', 'rna', 'fungal genomics', 'nucleic acids', 'sexual differentiation', 'genetics', 'transcriptome analysis', 'biology and life sciences', 'genomics', 'non-coding rna', 'computational biology', 'organisms']