Evidence of small interfering RNA in the fungus Fusarium fujikuroi (miRNA-Seq)
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ABSTRACT: RNA interference (RNAi) mechanisms play key regulatory roles in many biological systems, and components of the RNAi pathway are conserved in a wide range of eukaryotic genomes, including those of filamentous fungi. The biotechnological fungus Fusarium fujikuroi, widely used in secondary metabolism studies, contains the complete set of genes expected for RNAi pathways, including dcl1 and dcl2 dicer genes. We have analyzed by means of a high-throughput sequencing technology the content of sRNAs in F. fujikuroi grown in the dark or after one hour of illumination. For comparative purposes, the study was extended to the phytopathogenesis model Fusarium oxysporum, grown under the same conditions. Total RNA samples from each species and growth condition were used to construct RNA libraries, which were subjected to massive sequencing. sRNA preparations included a size cut-off below 150 nt, which covered sRNAs and their precursors. The size distributions and 5' nucleotide preferences of the sRNA reads showed a higher proportion of 5' uracil in the RNA samples of the expected sizes in both species, more noticeable in F. fujikuroi, indicating the occurrence of genuine sRNAs. Consistently, the number of sRNAs mapped at CDS loci was significantly higher in F. fujikuroi compared to F. oxysporum. F. fujikuroi carries at least one transcriptionally expressed copy of a Ty1/copia-like retrotransposable element, in which sRNAs were found in both sense and antisense DNA strands, whereas in F. oxysporum Skippy-like elements are expressed and show siRNA formation. The finding of sRNA in these mobile elements is an indication of an active siRNA-based RNAi pathway. The dcl2 deletion mutants did not show phenotypic alterations or changes in their global transcriptome, while no dcl1 deletion mutants could be obtained.
ORGANISM(S): Fusarium oxysporum f. sp. lycopersici Fusarium fujikuroi
Project description:RNA interference (RNAi) mechanisms play key regulatory roles in many biological systems, and components of the RNAi pathway are conserved in a wide range of eukaryotic genomes, including those of filamentous fungi. The biotechnological fungus Fusarium fujikuroi, widely used in secondary metabolism studies, contains the complete set of genes expected for RNAi pathways, including dcl1 and dcl2 dicer genes. We have analyzed by means of a high-throughput sequencing technology the content of sRNAs in F. fujikuroi grown in the dark or after one hour of illumination. For comparative purposes, the study was extended to the phytopathogenesis model Fusarium oxysporum, grown under the same conditions. Total RNA samples from each species and growth condition were used to construct RNA libraries, which were subjected to massive sequencing. sRNA preparations included a size cut-off below 150 nt, which covered sRNAs and their precursors. The size distributions and 5' nucleotide preferences of the sRNA reads showed a higher proportion of 5' uracil in the RNA samples of the expected sizes in both species, more noticeable in F. fujikuroi, indicating the occurrence of genuine sRNAs. Consistently, the number of sRNAs mapped at CDS loci was significantly higher in F. fujikuroi compared to F. oxysporum. F. fujikuroi carries at least one transcriptionally expressed copy of a Ty1/copia-like retrotransposable element, in which sRNAs were found in both sense and antisense DNA strands, whereas in F. oxysporum Skippy-like elements are expressed and show siRNA formation. The finding of sRNA in these mobile elements is an indication of an active siRNA-based RNAi pathway. The dcl2 deletion mutants did not show phenotypic alterations or changes in their global transcriptome, while no dcl1 deletion mutants could be obtained.
Project description:In the fungi Fusarium fujikuroi and Fusarium oxysporum, the induction of carotenogenesis by light and its deregulation in carS mutants, affected in a protein of the RING-finger family, are achieved on transcription of the structural genes of the pathway, some of them organized in a cluster. We have carried out global RNAseq transcriptomics analyses to investigate the relationship between the regulatory effects of light and the carS mutation. Either illumination or the absence of a functional carS gene exert wide effects on the transcriptome of F. fujikuroi, with a predominance of activated over repressed genes, and a greater functional diversity in the case of genes induced by light. The number of the latter decreases drastically in the carS mutant, indicating that the deregulation produced by the carS mutation affects the light response of many genes. In addition, light and CarS strongly influence the expression of some genes associated with stress responses. The effects of light and carS mutation on the F. oxysporum transcriptome were partially coincident with those in F. fujikuroi, indicating conservation of the objectives of their regulatory mechanisms. In conclusion, the CarS RING finger protein down-regulates many genes whose expression is up-regulated by light in the wild-type strains of the two Fusarium species investigated, indicating regulatory connections between the control by light and by the CarS protein.
Project description:Here we describe the identification and regulation of a novel dsRNA virus in Colletotrichum higginsianum. High throughput sequencing of small RNAs and strand-specific RNA-seq was performed on single gene knock-out mutants created for each RNAi component gene: rdr1, rdr2, rdr3, dcl1, dcl2, ago1, and ago2, and the double mutant: ∆dcl1∆dcl2. De novo assembly of the ∆dcl1 RNA-seq data identified two contigs that represented the forward and reverse strands of an uncharacterized dsRNA virus, designated here as Colletotrichum higginsianum non-segmented dsRNA virus 1 (ChNRV1). We found increased presence of the viral RNA in the RNA-seq datasets of the ∆dcl1, ∆dcl1dcl2, and ∆ago1 strains, suggesting that these genes are required for control of the virus. We show that viral small RNAs co-immunoprecipitate with a 6xFLAG-3xHIS-tagged AGO1 protein by sequencing the small RNAs from immunoprecipitated fractions. Additionally, analyses of the small RNA datasets from the RNAi mutants revealed control of the virus through small RNA-mediated silencing required both AGO1 and DCL1.
Project description:Here we describe the identification and regulation of a novel dsRNA virus in Colletotrichum higginsianum. High throughput sequencing of small RNAs and strand-specific RNA-seq was performed on single gene knock-out mutants created for each RNAi component gene: rdr1, rdr2, rdr3, dcl1, dcl2, ago1, and ago2, and the double mutant: ∆dcl1∆dcl2. De novo assembly of the ∆dcl1 RNA-seq data identified two contigs that represented the forward and reverse strands of an uncharacterized dsRNA virus, designated here as Colletotrichum higginsianum non-segmented dsRNA virus 1 (ChNRV1). We found increased presence of the viral RNA in the RNA-seq datasets of the ∆dcl1, ∆dcl1dcl2, and ∆ago1 strains, suggesting that these genes are required for control of the virus. We show that viral small RNAs co-immunoprecipitate with a 6xFLAG-3xHIS-tagged AGO1 protein by sequencing the small RNAs from immunoprecipitated fractions. Additionally, analyses of the small RNA datasets from the RNAi mutants revealed control of the virus through small RNA-mediated silencing required both AGO1 and DCL1.
Project description:Here we describe the identification and regulation of a novel dsRNA virus in Colletotrichum higginsianum. High throughput sequencing of small RNAs and strand-specific RNA-seq was performed on single gene knock-out mutants created for each RNAi component gene: rdr1, rdr2, rdr3, dcl1, dcl2, ago1, and ago2, and the double mutant: ∆dcl1∆dcl2. De novo assembly of the ∆dcl1 RNA-seq data identified two contigs that represented the forward and reverse strands of an uncharacterized dsRNA virus, designated here as Colletotrichum higginsianum non-segmented dsRNA virus 1 (ChNRV1). We found increased presence of the viral RNA in the RNA-seq datasets of the ∆dcl1, ∆dcl1dcl2, and ∆ago1 strains, suggesting that these genes are required for control of the virus. We show that viral small RNAs co-immunoprecipitate with a 6xFLAG-3xHIS-tagged AGO1 protein by sequencing the small RNAs from immunoprecipitated fractions. Additionally, analyses of the small RNA datasets from the RNAi mutants revealed control of the virus through small RNA-mediated silencing required both AGO1 and DCL1.
Project description:MicroRNAs (miRNAs) are a class of endogenous non-coding small RNAs that regulate targeted mRNAs by degrading or repressing translation, considered as post-transcrption regulators. So far, a large number of miRNAs have been discovered in model plants, but little information is available on miRNAs in banana. In this study, by sequencing the small RNA (sRNA) transcriptomes of Fusarium wilt resistant and susceptible banana varieties, 139 members in 38 miRNA families were discovered, and six out of eight new miRNAs were confirmed by RT-PCR. According to the analysis of sRNA transcriptome data and qRT-PCR verification, some miRNAs were differentially expressed between Fusarium wilt resistant and susceptible banana varieties. Two hundred and ninety-nine and 31 target genes were predicted based on the draft maps of banana B genome and Fusarium oxysporum (FOC1, FOC4) genomes respectively. Specifically, two important pathogenic genes in Fusarium oxysporum genomes, feruloyl esterase gene and proline iminopeptidase gene, were targeted by banana miRNAs. These novel findings may provide a new strategy for the prevention and control of Fusarium wilt in banana.
Project description:We sequenced Endogenous short RNAs in Mucor circinelloides fungus grown in standard liquid culture. Short RNAs were profiled in wild type, Dicer-like 1 mutant (dcl1-), Dicer-like 2 mutant (dcl2-) and double Dicer mutant (dcl1-/dcl2-) strains. We identified many loci that produced less short RNAs in the dcl2- strain suggesting that DCL2 is the major protein generating short RNAs in Mucor circinelloides.
Project description:We sequenced Endogenous short RNAs in Mucor circinelloides fungus grown in standard liquid culture. Short RNAs were profiled in wild type, Dicer-like 1 mutant (dcl1-), Dicer-like 2 mutant (dcl2-) and double Dicer mutant (dcl1-/dcl2-) strains. We identified many loci that produced less short RNAs in the dcl2- strain suggesting that DCL2 is the major protein generating short RNAs in Mucor circinelloides. Endogenous short RNAs were profiled in wild type, single and double dcl mutant strains.
Project description:Investigation of whole genome gene expression level differences of Fusarium fujikuroi between wild-type and a Ffvel1 (velvet) deletion mutant in liquid medium with minimal nitrogen between 24 hr, 72 hr and 120 hr of growth using an array based on a F. verticillioides gene call set. Fusarium fujikuroi produces a number of secondary metabolites including gibberellins, bikaverin, fumonisin and fusarin C that are influenced by nitrogen availability and the velvet global regulatory complex. A twelve chip study using total RNA recovered from six cultures of wild-type Fusarium fujikuroi and six cultures of Ffvel1 F. fujikuroi deletion mutant. Each chip measures the expression level of over 13,000 putative genes with twelve 60-mer probes per sequence.