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Gene silencing in the marine diatom Phaeodactylum tricornutum.

De Riso V, Raniello R, Maumus F, Rogato A, Bowler C, Falciatore A - Nucleic Acids Res. (2009)

Bottom Line: We report the successful silencing of a GUS reporter gene expressed in transgenic lines, as well as the knockdown of endogenous phytochrome (DPH1) and cryptochrome (CPF1) genes.Initial molecular analyses reveal that targeted downregulation likely occurs through transcriptional and post-transcriptional gene silencing mechanisms.Interestingly, molecular players involved in RNA silencing in other eukaryotes are only poorly conserved in diatoms.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Ecology and Evolution of Plankton, Stazione Zoologica Anton Dohrn, Villa Comunale, 80121, Napoli, Italy.

ABSTRACT
Diatoms are a major but poorly understood phytoplankton group. The recent completion of two whole genome sequences has revealed that they contain unique combinations of genes, likely recruited during their history as secondary endosymbionts, as well as by horizontal gene transfer from bacteria. A major limitation for the study of diatom biology and gene function is the lack of tools to generate targeted gene knockout or knockdown mutants. In this work, we have assessed the possibility of triggering gene silencing in Phaeodactylum tricornutum using constructs containing either anti-sense or inverted repeat sequences of selected target genes. We report the successful silencing of a GUS reporter gene expressed in transgenic lines, as well as the knockdown of endogenous phytochrome (DPH1) and cryptochrome (CPF1) genes. To highlight the utility of the approach we also report the first phenotypic characterization of a diatom mutant (cpf1). Our data open the way for reverse genetics in diatoms and represent a major advance for understanding their biology and ecology. Initial molecular analyses reveal that targeted downregulation likely occurs through transcriptional and post-transcriptional gene silencing mechanisms. Interestingly, molecular players involved in RNA silencing in other eukaryotes are only poorly conserved in diatoms.

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De novo cytosine methylation in silenced clones. Methylation analysis performed on the Pt/GUS expressing cells, and on selected silenced clones. (A) PCR amplifications performed on genomic DNA digested with McrBC, in the presence (+) and absence (–) of GTP, using primer sets specific for the GUS transgene and its regulatory regions. PCR analysis shows the amplification of the FcpBp region (PCR1), amplification of the first (PCR2) and second (PCR3) half of the GUS gene, the terminator region (PCR4), and amplification of the CPF1 gene used as control. Arrows indicate the bands corresponding to the expected amplification products. M, 1-kb DNA size marker. Schematic representation of the genomic region used for the analysis and the region targeted for silencing is indicated above. (B) Schematic representation of the methylation profile obtained by bisulfite sequencing of the fir-1 clone. Vertical bars show the distribution of mC in the sense and anti-sense strands. The targeted region is indicated.
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Figure 3: De novo cytosine methylation in silenced clones. Methylation analysis performed on the Pt/GUS expressing cells, and on selected silenced clones. (A) PCR amplifications performed on genomic DNA digested with McrBC, in the presence (+) and absence (–) of GTP, using primer sets specific for the GUS transgene and its regulatory regions. PCR analysis shows the amplification of the FcpBp region (PCR1), amplification of the first (PCR2) and second (PCR3) half of the GUS gene, the terminator region (PCR4), and amplification of the CPF1 gene used as control. Arrows indicate the bands corresponding to the expected amplification products. M, 1-kb DNA size marker. Schematic representation of the genomic region used for the analysis and the region targeted for silencing is indicated above. (B) Schematic representation of the methylation profile obtained by bisulfite sequencing of the fir-1 clone. Vertical bars show the distribution of mC in the sense and anti-sense strands. The targeted region is indicated.

Mentions: Small RNAs can also trigger transcriptional gene silencing (TGS) associated with DNA modification and chromatin remodelling (41,42). We therefore investigated the appearance of DNA methylation within the GUS gene of silenced clones using the McrBC methylation-restriction system (Figure 3A). The parental transgenic line did not show any difference in amplification of the GUS gene after McrBC treatment. By contrast, in silenced clones the partial or total failure to amplify the GUS gene from McrBC-treated genomic DNA indicated that the gene was indeed methylated. Reduction in PCR amplification was observed for the 5′ region (PCR2) and for the 3′ region (PCR3). As both the anti-sense and inverted-repeat constructs were only complementary to the 3′ end of the GUS gene, these results suggested a spreading of methylation along the gene sequence. Consequently, we also checked for possible methylation of the promoter and terminator regions of the chimeric GUS gene (PCR1 and 4, Figure 3A). While reduced amplification products were observed using primers specific for the terminator sequence (PCR4), no differences with the parental Pt/GUS strain were detected when amplifying the FcpBp promoter, suggesting that only the transcribed region was modified in the silenced clones.Figure 3.


Gene silencing in the marine diatom Phaeodactylum tricornutum.

De Riso V, Raniello R, Maumus F, Rogato A, Bowler C, Falciatore A - Nucleic Acids Res. (2009)

De novo cytosine methylation in silenced clones. Methylation analysis performed on the Pt/GUS expressing cells, and on selected silenced clones. (A) PCR amplifications performed on genomic DNA digested with McrBC, in the presence (+) and absence (–) of GTP, using primer sets specific for the GUS transgene and its regulatory regions. PCR analysis shows the amplification of the FcpBp region (PCR1), amplification of the first (PCR2) and second (PCR3) half of the GUS gene, the terminator region (PCR4), and amplification of the CPF1 gene used as control. Arrows indicate the bands corresponding to the expected amplification products. M, 1-kb DNA size marker. Schematic representation of the genomic region used for the analysis and the region targeted for silencing is indicated above. (B) Schematic representation of the methylation profile obtained by bisulfite sequencing of the fir-1 clone. Vertical bars show the distribution of mC in the sense and anti-sense strands. The targeted region is indicated.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2724275&req=5

Figure 3: De novo cytosine methylation in silenced clones. Methylation analysis performed on the Pt/GUS expressing cells, and on selected silenced clones. (A) PCR amplifications performed on genomic DNA digested with McrBC, in the presence (+) and absence (–) of GTP, using primer sets specific for the GUS transgene and its regulatory regions. PCR analysis shows the amplification of the FcpBp region (PCR1), amplification of the first (PCR2) and second (PCR3) half of the GUS gene, the terminator region (PCR4), and amplification of the CPF1 gene used as control. Arrows indicate the bands corresponding to the expected amplification products. M, 1-kb DNA size marker. Schematic representation of the genomic region used for the analysis and the region targeted for silencing is indicated above. (B) Schematic representation of the methylation profile obtained by bisulfite sequencing of the fir-1 clone. Vertical bars show the distribution of mC in the sense and anti-sense strands. The targeted region is indicated.
Mentions: Small RNAs can also trigger transcriptional gene silencing (TGS) associated with DNA modification and chromatin remodelling (41,42). We therefore investigated the appearance of DNA methylation within the GUS gene of silenced clones using the McrBC methylation-restriction system (Figure 3A). The parental transgenic line did not show any difference in amplification of the GUS gene after McrBC treatment. By contrast, in silenced clones the partial or total failure to amplify the GUS gene from McrBC-treated genomic DNA indicated that the gene was indeed methylated. Reduction in PCR amplification was observed for the 5′ region (PCR2) and for the 3′ region (PCR3). As both the anti-sense and inverted-repeat constructs were only complementary to the 3′ end of the GUS gene, these results suggested a spreading of methylation along the gene sequence. Consequently, we also checked for possible methylation of the promoter and terminator regions of the chimeric GUS gene (PCR1 and 4, Figure 3A). While reduced amplification products were observed using primers specific for the terminator sequence (PCR4), no differences with the parental Pt/GUS strain were detected when amplifying the FcpBp promoter, suggesting that only the transcribed region was modified in the silenced clones.Figure 3.

Bottom Line: We report the successful silencing of a GUS reporter gene expressed in transgenic lines, as well as the knockdown of endogenous phytochrome (DPH1) and cryptochrome (CPF1) genes.Initial molecular analyses reveal that targeted downregulation likely occurs through transcriptional and post-transcriptional gene silencing mechanisms.Interestingly, molecular players involved in RNA silencing in other eukaryotes are only poorly conserved in diatoms.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Ecology and Evolution of Plankton, Stazione Zoologica Anton Dohrn, Villa Comunale, 80121, Napoli, Italy.

ABSTRACT
Diatoms are a major but poorly understood phytoplankton group. The recent completion of two whole genome sequences has revealed that they contain unique combinations of genes, likely recruited during their history as secondary endosymbionts, as well as by horizontal gene transfer from bacteria. A major limitation for the study of diatom biology and gene function is the lack of tools to generate targeted gene knockout or knockdown mutants. In this work, we have assessed the possibility of triggering gene silencing in Phaeodactylum tricornutum using constructs containing either anti-sense or inverted repeat sequences of selected target genes. We report the successful silencing of a GUS reporter gene expressed in transgenic lines, as well as the knockdown of endogenous phytochrome (DPH1) and cryptochrome (CPF1) genes. To highlight the utility of the approach we also report the first phenotypic characterization of a diatom mutant (cpf1). Our data open the way for reverse genetics in diatoms and represent a major advance for understanding their biology and ecology. Initial molecular analyses reveal that targeted downregulation likely occurs through transcriptional and post-transcriptional gene silencing mechanisms. Interestingly, molecular players involved in RNA silencing in other eukaryotes are only poorly conserved in diatoms.

Show MeSH
Related in: MedlinePlus