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Analysis of hairpin RNA transgene-induced gene silencing in Fusarium oxysporum
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Titel: |
Analysis of hairpin RNA transgene-induced gene silencing in Fusarium oxysporum |
In: | Silence, 4, 2013, 1 |
veröffentlicht: |
Springer Science and Business Media LLC
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ISSN: |
1758-907X |
DOI: | 10.1186/1758-907x-4-3 |
Zusammenfassung: | <jats:title>Abstract</jats:title> <jats:sec> <jats:title>Background</jats:title> <jats:p>Hairpin RNA (hpRNA) transgenes can be effective at inducing RNA silencing and have been exploited as a powerful tool for gene function analysis in many organisms. However, in fungi, expression of hairpin RNA transcripts can induce post-transcriptional gene silencing, but in some species can also lead to transcriptional gene silencing, suggesting a more complex interplay of the two pathways at least in some fungi. Because many fungal species are important pathogens, RNA silencing is a powerful technique to understand gene function, particularly when gene knockouts are difficult to obtain. We investigated whether the plant pathogenic fungus <jats:italic>Fusarium oxysporum</jats:italic> possesses a functional gene silencing machinery and whether hairpin RNA transcripts can be employed to effectively induce gene silencing.</jats:p> </jats:sec> <jats:sec> <jats:title>Results</jats:title> <jats:p>Here we show that, in the phytopathogenic fungus <jats:italic>F. oxysporum</jats:italic>, hpRNA transgenes targeting either a β-glucuronidase (<jats:italic>Gus</jats:italic>) reporter transgene (<jats:italic>hpGus</jats:italic>) or the endogenous gene <jats:italic>Frp</jats:italic>1 (<jats:italic>hpFrp</jats:italic>) did not induce significant silencing of the target genes. Expression analysis suggested that the hpRNA transgenes are prone to transcriptional inactivation, resulting in low levels of hpRNA and siRNA production. However, the <jats:italic>hpGus</jats:italic> RNA can be efficiently transcribed by promoters acquired either by recombination with a pre-existing, actively transcribed <jats:italic>Gus</jats:italic> transgene or by fortuitous integration near an endogenous gene promoter allowing siRNA production. These siRNAs effectively induced silencing of a target <jats:italic>Gus</jats:italic> transgene, which in turn appeared to also induce secondary siRNA production. Furthermore, our results suggested that hpRNA transcripts without poly(A) tails are efficiently processed into siRNAs to induce gene silencing. A convergent promoter transgene, designed to express poly(A)-minus sense and antisense <jats:italic>G</jats:italic>us RNAs, without an inverted-repeat DNA structure, induced consistent <jats:italic>Gus</jats:italic> silencing in <jats:italic>F. oxysporum</jats:italic>.</jats:p> </jats:sec> <jats:sec> <jats:title>Conclusions</jats:title> <jats:p>These results indicate that <jats:italic>F. oxysporum</jats:italic> possesses functional RNA silencing machineries for siRNA production and target mRNA cleavage, but hpRNA transgenes may induce transcriptional self-silencing due to its inverted-repeat structure. Our results suggest that <jats:italic>F. oxysporum</jats:italic> possesses a similar gene silencing pathway to other fungi like fission yeast, and indicate a need for developing more effective RNA silencing technology for gene function studies in this fungal pathogen.</jats:p> </jats:sec> |
Format: | E-Article |
Quelle: | Springer Science and Business Media LLC (CrossRef) |
Sprache: | Englisch |