Abstract
BackgroundHairpin 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 Fusarium oxysporum possesses a functional gene silencing machinery and whether hairpin RNA transcripts can be employed to effectively induce gene silencing.ResultsHere we show that, in the phytopathogenic fungus F. oxysporum, hpRNA transgenes targeting either a β-glucuronidase (Gus) reporter transgene (hpGus) or the endogenous gene Frp1 (hpFrp) 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 hpGus RNA can be efficiently transcribed by promoters acquired either by recombination with a pre-existing, actively transcribed Gus transgene or by fortuitous integration near an endogenous gene promoter allowing siRNA production. These siRNAs effectively induced silencing of a target Gus 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 Gus RNAs, without an inverted-repeat DNA structure, induced consistent Gus silencing in F. oxysporum.ConclusionsThese results indicate that F. oxysporum 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 F. oxysporum 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.
Highlights
Hairpin RNA transgenes can be effective at inducing RNA silencing and have been exploited as a powerful tool for gene function analysis in many organisms
Transformation of Fusarium oxysporum with a hairpin RNA construct does not result in silencing of a β-glucuronidase reporter gene In order to develop a reporter gene system for studying RNA silencing in Fusarium oxysporum, the F. oxysporum strain 5176 was transformed with a Gus construct under the regulatory control of the gpdA promoter (Figure 1)
The results presented in this paper demonstrate that unlike Ascomycete fungi Hairpin RNA (hpRNA) transgenes do not reliably lead to the production of small interfering RNA (siRNA) in F. oxysporum
Summary
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. RNA silencing is an evolutionary conserved molecular mechanism that functions in genome defense and stability and plays an important role in developmental regulation [1,2,3] This process is characterized by the production of double-stranded RNA (dsRNA) molecules, which are cleaved by a Dicer-like protein (Dcl) into 20. Dicer-independent small interfering RNAs (disiRNAs) and DNA damage-induced Qde2-interacting siRNAs (qiRNAs) have been identified in Neurospora crassa [7,8] but their occurrence in other fungal species is undetermined In another well-studied fungus, Schizosaccharomyces pombe (fission yeast) only a single set of silencing machinery genes exists (for example, a single Dcl and Ago), which appear to function in both transcriptional and post-transcriptional silencing pathways [12,13,14]. Plant and animal RNA silencing mutants, the miRNA pathway mutants, often display severe developmental defects
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