Background: 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 as well as transcriptional gene silencing in some fungal species. This suggests a more complex interplay of the two pathways at least in some fungi. Because many fungal species are important pathogens, RNA silencing is a useful technique to study and 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.

Results: In the necrotrophic plant pathogen F. oxysporum, several different hpRNA transgenes targeting either a β-glucuronidase (Gus) reporter transgene or the endogenous gene Frp1 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 precursor transcript and consequently in low siRNA production. However, we show that 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. In such a case hairpin expression levels are high, allowing siRNA production. These siRNAs then 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.

Conclusions: These results indicate that F. oxysporum possesses functional RNA silencing machineries for siRNA production and target mRNA cleavage. However, hpRNA transgenes may induce transcriptional self-silencing due to their inverted-repeat structures. 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.