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Analysis of hairpin RNA transgene-induced gene silencing in Fusarium oxysporum.

Schumann U, Smith NA, Kazan K, Ayliffe M, Wang MB - Silence (2013)

Bottom Line: Here 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.These 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Commonwealth Scientific and Industrial Research Organisation Plant Industry, Clunies Ross Street, Canberra ACT 2601, Australia. ming-bo.wang@csiro.au.

ABSTRACT

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, 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.

Results: Here 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.

Conclusions: These 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.

No MeSH data available.


Related in: MedlinePlus

Schematic diagrams (not to scale) of transgenes introduced into Fusarium oxysporum. All Gus-derived sequences are shown in black, except a small region of the Gus ORF represented in grey, which is present in the full length Gus gene but absent in the hpGus constructs. The gpdA promoter is shown as an open arrow, while the transcription terminator sequence trpC is indicated as an open box. Sequences present in hairpin constructs of the Gfp and F. oxysporum Frp1 are shown as light grey regions in each construct, respectively. An intron from the pyruvate dehydrogenase kinase gene (pdk) is included in the hpGfp gene. The hygromycin phosphotransferase gene (hyg) and Streptomyces verticillius bleomycin gene (phle) were used as selectable markers for F. oxysporum transformation and are shown as dark grey arrows. The positions and expected fragment sizes of restriction endonuclease recognition sites used for DNA blot analyses are indicated.
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Figure 1: Schematic diagrams (not to scale) of transgenes introduced into Fusarium oxysporum. All Gus-derived sequences are shown in black, except a small region of the Gus ORF represented in grey, which is present in the full length Gus gene but absent in the hpGus constructs. The gpdA promoter is shown as an open arrow, while the transcription terminator sequence trpC is indicated as an open box. Sequences present in hairpin constructs of the Gfp and F. oxysporum Frp1 are shown as light grey regions in each construct, respectively. An intron from the pyruvate dehydrogenase kinase gene (pdk) is included in the hpGfp gene. The hygromycin phosphotransferase gene (hyg) and Streptomyces verticillius bleomycin gene (phle) were used as selectable markers for F. oxysporum transformation and are shown as dark grey arrows. The positions and expected fragment sizes of restriction endonuclease recognition sites used for DNA blot analyses are indicated.

Mentions: 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). Twenty independent lines were isolated and all exhibited varying degrees of Gus activity, as determined by the fluorimetrical assay using 4-methylumbelliferyl-β-D-glucuronide (MUG) (Figure 2). The majority of Gus lines contained a single T-DNA insertion and no correlation between transgene copy number and Gus activity was apparent (Figure 2).


Analysis of hairpin RNA transgene-induced gene silencing in Fusarium oxysporum.

Schumann U, Smith NA, Kazan K, Ayliffe M, Wang MB - Silence (2013)

Schematic diagrams (not to scale) of transgenes introduced into Fusarium oxysporum. All Gus-derived sequences are shown in black, except a small region of the Gus ORF represented in grey, which is present in the full length Gus gene but absent in the hpGus constructs. The gpdA promoter is shown as an open arrow, while the transcription terminator sequence trpC is indicated as an open box. Sequences present in hairpin constructs of the Gfp and F. oxysporum Frp1 are shown as light grey regions in each construct, respectively. An intron from the pyruvate dehydrogenase kinase gene (pdk) is included in the hpGfp gene. The hygromycin phosphotransferase gene (hyg) and Streptomyces verticillius bleomycin gene (phle) were used as selectable markers for F. oxysporum transformation and are shown as dark grey arrows. The positions and expected fragment sizes of restriction endonuclease recognition sites used for DNA blot analyses are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3733888&req=5

Figure 1: Schematic diagrams (not to scale) of transgenes introduced into Fusarium oxysporum. All Gus-derived sequences are shown in black, except a small region of the Gus ORF represented in grey, which is present in the full length Gus gene but absent in the hpGus constructs. The gpdA promoter is shown as an open arrow, while the transcription terminator sequence trpC is indicated as an open box. Sequences present in hairpin constructs of the Gfp and F. oxysporum Frp1 are shown as light grey regions in each construct, respectively. An intron from the pyruvate dehydrogenase kinase gene (pdk) is included in the hpGfp gene. The hygromycin phosphotransferase gene (hyg) and Streptomyces verticillius bleomycin gene (phle) were used as selectable markers for F. oxysporum transformation and are shown as dark grey arrows. The positions and expected fragment sizes of restriction endonuclease recognition sites used for DNA blot analyses are indicated.
Mentions: 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). Twenty independent lines were isolated and all exhibited varying degrees of Gus activity, as determined by the fluorimetrical assay using 4-methylumbelliferyl-β-D-glucuronide (MUG) (Figure 2). The majority of Gus lines contained a single T-DNA insertion and no correlation between transgene copy number and Gus activity was apparent (Figure 2).

Bottom Line: Here 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.These 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Commonwealth Scientific and Industrial Research Organisation Plant Industry, Clunies Ross Street, Canberra ACT 2601, Australia. ming-bo.wang@csiro.au.

ABSTRACT

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, 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.

Results: Here 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.

Conclusions: These 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.

No MeSH data available.


Related in: MedlinePlus