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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

Relative Gus activity of Fusarium oxysporum lines containing the Gus gene. Gus activity was measured by MUG assays of at least three biological replicates of each line. The Y-axis depicts Gus activity per μg of protein extract, while each column on the X-axis represents the activity of a single transgenic line. Standard deviations are indicated on each column. The number of Gus T-DNA insertions present in each line is indicated below the X-axis and was determined by DNA blot hybridization using a probe specific for the hygromycin selectable marker gene. Lines 0–1.3, 0–1.6 and 1–1.3 were used for subsequent hpGus transformation.
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Figure 2: Relative Gus activity of Fusarium oxysporum lines containing the Gus gene. Gus activity was measured by MUG assays of at least three biological replicates of each line. The Y-axis depicts Gus activity per μg of protein extract, while each column on the X-axis represents the activity of a single transgenic line. Standard deviations are indicated on each column. The number of Gus T-DNA insertions present in each line is indicated below the X-axis and was determined by DNA blot hybridization using a probe specific for the hygromycin selectable marker gene. Lines 0–1.3, 0–1.6 and 1–1.3 were used for subsequent hpGus transformation.

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)

Relative Gus activity of Fusarium oxysporum lines containing the Gus gene. Gus activity was measured by MUG assays of at least three biological replicates of each line. The Y-axis depicts Gus activity per μg of protein extract, while each column on the X-axis represents the activity of a single transgenic line. Standard deviations are indicated on each column. The number of Gus T-DNA insertions present in each line is indicated below the X-axis and was determined by DNA blot hybridization using a probe specific for the hygromycin selectable marker gene. Lines 0–1.3, 0–1.6 and 1–1.3 were used for subsequent hpGus transformation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Relative Gus activity of Fusarium oxysporum lines containing the Gus gene. Gus activity was measured by MUG assays of at least three biological replicates of each line. The Y-axis depicts Gus activity per μg of protein extract, while each column on the X-axis represents the activity of a single transgenic line. Standard deviations are indicated on each column. The number of Gus T-DNA insertions present in each line is indicated below the X-axis and was determined by DNA blot hybridization using a probe specific for the hygromycin selectable marker gene. Lines 0–1.3, 0–1.6 and 1–1.3 were used for subsequent hpGus transformation.
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