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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

Analysis of β-glucuronidase (Gus) 0–1.6 transgenics carrying the conP-Gus constructs. (A) Schematic diagram (not to scale) showing details of the T-DNA region of the conP-Gus construct. The Gus sequence consists of the 3′ 1.1 kb of the Gus ORF and is shown in black. The convergent promoters driving transcription are shown as open arrows. The Streptomyces noursei nouseothricin gene was used as selectable marker (clonNAT, Werner BioAgents, Germany) and is shown in grey. Total RNA (15 μg) was separated on 17% polyacrylamide gels and probed for Gus-derived small interfering RNAs (siRNAs) (upper panel). No small RNA species were detected in any of these lines. U6 transcripts are shown as loading control. To determine Gus transcript levels, total RNA (10μg) was separated by agarose gel electrophoresis and hybridized with a probe specific for the region unique to the Gus transgene, not present in the conP-Gus gene (middle panel). Most lines show reduced Gus mRNA levels. Detected fragments are likely either cleavage products (below the Gus fragment) or size shifted due to siRNA binding (above the Gus fragment). Ribosomal RNA bands are shown as loading control. All transgenic lines were analyzed for Gus activity, which was carried out by MUG assay in at least two independent biological replicates (bottom panel; error bars show standard deviation). All conP-Gus transformants showed significantly reduced Gus activity (*t-test: P < 0.003). (B)Gus transcription occurred from both transgenic promoters. Total RNA (500 ng) was reverse transcribed using Gus-specific primers Gus-RT2 or Gus-RT3 (see schematic). Fragments were amplified from cDNA or no RT control RNA using primers Gus-RT2 and A-RT2 (trpC transcript), or Gus-RT3 and A-RT3 (gpdA transcript). Products were separated on a 2% agarose gel. Fragments of the correct size were obtained for both promoters, indicating that dsRNA could be produced in these lines.
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Figure 8: Analysis of β-glucuronidase (Gus) 0–1.6 transgenics carrying the conP-Gus constructs. (A) Schematic diagram (not to scale) showing details of the T-DNA region of the conP-Gus construct. The Gus sequence consists of the 3′ 1.1 kb of the Gus ORF and is shown in black. The convergent promoters driving transcription are shown as open arrows. The Streptomyces noursei nouseothricin gene was used as selectable marker (clonNAT, Werner BioAgents, Germany) and is shown in grey. Total RNA (15 μg) was separated on 17% polyacrylamide gels and probed for Gus-derived small interfering RNAs (siRNAs) (upper panel). No small RNA species were detected in any of these lines. U6 transcripts are shown as loading control. To determine Gus transcript levels, total RNA (10μg) was separated by agarose gel electrophoresis and hybridized with a probe specific for the region unique to the Gus transgene, not present in the conP-Gus gene (middle panel). Most lines show reduced Gus mRNA levels. Detected fragments are likely either cleavage products (below the Gus fragment) or size shifted due to siRNA binding (above the Gus fragment). Ribosomal RNA bands are shown as loading control. All transgenic lines were analyzed for Gus activity, which was carried out by MUG assay in at least two independent biological replicates (bottom panel; error bars show standard deviation). All conP-Gus transformants showed significantly reduced Gus activity (*t-test: P < 0.003). (B)Gus transcription occurred from both transgenic promoters. Total RNA (500 ng) was reverse transcribed using Gus-specific primers Gus-RT2 or Gus-RT3 (see schematic). Fragments were amplified from cDNA or no RT control RNA using primers Gus-RT2 and A-RT2 (trpC transcript), or Gus-RT3 and A-RT3 (gpdA transcript). Products were separated on a 2% agarose gel. Fragments of the correct size were obtained for both promoters, indicating that dsRNA could be produced in these lines.

Mentions: Our analyses of the hpGus lines raised two possibilities. First, hpRNA transgenes in the F. oxysporum genome are highly susceptible to transcriptional inactivation, possibly due to the inverted repeat DNA structure, resulting in lack of siRNA production. Second, based on the analyses of lines S34 and S5, dsRNA transcribed from a terminatorless transgene, which would lack polyadenylation, may be more efficiently processed into siRNAs. To test these possibilities, a construct was generated (conP-Gus; Figure 8A), which contained two convergent promoters that bi-directionally transcribe a 1.1 kb sequence of the Gus ORF to generate dsRNA. This construct contained no terminator sequences and therefore both sense and antisense Gus transcripts, were expected to lack poly(A) tails. The construct was transformed into F. oxysporum line 0–1.6, which contains an actively expressed Gus gene.


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

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

Analysis of β-glucuronidase (Gus) 0–1.6 transgenics carrying the conP-Gus constructs. (A) Schematic diagram (not to scale) showing details of the T-DNA region of the conP-Gus construct. The Gus sequence consists of the 3′ 1.1 kb of the Gus ORF and is shown in black. The convergent promoters driving transcription are shown as open arrows. The Streptomyces noursei nouseothricin gene was used as selectable marker (clonNAT, Werner BioAgents, Germany) and is shown in grey. Total RNA (15 μg) was separated on 17% polyacrylamide gels and probed for Gus-derived small interfering RNAs (siRNAs) (upper panel). No small RNA species were detected in any of these lines. U6 transcripts are shown as loading control. To determine Gus transcript levels, total RNA (10μg) was separated by agarose gel electrophoresis and hybridized with a probe specific for the region unique to the Gus transgene, not present in the conP-Gus gene (middle panel). Most lines show reduced Gus mRNA levels. Detected fragments are likely either cleavage products (below the Gus fragment) or size shifted due to siRNA binding (above the Gus fragment). Ribosomal RNA bands are shown as loading control. All transgenic lines were analyzed for Gus activity, which was carried out by MUG assay in at least two independent biological replicates (bottom panel; error bars show standard deviation). All conP-Gus transformants showed significantly reduced Gus activity (*t-test: P < 0.003). (B)Gus transcription occurred from both transgenic promoters. Total RNA (500 ng) was reverse transcribed using Gus-specific primers Gus-RT2 or Gus-RT3 (see schematic). Fragments were amplified from cDNA or no RT control RNA using primers Gus-RT2 and A-RT2 (trpC transcript), or Gus-RT3 and A-RT3 (gpdA transcript). Products were separated on a 2% agarose gel. Fragments of the correct size were obtained for both promoters, indicating that dsRNA could be produced in these lines.
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Related In: Results  -  Collection

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Figure 8: Analysis of β-glucuronidase (Gus) 0–1.6 transgenics carrying the conP-Gus constructs. (A) Schematic diagram (not to scale) showing details of the T-DNA region of the conP-Gus construct. The Gus sequence consists of the 3′ 1.1 kb of the Gus ORF and is shown in black. The convergent promoters driving transcription are shown as open arrows. The Streptomyces noursei nouseothricin gene was used as selectable marker (clonNAT, Werner BioAgents, Germany) and is shown in grey. Total RNA (15 μg) was separated on 17% polyacrylamide gels and probed for Gus-derived small interfering RNAs (siRNAs) (upper panel). No small RNA species were detected in any of these lines. U6 transcripts are shown as loading control. To determine Gus transcript levels, total RNA (10μg) was separated by agarose gel electrophoresis and hybridized with a probe specific for the region unique to the Gus transgene, not present in the conP-Gus gene (middle panel). Most lines show reduced Gus mRNA levels. Detected fragments are likely either cleavage products (below the Gus fragment) or size shifted due to siRNA binding (above the Gus fragment). Ribosomal RNA bands are shown as loading control. All transgenic lines were analyzed for Gus activity, which was carried out by MUG assay in at least two independent biological replicates (bottom panel; error bars show standard deviation). All conP-Gus transformants showed significantly reduced Gus activity (*t-test: P < 0.003). (B)Gus transcription occurred from both transgenic promoters. Total RNA (500 ng) was reverse transcribed using Gus-specific primers Gus-RT2 or Gus-RT3 (see schematic). Fragments were amplified from cDNA or no RT control RNA using primers Gus-RT2 and A-RT2 (trpC transcript), or Gus-RT3 and A-RT3 (gpdA transcript). Products were separated on a 2% agarose gel. Fragments of the correct size were obtained for both promoters, indicating that dsRNA could be produced in these lines.
Mentions: Our analyses of the hpGus lines raised two possibilities. First, hpRNA transgenes in the F. oxysporum genome are highly susceptible to transcriptional inactivation, possibly due to the inverted repeat DNA structure, resulting in lack of siRNA production. Second, based on the analyses of lines S34 and S5, dsRNA transcribed from a terminatorless transgene, which would lack polyadenylation, may be more efficiently processed into siRNAs. To test these possibilities, a construct was generated (conP-Gus; Figure 8A), which contained two convergent promoters that bi-directionally transcribe a 1.1 kb sequence of the Gus ORF to generate dsRNA. This construct contained no terminator sequences and therefore both sense and antisense Gus transcripts, were expected to lack poly(A) tails. The construct was transformed into F. oxysporum line 0–1.6, which contains an actively expressed Gus gene.

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