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Two novel mitoviruses from a Canadian isolate of the Dutch elm pathogen Ophiostoma novo-ulmi (93-1224).

Hintz WE, Carneiro JS, Kassatenko I, Varga A, James D - Virol. J. (2013)

Bottom Line: Numerous mitigation strategies have been tried to eradicate this pathogen, but success has thus far been limited.An alternative approach might utilize double-stranded RNA (dsRNA) mycoviruses which have been reported to induce hypovirulence in other fungi.Naïve fungal hosts could be infected with both the engineered molecule and a helper mitovirus encoding an RdRp which would provide replication capacity for both molecules.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, University of Victoria, P.O. Box 3020 STN CSC, Victoria, BC V8W 3N5, Canada. whintz@uvic.ca

ABSTRACT

Background: Ophiostoma novo-ulmi is the causative agent of Dutch elm disease (DED). It is an ascomycetous filamentous fungus that ranks as the third most devastating fungal pathogen in Canada. The disease front has spread eastward and westward from the epicentre in Ontario and Quebec and is threatening elm populations across the country. Numerous mitigation strategies have been tried to eradicate this pathogen, but success has thus far been limited. An alternative approach might utilize double-stranded RNA (dsRNA) mycoviruses which have been reported to induce hypovirulence in other fungi.

Methods: Using a modified single primer amplification technique (SPAT) in combination with chromosomal walking, we have determined the genome sequence of two RdRp encoding dsRNA viruses from an O. novo-ulmi isolate (93-1224) collected from the disease front in Winnipeg.

Results: We propose that these viruses, which we have named OnuMV1c and OnuMV7 based on sequence similarity to other Ophiostoma mitoviruses, are two new members of the genus Mitovirus in the family Narnaviridae.

Conclusions: The discovery of such dsRNA elements raises the potential for engineering these viruses to include other genetic elements, such as anti-sense or interfering RNAs, to create novel and highly specific biological controls. Naïve fungal hosts could be infected with both the engineered molecule and a helper mitovirus encoding an RdRp which would provide replication capacity for both molecules.

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Northern blot hybridization using strand-specific probes of O. novo-ulmi 93–1224 mitovirus OnuMV1c. Total RNAs were extracted and probed with either (−) or (+) strand specific probes corresponding to positions 35 to 910 of the RdRP coding region of the OnuMV1c. Double stranded RNA (dsRNA) was detected with both probes and single stranded transcripts (ssRNA) was detected only when (+) stranded probes was used. The ssRNA ran further down the gel.
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Figure 5: Northern blot hybridization using strand-specific probes of O. novo-ulmi 93–1224 mitovirus OnuMV1c. Total RNAs were extracted and probed with either (−) or (+) strand specific probes corresponding to positions 35 to 910 of the RdRP coding region of the OnuMV1c. Double stranded RNA (dsRNA) was detected with both probes and single stranded transcripts (ssRNA) was detected only when (+) stranded probes was used. The ssRNA ran further down the gel.

Mentions: Northern hybridization analysis revealed that when total RNA was hybridized with (−) strand-specific probe derived from OnuMV1c there was a major discrete signal at 3.1 kbp, corresponding to the size of the dsRNA 01 observed by gel electrophoresis. When the blot was probed with the (+) strand-specific probe the same 3.1 kbp dsRNA signal was observed as well as a second signal migrating further down the gel corresponding to the single-stranded transcript (ssRNA). As estimated from the intensity of the hybdridization signal the single stranded transcript was in greater abundance than the dsRNA (Figure 5). A similar pattern was observed when the blots were probed with OnuMV7 probes except that the probes hybridized to a 2.8 kbp dsRNA (not shown).


Two novel mitoviruses from a Canadian isolate of the Dutch elm pathogen Ophiostoma novo-ulmi (93-1224).

Hintz WE, Carneiro JS, Kassatenko I, Varga A, James D - Virol. J. (2013)

Northern blot hybridization using strand-specific probes of O. novo-ulmi 93–1224 mitovirus OnuMV1c. Total RNAs were extracted and probed with either (−) or (+) strand specific probes corresponding to positions 35 to 910 of the RdRP coding region of the OnuMV1c. Double stranded RNA (dsRNA) was detected with both probes and single stranded transcripts (ssRNA) was detected only when (+) stranded probes was used. The ssRNA ran further down the gel.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750502&req=5

Figure 5: Northern blot hybridization using strand-specific probes of O. novo-ulmi 93–1224 mitovirus OnuMV1c. Total RNAs were extracted and probed with either (−) or (+) strand specific probes corresponding to positions 35 to 910 of the RdRP coding region of the OnuMV1c. Double stranded RNA (dsRNA) was detected with both probes and single stranded transcripts (ssRNA) was detected only when (+) stranded probes was used. The ssRNA ran further down the gel.
Mentions: Northern hybridization analysis revealed that when total RNA was hybridized with (−) strand-specific probe derived from OnuMV1c there was a major discrete signal at 3.1 kbp, corresponding to the size of the dsRNA 01 observed by gel electrophoresis. When the blot was probed with the (+) strand-specific probe the same 3.1 kbp dsRNA signal was observed as well as a second signal migrating further down the gel corresponding to the single-stranded transcript (ssRNA). As estimated from the intensity of the hybdridization signal the single stranded transcript was in greater abundance than the dsRNA (Figure 5). A similar pattern was observed when the blots were probed with OnuMV7 probes except that the probes hybridized to a 2.8 kbp dsRNA (not shown).

Bottom Line: Numerous mitigation strategies have been tried to eradicate this pathogen, but success has thus far been limited.An alternative approach might utilize double-stranded RNA (dsRNA) mycoviruses which have been reported to induce hypovirulence in other fungi.Naïve fungal hosts could be infected with both the engineered molecule and a helper mitovirus encoding an RdRp which would provide replication capacity for both molecules.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, University of Victoria, P.O. Box 3020 STN CSC, Victoria, BC V8W 3N5, Canada. whintz@uvic.ca

ABSTRACT

Background: Ophiostoma novo-ulmi is the causative agent of Dutch elm disease (DED). It is an ascomycetous filamentous fungus that ranks as the third most devastating fungal pathogen in Canada. The disease front has spread eastward and westward from the epicentre in Ontario and Quebec and is threatening elm populations across the country. Numerous mitigation strategies have been tried to eradicate this pathogen, but success has thus far been limited. An alternative approach might utilize double-stranded RNA (dsRNA) mycoviruses which have been reported to induce hypovirulence in other fungi.

Methods: Using a modified single primer amplification technique (SPAT) in combination with chromosomal walking, we have determined the genome sequence of two RdRp encoding dsRNA viruses from an O. novo-ulmi isolate (93-1224) collected from the disease front in Winnipeg.

Results: We propose that these viruses, which we have named OnuMV1c and OnuMV7 based on sequence similarity to other Ophiostoma mitoviruses, are two new members of the genus Mitovirus in the family Narnaviridae.

Conclusions: The discovery of such dsRNA elements raises the potential for engineering these viruses to include other genetic elements, such as anti-sense or interfering RNAs, to create novel and highly specific biological controls. Naïve fungal hosts could be infected with both the engineered molecule and a helper mitovirus encoding an RdRp which would provide replication capacity for both molecules.

Show MeSH
Related in: MedlinePlus