Limits...
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.

Show MeSH

Related in: MedlinePlus

Phylogenetic identification of OnuMV1c and OnuMV7. The fungal mitovirus RdRp sequences were obtained from the NCBI gene bank database. Multiple sequence alignments were performed using MUSCLE and the tree constructed using NJ method with 1000 bootstrap replicates. Bootstrap values are shown. The WAG substitution model was selected assuming an estimated proportion of invariant sites (of 0.025) and 4 gamma-distributed rate categories to account for rate heterogeneity across sites. Saccharomyces 23S RNA narnavirus [UniProt: Q07048] = RdRp Saccharomyces 23 S RNA narnavirus PE = 1 SV = 2 served as an outgroup. Virus notations were according to Figure 2 and as follows: Sclerotinia sclerotiorum MV2 [GenBank: AEX91879.1] = S. sclerotiorum mitovirus 2; Helicobasidium mompa MV1-18 [GenBank: BAD72871.1] = H. mompa mitovirus 1 – 18; Gremmeniella abietina GMV2 [GenBank: AEY76153.1] = G. abietina non-host-specific mitrochondrial RNA virus S1; Gremmeniella abietina GMV1 [GenBank: CCD32685.2] = RdRp Gremmeniella mitovirus; Sclerotinia sclerotiorum MV1 [GenBank: AEX91878.1] = S. sclerotiorum mitovirus 1; Thielaviopsis basicola MV01 [NCBI Reference Sequence: YP_002822229.1] = RdRp T. basicola mitovirus; Thielavopsis basicola MV02 [GenBank: AAZ99833.1] = RdRp T. basicola mitovirus; Sclerotinia sclerotiorum MV4 [GenBank: AGC24233.1] = RdRp S. sclerotiorum mitovirus 4; Glomus sp. Small MV [GenBank: BAJ23143.1] = Putative RdRp Glomus sp. RF1 small virus; Tuber excavatum MV [GenBank: AEP83726.1] = RdRp T. excavatum mitovirus; Cryphonectria parasitica MV1-NB631 [NCBI Reference Sequence: NP_660174.1] = RdRp C. parasitica mitovirus 1-NB631; Thanatephorus cucumeris MV [GenBank: AAD17381.1] = dsRNA viral RdRp T. cucumeris; Tuber aestivum MV [NCBI Reference Sequence: YP_004564622.1] = RdRp T. aestivum mitovirus; Clitocybe odora MV [NCBI Reference Sequence: YP_005352912.1] = RdRp C. odora virus; Botrytis cinerea dr MV [NCBI Reference Sequence: YP_002284334.1] = RdRp B. cinerea debilitation-related virus; Botrytis cinerea MV1 [GenBank: ABQ65153.3] = RdRp B. cinerea mitovirus 1; Sclerotinia sclerotiorum MV3 [GenBank: AGC24232.1] = RdRp S. sclerotiorum mitovirus 3; Sclerotinia homoeocarpa MV [GenBank: AAO21337.1] = S. homoeocarpa mitovirus.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3750502&req=5

Figure 3: Phylogenetic identification of OnuMV1c and OnuMV7. The fungal mitovirus RdRp sequences were obtained from the NCBI gene bank database. Multiple sequence alignments were performed using MUSCLE and the tree constructed using NJ method with 1000 bootstrap replicates. Bootstrap values are shown. The WAG substitution model was selected assuming an estimated proportion of invariant sites (of 0.025) and 4 gamma-distributed rate categories to account for rate heterogeneity across sites. Saccharomyces 23S RNA narnavirus [UniProt: Q07048] = RdRp Saccharomyces 23 S RNA narnavirus PE = 1 SV = 2 served as an outgroup. Virus notations were according to Figure 2 and as follows: Sclerotinia sclerotiorum MV2 [GenBank: AEX91879.1] = S. sclerotiorum mitovirus 2; Helicobasidium mompa MV1-18 [GenBank: BAD72871.1] = H. mompa mitovirus 1 – 18; Gremmeniella abietina GMV2 [GenBank: AEY76153.1] = G. abietina non-host-specific mitrochondrial RNA virus S1; Gremmeniella abietina GMV1 [GenBank: CCD32685.2] = RdRp Gremmeniella mitovirus; Sclerotinia sclerotiorum MV1 [GenBank: AEX91878.1] = S. sclerotiorum mitovirus 1; Thielaviopsis basicola MV01 [NCBI Reference Sequence: YP_002822229.1] = RdRp T. basicola mitovirus; Thielavopsis basicola MV02 [GenBank: AAZ99833.1] = RdRp T. basicola mitovirus; Sclerotinia sclerotiorum MV4 [GenBank: AGC24233.1] = RdRp S. sclerotiorum mitovirus 4; Glomus sp. Small MV [GenBank: BAJ23143.1] = Putative RdRp Glomus sp. RF1 small virus; Tuber excavatum MV [GenBank: AEP83726.1] = RdRp T. excavatum mitovirus; Cryphonectria parasitica MV1-NB631 [NCBI Reference Sequence: NP_660174.1] = RdRp C. parasitica mitovirus 1-NB631; Thanatephorus cucumeris MV [GenBank: AAD17381.1] = dsRNA viral RdRp T. cucumeris; Tuber aestivum MV [NCBI Reference Sequence: YP_004564622.1] = RdRp T. aestivum mitovirus; Clitocybe odora MV [NCBI Reference Sequence: YP_005352912.1] = RdRp C. odora virus; Botrytis cinerea dr MV [NCBI Reference Sequence: YP_002284334.1] = RdRp B. cinerea debilitation-related virus; Botrytis cinerea MV1 [GenBank: ABQ65153.3] = RdRp B. cinerea mitovirus 1; Sclerotinia sclerotiorum MV3 [GenBank: AGC24232.1] = RdRp S. sclerotiorum mitovirus 3; Sclerotinia homoeocarpa MV [GenBank: AAO21337.1] = S. homoeocarpa mitovirus.

Mentions: The nucleotide sequence of the dsRNA contigs were examined for the presence of open reading frames (ORFs) in all six reading frames. When the universal codon usage for cytoplasmically translated proteins was applied, there were no long ORFs however shorter segments of RdRp-like genes could be recognized. Because of the high similarity of these RdRp fragments to mitoviruses, a mitochondrial-specific codon usage pattern was applied. When the genetic code for mold, protozoan, coelenterate mitochondrial and mycoplasma (code 4) was employed, a single large ORF was found on the positive strand of both dsRNA 01 and dsRNA 02 (Figure 1). The ORF of dsRNA 01, which started with an AUG - start codon, and terminated with a UAG - stop codon, had the potential to encode a protein of 788 amino acids. Similarly a single large ORF was found on the positive strand of dsRNA 02, having an AUG - start codon and a TAA - stop codon, had the potential to encode a protein of 720 amino acids. According to Basic Local Alignment Search Tool (BLAST) analysis the dsRNA 01 ORF had a very high sequence similarity to the Ophiostoma RdRp encoded by the mitovirus OnuMV1b having a 70% of maximum amino acid identity for 97% of the query coverage. There was a significantly higher degree of sequence identity in the C-terminal region as compared to the N-terminal region. Alignment of the first 261 amino acids of OnuMV1c to the first 250 amino acids of OnuMV1b revealed only a 28% percent sequence identity while there was 88% sequence identity in the remaining 527 amino acid residues compared to a similar 526 amino acid region of OnuMV1b. Less significant but obvious identity existed in amino acid sequences with other mitoviruses clearly demonstrating a close relationship between this newly described dsRNA and other mitoviruses. Following a BLAST search for the ORF of dsRNA 02 it was found to be most similar to the RdRp of Gremmeniella mitovirus with a maximum identity of 30% for a 50% query cover. This molecule had only had a 29% identity for 49% query cover of Ophiostoma mitovirus OnuMV3a RdRp and a 35% identity for a 30% query cover of Ophstiostoma mitovirus OnuMV4, Construction of a phylogeny of all the mitovirus RdRp sequences for Ophiostoma, including those encoded by dsRNAs 01 and 02, demonstrated a close clustering of the dsRNA01 ORF with OnuMV1a and OnuMV1b hence we named this new mitovirus OnuMV1c (Figure 2). The RdRp encoded by the dsRNA 02 appeared to be unique and did not cluster with any other mitovirus previously described for O. novo-ulmi hence we named this second new mitovirus OnuMV7 following the numbering convention of dsRNAs that encode distinct RdRp-like proteins as proposed by Hong[17] and Doherty[11] (Figure 2). Phylogenetic comparison of the newly described OnuMV1c RdRp gene to a larger group of all characterized fungal mitoviruses indicated that this virus again grouped in a distinct clade containing Ophiostoma mitoviruses OnuMV1a, 1b, and also included OnuMV3a and 3b (Figure 3). Interestingly the RdRp of the Tuber aestivum MV clustered tightly with the OnuMV1a, 1b, 1c group. The RdRps of Sclerotinia sclerotiorum MV3 and Sclerotinia homoeocarpa MV clustered with OnuMV3a while those of Botrytis cinerea MV1 and Botrytis cinerea dr MV clustered tightly with OnuMV3b. While the newly described OnuMV7 did group in a clade containing OnuMV 4, 5, and 6 there was no close association to any of these Ophiostoma viral species nor with any other fungal virus species. The 729 bp ORF of dsRNA 03 had the potential to encode a polypeptide of 243 amino acids however there was no similarity of this ORF to any RdRp or to any other viral protein (Figure 1). There were four very small ORFs found on dsRNA 04 which shared limited sequence similarity to the 5′ ends of several mitovirus RdRps and had the highest similarity to the 5′ region of OnuMV1b. The RdRp homologous region of dsRNA 04 was, however, incomplete and interspersed with stop codons hence was unlikely to encode a functional enzyme (Figure 1). Both dsRNA 03 and dsRNA 04 are considered to be defective RNAs, the replication of which likely depends on a functional RdRp from some other source. Unlike dsRNAs 01 and 02, these molecules were not observed by gel electrophoresis and were much less abundant.


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)

Phylogenetic identification of OnuMV1c and OnuMV7. The fungal mitovirus RdRp sequences were obtained from the NCBI gene bank database. Multiple sequence alignments were performed using MUSCLE and the tree constructed using NJ method with 1000 bootstrap replicates. Bootstrap values are shown. The WAG substitution model was selected assuming an estimated proportion of invariant sites (of 0.025) and 4 gamma-distributed rate categories to account for rate heterogeneity across sites. Saccharomyces 23S RNA narnavirus [UniProt: Q07048] = RdRp Saccharomyces 23 S RNA narnavirus PE = 1 SV = 2 served as an outgroup. Virus notations were according to Figure 2 and as follows: Sclerotinia sclerotiorum MV2 [GenBank: AEX91879.1] = S. sclerotiorum mitovirus 2; Helicobasidium mompa MV1-18 [GenBank: BAD72871.1] = H. mompa mitovirus 1 – 18; Gremmeniella abietina GMV2 [GenBank: AEY76153.1] = G. abietina non-host-specific mitrochondrial RNA virus S1; Gremmeniella abietina GMV1 [GenBank: CCD32685.2] = RdRp Gremmeniella mitovirus; Sclerotinia sclerotiorum MV1 [GenBank: AEX91878.1] = S. sclerotiorum mitovirus 1; Thielaviopsis basicola MV01 [NCBI Reference Sequence: YP_002822229.1] = RdRp T. basicola mitovirus; Thielavopsis basicola MV02 [GenBank: AAZ99833.1] = RdRp T. basicola mitovirus; Sclerotinia sclerotiorum MV4 [GenBank: AGC24233.1] = RdRp S. sclerotiorum mitovirus 4; Glomus sp. Small MV [GenBank: BAJ23143.1] = Putative RdRp Glomus sp. RF1 small virus; Tuber excavatum MV [GenBank: AEP83726.1] = RdRp T. excavatum mitovirus; Cryphonectria parasitica MV1-NB631 [NCBI Reference Sequence: NP_660174.1] = RdRp C. parasitica mitovirus 1-NB631; Thanatephorus cucumeris MV [GenBank: AAD17381.1] = dsRNA viral RdRp T. cucumeris; Tuber aestivum MV [NCBI Reference Sequence: YP_004564622.1] = RdRp T. aestivum mitovirus; Clitocybe odora MV [NCBI Reference Sequence: YP_005352912.1] = RdRp C. odora virus; Botrytis cinerea dr MV [NCBI Reference Sequence: YP_002284334.1] = RdRp B. cinerea debilitation-related virus; Botrytis cinerea MV1 [GenBank: ABQ65153.3] = RdRp B. cinerea mitovirus 1; Sclerotinia sclerotiorum MV3 [GenBank: AGC24232.1] = RdRp S. sclerotiorum mitovirus 3; Sclerotinia homoeocarpa MV [GenBank: AAO21337.1] = S. homoeocarpa mitovirus.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Phylogenetic identification of OnuMV1c and OnuMV7. The fungal mitovirus RdRp sequences were obtained from the NCBI gene bank database. Multiple sequence alignments were performed using MUSCLE and the tree constructed using NJ method with 1000 bootstrap replicates. Bootstrap values are shown. The WAG substitution model was selected assuming an estimated proportion of invariant sites (of 0.025) and 4 gamma-distributed rate categories to account for rate heterogeneity across sites. Saccharomyces 23S RNA narnavirus [UniProt: Q07048] = RdRp Saccharomyces 23 S RNA narnavirus PE = 1 SV = 2 served as an outgroup. Virus notations were according to Figure 2 and as follows: Sclerotinia sclerotiorum MV2 [GenBank: AEX91879.1] = S. sclerotiorum mitovirus 2; Helicobasidium mompa MV1-18 [GenBank: BAD72871.1] = H. mompa mitovirus 1 – 18; Gremmeniella abietina GMV2 [GenBank: AEY76153.1] = G. abietina non-host-specific mitrochondrial RNA virus S1; Gremmeniella abietina GMV1 [GenBank: CCD32685.2] = RdRp Gremmeniella mitovirus; Sclerotinia sclerotiorum MV1 [GenBank: AEX91878.1] = S. sclerotiorum mitovirus 1; Thielaviopsis basicola MV01 [NCBI Reference Sequence: YP_002822229.1] = RdRp T. basicola mitovirus; Thielavopsis basicola MV02 [GenBank: AAZ99833.1] = RdRp T. basicola mitovirus; Sclerotinia sclerotiorum MV4 [GenBank: AGC24233.1] = RdRp S. sclerotiorum mitovirus 4; Glomus sp. Small MV [GenBank: BAJ23143.1] = Putative RdRp Glomus sp. RF1 small virus; Tuber excavatum MV [GenBank: AEP83726.1] = RdRp T. excavatum mitovirus; Cryphonectria parasitica MV1-NB631 [NCBI Reference Sequence: NP_660174.1] = RdRp C. parasitica mitovirus 1-NB631; Thanatephorus cucumeris MV [GenBank: AAD17381.1] = dsRNA viral RdRp T. cucumeris; Tuber aestivum MV [NCBI Reference Sequence: YP_004564622.1] = RdRp T. aestivum mitovirus; Clitocybe odora MV [NCBI Reference Sequence: YP_005352912.1] = RdRp C. odora virus; Botrytis cinerea dr MV [NCBI Reference Sequence: YP_002284334.1] = RdRp B. cinerea debilitation-related virus; Botrytis cinerea MV1 [GenBank: ABQ65153.3] = RdRp B. cinerea mitovirus 1; Sclerotinia sclerotiorum MV3 [GenBank: AGC24232.1] = RdRp S. sclerotiorum mitovirus 3; Sclerotinia homoeocarpa MV [GenBank: AAO21337.1] = S. homoeocarpa mitovirus.
Mentions: The nucleotide sequence of the dsRNA contigs were examined for the presence of open reading frames (ORFs) in all six reading frames. When the universal codon usage for cytoplasmically translated proteins was applied, there were no long ORFs however shorter segments of RdRp-like genes could be recognized. Because of the high similarity of these RdRp fragments to mitoviruses, a mitochondrial-specific codon usage pattern was applied. When the genetic code for mold, protozoan, coelenterate mitochondrial and mycoplasma (code 4) was employed, a single large ORF was found on the positive strand of both dsRNA 01 and dsRNA 02 (Figure 1). The ORF of dsRNA 01, which started with an AUG - start codon, and terminated with a UAG - stop codon, had the potential to encode a protein of 788 amino acids. Similarly a single large ORF was found on the positive strand of dsRNA 02, having an AUG - start codon and a TAA - stop codon, had the potential to encode a protein of 720 amino acids. According to Basic Local Alignment Search Tool (BLAST) analysis the dsRNA 01 ORF had a very high sequence similarity to the Ophiostoma RdRp encoded by the mitovirus OnuMV1b having a 70% of maximum amino acid identity for 97% of the query coverage. There was a significantly higher degree of sequence identity in the C-terminal region as compared to the N-terminal region. Alignment of the first 261 amino acids of OnuMV1c to the first 250 amino acids of OnuMV1b revealed only a 28% percent sequence identity while there was 88% sequence identity in the remaining 527 amino acid residues compared to a similar 526 amino acid region of OnuMV1b. Less significant but obvious identity existed in amino acid sequences with other mitoviruses clearly demonstrating a close relationship between this newly described dsRNA and other mitoviruses. Following a BLAST search for the ORF of dsRNA 02 it was found to be most similar to the RdRp of Gremmeniella mitovirus with a maximum identity of 30% for a 50% query cover. This molecule had only had a 29% identity for 49% query cover of Ophiostoma mitovirus OnuMV3a RdRp and a 35% identity for a 30% query cover of Ophstiostoma mitovirus OnuMV4, Construction of a phylogeny of all the mitovirus RdRp sequences for Ophiostoma, including those encoded by dsRNAs 01 and 02, demonstrated a close clustering of the dsRNA01 ORF with OnuMV1a and OnuMV1b hence we named this new mitovirus OnuMV1c (Figure 2). The RdRp encoded by the dsRNA 02 appeared to be unique and did not cluster with any other mitovirus previously described for O. novo-ulmi hence we named this second new mitovirus OnuMV7 following the numbering convention of dsRNAs that encode distinct RdRp-like proteins as proposed by Hong[17] and Doherty[11] (Figure 2). Phylogenetic comparison of the newly described OnuMV1c RdRp gene to a larger group of all characterized fungal mitoviruses indicated that this virus again grouped in a distinct clade containing Ophiostoma mitoviruses OnuMV1a, 1b, and also included OnuMV3a and 3b (Figure 3). Interestingly the RdRp of the Tuber aestivum MV clustered tightly with the OnuMV1a, 1b, 1c group. The RdRps of Sclerotinia sclerotiorum MV3 and Sclerotinia homoeocarpa MV clustered with OnuMV3a while those of Botrytis cinerea MV1 and Botrytis cinerea dr MV clustered tightly with OnuMV3b. While the newly described OnuMV7 did group in a clade containing OnuMV 4, 5, and 6 there was no close association to any of these Ophiostoma viral species nor with any other fungal virus species. The 729 bp ORF of dsRNA 03 had the potential to encode a polypeptide of 243 amino acids however there was no similarity of this ORF to any RdRp or to any other viral protein (Figure 1). There were four very small ORFs found on dsRNA 04 which shared limited sequence similarity to the 5′ ends of several mitovirus RdRps and had the highest similarity to the 5′ region of OnuMV1b. The RdRp homologous region of dsRNA 04 was, however, incomplete and interspersed with stop codons hence was unlikely to encode a functional enzyme (Figure 1). Both dsRNA 03 and dsRNA 04 are considered to be defective RNAs, the replication of which likely depends on a functional RdRp from some other source. Unlike dsRNAs 01 and 02, these molecules were not observed by gel electrophoresis and were much less abundant.

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