VIRUSES
crossm First Complete Genome Sequence of Tobacco necrosis virus D Isolated from Soybean and from North America Gustavo A. Díaz-Cruz, Charlotte M. Smith, Kiana F. Wiebe, Bryan J. Cassone Department of Biology, Brandon University, Brandon, Manitoba, Canada
We present the first complete genome sequence of the tombusvirus Tobacco necrosis virus D (TNV-D) from North America, obtained from an infected soybean plant. Compared with the three other TNV-D genomes isolated from different geographic regions and host plants, its nucleotide identities were between 83% and 93%.
ABSTRACT
T
obacco necrosis virus D (TNV-D) is a single-stranded positive-sense RNA virus in the family Tombusviridae (genus Necrovirus). First described in Nicotiana tabacum (1), the virus can be transmitted experimentally to at least 88 dicotyledonous and monocotyledon species in 37 families (2). While systemic infections are rare and limited to only a few host species, inoculated plants typically show necrotic local lesions (2). TNV-D is not insect vectored; rather, it is naturally transmitted by zoospores of the root-infecting chytrid fungus Olpidium brassicae, entering the root at the same time as the fungus (3–5). Although the virus has a broad host range and is likely distributed worldwide, its genome sequence is only available from three isolates: TNV-D infecting French bean (Phaseolus vulgaris cv. The Prince) from England (6, 7), TNV-DH infecting tobacco (Nicotiana clevelandii) from Hungary (8), and TNV-DP infecting olive trees (Olea europaea L.) from Portugal (9). Based on these assemblies, the genome is approximately 3.8 kb in length and contains six protein coding genes. In August 2016, a leaf sample was collected from a soybean (Glycine max [L.] Merr.) plant exhibiting necrotic lesions, growing in a natural field in Manitoba, Canada. Total RNA was extracted using the plant RNA/DNA purification kit (Norgen Biotek) and sent to the Génome Québec Innovation at McGill University for sequencing in 100-bp paired-end fashion on the Illumina HiSeq 2500 platform. After adapter removal and trimming for quality, a total of 50,927,028 reads were obtained. Preprocessed reads were de novo assembled using CLC Genomics Workbench 10.0.1 (CLC Bio) and the following parameters: bubble size, 500; similarity fraction, 0.9; length fraction, 0.65; and default settings herein. Functional annotation of the resultant contigs was carried out using BLASTn against the NCBI nonredundant database (E value ⬍ 1 ⫻ 10⫺50). The TNV-D (Manitoban isolate, TNV-DMB) genome length obtained was 3,742 nucleotides (nt) with an average coverage of 812⫻. The overall genome sequence identities between TNV-DMB and the other TNV-D isolates were 83.4% (TNV-D), 93.3% (TNV-DH), and 83.2% (TNV-DP). Gene prediction using FgenesV (Softberry, Inc.) and OrfPredictor (10) identified the same six protein coding genes found in the other sequenced isolates in the following order: 22-kDa and 82-kDa genes encoding products required for RNA replication (p22 and p82), three different 7-kDa genes (71, 7a, and 7b) thought to be involved in cell-to-cell movement, and a coat protein (CP) gene. Much like the other isolates, the genome organization is very compact. Only two noncoding nucleotides are present between 7b and CP, and several genes overlap one another (p82 and 71, 3 nt; 71 and 7a, 142 nt; 7a and 7b, 1 nt). This is the first complete nucleotide sequence of a TNV-D isolate from soybean, as well as from North America. Volume 5 Issue 32 e00781-17
Received 22 June 2017 Accepted 26 June 2017 Published 10 August 2017 Citation Díaz-Cruz GA, Smith CM, Wiebe KF, Cassone BJ. 2017. First complete genome sequence of Tobacco necrosis virus D isolated from soybean and from North America. Genome Announc 5:e00781-17. https://doi .org/10.1128/genomeA.00781-17. Copyright © 2017 Díaz-Cruz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Bryan J. Cassone, [email protected].
genomea.asm.org 1
Díaz-Cruz et al.
Accession number(s). The complete genome sequence of TNV-DMB has been deposited in GenBank under accession number MF158986. ACKNOWLEDGMENT We graciously thank Manitoba Pulse & Soybean Growers (MPSG) for financial support.
REFERENCES 1. Smith KM, Bald JG. 1935. A description of a necrotic virus disease affecting tobacco and other plants. Parasitology 27:231–245. https://doi .org/10.1017/S0031182000015109. 2. Price WC. 1938. Studies on the virus of tobacco necrosis. Am J Bot 25:603. https://doi.org/10.2307/2436520. 3. Teakle DS. 1962. Transmission of tobacco necrosis virus by a fungus, Olpidium brassicae. Virology 18:224 –231. https://doi.org/10.1016/0042 -6822(62)90008-9. 4. Teakle DS, Gold AH. 1963. Further studies of Olpidium as a vector of tobacco necrosis virus. Virology 19:310 –315. https://doi.org/10.1016/ 0042-6822(63)90069-2. 5. Kassanis B, Macfarlane I. 1964. Transmission of tobacco necrosis virus by zoospores of Olpidium brassicae. J Gen Microbiol 36:79 –93. https://doi .org/10.1099/00221287-36-1-79. 6. Coutts RHA, Rigden JE, Slabas AR, Lomonossoff GP, Wise PJ. 1991. The
Volume 5 Issue 32 e00781-17
7.
8.
9.
10.
complete nucleotide sequence of tobacco necrosis virus strain D. J Gen Virol 72:1521–1529. https://doi.org/10.1099/0022-1317-72-7-1521. Offel SK, Coutts RHA. 1996. Location of the 5= termini of tobacco necrosis virus strain D subgenomic mRNAs. J Phytopathol 144:13–17. https://doi .org/10.1111/j.1439-0434.1996.tb01481.x. Molnár A, Havelda Z, Dalmay T, Szutorisz H, Burgyán J. 1997. Complete nucleotide sequence of tobacco necrosis virus strain DH and genes required for RNA replication and virus movement. J Gen Virol 78: 1235–1239. https://doi.org/10.1099/0022-1317-78-6-1235. Cardoso JMS, Félix MR, Clara MIE, Oliveira S. 2009. Complete genome sequence of a Tobacco necrosis virus D isolate from olive trees. Arch Virol 154:1169 –1172. https://doi.org/10.1007/s00705-009-0414-9. Min XJ, Butler G, Storms R, Tsang A. 2005. OrfPredictor: predicting protein-coding regions in EST-derived sequences. Nucleic Acids Res 33:W677–W680. https://doi.org/10.1093/nar/gki394.
genomea.asm.org 2
crossm First Complete Genome Sequence of Tobacco necrosis virus D Isolated from Soybean and from North America Gustavo A. Díaz-Cruz, Charlotte M. Smith, Kiana F. Wiebe, Bryan J. Cassone Department of Biology, Brandon University, Brandon, Manitoba, Canada
We present the first complete genome sequence of the tombusvirus Tobacco necrosis virus D (TNV-D) from North America, obtained from an infected soybean plant. Compared with the three other TNV-D genomes isolated from different geographic regions and host plants, its nucleotide identities were between 83% and 93%.
ABSTRACT
T
obacco necrosis virus D (TNV-D) is a single-stranded positive-sense RNA virus in the family Tombusviridae (genus Necrovirus). First described in Nicotiana tabacum (1), the virus can be transmitted experimentally to at least 88 dicotyledonous and monocotyledon species in 37 families (2). While systemic infections are rare and limited to only a few host species, inoculated plants typically show necrotic local lesions (2). TNV-D is not insect vectored; rather, it is naturally transmitted by zoospores of the root-infecting chytrid fungus Olpidium brassicae, entering the root at the same time as the fungus (3–5). Although the virus has a broad host range and is likely distributed worldwide, its genome sequence is only available from three isolates: TNV-D infecting French bean (Phaseolus vulgaris cv. The Prince) from England (6, 7), TNV-DH infecting tobacco (Nicotiana clevelandii) from Hungary (8), and TNV-DP infecting olive trees (Olea europaea L.) from Portugal (9). Based on these assemblies, the genome is approximately 3.8 kb in length and contains six protein coding genes. In August 2016, a leaf sample was collected from a soybean (Glycine max [L.] Merr.) plant exhibiting necrotic lesions, growing in a natural field in Manitoba, Canada. Total RNA was extracted using the plant RNA/DNA purification kit (Norgen Biotek) and sent to the Génome Québec Innovation at McGill University for sequencing in 100-bp paired-end fashion on the Illumina HiSeq 2500 platform. After adapter removal and trimming for quality, a total of 50,927,028 reads were obtained. Preprocessed reads were de novo assembled using CLC Genomics Workbench 10.0.1 (CLC Bio) and the following parameters: bubble size, 500; similarity fraction, 0.9; length fraction, 0.65; and default settings herein. Functional annotation of the resultant contigs was carried out using BLASTn against the NCBI nonredundant database (E value ⬍ 1 ⫻ 10⫺50). The TNV-D (Manitoban isolate, TNV-DMB) genome length obtained was 3,742 nucleotides (nt) with an average coverage of 812⫻. The overall genome sequence identities between TNV-DMB and the other TNV-D isolates were 83.4% (TNV-D), 93.3% (TNV-DH), and 83.2% (TNV-DP). Gene prediction using FgenesV (Softberry, Inc.) and OrfPredictor (10) identified the same six protein coding genes found in the other sequenced isolates in the following order: 22-kDa and 82-kDa genes encoding products required for RNA replication (p22 and p82), three different 7-kDa genes (71, 7a, and 7b) thought to be involved in cell-to-cell movement, and a coat protein (CP) gene. Much like the other isolates, the genome organization is very compact. Only two noncoding nucleotides are present between 7b and CP, and several genes overlap one another (p82 and 71, 3 nt; 71 and 7a, 142 nt; 7a and 7b, 1 nt). This is the first complete nucleotide sequence of a TNV-D isolate from soybean, as well as from North America. Volume 5 Issue 32 e00781-17
Received 22 June 2017 Accepted 26 June 2017 Published 10 August 2017 Citation Díaz-Cruz GA, Smith CM, Wiebe KF, Cassone BJ. 2017. First complete genome sequence of Tobacco necrosis virus D isolated from soybean and from North America. Genome Announc 5:e00781-17. https://doi .org/10.1128/genomeA.00781-17. Copyright © 2017 Díaz-Cruz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Bryan J. Cassone, [email protected].
genomea.asm.org 1
Díaz-Cruz et al.
Accession number(s). The complete genome sequence of TNV-DMB has been deposited in GenBank under accession number MF158986. ACKNOWLEDGMENT We graciously thank Manitoba Pulse & Soybean Growers (MPSG) for financial support.
REFERENCES 1. Smith KM, Bald JG. 1935. A description of a necrotic virus disease affecting tobacco and other plants. Parasitology 27:231–245. https://doi .org/10.1017/S0031182000015109. 2. Price WC. 1938. Studies on the virus of tobacco necrosis. Am J Bot 25:603. https://doi.org/10.2307/2436520. 3. Teakle DS. 1962. Transmission of tobacco necrosis virus by a fungus, Olpidium brassicae. Virology 18:224 –231. https://doi.org/10.1016/0042 -6822(62)90008-9. 4. Teakle DS, Gold AH. 1963. Further studies of Olpidium as a vector of tobacco necrosis virus. Virology 19:310 –315. https://doi.org/10.1016/ 0042-6822(63)90069-2. 5. Kassanis B, Macfarlane I. 1964. Transmission of tobacco necrosis virus by zoospores of Olpidium brassicae. J Gen Microbiol 36:79 –93. https://doi .org/10.1099/00221287-36-1-79. 6. Coutts RHA, Rigden JE, Slabas AR, Lomonossoff GP, Wise PJ. 1991. The
Volume 5 Issue 32 e00781-17
7.
8.
9.
10.
complete nucleotide sequence of tobacco necrosis virus strain D. J Gen Virol 72:1521–1529. https://doi.org/10.1099/0022-1317-72-7-1521. Offel SK, Coutts RHA. 1996. Location of the 5= termini of tobacco necrosis virus strain D subgenomic mRNAs. J Phytopathol 144:13–17. https://doi .org/10.1111/j.1439-0434.1996.tb01481.x. Molnár A, Havelda Z, Dalmay T, Szutorisz H, Burgyán J. 1997. Complete nucleotide sequence of tobacco necrosis virus strain DH and genes required for RNA replication and virus movement. J Gen Virol 78: 1235–1239. https://doi.org/10.1099/0022-1317-78-6-1235. Cardoso JMS, Félix MR, Clara MIE, Oliveira S. 2009. Complete genome sequence of a Tobacco necrosis virus D isolate from olive trees. Arch Virol 154:1169 –1172. https://doi.org/10.1007/s00705-009-0414-9. Min XJ, Butler G, Storms R, Tsang A. 2005. OrfPredictor: predicting protein-coding regions in EST-derived sequences. Nucleic Acids Res 33:W677–W680. https://doi.org/10.1093/nar/gki394.
genomea.asm.org 2
