Recombination analysis of Soybean mosaic virus sequences reveals evidence of RNA recombination between distinct pathotypes
© Gagarinova et al; licensee BioMed Central Ltd. 2008
Received: 03 August 2008
Accepted: 26 November 2008
Published: 26 November 2008
RNA recombination is one of the two major factors that create RNA genome variability. Assessing its incidence in plant RNA viruses helps understand the formation of new isolates and evaluate the effectiveness of crop protection strategies. To search for recombination in Soybean mosaic virus (SMV), the causal agent of a worldwide seed-borne, aphid-transmitted viral soybean disease, we obtained all full-length genome sequences of SMV as well as partial sequences encoding the N-terminal most (P1 protease) and the C-terminal most (capsid protein; CP) viral protein. The sequences were analyzed for possible recombination events using a variety of automatic and manual recombination detection and verification approaches. Automatic scanning identified 3, 10, and 17 recombination sites in the P1, CP, and full-length sequences, respectively. Manual analyses confirmed 10 recombination sites in three full-length SMV sequences. To our knowledge, this is the first report of recombination between distinct SMV pathotypes. These data imply that different SMV pathotypes can simultaneously infect a host cell and exchange genetic materials through recombination. The high incidence of SMV recombination suggests that recombination plays an important role in SMV evolution. Obtaining additional full-length sequences will help elucidate this role.
Soybean mosaic virus (SMV) is a member of the genus Potyvirus, the family Potyviridae. It is one of the most devastating viral pathogens of soybean crops causing severe symptoms such as mosaic, mottling, chlorosis and rugosity in leaves, as well as reductions in plant growth with yield losses of up to 100% [1–4]. Like all potyviral genomes, the SMV genome is a single-stranded, positive-sense RNA molecule that is approximately 10 kb in length and contains a single open reading frame [4–6]. It encodes a large polyprotein that is co- and post-translationally cleaved into 11 final protein products [4–6]. SMV is found in all soybean-growing regions of the world. In the United States, at least 98 SMV isolates have been documented [7, 8]. Based on their differential interactions with SMV resistant cultivars, these isolates are classified into seven distinct strain groups, G1 through G7 [7, 8]. Similarly, five (A to E) and eight (Sa to Sh) SMV strains have been reported in Japan and China, respectively [9–11]. Though the pathotypic relationships between the SMV groups in the United States and the strains in China and Japan are not clear, these data clearly suggest a high genetic diversity of SMV.
The two major factors that contribute to the variability and evolution of RNA viruses are mutation introduced by the viral RNA-dependent RNA polymerase and recombination between different viral RNA molecules . The mutation rate varies between virus species, and the recombination frequency is dependent on the degree of sequence similarity between the sequences involved, the length of viral genome and the presence of recombination hot spots [12–14]. Mutation has been demonstrated to be responsible for the emergence of new SMV isolates that differentiate from their parental isolates by breaking resistance in soybean under both field and laboratory conditions [15–17]. However, the role of RNA recombination in SMV evolution still remains unknown.
We evaluated RNA recombination in SMV. All partial SMV sequences encoding the N-terminal most (P1 protease) and the C-terminal most (capsid protein; CP) as well as full-length SMV sequences were retrieved from GenBank [Additional file 1]. The alignments were performed using ClustalW . Subsequently, the automatic recombination scans of the sequence alignments were performed using Recombination Detection Program v.3.31 (RDP3) with default settings . RDP3 scans all possible triplet combinations of sequences to identify and statistically test the recombination signals . When two (parental) sequences are joined to form a recombinant (daughter) sequence, recombination signals may be detected in the parental, daughter, any descendant, and other closely related isolates. Thus, it is possible that a single recombination event can be counted several times. RDP3 overcomes this complication by automatically combining recombination signals to identify a minimum set of unique recombination events that account for the observed similarity patterns among sequences.
We further examined more recent, in evolutionary terms, recombination events in full-length SMV sequences. Putative non-recombinant and recombinant sequences were identified using Simplot  with 200 bp window and 20 bp step sizes. Location and significance of each putative recombination site was tested using the χ2 test, or the informative sites analysis, implemented in Simplot [21–23]. The recombination site was placed where the highest significant χ2 value was obtained. Two blocks of sequences, on either side of the recombination site, each from a single parent, were compared to assess the likelihood of recombination at the given site. First, non-SMV potyvirus and, subsequently, a distantly related isolate of SMV were used as outgroups in χ2 tests to increase the number of informative sites and to narrow down the location of the recombination. Essentially, locations of the putative recombination sites identified by the Simplot coincided with the locations of the unique recombination events identified by RDP3 (data not shown). Recombination sites in CN18, HZ, and HH5 sequences were supported with a P-value < 0.05 by the χ2 test. However, no recombination sites were manually assigned to these isolates since a large number of un-uniformly distributed informative sites supported grouping of these isolates with the outgroup in all tests, indicating that isolates CN18, HZ, and HH5 were too diverged from all other SMV sequences for the χ2 test (data not shown). Nevertheless, a number of significant recombination events were identified in G5, G7H, and G7f sequences.
Analyses of the G7f genome revealed six recombination events, 'a' through 'f', that occurred in the formation of this isolate. G7f was most similar to G7x and G7d isolates along most of its genome, but was more similar to G2 between recombination sites 'a' and 'b', 'c' and 'd', 'e' and 'f' [Additional file 5]. The following were the nucleotide position numbers of recombination event locations in respect to the G7f sequence: 'a' 5102–5114, 'b' 5252–5285, 'c' 6021–6026, 'd' 6140–6176, 'e' 8846–8858, 'f' 9008–9035. In χ2 tests, all 6 recombination sites were supported with P-value < 0.0025. However, either mutation, coupled with strong selection, or recombination could result in an isolate being most similar to two different isolates in the neighbouring regions . Selection of mutations would be expected to act at the amino acid sequence level as, to the best of our knowledge, avirulence determinants have only been reported to act at this level [26–32]. On the other hand, recombination may or may not affect the amino acid sequence of the resulting chimera. In this study, most sites that supported grouping of G7f with G2 were silent [Additional file 6] [Additional file 7], providing support for recombination rather than selection hypotheses to explain these similarities. All manually identified G5, G7H, and G7f recombination sites were recapitulated by manual GENECONV test implemented in RDP3 (data not shown).
The manually generated and verified results presented here provide the strong evidence of recombination in SMV. The most parsimonious output of RDP3 for full-length sequences [Additional file 2] partially, but better than other RDP3 outputs, coincided with the manual sequence comparisons results, emphasizing the need for the manual verification of automatically generated results. Furthermore, the G5 and G7H recombination analysis results suggest recombination analysis as a tool for directing experiments to identify avirulence determinants and develop novel crop protection strategies. However, utility of recombination detection in SMV is still limited by the lack of representative full-length genome sequences for most of SMV strains. Availability of representative sequences with associated pathogenicity profiles will allow elucidating the evolutionary history of SMV and deriving testable hypotheses about SMV-soybean interactions.
In spite of limitations to analyzing SMV recombination, application of conceptually different, complementary approaches allowed us to detect recombination sites previously missed by Chare and Holmes . Our work showed, for the first time, that recombination occurred during SMV evolution among distinct viral isolates and thus provided evidence that at least two distinct viral SMV pathotypes can simultaneously infect a host cell and exchange genetic materials through RNA recombination. The high frequency of recombination detected in SMV suggests that recombination plays an important role in SMV evolution and this should be considered when novel antiviral strategies are developed.
This work was supported by Ontario Soybean Growers, the AAFC Crop Genomics Initiative and the Natural Sciences and Engineering Research Council of Canada. AGG was a recipient of Ontario Graduate Scholarship and Western Graduate Research Scholarship.
- Arif M, Hassan S: Evaluation of resistance in soybean germplasm to Soybean mosaic potyvirus under field conditions. J Biol Sci 2002, 2: 601-604.View ArticleGoogle Scholar
- Gunduz I, Buss GR, Chen P, Tolin SA: Genetic and phenotypic analysis of Soybean mosaic virus resistance in PI 88788 soybean. Phytopathology 2004, 94: 687-692. 10.1094/PHYTO.2004.94.7.687View ArticlePubMedGoogle Scholar
- Liao L, Chen P, Buss GR, Yang Q, Tolin SA: Inheritance and allelism of resistance to Soybean mosaic virus in Zao18 soybean from China. J Hered 2002, 93: 447-452. 10.1093/jhered/93.6.447View ArticlePubMedGoogle Scholar
- Babu M, Gagarinova AG, Brandle JE, Wang A: Association of the transcriptional response of soybean plants with soybean mosaic virus systemic infection. J Gen Virol 2008, 89: 1069-1080. 10.1099/vir.0.83531-0View ArticlePubMedGoogle Scholar
- Urcuqui-Inchima S, Haenni AL, Bernardi F: Potyvirus proteins: a wealth of functions. Virus Res 2001, 74: 157-175. 10.1016/S0168-1702(01)00220-9View ArticlePubMedGoogle Scholar
- Chung BY-W, Miller WA, Atkins JF, Firth AE: An overlapping essential gene in the Potyviridae . Proc Natl Acad Sci USA 2008, 105: 5897-5902. 10.1073/pnas.0800468105PubMed CentralView ArticlePubMedGoogle Scholar
- Cho EK, Goodman RM: Strains of soybean mosaic virus classification based on virulence in resistanct soybean cultivars. Phytopathology 1979, 69: 467-470. 10.1094/Phyto-69-467View ArticleGoogle Scholar
- Cho EK, Goodman RM: Evaluation of resistance in soybeans to soybean mosaic virus strains. Crop Sci 1982, 22: 1133-1136.View ArticleGoogle Scholar
- Takahashi K, Tanaka T, Iida W, Tsuda Y: Studies on virus diseases and causal viruses of soybean in Japan. Tohoku Natl Agric Exp Stn Bull 1980, 62: 1-130.Google Scholar
- Chen YX, Xue BD, Hu YZ, Fang ZD: Identification of two new starins of soybean mosaic virus. Acta Phytophyl Sin 1986, 13: 221-226.Google Scholar
- Pu ZQ, Cao Q, Fang DC, Xi BD, Fang CT: Identification of strains of soybean mosaic virus. Acta Phytophyl Sin 1982, 9: 15-20.Google Scholar
- García-Arenal F, Fraile A, Malpica JM: Variation and evolution of plant virus populations. Int Microbiol 2003, 6: 225-232. 10.1007/s10123-003-0142-zView ArticlePubMedGoogle Scholar
- Lai MM: RNA recombination in animal and plant viruses. Microbiol Rev 1992, 56: 61-79.PubMed CentralPubMedGoogle Scholar
- Gallei A, Pankraz A, Thiel HJ, Becher P: RNA recombination in vivo in the absence of viral replication. J Virol 2004, 78: 6271-6281. 10.1128/JVI.78.12.6271-6281.2004PubMed CentralView ArticlePubMedGoogle Scholar
- Choi BK, Koo JM, Ahn HJ, Yum HJ, Choi CW, Ryu KH, Chen P, Tolin SA: Emergence of Rsv -resistance breaking Soybean mosaic virus isolates from Korean soybean cultivars. Virus Res 2005, 112: 42-51. 10.1016/j.virusres.2005.03.020View ArticlePubMedGoogle Scholar
- Gagarinova AG, Babu M, Poysa V, Hill JH, Wang A: Identification and molecular characterization of two naturally occurring Soybean mosaic virus isolates that are closely related but differ in their ability to overcome Rsv4 resistance. Virus Res 2008, in press.Google Scholar
- Hajimorad MR, Eggenberger AL, Hill JH: Evolution of Soybean mosaic virus -G7 molecularly cloned genome in Rsv1 -genotype soybean results in emergence of a mutant capable of evading Rsv1 -mediated recognition. Virology 2003, 314: 497-509. 10.1016/S0042-6822(03)00456-2View ArticlePubMedGoogle Scholar
- Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994, 22: 4673-4680. 10.1093/nar/22.22.4673PubMed CentralView ArticlePubMedGoogle Scholar
- Martin DP, Williamson C, Posada D: RDP2: recombination detection and analysis from sequence alignments. Bioinformatics 2005, 21: 260-262. 10.1093/bioinformatics/bth490View ArticlePubMedGoogle Scholar
- Lole KS, Bollinger RC, Paranjape RS, Gadkari D, Kulkarni SS, Novak NG, Ingersoll R, Sheppard HW, Ray SC: Full-length human immunodeficiency virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of inter subtype recombination. J Virol 1999, 73: 152-160.PubMed CentralPubMedGoogle Scholar
- Robertson DL, Hahn BH, Sharp PM: Recombination in AIDS viruses. J Mol Evol 1995, 40: 249-259. 10.1007/BF00163230View ArticlePubMedGoogle Scholar
- Robertson DL, Sharp PM, McCutchan FE, Hahn BH: Recombination in HIV-1. Nature 1995, 374: 124-126. 10.1038/374124b0View ArticlePubMedGoogle Scholar
- Smith JM: Analyzing the mosaic structure of genes. J Mol Evol 1992, 34: 126-129.PubMedGoogle Scholar
- Lim WS, Kim YH, Kim KH: Complete genome sequences of the genomic RNA of Soybean mosaic virus strains G7H and G5. Plant Pathol 2003, 19: 171-176.View ArticleGoogle Scholar
- Domingo E, Holland JJ: RNAvirus mutations and fitness for survival. Annu Rev Microbiol 1997, 51: 151-78. 10.1146/annurev.micro.51.1.151View ArticlePubMedGoogle Scholar
- Hajimorad MR, Eggenberger AL, Hill JH: Loss and gain of elicitor function of Soybean mosaic virus G7 provoking Rsv1 -mediated lethal systemic hypersensitive response maps to P3. J Virol 2005, 79: 1215-1222. 10.1128/JVI.79.2.1215-1222.2005PubMed CentralView ArticlePubMedGoogle Scholar
- Rajamaki ML, Valkonen JPT: The 6K2 protein and the VPg of potato virus A are determinants of systemic infection in Nicandra physaloides . Mol Plant-Microbe Interact 1999, 12: 1074-1081. 10.1094/MPMI.19184.108.40.2064View ArticlePubMedGoogle Scholar
- Jenner CE, Sanchez F, Nettleship SB, Foster GD, Ponz F, Walsh JA: The cylindrical inclusion gene of Turnip mosaic virus encodes a pathogenic determinant to the Brassica resistance gene TuRB01 . Mol Plant-Microbe Interact 2000, 13: 1102-1108. 10.1094/MPMI.2000.13.10.1102View ArticlePubMedGoogle Scholar
- Kühne T, Shi N, Proeseler G, Adams MJ, Kanyuka K: The ability of a bymovirus to overcome the rym4 -mediated resistance in barley correlates with a codon change in the VPg coding region on RNA1. J General Virol 2003, 84: 2853-2859. 10.1099/vir.0.19347-0View ArticleGoogle Scholar
- Ruffel S, Gallois JL, Lesage ML, Caranta C: The recessive potyvirus resistance gene pot-1 is the tomato orthologue of the pepper pvr2-eIF4E gene. Mol Gen Genomics 2005, 274: 346-353. 10.1007/s00438-005-0003-xView ArticleGoogle Scholar
- Hjulsager CK, Olsen BS, Kjaer Jensen DM, Cordea MI, Krath BN, Johansen IE, Lund OS: Multiple determinants in the coding region of Pea seed-borne mosaic virus P3 are involved in virulence against sbm-2 resistance. Virology 2006, 355: 52-61. 10.1016/j.virol.2006.07.016View ArticlePubMedGoogle Scholar
- Desbiez C, Gal-On A, Girard M, Wipf-Scheibel C, Lecoq H: Increase in Zucchini yellow mosaic virus symptom severity in tolerant zucchini cultivars is related to a point mutation in P3 protein and is associated with a loss of relative fitness on susceptible plants. Phytopathology 2003, 93: 1478-1484. 10.1094/PHYTO.2003.93.12.1478View ArticlePubMedGoogle Scholar
- Chare ER, Holmes EC: A phylogenetic survey of recombination frequency in plant RNA viruses. Arch Virol 2006, 151: 933-946. 10.1007/s00705-005-0675-xView ArticlePubMedGoogle Scholar
- Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG: The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 1997, 24: 4876-4882. 10.1093/nar/25.24.4876View ArticleGoogle Scholar
- Ronquist F, Huelsenbeck JP: MRBAYES 3:Bayesian phylogenetic inference under mixed models. Bioinformatics 2003, 19: 1572-1574. 10.1093/bioinformatics/btg180View ArticlePubMedGoogle Scholar
- Felsenstein J: PHYLIP – Phylogeny Inference Package (Version 3.2). Cladistics 1989, 5: 164-166.Google Scholar
- Guindon S, Gascuel O: A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 2003, 52: 696-704. 10.1080/10635150390235520View ArticlePubMedGoogle Scholar
- Shimodaira H, Hasegawa M: Multiple comparisons of log-likelihoods with applications to phylogenetic inference. Mol Biol Evol 1999, 16: 1114-1116.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.