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Evidence of a tomato spotted wilt virus resistance-breaking strain originated through natural reassortment between two evolutionary-distinct isolates
Virus Research 196 (2015) 157–161
Contents lists available at ScienceDirect
Virus Research journal homepage: www.elsevier.com/locate/virusres
Short communication
Evidence of a tomato spotted wilt virus resistance-breaking strain originated through natural reassortment between two evolutionary-distinct isolates P. Margaria a,b , M. Ciuffo a , C. Rosa b , M. Turina a,∗ a b
Istituto per la Protezione Sostenibile delle Piante, CNR, Strada delle Cacce 73, 10135 Torino, Italy Department of Plant Pathology and Environmental Microbiology, Pennsylvania State University, University Park, PA 16802, USA
a r t i c l e
i n f o
Article history: Received 30 September 2014 Received in revised form 11 November 2014 Accepted 12 November 2014 Available online 26 November 2014 Keywords: Tospovirus Reassortment Silencing suppressor Phylogeny
a b s t r a c t A Tsw resistance-breaking tomato spotted wilt virus field isolate (TSWV-p331) found in northern Italy originated via reassortment from two evolutionary distinct TSWV strains, as revealed by recombination and phylogenetic analysis. Compared to the closest isolate present in the database, p331 NSs protein carries an unusually high number of amino acid substitutions, but no differences in the nucleocapsid protein. Despite these substitutions, p331 NSs is a potent silencing suppressor. As shown by phylogenetic analyses of TSWV nucleocapsid sequences collected over fifteen years, one likely p331 parental lineage has never been detected in northern Italy, allowing speculations on the origin of TSWV-p331. © 2014 Elsevier B.V. All rights reserved.
Tomato spotted wilt virus is the type species of the genus Tospovirus in the family Bunyaviridae (Scholthof et al., 2011). Members of this species are able to infect more than 1000 plant species worldwide, and cause severe damage to many economically important crops including bean, lettuce, pepper, potato, tobacco, tomato and numerous ornamental species (Pappu et al., 2009). In nature, TSWV is transmitted exclusively by thrips species in the genera Frankliniella and Thrips; Frankliniella occidentalis Pergande, the western flower thrips, is its most efficient vector (Whitfield et al., 2005). TSWV has enveloped, quasi/spherical particles, 80–110 nm in size, containing three RNA genomic segments, designated large (L), medium (M) and small (S) (Goldbach and Peters, 1996). The L RNA possesses a single open reading frame (ORF) in the viral complementary sense, coding for the viral RNA dependent RNA polymerase (RdRp). The M and S RNAs encodes for two proteins each: specifically, in the viral sense for the non-structural proteins NSm and NSs, respectively, shown to be the movement protein and the silencing suppressor, and in the viral-complementary sense
∗ Corresponding author. Tel.: +39 0113977923; fax: +39 011343809. E-mail addresses: [email protected] (P. Margaria), [email protected] (M. Ciuffo), [email protected] (C. Rosa), [email protected] (M. Turina). http://dx.doi.org/10.1016/j.virusres.2014.11.012 0168-1702/© 2014 Elsevier B.V. All rights reserved.
for the glycoprotein precursor (Gn/Gc) and the nucleocapsid (N) protein, respectively (Goldbach and Peters, 1996). The tripartite genome offers the potential to exchange entire genomic segments among different isolates co-infecting the same plant, giving rise to new TSWV variants. This mechanism is designated as reassortment: first evidence of its occurrence for TSWV was provided by Best in 1961 (Best, 1961). Given the absence of a reverse-genetic system for this virus, reassortment has been used in experimental conditions to map the avirulence determinant necessary to overcome the Sw5 resistance gene in tomato (Hoffmann et al., 2001) and the Tsw resistance gene in pepper (Jahn et al., 2000; Margaria et al., 2007). The same approach was also used in F. occidentalis to map the Gn/Gc and NSs proteins as thrips vector transmission determinants (Sin et al., 2005; Margaria et al., 2014a). Reassortment was shown to occur spontaneously in TSWV (Qiu and Moyer, 1999). The occurrence of extensive reassortment events for isolates from Spain, France and Italy, showed that this mechanism plays an important role in TSWV emergence and epidemics (Tentchev et al., 2011). Reassortment exchanges have been predicted between Asiatic and European TSWV populations (Tentchev et al., 2011), as well as among isolates from Korea (Lian et al., 2013) and New Zealand (Timmerman-Vaughan et al., 2014), and are likely at the origin of one of the two TSWV lineages present in Italy (Margaria et al., 2014b). In this context, it should be mentioned that a broad analysis on reassortment events is still hampered by the
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P. Margaria et al. / Virus Research 196 (2015) 157–161
limited number of full-length TSWV genomic sequences available in GenBank. In fact, no evidence of reassortment or recombination was obtained in a previous analysis with natural TSWV isolates collected from several geographical locations in the United States and Europe (Tsompana et al., 2005), possibly due to the lack of complete genomic sequence data, especially for the L genomic segment. In Italy, a TSWV population survey carried out at the beginning of the twenty-first century in Apulia region grouped the isolates into two molecularly distinct subpopulation clusters, named TSWV-A or TSWV-D like, according to their similarity with type-isolates from the USA (TSWV-A) or the Netherlands (TSWV-D) (FinettiSialer et al., 2002). In this analysis, a collection of isolates sampled during virus epidemics in Apulia from 1999 to 2001 was considered, but no strains from northern Italy were included (Finetti-Sialer et al., 2002). A recent phylogenetic analysis based on the N gene of several isolates from the Mediterranean region, also showed that Italian isolates clustered in two distinct sub-populations, in contrast to other countries, such as France, Spain, and Bulgaria, where TSWV isolates grouped uniformly into one population (Turina et al., 2012). Despite the presence of evolutionary-distinct isolates in the same geographic area, reassortment between isolates from the two clades has not been reported so far in nature in Italy. Two TSWV isolates from Italy, p105 and p202/3WT, have been recently fully sequenced and characterized (Margaria et al., 2014b). Isolate p105 was collected from a pepper field in Liguria, northern Italy, while p202/3WT was collected in pepper in Sicily, southern Italy. Phylogenetic analyses grouped the two strains into the two separated clades previously reported in Italy (Finetti-Sialer et al., 2002), placing p105 in the TSWV-A clade and p202/3WT in the TSWV-D clade (Margaria et al., 2014b). In summer 2012, an outbreak of TSWV was observed in Carmagnola (Piedmont, Northern Italy), a district where sweet pepper crops are intensively cultivated and where the use of pepper varieties carrying the Tsw resistance gene is still limited. Isolate p331 was collected in a field where resistant peppers were grown. This isolate was passaged three times through single-local lesion transfer on Nicotiana tabacum cv. White Burley and then on Capsicum chinensis PI152225 carrying the Tsw gene, where it was still able to overcome resistance as the original field isolate. In this work, we characterized the p331 genome, and we found that molecular markers for the L and S segment grouped consistently with p202/3WT-like isolates (TSWV-D clade), while those for the M segment grouped with p105-like isolates (TSWV-A clade). In order to characterize p331 genomic segments, we used five molecular markers located along the whole genome: the Gn/Gc, NSs and N markers consisted of a fragment in the open reading frame (ORF) of the corresponding gene, being 692, 587 and 774 nt in length, respectively; the RdRp and NSm markers, beside a fragment of the corresponding gene, included also a portion of the 3 untranslated region and intergenic region, respectively, and were 310 and 878 nt in length. Total RNA was extracted from systemically infected N. benthamiana leaves using the “SpectrumTM Plant Total RNA Kit” (Sigma–Aldrich, St. Loius, MO, USA). Reverse transcription was performed using the specific reverse primer (Supplementary Table 1) with the “ThermoscriptTM RT-PCR” kit (Invitrogen, Grand Island, NY, USA), and followed by PCR using PolyTaq polymerase (Polymed, Florence, Italy) and primers reported in Supplementary Table 1. Amplification products were cloned using the “pGEM-T Easy vector” system (Promega, Madison, WI, USA) and sequenced (see Supplementary Table 1 for GenBank accession numbers). A first analysis of possible recombination events was performed using Recombination Detection Program 4.16 (RDP4) software (Martin and Rybicki, 2010). For this purpose, the sequences of the five markers of each isolate were concatenated together in a single sequence file, in the following order: RdRp, NSm, Gn/Gc, NSs and N marker, giving rise to an artificial full-length contig
of about 3300 nt. Markers for p105 and p202/3WT isolates were retrieved by selecting the same target regions from the sequences available in GenBank (Margaria et al., 2007, 2014a,b). Isolates p105 and p202/3WT were chosen for comparison because they are the closest isolates present in the database for which full length genomic sequence is available (Margaria et al., 2014b). Sequences were aligned using MEGA6 software according to the Clustal W algorithm (Tamura et al., 2013), and then run in RPD software, at default parameter values. Three recombination events were consistently found by different detection algorithms: recombination event number 1 concerned the whole S segment markers (N and NSs), and event number 2 concerned the whole L segment marker (Table 1). Both events were detected at stringent P-value scores (lowest confidence was at E-05), and identified p202/3WT as the contributing parental for p331 for the L and S genomic segments. A third recombination event identified the p105 as the major parent contributing to p331 for the whole M segment, at stringent P values (Table 1). Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres. 2014.11.012. Similarity percentages and potential reassortment analysis among the isolates in study was further performed by scanning of the concatenated marker sequences with SimPlot v. 3.5.1 (Lole et al., 1999), at default values. As shown in Fig. 1, p331 high sequence similarity was found with p202/3WT in the L (RdRp marker) and S (NSs and N marker) segments, while for the M segment, the highest similarity was between p331 and p105. We further obtained similarity matrix scores for the markers in study using MatGat v. 2.03 (Campanella et al., 2003): sequence analysis confirmed higher percentage scores between p331 and p202/3WT for the L and S segment markers (98.4, 99.5 and 99.6% for RdRp, NSs and N markers, respectively), and between p331 and p105 for the M markers (97.8 and 90.5%, for Gn/Gc and NSm, respectively) (Supplementary Table 2). The nucleotide alignments of the five markers are provided in Supplementary Fig. 1. Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres. 2014.11.012. Phylogenetic analyses according to the Neighbor-Joining method (Saitou and Nei, 1987) using the alignment of the concatenated markers for each genomic segment, grouped p331 in the p202/3WT clade for the L and S segment, and in the p105 clade for the M segment (Fig. 1). Together, these results support different origin for the genomic segments of p331 and consistently provide direct evidence that p331 was derived through reassortment between two evolutionary distinct TSWV isolates; the two possible parental isolates used for comparison are both unable to overcome Tsw resistance. Presence of a TSWV-D-like strain was never witnessed before in northern Italy, and therefore we decided to investigate a number of isolates from northern Italy present in the PLAVIT (Plant Viruses Italy, http://www.wfcc.info/ccinfo/index.php/collection/ by id/1057/) collection. For this study, we newly sequenced the N gene of seven isolates collected during TSWV outbreaks in northern Italy (Piedmont, Liguria and Lombardy regions) from 1997 to 2014. Sequences were used for phylogenetic analysis, together with the sequence of TSWV isolates from northern and southern Italy previously characterized (Margaria et al., 2007; Turina et al., 2012; Zindovic et al., 2014). Nucleocapsid protein sequences showed high similarity among all isolates (not shown), as expected; in fact, amino acid changes in this protein are thought to be deleterious for the virus (Tentchev et al., 2011). However, phylogenetic analysis on the nucleotide sequences showed that all the isolates from northern Italy grouped with p105, with the exception of isolate p331 which grouped in the p202/3WT clade (Fig. 2), suggesting that this
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Table 1 Prediction of potential recombination/reassortment events among tomato spotted wilt virus (TSWV) isolates p331, p105 and p202/3WT by different detection algorithms implemented in the Recombination Detection Program (RDP4) software, using as input the concatenated sequence of five genomic markers on TSWV genomic segments. Event number
1 2 3
Breakpoint position Begin
End
1954 1 256
3286 313 1954
Corresponding marker
NSs + N RdRp NSm + Gn/Gc
Genomic segment
S L M
Recombinant
p331 p331 p331
Parental
p202/3WT p202/3WT p105
Score of detection algorithms
GENECONV
Bootscan
MaxChi
Chimaera
RDP
3Seq
2.98E−05 2.71E−06 5.51E−03
8.97E−11 2.64E−07 –
3.61E−12 – 2.67E−14
8.42E−12 – 1.63E−14
5.50E−12 3.25E−07 5.60E−08
– 1.60E−06 2.25E−20
RdRp, RNA dependent RNA polymerase; NSm, non-structural m protein; Gn/Gc, glycoprotein precursor; NSs non-structural s protein; N, nucleocapsid protein; L, large genomic segment; M, medium genomic segment; S, small genomic segment.
evolutionary lineage in northern Italy is not widespread, and that isolate p331 shows unique features. Given that p331 was able to overcome the Tsw resistance gene in pepper and that substitutions in the NSs protein are sufficient to provide the resistance-breaking (RB) phenotype (Margaria et al., 2007), we fully characterized the NSs gene, according to previous protocols (Margaria et al., 2014a). Three independent PCR reactions and sequencing analysis were performed. The NSs ORF of p331 is 1401 nt in length and encodes a 467aa-long protein (GenBank accession KM213989), similar to other Italian wild-type and RB TSWV isolates previously characterized (Margaria et al., 2007).
While we and other authors have shown that one or two amino acid changes in the NSs protein were enough to confer the RB phenotype on Tsw resistant pepper plants (Margaria et al., 2007; de Ronde et al., 2013), seven amino acid substitutions are present in p331 in comparison to the p202/3WT NSs protein, and five against the NSs protein of p170, a wild-type TSWV isolate also collected in Sicily, southern Italy (as for p202/3WT) and partially characterized (Margaria et al., 2007) (Supplementary Fig. 2). Remarkably, the p331 nucleocapsid protein was found to be conserved without changes (not shown). None of the observed NSs-substitutions were present in other Tsw-gene RB isolates previously characterized
Fig. 1. Isolates sequence comparison. (A) Phylogenetic trees of the concatenated markers in each genomic segment inferred using the Neighbor-Joining method, showing evolutionary relationships between the three isolates in study. (B) Similarity plot between tomato spotted wilt virus isolates p331, p105 and p202/3WT generated using SimPlot v. 3.5.1 software. The concatenated nucleotide sequences of the five genomic markers (one in each viral gene) were first aligned using ClustalW, and identities between the query sequence (p331) and each putative parental sequences (p105 and p202/3WT) were plotted. Each point plotted is the percent identity within a sliding window 200 bp wide centred on the position plotted, with a step size between points of 20 bp. The horizontal bar above the curves shows the open reading frames of the TSWV genome corresponding to the markers and the brackets show the three genomic segments. RdRp, RNA dependent RNA polymerase; NSm, non-structural m protein; Gn/Gc, glycoprotein precursor; NSs non-structural s protein; N, nucleocapsid; L, large genomic segment; M, medium genomic segment; S, small genomic segment.
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Fig. 2. Phylogenetic tree obtained from the alignment of nucleocapsid (N) gene sequences of tomato spotted wilt virus (TSWV) isolates from different regions (in parenthesis) in northern and southern Italy. Tree was drawn according to the Neighbor-Joining method allowing 1000 bootstrap replicates. Bootstrap values above 60% are indicated for each node. The evolutionary distances are expressed in the units of the number of base differences per site. Asterisks mark TSWV isolates newly sequenced in this work. The virus isolates and GenBank accession numbers are as follows: p302 (KM096541), p166 (DQ376179), p322 (KM096538), p207 (KM096539), p105 (DQ376178), p231 (KM096536), p272 (DQ376181), p252 (KM096537), p147 (KM096535), p267(DQ376180), T-1081 (KM096542), p331 (KM213988), p170 (DQ431237), I-186 (GU369719), Miz3 (GU369720), T-1003 (GU369725), p202/3WT, p202/1 (GU369721), p240 (GU369722).
(Margaria et al., 2007; de Ronde et al., 2013), further confirming that different substitutions in different region of the NSs protein are sufficient to provide the RB phenotype (Margaria et al., 2007; de Ronde et al., 2013). Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres. 2014.11.012. Recent works have outlined the possibility that some RB isolates might lose the silencing suppression ability provided by the NSs protein (Margaria et al., 2007, 2014a). A recent study showed the presence of essential residues in the N-terminal domain of the NSs protein for silencing suppression activity (de Ronde et al., 2014).
Similarly, other authors performed site-directed mutagenesis of highly conserved amino acids in the NSs protein and identified two sites which are critical for silencing suppression activity (Zhai et al., 2014). Given the unusually high number of amino acid substitutions present in the p331 reassortant RB isolate, we further investigated the silencing suppressor ability of p331 NSs, by in vivo assays in N. benthamiana line 16C, as previously described (Margaria et al., 2007). The p331 NSs proved to be a strong silencing suppressor, not distinguishable in the bioassay from a control NSs from a closely related wild-type isolate (p105); as negative control, we used the p170RB NSs allelic version, which was previously shown to have lost silencing suppressor activity (Margaria et al., 2007) (Fig. 3).
Fig. 3. In planta bioassays for silencing suppression activity of p331 NSs allelic variants. Full-length NSs allels were cloned in pBin61 vector and transiently expressed through agroinfiltration together with pBin-GFP in 16C transgenic Nicotiana benthamiana. Controls consisted of vector only (pBin61), p105 NSs allele (positive control) and p170RB NSs allele (not functional in silencing suppression, negative control). Pictures were taken by using a hand-held UV light 3 days post-infiltration.
P. Margaria et al. / Virus Research 196 (2015) 157–161
Apart from the uniqueness of our finding of an RB isolate derived through reassortment of evolutionary distinct parental strains, we can speculate about the possible origin of p331: either the two parental isolates (A-like and D-like) were already present in the area and the reassortant isolate might have evolved in a possible adaptation step, or alternatively, the reassortant isolate was imported together with pepper plantlets from nurseries located in areas where the two kinds of parental isolates already coexist. Future surveys in the Piedmont area will help us establishing if a mix of TSWV populations already coexisted, or if a clonal population with characteristics similar to p331 originated from genetic drift and is expanding in the area in the absence of one of the likely parental isolates. References Best, R.J., 1961. Recombination experiments with strains A and E of tomato spotted wilt virus. Virology 15, 327–339. Campanella, J.J., Bitincka, L., Smalley, J., 2003. MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences. BMC Bioinform. 4, 29. de Ronde, D., Pasquier, A., Ying, S., Butterbach, P., Lohuis, D., Kormelink, R., 2014. Analysis of tomato spotted wilt virus NSs protein indicates the importance of the N-terminal domain for avirulence and RNA silencing suppression. Mol. Plant Pathol. 15, 185–195. de Ronde, D., Butterbach, P., Lohuis, D., Hedil, M., Van Lent, J.W.M., Kormelink, R., 2013. Tsw gene-based resistance is triggered by a functional RNA silencing suppressor protein of the tomato spotted wilt virus. Mol. Plant Pathol. 14, 405–415. Finetti-Sialer, M.M., Lanace, V., Padula, M., Vovlas, C., Gallitelli, D., 2002. Occurrence of two distinct tomato spotted wild virus (TSWV) subgroups in Southern Italy. J. Plant Pathol. 84, 145–152. Goldbach, R., Peters, D., 1996. Molecular and biological aspects of tospoviruses. In: Elliot, R.M. (Ed.), The Bunyaviridae. Plenum Press, New York, NY, USA, pp. 129–157. Hoffmann, K., Qiu, W.P., Moyer, J.W., 2001. Overcoming host- and pathogenmediated resistance in tomato and tobacco maps to the M RNA of tomato spotted wilt virus. Mol. Plant Microbe Interact. 14, 242–249. Jahn, M., Paran, I., Hoffman, K., Radwanski, E.R., Livingstone, K.D., Grube, R.C., Aftergoot, E., Lapidot, M., Moyer, J., 2000. Genetic mapping of the Tsw locus for resistance to the tospovirus tomato spotted wilt virus in Capsicum spp. and its relationship to the Sw-5 gene for resistance to the same pathogen in tomato. Mol. Plant Microbe Interact. 13, 673–682. Lian, S., Lee, J.S., Cho, W.K., Yu, J., Kim, M.K., Choi, H.S., Kim, K.H., 2013. Phylogenetic and recombination analysis of tomato spotted wilt virus. PLoS ONE 8, e63380. Lole, K.S., Bollinger, R.C., Paranjape, R.S., Gadkari, D., Kulkarni, S.S., Novak, N.G., Ingersoll, R., Sheppard, H.W., Ray, S.C., 1999. Full length human immunodeficiency
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virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination. J. Virol. 73, 152–160. Margaria, P., Ciuffo, M., Pacifico, D., Turina, M., 2007. Evidence that the nonstructural protein of tomato spotted wilt virus is the avirulence determinant in the interaction with resistant pepper carrying the Tsw gene. Mol. Plant Microbe Interact. 20, 547–558. Margaria, P., Bosco, L., Vallino, M., Ciuffo, M., Mautino, G.C., Tavella, L., Turina, M., 2014a. The NSs protein of tomato spotted wilt virus is required for persistent infection and transmission by Frankliniella occidentalis. J. Virol. 88, 5788–5802. Margaria, P., Miozzi, L., Ciuffo, M., Pappu, H., Turina, M., 2014b. The first complete genome sequences of two distinct European tomato spotted wilt virus isolates. Arch. Virol., http://dx.doi.org/10.1007/s00705-014-2256-3 (in press). Martin, D., Rybicki, E., 2010. RDP: detection of recombination amongst aligned sequences. Bioinformatics 26, 2462–2463. Pappu, H.R., Jones, R.A.C., Jain, R.K., 2009. Global status of tospovirus epidemics in diverse cropping systems: successes achieved and challenges ahead. Virus Res. 141, 219–236. Qiu, W., Moyer, J., 1999. Tomato spotted wilt tospovirus adapts to the TSWV N gene-derived resistance by genome reassortment. Phytopathology 89, 575–582. Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425. Scholthof, K.B.G., Adkins, S., Czosnek, H., Palukaitis, P., Jacquot, E., Hohn, T., Hohn, B., Saunders, K., Candresse, T., Ahlquist, P., Hemenway, C., Foster, G.D., 2011. Top 10 plant viruses in molecular plant pathology. Mol. Plant Pathol. 12, 938–954. Sin, S.H., McNulty, B.C., Kennedy, G.G., Moyer, J.W., 2005. Viral genetic determinants for thrips transmission of tomato spotted wilt virus. Proc. Natl. Acad. Sci. U.S.A. 102, 5168–5173. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: molecular evolutionary genetics analysis Version 6.0. Mol. Biol. Evol. 30, 2725–2729. Tentchev, D., Verdin, E., Marchal, C., Jacquet, M., Aguilar, J., Moury, B., 2011. Evolution and structure of tomato spotted wilt virus populations: evidence of extensive reassortment and insights into emergence processes. J. Gen. Virol. 92, 961–973. Timmerman-Vaughan, G.M., Lister, R., Cooper, R., Tang, J., 2014. Phylogenetic analysis of New Zealand tomato spotted wilt virus isolates suggests likely incursion history scenarios and mechanisms for population evolution. Arch. Virol. 159, 993–1003. Tsompana, M., Abad, J., Purugganan, M., Moyer, J.W., 2005. The molecular population genetics of the tomato spotted wilt virus (TSWV) genome. Mol. Ecol. 14, 53–66. Turina, M., Tavella, L., Ciuffo, M., 2012. Tospoviruses in the Mediterranean area. In: Loebenstein, G., Lecoq, H. (Eds.), Viruses and Virus Diseases of Vegetables in the Mediterranean Basin-Advances in Virus Research, vol. 84. Elsevier Academic Press Inc, San Diego, CA, pp. 403–437. Whitfield, A.E., Ullman, D.E., German, T.L., 2005. Tospovirus-thrips interactions. Annu. Rev. Phytopathol. 43, 459–489. Zhai, Y., Bag, S., Mitter, N., Turina, M., Pappu, H.R., 2014. Mutational analysis of two highly conserved motifs in the silencing suppressor encoded by tomato spotted wilt virus (genus Tospovirus, family Bunyaviridae). Arch. Virol. 159, 1499–1504. Zindovic, J., Ciuffo, M., Turina, M., 2014. Molecular characterization of tomato spotted wilt virus in Montenegro. J. Plant Pathol. 96, 201–205.
Contents lists available at ScienceDirect
Virus Research journal homepage: www.elsevier.com/locate/virusres
Short communication
Evidence of a tomato spotted wilt virus resistance-breaking strain originated through natural reassortment between two evolutionary-distinct isolates P. Margaria a,b , M. Ciuffo a , C. Rosa b , M. Turina a,∗ a b
Istituto per la Protezione Sostenibile delle Piante, CNR, Strada delle Cacce 73, 10135 Torino, Italy Department of Plant Pathology and Environmental Microbiology, Pennsylvania State University, University Park, PA 16802, USA
a r t i c l e
i n f o
Article history: Received 30 September 2014 Received in revised form 11 November 2014 Accepted 12 November 2014 Available online 26 November 2014 Keywords: Tospovirus Reassortment Silencing suppressor Phylogeny
a b s t r a c t A Tsw resistance-breaking tomato spotted wilt virus field isolate (TSWV-p331) found in northern Italy originated via reassortment from two evolutionary distinct TSWV strains, as revealed by recombination and phylogenetic analysis. Compared to the closest isolate present in the database, p331 NSs protein carries an unusually high number of amino acid substitutions, but no differences in the nucleocapsid protein. Despite these substitutions, p331 NSs is a potent silencing suppressor. As shown by phylogenetic analyses of TSWV nucleocapsid sequences collected over fifteen years, one likely p331 parental lineage has never been detected in northern Italy, allowing speculations on the origin of TSWV-p331. © 2014 Elsevier B.V. All rights reserved.
Tomato spotted wilt virus is the type species of the genus Tospovirus in the family Bunyaviridae (Scholthof et al., 2011). Members of this species are able to infect more than 1000 plant species worldwide, and cause severe damage to many economically important crops including bean, lettuce, pepper, potato, tobacco, tomato and numerous ornamental species (Pappu et al., 2009). In nature, TSWV is transmitted exclusively by thrips species in the genera Frankliniella and Thrips; Frankliniella occidentalis Pergande, the western flower thrips, is its most efficient vector (Whitfield et al., 2005). TSWV has enveloped, quasi/spherical particles, 80–110 nm in size, containing three RNA genomic segments, designated large (L), medium (M) and small (S) (Goldbach and Peters, 1996). The L RNA possesses a single open reading frame (ORF) in the viral complementary sense, coding for the viral RNA dependent RNA polymerase (RdRp). The M and S RNAs encodes for two proteins each: specifically, in the viral sense for the non-structural proteins NSm and NSs, respectively, shown to be the movement protein and the silencing suppressor, and in the viral-complementary sense
∗ Corresponding author. Tel.: +39 0113977923; fax: +39 011343809. E-mail addresses: [email protected] (P. Margaria), [email protected] (M. Ciuffo), [email protected] (C. Rosa), [email protected] (M. Turina). http://dx.doi.org/10.1016/j.virusres.2014.11.012 0168-1702/© 2014 Elsevier B.V. All rights reserved.
for the glycoprotein precursor (Gn/Gc) and the nucleocapsid (N) protein, respectively (Goldbach and Peters, 1996). The tripartite genome offers the potential to exchange entire genomic segments among different isolates co-infecting the same plant, giving rise to new TSWV variants. This mechanism is designated as reassortment: first evidence of its occurrence for TSWV was provided by Best in 1961 (Best, 1961). Given the absence of a reverse-genetic system for this virus, reassortment has been used in experimental conditions to map the avirulence determinant necessary to overcome the Sw5 resistance gene in tomato (Hoffmann et al., 2001) and the Tsw resistance gene in pepper (Jahn et al., 2000; Margaria et al., 2007). The same approach was also used in F. occidentalis to map the Gn/Gc and NSs proteins as thrips vector transmission determinants (Sin et al., 2005; Margaria et al., 2014a). Reassortment was shown to occur spontaneously in TSWV (Qiu and Moyer, 1999). The occurrence of extensive reassortment events for isolates from Spain, France and Italy, showed that this mechanism plays an important role in TSWV emergence and epidemics (Tentchev et al., 2011). Reassortment exchanges have been predicted between Asiatic and European TSWV populations (Tentchev et al., 2011), as well as among isolates from Korea (Lian et al., 2013) and New Zealand (Timmerman-Vaughan et al., 2014), and are likely at the origin of one of the two TSWV lineages present in Italy (Margaria et al., 2014b). In this context, it should be mentioned that a broad analysis on reassortment events is still hampered by the
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limited number of full-length TSWV genomic sequences available in GenBank. In fact, no evidence of reassortment or recombination was obtained in a previous analysis with natural TSWV isolates collected from several geographical locations in the United States and Europe (Tsompana et al., 2005), possibly due to the lack of complete genomic sequence data, especially for the L genomic segment. In Italy, a TSWV population survey carried out at the beginning of the twenty-first century in Apulia region grouped the isolates into two molecularly distinct subpopulation clusters, named TSWV-A or TSWV-D like, according to their similarity with type-isolates from the USA (TSWV-A) or the Netherlands (TSWV-D) (FinettiSialer et al., 2002). In this analysis, a collection of isolates sampled during virus epidemics in Apulia from 1999 to 2001 was considered, but no strains from northern Italy were included (Finetti-Sialer et al., 2002). A recent phylogenetic analysis based on the N gene of several isolates from the Mediterranean region, also showed that Italian isolates clustered in two distinct sub-populations, in contrast to other countries, such as France, Spain, and Bulgaria, where TSWV isolates grouped uniformly into one population (Turina et al., 2012). Despite the presence of evolutionary-distinct isolates in the same geographic area, reassortment between isolates from the two clades has not been reported so far in nature in Italy. Two TSWV isolates from Italy, p105 and p202/3WT, have been recently fully sequenced and characterized (Margaria et al., 2014b). Isolate p105 was collected from a pepper field in Liguria, northern Italy, while p202/3WT was collected in pepper in Sicily, southern Italy. Phylogenetic analyses grouped the two strains into the two separated clades previously reported in Italy (Finetti-Sialer et al., 2002), placing p105 in the TSWV-A clade and p202/3WT in the TSWV-D clade (Margaria et al., 2014b). In summer 2012, an outbreak of TSWV was observed in Carmagnola (Piedmont, Northern Italy), a district where sweet pepper crops are intensively cultivated and where the use of pepper varieties carrying the Tsw resistance gene is still limited. Isolate p331 was collected in a field where resistant peppers were grown. This isolate was passaged three times through single-local lesion transfer on Nicotiana tabacum cv. White Burley and then on Capsicum chinensis PI152225 carrying the Tsw gene, where it was still able to overcome resistance as the original field isolate. In this work, we characterized the p331 genome, and we found that molecular markers for the L and S segment grouped consistently with p202/3WT-like isolates (TSWV-D clade), while those for the M segment grouped with p105-like isolates (TSWV-A clade). In order to characterize p331 genomic segments, we used five molecular markers located along the whole genome: the Gn/Gc, NSs and N markers consisted of a fragment in the open reading frame (ORF) of the corresponding gene, being 692, 587 and 774 nt in length, respectively; the RdRp and NSm markers, beside a fragment of the corresponding gene, included also a portion of the 3 untranslated region and intergenic region, respectively, and were 310 and 878 nt in length. Total RNA was extracted from systemically infected N. benthamiana leaves using the “SpectrumTM Plant Total RNA Kit” (Sigma–Aldrich, St. Loius, MO, USA). Reverse transcription was performed using the specific reverse primer (Supplementary Table 1) with the “ThermoscriptTM RT-PCR” kit (Invitrogen, Grand Island, NY, USA), and followed by PCR using PolyTaq polymerase (Polymed, Florence, Italy) and primers reported in Supplementary Table 1. Amplification products were cloned using the “pGEM-T Easy vector” system (Promega, Madison, WI, USA) and sequenced (see Supplementary Table 1 for GenBank accession numbers). A first analysis of possible recombination events was performed using Recombination Detection Program 4.16 (RDP4) software (Martin and Rybicki, 2010). For this purpose, the sequences of the five markers of each isolate were concatenated together in a single sequence file, in the following order: RdRp, NSm, Gn/Gc, NSs and N marker, giving rise to an artificial full-length contig
of about 3300 nt. Markers for p105 and p202/3WT isolates were retrieved by selecting the same target regions from the sequences available in GenBank (Margaria et al., 2007, 2014a,b). Isolates p105 and p202/3WT were chosen for comparison because they are the closest isolates present in the database for which full length genomic sequence is available (Margaria et al., 2014b). Sequences were aligned using MEGA6 software according to the Clustal W algorithm (Tamura et al., 2013), and then run in RPD software, at default parameter values. Three recombination events were consistently found by different detection algorithms: recombination event number 1 concerned the whole S segment markers (N and NSs), and event number 2 concerned the whole L segment marker (Table 1). Both events were detected at stringent P-value scores (lowest confidence was at E-05), and identified p202/3WT as the contributing parental for p331 for the L and S genomic segments. A third recombination event identified the p105 as the major parent contributing to p331 for the whole M segment, at stringent P values (Table 1). Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres. 2014.11.012. Similarity percentages and potential reassortment analysis among the isolates in study was further performed by scanning of the concatenated marker sequences with SimPlot v. 3.5.1 (Lole et al., 1999), at default values. As shown in Fig. 1, p331 high sequence similarity was found with p202/3WT in the L (RdRp marker) and S (NSs and N marker) segments, while for the M segment, the highest similarity was between p331 and p105. We further obtained similarity matrix scores for the markers in study using MatGat v. 2.03 (Campanella et al., 2003): sequence analysis confirmed higher percentage scores between p331 and p202/3WT for the L and S segment markers (98.4, 99.5 and 99.6% for RdRp, NSs and N markers, respectively), and between p331 and p105 for the M markers (97.8 and 90.5%, for Gn/Gc and NSm, respectively) (Supplementary Table 2). The nucleotide alignments of the five markers are provided in Supplementary Fig. 1. Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres. 2014.11.012. Phylogenetic analyses according to the Neighbor-Joining method (Saitou and Nei, 1987) using the alignment of the concatenated markers for each genomic segment, grouped p331 in the p202/3WT clade for the L and S segment, and in the p105 clade for the M segment (Fig. 1). Together, these results support different origin for the genomic segments of p331 and consistently provide direct evidence that p331 was derived through reassortment between two evolutionary distinct TSWV isolates; the two possible parental isolates used for comparison are both unable to overcome Tsw resistance. Presence of a TSWV-D-like strain was never witnessed before in northern Italy, and therefore we decided to investigate a number of isolates from northern Italy present in the PLAVIT (Plant Viruses Italy, http://www.wfcc.info/ccinfo/index.php/collection/ by id/1057/) collection. For this study, we newly sequenced the N gene of seven isolates collected during TSWV outbreaks in northern Italy (Piedmont, Liguria and Lombardy regions) from 1997 to 2014. Sequences were used for phylogenetic analysis, together with the sequence of TSWV isolates from northern and southern Italy previously characterized (Margaria et al., 2007; Turina et al., 2012; Zindovic et al., 2014). Nucleocapsid protein sequences showed high similarity among all isolates (not shown), as expected; in fact, amino acid changes in this protein are thought to be deleterious for the virus (Tentchev et al., 2011). However, phylogenetic analysis on the nucleotide sequences showed that all the isolates from northern Italy grouped with p105, with the exception of isolate p331 which grouped in the p202/3WT clade (Fig. 2), suggesting that this
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Table 1 Prediction of potential recombination/reassortment events among tomato spotted wilt virus (TSWV) isolates p331, p105 and p202/3WT by different detection algorithms implemented in the Recombination Detection Program (RDP4) software, using as input the concatenated sequence of five genomic markers on TSWV genomic segments. Event number
1 2 3
Breakpoint position Begin
End
1954 1 256
3286 313 1954
Corresponding marker
NSs + N RdRp NSm + Gn/Gc
Genomic segment
S L M
Recombinant
p331 p331 p331
Parental
p202/3WT p202/3WT p105
Score of detection algorithms
GENECONV
Bootscan
MaxChi
Chimaera
RDP
3Seq
2.98E−05 2.71E−06 5.51E−03
8.97E−11 2.64E−07 –
3.61E−12 – 2.67E−14
8.42E−12 – 1.63E−14
5.50E−12 3.25E−07 5.60E−08
– 1.60E−06 2.25E−20
RdRp, RNA dependent RNA polymerase; NSm, non-structural m protein; Gn/Gc, glycoprotein precursor; NSs non-structural s protein; N, nucleocapsid protein; L, large genomic segment; M, medium genomic segment; S, small genomic segment.
evolutionary lineage in northern Italy is not widespread, and that isolate p331 shows unique features. Given that p331 was able to overcome the Tsw resistance gene in pepper and that substitutions in the NSs protein are sufficient to provide the resistance-breaking (RB) phenotype (Margaria et al., 2007), we fully characterized the NSs gene, according to previous protocols (Margaria et al., 2014a). Three independent PCR reactions and sequencing analysis were performed. The NSs ORF of p331 is 1401 nt in length and encodes a 467aa-long protein (GenBank accession KM213989), similar to other Italian wild-type and RB TSWV isolates previously characterized (Margaria et al., 2007).
While we and other authors have shown that one or two amino acid changes in the NSs protein were enough to confer the RB phenotype on Tsw resistant pepper plants (Margaria et al., 2007; de Ronde et al., 2013), seven amino acid substitutions are present in p331 in comparison to the p202/3WT NSs protein, and five against the NSs protein of p170, a wild-type TSWV isolate also collected in Sicily, southern Italy (as for p202/3WT) and partially characterized (Margaria et al., 2007) (Supplementary Fig. 2). Remarkably, the p331 nucleocapsid protein was found to be conserved without changes (not shown). None of the observed NSs-substitutions were present in other Tsw-gene RB isolates previously characterized
Fig. 1. Isolates sequence comparison. (A) Phylogenetic trees of the concatenated markers in each genomic segment inferred using the Neighbor-Joining method, showing evolutionary relationships between the three isolates in study. (B) Similarity plot between tomato spotted wilt virus isolates p331, p105 and p202/3WT generated using SimPlot v. 3.5.1 software. The concatenated nucleotide sequences of the five genomic markers (one in each viral gene) were first aligned using ClustalW, and identities between the query sequence (p331) and each putative parental sequences (p105 and p202/3WT) were plotted. Each point plotted is the percent identity within a sliding window 200 bp wide centred on the position plotted, with a step size between points of 20 bp. The horizontal bar above the curves shows the open reading frames of the TSWV genome corresponding to the markers and the brackets show the three genomic segments. RdRp, RNA dependent RNA polymerase; NSm, non-structural m protein; Gn/Gc, glycoprotein precursor; NSs non-structural s protein; N, nucleocapsid; L, large genomic segment; M, medium genomic segment; S, small genomic segment.
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Fig. 2. Phylogenetic tree obtained from the alignment of nucleocapsid (N) gene sequences of tomato spotted wilt virus (TSWV) isolates from different regions (in parenthesis) in northern and southern Italy. Tree was drawn according to the Neighbor-Joining method allowing 1000 bootstrap replicates. Bootstrap values above 60% are indicated for each node. The evolutionary distances are expressed in the units of the number of base differences per site. Asterisks mark TSWV isolates newly sequenced in this work. The virus isolates and GenBank accession numbers are as follows: p302 (KM096541), p166 (DQ376179), p322 (KM096538), p207 (KM096539), p105 (DQ376178), p231 (KM096536), p272 (DQ376181), p252 (KM096537), p147 (KM096535), p267(DQ376180), T-1081 (KM096542), p331 (KM213988), p170 (DQ431237), I-186 (GU369719), Miz3 (GU369720), T-1003 (GU369725), p202/3WT, p202/1 (GU369721), p240 (GU369722).
(Margaria et al., 2007; de Ronde et al., 2013), further confirming that different substitutions in different region of the NSs protein are sufficient to provide the RB phenotype (Margaria et al., 2007; de Ronde et al., 2013). Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres. 2014.11.012. Recent works have outlined the possibility that some RB isolates might lose the silencing suppression ability provided by the NSs protein (Margaria et al., 2007, 2014a). A recent study showed the presence of essential residues in the N-terminal domain of the NSs protein for silencing suppression activity (de Ronde et al., 2014).
Similarly, other authors performed site-directed mutagenesis of highly conserved amino acids in the NSs protein and identified two sites which are critical for silencing suppression activity (Zhai et al., 2014). Given the unusually high number of amino acid substitutions present in the p331 reassortant RB isolate, we further investigated the silencing suppressor ability of p331 NSs, by in vivo assays in N. benthamiana line 16C, as previously described (Margaria et al., 2007). The p331 NSs proved to be a strong silencing suppressor, not distinguishable in the bioassay from a control NSs from a closely related wild-type isolate (p105); as negative control, we used the p170RB NSs allelic version, which was previously shown to have lost silencing suppressor activity (Margaria et al., 2007) (Fig. 3).
Fig. 3. In planta bioassays for silencing suppression activity of p331 NSs allelic variants. Full-length NSs allels were cloned in pBin61 vector and transiently expressed through agroinfiltration together with pBin-GFP in 16C transgenic Nicotiana benthamiana. Controls consisted of vector only (pBin61), p105 NSs allele (positive control) and p170RB NSs allele (not functional in silencing suppression, negative control). Pictures were taken by using a hand-held UV light 3 days post-infiltration.
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