Abstract
Sw-5b is a widely used resistance gene in tomato breeding to control tomato spotted wilt virus (TSWV). The NSm protein encoded by TSWV is identified as the avirulence (AVR) determinant in Sw-5b-mediated resistance. In the last decades, Sw-5b resistance breaking (RB) isolates were found and identified in many locations around the world. The resistance-breaking phenotype in all the previously verified TSWV Sw-5b RB strains is associated with the NSmC118Y or NSmT120N mutations. In the summer of 2022, a Sw-5b RB TSWV strain was recognized in a greenhouse in Hungary. In inoculation experiments this strain was able to infect tomato plants with the Sw-5b resistance gene. Molecular analysis of the NSm avirulence determinant revealed a single alteration in the NSm protein, D122G mutation was identified. To our knowledge, this is the first report to identify this amino acid alteration associated with resistance-breaking phenotype in Sw-5b resistant tomato plants.
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Among plant viruses, the tomato spotted wilt virus (TSWV) has a great impact on economically important vegetables and ornamental plants worldwide. TSWV has an extremely broad host range with more than 1000 plant species (Kormelink et al., 2011; Oliver & Whitfield, 2016; Pappu et al., 2009; Parrella et al., 2003; Scholthof et al., 2011). The virus spreads primarily by thrips vectors in a persistent propagative manner, therefore, it is a great challenge to maintain control against it (Gilbertson et al., 2015; Hogenhout et al., 2008; Oliver & Whitfield, 2016; Whitfield et al., 2005). TSWV is the type member of the genus Orthotospovirus in the family Tospoviridae of the order Bunyavirales. Among the three genomic RNAs of TSWV the large (L) RNA is negative sense and encodes an RNA-dependent RNA polymerase (RdRp). The medium (M) and the small (S) RNAs are ambisense. The M RNA encodes a non-structural protein in sense orientation which has a basic role in cell-to-cell and long-distance movement. In ambisense orientation, the M RNA encodes precursors of glycoproteins that are further processed into two mature glycoproteins. The S RNA codes for the non-structural protein NSs that has RNA silencing suppressor function in sense orientation, and in the antisense strand, the S RNA encodes the nucleocapsid (N) protein (Adkins, 2000).
One prevalent defence strategy against plant viruses is resistance breeding. There are two dominant resistance genes available against TSWV: the Tsw gene deployed in pepper and Sw-5b in tomato cultivars (Boiteux & Ávila, 1994; Stevens et al., 1991). Both of them belong to the intracellular nucleotide-binding leucine-rich repeat (NLR) immune receptors that represent the largest group of resistance (R) proteins in plants, consisting of an N-terminal CC domain, a central nucleotide-binding domain, and the C-terminal LRR domain (de Ronde et al, 2014; Dodds & Rathjen, 2010; Jones & Dangl, 2006; Jones et al., 2016; Kapos et al., 2019; Meier et al., 2019; van Wersch et al., 2019). These types of R proteins have a central role in pathogen perception and host immune response activation. The avirulence factors (Avr) of these resistance genes are different, and they are located on distinct RNAs of the TSWV genome; NSs, the Avr factor for Tsw in pepper is encoded on the S RNA, while the NSm or movement protein (MP), the Sw-5b Avr factor in tomato is located on the M RNA (Margaria et al., 2007; Peiro et al., 2014). The Sw-5b encodes an NLR protein with an N-terminal Solanaceae-specific domain (SD). For activation, Sw-5b uses a two-step recognition mechanism involving both the SD and the LRR domain in the direct detection of viral NSm (Chen et al., 2021).
The large-scale use of resistant cultivars leads to the rapid emergence and spread of resistance-breaking strains of TSWV in pepper and in tomatoes (Ciuffo et al., 2005; Crescenzi et al., 2013). In the case of different TSWV RB strains on pepper, no common amino acid (aa) alteration can be observed in the NSs Avr protein, but in the case of the Sw-5b gene in tomato, two distinct aa alterations were identified in all the RB TSWV strains. The RB phenotype was linked to one of the two single specific aa substitutions in the Avr determinant NSm, namely C118Y or T120N (Huang et al., 2022; Lopez et al., 2011).
This year in a significant Hungarian vegetable production region, TSWV infection was detected in a tomato cultivar bearing the Sw-5b resistance gene. Molecular analysis of the NSm coding region of the virus isolate demonstrated that there was no aa alteration present in the two previously identified aa residues, but in position 122, a novel aa substitution was detected: D122G.
In the tomato production season of 2022, tomato (Solanum lycopersicum cv. Janus F1) fruit samples with necrotic patch symptoms (Fig. 1) were collected in a greenhouse near Kecskemét in central Hungary. To verify potential virus infection of the tomato samples, multiplex RT-PCR assay was carried out for the most prevalent tomato pathogen viruses in Hungary, namely TSWV, cucumber mosaic virus (CMV), tobamoviruses and potato virus Y (PVY) according to Nemes and Salánki (2020). The RT-PCR analysis proved the presence of TSWV and the lack of CMV, PVY and tobamoviruses in the samples.
For further characterization of the virus infection, inoculation experiments were carried out in an environmentally controlled growth chamber (16-h light period at 25 °C, 8-h dark period at 20 °C) on test plants and on different tomato cultivars.
Sap of the tomato sample was inoculated mechanically on Nicotiana tabacum cv. Xanthi-nc and N. benthamiana. Both test plants showed macroscopic symptoms typical for TSWV infection. Symptoms have been observed until the 10th day after inoculation (dai). On the leaves of N. tabacum cv. Xanthi-nc chlorotic and necrotic ringspots were observed, and on the inoculated leaves of N. benthamiana necrotic and chlorotic spots and ringspots, local chlorosis and on the upper, non-inoculated leaves mild mosaic and leaf distortion were detected.
Inoculation experiments on different tomato varieties were also carried out to verify the resistance phenology of the virus. The infection phenotype was evaluated on resistant (S. lycopersicum cv. Janus F1) and susceptible (S. lycopersicum cv. H1015 F1) tomato varieties. The tomato plants were inoculated mechanically. As control, the same tomato varieties were inoculated with HUP2 TSWV strain isolated earlier from pepper, which was observed to elicit an RB phenotype on pepper varieties bearing the Tsw gene (Almási et al., 2015). In the case of the TSWV T-37 strain isolated in the present study, no local lesions were detected on the inoculated leaves of the two tomato cultivars (Fig. 2A, B). Similarly, no hypersensitive reaction (HR) was detected on the susceptible tomato cultivar inoculated with the pepper strain (TSWV HUP2), but four dai TSWV HUP2 elicited local necrosis (hypersensitive reaction, HR) on the inoculated leaves of the resistant tomato cultivar (S. lycopersicum cv. Janus F1) (Fig. 2C, D). Ten dai systemic symptoms were recognized on both cultivars when inoculated with TSWV T-37 (Fig. 3A, B), while TSWV HUP2 strain only elicited systemic symptoms on the susceptible variety. On the resistant cultivar inoculated with TSWV HUP2, no systemic symptoms were observed during the monitoring period (Fig. 3C, D). The systemic symptoms induced by the tomato RB strain (TSWV T-37) were mild mosaic, bronzing of the leaves, stunting and wilting. The symptom development was more progressive and led to the decay of the complete plant (Fig. 3A, D). In the case of the susceptible tomato plants infected with the TSWV HUP2 strain, symptoms (dwarfing, mild mosaic, chlorosis and bronzing) were milder, and the symptom formation was delayed. The virus accumulation in the systemic, non-inoculated upper leaves of all the studied tomato plants were verified by RT-PCR. TSWV T-37 was detected in both of the inoculated cultivars, but TSWV HUP2 was detected just in the susceptible tomato cultivar, and not in the resistant variety.
Since the Avr factor of the Sw-5b resistance gene is the NSm, the nucleotide sequence of the NSm gene of TSWV T-37 strain was determined. Total nucleic acid was extracted using Spectrum™ Plant Total RNA Kit (Sigma Aldrich/Merck) according to the supplier’s instructions. cDNA was produced with TSWV MP reverse oligo (5’-GGTTTTCGAATTAAATGCAAAATTAACAG-3’), then RT-PCR was carried out using TSWV MP forward (5’-CACAAGCTCCTCTACCTTAGGC-3’) and reverse primer pair using an Eppendorf Mastercycler as follows: 95 °C for 5 min, 30 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 90 s, and a final 72 °C for 10 min. The PCR product was separated on 1% agarose gel, and after purification (GeneJet Gel Extraction Kit, Thermo Fisher Scientific) its nucleotide sequence was determined (Biomi Ltd.), and the sequence data were deposited in the NCBI GenBank database (accession number OP603958). The nucleotide sequence of the NSm coding region was aligned and analysed by MEGA X software. The evolutionary history was inferred by using the Maximum Likelihood method and Tamura-Nei model (Tamura & Nei, 1993). The tree with the highest log likelihood (-2886.37) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Tamura-Nei model, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 17 nucleotide sequences of the NSm coding region of the M RNA. There were a total of 912 positions in the final dataset. Evolutionary analyses were conducted in MEGA X (Kumar et al., 2018). Maximum likelihood phylogenetic tree was composed of representatives of different TSWV clades retrieved from the NCBI GenBank database. Tomato chlorotic spot virus (TCSV) was chosen as an outgroup. According to the phylogenetic analysis, the TSWV T-37 strain belongs to the TSWV clade 1 together with another RB strain from the USA and with non-resistance breaking strains from different locations (Spain, Korea) (Fig. 4).
The comparison of the TSWV T-37 strain NSm protein aa sequence to other strains revealed an unexpected result, since neither of the previously described Sw-5b resistance breaking mutations (C118Y and T120N) were present. Recently, a 21 aa peptide region (aa 115–135) of the NSm protein was identified as a critical domain for Sw-5b recognition (Zhu et al., 2017). The sequence comparison of this specific region between the TSWV T-37 strain and other RB and NI strains retrieved from the GenBank revealed a novel single aa alteration of TSWV T-37, at aa position 122 (D122G) (Fig. 5).
This aa mutation was detected exclusively in the RB isolate presented in this study, namely TSWV T-37. The key residues in the aa 115–135 region required for the recognition of Sw-5b were analysed recently in detail, using alanine-scanning mutants coexpressed with Sw-5b in N. benthamiana leaves. In that study, NSmP119A, NSmW121A, NSmD122A, NSmR124A and NSmQ126A failed to induce HR, and as a result, these amino acids were proposed as potential critical sites for Sw-5b recognition (Huang et al., 2021). Based on the results presented in this study, in the case of the TSWV T-37 isolate, the D122G mutation is proposed to be responsible for the Sw-5b resistance breaking phenotype. This is the first time in Hungary to identify a Sw-5b resistance breaking isolate of TSWV, and the origin of this strain is in question. It could have emerged in the local tomato breeding region, or it could have been transported from other regions of Hungary, or even abroad. Since this type of Sw-5b resistance breaking TSWV strain was not identified previously, the verification of this question is problematic. Nevertheless, these results are consistent with the suggestion of Huang et al. (2021), that further aa mutation in the NSm protein may evade the recognition of Sw-5b, resulting in new RB TSWV isolate.
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All data underlying the results are available as part of the article and the supplementary materials and no additional source data are required.
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Almási, A., Pinczés, D., Tímár, Z. et al. Identification of a new type of resistance breaking strain of tomato spotted wilt virus on tomato bearing the Sw-5b resistance gene. Eur J Plant Pathol 166, 219–225 (2023). https://doi.org/10.1007/s10658-023-02656-5
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DOI: https://doi.org/10.1007/s10658-023-02656-5