Integration of Ty-1/Ty-3 and Ty-6 confers improved and durable resistance to highly pathogenic begomoviruses in tomato

Integration of Ty-1/Ty-3 and Ty-6 confers improved and durable resistance to highly pathogenic begomoviruses in tomato | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Integration of Ty-1/Ty-3 and Ty-6 confers improved and durable resistance to highly pathogenic begomoviruses in tomato Moeno Shimoide, Misaki Nakajima, Nadya Syafira Pohan, Elly Kesumawati, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8207444/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Mar, 2026 Read the published version in Euphytica → Version 1 posted 10 You are reading this latest preprint version Abstract Begomoviruses are among the most destructive pathogens of tomato worldwide, and the introgression of Ty -genes in tomato is a key strategy for disease management. However, a single Ty -gene often provides incomplete protection against highly virulent begomovirus species. Here, commercial tomato cultivars and breeding lines were inoculated with one of three begomovirus species that differed in virulence. The presence of Ty-1 , Ty-2 , Ty-3 , Ty-3a , ty-5 , and Ty-6 was validated using in-gene markers. Tomato plants carrying only Ty-2 were susceptible to all viruses, whereas those with Ty-1 were resistant to a less-virulent begomovirus, but susceptible to more-virulent viruses. Among the seven AVTO breeding lines, 1919 ( Ty-1 / Ty-1 , Ty-6 / Ty-6 ) and 1920 ( Ty-3 / Ty-3 , Ty-6 / Ty-6 ) were symptomless in most cases and the lowest viral DNA accumulation across all lines tested indicating strong begomovirus resistance. The resistance of 1919 was comparable to or greater than that of 1701, a line homozygous for Ty-2 , Ty-3 , ty-5 , and Ty-6 . Furthermore, F₁ plants derived from crosses between 1919 or 1920 and susceptible Moneymaker exhibited reduced disease tolerance, indicating the importance of homozygosity for Ty-1 or Ty-3 combined with Ty-6 . These results demonstrate that Ty-1/Ty-3 and Ty-6 integration in homozygous states can provide robust and broad-spectrum resistance to begomoviruses and is a valuable strategy for breeding durable resistant tomato cultivars. Begomovirus DNA marker Geminivirus Pyramiding resistance genes Resistance breeding Tomato yellow leaf curl disease Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Tomato yellow leaf curl disease (TYLCD), caused by several species of the genus Begomovirus within the Geminiviridae family, is one of the most destructive diseases of tomato ( Solanum lycopersicum ). Virus-infected plants exhibit yellowing of young leaves, upward leaf curling, smaller leaflets, stunted growth, and aborted flowers (Moriones and Navas-Castillo 2000 ). The disease severely limits tomato production, often causing complete yield loss in virus-susceptible cultivars (Czosnek and Laterrot 1997 ). Among the associated viruses, tomato yellow leaf curl virus (TYLCV) is particularly widespread, affecting tomato crops. Begomoviruses are persistently and circulatively transmitted by the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae). Although complete control of this vector remains difficult, whitefly populations can be reduced through basic measures such as insecticide use and fine-mesh screening of greenhouse openings (Rojas et al. 2018 ). In addition to these approaches, the primary means of managing TYLCD has been the use of TYLCV resistant cultivars carrying Ty genes. Extensive screening of tomato germplasm has identified several wild relatives exhibiting resistance to TYLCV. Multiple resistance loci ( Ty-1 to Ty-6 ) have been introgressed into cultivated tomato ( S. lycopersicum ) (Agrama and Scott 2006 ; Anbinder et al. 2009 ; Hutton and Scott 2014 ; Ji et al. 2009a , b ; Zamir et al. 1994 ). To date, all Ty genes except Ty-4 have been cloned. Specifically, Ty-1 , Ty-3 , and Ty-3a encode RNA-dependent RNA polymerase (RDR) (Verlaan et al. 2013 ); Ty-2 encodes an NB-LRR protein (Yamaguchi et al. 2018 ); ty-5 encodes pelota (Lapidot et al. 2015 ); and Ty-6 encodes DNA polymerase delta subunit 1 (POLD1) (Shen et al. 2025 ). Among these Ty genes, Ty-1 in the heterozygous state is the most widely used in commercial F₁ hybrid tomato cultivars released in Europe, North America, and East Asia (Fortes et al. 2023 ; Koeda et al. 2020 ; Koeda and Kitawaki 2024 ; Lapidot et al. 2014 ). However, Ty-1 -mediated resistance to TYLCV has been reported to break down due to begomoviral evolution, coinfection with a beta-satellite or crinivirus, or cultivation at high temperatures (Belabess et al. 2015 , 2016 ; Fortes et al. 2023 ; Jammes et al. 2024 ; Koeda and Kitawaki 2024 ; Voorburg et al. 2020 ). Moreover, predominant begomovirus species in subtropical and tropical regions are generally more virulent than TYLCV, and Ty-1 -mediated resistance is not fully effective (Koeda et al. 2020 ; Pohan et al. 2024 ). Such challenges have driven breeding efforts toward the development of tomato cultivars with multiple Ty resistance genes. The transfer of resistance from wild relatives to cultivated species is a cornerstone of modern breeding. Yet, linkage drag caused by pleiotropic effects of resistance genes or neighboring wild genomic regions can impair key agronomic traits (Kashyap et al. 2022 ). Consequently, breeders aim to introgress the minimal number of resistance genes required for effective protection. To explore this concept, we assessed the resistance of tomato lines carrying different combinations of Ty genes against begomoviruses with varying virulence originating from tropical area. The results provide valuable insights and a conceptual basis for developing tomato cultivars with durable resistance. Materials and methods Plant material The tomato F 1 hybrid cultivars Momotaro (M: no Ty -genes, susceptible to begomoviruses) and TYLCV-resistant cultivars Momotaro Sakura (MS) and Momotaro Peace (MP) (Takii Seeds, Kyoto, Japan) were used in the study. Momotaro is one of the most popular fresh tomato cultivars in Japan. MS and MP have the same genetic background as M. AVTO lines were obtained from the World Vegetable Center: 1919, 1920, 1314, 1429, 1954, 1315, and 1701. These fresh market tomato lines, which have different Ty -gene combinations, were selected for the study. The officially described Ty -gene combinations and parentage of each AVTO line are listed in Table S1 . AVTO 1919 and 1920 were crossed with Moneymaker (MM: no Ty -genes, susceptible to begomoviruses) in both forward and reciprocal directions to obtain the F 1 populations. Genotyping of Ty -genes using in-gene DNA markers The presence of Ty-1 , Ty-3 , and Ty-3a was verified using the previously reported codominant in-gene DNA markers and methods (Koeda and Kitawaki 2024 ). Ty-2 and ty-2 were verified using dominant in-gene DNA-markers reported by Yamaguchi et al. ( 2018 ). An in-gene derived cleaved amplified polymorphic sequences (dCAPS) marker for ty-5 and a cleaved amplified polymorphic sequences (CAPS) marker for Ty-6 were developed in this study to detect the causal single nucleotide polymorphisms (SNPs) reported (Lapidot et al. 2015 ; Shen et al. 2025 ). The primer sequences and PCR conditions are listed in Table S2 and S3. For Ty-1 , Ty-3 , Ty-3a , ty-5 , and Ty-6 markers, reaction was conducted in a 10-µL reaction mixture containing 5 µL of Emerald Amp PCR Master Mix (Takara Bio, Shiga, Japan), 0.1 µL of each primer (20 pmol/µL), 3.8 µL of water, and 1 µL of DNA template (30 ng/µL). For Ty-2 and ty-2 markers, reaction was conducted in a 10-µL reaction mixture containing 0.1 µL of BlendTaq-Plus (Toyobo, Osaka, Japan), 1 µL of 10×buffer, 1 µL of dNTPs (2mM), 0.1 µL of each primer (20 pmol/µL), 6.8 µL of water, and 1 µL of DNA template (30 ng/µL). The PCRs were run in a GeneAtlas G02 Thermal Cycler (Astec, Fukuoka, Japan). For ty-5 dCAPS and Ty-6 CAPS markers, amplicons were digested with the restriction enzyme in a 10-µL mixture containing 4 µL of the PCR amplicons, 1 U of NcoI (Takara Bio) or AanI (PsiI) (Thermo Scientific, MA, USA), 1 µL of 10×buffer, 1 µL of BSA if required, and incubated at 37°C for 1 h. The entire digested product was electrophoresed in 1.5% (w/v) agarose gels for 30 min at 100 V. Virus inoculation and symptom evaluation The infectious clones of pepper yellow leaf curl Indonesia virus (PepYLCIV) isolate BA_D1-1 (GenBank accessions LC051114 [DNA-A] and LC314794 [DNA-B]) (Koeda et al. 2016 , 2018 ), pepper yellow leaf curl Aceh virus (PepYLCAV) isolate BAPep-V2 (GenBank LC387327 [DNA-A] and LC387329 [DNA-B]) (Kesumawati et al. 2019 ; Koeda et al. 2021 ), and tomato yellow leaf curl Kanchanaburi virus (TYLCKaV) isolate BA_B6 (GenBank LC051116 [DNA-A] and LC177332 [DNA-B]) (Koeda et al. 2016 , 2017 ) have been described elsewhere. We selected these bipartite begomovirus species, which are predominant in Southeast Asia, for this study because they are more virulent than the monopartite TYLCV (Koeda et al. 2020 ; Taniguchi et al. 2023 ). Various tomato lines were graft inoculated with higher inoculum pressure to evaluate their begomovirus resistance because agroinoculation was occasionally less effective because of the high virus resistance conferred by introgressed Ty -genes. First, tomato M plants were agroinoculated using the method of Koeda et al. ( 2020 ) with either of the three begomoviruses and used as the rootstock. Plants were transplanted into plastic pots containing horticultural soil mix (Japan Agricultural Cooperatives, Tokyo, Japan) and were grown in a growth room at 25–30°C day/23–25°C night with 13 h light/11 h dark. The virus-free healthy tomato lines were used as the scion and grafted onto virus-infected M plants using grafting tubes (Nasunics Co., Ltd., Tochigi, Japan). The grafted plants were kept in humid conditions for about 2 weeks. After plants were acclimatized, growth of the scion was promoted. We conducted experiments in five different time periods because we grafted more than 600 plants in total. However, because we cultivated the plants in the aforementioned growth room, the climate conditions were uniform for all experiments. At 42 days after inoculation (dpi) by grafting, symptoms were evaluated, and newly developed upper young leaves were collected to extract DNA. Graft-inoculated plants took a longer time of 42 days to evaluate because more time was required for the scions to elongate to a size large enough for symptom observation. In our subsequent experiment, we evaluated the symptoms of the PepYLCAV agroinoculated tomato cultivar, line, and F 1 progeny at 28 dpi. Symptoms on each plant were scored using a disease severity index (DSI) as shown in Fig. 1 : 0, no symptoms; 1, slight leaf yellowing symptoms; 2, mild leaf yellowing symptoms; 3, moderate leaf yellowing and curing symptoms; and 4, severe leaf yellowing and curing symptoms. Virus DNA detection and quantification The Nucleon PhytoPure Kit (Cytiva, Malborough, MA, USA) was used to extract DNA from tomato leaves. Primer pairs specific for each virus species were used for PCR (Supplemental Table S1 ) in a 10-µL reaction mixture containing 5 µL of Emerald Amp PCR Master Mix (Takara Bio), 0.1 µL of each primer (20 pmol/µL), 3.8 µL of water, and 1 µL of DNA template (30 ng/µL). The PCRs were run in a GeneAtlas G02 Thermal Cycler (Astec). The primer sequences and PCR conditions are listed in Table S2 and S3. The amplicons were electrophoresed in a 1.0% (w/v) agarose gel. The CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) was used for quantitative PCR (qPCR) with primer pairs specific for each virus species (Supplemental Table S1 ). The multicopy 25S ribosomal RNA gene was detected by qPCR using the 25s-rRNA 2F and 2R primers to normalize viral DNA accumulation data. The 10-µL reaction mixture contained 5 µL of SsoAdvanced Universal SYBR Green Supermix (Bio-Rad Laboratories), 0.125 µL of each primer (20 pmol/µL), 2.75 µL of water, and 2 µL of DNA template (30 ng/µL). The primer sequences and PCR conditions are listed in Table S2 and S3. Samples were analyzed in duplicate to calculate the average threshold cycle. The 2 −ΔΔCT comparative method (Livak and Schmittgen 2001 ) was applied to calculate the relative quantity of each virus. We calculated the Cq values for the mock-inoculated plants using virus-specific and 25s-rRNA 2F and 2R primers. If the calculated amount of DNA in the virus inoculated samples was lower than in the mock-inoculated samples, we considered the plants uninfected and excluded the data when calculating the mean amount of viral DNA for each tomato cultivar and line. In the first experiment, which aimed to assess the amount of viral DNA in each tomato cultivar and line, we conducted cultivation on five different dates/blocks, as mentioned above. For the analysis, we combined the raw data obtained from qPCR. Statistical analyses SPSS Statistics (Version 22.0; IBM, Armonk, NY, USA) was used for all statistical analyses. Mean values for the accumulated begomoviral DNA were analyzed for significant differences using a nonparametric one-way ANOVA, with subsequent comparison of multiple means using Fisher’s LSD test, with P < 0.05 considered statistically significant. Spearman’s rank correlation coefficient was used to assess correlations between disease severity and viral DNA accumulation because disease severity was recorded as ordinal data, with P < 0.05 considered statistically significant. Results Validation of Ty -genes using in-gene DNA markers The presence and status of Ty -genes were assessed using in-gene DNA markers (Fig. 2 A). Genotyping with the SK-F1 and R1 and SK-F3 and R4 markers detected the resistance-type RDR allele ( Ty-1 / Ty-3 / Ty-3a ), indicating that MP is heterozygous, whereas AVTO 1919, 1920, 1429, 1954, 1315, and 1701 are homozygous. Further analysis using the SK-F2 and R3 markers revealed the presence of Ty-3 in AVTO 1920, 1429, 1954, 1315, and 1701, while the SK-F4 and R5 markers identified Ty-1 in MP and AVTO 1919. No Ty-3a allele was detected in any lines. Using markers specific for Ty-2 and ty-2 , MS was found to be heterozygous for Ty-2 , whereas AVTO 1429, 1954, 1315, and 1701 were homozygous. The ty-5 -specific marker showed that AVTO 1314 and 1701 were homozygous for ty-5 , and Ty-6 -specific marker revealed that AVTO 1919, 1920, 1314, 1429, 1315, and 1701 were homozygous for Ty-6 . A summary of the Ty gene combinations in each cultivar or line is presented in Fig. 2 B. Assessment of begomovirus resistance in tomato commercial cultivars and breeding lines In classical virus–host interactions, such as the tobacco mosaic virus (TMV)– N gene system in tobacco, pathogen recognition by the resistance gene activates a hypersensitive response, resulting in localized cell death as an effective immune defense (Whitham et al. 1994 ). However, unlike this immune-based resistance, all begomovirus resistance genes characterized to date confer disease tolerance and restrict viral DNA accumulation to levels that are significantly lower than in the susceptible genotype (Koeda et al. 2021 , 2022 , 2024 ; Lapidot et al. 2015 ; Lim et al. 2022 ; Pohan et al. 2025 ; Shen et al. 2025 ; Verlaan et al. 2013 ; Yamaguchi et al. 2018 ). In the present study, tolerance was defined as the absence of visible symptoms or the presence of slight symptoms following infection, while virus resistance was defined as a significant restriction of viral DNA accumulation in these plants. In this study, susceptible M plants were inoculated with one of the three viruses, and tomato cultivars or lines carrying different Ty genes were grafted onto M rootstocks. Virus-infected plants were diagnosed using PCR, and resistance was evaluated based on the disease severity index (DSI) (Fig. 1 , Table 1 ) and viral DNA accumulation at 42 dpi (Fig. 3 ). Table 1 Begomovirus resistance of tomato cultivars and lines with different Ty gene combinations Tomato cultivars or lines used as scions Inoculum Number of plants DSI x Grafted Infected z Symptomatic (%) y M (Without Ty gene) PepYLCIV 28 28 28 (100) 4 TYLCKaV 24 24 24 (100) 4 PepYLCAV 24 24 24 (100) 4 MS ( Ty-2 / ty-2 ) PepYLCIV 14 14 14 (100) 4 TYLCKaV 20 20 20 (100) 4 PepYLCAV 19 19 19 (100) 4 MP ( Ty-1 / ty-1 ) PepYLCIV 11 9 0 (0) 0 TYLCKaV 23 21 21 (100) 3 PepYLCAV 20 20 20 (100) 3 AVTO1920 ( Ty-3 / Ty-3, Ty-6 / Ty-6 ) PepYLCIV 25 25 0 (0) 0 TYLCKaV 10 8 8 (100) 1 PepYLCAV 19 19 0 (0) 0 AVTO1919 ( Ty-1 / Ty-1, Ty-6 / Ty-6 ) PepYLCIV 20 10 0 (0) 0 TYLCKaV 26 25 0 (0) 0 PepYLCAV 14 14 0 (0) 0 AVTO1314 ( ty-5 / ty-5, Ty-6 / Ty-6 ) PepYLCIV 30 30 30 (100) 1 TYLCKaV 24 24 24 (100) 3 PepYLCAV 27 27 27 (100) 2 AVTO1429 ( Ty-2 / Ty-2, Ty-3 / Ty-3, Ty-6/Ty-6 ) PepYLCIV 29 29 0 (0) 0 TYLCKaV 27 27 27 (100) 2 PepYLCAV 25 25 25 (100) 2 AVTO1954 ( Ty-2 / Ty-2, Ty-3 / Ty-3, Ty-6/Ty-6 ) PepYLCIV 18 18 0 (0) 0 TYLCKaV 11 11 11 (100) 2 PepYLCAV 13 13 13 (100) 3 AVTO1315 ( Ty-2 / Ty-2, Ty-3 / Ty-3, Ty-6/Ty-6 ) PepYLCIV 27 27 0 (0) 0 TYLCKaV 27 26 26 (100) 2 PepYLCAV 21 21 21 (100) 3 AVTO1701 ( Ty-2 / Ty-2, Ty-3 / Ty-3, ty-5/ty-5, Ty-6/Ty-6 ) PepYLCIV 19 17 0 (0) 0 TYLCKaV 23 23 0 (0) 0 PepYLCAV 21 21 0 (0) 0 z Virus detected by PCR y (Number of symptomatic plants / Number of infected plants) × 100 x Symptom severity on each plant was scored using a disease severity index (DSI): 0, no symptoms; 1, slight leaf yellowing symptoms; 2, mild leaf yellowing symptoms; 3, moderate leaf yellowing and curing symptoms; and 4, severe leaf yellowing and curing symptoms. In tomato lines that has been tested in previous studies, TYLCKaV and PepYLCAV were more virulent than PepYLCIV (Koeda et al. 2020 ; Pohan et al. 2024 ). In response to the less-virulent PepYLCIV, severe leaf yellowing, curling, and stunting (DSI = 4) were observed only on M and MS plants. AVTO 1314 showed mild symptoms (DSI = 1), while all other lines remained asymptomatic. When challenged with the more-virulent TYLCKaV, M and MS had severe symptoms (DSI = 4), MP and AVTO 1314 had moderate symptoms (DSI = 3), and AVTO 1429, 1954, and 1315 had mild symptoms (DSI = 2). AVTO 1920 exhibited slight symptoms (DSI = 1), while AVTO 1919 and 1701 were asymptomatic. When infected by the most virulent PepYLCAV, M and MS had severe symptoms (DSI = 4), MP, AVTO 1954, and 1315 had moderate symptoms (DSI = 3), and AVTO 1314 and AVTO 1429 had mild symptoms (DSI = 2). AVTO 1919, 1920, and 1701 remained symptomless. Overall, AVTO 1919 ( Ty-1/Ty-1 , Ty-6/Ty-6 ), 1920 ( Ty-3/Ty-3 , Ty-6/Ty-6 ), and 1701 ( Ty-2/Ty-2 , Ty-3/Ty-3 , ty-5/ty-5 , Ty-6/Ty-6 ) exhibited the strongest TYLCD tolerance. To assess the association between symptom expression and accumulation of viral DNA, we quantified viral DNAs using qPCR and DNA extracted from young upper leaves collected at 42 dpi. Viral DNA levels were significantly higher in susceptible M and Ty-2 -bearing MS plants. In contrast, PepYLCIV DNA levels were markedly lower in other tomato lines harboring Ty genes. However, accumulation of the more virulent TYLCKaV and PepYLCAV was not effectively suppressed in MP, AVTO 1314, 1429, 1954, and 1315. In contrast, AVTO 1919, 1920, and 1701 had significantly lower level of TYLCKaV and PepYLCAV DNA compared with other lines. A strong positive correlation was found between symptom severity and viral DNA accumulation ( r ≥ 0.84). AVTO 1919 ( Ty-1/Ty-1, Ty-6/Ty-6 ), 1920 ( Ty-3/Ty-3, Ty-6/Ty-6 ), and 1701 ( Ty-3/Ty-3, Ty-2/Ty-2, ty-5/ty-5, Ty-6/Ty-6 ) had resistance to all three viruses, effectively suppressing viral DNA accumulation. These Ty gene combinations therefore appear to confer broad-spectrum and stable resistance against begomoviruses. Inheritance of begomovirus resistance in F generation Preliminary assays indicated that the infection rate and virulence was high for PepYLCAV after agroinoculation (Table S4 ), so we used it for subsequent experiments (Fig. 4 ). AVTO 1919 and 1920 were crossed with begomovirus susceptible MM in both forward and reciprocal directions to generate F₁ populations, which were then agroinoculated with PepYLCAV to evaluate resistance. In MM, all plants exhibited severe symptoms—leaf yellowing, curling, and stunting—and had a DSI score of 4 at 28 dpi. In contrast, 1919 and 1920 remained symptomless, confirming their TYLCD tolerance. The F₁ progenies of MM×1919 (mean DSI = 1.4), MM×1920 (mean DSI = 1.5), 1919×MM (mean DSI = 1.6), and 1920×MM (mean DSI = 1.3) exhibited mild symptoms. The qPCR showed that viral DNA accumulation in the resistant AVTO 1919 and 1920 was reduced to approximately 1/9–1/12 the level in susceptible MM, which had severe symptoms. In contrast, the mildly symptomatic F₁ hybrids (MM×1919, 1919×MM, 1920×MM) accumulated viral DNA at comparable levels to those in MM. Only MM×1920 F₁ had moderate reduction in viral DNA, to about 1/4 of the MM level, yet the level was still higher than in the resistant parental lines. These findings indicate that heterozygosity for Ty-1 or Ty-3 and Ty-6 does not provide resistance like homozygosity but confer partial TYLCD tolerance. Discussion Many studies have evaluated the effects of Ty -gene pyramiding on begomovirus resistance (Elbaz et al. 2016 ; Hanson et al. 2016 ; Hutton et al. 2015 ; Kenyon et al. 2019 ; Mejia et al. 2010 ; Prabhandakavi et al. 2020 , 2021 ; Prasanna et al. 2015a , b ; Tabein et al. 2017 ; Vidavski et al. 2008 ). In particular, most of these studies relied on Ty -gene-linked markers rather than in-gene markers because some of the studies were done before the resistance genes were cloned. At the outset of this study, we found that AVTO 1919 and 1920 exhibited strong resistance to begomoviruses, despite being reported by the World Vegetable Center to carry only Ty-1/Ty-3 . Genotyping with in-gene markers confirmed the presence of Ty-1 and Ty-3 , respectively, but Ty-2 and ty-5 were not detected, suggesting that these lines contained an additional, unidentified Ty gene. Most recently, after significant effort, Shen et al. ( 2025 ) cloned Ty-6 as POLD1. Subsequent analysis using the in-gene marker for Ty-6 revealed that most AVTO lines, including AVTO 1919 and 1920, indeed harbor Ty-6 . We therefore emphasize the importance of using in-gene markers to confirm the presence of Ty genes and thus determine their true effects on begomovirus resistance and develop effective breeding strategies. Comparison among the AVTO lines revealed that combining Ty-2 with Ty-1/Ty-3 and Ty-6 did not enhance resistance; instead, it appeared to have a negative effect, reducing resistance to begomoviruses. It has been reported that recombination in the genomic region containing the Ty-2 gene is severely suppressed when introgressed from the wild tomato relative Solanum habrochaites into cultivated tomato ( S. lycopersicum ) (Yamaguchi et al. 2018 ; Yang et al. 2014 ). Under our assay conditions, Ty-2 itself, or a neighboring gene, appeared to negatively affect begomovirus resistance when combined with Ty-3 and/or Ty-6 . In addition, although Ty-2 -mediated resistance is fully effective against the Israel strain of TYLCV, it is largely ineffective against most other monopartite and bipartite begomoviruses (Barbieri et al. 2010 ; Hanson et al. 2000 ; Ohnishi et al. 2016 ; Prasanna et al. 2015b ; Shahid et al. 2013 ; Tsai et al. 2011 ). Therefore, the utility of Ty-2 in pyramiding strategies, particularly in regions where these viruses predominate, remains questionable. Another possibility is that AVTO 1919 and 1920 carry the Ty-4 gene, whereas 1429 and 1315 do not. Cloning Ty-4 is necessary to evaluate this hypothesis. Because a tomato line carrying only Ty-6 was not available in this study, we could not directly evaluate the resistance conferred solely by Ty-6 against the tested begomoviruses. However, previous work has shown that Ty-6 provides stronger resistance when combined with other Ty genes such as Ty-3 and ty-5 , which is consistent with our current findings (Gill et al. 2019 ). Our results suggest that combining Ty-1 or Ty-3 with Ty-6 in the homozygous state may represent the minimal number of resistance genes required for effective protection against TYLCD. To further validate this hypothesis, Ty-1/Ty-3 and Ty-6 should be introgressed into the same genetic background in various combinations. Declarations Conflict of interest The authors declare that they have no conflict of interest. Funding This study was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 19H02950, 21KK0109, 23K26900 to S. Koeda. Author Contribution MS and SK designed the experiments; analyzed the data; interpreted the results and wrote the manuscript. MS, MN, and NSP performed the experiments. NSP conducted the statistical analyses. EK prepared the virus material. All authors read and approved the final manuscript. Acknowledgement We thank Takii for providing MS seeds and preparing crossing populations. 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Hort J 87:364–371 Koeda S, Kesumawati E, Tanaka Y, Hosokawa M, Doi M, Kitajima A (2016) Mixed infection of begomoviruses on pepper plants at Northern Sumatra, Indonesia. Trop Agric Dev 60:59–64 Koeda S, Kitawaki A (2024) Breakdown of Ty-1 -based resistance to tomato yellow leaf curl virus in tomato plants at high temperatures. Phytopathology 114:294–303 Koeda S, Onouchi M, Mori N, Pohan NS, Nagano AJ, Kesumawati E (2021) A recessive gene pepy-1 encoding Pelota confers resistance to begomovirus isolates of PepYLCIV and PepYLCAV in Capsicum annuum . Theor Appl Genet 134:2947–2964 Koeda S, Mori N, Horiuchi R, Watanabe C, Nagano AJ, Shiragane H (2022) PepYLCIV and PepYLCAV resistance gene Pepy-2 encodes DFDGD-class RNA-dependent RNA polymerase in Capsicum . Theor Appl Genet 135:2437–2452 Koeda S, Yamamoto C, Yamamoto H, Fujishiro K, Mori R, Okamoto M, Nagano AJ, Mashiko T (2024) Cy-1 , a major QTL for tomato leaf curl New Delhi virus resistance, harbours a gene encoding a DFDGD-class RNA-dependent RNA polymerase in cucumber ( Cucumis sativus ). BMC Plant Biol 24:879 Lapidot M, Karniel U, Gelbart D, Fogel D, Evenor D, Kutsher Y, Makhbash Z, Nahon S, Shlomo H, Chen L (2015) A novel route controlling begomovirus resistance by the messenger RNA surveillance factor Pelota. Plos Genet 11:e1005538 Lapidot M, Legg JP, Wintermantel WM, Polston JE (2014) Chapter Three - Management of whitefly-transmitted viruses in open-field production systems. In: Loebenstein G, Katis N (eds) Advances in Virus Research. Academic Press, 147–206 Lim YW, Mansfeld BN, Schläpfer P et al (2022) Mutations in DNA polymerase δ subunit 1 co-segregate with CMD2 -type resistance to Cassava Mosaic Geminivirus. 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Theor Appl Genet 139:20 Pohan NS, Okuno K, Okabe S, Kesumawati E, Koeda S (2024) Pepper yellow leaf curl Aceh virus: a recombinant replacing the parental begomovirus is highly virulent and breaks Ty-1 -mediated resistance in tomato. J Gen Plant Pathol 90:333–343 Prabhandakavi P, Kumar R, Acharya S, Chakraborty M, Rambabu P, Palicherla SR, Pinnamaneni R (2021) Evaluation of tomato inbred lines harboring Ty gene(s) for resistance against monopartite and bipartite begomoviruses. Proc Natl Acad Sci India Sect B Biol Sci 91:45–52 Prabhandakavi P, Pogiri R, Kumar R, Acharya S, Esakky R, Chakraborty, Pinnamaneni, Sairam, Palicherla S (2020) Pyramiding Ty-1 / Ty-3 , Ty-2 , ty-5 and ty-6 genes into tomato hybrid to develop resistance against tomato leaf curl viruses and recurrent parent genome recovery by ddRAD sequencing method. J Plant Biochem Biotechnol 30:462–476 Prasanna HC, Kashyap SP, Krishna R, Sinha DP, Reddy S, Malathi VG (2015a) Marker assisted selection of Ty-2 and Ty-3 carrying tomato lines and their implications in breeding tomato leaf curl disease resistant hybrids. Euphytica 204:407–418 Prasanna HC, Sinha DP, Rai GK, Krishna R, Kashyap SP, Singh NK, Singh M, Malathi VG (2015b) Pyramiding Ty-2 and Ty-3 genes for resistance to monopartite and bipartite tomato leaf curl viruses of India. Plant Pathol 64:256–264 Rojas MR, Macedo MA, Maliano MR, Soto-Aguilar M, Souza JO, Briddon RW, Kenyon L, Rivera Bustamante RF, Zerbini FM, Adkins S, Legg JP, Kvarnheden A, Wintermantel WM, Sudarshana MR, Peterschmitt M, Lapidot M, Martin DP, Moriones E, Inoue-Nagata AK, Gilbertson RL (2018) World management of geminiviruses. Annu Rev Phytopathol 56:637–677 Shahid MS, Ito T, Kimbara J, Onozato A, Natsuaki KT, Ikegami M (2013) Evaluation of tomato hybrids carrying Ty-1 and Ty-2 loci to Japanese monopartite begomovirus species. J Phytopathol 161:205–209 Shen X, Gill U, Arens M, Yan Z, Bai Y, Hutton SF, Wolters A-MA (2025) The tomato gene Ty-6 , encoding DNA polymerase delta subunit 1, confers broad resistance to Geminiviruses. Theor Appl Genet 138:22 Tabein S, Behjatnia SAA, Laviano L, Pecchioni N, Accotto GP, Noris E, Miozzi L (2017) Pyramiding Ty-1 / Ty-3 and Ty-2 in tomato hybrids dramatically inhibits symptom expression and accumulation of tomato yellow leaf curl disease inducing viruses. Arch Phytopathol Plant Prot 50:213–227 Taniguchi M, Sekine KT, Koeda S (2023) Lisianthus enation leaf curl virus, a begomovirus new to Japan, is more virulent than the prevalent tomato yellow leaf curl virus in Ty -gene-mediated resistant tomato cultivars. J Gen Plant Pathol 89:35–46 Tsai WS, Shih SL, Kenyon L, Green SK, Jan FJ (2011) Temporal distribution and pathogenicity of the predominant tomato-infecting begomoviruses in Taiwan. Plant Pathol 60:787–799 Verlaan MG, Hutton SF, Ibrahem RM, Kormelink R, Visser RGF, Scott JW, Edwards JD, Bai YL (2013) The tomato yellow leaf curl virus resistance genes Ty-1 and Ty-3 are allelic and code for DFDGD-class RNA-dependent RNA polymerases. Plos Genet 9:e1003399 Vidavski F, Czosnek H, Gazit S, Levy D, Lapidot M (2008) Pyramiding of genes conferring resistance to Tomato yellow leaf curl virus from different wild tomato species. Plant Breed 127:625–631 Voorburg CM, Yan Z, Bergua-Vidal M, Wolters AA, Bai Y, Kormelink R. (2020) Ty-1 , a universal resistance gene against geminiviruses that is compromised by co-replication of a betasatellite. Mol Plant Pathol 21:160–172 Whitham S, Dinesh-Kumar SP, Choi D, Hehl R, Corr C, Baker B (1994) The product of the tobacco mosaic virus resistance gene N : similarity to toll and the interleukin-1 receptor. Cell 78:1101–1115 Yamaguchi H, Ohnishi J, Saito A, Ohyama A, Nunome T, Miyatake K, Fukuoka H (2018) An NB-LRR gene, TYNBS1 , is responsible for resistance mediated by the Ty-2 Begomovirus resistance locus of tomato. Theor Appl Genet 131:1345–1362 Yang X, Caro M, Hutton SF, Scott JW, Guo Y, Wang X, Rashid MH, Szinay D, de Jong H, Visser RGF, Bai Y, Du Y (2014) Fine mapping of the tomato yellow leaf curl virus resistance gene Ty-2 on chromosome 11 of tomato. Mol Breed 34:749–760 Zamir D, Eksteinmichelson I, Zakay Y, Navot N, Zeidan M, Sarfatti M, Eshed Y, Harel E, Pleban T, Vanoss H, Kedar N, Rabinowitch HD, Czosnek H (1994) Mapping and introgression of a tomato yellow leaf curl virus tolerance gene, Ty-1 . Theor Appl Genet 88:141–146 Additional Declarations No competing interests reported. Supplementary Files TableS1.pdf TableS2.pdf TableS3.pdf TableS4.pdf Cite Share Download PDF Status: Published Journal Publication published 30 Mar, 2026 Read the published version in Euphytica → Version 1 posted Editorial decision: Revision requested 04 Mar, 2026 Reviews received at journal 03 Mar, 2026 Reviews received at journal 18 Feb, 2026 Reviewers agreed at journal 28 Jan, 2026 Reviews received at journal 27 Jan, 2026 Reviewers agreed at journal 27 Jan, 2026 Reviewers agreed at journal 26 Jan, 2026 Reviewers invited by journal 26 Jan, 2026 Submission checks completed at journal 23 Jan, 2026 First submitted to journal 22 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8207444","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":581615716,"identity":"0100eac4-32d5-4999-bbdf-1cf2884ca7d6","order_by":0,"name":"Moeno Shimoide","email":"","orcid":"","institution":"Kindai University","correspondingAuthor":false,"prefix":"","firstName":"Moeno","middleName":"","lastName":"Shimoide","suffix":""},{"id":581615717,"identity":"706d8410-9dc2-4f50-b90f-ab104b41621c","order_by":1,"name":"Misaki Nakajima","email":"","orcid":"","institution":"Kindai University","correspondingAuthor":false,"prefix":"","firstName":"Misaki","middleName":"","lastName":"Nakajima","suffix":""},{"id":581615722,"identity":"9b3ecc73-8dbb-44f2-b064-25be4d9cad07","order_by":2,"name":"Nadya Syafira Pohan","email":"","orcid":"","institution":"Kindai University","correspondingAuthor":false,"prefix":"","firstName":"Nadya","middleName":"Syafira","lastName":"Pohan","suffix":""},{"id":581615729,"identity":"6d880c69-953e-4035-bec9-5566dbaf71bc","order_by":3,"name":"Elly Kesumawati","email":"","orcid":"","institution":"Syiah Kuala University","correspondingAuthor":false,"prefix":"","firstName":"Elly","middleName":"","lastName":"Kesumawati","suffix":""},{"id":581615731,"identity":"5d6d2c8c-09c9-4743-be0c-868bed937be8","order_by":4,"name":"Sota Koeda","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEklEQVRIie2QMUvDQBTHX3iQLrFZ71D0K1zJYIXSz3JHIJnq4mjRAweX4ly/RTZxCxykSyTrrbd0cjhxySKY2A46XKibyP3g8d4NP97/HYDH8weJEcu+n+6eJekqkP2ILoXehxyAQ3K4wpqI9YqQO2VfQ9C7MLVv7Tx/Gr0YC/X0crxREtoljM7dt1R0zdPF8ypPCGhyRWshg1UFeCGdWzKMOC6KMgMCloii7EIeSUDmSMhUlLxH/DZnzRbbL6UxMvgYVthxxBVnOgv7YKLQQuLQlj4YXWebSaG34ZTXRDxqI9VJRZy3xLFS1s6uz1iTobbVjXhocmNel7PU9WM/4fveRSIpO0j5zvz3isfj8fxTPgHI+19o0WjQ5gAAAABJRU5ErkJggg==","orcid":"","institution":"Kindai University","correspondingAuthor":true,"prefix":"","firstName":"Sota","middleName":"","lastName":"Koeda","suffix":""}],"badges":[],"createdAt":"2025-11-26 01:38:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8207444/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8207444/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10681-026-03711-y","type":"published","date":"2026-03-30T15:59:02+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":102597908,"identity":"c82e726d-60ea-491a-8359-a5b17c2019ce","added_by":"auto","created_at":"2026-02-13 12:26:54","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":247869,"visible":true,"origin":"","legend":"\u003cp\u003eSymptoms for different disease severity index (DSI) scores at 42 days after graft inoculation of tomato cultivars and lines with begomovirus.\u003c/p\u003e\n\u003cp\u003eThe susceptible Momotaro (M) plants were inoculated with one of the three viruses, pepper yellow leaf curl Indonesia virus (PepYLCIV), tomato yellow leaf curl Kanchanaburi virus (TYLCKaV), and pepper yellow leaf curl Aceh virus (PepYLCAV), and tomato cultivars or lines carrying different \u003cem\u003eTy\u003c/em\u003e genes were grafted onto virus-infected M rootstocks. Virus-infected scions were identified by PCR-based diagnosis, and resistance was evaluated based on disease severity index (DSI). Symptoms of each plant were scored using a disease severity index (DSI): 0, no symptoms; 1, slight leaf yellowing symptoms; 2, mild leaf yellowing symptoms; 3, moderate leaf yellowing and curing symptoms; and 4, severe leaf yellowing and curing symptoms.\u003c/p\u003e","description":"","filename":"11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8207444/v1/da6fe5ea2e2937d0c1b35ae6.jpg"},{"id":102597840,"identity":"7036b9b3-809c-49db-9de2-d148eaba5ecc","added_by":"auto","created_at":"2026-02-13 12:26:38","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":479861,"visible":true,"origin":"","legend":"\u003cp\u003eValidation of \u003cem\u003eTy-1\u003c/em\u003e, \u003cem\u003eTy-2\u003c/em\u003e, \u003cem\u003eTy-3\u003c/em\u003e, \u003cem\u003eTy-3a\u003c/em\u003e, \u003cem\u003ety-5\u003c/em\u003e, and \u003cem\u003eTy-6\u003c/em\u003e presence in tomato cultivars and lines using allele-specific in-gene markers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e The susceptible (S) allele and \u003cem\u003eTy-1\u003c/em\u003e/\u003cem\u003eTy-3\u003c/em\u003e/\u003cem\u003eTy-3a\u003c/em\u003e resistance (R) alleles were distinguished using marker SK-F1 and R1 (S: 92 bp, R: 104 bp) and marker SK-F3 and R4 (S: 159 bp, R: 215 bp). The susceptible allele (264 bp), \u003cem\u003eTy-1\u003c/em\u003e/\u003cem\u003eTy-3\u003c/em\u003e(276 bp), and \u003cem\u003eTy-3a\u003c/em\u003e (238 bp) were distinguished using marker SK-F1 and R2. The susceptible/\u003cem\u003eTy-1\u003c/em\u003e/\u003cem\u003eTy-3a\u003c/em\u003e alleles (96 bp) and \u003cem\u003eTy-3\u003c/em\u003e(113 bp) were distinguished using marker SK-F2 and R3. The susceptible/\u003cem\u003eTy-3\u003c/em\u003e/\u003cem\u003eTy-3a\u003c/em\u003ealleles (136 bp) and \u003cem\u003eTy-1\u003c/em\u003e (106 bp) were distinguished using marker SK-F4 and R5. The \u003cem\u003eTy-2\u003c/em\u003e resistance allele (approximately 3000 bp) was distinguished using marker Ty-2 F and R and the susceptible allele (approximately 2800 bp) was distinguished using marker ty-2 F and R. The susceptible (S) \u003cem\u003eTy-5\u003c/em\u003eallele and resistance (R) \u003cem\u003ety-5\u003c/em\u003e alleles were distinguished using marker ty-5 F and R (S: 312 bp, R: 31 + 281 bp). The susceptible (S) \u003cem\u003ety-6\u003c/em\u003eallele and resistance (R) \u003cem\u003eTy-6\u003c/em\u003e alleles were distinguished using marker Ty-6 F and R (S: 223 + 567 bp, R: 790 bp). Asterisks indicate resistance alleles. L, DNA ladder; M, begomovirus-susceptible tomato Momotaro; MS, TYLCV-resistant tomato Momotaro Sakura; MP, TYLCV-resistant tomato Momotaro Peace. \u003cstrong\u003eB\u003c/strong\u003e Summary of genotyped \u003cem\u003eTy\u003c/em\u003e-gene combinations. Details on molecular markers and PCR conditions are in Table S2 and S3.\u003c/p\u003e","description":"","filename":"12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8207444/v1/8c15defcf2689cf663a898e2.jpg"},{"id":102598014,"identity":"81a22f39-4c63-42aa-b0a4-3565614203ce","added_by":"auto","created_at":"2026-02-13 12:27:01","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":318113,"visible":true,"origin":"","legend":"\u003cp\u003eViral DNA levels at 42 days after graft inoculation of tomato cultivars and lines with pepper yellow leaf curl Indonesia virus (PepYLCIV), tomato yellow leaf curl Kanchanaburi virus (TYLCKaV), or pepper yellow leaf curl Aceh virus (PepYLCAV).\u003c/p\u003e\n\u003cp\u003eDNA extracted from young upper leaves was used for qPCR. Viral DNA values were normalized against the 25S rRNA gene values. Relative amount of viral DNA was calculated using the 2\u003csup\u003e−ΔΔCT\u003c/sup\u003e method (Livak and Schmittgen 2001). M, begomovirus-susceptible tomato Momotaro; MS, TYLCV-resistant tomato Momotaro Sakura; MP, TYLCV-resistant tomato Momotaro Peace. Each dot indicates viral DNA amount of each individual. \u003cem\u003eN\u003c/em\u003e indicates number of individuals used for analysis in each tomato genotype. Different letters above means indicate a significant difference between means (Fisher’s LSD test, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05). Spearman’s rank correlation coefficient was used to calculate correlations value \u003cem\u003er\u003c/em\u003e between disease severity and viral DNA accumulation (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"13.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8207444/v1/9a7ab0327ef944c2788b1d2e.jpg"},{"id":102597841,"identity":"4c34dccd-41d3-4e50-8b4d-43e16a1ce6da","added_by":"auto","created_at":"2026-02-13 12:26:39","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":281230,"visible":true,"origin":"","legend":"\u003cp\u003eSymptoms and accumulation of pepper yellow leaf curl Aceh virus (PepYLCAV) DNA at 28 days after agroinoculation of tomato cultivar, line, and F\u003csub\u003e1\u003c/sub\u003e progeny.\u003c/p\u003e\n\u003cp\u003eAVTO 1919 and 1920 were crossed with begomovirus-susceptible Moneymaker (MM) in both forward and reciprocal directions to generate F₁ populations. The number above each leaf is the mean disease severity index (DSI): 0, no symptoms; 1, slight leaf yellowing symptoms; 2, mild leaf yellowing symptoms; 3, moderate leaf yellowing and curing symptoms; and 4, severe leaf yellowing and curing symptoms. DNA extracted from young upper leaves was used for qPCR. Viral DNA values were normalized against the 25S rRNA gene values. Relative amount of viral DNA was calculated using the 2\u003csup\u003e−ΔΔCT\u003c/sup\u003e method (Livak and Schmittgen 2001). Each dot indicates viral DNA amount of each individual. \u003cem\u003eN\u003c/em\u003e indicates number of individuals used for analysis in each tomato genotype. Different letters above means indicate a significant difference between means (Fisher’s LSD test, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"14.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8207444/v1/77b5c513928fd1d6df26ab1f.jpg"},{"id":106344481,"identity":"12deec26-e99d-4ed4-a4c6-ccf2c6c8312c","added_by":"auto","created_at":"2026-04-07 16:15:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2248204,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8207444/v1/a66741ca-ea5d-4718-b536-6d82fce1accc.pdf"},{"id":102598015,"identity":"d786d10f-7633-49df-9039-3d73c6916af6","added_by":"auto","created_at":"2026-02-13 12:27:01","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":64629,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8207444/v1/c6bfbe977c108a497f800b8a.pdf"},{"id":102597851,"identity":"8fcc7cdd-f45e-4cb5-bb49-adc79fa43327","added_by":"auto","created_at":"2026-02-13 12:26:43","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":84011,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8207444/v1/5785888fb584bdb79e927b8c.pdf"},{"id":102597848,"identity":"1f40f524-ec72-49ba-b0b5-6f0413e16553","added_by":"auto","created_at":"2026-02-13 12:26:42","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":60762,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8207444/v1/923bc246e12a962f93eca921.pdf"},{"id":102597927,"identity":"b501edff-e23b-4846-bdcc-0508039d40ab","added_by":"auto","created_at":"2026-02-13 12:26:55","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":41312,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8207444/v1/ef8bbb4689e34fab86216b24.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Integration of Ty-1/Ty-3 and Ty-6 confers improved and durable resistance to highly pathogenic begomoviruses in tomato","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTomato yellow leaf curl disease (TYLCD), caused by several species of the genus \u003cem\u003eBegomovirus\u003c/em\u003e within the \u003cem\u003eGeminiviridae\u003c/em\u003e family, is one of the most destructive diseases of tomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e). Virus-infected plants exhibit yellowing of young leaves, upward leaf curling, smaller leaflets, stunted growth, and aborted flowers (Moriones and Navas-Castillo \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). The disease severely limits tomato production, often causing complete yield loss in virus-susceptible cultivars (Czosnek and Laterrot \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Among the associated viruses, tomato yellow leaf curl virus (TYLCV) is particularly widespread, affecting tomato crops. Begomoviruses are persistently and circulatively transmitted by the whitefly \u003cem\u003eBemisia tabaci\u003c/em\u003e (Hemiptera: Aleyrodidae). Although complete control of this vector remains difficult, whitefly populations can be reduced through basic measures such as insecticide use and fine-mesh screening of greenhouse openings (Rojas et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In addition to these approaches, the primary means of managing TYLCD has been the use of TYLCV resistant cultivars carrying \u003cem\u003eTy\u003c/em\u003e genes.\u003c/p\u003e \u003cp\u003eExtensive screening of tomato germplasm has identified several wild relatives exhibiting resistance to TYLCV. Multiple resistance loci (\u003cem\u003eTy-1\u003c/em\u003e to \u003cem\u003eTy-6\u003c/em\u003e) have been introgressed into cultivated tomato (\u003cem\u003eS. lycopersicum\u003c/em\u003e) (Agrama and Scott \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Anbinder et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Hutton and Scott \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Ji et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2009a\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003eb\u003c/span\u003e; Zamir et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). To date, all \u003cem\u003eTy\u003c/em\u003e genes except \u003cem\u003eTy-4\u003c/em\u003e have been cloned. Specifically, \u003cem\u003eTy-1\u003c/em\u003e, \u003cem\u003eTy-3\u003c/em\u003e, and \u003cem\u003eTy-3a\u003c/em\u003e encode RNA-dependent RNA polymerase (RDR) (Verlaan et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2013\u003c/span\u003e); \u003cem\u003eTy-2\u003c/em\u003e encodes an NB-LRR protein (Yamaguchi et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2018\u003c/span\u003e); \u003cem\u003ety-5\u003c/em\u003e encodes pelota (Lapidot et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e); and \u003cem\u003eTy-6\u003c/em\u003e encodes DNA polymerase delta subunit 1 (POLD1) (Shen et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Among these \u003cem\u003eTy\u003c/em\u003e genes, \u003cem\u003eTy-1\u003c/em\u003e in the heterozygous state is the most widely used in commercial F₁ hybrid tomato cultivars released in Europe, North America, and East Asia (Fortes et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Koeda et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Koeda and Kitawaki \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Lapidot et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). However, \u003cem\u003eTy-1\u003c/em\u003e-mediated resistance to TYLCV has been reported to break down due to begomoviral evolution, coinfection with a beta-satellite or crinivirus, or cultivation at high temperatures (Belabess et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Fortes et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Jammes et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Koeda and Kitawaki \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Voorburg et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Moreover, predominant begomovirus species in subtropical and tropical regions are generally more virulent than TYLCV, and \u003cem\u003eTy-1\u003c/em\u003e-mediated resistance is not fully effective (Koeda et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Pohan et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Such challenges have driven breeding efforts toward the development of tomato cultivars with multiple \u003cem\u003eTy\u003c/em\u003e resistance genes.\u003c/p\u003e \u003cp\u003eThe transfer of resistance from wild relatives to cultivated species is a cornerstone of modern breeding. Yet, linkage drag caused by pleiotropic effects of resistance genes or neighboring wild genomic regions can impair key agronomic traits (Kashyap et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Consequently, breeders aim to introgress the minimal number of resistance genes required for effective protection. To explore this concept, we assessed the resistance of tomato lines carrying different combinations of \u003cem\u003eTy\u003c/em\u003e genes against begomoviruses with varying virulence originating from tropical area. The results provide valuable insights and a conceptual basis for developing tomato cultivars with durable resistance.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant material\u003c/h2\u003e \u003cp\u003eThe tomato F\u003csub\u003e1\u003c/sub\u003e hybrid cultivars Momotaro (M: no \u003cem\u003eTy\u003c/em\u003e-genes, susceptible to begomoviruses) and TYLCV-resistant cultivars Momotaro Sakura (MS) and Momotaro Peace (MP) (Takii Seeds, Kyoto, Japan) were used in the study. Momotaro is one of the most popular fresh tomato cultivars in Japan. MS and MP have the same genetic background as M. AVTO lines were obtained from the World Vegetable Center: 1919, 1920, 1314, 1429, 1954, 1315, and 1701. These fresh market tomato lines, which have different \u003cem\u003eTy\u003c/em\u003e-gene combinations, were selected for the study. The officially described \u003cem\u003eTy\u003c/em\u003e-gene combinations and parentage of each AVTO line are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. AVTO 1919 and 1920 were crossed with Moneymaker (MM: no \u003cem\u003eTy\u003c/em\u003e-genes, susceptible to begomoviruses) in both forward and reciprocal directions to obtain the F\u003csub\u003e1\u003c/sub\u003e populations.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGenotyping of\u003c/b\u003e \u003cb\u003eTy\u003c/b\u003e\u003cb\u003e-genes using in-gene DNA markers\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe presence of \u003cem\u003eTy-1\u003c/em\u003e, \u003cem\u003eTy-3\u003c/em\u003e, and \u003cem\u003eTy-3a\u003c/em\u003e was verified using the previously reported codominant in-gene DNA markers and methods (Koeda and Kitawaki \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). \u003cem\u003eTy-2\u003c/em\u003e and \u003cem\u003ety-2\u003c/em\u003e were verified using dominant in-gene DNA-markers reported by Yamaguchi et al. (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). An in-gene derived cleaved amplified polymorphic sequences (dCAPS) marker for \u003cem\u003ety-5\u003c/em\u003e and a cleaved amplified polymorphic sequences (CAPS) marker for \u003cem\u003eTy-6\u003c/em\u003e were developed in this study to detect the causal single nucleotide polymorphisms (SNPs) reported (Lapidot et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Shen et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The primer sequences and PCR conditions are listed in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e and S3. For \u003cem\u003eTy-1\u003c/em\u003e, \u003cem\u003eTy-3\u003c/em\u003e, \u003cem\u003eTy-3a\u003c/em\u003e, \u003cem\u003ety-5\u003c/em\u003e, and \u003cem\u003eTy-6\u003c/em\u003e markers, reaction was conducted in a 10-\u0026micro;L reaction mixture containing 5 \u0026micro;L of Emerald Amp PCR Master Mix (Takara Bio, Shiga, Japan), 0.1 \u0026micro;L of each primer (20 pmol/\u0026micro;L), 3.8 \u0026micro;L of water, and 1 \u0026micro;L of DNA template (30 ng/\u0026micro;L). For \u003cem\u003eTy-2\u003c/em\u003e and \u003cem\u003ety-2\u003c/em\u003e markers, reaction was conducted in a 10-\u0026micro;L reaction mixture containing 0.1 \u0026micro;L of BlendTaq-Plus (Toyobo, Osaka, Japan), 1 \u0026micro;L of 10\u0026times;buffer, 1 \u0026micro;L of dNTPs (2mM), 0.1 \u0026micro;L of each primer (20 pmol/\u0026micro;L), 6.8 \u0026micro;L of water, and 1 \u0026micro;L of DNA template (30 ng/\u0026micro;L). The PCRs were run in a GeneAtlas G02 Thermal Cycler (Astec, Fukuoka, Japan). For \u003cem\u003ety-5\u003c/em\u003e dCAPS and \u003cem\u003eTy-6\u003c/em\u003e CAPS markers, amplicons were digested with the restriction enzyme in a 10-\u0026micro;L mixture containing 4 \u0026micro;L of the PCR amplicons, 1 U of NcoI (Takara Bio) or AanI (PsiI) (Thermo Scientific, MA, USA), 1 \u0026micro;L of 10\u0026times;buffer, 1 \u0026micro;L of BSA if required, and incubated at 37\u0026deg;C for 1 h. The entire digested product was electrophoresed in 1.5% (w/v) agarose gels for 30 min at 100 V.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eVirus inoculation and symptom evaluation\u003c/h3\u003e\n\u003cp\u003eThe infectious clones of pepper yellow leaf curl Indonesia virus (PepYLCIV) isolate BA_D1-1 (GenBank accessions LC051114 [DNA-A] and LC314794 [DNA-B]) (Koeda et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), pepper yellow leaf curl Aceh virus (PepYLCAV) isolate BAPep-V2 (GenBank LC387327 [DNA-A] and LC387329 [DNA-B]) (Kesumawati et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Koeda et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and tomato yellow leaf curl Kanchanaburi virus (TYLCKaV) isolate BA_B6 (GenBank LC051116 [DNA-A] and LC177332 [DNA-B]) (Koeda et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) have been described elsewhere. We selected these bipartite begomovirus species, which are predominant in Southeast Asia, for this study because they are more virulent than the monopartite TYLCV (Koeda et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Taniguchi et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eVarious tomato lines were graft inoculated with higher inoculum pressure to evaluate their begomovirus resistance because agroinoculation was occasionally less effective because of the high virus resistance conferred by introgressed \u003cem\u003eTy\u003c/em\u003e-genes. First, tomato M plants were agroinoculated using the method of Koeda et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) with either of the three begomoviruses and used as the rootstock. Plants were transplanted into plastic pots containing horticultural soil mix (Japan Agricultural Cooperatives, Tokyo, Japan) and were grown in a growth room at 25\u0026ndash;30\u0026deg;C day/23\u0026ndash;25\u0026deg;C night with 13 h light/11 h dark. The virus-free healthy tomato lines were used as the scion and grafted onto virus-infected M plants using grafting tubes (Nasunics Co., Ltd., Tochigi, Japan). The grafted plants were kept in humid conditions for about 2 weeks. After plants were acclimatized, growth of the scion was promoted. We conducted experiments in five different time periods because we grafted more than 600 plants in total. However, because we cultivated the plants in the aforementioned growth room, the climate conditions were uniform for all experiments. At 42 days after inoculation (dpi) by grafting, symptoms were evaluated, and newly developed upper young leaves were collected to extract DNA. Graft-inoculated plants took a longer time of 42 days to evaluate because more time was required for the scions to elongate to a size large enough for symptom observation. In our subsequent experiment, we evaluated the symptoms of the PepYLCAV agroinoculated tomato cultivar, line, and F\u003csub\u003e1\u003c/sub\u003e progeny at 28 dpi. Symptoms on each plant were scored using a disease severity index (DSI) as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e: 0, no symptoms; 1, slight leaf yellowing symptoms; 2, mild leaf yellowing symptoms; 3, moderate leaf yellowing and curing symptoms; and 4, severe leaf yellowing and curing symptoms.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eVirus DNA detection and quantification\u003c/h3\u003e\n\u003cp\u003eThe Nucleon PhytoPure Kit (Cytiva, Malborough, MA, USA) was used to extract DNA from tomato leaves. Primer pairs specific for each virus species were used for PCR (Supplemental Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) in a 10-\u0026micro;L reaction mixture containing 5 \u0026micro;L of Emerald Amp PCR Master Mix (Takara Bio), 0.1 \u0026micro;L of each primer (20 pmol/\u0026micro;L), 3.8 \u0026micro;L of water, and 1 \u0026micro;L of DNA template (30 ng/\u0026micro;L). The PCRs were run in a GeneAtlas G02 Thermal Cycler (Astec). The primer sequences and PCR conditions are listed in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e and S3. The amplicons were electrophoresed in a 1.0% (w/v) agarose gel.\u003c/p\u003e \u003cp\u003eThe CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) was used for quantitative PCR (qPCR) with primer pairs specific for each virus species (Supplemental Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The multicopy 25S ribosomal RNA gene was detected by qPCR using the 25s-rRNA 2F and 2R primers to normalize viral DNA accumulation data. The 10-\u0026micro;L reaction mixture contained 5 \u0026micro;L of SsoAdvanced Universal SYBR Green Supermix (Bio-Rad Laboratories), 0.125 \u0026micro;L of each primer (20 pmol/\u0026micro;L), 2.75 \u0026micro;L of water, and 2 \u0026micro;L of DNA template (30 ng/\u0026micro;L). The primer sequences and PCR conditions are listed in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e and S3. Samples were analyzed in duplicate to calculate the average threshold cycle. The 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e comparative method (Livak and Schmittgen \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) was applied to calculate the relative quantity of each virus. We calculated the Cq values for the mock-inoculated plants using virus-specific and 25s-rRNA 2F and 2R primers. If the calculated amount of DNA in the virus inoculated samples was lower than in the mock-inoculated samples, we considered the plants uninfected and excluded the data when calculating the mean amount of viral DNA for each tomato cultivar and line. In the first experiment, which aimed to assess the amount of viral DNA in each tomato cultivar and line, we conducted cultivation on five different dates/blocks, as mentioned above. For the analysis, we combined the raw data obtained from qPCR.\u003c/p\u003e\n\u003ch3\u003eStatistical analyses\u003c/h3\u003e\n\u003cp\u003eSPSS Statistics (Version 22.0; IBM, Armonk, NY, USA) was used for all statistical analyses. Mean values for the accumulated begomoviral DNA were analyzed for significant differences using a nonparametric one-way ANOVA, with subsequent comparison of multiple means using Fisher\u0026rsquo;s LSD test, with \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered statistically significant. Spearman\u0026rsquo;s rank correlation coefficient was used to assess correlations between disease severity and viral DNA accumulation because disease severity was recorded as ordinal data, with \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eValidation of\u003c/b\u003e \u003cb\u003eTy\u003c/b\u003e\u003cb\u003e-genes using in-gene DNA markers\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe presence and status of \u003cem\u003eTy\u003c/em\u003e-genes were assessed using in-gene DNA markers (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Genotyping with the SK-F1 and R1 and SK-F3 and R4 markers detected the resistance-type RDR allele (\u003cem\u003eTy-1\u003c/em\u003e/\u003cem\u003eTy-3\u003c/em\u003e/\u003cem\u003eTy-3a\u003c/em\u003e), indicating that MP is heterozygous, whereas AVTO 1919, 1920, 1429, 1954, 1315, and 1701 are homozygous. Further analysis using the SK-F2 and R3 markers revealed the presence of \u003cem\u003eTy-3\u003c/em\u003e in AVTO 1920, 1429, 1954, 1315, and 1701, while the SK-F4 and R5 markers identified \u003cem\u003eTy-1\u003c/em\u003e in MP and AVTO 1919. No \u003cem\u003eTy-3a\u003c/em\u003e allele was detected in any lines. Using markers specific for \u003cem\u003eTy-2\u003c/em\u003e and \u003cem\u003ety-2\u003c/em\u003e, MS was found to be heterozygous for \u003cem\u003eTy-2\u003c/em\u003e, whereas AVTO 1429, 1954, 1315, and 1701 were homozygous. The \u003cem\u003ety-5\u003c/em\u003e-specific marker showed that AVTO 1314 and 1701 were homozygous for \u003cem\u003ety-5\u003c/em\u003e, and \u003cem\u003eTy-6\u003c/em\u003e-specific marker revealed that AVTO 1919, 1920, 1314, 1429, 1315, and 1701 were homozygous for \u003cem\u003eTy-6\u003c/em\u003e. A summary of the \u003cem\u003eTy\u003c/em\u003e gene combinations in each cultivar or line is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of begomovirus resistance in tomato commercial cultivars and breeding lines\u003c/h2\u003e \u003cp\u003eIn classical virus\u0026ndash;host interactions, such as the tobacco mosaic virus (TMV)\u0026ndash;\u003cem\u003eN\u003c/em\u003e gene system in tobacco, pathogen recognition by the resistance gene activates a hypersensitive response, resulting in localized cell death as an effective immune defense (Whitham et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). However, unlike this immune-based resistance, all begomovirus resistance genes characterized to date confer disease tolerance and restrict viral DNA accumulation to levels that are significantly lower than in the susceptible genotype (Koeda et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Lapidot et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Lim et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Pohan et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Shen et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Verlaan et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Yamaguchi et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In the present study, tolerance was defined as the absence of visible symptoms or the presence of slight symptoms following infection, while virus resistance was defined as a significant restriction of viral DNA accumulation in these plants. In this study, susceptible M plants were inoculated with one of the three viruses, and tomato cultivars or lines carrying different \u003cem\u003eTy\u003c/em\u003e genes were grafted onto M rootstocks. Virus-infected plants were diagnosed using PCR, and resistance was evaluated based on the disease severity index (DSI) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and viral DNA accumulation at 42 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBegomovirus resistance of tomato cultivars and lines with different Ty gene combinations\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTomato cultivars or lines\u003c/p\u003e \u003cp\u003eused as scions\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eInoculum\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eNumber of plants\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eDSI\u003csup\u003ex\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGrafted\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInfected\u003csup\u003ez\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSymptomatic (%)\u003csup\u003ey\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003cp\u003e(Without \u003cem\u003eTy\u003c/em\u003e gene)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePepYLCIV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e28 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTYLCKaV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePepYLCAV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eMS\u003c/p\u003e \u003cp\u003e(\u003cem\u003eTy-2\u003c/em\u003e/\u003cem\u003ety-2\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePepYLCIV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e14 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTYLCKaV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePepYLCAV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eMP\u003c/p\u003e \u003cp\u003e(\u003cem\u003eTy-1\u003c/em\u003e/\u003cem\u003ety-1\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePepYLCIV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTYLCKaV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePepYLCAV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eAVTO1920\u003c/p\u003e \u003cp\u003e(\u003cem\u003eTy-3\u003c/em\u003e/\u003cem\u003eTy-3, Ty-6\u003c/em\u003e/\u003cem\u003eTy-6\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePepYLCIV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTYLCKaV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePepYLCAV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eAVTO1919\u003c/p\u003e \u003cp\u003e(\u003cem\u003eTy-1\u003c/em\u003e/\u003cem\u003eTy-1, Ty-6\u003c/em\u003e/\u003cem\u003eTy-6\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePepYLCIV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTYLCKaV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePepYLCAV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eAVTO1314\u003c/p\u003e \u003cp\u003e(\u003cem\u003ety-5\u003c/em\u003e/\u003cem\u003ety-5, Ty-6\u003c/em\u003e/\u003cem\u003eTy-6\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePepYLCIV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e30 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTYLCKaV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePepYLCAV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e27 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eAVTO1429\u003c/p\u003e \u003cp\u003e(\u003cem\u003eTy-2\u003c/em\u003e/\u003cem\u003eTy-2, Ty-3\u003c/em\u003e/\u003cem\u003eTy-3, Ty-6/Ty-6\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePepYLCIV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTYLCKaV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e27 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePepYLCAV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eAVTO1954\u003c/p\u003e \u003cp\u003e(\u003cem\u003eTy-2\u003c/em\u003e/\u003cem\u003eTy-2, Ty-3\u003c/em\u003e/\u003cem\u003eTy-3, Ty-6/Ty-6\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePepYLCIV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTYLCKaV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePepYLCAV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eAVTO1315\u003c/p\u003e \u003cp\u003e(\u003cem\u003eTy-2\u003c/em\u003e/\u003cem\u003eTy-2, Ty-3\u003c/em\u003e/\u003cem\u003eTy-3, Ty-6/Ty-6\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePepYLCIV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTYLCKaV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e26 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePepYLCAV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eAVTO1701\u003c/p\u003e \u003cp\u003e(\u003cem\u003eTy-2\u003c/em\u003e/\u003cem\u003eTy-2, Ty-3\u003c/em\u003e/\u003cem\u003eTy-3, ty-5/ty-5, Ty-6/Ty-6\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePepYLCIV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTYLCKaV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePepYLCAV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003e\u003csup\u003ez\u003c/sup\u003e Virus detected by PCR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003e\u003csup\u003ey\u003c/sup\u003e (Number of symptomatic plants / Number of infected plants) \u0026times; 100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003ex\u003c/sup\u003e Symptom severity on each plant was scored using a disease severity index (DSI): 0, no symptoms; 1, slight leaf yellowing symptoms; 2, mild leaf yellowing symptoms; 3, moderate leaf yellowing and curing symptoms; and 4, severe leaf yellowing and curing symptoms.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn tomato lines that has been tested in previous studies, TYLCKaV and PepYLCAV were more virulent than PepYLCIV (Koeda et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Pohan et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In response to the less-virulent PepYLCIV, severe leaf yellowing, curling, and stunting (DSI\u0026thinsp;=\u0026thinsp;4) were observed only on M and MS plants. AVTO 1314 showed mild symptoms (DSI\u0026thinsp;=\u0026thinsp;1), while all other lines remained asymptomatic. When challenged with the more-virulent TYLCKaV, M and MS had severe symptoms (DSI\u0026thinsp;=\u0026thinsp;4), MP and AVTO 1314 had moderate symptoms (DSI\u0026thinsp;=\u0026thinsp;3), and AVTO 1429, 1954, and 1315 had mild symptoms (DSI\u0026thinsp;=\u0026thinsp;2). AVTO 1920 exhibited slight symptoms (DSI\u0026thinsp;=\u0026thinsp;1), while AVTO 1919 and 1701 were asymptomatic. When infected by the most virulent PepYLCAV, M and MS had severe symptoms (DSI\u0026thinsp;=\u0026thinsp;4), MP, AVTO 1954, and 1315 had moderate symptoms (DSI\u0026thinsp;=\u0026thinsp;3), and AVTO 1314 and AVTO 1429 had mild symptoms (DSI\u0026thinsp;=\u0026thinsp;2). AVTO 1919, 1920, and 1701 remained symptomless. Overall, AVTO 1919 (\u003cem\u003eTy-1/Ty-1\u003c/em\u003e, \u003cem\u003eTy-6/Ty-6\u003c/em\u003e), 1920 (\u003cem\u003eTy-3/Ty-3\u003c/em\u003e, \u003cem\u003eTy-6/Ty-6\u003c/em\u003e), and 1701 (\u003cem\u003eTy-2/Ty-2\u003c/em\u003e, \u003cem\u003eTy-3/Ty-3\u003c/em\u003e, \u003cem\u003ety-5/ty-5\u003c/em\u003e, \u003cem\u003eTy-6/Ty-6\u003c/em\u003e) exhibited the strongest TYLCD tolerance.\u003c/p\u003e \u003cp\u003eTo assess the association between symptom expression and accumulation of viral DNA, we quantified viral DNAs using qPCR and DNA extracted from young upper leaves collected at 42 dpi. Viral DNA levels were significantly higher in susceptible M and \u003cem\u003eTy-2\u003c/em\u003e-bearing MS plants. In contrast, PepYLCIV DNA levels were markedly lower in other tomato lines harboring \u003cem\u003eTy\u003c/em\u003e genes. However, accumulation of the more virulent TYLCKaV and PepYLCAV was not effectively suppressed in MP, AVTO 1314, 1429, 1954, and 1315. In contrast, AVTO 1919, 1920, and 1701 had significantly lower level of TYLCKaV and PepYLCAV DNA compared with other lines. A strong positive correlation was found between symptom severity and viral DNA accumulation (\u003cem\u003er\u003c/em\u003e\u0026thinsp;\u0026ge;\u0026thinsp;0.84). AVTO 1919 (\u003cem\u003eTy-1/Ty-1, Ty-6/Ty-6\u003c/em\u003e), 1920 (\u003cem\u003eTy-3/Ty-3, Ty-6/Ty-6\u003c/em\u003e), and 1701 (\u003cem\u003eTy-3/Ty-3, Ty-2/Ty-2, ty-5/ty-5, Ty-6/Ty-6\u003c/em\u003e) had resistance to all three viruses, effectively suppressing viral DNA accumulation. These \u003cem\u003eTy\u003c/em\u003e gene combinations therefore appear to confer broad-spectrum and stable resistance against begomoviruses.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eInheritance of begomovirus resistance in F generation\u003c/h3\u003e\n\u003cp\u003ePreliminary assays indicated that the infection rate and virulence was high for PepYLCAV after agroinoculation (Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e), so we used it for subsequent experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). AVTO 1919 and 1920 were crossed with begomovirus susceptible MM in both forward and reciprocal directions to generate F₁ populations, which were then agroinoculated with PepYLCAV to evaluate resistance. In MM, all plants exhibited severe symptoms\u0026mdash;leaf yellowing, curling, and stunting\u0026mdash;and had a DSI score of 4 at 28 dpi. In contrast, 1919 and 1920 remained symptomless, confirming their TYLCD tolerance. The F₁ progenies of MM\u0026times;1919 (mean DSI\u0026thinsp;=\u0026thinsp;1.4), MM\u0026times;1920 (mean DSI\u0026thinsp;=\u0026thinsp;1.5), 1919\u0026times;MM (mean DSI\u0026thinsp;=\u0026thinsp;1.6), and 1920\u0026times;MM (mean DSI\u0026thinsp;=\u0026thinsp;1.3) exhibited mild symptoms.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe qPCR showed that viral DNA accumulation in the resistant AVTO 1919 and 1920 was reduced to approximately 1/9\u0026ndash;1/12 the level in susceptible MM, which had severe symptoms. In contrast, the mildly symptomatic F₁ hybrids (MM\u0026times;1919, 1919\u0026times;MM, 1920\u0026times;MM) accumulated viral DNA at comparable levels to those in MM. Only MM\u0026times;1920 F₁ had moderate reduction in viral DNA, to about 1/4 of the MM level, yet the level was still higher than in the resistant parental lines. These findings indicate that heterozygosity for \u003cem\u003eTy-1\u003c/em\u003e or \u003cem\u003eTy-3\u003c/em\u003e and \u003cem\u003eTy-6\u003c/em\u003e does not provide resistance like homozygosity but confer partial TYLCD tolerance.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMany studies have evaluated the effects of \u003cem\u003eTy\u003c/em\u003e-gene pyramiding on begomovirus resistance (Elbaz et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Hanson et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Hutton et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Kenyon et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mejia et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Prabhandakavi et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Prasanna et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2015a\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003eb\u003c/span\u003e; Tabein et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Vidavski et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In particular, most of these studies relied on \u003cem\u003eTy\u003c/em\u003e-gene-linked markers rather than in-gene markers because some of the studies were done before the resistance genes were cloned. At the outset of this study, we found that AVTO 1919 and 1920 exhibited strong resistance to begomoviruses, despite being reported by the World Vegetable Center to carry only \u003cem\u003eTy-1/Ty-3\u003c/em\u003e. Genotyping with in-gene markers confirmed the presence of \u003cem\u003eTy-1\u003c/em\u003e and \u003cem\u003eTy-3\u003c/em\u003e, respectively, but \u003cem\u003eTy-2\u003c/em\u003e and \u003cem\u003ety-5\u003c/em\u003e were not detected, suggesting that these lines contained an additional, unidentified \u003cem\u003eTy\u003c/em\u003e gene. Most recently, after significant effort, Shen et al. (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) cloned \u003cem\u003eTy-6\u003c/em\u003e as POLD1. Subsequent analysis using the in-gene marker for \u003cem\u003eTy-6\u003c/em\u003e revealed that most AVTO lines, including AVTO 1919 and 1920, indeed harbor \u003cem\u003eTy-6\u003c/em\u003e. We therefore emphasize the importance of using in-gene markers to confirm the presence of \u003cem\u003eTy\u003c/em\u003e genes and thus determine their true effects on begomovirus resistance and develop effective breeding strategies.\u003c/p\u003e \u003cp\u003eComparison among the AVTO lines revealed that combining \u003cem\u003eTy-2\u003c/em\u003e with \u003cem\u003eTy-1/Ty-3\u003c/em\u003e and \u003cem\u003eTy-6\u003c/em\u003e did not enhance resistance; instead, it appeared to have a negative effect, reducing resistance to begomoviruses. It has been reported that recombination in the genomic region containing the \u003cem\u003eTy-2\u003c/em\u003e gene is severely suppressed when introgressed from the wild tomato relative \u003cem\u003eSolanum habrochaites\u003c/em\u003e into cultivated tomato (\u003cem\u003eS. lycopersicum\u003c/em\u003e) (Yamaguchi et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Under our assay conditions, \u003cem\u003eTy-2\u003c/em\u003e itself, or a neighboring gene, appeared to negatively affect begomovirus resistance when combined with \u003cem\u003eTy-3\u003c/em\u003e and/or \u003cem\u003eTy-6\u003c/em\u003e. In addition, although \u003cem\u003eTy-2\u003c/em\u003e-mediated resistance is fully effective against the Israel strain of TYLCV, it is largely ineffective against most other monopartite and bipartite begomoviruses (Barbieri et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Hanson et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Ohnishi et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Prasanna et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2015b\u003c/span\u003e; Shahid et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Tsai et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Therefore, the utility of \u003cem\u003eTy-2\u003c/em\u003e in pyramiding strategies, particularly in regions where these viruses predominate, remains questionable. Another possibility is that AVTO 1919 and 1920 carry the \u003cem\u003eTy-4\u003c/em\u003e gene, whereas 1429 and 1315 do not. Cloning \u003cem\u003eTy-4\u003c/em\u003e is necessary to evaluate this hypothesis.\u003c/p\u003e \u003cp\u003eBecause a tomato line carrying only \u003cem\u003eTy-6\u003c/em\u003e was not available in this study, we could not directly evaluate the resistance conferred solely by \u003cem\u003eTy-6\u003c/em\u003e against the tested begomoviruses. However, previous work has shown that \u003cem\u003eTy-6\u003c/em\u003e provides stronger resistance when combined with other \u003cem\u003eTy\u003c/em\u003e genes such as \u003cem\u003eTy-3\u003c/em\u003e and \u003cem\u003ety-5\u003c/em\u003e, which is consistent with our current findings (Gill et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Our results suggest that combining \u003cem\u003eTy-1\u003c/em\u003e or \u003cem\u003eTy-3\u003c/em\u003e with \u003cem\u003eTy-6\u003c/em\u003e in the homozygous state may represent the minimal number of resistance genes required for effective protection against TYLCD. To further validate this hypothesis, \u003cem\u003eTy-1/Ty-3\u003c/em\u003e and \u003cem\u003eTy-6\u003c/em\u003e should be introgressed into the same genetic background in various combinations.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 19H02950, 21KK0109, 23K26900 to S. Koeda.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMS and SK designed the experiments; analyzed the data; interpreted the results and wrote the manuscript. MS, MN, and NSP performed the experiments. NSP conducted the statistical analyses. EK prepared the virus material. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank Takii for providing MS seeds and preparing crossing populations. We thank World Vegetable Center for providing seeds of AVTO lines. We thank Shinya Kanzaki (Kindai University, Japan) for useful discussion. We thank Beth E. Hazen for editing a draft of the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAgrama HA, Scott JW (2006) Quantitative trait loci for \u003cem\u003eTomato yellow leaf curl virus\u003c/em\u003e and \u003cem\u003eTomato mottle virus\u003c/em\u003e resistance in tomato. 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Theor Appl Genet 131:1345\u0026ndash;1362\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang X, Caro M, Hutton SF, Scott JW, Guo Y, Wang X, Rashid MH, Szinay D, de Jong H, Visser RGF, Bai Y, Du Y (2014) Fine mapping of the tomato yellow leaf curl virus resistance gene \u003cem\u003eTy-2\u003c/em\u003e on chromosome 11 of tomato. Mol Breed 34:749\u0026ndash;760\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZamir D, Eksteinmichelson I, Zakay Y, Navot N, Zeidan M, Sarfatti M, Eshed Y, Harel E, Pleban T, Vanoss H, Kedar N, Rabinowitch HD, Czosnek H (1994) Mapping and introgression of a tomato yellow leaf curl virus tolerance gene, \u003cem\u003eTy-1\u003c/em\u003e. Theor Appl Genet 88:141\u0026ndash;146\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"euphytica","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"euph","sideBox":"Learn more about [Euphytica](https://www.springer.com/journal/10681)","snPcode":"10681","submissionUrl":"https://submission.springernature.com/new-submission/10681/3","title":"Euphytica","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Begomovirus, DNA marker, Geminivirus, Pyramiding resistance genes, Resistance breeding, Tomato yellow leaf curl disease","lastPublishedDoi":"10.21203/rs.3.rs-8207444/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8207444/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBegomoviruses are among the most destructive pathogens of tomato worldwide, and the introgression of \u003cem\u003eTy\u003c/em\u003e-genes in tomato is a key strategy for disease management. However, a single \u003cem\u003eTy\u003c/em\u003e-gene often provides incomplete protection against highly virulent begomovirus species. Here, commercial tomato cultivars and breeding lines were inoculated with one of three begomovirus species that differed in virulence. The presence of \u003cem\u003eTy-1\u003c/em\u003e, \u003cem\u003eTy-2\u003c/em\u003e, \u003cem\u003eTy-3\u003c/em\u003e, \u003cem\u003eTy-3a\u003c/em\u003e, \u003cem\u003ety-5\u003c/em\u003e, and \u003cem\u003eTy-6\u003c/em\u003e was validated using in-gene markers. Tomato plants carrying only \u003cem\u003eTy-2\u003c/em\u003e were susceptible to all viruses, whereas those with \u003cem\u003eTy-1\u003c/em\u003e were resistant to a less-virulent begomovirus, but susceptible to more-virulent viruses. Among the seven AVTO breeding lines, 1919 (\u003cem\u003eTy-1\u003c/em\u003e/\u003cem\u003eTy-1\u003c/em\u003e, \u003cem\u003eTy-6\u003c/em\u003e/\u003cem\u003eTy-6\u003c/em\u003e) and 1920 (\u003cem\u003eTy-3\u003c/em\u003e/\u003cem\u003eTy-3\u003c/em\u003e, \u003cem\u003eTy-6\u003c/em\u003e/\u003cem\u003eTy-6\u003c/em\u003e) were symptomless in most cases and the lowest viral DNA accumulation across all lines tested indicating strong begomovirus resistance. The resistance of 1919 was comparable to or greater than that of 1701, a line homozygous for \u003cem\u003eTy-2\u003c/em\u003e, \u003cem\u003eTy-3\u003c/em\u003e, \u003cem\u003ety-5\u003c/em\u003e, and \u003cem\u003eTy-6\u003c/em\u003e. Furthermore, F₁ plants derived from crosses between 1919 or 1920 and susceptible Moneymaker exhibited reduced disease tolerance, indicating the importance of homozygosity for \u003cem\u003eTy-1\u003c/em\u003e or \u003cem\u003eTy-3\u003c/em\u003e combined with \u003cem\u003eTy-6\u003c/em\u003e. These results demonstrate that \u003cem\u003eTy-1/Ty-3\u003c/em\u003e and \u003cem\u003eTy-6\u003c/em\u003e integration in homozygous states can provide robust and broad-spectrum resistance to begomoviruses and is a valuable strategy for breeding durable resistant tomato cultivars.\u003c/p\u003e","manuscriptTitle":"Integration of Ty-1/Ty-3 and Ty-6 confers improved and durable resistance to highly pathogenic begomoviruses in tomato","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-13 12:26:14","doi":"10.21203/rs.3.rs-8207444/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-04T09:54:18+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-03T14:28:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-18T08:45:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"291678284405842274240434459872511252576","date":"2026-01-28T07:53:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-28T00:59:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"315979164714895888219787348597534315925","date":"2026-01-28T00:35:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"126504512835078728059959757740842239578","date":"2026-01-26T14:41:03+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-26T14:36:34+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-23T10:06:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"Euphytica","date":"2026-01-22T08:52:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"euphytica","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"euph","sideBox":"Learn more about [Euphytica](https://www.springer.com/journal/10681)","snPcode":"10681","submissionUrl":"https://submission.springernature.com/new-submission/10681/3","title":"Euphytica","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5ab0d15a-f861-44bd-90c6-6b6672609fba","owner":[],"postedDate":"February 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-07T16:10:38+00:00","versionOfRecord":{"articleIdentity":"rs-8207444","link":"https://doi.org/10.1007/s10681-026-03711-y","journal":{"identity":"euphytica","isVorOnly":false,"title":"Euphytica"},"publishedOn":"2026-03-30 15:59:02","publishedOnDateReadable":"March 30th, 2026"},"versionCreatedAt":"2026-02-13 12:26:14","video":"","vorDoi":"10.1007/s10681-026-03711-y","vorDoiUrl":"https://doi.org/10.1007/s10681-026-03711-y","workflowStages":[]},"version":"v1","identity":"rs-8207444","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8207444","identity":"rs-8207444","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}