Molecular characterization of tomato spotted wilt virus (Orthotospovirus tomatomaculae) and development of resistant tomato (Solanum lycopersicum L.) lines using RNAi technology
Küçük Resim Yok
Tarih
2024
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Niğde Ömer Halisdemir Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Domates lekeli solgunluk virüsü (Orthotospovirus tomatomaculae, TSWV), özellikle domates ve biber başta olmak üzere Solanaceae familyası grubu bitkileri için önemli bir tehdit oluşturmaktadır. Dayanıklı çeşitlerin geliştirilmesi, viral hastalıkların etkisiz hale getirilmesi için önemli mücadele yöntemlerinden biridir. Bununla birlikte, dayanıklı çeşitlerin yaygın bir şekilde kullanılmasından dolayı dayanıklılık kıran virüs izolatları ortaya çıkmıştır. Bu çalışmada, 2020-2021 yıllarında Türkiye'nin çeşitli bölgelerinden simptomatik domates ve biber örnekleri toplanmış ve virüs enfeksiyonu bakımından hem DAS-ELISA hem de RT-PCR ile testlenmiştir. Domates ve biber bitkilerindeki viral dayanıklılık genlerini taramak için sequence-characterized amplified region (SCAR) ve cleaved amplified polymorphic sequence (CAPS) belirteçleri kullanılmıştır. Bazı çeşitlerin şiddetli viral enfeksiyonlar göstermelerine rağmen dayanıklılık genleri taşıdıkları tespit edilmiştir. Bu bitkilerden ekstrakte edilen TSWV izolatları, dayanıklılık kıran izolat olup olmadıklarını testlemek için susturucu baskılayıcı geni (silencing suppressor gene, NSs) ve hareketlilik proteini (movement protein, NSm) genlerinde rapor edilen mutasyonların tespiti için kısmen dizilenmiştir. DNA dizi karşılaştırmaları ve filogenetik analizler, virüsün NSs ve NSm genomik bölgelerinin bildirilen mutasyonları içermediğini ve aynı genetik havuzda toplandığını, N genine ait Türk izolatlarının ise ayrı bir grupta kümelendiğini göstermiştir. Ayrıca bu çalışmada, orthotospovirüslere karşı dayanıklılık kazandırmak amacı ile NSs genini hedefleyen bitki aracılı RNAi yaklaşımının kullanımı da araştırılmıştır. İntegral 35S promotör bölgesinin 320 bp'lik korunmuş ters bir bölgesini ifade eden transgenik domates (Solanum lycopersicum cv. Rio Grande) hatları geliştirilmiştir. Bitki ekspresyon vektörü pFGC5941'de hem sense hem de antisense oryantasyon içeren Orthotospovirus NSs geni ile bir saç tokası (hairpin) yapısında ekspresyon vektörlerinin oluşturulması için moleküler klonlama yapılmıştır. Transformasyon çalışmaları için doku kültürü deneylerinde 3 farklı eksplant türü (hipokotil, kotiledon node ve kotiledon) kullanılmıştır. Viral yapılara (TSWV-NSs_tüm (pFGC5941+sense+antisense), TSWV-NSs_sense) sahip Agrobacterium EHA-105 suşu kullanılarak enfeksiyonlar yapılmıştır. Transgenik bitkileri doğrulamak için TSWV genine özgü primerler kullanılarak moleküler analizler yapılmıştır. Agrobacterium EHA105 suşu ve T-DNA bölgesinde NSs ve nptII genlerini taşıyan pFGC5941 vektörüne ait sonuçlar, rejenere transgenik bitkilere kanamisin dayanıklılığının eklendiğini doğrulamaktadır. Ayrıca, doğrulama için transgenik bitkiler TSWV ile inokule edilmiş ve kantitatif gerçek zamanlı polimeraz zincir reaksiyonu (qRT-PCR) deneyleri gerçekleştirilmiştir. TSWV-NSs_tüm yapısını barındıran transgenik bitkilerde patojenite ile ilişkili (PR-1) genlerin ve NSs geninin ifade seviyelerinde kontrol gruplarına (transgenik olmayan kontrol, boş vektör ve TSWV-NSs_sense) kıyasla önemli bir azalma olduğunu göstermektedir. RNAi etkinliğinin doğrulanması için, tütün bitkileri de viral yapılarla inokule edilmiş ve , boş vektör içeren bitkilerde ve transgenik olmayan domates bitkilerinde TSWV'nin şiddetli simptomları görülürken TSWV-NSs_tüm yapılarını içeren bitkilerde herhangi bir simptom gözlenmemiştir, bu da RNAi'nin etkinliğini göstererek bu çalışmanın hipotezini doğrulamıştır. Bu araştırma, domateslerde uzun süreli antiviral koruma elde etmek için güvenilir bir teknik olarak NSs genini hedefleyerek RNA interferans etkinliğini göstermektedir. Bulgular pratik öneme sahiptir ve dünya çapında TSWV'nin domates ürünlerinde neden olduğu kayıpları kontrol etmek veya azaltmak için bir yöntem olma potansiyeline sahiptir.
Tomato spotted wilt virus (Orthotospovirus tomatomaculae, TSWV) poses a devastating threat to Solanaceous crops specially tomato and pepper. The development of resistant cultivars is one of the important management tools for incapacitating viral diseases. However, due to the widespread utilization of resistance cultivars, the resistant breaking virus isolates have emerged. In this study, symptomatic tomato and pepper samples were collected from diverse locations of Türkiye in 2020-2021, and incidence of virus was tested by both DAS-ELISA and RT-PCR. The sequence-characterized amplified region (SCAR) and cleaved amplified polymorphic sequence (CAPS) markers were used to screen viral resistance genes in tomato and pepper plants. Some of the cultivars were found to carry resistance genes even though they displayed severe viral infections. The extracted TSWV isolates from these plants were partially sequenced for the detection of reported mutations in the silencing suppressor gene (NSs) and movement protein (NSm) genes to test whether they were resistance-breaking (RB) isolates. Sequence comparisons and phylogenetic analyses revealed that the NSs and NSm genomic regions of virus did not show reported mutations and they were distributed in the same genetic pool while Turkish isolates of N gene were clustered in a separate clade. Moreover, in this study the prospective use of plant-mediated RNAi approach by targeting the NSs gene for the conferring resistance to orthotospoviruses was investigated. Transgenic tomato (Solanum lycopersicum cv. Rio Grande) lines expressing a 320 bp conserved inverted region of the integral 35S promoter region were developed. The molecular cloning was done for the construction of expression vectors in a hairpin structure by the Orthotospovirus NSs gene which contains both sense and antisense orientation in plant expression vector pFGC5941. For the transformation studies, 3 different types of explants (hypocotyls, cotyledonary nodes, and cotyledons) were used in tissue culture experiments. Co-infection was done by using Agrobacterium strain EHA-105 having viral constructs (TSWV-NSs_full (pFGC5941+sense+antisense), TSWV-NSs_sense). To confirm the transgenic plants, molecular analysis was performed using TSWV gene-specific primers. Based on the EHA105 Agrobacterium strain and the pFGC5941 vector carrying NSs and nptII genes in the T-DNA region, the results validate the insertion of kanamycin resistance into the regenerated transgenic plants. Moreover, for the confirmation, transgenic plants were inoculated with TSWV and quantitative real-time polymerase chain reaction (qRT-PCR) assays were performed which demonstrates a significant down-regulation in the gene expression levels of pathogenicity-related genes (PR-1) and NSs genes in transgenic plants harboring the TSWV-NSs_full constructs compared to control groups (non-transgenic wild type control, empty vector and the TSWV-NSs_sense constructs). For the validation of RNAi effectiveness, tobacco plants were also inoculated with viral constructs and they displayed severe symptoms of TSWV in plants containing the TSWV-NSs_sense constructs, empty vector, and in wild-type tomato plants while no any symptoms were observed with TSWV-NSs_full constructs that confirmed the hypothesis of this study by showing the efficacy of RNAi. This research demonstrates the RNA interference effectiveness by targeting NSs gene as a reliable technique for achieving long lasting antiviral protection in tomatoes. The findings have practical significance and the potential to be a tool for controlling or mitigating the losses in tomato crops caused by TSWV around the world.
Tomato spotted wilt virus (Orthotospovirus tomatomaculae, TSWV) poses a devastating threat to Solanaceous crops specially tomato and pepper. The development of resistant cultivars is one of the important management tools for incapacitating viral diseases. However, due to the widespread utilization of resistance cultivars, the resistant breaking virus isolates have emerged. In this study, symptomatic tomato and pepper samples were collected from diverse locations of Türkiye in 2020-2021, and incidence of virus was tested by both DAS-ELISA and RT-PCR. The sequence-characterized amplified region (SCAR) and cleaved amplified polymorphic sequence (CAPS) markers were used to screen viral resistance genes in tomato and pepper plants. Some of the cultivars were found to carry resistance genes even though they displayed severe viral infections. The extracted TSWV isolates from these plants were partially sequenced for the detection of reported mutations in the silencing suppressor gene (NSs) and movement protein (NSm) genes to test whether they were resistance-breaking (RB) isolates. Sequence comparisons and phylogenetic analyses revealed that the NSs and NSm genomic regions of virus did not show reported mutations and they were distributed in the same genetic pool while Turkish isolates of N gene were clustered in a separate clade. Moreover, in this study the prospective use of plant-mediated RNAi approach by targeting the NSs gene for the conferring resistance to orthotospoviruses was investigated. Transgenic tomato (Solanum lycopersicum cv. Rio Grande) lines expressing a 320 bp conserved inverted region of the integral 35S promoter region were developed. The molecular cloning was done for the construction of expression vectors in a hairpin structure by the Orthotospovirus NSs gene which contains both sense and antisense orientation in plant expression vector pFGC5941. For the transformation studies, 3 different types of explants (hypocotyls, cotyledonary nodes, and cotyledons) were used in tissue culture experiments. Co-infection was done by using Agrobacterium strain EHA-105 having viral constructs (TSWV-NSs_full (pFGC5941+sense+antisense), TSWV-NSs_sense). To confirm the transgenic plants, molecular analysis was performed using TSWV gene-specific primers. Based on the EHA105 Agrobacterium strain and the pFGC5941 vector carrying NSs and nptII genes in the T-DNA region, the results validate the insertion of kanamycin resistance into the regenerated transgenic plants. Moreover, for the confirmation, transgenic plants were inoculated with TSWV and quantitative real-time polymerase chain reaction (qRT-PCR) assays were performed which demonstrates a significant down-regulation in the gene expression levels of pathogenicity-related genes (PR-1) and NSs genes in transgenic plants harboring the TSWV-NSs_full constructs compared to control groups (non-transgenic wild type control, empty vector and the TSWV-NSs_sense constructs). For the validation of RNAi effectiveness, tobacco plants were also inoculated with viral constructs and they displayed severe symptoms of TSWV in plants containing the TSWV-NSs_sense constructs, empty vector, and in wild-type tomato plants while no any symptoms were observed with TSWV-NSs_full constructs that confirmed the hypothesis of this study by showing the efficacy of RNAi. This research demonstrates the RNA interference effectiveness by targeting NSs gene as a reliable technique for achieving long lasting antiviral protection in tomatoes. The findings have practical significance and the potential to be a tool for controlling or mitigating the losses in tomato crops caused by TSWV around the world.
Açıklama
Fen Bilimleri Enstitüsü, Bitkisel Üretim ve Teknolojileri Ana Bilim Dalı
Anahtar Kelimeler
Ziraat, Agriculture
