{"title":"复制酶基因介导的 RNA 沉默机制赋予番茄(Lycopersicon esculentum Mill.)","authors":"Karthikeyan Gandhi, Suganyadevi Murugesan, Rajamanickam Suppaiah","doi":"10.1007/s10658-024-02908-y","DOIUrl":null,"url":null,"abstract":"<p>Groundnut bud necrosis virus (GBNV), a member of the genus <i>Orthotospovirus</i>, is the most devastating pathogen causing bud blight of tomato and causes substantial crop losses in India. Current management strategies rely upon the use of virus tolerant cultivars, control of insect vectors, and other cultural practices. Under field conditions, these methods are ineffective in reducing the disease. Control can be achieved with RNA silencing, which regulates the homologous specific degradation of targeted genes, resulting in reduced virus multiplication. In the present study, virus infected tomato plant samples were collected from different parts of Tamil Nadu, and infection was confirmed through DAC-ELISA using a polyclonal antibody specific to GBNV. The virus inoculum was propagated on the local lesion host, cowpea (<i>Vigna unguiculata</i>), resulting in the production of chlorotic and necrotic spots on inoculated primary leaves. Our results demonstrated that the complete nucleotide sequence of the replicase gene identified using PCR shared an identity of 94.7 to 97.7% with other isolates of GBNV. To investigate the virus suppression mechanism, an effective RNAi construct was developed with the conserved sequence of the replicase gene for GBNV. A 3674 bp hpRNA cassette, comprising the sense and antisense fragments of 357 bp along with the flanking sequence, inserted in a pHANNIBAL vector, generated transgenic tomato plants using shoot apical meristem explants through <i>Agrobacterium</i> harboring the gene construct. The presence of the transgene in the developed putative transformants was assessed by PCR analysis using <i>nptII</i> and <i>Rep</i> genes and dot blot hybridization using a DIG luminescent detection kit. The expression of the replicase hpRNA construct revealed reduced symptom development upon artificial inoculation of GBNV. Further analysis of the transgenic tomato plants using DAC-ELISA confirmed the reduced level of virus titer. We propose that the RNAi construct, established with a conserved sequence of the replicase gene, showed a gene silencing mechanism as evidenced by reduced virus accumulation in putative transgenic lines, and this could be used as an effective strategy in the management of GNBV in tomato.</p>","PeriodicalId":12052,"journal":{"name":"European Journal of Plant Pathology","volume":null,"pages":null},"PeriodicalIF":1.7000,"publicationDate":"2024-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Replicase gene mediated RNA silencing mechanism confers resistance against groundnut bud necrosis virus in tomato (Lycopersicon esculentum Mill.)\",\"authors\":\"Karthikeyan Gandhi, Suganyadevi Murugesan, Rajamanickam Suppaiah\",\"doi\":\"10.1007/s10658-024-02908-y\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>Groundnut bud necrosis virus (GBNV), a member of the genus <i>Orthotospovirus</i>, is the most devastating pathogen causing bud blight of tomato and causes substantial crop losses in India. Current management strategies rely upon the use of virus tolerant cultivars, control of insect vectors, and other cultural practices. Under field conditions, these methods are ineffective in reducing the disease. Control can be achieved with RNA silencing, which regulates the homologous specific degradation of targeted genes, resulting in reduced virus multiplication. In the present study, virus infected tomato plant samples were collected from different parts of Tamil Nadu, and infection was confirmed through DAC-ELISA using a polyclonal antibody specific to GBNV. The virus inoculum was propagated on the local lesion host, cowpea (<i>Vigna unguiculata</i>), resulting in the production of chlorotic and necrotic spots on inoculated primary leaves. Our results demonstrated that the complete nucleotide sequence of the replicase gene identified using PCR shared an identity of 94.7 to 97.7% with other isolates of GBNV. To investigate the virus suppression mechanism, an effective RNAi construct was developed with the conserved sequence of the replicase gene for GBNV. A 3674 bp hpRNA cassette, comprising the sense and antisense fragments of 357 bp along with the flanking sequence, inserted in a pHANNIBAL vector, generated transgenic tomato plants using shoot apical meristem explants through <i>Agrobacterium</i> harboring the gene construct. The presence of the transgene in the developed putative transformants was assessed by PCR analysis using <i>nptII</i> and <i>Rep</i> genes and dot blot hybridization using a DIG luminescent detection kit. The expression of the replicase hpRNA construct revealed reduced symptom development upon artificial inoculation of GBNV. Further analysis of the transgenic tomato plants using DAC-ELISA confirmed the reduced level of virus titer. We propose that the RNAi construct, established with a conserved sequence of the replicase gene, showed a gene silencing mechanism as evidenced by reduced virus accumulation in putative transgenic lines, and this could be used as an effective strategy in the management of GNBV in tomato.</p>\",\"PeriodicalId\":12052,\"journal\":{\"name\":\"European Journal of Plant Pathology\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":1.7000,\"publicationDate\":\"2024-07-11\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"European Journal of Plant Pathology\",\"FirstCategoryId\":\"97\",\"ListUrlMain\":\"https://doi.org/10.1007/s10658-024-02908-y\",\"RegionNum\":3,\"RegionCategory\":\"农林科学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"AGRONOMY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"European Journal of Plant Pathology","FirstCategoryId":"97","ListUrlMain":"https://doi.org/10.1007/s10658-024-02908-y","RegionNum":3,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"AGRONOMY","Score":null,"Total":0}
Replicase gene mediated RNA silencing mechanism confers resistance against groundnut bud necrosis virus in tomato (Lycopersicon esculentum Mill.)
Groundnut bud necrosis virus (GBNV), a member of the genus Orthotospovirus, is the most devastating pathogen causing bud blight of tomato and causes substantial crop losses in India. Current management strategies rely upon the use of virus tolerant cultivars, control of insect vectors, and other cultural practices. Under field conditions, these methods are ineffective in reducing the disease. Control can be achieved with RNA silencing, which regulates the homologous specific degradation of targeted genes, resulting in reduced virus multiplication. In the present study, virus infected tomato plant samples were collected from different parts of Tamil Nadu, and infection was confirmed through DAC-ELISA using a polyclonal antibody specific to GBNV. The virus inoculum was propagated on the local lesion host, cowpea (Vigna unguiculata), resulting in the production of chlorotic and necrotic spots on inoculated primary leaves. Our results demonstrated that the complete nucleotide sequence of the replicase gene identified using PCR shared an identity of 94.7 to 97.7% with other isolates of GBNV. To investigate the virus suppression mechanism, an effective RNAi construct was developed with the conserved sequence of the replicase gene for GBNV. A 3674 bp hpRNA cassette, comprising the sense and antisense fragments of 357 bp along with the flanking sequence, inserted in a pHANNIBAL vector, generated transgenic tomato plants using shoot apical meristem explants through Agrobacterium harboring the gene construct. The presence of the transgene in the developed putative transformants was assessed by PCR analysis using nptII and Rep genes and dot blot hybridization using a DIG luminescent detection kit. The expression of the replicase hpRNA construct revealed reduced symptom development upon artificial inoculation of GBNV. Further analysis of the transgenic tomato plants using DAC-ELISA confirmed the reduced level of virus titer. We propose that the RNAi construct, established with a conserved sequence of the replicase gene, showed a gene silencing mechanism as evidenced by reduced virus accumulation in putative transgenic lines, and this could be used as an effective strategy in the management of GNBV in tomato.
期刊介绍:
The European Journal of Plant Pathology is an international journal publishing original articles in English dealing with fundamental and applied aspects of plant pathology; considering disease in agricultural and horticultural crops, forestry, and in natural plant populations. The types of articles published are :Original Research at the molecular, physiological, whole-plant and population levels; Mini-reviews on topics which are timely and of global rather than national or regional significance; Short Communications for important research findings that can be presented in an abbreviated format; and Letters-to-the-Editor, where these raise issues related to articles previously published in the journal. Submissions relating to disease vector biology and integrated crop protection are welcome. However, routine screenings of plant protection products, varietal trials for disease resistance, and biological control agents are not published in the journal unless framed in the context of strategic approaches to disease management.