Arunabha Mitra, Sridhar Jarugula, Rayapeti A Naidu
{"title":"葡萄叶卷相关病毒1微型复制子的研制及复制所需5'非翻译区序列的遗传分析。","authors":"Arunabha Mitra, Sridhar Jarugula, Rayapeti A Naidu","doi":"10.1094/PHYTO-12-24-0380-R","DOIUrl":null,"url":null,"abstract":"<p><p>Grapevine leafroll-associated virus 1 (GLRaV-1, genus <i>Ampelovirus</i>, family <i>Closteroviridae</i>) has a monopartite RNA genome with size varying among genetic variants between 18,731 and 18,946 nucleotides (nt) and a 5' non-translated region (5'-NTR), varying in length between 857 and 922 nt. This study was undertaken to examine the role of the 5'-NTR in GLRaV-1 replication. For this purpose, a minireplicon cDNA clone, consisting of the 5'-NTR, the replicase gene module, the green fluorescent protein (GFP) reporter gene, and the 3'-NTR, was constructed. Functionality of the minireplicon was validated by GFP fluorescence and the presence of GFP-specific mRNA transcripts by Northern blot hybridization and RT-qPCR assays in <i>Nicotiana benthamiana</i> leaves agro-coinfiltrated with silencing suppressors. The minireplicon retained functionality when its 5'-NTR was exchanged with corresponding sequences from distinct genetic variants of GLRaV-1. In contrast, the minireplicon of GLRaV-1 was non-functional when its 5'-NTR sequence was swapped with corresponding sequences from other GLRaV species. Deletion mutations in the 5'-NTR indicated that the first 32 nt at the 5'-terminus of the genome are essential for replication of the minireplicon. <i>In silico</i>-predicted secondary structure of the 5'-terminal 32-nt sequence showed two stem-loop structures and mutations that altered these secondary structures or compensatory mutations preserving the secondary structures failed to retain functionality of the minireplicon, suggesting that the nucleotide sequence, rather than any higher order secondary structures in this genomic region, is important for replication.</p>","PeriodicalId":20410,"journal":{"name":"Phytopathology","volume":" ","pages":""},"PeriodicalIF":3.1000,"publicationDate":"2025-05-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Development of a Minireplicon for <i>Grapevine leafroll-associated virus 1</i> and Genetic Analyses of Sequences in the 5' Non-Translated Region Required for Replication.\",\"authors\":\"Arunabha Mitra, Sridhar Jarugula, Rayapeti A Naidu\",\"doi\":\"10.1094/PHYTO-12-24-0380-R\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Grapevine leafroll-associated virus 1 (GLRaV-1, genus <i>Ampelovirus</i>, family <i>Closteroviridae</i>) has a monopartite RNA genome with size varying among genetic variants between 18,731 and 18,946 nucleotides (nt) and a 5' non-translated region (5'-NTR), varying in length between 857 and 922 nt. This study was undertaken to examine the role of the 5'-NTR in GLRaV-1 replication. For this purpose, a minireplicon cDNA clone, consisting of the 5'-NTR, the replicase gene module, the green fluorescent protein (GFP) reporter gene, and the 3'-NTR, was constructed. Functionality of the minireplicon was validated by GFP fluorescence and the presence of GFP-specific mRNA transcripts by Northern blot hybridization and RT-qPCR assays in <i>Nicotiana benthamiana</i> leaves agro-coinfiltrated with silencing suppressors. The minireplicon retained functionality when its 5'-NTR was exchanged with corresponding sequences from distinct genetic variants of GLRaV-1. In contrast, the minireplicon of GLRaV-1 was non-functional when its 5'-NTR sequence was swapped with corresponding sequences from other GLRaV species. Deletion mutations in the 5'-NTR indicated that the first 32 nt at the 5'-terminus of the genome are essential for replication of the minireplicon. <i>In silico</i>-predicted secondary structure of the 5'-terminal 32-nt sequence showed two stem-loop structures and mutations that altered these secondary structures or compensatory mutations preserving the secondary structures failed to retain functionality of the minireplicon, suggesting that the nucleotide sequence, rather than any higher order secondary structures in this genomic region, is important for replication.</p>\",\"PeriodicalId\":20410,\"journal\":{\"name\":\"Phytopathology\",\"volume\":\" \",\"pages\":\"\"},\"PeriodicalIF\":3.1000,\"publicationDate\":\"2025-05-19\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Phytopathology\",\"FirstCategoryId\":\"97\",\"ListUrlMain\":\"https://doi.org/10.1094/PHYTO-12-24-0380-R\",\"RegionNum\":2,\"RegionCategory\":\"农林科学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"PLANT SCIENCES\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Phytopathology","FirstCategoryId":"97","ListUrlMain":"https://doi.org/10.1094/PHYTO-12-24-0380-R","RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"PLANT SCIENCES","Score":null,"Total":0}
Development of a Minireplicon for Grapevine leafroll-associated virus 1 and Genetic Analyses of Sequences in the 5' Non-Translated Region Required for Replication.
Grapevine leafroll-associated virus 1 (GLRaV-1, genus Ampelovirus, family Closteroviridae) has a monopartite RNA genome with size varying among genetic variants between 18,731 and 18,946 nucleotides (nt) and a 5' non-translated region (5'-NTR), varying in length between 857 and 922 nt. This study was undertaken to examine the role of the 5'-NTR in GLRaV-1 replication. For this purpose, a minireplicon cDNA clone, consisting of the 5'-NTR, the replicase gene module, the green fluorescent protein (GFP) reporter gene, and the 3'-NTR, was constructed. Functionality of the minireplicon was validated by GFP fluorescence and the presence of GFP-specific mRNA transcripts by Northern blot hybridization and RT-qPCR assays in Nicotiana benthamiana leaves agro-coinfiltrated with silencing suppressors. The minireplicon retained functionality when its 5'-NTR was exchanged with corresponding sequences from distinct genetic variants of GLRaV-1. In contrast, the minireplicon of GLRaV-1 was non-functional when its 5'-NTR sequence was swapped with corresponding sequences from other GLRaV species. Deletion mutations in the 5'-NTR indicated that the first 32 nt at the 5'-terminus of the genome are essential for replication of the minireplicon. In silico-predicted secondary structure of the 5'-terminal 32-nt sequence showed two stem-loop structures and mutations that altered these secondary structures or compensatory mutations preserving the secondary structures failed to retain functionality of the minireplicon, suggesting that the nucleotide sequence, rather than any higher order secondary structures in this genomic region, is important for replication.
期刊介绍:
Phytopathology publishes articles on fundamental research that advances understanding of the nature of plant diseases, the agents that cause them, their spread, the losses they cause, and measures that can be used to control them. Phytopathology considers manuscripts covering all aspects of plant diseases including bacteriology, host-parasite biochemistry and cell biology, biological control, disease control and pest management, description of new pathogen species description of new pathogen species, ecology and population biology, epidemiology, disease etiology, host genetics and resistance, mycology, nematology, plant stress and abiotic disorders, postharvest pathology and mycotoxins, and virology. Papers dealing mainly with taxonomy, such as descriptions of new plant pathogen taxa are acceptable if they include plant disease research results such as pathogenicity, host range, etc. Taxonomic papers that focus on classification, identification, and nomenclature below the subspecies level may also be submitted to Phytopathology.