Grapevine leafroll-associated virus 3 (GLRaV-3), the main viral species of the grapevine leafroll complex, causes yield and quality reduction in grapes (Vitis spp.). The coat protein gene was RT-PCR-amplified from total RNA extracted from infected grapevine leaves and the amplified fragment was cloned and completely sequenced. The fragment was subsequently subcloned into the pRSET-C expression vector. The recombinant plasmid was used to transform Escherichia coli BL21:DE3 and express the capsid protein. The coat protein, fused to a 6 His-tag, was purified by affinity chromatography using an Ni-NTA resin. The identity of the purified protein was confirmed by SDS-PAGE and Western blot. The in vitro-expressed protein was quantified and used for rabbit immunizations. The antiserum was shown to be sensitive and specific for the detection of GLRaV-3 in grapevine extracts in Western blot and DAS-ELISA assays, with no unspecific or heterologous reactions against other non-serologically related viruses being observed.Additional keywords: grapevine, GLRaV-3, recombinant protein, serology, antibodies. RESUMO Expressão da proteína capsidial do Grapevine leafroll-associated virus 3 em Escherichia coli e produção de anticorpos policlonaisGrapevine leafroll-associated virus 3 (GLRaV-3), a principal espécie viral do complexo do enrolamento da folha da videira (Vitis spp.), causa reduções no rendimento e na qualidade da uva. O gene da proteína capsidial foi amplificado via RT-PCR a partir de RNA total, extraído de folhas de videira infectadas. O fragmento amplificado foi clonado e completamente seqüenciado. Em seguida, o fragmento foi subclonado no vetor de expressão pRSET-C. O plasmídeo recombinante foi utilizado para a expressão da proteína capsidial em Escherichia coli BL21:DE3. A proteína capsidial, ligada a uma cauda de 6-His, foi purificada por cromatografia de afinidade em coluna de Ni-NTA. A identidade da proteína purificada foi confirmada em SDS-PAGE e Western blot e, após quantificação, foi utilizada para imunizar coelhos. O anti-soro mostrou-se sensível e específico para a detecção do GLRaV-3 em extratos de videira por Western blot e DAS-ELISA, não tendo sido observadas reações inespecíficas ou heterólogas contra outros vírus sorologicamente não relacionados.Palavras-chave adicionais: videira, GLRaV-3, proteína recombinante, sorologia, anticorpos.
The application of high-throughput sequencing technologies (HTS) enables the recovery of many nucleotide sequence fragments from diseased plants and may help in pathogen identification. This study was designed to identify viruses infecting 15 grapevine (Vitis spp.) samples collected from experimental fields and vine collections and assess the genetic variability of the identified viruses. The virus-enriched dsRNAs were extracted from bark scrapings and sequenced using an Illumina platform. The paired-end reads were analyzed, assembled contigs were generated and identified as related to viruses. Contigs of 14 viruses have been identified, some of them covering large extensions of viral genomes or resulting in assembly of near-complete or complete genomes. Grapevine virus infections are usually mixed and the HTS assays were suitable to identify ten viruses already reported that traditionally infect grapevines in Brazil, one that has been recently identified (Grapevine Syrah virus 1) and others (Grapevine Cabernet Sauvignon reovirus, Grapevine Red Globe virus and Grapevine vein clearing virus) not previously reported in this country. Nucleotide identities among Brazilian isolates identified by HTS and homologous grapevine virus sequences in GenBank were high, ranging from 77% to 99%. Genetic variability analysis of viral sequences obtained by HTS and sequences available in GenBank indicated that the coding regions in the different viral species are under purifying selection, and that recombination events occurred in the majority of the viral species analyzed. The coat protein genes, generally, had lower genetic variability than the replicase and movement protein genes.
Presently, Hop stunt viroid (HSVd) and Citrus exocortis viroid (CEVd) are the only viroids reported to infect grapevines (Vitis spp.) in Brazil, among the seven viroid species already reported infecting this host in other countries. All grapevine viroid diseases are graft-transmissible and can induce losses especially when associated with viruses. The aim of this work was to confirm infection by Grapevine yellow speckle viroid 1 (GYSVd-1) in grapevine samples exhibiting yellow speckle symptoms in the leaves and in asymptomatic samples sequenced by next generation sequencing (NGS). The occurrence of this viroid in Brazil was further investigated in a second study. Total RNAs and dsRNAs were extracted from five symptomatic plants and 16 asymptomatic samples, respectively. Specific primers were used for RT-PCR and amplified DNA fragments were cloned and sequenced by the Sanger method. Eleven complete nucleotide sequences of GYSVd-1 isolates (366-367 nt) were obtained from NGS and from RT-PCR amplicons. Comparisons showed high identities (95.9-100 %) among ten isolates and an identity of 87.2-90.4 % with a divergent isolate (RM-BR). Phylogenetic analyses placed GYSVd-1 isolates in four clusters (types 1, 2, 3 and 4). All GYSVd-1 infections were confirmed by conventional RT-PCR and RT-qPCR using specific oligonucleotides and a labeled probe. This is the first report and molecular characterization of GYSVd-1 infecting grapevines in Brazil, and our survey indicates that this viroid could be widespread in the major grape producing regions of Brazil.
Apple stem grooving virus (ASGV) is one of the most important viruses infecting fruit trees. This study aimed at the molecular characterization of ASGV infecting apple (Malus domestica) plants in Santa Catarina (SC). RNA extracted from plants infected with isolate UV01 was used as a template for RT-PCR using specific primers. An amplified DNA fragment of 755 bp was sequenced. The coat protein gene of ASGV isolate UV01 contains 714 nucleotides, coding for a protein of 237 amino acids with a predicted Mr of approximately 27 kDa. The nucleotide and the deduced amino acid sequences of the coat protein gene showed identities of 90.9% and 97.9%, respectively, with a Japanese isolate of ASGV. Very high amino acid homologies (98.7%) were also found with Citrus tatter leaf capillovirus (CTLV), a very close relative of ASGV. These results indicate low coat protein gene variability among Capillovirus isolates from distinct regions. In a restricted survey, mother stocks in orchards and plants introduced into the country for large scale fruit production were indexed and shown to be infected by ASGV (20%), usually in a complex with other (latent) apple viruses (80%).
Apple stem pitting virus (ASPV), Apple chlorotic leaf spot virus (ACLSV) and Apple stem grooving virus (ASGV) are several major viral pathogens of apple trees, responsible for substantial damage to the world's apple industry. This study aimed to evaluate the effectiveness of the encapsulation-dehydration cryopreservation technique to eradicate these viral pathogens from in vitro shoot tips excised from 'Marubakaido' apple rootstock cultures. Axillary shoot tips were excised from in vitro cultures, encapsulated in alginate beads, precultured in MS salts, dehydrated in a laminar flow hood, immersed in liquid nitrogen, then warmed and recovered on medium. After LN exposure, in vitro rooting and acclimatization, recovered 'Marubakaido' plants exhibited 52% survival and 35% regrowth without callus formation. After 8 months of regrowth, PCR analyses revealed that all the plants were free of ACLSV and ASPV, but 2 out of 20 recovered plants were still infected with ASGV. This is the first report in Brazil of the application of cryotherapy to eradicate viral complexes in Malus. Cryotherapy can facilitate the production of virus-free plants by producing high quality plant material.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.