DOCSLIB.ORG

Replication of Cauliflower Mosaic Virus ORF I Mutants in Turnip Protoplasts

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Replication of Cauliflower Mosaic Virus ORF I Mutants in Turnip Protoplasts 日木直病 報 60: 27-35 (1994) Ann. Phytopath. Soc. Japan 60: 27-35 (1994) Replication of Cauliflower Mosaic Virus ORF I Mutants in Turnip Protoplasts Seiji TSUGE*,•õ, Kappei KOBAYASHI*,•õ•õ, Hitoshi NAKAYASHIKI*, Tetsuro OKUNO* and Iwao FURUSAWA* Abstract We succeeded in infecting turnip protoplasts with a cloned cauliflower mosaic virus (CaMV) DNA, pCa122, which contains 1.2 copy of CaMV genomic DNA and a plasmid expressing open reading frame (ORF) VI products (pEXP6) using polyethylene glycol. Fluorescent antibody staining showed that up to 50% of protoplasts were infected. It was difficult to detect the progeny DNA and the viral protein in protoplasts inoculated with pCa122 alone. Co-transfection with the plasmid pEXP6 produced larger fluorescing specks in each infected protoplasts and increased the accumulation of the progeny DNA and some other viral proteins to detectable levels. Using this protoplast system, three CaMV ORF I insertional mutants which were not infectious on turnip plants were tested for their infectivity on turnip protoplasts. Viral DNA and products accumulated in infected proto- plasts to the same extent of the wild type DNA-infected protoplasts. These results indicate that ORF I product is not required for multiplication of CaMV in protoplasts, but is indispensable for infection on whole plants, strongly supporting that ORF I product is involved in cell-to-cell movement of CaMV. (Received May 6, 1993) Key words: cauliflower mosaic virus, protoplasts, movement protein. INTRODUCTION Cauliflower mosaic virus (CaMV) has an 8kb circular double stranded DNA genome which encodes six major open reading frames (ORFs) I to VI on the same DNA strand17). Functions have been assigned to these ORF products except for the product of ORF III. It is tempting to study the gene expression of CaMV in relation to the functions of genes in protoplasts8). However, no one has been successful in infecting protoplasts with cloned CaMV DNAs although infection of turnip protoplasts with virion DNA has been reported22). We found that co-inoculation with a plasmid which expresses the CaMV ORF VI product (P6) under the control of the CaMV 35S promoter by means of polyethylene glycol (PEG) led to efficient infection of turnip protoplasts with cloned CaMV DNAs. P6 has been shown to function as a trans-activator for other genes of CaMV2) and also to be a major component of viroplasm where viral replication occurs3,18). Therefore, gene expression and viral replication are thought to be activated by P6 in protoplasts inoculated with the cloned CaMV DNA. Using this protoplast system we analyzed three CaMV mutants having 27bp (in-frame) and 31bp (frame-shift) non-viral sequence inserted in ORF I which was thought to encode a cell-to-cell movement protein of CaMV21). We report here that CaMV ORF I mutant DNAs, which cannot infect systemically on turnip plants, replicated in protoplasts to the * Laboratory of Plant Pathology , Faculty of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan 京 都 大 学 農 学 部 † Present address: Laboratory of Plant Pathology , Faculty of Agriculture, Kyoto Prefectural University, Sakyo- ku, Kyoto 606, Japan 京 都 府 立 大 学 農 学 部 †† Present address: Department of Microbiology , Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602, Japan 京 都 府 立 医 科 大 学 28 日本植 物 病 理 学会 報 第60巻 第1号 平 成6年2月 same level of the wild type DNA. This indicates that the ORF I product (P1) is not essential for the replication of CaMV in single cells and supports the recent findings that CaMV ORF I encodes a cell-to-cell movement protein21). MATERIALS AND METHODS Plasmids. The plasmid pCa122 has an 1.2 copy of CaMV CM1841 DNA which comprises a complete CaMV DNA and an additional PvuII-BstEII fragment (#6,321-#8,031/#0-#127: nucleotide number refers to Gardner et al. 10)). The plasmid was constructed by using pCaMV10 which has one copy of CM1841 DNA inserted in pBR322 at its unique SalI site (#4,834)10. The plasmid pCaMV10 was digested with PvuII and religated for removing the shorter PvuII fragment, creating pCa0.8. Large SalI-SphI (filled out) fragment of pCa0.8 was ligated with the shortest SalI-BstEII (filled in) fragment (#4,834-#8,031/#0-#127) of pCaMV10, creating the pCa122 (Fig. 1). Plasmid pEXP6 which has CaMV ORF VI region 3•Œ to the CaMV 35S promoter region was constructed as follows; the PvuII-ScaI fragment (#6,321-#7,640) of CaMV DNA was inserted into pUC118 at HindIII site after filling in, and a new SaiI site was introduced to the 35S RNA cap site (#7,433) by site-directed mutagenesis using oligonucleotide (5•Œ-CATTTGGAGTCGACACGCTGAA-3•Œ)14, to obtain pMC. The 3•Œend sequence of nopaline synthase gene was excised from pBI221 (Clontech Laboratories), and inserted into SacI-EcoRI sites of pMC, to create pMCT. The EcoRV fragment of CaMV DNA (#5,711-#7,343) was inserted into the respective site of pBluescript II SK-. A SalI site was introduced to the cap site of 19S RNA by site-directed mutagenesis using oligonucleotide (5•Œ-CTGAGAAAGTCGA- CCTCCAAGC-3•Œ), and the SalI-EcoRV fragment of the plasmid was inserted into pMCT which had been digested with SalI and Smal to create pEXP6. This plasmid was used for co-inoculation of turnip protoplasts with cloned CaMV DNA. Construction of ORF I mutants. First, we constructed a plasmid pUCK191 (a derivative of pUC19) which has kanamycin resistance (Kmr) gene (derived from Tn903) with KpnI and SmaI (XmaI) Fig. 1. Schematic representation of pCa122. CaMV CM1841 DNA is shown by a thin line with ORFs indicated by arrows on it, and DNA derived from pBR322 is shown by an open box. Thin arrows inside the DNA represent RNA transcripts of CaMV. Zero are the start positions of a nucleotide number of CM1841 (refer to Gardner et al.10)). Black and shaded arrowheads indicate CIaI and XbaI sites, respectively. Ann. Phytopath. Soc. Japan 60 (1). February, 1994 29 Fig. 2. Restriction sites flanking on both sides of kanamycin resistance gene (Kmr) in pUCK191. Kmr is derived from Tn903, and the other region of this plasmid is derived from pUC19 in which DraI fragment, containing ampicillin resistance gene, is deleted. Thick and thin arrows indicate that each regions are located in the same orientation, respectively. sites flanking on either side in the same orientation, and SalI (AccI), XbaI and Bam HI sites flanking on either outside of the KpnI and SmaI (XmaI) sites in the same orientation (Fig. 2). Dral fragment containing a part of ampicillin resistance gene was removed from the plasmid. CaMV mutants having an inserted sequence in ORF I region were constructed in a similar way as described by Dixon et al.4) The plasmid pCa122 was partially digested with ClaI in the presence of 80ƒÊg/ml ethidium bromide to obtain full length linear DNA molecules. The DNAs were then ligated with the Kmr gene excised from pUCK191 by digestion with AccI, and introduced into Escherichia coli. The transformants were selected on medium containing both ampicillin and kanamycin. The Kmr gene was removed from the recom- binant CaMV DNA by digestion with KpnI and relegation for generating insertional mutants containing a 27bp inserted sequence including KpnI, SmaI BamHI and XbaI sites at any one of ClaI sites of pCa122. Alternatively, the Kmr gene was removed by XmaI digestion followed by filling-in and relegation, resulting in mutants having a 31bp inserted sequence containing KpnI, BamHI, XbaI and Eco52I sites. The plasmids having a 27bp and a 31bp inserted sequence at the ClaI site in the ORF I of CaMV (#822) were identified by restriction enzyme analysis, and designated pCaMV822-1 and pCaMV822-2, respec- tively. Similarly, we constructed other insertional mutants which have a 31bp inserted sequence at the XbaI site in the ORF I (#839) and named pCaMV839-2. Nucleotide sequences in the ORF I region of mutant DNAs were determined using Sequenase kit (USB) according to the manufacturer's instruction. Inoculation of turnip protoplasts with cloned CaMV DNA. Turnip protoplasts were prepared as described by Furusawa and Okuno6). To the pellet of protoplasts (1•~106) added were 0.5ml of 10mM MES buffer (pH 5.8) containing 0.5M mannitol, 40mM CaCl2, 50ƒÊg of cloned CaMV DNA, 20 ƒÊg of pEXP6 and 50ƒÊg of salmon sperm DNA (as a carrier), followed by mixing with 0.9ml of 0.5M mannitol containing 40% PEG, MW. 4000 (SIGMA) and 40mM CaCl2. Protoplasts were incubated for 30min on ice with gentle shaking, diluted with 10ml of ice cold 0.5M mannitol containing 40mM CaCl2, and incubated for additional 30min on ice. After washing three times with 0.5M mannitol containing 50mM glycine and 50mM CaCl2, pH 8.513), protoplasts were cultured as described previously7). Infection of protoplasts was determined by fluorescent antibody staining7). Inoculation of turnip plants with cloned CaMV DNA. Turnip plants (Brassica raga L. cv. Marubakomatsuna) were grown under natural daylight in a greenhouse at controlled temperature of 22•}3•Ž. Ten microlitter of pCa122 or its derivatives (1ƒÊg/ƒÊl) was mixed with an equal volume of sterilized water containing carborundum (50% <v/v>) and the mixture was rubbed on the surface of primary leaves of two week-old plants with a sterilized glass rod with spherical end. Electron microscopic observation. Protoplasts were fixed with 50mM phosphate buffer, pH 6.8, containing 5% glutaraldehyde and 0.5M mannitol at 0•Ž overnight. After washing with the phos- phate buffer, protoplasts were post-fixed with 1% OsO4 at 0•Ž overnight and washed again with phosphate buffer. Samples were dehydrated by ethanol series and embedded in a LR-White (The London Resin).
Load more

Recommended publications
  • TESTS of the Associanon of HEAT SHOCK PROTEIN 90 with A
    TESTS OF THE ASSOCIAnON OF HEAT SHOCK PROTEIN 90 WITH A CAULIFLOWER MOSAIC VIRUS REVERSE TRANSCRIPTASE By BRESHANA QUIENE JOHNSON Bachelor of Science Xavier University of Louisiana New Orleans, Louisiana May 1997 Submitted to the Faculty of the Graduate College of the Oklahoma State University in partial fulfillment of the requirements for the Degree of MASTER OF SCIENCE July, 2000 Ok/ahoma State University Library TESTS OF THE ASSOCIAnON OF HEAT SHOCK PROTEIN 90 WITH A CAULIFLOWER MOSAIC VIRUS REVERSE TRANSCRIPTASE Thesis Approved: ~~-- Thesis Advisor ~~-~---- 11 ACKNOWLEDGEMENTS I sincerely thank my thesis advisor, Dr. Ulrich K. Melcher, for his guidance, supervision, expertise, and friendship. It was a pleasure to be a part of his Jab and I will always appreciate his dedication to his students and encouragement. I thank my committee members Dr. Richard C. Essenberg and Dr. Robelt L. Matts for their support and expertise. I also thank Dr. James B. Blair and the Department of Biochemistry and Molecular Biology for providing me with the opportunity to participate in the NIH Biomedical Graduate Students program and their financial support. I express my sincere gratitude to Dr. Steven D. Hartson for his guidance, suggestions, and inspiration throughout this project. I would like to thank staff and students of the Department of Biochemistry and Molecular Biology, who provided me with assistance: Ann Williams, Dr. Ba am Fraij, Dr. Jerry Merz, Wenhao Wang, Qi Jiang, Kenji Onodera, Wenjun Huang, Jieya Shao, Abdel Bior, and Thomas Prince I truly thank my parents for encouraging and supporting me throughout my academic pursuits. I am very grateful to them and blessed to have their love and concern.
    [Show full text]
  • CRISPR/Cas9-Mediated Resistance to Cauliflower Mosaic Virus Haijie Liu1
    bioRxiv preprint doi: https://doi.org/10.1101/191809; this version posted September 23, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. CRISPR/Cas9-mediated resistance to cauliflower mosaic virus Haijie Liu1,*, Cara L. Soyars2,7, *, Jianhui Li1, *, Qili Fei4,5, Guijuan He1, Brenda A. Peterson2,7, Blake C. Meyers4,5,6, Zachary L. Nimchuk2,3,7,8, and Xiaofeng Wang1,#. 1Department of Plant Pathology, Physiology and Weed Science; 2Department of Biological Sciences; 3Faculty of Health Sciences; Virginia Tech, Blacksburg, VA, USA; 4Department of Plant & Soil Sciences and Delaware Biotechnology Institute, University of Delaware, Newark, Delaware, USA; 5Donald Danforth Plant Science Center, St. Louis, Missouri, USA; 6University of Missouri – Columbia, Division of Plant Sciences, Columbia, MO; 7Department of Biology, University of North Carolina at Chapel Hill, and 8Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; *These authors contributed equally to this project. # Author for correspondence: Xiaofeng Wang, 549 Latham Hall, 220 Ag Quad Lane, Blacksburg, VA 24061. Telephone: 1-540-231-1868, Fax: 1-540-231-7477, Email: [email protected]. H. Liu: [email protected]; C.L. Soyars: [email protected]; J. Li: [email protected]; Q. Fei: [email protected]; G. He: [email protected]; B.A. Peterson: [email protected]; B.C. Meyers: [email protected]; Z.L. Nimchuk: [email protected] Running title: CRISPR-Cas9-conferred resistance to CaMV Key words: CRISPR-Cas9, cauliflower mosaic virus, virus resistance, virus escape, small RNA Word count: Summary, 216 and text, 4201 1 bioRxiv preprint doi: https://doi.org/10.1101/191809; this version posted September 23, 2017.
    [Show full text]
  • Cauliflower Mosaic Virus (Camv)
    Cauliflower Mosaic Virus (CaMV) Dr.Ramesh C.K Cauliflower Mosaic Virus (CaMV) • Cauliflower mosaic virus (CaMV) is a member of the genus Caulimovirus, one of the six genera in the family Caulimoviridae which are pararetroviruses that infect plants. • Pararetroviruses group due to its mode of replication via reverse transcription of a pre- genomic RNA intermediate. just like retroviruses but the viral particles contain DNA instead of RNA. • True retroviruses are not known in plants; however, plant pararetroviruses (caulimoviridae) share many retroviral properties, replicating by transcription in the nucleus followed by reverse transcription in the cytoplasm. • Pararetroviruses have circular DNA genomes that do not integrate into the host genome, and display several unique expression strategies. • CaMV infects mostly plants of the family Brassicaceae (such as cauliflower and turnip) but some CaMV strains are also able to infect Solanaceae species of the genera Datura and Nicotiana. • CaMV induces a variety of systemic symptoms such as mosaic, necrotic lesions on leaf surfaces, stunted growth, and deformation of the overall plant structure. • CaMV is transmitted by aphid species such as Myzus persicae. Once introduced within a plant host cell, virions migrate to the nuclear envelope of the plant cell. Structure • The CaMV particle is an icosahedron with a diameter of 52 nm built from 420 capsid protein (CP), which surrounds a solvent-filled central cavity. • In addition to capsid proteins, caulimoviruses are also surrounded by virus associated proteins. These proteins are responsible for assisting in the binding of the virus to DNA • CaMV contains a circular double-stranded DNA molecule of about 8.0 kilobases, interrupted by nicks that result from the actions of RNAse H during reverse transcription.
    [Show full text]
  • Ribosome Shunting, Polycistronic Translation, and Evasion of Antiviral Defenses in Plant Pararetroviruses and Beyond Mikhail M
    Ribosome Shunting, Polycistronic Translation, and Evasion of Antiviral Defenses in Plant Pararetroviruses and Beyond Mikhail M. Pooggin, Lyuba Ryabova To cite this version: Mikhail M. Pooggin, Lyuba Ryabova. Ribosome Shunting, Polycistronic Translation, and Evasion of Antiviral Defenses in Plant Pararetroviruses and Beyond. Frontiers in Microbiology, Frontiers Media, 2018, 9, pp.644. 10.3389/fmicb.2018.00644. hal-02289592 HAL Id: hal-02289592 https://hal.archives-ouvertes.fr/hal-02289592 Submitted on 16 Sep 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Distributed under a Creative Commons Attribution - ShareAlike| 4.0 International License fmicb-09-00644 April 9, 2018 Time: 16:25 # 1 REVIEW published: 10 April 2018 doi: 10.3389/fmicb.2018.00644 Ribosome Shunting, Polycistronic Translation, and Evasion of Antiviral Defenses in Plant Pararetroviruses and Beyond Mikhail M. Pooggin1* and Lyubov A. Ryabova2* 1 INRA, UMR Biologie et Génétique des Interactions Plante-Parasite, Montpellier, France, 2 Institut de Biologie Moléculaire des Plantes, Centre National de la Recherche Scientifique, UPR 2357, Université de Strasbourg, Strasbourg, France Viruses have compact genomes and usually translate more than one protein from polycistronic RNAs using leaky scanning, frameshifting, stop codon suppression or reinitiation mechanisms.
    [Show full text]
  • An Abstract of the Dissertation Of
    AN ABSTRACT OF THE DISSERTATION OF Alfredo Diaz Lara for the degree of Doctor of Philosophy in Botany and Plant Pathology presented on December 16, 2016. Title: Identification of Endogenous and Exogenous Pararetroviruses in Red Raspberry (Rubus idaeus L.) and Blueberry (Vaccinium corymbosum L.). Abstract approved: ______________________________________________________ Robert R. Martin The Pacific Northwest (Oregon and Washington in the United States and British Columbia in Canada) is one of the major producers of red raspberry (Rubus idaeus L.) and blueberry (Vaccinium corymbosum L.) in the world. The expansion of growing area with these crops has resulted in the emergence of new virus diseases that cause serious economic losses. The majority of viruses affecting plants (including blueberry and red raspberry) contain RNA genomes. In contrast, plant viruses with DNA genomes are relatively rare and most of the time ignored in virus surveys. The family Caulimoviridae is a group of plant pararetroviruses (reverse-transcribing viruses) with the ability to integrate their DNA into the host genome, resulting in complex molecular interactions that lead to inconsistencies in terms of detection and disease symptoms. Albeit, few studies have been conducted to determine the nature of plant pararetroviruses and their relationships with the associated host. To investigate the presence of pararetroviruses in blueberry and red raspberry, and their possible integration events, different plant material suspected to be infected with viruses was collected in nurseries, commercial fields and clonal germplasm repositories for a period of four years. For blueberry, using rolling circle amplification (RCA) a new virus was identified and named Blueberry fruit drop-associated virus (BFDaV) because of its association with fruit-drop disorder.
    [Show full text]
  • A New and Distinct Species in the Genus Caulimovirus Exists As an Endogenous Plant Pararetroviral Sequence in Its Host, Dahlia Variabilis
    Virology 376 (2008) 253–257 Contents lists available at ScienceDirect Virology journal homepage: www.elsevier.com/locate/yviro Rapid Communication A new and distinct species in the genus Caulimovirus exists as an endogenous plant pararetroviral sequence in its host, Dahlia variabilis Vihanga Pahalawatta, Keri Druffel, Hanu Pappu ⁎ Department of Plant Pathology, Washington State University, Pullman, WA, USA article info abstract Article history: Viruses in certain genera in family Caulimoviridae were shown to integrate their genomic sequences into their Received 6 August 2007 host genomes and exist as endogenous pararetroviral sequences (EPRV). However, members of the genus Returned to author for revision Caulimovirus remained to be the exception and are known to exist only as episomal elements in the 13 September 2007 infected cell. We present evidence that the DNA genome of a new and distinct Caulimovirus species, associated Accepted 4 March 2008 with dahlia mosaic, is integrated into its host genome, dahlia (Dahlia variabilis). Using cloned viral genes as Available online 7 May 2008 probes, Southern blot hybridization of total plant DNA from dahlia seedlings showed the presence of viral DNA in the host DNA. Fluorescent in situ hybridization using labeled DNA probes from the D10 genome localized Keywords: the viral sequences in dahlia chromosomes. The natural integration of a Caulimovirus genome into its host and Pararetrovirus – Dahlia mosaic virus its existence as an EPRV suggests the co-evolution of this plant virus pathosystem. Caulimovirus © 2008 Elsevier Inc. All rights reserved. Introduction genome, dahlia (Dahlia variabilis) and thus exists as an endogenous plant pararetrovirus (EPRV). Dahlia mosaic caulimovirus (DMV) is an important viral pathogen of dahlia in the US and several parts of the world.
    [Show full text]
  • Cauliflower Mosaic Virus P6 Dysfunctions Histone Deacetylase
    cells Article Cauliflower mosaic virus P6 Dysfunctions Histone Deacetylase HD2C to Promote Virus Infection Shun Li 1,2, Shanwu Lyu 1, Yujuan Liu 2, Ming Luo 1,3, Suhua Shi 4 and Shulin Deng 1,3,5,* 1 Guangdong Provincial Key Laboratory of Applied Botany & CAS Key Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China; [email protected] (S.L.); [email protected] (S.L.); [email protected] (M.L.) 2 School of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China; [email protected] 3 Center of Economic Botany, Core Botanical Gardens, Chinese Academy of Sciences, Guangzhou 510650, China 4 State Key Laboratory of Biocontrol, Guangdong Provincial Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, China; [email protected] 5 National Engineering Research Center of Navel Orange, School of Life Sciences, Gannan Normal University, Ganzhou 341000, China * Correspondence: [email protected] Abstract: Histone deacetylases (HDACs) are vital epigenetic modifiers not only in regulating plant development but also in abiotic- and biotic-stress responses. Though to date, the functions of HD2C— an HD2-type HDAC—In plant development and abiotic stress have been intensively explored, its function in biotic stress remains unknown. In this study, we have identified HD2C as an interaction partner of the Cauliflower mosaic virus (CaMV) P6 protein. It functions as a positive regulator in defending against CaMV infection. The hd2c mutants show enhanced susceptibility to CaMV infection.
    [Show full text]
  • Review Article How to Deal with the Upcoming Challenges in GMO Detection in Food and Feed
    Hindawi Publishing Corporation Journal of Biomedicine and Biotechnology Volume 2012, Article ID 402418, 11 pages doi:10.1155/2012/402418 Review Article How to Deal with the Upcoming Challenges in GMO Detection in Food and Feed Sylvia R. M. Broeders, Sigrid C. J. De Keersmaecker, and Nancy H. C. Roosens Platform Biotechnology and Molecular Biology, Wetenschappelijk Instituut Volksgezondheid-Institut Scientifique de Sant´e Publique (WIV-ISP), J. Wytsmanstraat 14, 1050 Brussel, Belgium Correspondence should be addressed to Nancy H. C. Roosens, [email protected] Received 30 March 2012; Accepted 13 September 2012 Academic Editor: Joel W. Ochieng Copyright © 2012 Sylvia R. M. Broeders et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Biotech crops are the fastest adopted crop technology in the history of modern agriculture. The commercialisation of GMO is in many countries strictly regulated laying down the need for traceability and labelling. To comply with these legislations, detection methods are needed. To date, GM events have been developed by the introduction of a transgenic insert (i.e., promoter, coding sequence, terminator) into the plant genome and real-time PCR is the detection method of choice. However, new types of genetic elements will be used to construct new GMO and new crops will be transformed. Additionally, the presence of unauthorised GMO in food and feed samples might increase in the near future. To enable enforcement laboratories to continue detecting all GM events and to obtain an idea of the possible presence of unauthorised GMO in a food and feed sample, an intensive screening will become necessary.
    [Show full text]
  • Cauliflower Mosaic Virus (Camv) Biology, Management, And
    MINI REVIEW published: 31 March 2020 doi: 10.3389/fsufs.2020.00021 Cauliflower mosaic virus (CaMV) Biology, Management, and Relevance to GM Plant Detection for Sustainable Organic Agriculture Aurélie Bak and Joanne B. Emerson* Department of Plant Pathology, University of California, Davis, Davis, CA, United States In today’s global market, some organic farmers must meet regulatory requirements to demonstrate that their plants and feedstocks are genetically modified organism (GMO)-free. Many GM plants are engineered to contain a promoter from the plant virus, Cauliflower mosaic virus (CaMV), in order to facilitate expression of an engineered target gene. The relative ubiquity of this CaMV 35S promoter (P35S) in GM constructs means that assays designed to detect GM plants often target the P35S DNA sequence, but these detection assays can yield false-positives from plants that are infected by naturally-occurring CaMV or its relatives within the Caulimoviridae. This review places Edited by: CaMV infection and these ambiguous GM plant detection assays in context, serving Avtar Krishan Handa, Purdue University, United States as a resource for industry professionals, regulatory bodies, and researchers at the Reviewed by: nexus of organic farming and global commerce. We first briefly introduce GM plants James Schoelz, from a regulatory perspective, and then we describe CaMV biology, transmission, and University of Missouri, United States Tahira Fatima, management practices, highlighting the relatively widespread nature of CaMV infection Purdue University, United States in both GM and non-GM crops within the Brassicaceae and Solanaceae families. Finally, *Correspondence: we discuss current knowledge of public food safety related to the consumption of Joanne B.
    [Show full text]
  • Criteria for Identification of Cauliflower Mosaic Virus's of the Far Eastern
    Plant Protection Science – 2002 Plant Protection Science – 2002 Vol. 38, Special Issue 2: 258–260 Criteria for Identification of Cauliflower Mosaic Virus’s of the Far Eastern Strains R. V. GNUTOVA*, V. F. TOLKACH and JU. V. BOGUNOV Institute of Biology & Soil Science Far Est Branch, 690022 Vladivostok, Russia *E-mail: [email protected] Abstract On the base of the present-day principles to classify plant viruses the identification of Cauliflower mosaic virus (CaMV), a new virus for the Russian Federation, is carried out. Biological properties of 7 isolates have been studied. Symptoma- tology, range of host-plants and physical properties of virions of studied strains differ. The least thermostable strain is CaMV-B3 (TIP – 75°C) and the highest TIP (85°C) is CaMV-B1. The highest virus concentration in sap was observed for CaMV-B2 (DEP – 10–6), and lowest – CaMV-R1 (10–1–10–2). CaMV-B2 and CaMV-C2 lost infection during 4 days in room conditions, CaMV-B3 – 1 day. A virus proteins were isolated (42 and 44 kD). The native nucleic acid of CaMV have been extracted. The DNA was separated into mixtures of circular and linear molecules. Size of the DNA is about 8000 base pairs. Keywords: Cauliflower mosaic virus; Brassicaceae; identification INTRODUCTION Primorye Territory. CaMV is the first DNA-containing phytovirus, discovered in the territory of Russia. The fundamental investigations are essential for study of properties virus-specific proteins and nu- MATERIALS AND METHODS cleic acids of phytoviruses and their strains not only described in literature but also recent for description The virus isolates with virus-like symptoms were to a new species according to the latest requirement used, isolated from cauliflower (CaMV-C1, CaMV-C2), of classification.
    [Show full text]
  • The Expression of Foreign Gene Under the Control of Cauliflower Mosaic Virus 35S RNA Promoter
    Cell Research (1990), 1, 1-10 The expression of foreign gene under the control of cauliflower mosaic virus 35s RNA promoter Wang Hao and Bai Yongyan Shanghai Institute of Plant Physiology, Academia Sinica. 300 Fenglin Road, Shanghai 200032, China ABSTRACT The promoter region of cauliflower mosaic virus (CaMV) 35s RNA was employed to construct an intermediate expres- sion vector which can be used in Ti plasmid system of Agro- bacterium tumefaciens. The original plasmid, which contains a polylinker between CaMV 35s RNA and its 3' termination signal in pUC18 was modified to have another antibiotic resistance marker (kanamycin resistance gene Kmr) to facili- tate the selection of recombinant with Ti plasmid. Octopine synthase (ocs) structural gene was inserted into this vector downstream of CaMV 35s RNA promoter. This chimaeric gene was introduced into integrative Ti plasmid vector pGV3850, and then transformed into Nicotiana tobaccum cells. A binary plasmid vector was also used to introduce the chimaeric gene into tobacco cells. In both cases, the expression of ocs gene was demonstrated. The amount of oc- topine was much more than the nopaline synthesized by no- paline synthase (nos) gene transferred at the same time with Ti plasmid vector. This demonstrated that CaMV 35s RNA promoter is stronger in transcriptional function than the pro- moter of nos in tobacco cells. Key words: Agrobacterium tumefaciens, gene expression, cauliflower mosaic Cirus 35s RNA promoter. INTRODUCTION During the past decade, extensive research on the Ti plasmid of Agrobacterium tumefaciens has brought development of useful and efficient vectors to introduce fo- reign genes into dicotyledonous plants.
    [Show full text]
  • Bt BRINJAL Primer
    National Consultations on Bt Brinjal A primer on concerns, issues and prospects National Consultations on Bt Brinjal A primer on concerns, issues and prospects Contents 1. Introduction 1 2. Brinjals in India 3 3. Genetically Modified Crops and the Brinjal 5 4. Bt Brinjal: Prospects and Concerns 13 5. Glossary 19 6. References 20 Disclaimer: This booklet has been compiled by the Centre for Environment Education (CEE) as a primer for discussants at the national consultations and does not in any way express the views of the Ministry of Environment and Forests, Government of India or of the Centre for Environment Education. Introduction What is Bt Brinjal? Bt Brinjal is a transgenic brinjal created by inserting a gene cry1Ac from the soil bacterium Bacillus thuringiensis into Brinjal. This is said to give the Brinjal plant resistance against lepidopteran insects like the Brinjal Fruit and Shoot Borer Leucinodes orbonalis and Fruit Borer Helicoverpa armigera. What is the controversy about? Bt Brinjal has generated much debate in India. The promoters say that Bt Brinjal will be beneficial to small farmers because it is insect resistant, increases yields, is more cost-effective and will have minimal environmental impact. On the other hand, concerns about Bt Brinjal relate to its possible adverse impact on human health and bio-safety, livelihoods and biodiversity. What is the official response to the controversy? The Ministry of Environment and Forests (MoEF) has a statutory body called the Genetic Engineering Approval Committee (GEAC) which has recommended the environmental release of Bt Brinjal in India based on the recommendations of the Review Committee on Genetic Manipulation (RCGM), a statutory body and two expert committees constituted by the GEAC between 2006 and 2009.
    [Show full text]