Citrus tristeza virus, 1st ed , antonino f catara, moshe bar joseph, grazia licciardello, 2019 99

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Methods in Molecular Biology 2015 Antonino F Catara · Moshe Bar-Joseph Grazia Licciardello Editors Citrus Tristeza Virus Methods and Protocols METHODS IN MOLECULAR BIOLOGY Series Editor John M Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, UK For further volumes: http://www.springer.com/series/7651 For over 35 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing Each protocol is provided in readily-reproducible step-bystep fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice These hallmark features were introduced by series editor Dr John Walker and constitute the key ingredient in each and every volume of the Methods in Molecular Biology series Tested and trusted, comprehensive and reliable, all protocols from the series are indexed in Pub Med Citrus Tristeza Virus Methods and Protocols Edited by Antonino F Catara Department of Phytosanitary Sciences, University of Catania, Science and Technology Park of Sicily, Catania, Italy Moshe Bar-Joseph The S Talkowski Laboratory, Department of Plant Pathology and Weed Research, The Volcani Center, Agricultural Research Organization, Bet Dagan, Israel Grazia Licciardello Consiglio per la Ricerca in agricoltura e l’analisi dell’Economia Agraria, Centro di Olivicoltura, Frutticoltura e Agrumicoltura (CREA-OFA), Acireale (Catania), Italy Editors Antonino F Catara Department of Phytosanitary Sciences University of Catania Science and Technology Park of Sicily Catania, Italy Moshe Bar-Joseph The S Talkowski Laboratory Department of Plant Pathology and Weed Research The Volcani Center Agricultural Research Organization Bet Dagan, Israel Grazia Licciardello Consiglio per la Ricerca in agricoltura e l’analisi dell’Economia Agraria Centro di Olivicoltura Frutticoltura e Agrumicoltura (CREA-OFA) Acireale (Catania), Italy ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-9557-8 ISBN 978-1-4939-9558-5 (eBook) https://doi.org/10.1007/978-1-4939-9558-5 © Springer Science+Business Media, LLC, part of Springer Nature 2019 This work is subject to copyright All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed The use of general descriptive names, registered names, trademarks, service marks, etc in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer Nature The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A Preface Citrus tristeza virus (CTV) is one of the most destructive plant viruses that replicates in the cytoplasm of companion or phloem parenchyma cells of Citrus, Poncirus, and Fortunella It causes a variety of symptoms depending on the host species, the cultivar, and the CTV isolate involved Given that this virus is associated worldwide with citrus, many plant pathologists and virologists have been involved in the study of this complex virus and the diseases it causes Few, however, have spent as long as 50 years like Moshe Bar-Joseph His pioneering efforts led to the development of new methods of CTV diagnosis (1970) based on the electron microscope observation of partially purified particles and enabled other groups to develop rapid serological assays (Chapter 1) With some modifications, his method was also useful for Beet yellows virus (BYV) particles that cause Closterovirus To demonstrate the genetic diversity of CTV isolates, Moshe Bar-Joseph developed strain-specific assays using CTV-VT cDNA fragments as hybridization probes He also developed a CTV-dsRNA cloning method and used complementary oligonucleotides for cDNA synthesis and PCR amplification To honor his invaluable dedication and contribution to the study of the virus, 45 authors have contributed to this laboratory methods and protocols book on the Citrus tristeza virus, which is one of the most complex viruses, as well one of the most scientifically attractive research topics Thanks to the highly sensitive and specific diagnostic procedures developed, knowledge of the molecular characteristics, expression strategies, genetic variability, and epidemiology of the virus have improved significantly Since deep sequencing opened new doors to reconstructing viral populations in a high-throughput and cost-effective manner, many of the past grouping criteria have now been revisited Today, 67 complete sequences of CTV genomes from different countries are in the GenBank Unfortunately, not all of them have been associated with a phenotypic profile Reports from all over the world show that several destructive isolates of CTV, not dependent on sensitive rootstocks, may suddenly appear as a result of rearrangements or mutations of the genome The rapid identification of the genetic diversity of the virus remains critical for surveying specific land areas This book provides methods and clear protocols for the various technologies available to detect, characterize, and study CTV, a member of the genus Closterovirus family Closteroviridae (Chapter 2) Despite the fact that new detection methods have strengthened the discrimination potential of genotypes of the isolates, biological indexing remains invaluable in order to phenotype the biological properties of isolates in terms of their aggressiveness on various hosts (Chapter 3) Enzyme immunoassays and PCR-based assays, which are frequently used in combination, have revealed the worldwide rapid diffusion of the virus, even in symptomless infected citrus trees The relationships of vectors with the virus and its host plants, which are mostly based on host plant inoculation, have been highlighted by sensitive detection technologies (Chapter 4) The potential of CTV detection by RT-PCR was strengthened after the development of direct systems of sample preparation and real-time RT-PCR (Chapter 5) Fast, reliable, and specific detection methods based on real-time PCR protocols have been designed to simultaneously detect CTV, HSVd, and CEVd (Chapter 6) The analysis of single-strand v vi Preface conformation polymorphism of RT-PCR product by polyacrylamide gel or capillary electrophoresis is still now the most common and informative method to investigate the structure of CTV isolate populations (Chapter 7) Integrating molecular assays and biological tests has made it possible to identify RB CTV isolates which overcome the resistance of trifoliate orange and its hybrids (Chapter 8), whereas sequential RT-PCR and microarray hybridization, on a lab-on chip device, enable a fast characterization of virus genotypes (Chapter 9), which is very useful for the CTV surveillance of territories At the same time, the fast and sensitive field detection of CTV has recently been achieved by reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay (Chapter 10) A strategy to clone the entire genome of CTV obtained from two RT-PCR amplified products has also been developed (Chapter 11) After high-throughput sequencing (HTS) was developed, bioinformatics started to be applied to analyze the genome of the virus (Chapter 12), to differentiate between isolates based on genotype composition which has been used to select candidate cross protective isolates (Chapter 13), and to study host RNA silencing and virus attack (Chapter 14) The study of proteins involved in CTV infection has been made possible by techniques such as proteomics (Chapter 15), whereas transient expression of the virus proteins is possible by biolistic bombardment (Chapter 16) Methods are now available for producing transgenic plants resistant to CTV (Chapter 17) This book will be of interest to plant pathologists, plant virologists, molecular biologists, and graduate students, as a guide to performing qualitative and quantitative tests as well as recently developed diagnostic methods We hope the methods and protocols reported here will be helpful to find new solutions to improve the management of the disease, and we wish to thank all the authors who have contributed Catania, Italy Bet Dagan, Israel Acireale (Catania), Italy Antonino F Catara Moshe Bar-Joseph Grazia Licciardello Contents Preface Contributors A Short Note on Reflections and Publications on Citrus tristeza virus (CTV) Methodologies Moshe Bar-Joseph A Brief Historical Account of the Family Closteroviridae Giovanni P Martelli Phenotyping Biological Properties of CTV Isolates Marcella Russo and Antonino F Catara CTV Vectors and Interactions with the Virus and Host Plants Raymond Yokomi Tissue-Print and Squash Capture Real-Time RT-PCR Method for Direct Detection of Citrus tristeza virus (CTV) in Plant or Vector Tissues Mariano Cambra, Eduardo Vidal, Carmen Martı´nez, and Edson Bertolini Detection of Citrus tristeza virus and Coinfecting Viroids Maria Saponari, Stefania Zicca, Giuliana Loconsole, Beatriz Navarro, and Francesco Di Serio Assessment of Genetic Variability of Citrus tristeza virus by SSCP and CE-SSCP Elisavet K Chatzivassiliou and Grazia Licciardello Identification and Characterization of Resistance-Breaking (RB) Isolates of Citrus tristeza virus Maria Saponari, Annalisa Giampetruzzi, Vijayanandraj Selvaraj, Yogita Maheshwari, and Raymond Yokomi Genotyping Citrus tristeza virus Isolates by Sequential Multiplex RT-PCR and Microarray Hybridization in a Lab-on-Chip Device Giuseppe Scuderi, Antonino F Catara, and Grazia Licciardello 10 Rapid and Sensitive Detection of Citrus tristeza virus Using Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay Dilip Kumar Ghosh, Ashish Warghane, and Kajal Kumar Biswas 11 Amplification and Cloning of Large cDNA Fragments of the Citrus tristeza virus Genome Munir Mawassi, Sabrina Haviv, and Ludmila Maslenin 12 Bioinformatic Tools and Genome Analysis of Citrus tristeza virus Ana Bele´n Ruiz-Garcı´a, Rachelle Bester, Antonio Olmos, and Hans Jacob Maree vii v ix 15 29 55 67 79 105 127 143 151 163 viii Contents Analysis of Genotype Composition of Citrus tristeza virus Populations Using Illumina Miseq Technology David A Read and Gerhard Pietersen 14 Citrus tristeza virus: Host RNA Silencing and Virus Counteraction ˜ a, Luis Navarro, Susana Ruiz-Ruiz, Beatriz Navarro, Leandro Pen Pedro Moreno, Francesco Di Serio, and Ricardo Flores 15 Proteomic Response of Host Plants to Citrus tristeza virus Milena Santos Doria and Carlos Priminho Pirovani 16 Gene Expression in Citrus Plant Cells Using Helios® Gene Gun System for Particle Bombardment Yosvanis Acanda, Chunxia Wang, and Amit Levy 17 Methods for Producing Transgenic Plants Resistant to CTV Nuria Soler, Montserrat Plomer, Carmen Fagoaga, Pedro Moreno, ˜a Luis Navarro, Ricardo Flores, and Leandro Pen 13 Index 179 195 209 219 229 245 Contributors YOSVANIS ACANDA  Department of Plant Pathology, Citrus Research and Education Center, University of Florida, Lake Alfred, FL, USA MOSHE BAR-JOSEPH  The S Talkowski Laboratory, Department of Plant Pathology and Weed Research, The Volcani Center, Agricultural Research Organization, Bet Dagan, Israel EDSON BERTOLINI  Departamento de Fitossanidade, Faculdade de Agronomia, Universidade Federal Rio Grande Sul (UFRGS), Porto Alegre, RS, Brazil RACHELLE BESTER  Department of Genetics, Stellenbosch University, Matieland, South Africa; Agricultural Research Council, Infruitec-Nietvoorbij: Institute for Deciduous Fruit, Vines and Wine, Stellenbosch, South Africa KAJAL KUMAR BISWAS  Division of Plant Pathology, Advanced Centre for Plant Virology, ICAR Indian Agricultural Research Institute, New Delhi, India MARIANO CAMBRA  Plant Protection and Biotechnology, Department of Virology and Immunology, Centre of Moncada, Instituto Valenciano de Investigaciones Agrarias (IVIA), Valencia, Spain ANTONINO F CATARA  Department of Phytosanitary Sciences, University of Catania, Catania, Italy; Science and Technology Park of Sicily, Catania, Italy ELISAVET K CHATZIVASSILIOU  Plant Pathology Laboratory, Faculty of Crop Science, Agricultural University of Athens, Athens, Greece FRANCESCO DI SERIO  Dipartimento di Scienze del Suolo, della Pianta e degli Alimenti, ` di Bari “Aldo Moro”, Bari, Italy; Istituto per la Protezione Sostenibile delle Universita Piante, Consiglio Nazionale delle Ricerche, Bari, Italy MILENA SANTOS DO´RIA  Centro de Biotecnologia e Gene´tica, Universidade Estadual de Santa Cruz, Ilhe´us, BA, Brazil CARMEN FAGOAGA  Facultad de Veterinaria y Ciencias Experimentales, Universidad Catolica de Valencia (UCV), Valencia, Spain RICARDO FLORES  Instituto de Biologı´a Molecular y Celular de Plantas, Universidad Polite´ cnica de Valencia-Consejo Superior de Investigaciones Cientı´ficas, Valencia, Spain DILIP KUMAR GHOSH  Plant Virology Laboratory, ICAR-Central Citrus Research Institute, Nagpur, India ANNALISA GIAMPETRUZZI  Istituto per la Protezione Sostenibile delle Piante, Consiglio Nazionale delle Ricerche, Bari, Italy SABRINA HAVIV  The S Tolkowsky Laboratory, Department of Plant Pathology and Weed Research, The Volcani Center, Agricultural Research Organization, Bet Dagan, Israel AMIT LEVY  Department of Plant Pathology, Citrus Research and Education Center, University of Florida, Lake Alfred, FL, USA GRAZIA LICCIARDELLO  Consiglio per la Ricerca in agricoltura e l’Analisi dell’Economia Agraria, Centro di Olivicoltura, Frutticoltura e Agrumicoltura (CREA-OFA), Acireale (Catania), Italy GIULIANA LOCONSOLE  Dipartimento di Scienze del Suolo, della Pianta e degli Alimenti, ` di Bari “Aldo Moro”, Bari, Italy Universita YOGITA MAHESHWARI  San Joaquin Valley Agricultural Sciences Center, USDA-ARS, Parlier, CA, USA ix 232 Nuria Soler et al 19 Aluminum foils 2.2 Plants For juvenile transformation: 12-month-old citrus seedlings grown in a temperature-controlled greenhouse for transgenic plants with 24–27/18–20  C day/night temperature and a relative humidity between 60 and 80% For mature transformation, plants propagated in the greenhouse (18–27  C) by grafting of buds from adult citrus trees on a vigorous rootstock, such as rough lemon (C jambhiri Lush.) or C volkameriana Ten and Pasq (see Note 1) For in vitro grafting: seedlings of Troyer citrange germinated in vitro on seed germination medium (SGM) and grown in the dark for weeks (see Note 2) For greenhouse grafting: seedlings of a vigorous rootstock, such as Carrizo citrange or rough lemon germinated in nursery and grown under greenhouse conditions (18–27  C) for approx months 2.3 Tissue Culture Media and Components Inoculation medium (IM) (pH 5.7) MS salts [13] (see Note 3), 100 mg/L myoinositol, 0.2 mg/L thiamine hydrochloride, mg/L pyridoxine hydrochloride, mg/L nicotinic acid (see Note 4), and 3% (w/v) sucrose Co-cultivation medium (CM) (pH 5.7): MS salts [13] (see Note 3), 100 mg/L myoinositol, 0.2 mg/L thiamine hydrochloride, mg/L pyridoxine hydrochloride, mg/L nicotinic acid (see Note 4), 3% (w/v) sucrose, mg/L indole-3-acetic acid, mg/L 2-isopentenyl-adenine, and mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) (see Note 5), 0.8% (w/v) agar Shoot regeneration medium (SRM) (pH 5.7): MS salts [13] (see Note 3), 100 mg/L myoinositol, 0.2 mg/L thiamine hydrochloride, mg/L pyridoxine hydrochloride, mg/L nicotinic acid (see Note 4), and 3% (w/v) sucrose Add the hormones according to the citrus type used, for example, mg/L benzylaminopurine (BAP) (see Note 5) for Mexican lime, mg/L benzylaminopurine (BAP) plus 0.3 mg/L naphthalene-acetic acid (NAA) for sour orange, and mg/L benzylaminopurine (BAP) plus 0.5 mg/L naphthalene-acetic acid (NAA) for navel sweet orange, 1% (w/v) agar After the sterilization, add 100 mg/L kanamycin, 250 mg/L vancomycin and 250 mg/L cefotaxime (see Note 6) Seed germination medium (SGM) (pH 5.7): MS salts [13] (see Note 3), 1% (w/v) agar Cover the flask with aluminum foil, and melt the medium in an autoclave during min, at 121  C Distribute on 25  125 mm glass tubes, 25 mL of medium per tube, and cover the tubes with caps Then, proceed to Transgenic Plants Resistant to CTV 233 sterilization of the tubes with the medium in an autoclave during 15 min, at 121  C Micrografting medium (pH 5.7): MS salts [13] (see Note 3), 100 mg/L myoinositol, 0.2 mg/L thiamine hydrochloride, mg/L pyridoxine hydrochloride, mg/L nicotinic acid (see Note 4), and 7.5% (w/v) sucrose Shake the flask and its contents gently by hand, and distribute on glass tubes, 25 mL of medium per tube Prepare pieces of circular papers with the diameter a bit larger than that of the test tubes (90 mm), and make two small holes in the center of each These pieces of paper should serve as brackets for the grafts Insert the paper inside each tube and cover the tubes with caps Then, proceed to the sterilization of the tubes with the medium and paper pieces in an autoclave during 15 min, at 121  C mg/100 mL 2,4-dichlorophenoxyacetic acid (2,4-D) stock solution: dissolve in few drops of dimethyl sulfoxide (DMSO) Adjust volume with double-distilled water Store at  C (see Note 5) mg/100 mL indole-3-acetic acid (IAA) stock solution (see Note 5) mg/100 mL 2-isopentenyl-adenine stock solution (see Note 5) mg/100 mL benzylaminopurine (BAP) stock solution: dissolve the powder in a few drops of M NaOH Complete final volume with double-distilled water Store at  C (see Note 5) 10 mg/100 mL naphthalene-acetic acid (NAA) stock solution: dissolve the powder in a few drops of M NaOH Complete final volume with double-distilled water Store at  C (see Note 5) 11 100 mg/mL kanamycin sulfate stock solution: dissolve g of powder in 10 mL of double-distilled water Sterilize by filtration through a 0.2 μm membrane, make mL aliquots in sterile Eppendorf tubes, and store at À20  C (see Note 6) 12 250 mg/mL cefotaxime stock solution: dissolve g of powder in mL of double-distilled water Sterilize by filtration through a 0.2 μm membrane, make mL aliquots in sterile Eppendorf tubes, and store at À20  C (see Note 6) 13 250 mg/mL vancomycin stock solution (see Note 6) 2.4 Bacterial Strain and Vector Bacterial strain: Agrobacterium tumefaciens EHA105, which is a disarmed derivative of A tumefaciens A281 (see Note 7) This strain holds chromosomic resistance to nalidixic acid and rifampicin Binary vector: the T-DNA of the binary plasmid usually contains, apart from the expression cassette/s of interest, a 234 Nuria Soler et al selectable marker gene, such as neomycin phosphotransferase II (nptII), which confers resistance to kanamycin, and a reporter marker gene, such as β-D-glucuronidase (uidA) or green fluorescent protein (gfp), under the control of constitutive promoter and terminator sequences The binary plasmid is introduced into Agrobacterium by electroporation (see Note 8) 2.5 Culture Media for A tumefaciens Luria broth (LB) medium: dissolve 20 g of LB medium (see Note 9) in L of distilled water solution Distribute 100 mL of the medium on 200 mL flasks, cover with aluminum foil, and proceed to the sterilization in an autoclave during 15 min, at 121  C Before using it, add 25 mg/L each of kanamycin and nalidixic acid (or rifampicin) (see Note 6), under sterile conditions in a laminar flow cabinet (see Note 10) In the case of solid LB medium to be distributed in plates, transfer the LB solution to a L flask, and add 1% (w/v) agar 100 mg/mL kanamycin sulfate stock solution (see Note 6) 25 mg/mL nalidixic acid stock solution: dissolve 250 mg of powder in a few drops of M NaOH, and then add water to complete 10 mL Sterilize by filtration, make mL aliquots in sterile Eppendorf tubes, and store at –20  C (see Note 6) 2.6 Other Solutions Disinfection solution: To prepare L of solution, add about 600 mL of distilled water to a L glass, and add 2% (v/v) (stems) or 0.5% (v/v) (seeds) sodium hypochlorite plus 0.1% (v/v) Tween 20 Transfer the solution to a L test tube, and make up to L with water Mix the solution in the L glass with a magnetic stirrer X-Gluc (5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid) solution: 100 mM Tris–HCL buffer pH 7, 50 mM NaCl, 0.01% Triton X-100, 0.65 g/L potassium ferricyanide, and 10.41 g/L X-Gluc substrate (dissolved in 40 mL/L of dimethylformamide) 2.7 Equipments Laminar flow cabinet Culture chamber allowing temperature, humidity, and illumination control Standard conditions are fixed at 26  C, 60% relative humidity, and a 16-h photoperiod at 45 μEmÀ2 sÀ1 illumination Incubators allowing temperature control at 26–28  C Incubators allowing low irradiance (20 μmol mÀ2 sÀ1), 16-h photoperiod, 60% relative humidity, and 24  C Orbital shaker allowing temperature and speed control Spectrophotometer Transgenic Plants Resistant to CTV 235 Electroporator Stereomicroscope Stereomicroscope equipped with a 480/40 nm (460–500 nm) exciter filter, a 505 nm dichromatic beam splitter, and a 510 nm barrier to check GFP fluorescence Methods 3.1 Agrobacterium Preparation Carry out the procedures with the bacterium under sterile conditions in a laminar flow cabinet From a stock of Agrobacterium cultured with the corresponding construct in a binary plasmid, take a loopful of bacteria, and streak in a Petri dish with solid LB medium plus kanamycin and nalidixic acid Cultivate Agrobacterium during 48 h, at 28  C Sub-cultivate an isolated bacterial colony in 100 mL of liquid LB medium with kanamycin and nalidixic acid, during 48 h in an orbital shaker at 200 rpm and 28  C Repeat sub-cultivation of an aliquot Read the absorbance (OD) at 600 nm in a spectrophotometer until the exponential growth reaches values between 0.4 and 0.8 Calculate bacterial concentration using the growth curve (see Note 11) Pellet the bacterial culture at 1900  g for 10 in 40 mL sterile centrifuge tubes with cap, discard the supernatant, and resuspend and dilute the pellet with IM to a concentration of approximately  107 cells/mL 3.2 Plant Material Preparation Cut stem pieces of about 20 cm in length, and strip them of leaves and thorns (see Note 12) Rub the stem pieces with a brush in soapy water, and rinse with water Transfer the stem pieces to a L test tube Carry out the following procedures under sterile conditions in a laminar flow cabinet For disinfection, add the disinfection solution with sodium hypochlorite plus Tween 20, cover the test tube with Parafilm, shake it gently by hand several times during 10 min, and rinse three times with sterile distilled water Cut each stem piece transversely in internodal stem segments of cm, using tweezers and small garden scissors (or scalpel) on sterile paper, and keep explants in sterile humid plates until all stem pieces have been prepared 236 Nuria Soler et al 3.3 Inoculation, Co-cultivation, and Selection Carry out the following procedures under sterile conditions in a laminar flow cabinet, using sterile tweezers Place the internodal stem segments in sterile 100 mm diameter Petri dishes containing 15 mL of the bacterial suspension in inoculation medium, using sterile tweezers Incubate with gentle shaking for 15 at room temperature Blot dry the infected explants on sterile filter paper (see Note 13) Place the explants horizontally on 90 mm Petri dishes containing CM, and seal the plates with Parafilm Incubate for a 3-day co-cultivation period, under low irradiance (20 μmol mÀ2 sÀ1), 16-h photoperiod, 60% relative humidity, and 24  C After the 3-day co-cultivation period, transfer the explants to Petri dishes with SRM (10 explants per dish), at 26  C for weeks under dark conditions (see Note 14) Transfer the Petri dishes to 16-h photoperiod, 45 μmol mÀ2 sÀ1 illumination, 60% RH, and 26  C Transfer the explants to fresh SRM each 3–4 weeks Any contaminated explant should be discarded 3.4 Recovery of Whole Transgenic Plants Shoots should develop from cambial callus at the cut ends of explants about 3–5 weeks after co-cultivation (see Fig 1a) Screen the transgenic nature of the regenerated shoots according to the reporter marker included in the binary vector, β-Dglucuronidase (uidA), or green fluorescent protein (gfp): (a) Histochemical β-D-glucuronidase (GUS) assay: l Cut a small piece of the regenerated shoot under sterile conditions Perform a GUS assay incubating the plant material at 37  C in mM X-Gluc solution, overnight [14] After that, rinse the piece with 100 mM Tris–HCL buffer, pH 7, in order to stop the reaction l Fix the tissue with 1% glutaraldehyde in the same buffer during 2–3 h l Rinse three times with 100 mM Tris–HCl buffer, pH l Rinse with ethanol dilutions (30%, 50%, 70%, 90%, 100%) l Examine the explants under a stereomicroscope, and select the shoots exhibiting blue color (see Fig 1b) (b) Testing green fluorescent protein (GFP) expression: l Examine under a stereomicroscope equipped with a GFP-Plus Fluorescence module Select the shoots exhibiting bright green fluorescence (see Fig 1c) Transgenic Plants Resistant to CTV 237 Fig (a) Callus formation at the cut ends of explants; (b) after performing the histochemical β-D-glucuronidase (GUS) assay, each blue spot indicates a transgenic event at the cambial callus; (c) GFP-positive shoot exhibiting bright green fluorescence; (d) shoot grafted in vitro onto a Troyer citrange decapitated seedling GUS- or GFP-negative shoots are considered as non-transformed or transgene silenced (see Note 15) Graft in vitro apical portions of the GUS- or GFP-positive shoots onto decapitated seedlings of Troyer citrange (see Fig 1d) Rootstock preparation is as follows: peel seeds, remove both seed coats, disinfect for 10 in disinfection solution, and rinse three times with sterile distilled water Sow individual seeds onto 25 mL aliquots of SGM contained in 25  150 mm glass tubes, and incubate at 27  C in the dark for weeks Decapitate seedlings leaving 1–1.5 cm of the epicotyls Shorten the roots to 4–6 cm, and remove the cotyledons and their axillary buds, and insert the decapitated seedlings in one of the holes of the piece of paper leaving just the roots immersed in the SGM below the paper Place the regenerated shoot onto the apical end of the cut surface of the decapitated epicotyls (see Note 16) Culture grafted plants in micrografting medium, and maintain at 25  C, 16 h photoperiod, and 45 μEmÀ2 sÀ1 of illumination 238 Nuria Soler et al (see Note 17) Scions develop two to four expanded leaves 3–4 weeks after grafting Graft the in vitro grown plants onto vigorous rootstocks germinated in nursery and grown under greenhouse conditions (see Note 18) Putative transgenic plants should be assayed by polymerase chain reaction (PCR) to detect the presence of the transgene (s) (see Note 19) Southern blot analyses must be performed to confirm the stable integration of the transgene(s), as well as the number of copies integrated in the genome In the case of the intron-hairpin construction carrying full-length, untranslatable versions of the genes p25, p20, and p23 from CTV, analysis of the accumulation of transgene-derived siRNAs by northern blot is recommendable in order to determinate the siRNA accumulation levels derived from p25, p20, and p23 transgenes (see Note 20) 3.5 Virus Resistance Analyses Propagate 10–20 buds from each transgenic line by grafting onto Carrizo citrange seedlings, and keep in a greenhouse at 24–26  C/16–18  C (day/night), 60–80% relative humidity, and natural light When new shoots are 30–40 cm long, graft-inoculate the homogeneous propagations from each transgenic line plus empty-vector and non-transgenic controls with two bark chips of 0.75–1 cm2 in size from a CTV-infected source plant (e.g., infected with CTV T36) (see Note 21) Three months after challenge inoculation, remove one inoculum bark chipper challenged plant Confirm the presence of the virus in the inoculum bark chip by RT-qPCR with specific primers [15] Evaluate virus accumulation in leaves by DAS-ELISA with the monoclonal antibodies 3DF1 + 3CA5 [16] and RT-qPCR in at least three consecutive flushes spanning over a 1-year period A plant is considered CTV-infected when the absorbance at 405 nm is at least twofold that of non-inoculated controls in DAS-ELISA tests Monitor CTV symptoms in at least three consecutive flushes spanning over a 1-year period Symptom intensity in Mexican lime can be rated on a 0–3 scale in which indicates a complete absence of symptoms, mild vein clearing, moderate vein clearing with young leaf epinasty and adult leaf cupping/distortion, and severe symptoms including vein corking and stunting (see Fig 2) Transgenic Plants Resistant to CTV 239 Fig (a, b) CTV symptoms in leaves and shoots from control Mexican lime lines graft-inoculated with (a) CTV-T36 and (b) CTV-T318a, showing vein clearing (top), vein corking (middle), and leaf distortion (bottom right) or epinasty (bottom left); (c) asymptomatic leaves and shoots from resistant transgenic Mexican lime lines carrying full untranslatable versions of genes p25, p20, and p23 plus the 30 -UTR in sense and antisense orientation separated by an intron Notes From these invigorated mature plants, it is recommended to use the newly elongated first flushes, in order to achieve the maximum transformation and regeneration frequency The seeds are peeled and put in small nets before the disinfection process, to facilitate manipulation After weeks in the dark at 27  C, the seedlings should be transferred to the refrigerator at 4–8  C to slow growth They can be used within 15 days to a month In order to make easier the preparation of the media, we usually maintain separate stock solutions of MS macroelements, microelements, and FeNa-EDTA These stock solutions are maintained at  C, and check for contamination before each use A 240 Nuria Soler et al convenient alternative is using commercial MS salts, weighed and presented in individual bags for L We usually prepare a vitamin stock solution with the mix of thiamine hydrochloride, pyridoxine hydrochloride, and nicotinic acid at the proper concentration This stock solution is maintained at  C and checked for contamination before each use The hormone stock solutions are maintained at  C and checked for contamination before each use It is not recommended to maintain the hormone stock solutions more than month The concentration of auxins in CM can be increased slightly if no or low cambial callus is induced in the explants, depending of the citrus type used Hormones in SRM could be added (NAA) or their concentration increased (BAP) if shoot regeneration from the cambial callus is insufficient, depending on the citrus type used Kanamycin is used for the selection of transgenic events, whereas vancomycin and cefotaxime to control bacterial growth We prefer to make mL aliquots of antibiotic solutions to avoid possible contaminations In the case of tissue culture media, one or two full aliquots will serve to reach the final desired concentration for L of medium Agrobacterium strain A281 was shown to be the most virulent in the infection of citrus types This is the reason of using a disarmed derivative of A281 for our transformation experiments Bacterial resistance to kanamycin (or any other present in the corresponding binary plasmid), together with the chromosomic resistance of EHA105 to nalidixic acid (or rifampicin), is used to select the bacteria As example of expression cassette with the aim of achieving resistance to CTV, the preferred strategy adopted was the transformation with an intron-hairpin vector carrying full-length, untranslatable versions of the genes p25, p20, and p23 from CTV strain T36 to silence the expression of these viral genes in CTV-infected cells [8].This expression cassette was cloned in a pCAMBIA binary vector, carrying the selectable marker gene neomycin phosphotransferase II (nptII) and the reporter marker gene β-D-glucuronidase (uidA) Luria broth (LB) medium: 10 g/L of tryptone, g/L of yeast extract, 10 g/L of NaCl, and pH 7.5 10 The LB liquid medium can be maintained at RT and checked for contamination before each use Before use, add the antibiotics to select for binary plasmid and Agrobacterium resistance Transgenic Plants Resistant to CTV 241 11 It is convenient to determine the growth curve (A600 vs bacterial cell concentration) for the bacterial strain used in the transformation experiment Bacterial culture should grow to the exponential phase to play all its infectious potential (A600 between 0.1 and 1.0 in the case of strain EHA105) 12 Flushes should be in a good ontological state, neither too tender (they would not bear Agrobacterium infection) nor too lignified, as to keep an acceptable regenerative potential 13 We use sterile soft paper towels to help explants to dry It is important to eliminate any bacterial liquid residue, as it can be a source of bacterial overgrowth during co-cultivation 14 Culture of explants in the dark improves callus formation and the progress of transformation events to regenerate transgenic shoots Four weeks in the dark is usually the most frequent period for callus formation, but it depends on the citrus genotype used It is recommended to keep the explants in darkness until they develop a prominent visible callus formed at the cambial ring 15 We must take into consideration that GUS- or GFP-positive explants could show different levels of expression, chimeric tissues, and false negatives (with gene silencing affecting to the reporter transgene) PCR analysis could provide accurate results, but it may also involve selection of regenerants with silenced transgenes For a first screening, GUS and GFP are the most appropriate reporters 16 For the in vitro grafting of long shoots (0.5–1 cm), cutting the basal end as a wedge and introducing it into a small longitudinal incision practiced on the upper part of the rootstock can also be helpful to facilitate vascular contact and success of the graft 17 During development of the grafts, it is necessary to check them periodically and to remove, by using sterile small scissors, any shoot coming from the rootstock The growth of other shoots could weaken the connection between rootstock and transgenic scion 18 To ensure a rapid and successful acclimatization, it is important to follow good greenhouse practices We recommend working with sterile potting substrate, vigorous seedlings, and keeping grafted plants in plastic bags that will be progressively opened over approximately month This will help to maintain optimal moisture and temperature and will facilitate a gradual process of acclimatization 19 PCR analysis could be carried before grafting of the in vitro grown plants onto vigorous rootstocks in the greenhouse 242 Nuria Soler et al After that process, PCR analysis could be repeated in order to assure the transgenic nature of each line after the in vitro growth 20 The extent of transgene silencing is assessed by Northern blot analysis of siRNAs derived from p25, p20, and p23, with transformants of interest usually requiring high siRNA levels of the three transgene fragments This strategy shows that targeting by RNAi the three viral silencing suppressors simultaneously is critical for developing transgenic resistance to CTV, although the involvement of other concurrent mechanisms cannot be excluded [8] 21 Resistance to CTV of a transgenic Mexican lime line carrying an intron-hairpin construct is very much influenced by the sequence identity between the transgene and the challenging CTV strain used [8] Moreover, CTV causes different symptoms depending on the strain used for challenge inoculation (see Fig 2) [8] References Moreno P, Ambro´s S, Albiach-Martı´ MR et al (2008) Citrus tristeza virus: a pathogen that changed the course of the citrus industry Mol Plant Pathol 9:251–268 Yoshida Y (1985) Inheritance of susceptibility to Citrus tristeza virus in trifoliate orange Bull Fruit Tree Res Stn 12:17–25 Yoshida T (1993) Inheritance of immunity to Citrus tristeza virus of trifoliate orange in some citrus intergeneric hybrids Bull Fruit Tree Res Stn 25:33–43 Gmitter FG, Xiao SY, Huang S et al (1996) A localized linkage map of the Citrus tristeza virus resistance gene region Theor Appl Genet 92:688–695 Fire A, Xu S, Montgomery MK et al (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans Nature 391:806–811 Smith NA, Singh SP, Wang MB et al (2000) Total silencing by intron-spliced hairpin RNAs Nature 407:319–320 Soler N, Fagoaga C, Chiibi S et al (2011) RNAi-mediated protection against Citrus tristeza virus in transgenic Citrus plants In: Erdmann V, Barciszewski J (eds) Non coding RNAs in plants RNA Technologies Springer, Berlin, Heidelberg Soler N, Plomer M, Fagoaga C et al (2012) Transformation of Mexican lime with an intron-hairpin construct expressing untranslatable versions of the genes coding for the three silencing suppressors of Citrus tristeza virus confers complete resistance to the virus Plant Biotechnol J 10:597–608 Fagoaga C, Lo´pez C, Hermoso de Mendoza A et al (2006) Post-transcriptional gene silencing of the p23 silencing suppressor of Citrus tristeza virus confers resistance to the virus in transgenic Mexican lime Plant Mol Biol 60:153–165 10 Ruiz-Ruiz S, Navarro B, Gisel A et al (2011) Citrus tristeza virus infection induces the accumulation of viral small RNAs (21–24-nt) mapping preferentially at the 30 -terminal region of the genomic RNA and affects the host small RNA profile Plant Mol Biol 75:607–619 11 Lu R, Folimonov A, Shintaku M et al (2004) Three distinct suppressors of RNA silencing encoded by a 20-kb viral RNA genome Proc Natl Acad Sci U S A 101:15742–15747 ˜ a L, Cervera M, Fagoaga C et al (2008) 12 Pen Citrus In: Kole C, Hall TC (eds) Compendium of transgenic crop plants: tropical and subtropical fruits and nuts Blackwell Publishing, Oxford, pp 1–62 13 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures Physiol Plant 15:473–479 14 Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: b-glucuronidase as a Transgenic Plants Resistant to CTV sensitive and versatile gene fusion marker in higher plants EMBO J 6:3901–3907 15 Domı´nguez A, Hermoso de Mendoza A, Guerri J et al (2002) Pathogen-derived resistance to Citrus tristeza virus (CTV) in transgenic Mexican lime (Citrus aurantifolia 243 (Christ.) swing.) plants expressing its p25 coat protein gene Mol Breed 10:1–10 16 Cambra M, Garnsey SM, Permar TA et al (1990) Detection of Citrus tristeza virus (CTV) with a mixture of monoclonal antibodies Phytopathology 80:103 INDEX A Antibodies MCA13 16, 106 monoclonal 16, 55, 57, 98, 106, 237 polyclonal 98 Aphid brown citrus aphid (Toxoptera citricidus) 29, 143 capturing and identification 38 cotton aphid (Aphis gossypii) 29, 30, 32, 40 identification 38, 39 rearing aphid colonies 40 RT-qPCR from aphids 47, 48 total RNA extraction from aphids 47 vector transmission 31, 32, 42, 43 B Bioinformatic analysis 121, 163–178, 182, 185–188, 190 pipelines 164, 172 software 111 tool 163–178 Biological indexing v, 55, 80, 122, 144 seedling yellows (SY-CTV) 15, 16, 19, 23, 67, 79, 121, 127, 151, 203 severe strain 179, 197 stem pitting (SP-CTV) 19 Closteroviridae family v, 2, 7–9, 127, 143, 152, 195, 219, 230 Cross-protection 16, 25, 80, 179, 197 CTV strain characterization capillary electrophoresis-single-strand conformation polymorphisms (CE-SSCP) data analysis 97 primer design .94, 95 multiple molecular marker (MMM) 129 phylogenetic markers 180, 186 restriction fragment length polymorphism (RFLP) 128, 180 single nucleotide polymorphisms (SNPs) 175 single-strand conformation polymorphism (SSCP) vi, 80, 81, 89–94, 128, 180 E Electron microscopy 144 G C cDNA fragments v, 2, 80, 151–161 probes v synthesis v, 3, 36, 37, 45, 71, 72, 74, 80, 82, 88, 89, 107, 113, 153, 160 Citrus tristeza virus (CTV) complex 79, 219, 230 detection .v, 2, 43–48, 56, 57, 64, 82, 106, 128, 144, 164 diagnosis .v, 2, 56, 57, 163 exotic isolates 56 genome v, 105, 115, 127, 132, 143, 152, 153, 157–159, 164, 175, 177, 180, 186, 187, 201 genotypes 16, 43, 106, 129, 134, 140, 152, 160, 180 interference 195–204 mild strain 152, 197 resistance breaking (RB) 15, 105–125 Gel electrophoresis agarose gel electrophoresis 82, 90, 91, 145, 147, 148 capillary electrophoresis vi, 81, 83, 87, 88, 96 non-denaturing polyacrylamide gel preparation 83, 91–92 polyacrylamide gel electrophoresis vi, 91–92, 210 silver staining of SSCP gel 83, 92–93 SSCP gel analysis of electrophoretic patterns 93, 94 2D PAGE 212 H Host plants alemow (C macrophylla) 16, 17, 21, 22, 25, 32, 43 citranges 15, 230, 231 citron (C.medica) 17, 21, 22, 25 Antonino F Catara et al (eds.), Citrus Tristeza Virus: Methods and Protocols, Methods in Molecular Biology, vol 2015, https://doi.org/10.1007/978-1-4939-9558-5, © Springer Science+Business Media, LLC, part of Springer Nature 2019 245 CITRUS TRISTEZA VIRUS: METHODS 246 Index AND PROTOCOLS Host plants (cont.) citrumelo 15, 230, 231 Citrus excelsa 17 Duncan grapefruit (C paradisi) .38, 49, 109, 122, 225, 230 Eureka lemon 23 Fortunella .v, 15 lisbon lemon 23 Madam Vinous sweet orange (C sinensis) 38, 109 mandarin (C reticulate) 127, 231 mexican lime (C aurantifolia) 32, 33, 38, 49, 68, 109, 196–198, 201, 203, 230, 237, 239, 242 Poncirus trifoliata 32, 105, 109, 196, 230 rough lemon (C jambhiri) .16, 17, 232 sour orange (C aurantium) 1, 2, 15–17, 19, 21–24, 38, 49, 109, 127, 144, 151, 196, 198, 201, 203, 231 trifoliate orange vi, 15, 105, 122, 196, 201 volkameriana lemon (C volkameriana) 17 Host RNA silencing vi, 195–204 L Lab-on-chip (LoC) microarray hybridization vi, 127 optical detection 137 primer and probe design 132–135 Loop mediated isothermal amplification (LAMP) 144, 145, 147–150 M MicroRNAs (miRNAs) 67, 197, 201 N Next generation sequencing (NGS) bioinformatic data analysis 163, 168–172 contig identification 171, 176 de novo assembly 169–171, 174, 176 high throughput sequencing (HTS) .vi, 115, 152, 163 Illumina Miseq technology 179–193 raw data analysis and clean-up 168–170 reads mapping 171–173, 186 separation of low and high molecular weight RNAs 110, 117–118 small RNA library construction 120 small RNA purification and sequencing 115–121 P PCR amplification of large CTV cDNAs 156–159 asymmetric labeled RT-PCR 136 colony PCR 89 IC Nested RT-PCR 56 multiplex Real-Time RT-PCR 73, 74 multiplex RT-PCR 127, 140 overlapped-PCR 153, 158 quadruplex RT-PCR 134, 136 quantitative Real-Time RT PCR (RT-qPCR) .37, 45, 47, 48, 69, 107, 108, 112, 122, 198, 237 reverse-transcription polymerase chain (RT-PCR) v, vi, 45, 55–64, 71–77, 80, 81, 87–89, 95, 100, 107, 113–115, 127, 144, 152–155, 157, 172 Phenotyping, see Biological indexing Plant inoculation grafting and budding inoculation 17–19 graft inoculation 21, 37, 48 growing plants 42 leaf disc inoculation 20, 21 leaf grafting 20, 21 leaf-piece graft 19, 20, 37, 42, 122 mechanical transmission .18, 21 micro-grafting 233, 237 seedling preparation 18 soil and fertigation system 42 symptom assessment 22–23 testing cross protective isolates 21 Probe hybridization v, 129, 131, 132, 135, 203 TaqMan 46, 48, 59, 64, 68, 73, 76, 106, 112, 113 Protein expression 220, 223 extraction 211–214 host proteome analysis 209–218 isoelectric focusing 210 proteomic response 209–218 separation of proteins 214–215 treatment of spots 212, 215 R RNA double strands (ds)-RNA 2, 3, 87, 92, 98, 164, 172, 197, 199, 203, 230 extraction/isolation 36, 37, 47, 82, 108–109, 115–117, 132, 145, 146, 148, 149, 153–156, 160, 166–168 interference (RNAi) 230, 242 RNA silencing vi, 67, 68, 140, 195–204, 230 silencing suppressor 67, 68, 202 CITRUS TRISTEZA VIRUS: METHODS S Sanger sequencing 172, 178, 182, 184, 185, 191 Serological assay DASI-ELISA 43 DTBIA 87, 89 enzyme-linked immunosorbent assay (ELISA) 43, 55 immunocapture (IC) 33, 45, 51, 56 virus release procedure after ELISA or DTBIA 87 Silencing suppressor proteins 67, 230 Small interfering RNA (siRNAs) 115–117, 164, 172, 197, 202, 203, 220, 238, 242 Super-Infection exclusion, see Cross-protection T Tissue print and squash 55–64 Total nucleic acid (TNA) extraction 69, 72, 73, 106, 111 Transformation agrobacterium-mediated transformation 231 biolistic bombardment vi electroporation 234 Escherichia coli transformation 159, 160 green fluorescent protein (GFP) 234 Gus (beta-glucuronidase) 202 intron-hairpin vector 240 mature tissue transformation 232 AND PROTOCOLS Index 247 transgenic plants resistant to CTV vi, 229–242 transient protein expression vi, 220 V Viroid Apscaviroid 68 Citrus bark cracking viroid (CBCVd) 68 Citrus bent leaf viroid (CBLVd) 68 Citrus dwarfing viroid (CDVd) 68 Citrus exocortis viroid (CEVd) v, 67–69, 71, 73, 74 Citrus viroid V (CVd-V) 68 Citrus viroid VI (CVd-VI) 68 differentiation of HSVd variants 68, 71, 72, 74–77 Hop stunt viroid (HSVd) v, 68–77 Hostuviroid 68 Pospiviroid 67 viroid detection 67–77 Virus beet yellows virus (BYV) v, 2, 7, 11 Crinivirus 8–11 Grapevine leafroll-associated virus (GLRaV-2) .8, 11 Grapevine leafroll-associated virus (GLRaV-7) 8, 10, 12 plum pox virus (PPV) 81 velarivirus 8, 10, 12 ... (Catania), Italy ISSN 106 4-3 745 ISSN 194 0-6 029 (electronic) Methods in Molecular Biology ISBN 97 8-1 -4 93 9-9 55 7-8 ISBN 97 8-1 -4 93 9-9 55 8-5 (eBook) https://doi.org/10.1007/97 8-1 -4 93 9-9 55 8-5 © Springer Science+Business... Genotyping Citrus tristeza virus Isolates by Sequential Multiplex RT-PCR and Microarray Hybridization in a Lab-on-Chip Device Giuseppe Scuderi, Antonino F Catara, and Grazia Licciardello... MV-1 v i rin V-2 Ra SV GL BY TICV LIYV C CTV BYVaV RLMoV BPYV PYVV SCFaV LCV 1000 1000 BYDV 997 CYSDV MVBaV 976 1000 ToCV Outgroup LChV-1 PBNSPaV GLRaV-4 CoV-1 Ve GLRaV-7 lar ivir us PMWaV-1
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