LARISSA GOULART ZANARDO
NEW INSIGHTS OF Cowpea mild mottle virus (CPMMV) INFECTION IN SOYBEAN: THE INVOLVEMENT OF VIRAL REPLICASE IN SYMPTOMS INDUCTION AND ITS INTERACTION WITH HOST FACTORS
Tese apresentada à Universidade Federal de Viçosa, como parte das exigências do Programa de Pós-Graduação em Genética e Melhoramento, para obtenção do título de Doctor Scientiae.
VIÇOSA MINAS GERAIS-BRASIL 2017
LARISSA GOULART ZANARDO
NEW INSIGHTS OF Cowpea mild mottle virus (CPMMV) INFECTION IN SOYBEAN: THE INVOLVEMENT OF VIRAL REPLICASE IN SYMPTOMS INDUCTION AND ITS INTERACTION WITH HOST FACTORS
Tese apresentada à Universidade Federal de Viçosa, como parte das exigências do Programa de Pós-Graduação em Genética e Melhoramento, para obtenção do título de Doctor Scientiae.
______Pedro Augusto Braga dos Reis Sérgio Hermínio Brommonschenkel
______Danielle Ribeiro de Barros Claudine Márcia Carvalho (Coorientadora)
______Francisco Murilo Zerbini Júnior (Orientador) AGRADECIMENTOS
Primeiramente à Deus pelo dom da vida, pelas oportunidades e possibilidade de recomeço a cada dificuldade.
Aos meus pais por serem um porto seguro, por me ouvirem e me apoiarem a cada dia, e principalmente, por me fazerem acreditar que era possível!
Aos meus irmãos pelo apoio, incentivo, conselhos e brincadeiras durante todos os anos de estudo.
Ao Bruno pelo amor e cuidado, pelo incentivo e paciência, pelos conselhos e por sonhar esse sonho comigo.
À Universidade Federal de Viçosa (UFV) pela formação e oportunidade.
Ao Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) e à
Comissão de Aperfeiçoamento de Pessoal do Nível Superior (CAPES) pela concessão das bolsas de estudos.
À Prof. Claudine Márcia de Carvalho por compartilhar seus conhecimentos e experiências, por acreditar e incentivar os trabalhos, pela dedicação, amizade e orientação valiosa.
Ao Prof. Francisco Murilo Zerbini, pela oportunidade e confiança, pelos ensinamentos, pela disponibilidade e suporte durante todo o curso.
Ao Prof. Eduardo S. G. Mizubuti pela paciência, atenção e disponibilidade, pelos ensinamentos e por compartilhar um pouco do seu conhecimento.
Aos amigos Talita, Fernanda, Diogo, César e Sarah, em especial, pelos incentivos constantes, pelos conselhos, conversas e brincadeiras diárias, pela ajuda nos trabalhos e por compartilharem suas experiências. Muito Obrigada!
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Ao Dr. Murilo Siqueira Alves por todo apoio e amizade, pelas repetições experimentais e biológicas e por acreditar e incentivar todos os trabalhos.
Aos amigos do Laboratório de Virologia Vegetal Adriana, Alison, Ana Carolina,
André, Angélica, Camila, Fábio, Glória, Hermano, Igor, João Paulo, Josiane, Márcio,
Osvaldo, Patrícia, Renan, Roberta, Silvia e Tarsiane pela amizade, companheirismo, brincadeiras e por tornarem o ambiente de trabalho mais agradável.
A todos que contribuíram e torceram por mim durante toda essa etapa, minha sincera gratidão.
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SUMÁRIO
ABSTRACT ...... vii RESUMO ...... ix INTRODUÇÃO...... 1 CHAPTER 1...... 3 Cowpea mild mottle virus – an emerging carlavirus ...... 4 Abstract ...... 4 Biology of the pathogen Cowpea mild mottle virus (CPMMV) ...... 7 Genomic organization ...... 7 Viral genome expression ...... 9 Taxonomy...... 11 Transmission mode...... 14 The emergence of CPMMV as threat to crops ...... 16 Host range and symptomatology ...... 17 The spread of CPMMV globally and in Brazil ...... 20 Phylogenetic relationship and viral strains...... 23 The recent emergence of CPMMV infecting transgenic beans in Brazil ...... 25 Control mechanisms and resistance...... 28 Perpectives ...... 31 References ...... 33 Figure Legends ...... 40 Figures...... 43
CHAPTER 2...... 47 Experimental Evolution of a Plant Virus Reveals Recombination-Driven Reduction in Virulence Accompanied by Increases in Diversity and Viral Fitness...... 47 Abstract ...... 49 Author summary ...... 50 Introduction ...... 50 Results and Discussion ...... 54 Serial Passages Lead to Reduced Virulence ...... 54 iv
Viral Accumulation and Vector Performance Increase once CPMMV Becomes Less Virulent ...... 55 Systemic Necrosis Has Features of the Hypersensitive Response (HR) ...... 57 ORF 1 Has High Genetic Polymorphism and is The Region Involved in Population Subdivision ...... 59 ORF1 is a Recombination Hotspot ...... 62 Some Codons Are Under Positive Selection in the Replicase Gene, But Adaptive Selection is Not Responsible for Maintaining the Diversity in ORF1 ...... 63 The Sites Associated with Phenotypic Change Differ Between Experiments 1 and 2, but are Propagated Across Extant Population by Recombination ...... 65 Material and methods ...... 69 References ...... 78 Figure Captions ...... 83 Figures ...... 94 CHAPTER 3...... 171 The proteins early-responsive to dehydration stress protein (GmERD4) and an inositol methyltransferase (GmIMT) are recruited during the replication of a Betaflexivirus in soybean...... 171 Abstract ...... 172 Introduction ...... 173 Material and Methods ...... 176 Results ...... 186 Isolation of soybean factors interacting with CPMMV RdRp domain using yeast two- hybrid system and protein identification ...... 186 RdRp domain is able to interact with GmERD4 and GmIMT by yeast two-hybrid ....187 The RdRp domain interacts in vivo with GmERD4 and GmIMT ...... 188 GmERD4 and GmIMT have a nucleic acid binding domain ...... 189 Individual localization of the RdRp domain, GmERD4 and GmIMT showed that they are in different subcellular compartment in the absence of infection ...... 190 GmERD4 and GmIMT expression is induced three and seven days after CPMMV infection ...... 192
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Overexpression of GmERD4, but not GmIMT, increased the viral accumulation of CPMMV ...... 192 Discussion...... 193 References ...... 198 Figure legends ...... 202 Figures ...... 206 CONCLUSÕES GERAIS...... 218
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ABSTRACT ZANARDO, Larissa Goulart, D.Sc., Universidade Federal de Viçosa, February, 2017. New insights of Cowpea mild mottle virus (CPMMV) infection in soybean: The involvement of viral replicase in symptoms induction and its interaction with host factors. Advisor: Francisco Murilo Zerbini Júnior. Co-advisers: Claudine Márcia Carvalho and Murilo Siqueira Alves.
Cowpea mild mottle virus (CPMMV, family Betaflexiviridae and genus Carlavirus) is an emerging virus in Brazil. The virus is transmitted by the whitefly Bemisia tabaci and it infects hosts preferentially of Fabaceae family, being found in different crops in almost all the continents in the world. In this work, several aspects about biology and genetics of CPMMV were dicussed and the CPMMV-host relationship was explored. CPMMV causes varied symptoms in plants in the fields and in greenhouse. The nature of this symptoms variation was determinate in this work using successive inoculations of a CPMMV Brazilian isolate causing necrosis in the CD206 soybean cultivar. Successive inoculations led to the isolate, which previously had caused necrosis to induce mild symptoms (mosaic and vein clearing). This was verified after six successive inoculations of a soybean sample from the field and also from a local lesion of a viral isolate, and thus submitted to a genetic bottleneck, in Nicotiana benthamiana leaves. We have shown that altering of symptoms pattern increased the fitness of the virus and vector, suggesting that the induction of different symptoms by viral variants is an adaptive advantage. The viral region involved in the induction of CPMMV symptoms was viral replicase (encoded by ORF1), different sites distributed throughout the recombinant blocks of ORF1 were associated with phenotype changes. In this work, also shought to the understanding about