Citrus leprosis : a model for the study of the plant/mite/virus interaction

Orientadores: Marcos Antonio Machado, Pedro Luis Ramos GonzálezTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A leprose dos citros, causada pelo citrus leprosis virus C (CiLV-C) e transmitida pelo ácaro Brevipalpus yothersi é a principal doença viral dos citros no Brasil. Além dos prejuízos econômicos à cultura, a leprose desperta interesse científico por ser uma doença atípica na qual o vírus permanece restrito nos tecidos vegetais ao redor do ponto de alimentação do vetor, assemelhando-se a uma reação de incompatibilidade do tipo hipersensibilidade (HR). Com este trabalho, objetivou-se revelar os mecanismos moleculares envolvidos na interação planta/CiLV-C/B. yothersi, ainda amplamente desconhecidos. Para o estudo da interação planta/ácaro foram analisados o transcriptoma de Arabidopsis thaliana em resposta ao B. yothersi via RNA-Seq, os níveis de ácido salicílico (SA) e jasmônico (JA) através de LC-MS/MS, e a relevância das vias hormonais com o uso de plantas mutantes de Arabidopsis. As plantas selvagens infestadas induziram ambas as vias de SA e JA, enquanto os processos envolvidos no seu crescimento e desenvolvimento foram reprimidos. A oviposição dos ácaros fui reduzida nas mutantes sid2 e npr1, sugerindo que o B. yothersi manipula a planta tornando-a mais suscetível à colonização. Para o estudo da interação planta/vírus foram analisados os principais eventos da replicação do CiLV-C através da quantificação da relação RNA1/p29sgRNA, a resposta transcricional de Arabidopsis infectada com CiLV-C via RNA-Seq, a presença de células mortas e a produção de espécies reativas de oxigênio (ROS) em tecidos afetados através de ensaios histoquímicos, e a expressão transiente das proteínas virais em Nicotiana benthamiana. Em resposta à infecção pelo CiLV-C as plantas apresentaram uma explosão de ROS e uma indução de genes associados ao crescimento celular, à via do SA, e aos processos de morte celular e HR; enquanto reprimiram a via de JA e o metabolismo primário. A expressão da proteína viral P61 mimetizou respostas que ocorrem durante a infecção viral, indicando-a como uma elicitora do sistema imune vegetal. O CiLV-C favoreceu a oviposição do ácaro vetor nas folhas infectadas, sugerindo que o vírus contribui com a infestação do seu vetor. Finalmente, a expressão de alguns genes modulados durante a interação Arabidopsis/CiLV-C/B. yothersi foi confirmada em plantas de laranja-doce (Citrus sinensis), validando o uso da planta-modelo no estudo do patosistema da leprose. Foi demonstrado ainda que outros vírus transmitidos por Brevipalpus (VTBs) são capazes de infectar Arabidopsis, o que permite o seu uso como hospedeira alternativa em estudos de interação com VTBs filogeneticamente distantes ao CiLV-C. Os resultados apresentados, além de proporcionar uma melhor compreensão dos processos fisiopatológicos da leprose, a longo prazo poderão contribuir para o estabelecimento de estratégias de controle mais sustentáveis da doençaAbstract: Citrus leprosis is caused by citrus leprosis virus C (CiLV-C), transmitted by Brevipalpus yothersi mites, and is the main viral disease of citrus in Brazil. Differently from other plant viruses, CiLV-C is unable to accomplish systemic infection in its hosts, remaining restricted to cells around the inoculation sites, where symptoms of viral infection develop. Phenotypically, these features resemble the outcome a hypersensitivity response (HR). In this study, we attempted to unravel the molecular mechanisms involved in the plant/CiLV-C/B. yothersi interaction, which are still poorly understood. To disentangle the plant/mite interaction, we analyzed the Arabidopsis thaliana transcriptome in response to B. yothersi by RNA-Seq, the levels of salicylic acid (SA) and jasmonic acid (JA) in infested plants by LC-MS/MS, and the role of the hormonal pathways during mite infestation using Arabidopsis mutant plants. Infested wild type plants induced both SA e JA pathways, whilst processes involved in plant growth and defense were repressed. sid2 e npr1 mutant plants reduced the oviposition of mites, suggesting that B. yothersi manipulate plant response to render it more susceptible to its colonization. To uncover plant/virus interaction, we identified the main events of viral replication by quantifying the CiLV-C RNA1/p29sgRNA ratio during the infection, the transcriptional Arabidopsis response to CiLV-C using RNA-Seq, the presence of dead cells and reactive oxygen species (ROS) in infected tissues through histochemical assays, and the elicitor activity of viral proteins by transient expression in Nicotiana benthamiana. Plants responded to CiLV-C with a ROS burst and the induction of genes related to cell growth, SA pathway, cell death and HR. Conversely, infected plants repressed the SA pathway and the primary metabolism. The expression of the P61 viral protein mimicked responses observed during CiLV-C infection, indicating P61 as a viral component that elicits the plant immune system. CiLV-C presence favored mite ovoposition in virus-infected leaves, suggesting that the virus benefit mite infestation. Finally, the expression of selected genes modulated during the interaction with Arabidopsis was confirmed in sweet orange (Citrus sinensis), validating the use of this model plant in the study of citrus leprosis. We also showed that other Brevipalpus-transmitted viruses (BTVs) infect Arabidopsis, which thus can be used as an alternative host in the studies of plant interaction with CiLV-C-phylogenetically distant BTVs. The results presented here provided a better understanding of the processes developed during citrus leprosis disease and will be helpful to the establishment of sustainable strategies of the disease controlDoutoradoGenetica Vegetal e MelhoramentoDoutora em Genética e Biologia Molecular2014/00366-8FAPES

Gene expression analysis of Arabidopsis thaliana in response to Citrus leprosis virus C and its vector Brevipalpus phoenicis

A leprose dos citros, principal doença viral que afeta a citricultura no Brasil, é causada pelo Citrus leprosis virus C (CiLV-C, gênero Cilevirus). CiLV-C possui um genoma bipartido de RNA de fita simples, polaridade positiva, que codifica para seis proteínas. O vírus é transmitido de planta a planta por ácaros Brevipalpus phoenicis e pode infectar mais de 40 espécies vegetais, produzindo lesões localizadas cloróticas ou necróticas ao redor do sítio de inoculação pelo ácaro. Invariavelmente, o patógeno não realiza movimento sistêmico em nenhuma de suas hospedeiras conhecidas. Para se revelar os mecanismos moleculares que determinam a atípica interação vírus/ácaro/planta, as atividades das principais vias de defesa foram avaliadas durante a infestação de A. thaliana com ácaros avirulíferos e virulíferos para o CiLV-C. A expressão de 19 genes marcadores associados às respostas de defesa do hospedeiro foi verificada mediante PCR quantitativo (RT-qPCR) em um experimento de time course (6, 12 e 24 horas após a infestação, e no momento do aparecimento dos sintomas de leprose). As análises demostraram que os genes envolvidos na via do ácido salicílico (SA) foram induzidos durante a interação com o ácaro e com o vírus. O perfil de expressão dos genes desta via durante a infestação com ácaros virulíferos foi similar ao observado com ácaros avirulíferos, porém a resposta da planta a ambos os estímulos foi mais intensa. Ademais, ambas as vias do ácido jasmônico e etileno foram ativadas durante a interação com o ácaro e reprimidas ao longo da infecção com o vírus, sugerindo uma interferência antagonística mediada pela via do SA. O mecanismo de silenciamento de RNA foi regulado de maneira diferencial em resposta à interação com ácaros avirulíferos e virulíferos. Diante da infecção viral, em tempos iniciais da infecção, as plantas responderam com a ativação de uma primeira linha de defesa mediada por AGO1, e depois alternaram para uma segunda linha de defesa mediada por AGO2. Os resultados indicam a ativação de um processo multifatorial em resposta ao CiLV-C e ao ácaro B. phoenicis em A. thaliana.Citrus leprosis, the main viral disease affecting citrus orchards in Brazil, is caused by Citrus leprosis virus C (CiLV-C, genus Cilevirus). CiLV-C has a bipartite genome of singled stranded positive RNA, which encodes six proteins. CiLV-C is plant-to-plant transmitted by Brevipalpus phoenicis mites and can infect more than 40 plant species, invariably producing localized chlorotic or necrotic lesions around the site of feeding of the viruliferous mites. Viral long distance movement in its hosts is not accomplished. To unveil the mechanisms determining the unique characteristic of the virus/mite/plant interaction, activities of main plant defense pathways were evaluated during aviruliferous and CiLV-C viruliferous mite infestation in Arabidopsis thaliana. The expression of 19 marker genes involved in defense responses along a time course experiment (6, 12 and 24 hours after infestation, and after appearance of leprosis symptoms) was assessed by RT-qPCR. Analyses showed that genes involved in the salicylic acid (SA) pathway were up-regulated during plant interaction with mite and virus. The SA pathway expression profile observed at the infestation by viruliferous mites resembled those observed for the aviruliferous mites, but plant response to both stimuli was stronger. Both the jasmonic acid and ethylene pathways were activated during mite/plant interaction and were repressed at the course of infection with CiLV-C, suggesting an antagonistic effect mediated by the activated SA pathway. Gene silencing mechanism was differentially regulated in response to both aviruliferous and viruliferous mites. Upon viral infection, plants responded with the activation of an AGO1-mediated first defense line, in early times of infection; and then switched to an AGO2-mediated defense. Results indicate the activation of a multifactorial process in response to CiLV-C and B. phoenicis mites in A. thaliana

Citrus leprosis virus C infection results in hypersensitive-like response, suppression of the JA/ET plant defense pathway and promotion of the colonization of its mite vector.

Leprosis is a serious disease of citrus caused by Citrus leprosis virus C (CiLV-C, genus Cilevirus) whose transmission is mediated by false-spider mites of the genus Brevipalpus. CiLV-C infection does not systemically spread in any of its known host plants, thus remaining restricted to local lesions around the feeding sites of viruliferous mites. To get insight into this unusual pathosystem, we evaluated the expression profiles of genes involved in defense mechanisms of Arabidopsis thaliana and Citrus sinensis upon infestation with non-viruliferous and viruliferous mites by using reverse transcriptase-qPCR. These results were analyzed together with the production of reactive oxygen species (ROS) and the appearance of dead cells as assessed by histochemical assays. After interaction with non-viruliferous mites, plants locally accumulated ROS and triggered the salicylic acid (SA) and jasmonate/ethylene (JA/ET) pathways. ERF branch of the JA/ET pathways was highly activated. In contrast, JA pathway genes were markedly suppressed upon the CiLV-C infection mediated by viruliferous mites. Viral infection also intensified the ROS burst and cell death, and enhanced the expression of genes involved in the RNA silencing mechanism and SA pathway. After 13 days of infestation of two sets of Arabidopsis plants with non-viruliferous and viruliferous mites, the number of mites in the CiLV-C infected Arabidopsis plants was significantly higher than in those infested with the non-viruliferous ones. Oviposition of the viruliferous mites occurred preferentially in the CiLV-C infected leaves. Based on these results, we postulated the first model of plant/Brevipalpus mite/cilevirus interaction in which cells surrounding the feeding sites of viruliferous mites typify the outcome of a hypersensitive-like response, whereas viral infection induces changes in the behavior of its vector

Arabidopsis thaliana as a model host for Brevipalpus mite-transmitted viruses

Brevipalpus-transmitted viruses (BTV) are a taxonomically diverse group of plant viruses which severely affect a number of major crops. Members of the group can be sub-classified into cytoplasmic (BTV-C) or nuclear type (BTV-N) according to the accumulation sites of virions in the infected plant cells. Both types of BTV produce only local infections near the point of inoculation by viruliferous mites. Features of BTV-plant interactions such as the failure of systemic spread in their natural hosts are poorly understood. In this study we evaluated Arabidopsis thaliana, a model plant commonly used for the study of plant-virus interactions, as an alternative host for BTV. Infection of Arabidopsis with the BTV-N Coffee ringspot virus and Clerodendrum chlorotic spot virus, and the BTV-C Solanum violaefolium ringspot virus, were mediated by viruliferous Brevipalpus mites collected in the wild. Upon infestation, local lesions appeared in 7 to 10 days on leaves of, at least, 80 % of the assayed plants. Presence of viral particles and characteristic cytopathic effects were detected by transmission electron microscopy (TEM) and the viral identities confirmed by specific reverse-transcriptase polymerase chain reaction (RT-PCR) and further amplicon sequencing. The high infection rate and reproducibility of symptoms of the three different viruses assayed validate A. thaliana as a feasible alternative experimental host for BTV

Phylogenetic and Molecular Variability Studies Reveal a New Genetic Clade of Citrus leprosis virus C

Citrus leprosis virus C (CiLV-C) causes a severe disease affecting citrus orchards in the Western hemisphere. This study reveals the molecular variability of the virus by analyzing four genomic regions (p29, p15, MP and RNA2-intergenic region) distributed over its two RNAs. Nucleotide diversity (π) values were relatively low but statistically different over the analyzed genes and subpopulations, indicating their distinct evolutionary history. Values of πp29 and πMP were higher than those of πp15 and πRNA2–IR, whereas πMP was increased due to novel discovered isolates phylogenetically clustered in a divergent clade that we called SJP. Isolate BR_SP_SJP_01 RNA1 and RNA2 sequences, clade SJP, showed an identity of 85.6% and 88.4%, respectively, with those corresponding to CiLV-C, the type member of the genus Cilevirus, and its RNA2 5′-proximal region was revealed as a minor donor in a putative inter-clade recombination event. In addition to citrus, BR_SP_SJP_01 naturally infects the weed Commelina benghalensis and is efficiently transmitted by Brevipalpus yothersi mites. Our data demonstrated that negative selection was the major force operating in the evaluated viral coding regions and defined amino acids putatively relevant for the biological function of cilevirus proteins. This work provides molecular tools and sets up a framework for further epidemiological studies

Arabidopsis thaliana as a model host for Brevipalpus mite-transmitted viruses

ABSTRACT: Brevipalpus-transmitted viruses (BTV) are a taxonomically diverse group of plant viruses which severely affect a number of major crops. Members of the group can be sub-classified into cytoplasmic (BTV-C) or nuclear type (BTV-N) according to the accumulation sites of virions in the infected plant cells. Both types of BTV produce only local infections near the point of inoculation by viruliferous mites. Features of BTV-plant interactions such as the failure of systemic spread in their natural hosts are poorly understood. In this study we evaluated Arabidopsis thaliana, a model plant commonly used for the study of plant-virus interactions, as an alternative host for BTV. Infection of Arabidopsis with the BTV-N Coffee ringspot virus and Clerodendrum chlorotic spot virus, and the BTV-C Solanum violaefolium ringspot virus, were mediated by viruliferous Brevipalpus mites collected in the wild. Upon infestation, local lesions appeared in 7 to 10 days on leaves of, at least, 80 % of the assayed plants. Presence of viral particles and characteristic cytopathic effects were detected by transmission electron microscopy (TEM) and the viral identities confirmed by specific reverse-transcriptase polymerase chain reaction (RT-PCR) and further amplicon sequencing. The high infection rate and reproducibility of symptoms of the three different viruses assayed validate A. thaliana as a feasible alternative experimental host for BTV