Estudo funcional da proteína de movimento (NSm) de distintos Tospovirus

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, 2015.Vírus de planta desenvolveram uma classe de proteínas responsáveis por garantir a infecção e disseminação viral. Estudos vêm demonstrando que essa classe de proteínas de movimento (MPs), interage com o plasmodesma (PD), aumentando o seu limite de exclusão (LEP), possibilitando a passagem dos vírus. Várias estratégias são utilizadas para o estudo de movimento viral. Alguns sistemas baseiam-se na utilização de clones infecciosos que possibilitam a inserção de genes heterólogos de MPs. Para o estudo do movimento célula à célula e sistêmico utilizamos o vetor viral Alfalfa mosaic virus (AMV). O sistema consiste na utilização de transgênicos P12 em combinação com transcritos do RNA 3 adaptados para a inserção de genes heterólogos. Com base neste sistema, demonstramos que a proteína de movimento NSm, pode ter relação com a limitação ou amplitude no espectro de infecção viral devido às diferenças significativas observadas no movimento célula à célula e sistêmico de tospovírus que apresentam características moleculares e biológicas distintas (Capítulo II). Concluímos que a movimentação das espécies do gênero tospovírus é dependente da formação de vírions baseado no contexto AMV de infecção e movimentação (Capítulo II). Também concluímos que as MPs de 4 espécies de tospovírus estudadas, são eficientes na formação de túbulos. Em ensaios de truncamento das MPs observamos que para o BeNMV, o extremo C-terminal não é essencial para o movimento, a partir da identificação do limite de aminoácidos que poderiam ser deletados da MP sem inibição do movimento célula à célula. Contrastantemente, as MPs dos vírus CSNV, TCSV e TSWV demonstraram a necessidade da integridade da proteína para garantia do movimento (Capítulo II). Com a utilização das metodologias de “biomolecular fluorescence complementation” (BiFC) e tratamentos químicos com Na2CO3 e Ureia, a partir das MPs, sugerimos que as NSm se associam a membrana de forma periférica. Baseado em predições de hidrofobicidade e orientação topológicas (BiFC topology) sugerimos o modelo de interação da NSm com membrana celular, onde a região central hidrofóbica está mergulhada na periferia das membranas lipídicas e os extremos C e N-terminal livres apontados ao citoplasma (Capítulo III). Finalmente, com a metodologia de BiFC de interação, a partir de ensaios de dímeros, heterodímeros, intra-associação e inter-associação de homólogos e heterólogos de MP e NP (nucleoproteína) observamos que: i) todas NSm e N são eficientes na formação de dímeros ii) as NSm interagem com a sua N cognata e iii) nós observamos interação entre NSm, N e NSm-N entre espécies distintas de tospovírus. Estes resultados podem facilitar uma melhor interpretação das interações de proteína viral com especial ênfase na compreensão das questões de infecção mista. Além do uso de transgênicos na expressão de proteínas heterólogas para geração de resistência, abordagens através do uso de resistência natural também vem sendo exploradas contra espécies do gênero Tospovirus. O presente estudo avaliou o envolvimento de 4 NSm de distintos tospovírus com a resposta de resistência (reação de hipersensibilidade e necrose) mediada pelo gene Sw5-b inserido em plantas de Nicotiana benthamiana. Substituições dos aminoácidos de posição C118Y e T120N da proteína NSm, caracterizados como cruciais para resistência foram realizados. BeNMV, CSNV, TCSV e TSWV NSm wt fusionadas a HA (Hemagglutinin), em processo de agroinfiltração, foram desafiadas contra plantas de N. benthamiana transformadas com gene Sw5-b. Observamos a possível quebra de resistência a partir da NSm do BeNMV, onde não observou-se reação necrótica. Plantas infiltradas com as demais NSm reagiram com resposta necrótica (Capítulo IV). Novo ensaio com vetores contendo as NSm com modificação dos aminoácidos C118 por Y e T120 por N foi desenvolvido. Baseado no resultado do ensaio anterior, as MPs que continham os aminoácidos essenciais para ruptura da resistência, foram modificadas por aminoácidos que não garantem a ruptura e o contrário foi feito para aquelas MPs que não ultrapassaram a resistência. Com base nessa abordagem observamos que CSNV, TCSV e TSWV NSm superaram a resistência sem a formação de necrose foliar, resultado que difere do observado com as mesmas MPs sem mutação, e intrigantemente, a BeNMV NSm aparentemente também superou a resistência sem a formação de lesões locais e necrose foliar após a modificação dos aminoácidos, dado contrastante. No contexto de infecção viral utilizando o sistema AMV, não obtivemos resultados satisfatórios de manutenção ou ruptura da resistência. (Capítulo V). No gênero Tospovirus, pouco se sabe sobre o sítio de replicação ou sobre envolvimento de organelas celulares com o processo de infecção. Existem apenas evidências de expressão isolada das proteínas virais co-localizando com filamentos de Actina e miosina, Membrana, ER e Complexo de Golgi. No presente estudo, com auxílio de metodologia de BiFC, além de ensaio com expressão heteróloga da NSm no contexto de infecção do vírus Potato virus X (PVX), visualizamos a associação da NSm de tospovírus com cloroplatos. Hipotetizamos o possível envolvimento dessa organela com processo de infecção dos tospovírus (Capítulo IV)

Orchid fleck dichorhavirus movement protein shows RNA silencing suppressor activity.

[EN] To counteract RNA interference-mediated antiviral defence, virus genomes evolved to express proteins that inhibit this plant defence mechanism. Using six independent biological approaches, we show that orchid fleck dichorhavirus citrus strain (OFV-citrus) movement protein (MP) may act as a viral suppressor of RNA silencing (VSR). By using the alfalfa mosaic virus (AMV) RNA 3 expression vector, it was observed that the MP triggered necrosis response in transgenic tobacco leaves and increased the viral RNA (vRNA) accumulation. The use of the potato virus X (PVX) expression system revealed that the cis expression of MP increased both the severity of the PVX infection and the accumulation of PVX RNAs, further supporting that MP could act as an RNA silencing suppressor (RSS). From the analysis of the RSS-defective turnip crinkle virus (TCV), we do not find local RSS activity for MP, suggesting a link between MP suppressor activity and the prevention of systemic silencing. In the analysis of local suppressive activity using the GFP-based agroinfiltration assay in Nicotiana benthamiana (16 c line), we do not identify local RSS activity for the five OFV RNA1-encoded proteins. However, when evaluating the small interfering RNA (siRNA) accumulation, we find that the expression of MP significantly reduces the accumulation of GFP-derived siRNA. Finally, we examine whether the MP can prevent systemic silencing in 16c plants. Our findings show that MP inhibits the long-distance spread of RNA silencing, but does not affect the short-distance spread. Together, our findings indicate that MP is part of OFV's counter-defence mechanism, acting mainly in the prevention of systemic long-distance silencing. This work presents the first report of a VSR for a member of the genus Dichorhavirus.This work was supported by grant PID2020-115571RB-100 from the Spanish MCIN/AEI/10.13039/501100011033 granting agency and Fondo Europeo de Desarrollo Regional (FEDER), and by the company INVESTIR IMOVEIS LDTA from Brasilia, Brazil.Oliveira Leastro, M.; Pallás Benet, V.; Sánchez-Navarro, JÁ. (2022). Orchid fleck dichorhavirus movement protein shows RNA silencing suppressor activity. Journal of General Virology. 103(11):1-14. https://doi.org/10.1099/jgv.0.0018051141031

Rescue of a Cilevirus from infectious cDNA clones

[EN] Reverse genetics systems represent an important tool for studying the molecular and functional processes of viral infection. Citrus leprosis virus C (CiLV-C) (genus Cilevirus, family Kitaviridae) is the main pathogen responsible for the citrus leprosis (CL) disease in Latin America, one of the most economically important diseases of the citrus industry. Molecular studies of this pathosystem are limited due to the lack of infectious clones. Here, we report the construction and validation of a CiLV-C infectious cDNA clone based on an agroinfection system. The two viral RNA segments (RNA1 and RNA2) were assembled into two binary vectors (pJL89 and pLXAS). Agro-infiltrated Nicotiana benthamiana plants showed a response similar to that observed in the natural infection process with the formation of localized lesions restricted to the inoculated leaves. The virus recovered from the plant tissue infected with the infectious clones can be mechanically transmitted between N. benthamiana plants. Detection of CiLV-C subgenomic RNAs (sgRNAs) from agroinfiltrated and mechanically inoculated leaves further confirmed the infectivity of the clones. Finally, partial particle-purification preparations or sections of CiLV-C-infected tissue followed by transmission electron microscopy (TEM) analysis showed the formation of CiLV-C virions rescued by the infectious clone. The CiLV-C reverse genetic system now provides a powerful molecular tool to unravel the peculiarities of the CL pathosystem.This work was supported by grant PID2020-115571RB-100 from the Spanish MCIN/AEI/10.13039/501100011033 granting agency.Leastro, MO.; Kitajima, EW.; Pallás Benet, V.; Sanchez Navarro, JA. (2024). Rescue of a Cilevirus from infectious cDNA clones. Virus Research. 339. https://doi.org/10.1016/j.virusres.2023.19926433

Membrane Association and Topology of Citrus Leprosis Virus C2 Movement and Capsid Proteins

[EN] Although citrus leprosis disease has been known for more than a hundred years, one of its causal agents, citrus leprosis virus C2 (CiLV-C2), is poorly characterized. This study described the association of CiLV-C2 movement protein (MP) and capsid protein (p29) with biological membranes. Our findings obtained by computer predictions, chemical treatments after membrane fractionation, and biomolecular fluorescence complementation assays revealed that p29 is peripherally associated, while the MP is integrally bound to the cell membranes. Topological analyses revealed that both the p29 and MP expose their N- and C-termini to the cell cytoplasmic compartment. The implications of these results in the intracellular movement of the virus were discussed.This work was supported by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), proc. 2014/0845-9, 2017/50222-0, 2015/10249-1 and 2017/19898-8. This work was also supported by grant BIO2017-88321-R from the Spanish Agencia Estatal de Investigacion (AEI) and Fondo Europeo de Desarrollo Regional (FEDER), and the Prometeo Program GV2015/010 from the Generalitat Valenciana.Oliveira Leastro, M.; Freitas-Astua, J.; Watanabe Kitajima, E.; Pallás Benet, V.; Sanchez Navarro, JA. (2021). Membrane Association and Topology of Citrus Leprosis Virus C2 Movement and Capsid Proteins. Microorganisms. 9(2):1-9. https://doi.org/10.3390/microorganisms9020418199

Spontaneous Mutation in the Movement Protein of Citrus Leprosis Virus C2, in a Heterologous Virus Infection Context, Increases Cell-to-Cell Transport and Generates Fitness Advantage

[EN] Previous results using a movement defective alfalfa mosaic virus (AMV) vector revealed that citrus leprosis virus C (CiLV-C) movement protein (MP) generates a more efficient local movement, but not more systemic transport, than citrus leprosis virus C2 (CiLV-C2) MP, MPs belonging to two important viruses for the citrus industry. Here, competition experiment assays in transgenic tobacco plants (P12) between transcripts of AMV constructs expressing the cilevirus MPs, followed by several biological passages, showed the prevalence of the AMV construct carrying the CiLV-C2 MP. The analysis of AMV RNA 3 progeny recovered from P12 plant at the second viral passage revealed the presence of a mix of progeny encompassing the CiLV-C2 MP wild type (MPWT) and two variants carrying serines instead phenylalanines at positions 72 (MPS72F) or 259 (MPS259F), respectively. We evaluated the effects of each modified residue in virus replication, and cell-to-cell and long-distance movements. Results indicated that phenylalanine at position 259 favors viral cell-to-cell transport with an improvement in viral fitness, but has no effect on viral replication, whereas mutation at position 72 (MPS72F) has a penalty in the viral fitness. Our findings indicate that the prevalence of a viral population may be correlated with its greater efficiency in cell-to-cell and systemic movements.This research was funded by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), grant numbers 2014/0845-9, 2017/50222-0, 2015/10249-1, 2017/19898-8 and by the Spanish Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER), grant number PID2020-115571RB-100.Oliveira Leastro, M.; Villar-Álvarez, D.; Freitas-Astúa, J.; Watanabe Kitajima, E.; Pallás Benet, V.; Sanchez Navarro, JA. (2021). Spontaneous Mutation in the Movement Protein of Citrus Leprosis Virus C2, in a Heterologous Virus Infection Context, Increases Cell-to-Cell Transport and Generates Fitness Advantage. Viruses. 13(12):1-16. https://doi.org/10.3390/v13122498S116131

Citrus Leprosis Virus C Encodes Three Proteins With Gene Silencing Suppression Activity

[EN] Citrus leprosis virus C (CiLV-C) belongs to the genusCilevirus, familyKitaviridae, and is considered the most devastating virus infecting citrus in Brazil, being the main viral pathogen responsible for citrus leprosis (CL), a severe disease that affects citrus orchards in Latin America. Here, proteins encoded by CiLV-C genomic RNA 1 and 2 were screened for potential RNA silencing suppressor (RSS) activity by five methods. Using the GFP-based reporter agroinfiltration assay, we have not found potential local suppressor activity for the five CiLV-C encoded proteins. However, when RSS activity was evaluated using the alfalfa mosaic virus (AMV) system, we found that the p29, p15, and p61 CiLV-C proteins triggered necrosis response and increased the AMV RNA 3 accumulation, suggesting a suppressive functionality. From the analysis of small interfering RNAs (siRNAs) accumulation, we observed that the ectopic expression of the p29, p15, and p61 reduced significantly the accumulation of GFP derived siRNAs. The use of the RSS defective turnip crinkle virus (TCV) system revealed that only thetrans-expression of the p15 protein restored the cell-to-cell viral movement. Finally, the potato virus X (PVX) system revealed that the expression of p29, p15, and p61 increased the PVX RNA accumulation; in addition, the p29 and p15 enhanced the pathogenicity of PVX resulting in the death of tobacco plants. Furthermore, PVX-p61 infection resulted in a hypersensitive response (HR), suggesting that p61 could also activate a plant defense response mechanism. This is the first report describing the RSS activity for CiLV-C proteins and, moreover, for a member of the familyKitaviridae.This work was supported by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), proc. 2014/0845-9, 2017/50222-0, 2015/10249-1, and 2017/19898-8. This work was also supported by Instituto para la Formacion y Aprovechamiento de Recursos Humanos, Becas IFARHU-SENACYT, contrato 270-2018-361, grant BIO2017-88321-R from the Spanish Agencia Estatal de Investigacion (AEI), Fondo Europeo de Desarrollo Regional (FEDER), and the Prometeo Program GV2015/010 from the Generalitat Valenciana.Leastro, MO.; Ortega Castro, DY.; Freitas-Astúa, J.; Kitajima, EW.; Pallás Benet, V.; Sanchez Navarro, JA. (2020). Citrus Leprosis Virus C Encodes Three Proteins With Gene Silencing Suppression Activity. Frontiers in Microbiology. 11:1-16. https://doi.org/10.3389/fmicb.2020.01231S11611Anandalakshmi, R., Pruss, G. J., Ge, X., Marathe, R., Mallory, A. C., Smith, T. H., & Vance, V. 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Long-Distance Movement, Virulence, and RNA Silencing Suppression Controlled by a Single Protein in Hordei- and Potyviruses: Complementary Functions between Virus Families. Journal of Virology, 76(24), 12981-12991. doi:10.1128/jvi.76.24.12981-12991.200

Biological and molecular studies approaches to Zucchini lethal chlorosis vírus (ZLCV) investigation, a differentiated species of the American group of Tospovirus

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Fitopatologia, Programa de Pós-Graduação em Fitopatologia, 2011.O gênero Tospovirus pertence à família Bunyaviridae que tem como espécie tipo Tomato spotted wilt virus (TSWV). Espécies deste gênero possuem grande relevância para as hortaliças, principalmente, para as culturas do tomate e do pimentão, nas quais causam perdas significativas. Além de TSWV, várias outras espécies de Tospovirus causam perdas de impacto na produção agrícola no País, como Zucchini lethal chlorosis virus (ZLCV) que infecta abobrinha-da-moita, melancia e melão, Groundnut ringspot virus (GRSV) em tomate, pimentão e alface, Tomato chlorotic spot virus (TCSV) em tomate e pimentão, Chrysanthemum stem necrosis virus (CNSV) em plantas ornamentais e íris yellow spot virus (IYSV) em cebola. Estas espécies apresentam um espectro de hospedeiras de tamanho relevante. A doença tipo é chamada de "vira-cabeça" e está entre os principais problemas fitossanitários em variedades destes grupos de plantas. Em cucurbitáceas a doença é denominada de amarelo letal da abobrinha-da-moita. O Zucchini lethal chlorosis virus é uma espécie que foi descrita apenas no Brasil, principalmente nas regiões produtoras de cucurbitáceas dos Estados de São Paulo e de Tocantins, infectando, especialmente, abobrinha-da-moita,melão e melancia. Em relação às demais espécies do gênero Tospovirus, ZLCV é a única que demonstra uma restrição em relação ao espectro de hospedeiras, fato este que pode estar envolvido com a proteína de movimento viral (NSm) e a proteína supressora de silenciamento gênico (NSs). Estes pontos reforçam o principal objetivo do presente trabalho que é a caracterização da proteína supressora de silenciamento NSs e um comparativo molecular desta com as demais NSs caracterizadas até o momento dentro do gênero e dentro da família do vírus. Além desse aspecto, ZLCV vem demonstrando um aumento de incidência infectando cucurbitáceas, ficando atrás apenas de Papaya ringspot virus (PRSV-W) e Zucchini yellow mosaic virus (ZYMV). Diante dessa problemática surgem mais dois objetivos no trabalho, o entendimento mais amplo em relação ao espectro de hospedeiras do vírus e o desenvolvimento de um antisoro eficiente para a diagnose do vírus. Para a caracterização molecular da NSs do ZLCV, foi utilizado um primer (Oligonucleotídeo) degenerado sentido senso que se anelou após o códon iniciador e um antisenso, que se anelou na região hairpin (em formato de grampo) do segmento S do genoma viral. Esta combinação resultou na amplificação de um fragmento de aproximadamente 2.000 pb da NSs do vírus e parte da região intergênica do segmento S, faltando apenas um pequeno fragmento para o sequenciamento total da NSs. Foi desenhado então outro primer antisenso que se anelou no início do genoma e que em combinação com o primer J13 que se anela na região consenso de todos os segmentos do genoma de vírus deste gênero, foi obtido o restante da seqüência. Comparando-se a NSs do ZLCV com segmentos correspondentes das demais espécies do grupo Americano e do grupo Euro- Asiático obteve-se identidade máxima de 73,2% com TSWV. Com base na análise de identidade e na análise fílogenética da proteína NSs de Tospovirus foi levantada a hipótese de que a restrição do círculo de hospedeiras do ZLCV poderia estar relacionada com a diferenciação de regiões da proteína NSs que tenham relação com a supressão do silenciamento. O que poderia estar causando a sua restrição em relação à infecção de novas hospedeiras. A demais é sugerida a utilização das proteínas NSs como um valor adicional na classificação das espécies do gênero Tospovirus. Para a produção do anti-soro foram imunizados dois coelhos. O anticorpo produzido apresentou reação cruzada com TCSV e GRSV, entretanto, em uma concentração de 1:500 foi possível diagnosticar a presença do ZLCV na planta e a ausência de reação cruzada. No círculo de hospedeiras foram testadas 23 diferentes cultivares de cucurbitáceas (um aumento relevante em relação aos outros ensaios já feitos de espectro de hospedeiras do vírus), além de plantas pertencentes a outras famílias como Solanaceae, Convolvulaceae, Amaranthaceae, Fabaceae e Chenopodiaceae. Foi identificada por sorologia e/ou reação da polimerase em cadeia (PCR) a presença do ZLCV em um número pequeno de plantas o que não representa uma co-evolução do vírus para à infecção de hospedeiras alternativas. O ensaio sorológico contribuirá para otimizar a diagnose viral, considerando que ZLCV vem ocorrendo em maior incidência em plantações de cucurbitáceas. O ensaio biológico e o ensaio molecular geraram informações que contribuíram ao melhor entendimento da relação patógeno x hospedeira, considerando-se que ZLCV é uma espécie dentro do gênero Tospovirus que apresenta restrição em relação a plantas hospedeiras. O entendimento dos eventos que induzem a essa restrição podem, de alguma forma, ser empregados na determinação de medidas a serem empregadas no controle das espécies de Tospovirus.The genus Tospovirus belongs to the Bunyaviridae family and has Tomato spotted wilt vírus (TSWV) as type species. Species within this genus are of great relevance to vegetable crops, especially, on tomatoes and peppers on which these viruses can cause severe economical losses. In addition to TSWV, there are other Topovirus species which have an impact on vegetable production in the country, such as Zucchini lethal chlorosis virus (ZLCV) infecting cucurbits, especially squash, Groundnut ringspot virus (GRSV) on tomato, pepper and lettuce, Tomato chlorotic spot virus (TCSV) on tomato and pepper, Chrysanthemum stem necrosis virus (CNSV) on ornamental plants and íris yellow spot virus (IYSV) on onion. The disease caused by these viruses is commonly called a "top distortion of tomato planf, being among the major diseases on various groups of plants. In cucurbitaceous the disease is known as lethal yellowing of squash. Zucchini lethal chlorosis virus has been detected and characterized only in Brazil, mainly in cucurbit producing areas of the State of São Paulo and State of Tocantins. This virus infects squash, melon and watermelon crops. Comparing to other Tospovirus species found in Brazil, ZLCV is reported to infect just a few plant species, which may be related to the viral movement protein (NSm) and/ or to the gene silencing suppressor protein (NSs). From this premise, the main objective of this work was to perform the molecular characterization of the NSs, putative silencing suppressor protein and also to perform a comparative analysis with other NSs proteins characterized until now within the genus Tospovirus and within the Bunyaviridae family. Also, considering that ZLCV incidence in cucurbit species has increased in the last years, the present work includes an investigation on the host range of ZLCV and also the development of an antiserum to be used in lhe ZLCV detection. To obtain the sequence of ZLCV NSs to perform the molecular characterization, a pair of primers was used in an RT-PCR reaction. A forward degenerate primer was designed which annealed after the start codon onto the viral sequence and a reverse primer (UHP) that annealed onto the "hairpin" region of the genome sequence of the S RNA segment. That primer combination amplified a 2000 bp fragment which corresponded to the NSs partial sequence and part of the intergenic region of the S segment. In order to obtain the complete NSs sequence including the C-terminal sequence of S RNA, another reverse primer was designed to anneal onto the 5' end region of the NSs sequence. The reverse primer in combination with J13 primer (anneal onto the consensus region present in ali three segments of the viral genome within the genus) made it possible to amplify the remaining fragment and complete the NSs sequence. Comparative analysis of nucleotide and amino acid sequences of the NSs ZTCV with the corresponding sequences of other tospovirus clustered in the American and in the Asian groups indicated a maximum identity of 73.2% with TSWV NSs sequence. Based on the identity and on the phylogenetic analysis of the NSs and NSm proteins of tospoviruses it was hypothesized that the restriction of the host range of ZTCV may be related to the gene silencing suppressor protein (NSs) and viral movement protein (NSm) influencing virus/ host interactions. Analysis of amino acid sequences of the NSm protein showed that ZTCV and INSV differ most from other species in the American group. In addition, larger identity value of 71% was observed between ZTCV and of CSNV NSm proteins. Considering that, it is likely that non-structural proteins of tospoviruses may be associated to virus pathogenicity and having some iníluence on the size of its host range. Beyond the hypothesis suggested the use of NSs proteins as an additional parameter in the classification of the genus Tospovirus. Antiserum produetion was carried out using two rabbits. By dot- EL1SA, the ZTCV antibodies cross-reacted with TCSV and GRSV and at 1:500 dilution it was possible to detect just ZTCV presence in infected plants. To investigate the host range of ZTCV, 23 cucurbit plants including different cultivars and species, and also species from Solanaceae, Convolvulaceae, Amaranthaceae, Fabaceae and Chenopodiaceae families were tested in this assay. Virus detection on inoculated plants was done by serology and by PCR and the results indicated ZTCV presence in just a few plant species and/or cultivars, indicating no evolution in virus infection of new hosts, therefore confirming the restrict host range nature of the ZTCV. Considering that ZLCV has been occurring in an increasing incidence in cucurbit crops, within the last years, the antiserum produced in this work will contribute to a better viral diagnosis. The biological and the molecular data generated will contribute to a better understanding of host pathogen interaction, considering that ZLCV has a restricted host range when compared to other Tospovirus species. These data ali together will help to understand the host range restriction of this Tospovirus species which may be useful to search for strategies virus control

The capsid protein of citrus leprosis virus C shows a nuclear distribution and interacts with the nucleolar fibrillarin protein

[EN] Brevipalpus-transmitted viruses (BTVs) have a significant negative economic impact on the citrus industry in Central and South America. Until now, only a few studies have explored the intracellular distribution and interaction of BTVs-encoded proteins with host factors, particularly for cileviruses, the main BTV responsible for the Citrus Leprosis (CL) disease. This study describes the nuclear localization of citrus leprosis virus C (CiLV-C) capsid protein (p29) and its interaction with the fibrillarin (Fib2) within the nucleolar compartment and cell cytoplasm. Our results, obtained by computer predictions and laser scanning confocal microscopy analyses, including colocalization and bimolecular fluorescence complementation (BiFC) approaches, revealed that a fraction of the p29 is localized in the nucleus and colocalizes with the Fib2 in both the nucleolus and cytosol. The nuclear localization of p29 correlated with a smaller nucleus size. Furthermore, co-immunoprecipitation (Co-IP) assays confirmed the interactions between p29 and Fib2. The implications of these findings for the functionalities of the cilevirus capsid protein are discussed.This work was supported by grant PID2020-115571RB-100 from the Spanish MCIN/AEI/10.13039/501100011033 granting agency and from Prometeo project CIPROM/2022/31 funded by Generalitat Valenciana.Leastro, MO.; Pallás Benet, V.; Sanchez Navarro, JA. (2024). The capsid protein of citrus leprosis virus C shows a nuclear distribution and interacts with the nucleolar fibrillarin protein. Virus Research. 340. https://doi.org/10.1016/j.virusres.2023.19929734

Orchid fleck dichorhavirus movement protein shows RNA silencing suppressor activity

To counteract RNA interference-mediated antiviral defence, virus genomes evolved to express proteins that inhibit this plant defence mechanism. Using six independent biological approaches, we show that orchid fleck dichorhavirus citrus strain (OFV-citrus) movement protein (MP) may act as a viral suppressor of RNA silencing (VSR). By using the alfalfa mosaic virus (AMV) RNA 3 expression vector, it was observed that the MP triggered necrosis response in transgenic tobacco leaves and increased the viral RNA (vRNA) accumulation. The use of the potato virus X (PVX) expression system revealed that the cis expression of MP increased both the severity of the PVX infection and the accumulation of PVX RNAs, further supporting that MP could act as an RNA silencing suppressor (RSS). From the analysis of the RSS-defective turnip crinkle virus (TCV), we do not find local RSS activity for MP, suggesting a link between MP suppressor activity and the prevention of systemic silencing. In the analysis of local suppressive activity using the GFP-based agroinfiltration assay in Nicotiana benthamiana (16 c line), we do not identify local RSS activity for the five OFV RNA1-encoded proteins. However, when evaluating the small interfering RNA (siRNA) accumulation, we find that the expression of MP significantly reduces the accumulation of GFP-derived siRNA. Finally, we examine whether the MP can prevent systemic silencing in 16c plants. Our findings show that MP inhibits the long-distance spread of RNA silencing, but does not affect the short-distance spread. Together, our findings indicate that MP is part of OFV's counter-defence mechanism, acting mainly in the prevention of systemic long-distance silencing. This work presents the first report of a VSR for a member of the genus Dichorhavirus.Peer reviewe

The NSm proteins of phylogenetically related tospoviruses trigger Sw-5b-mediated resistance dissociated of their cell-to-cell movement function

[EN] The cell-to-cell movement protein (NSM) in members of the tospovirus species Tomato spotted wilt virus (TSWV) has been recently identified as the effector of the single dominant Sw-5b resistance gene from tomato (Solanum lycopersicum L.). Although most TSWV isolates shows a resistance-inducing (RI) phenotype, regular reports have appeared on the emergence of resistance-breaking (RB) isolates in tomato fields, and suggested a strong association with two point mutations (C118Y and T120N) in the NSM protein. In this study the Sw-5b gene has been demonstrated to confer not only resistance against TSWV but to members of five additional, phylogeneticallyrelated tospovirus species classified within the so-called ¿American¿ evolutionary clade, i.e. Alstroemeria necrotic streak virus (ANSV), Chrysanthemum stem necrosis virus (CSNV), Groundnut ringspot virus (GRSV), Impatiens necrotic spot virus (INSV) and Tomato chlorotic spot virus (TCSV). Remarkably, a member of the species Bean necrotic mosaic virus (BeNMV), a recently discovered tospovirus classified in a distinct American subclade and circulating on the American continent, did not trigger a Sw-5b-mediated hypersensitive (HR) response. Introduction of point mutations C118Y and T120N into the NSM protein of TSWV, TCSV and CSNV abrogated the ability to trigger Sw-5b-mediated HR in both transgenic-N. benthamiana and tomato isolines harboring the Sw-5b gene whereas it had no effect on BeNMV NSM. Truncated versions of TSWV NSM lacking motifs associated with tubule formation, cell-to-cell or systemic viral movement were made and tested for triggering of resistance. HR was still observed with truncated NSM proteins lacking 50 amino acids (out of 301) from either the amino- or carboxy-terminal end. These data altogether indicate the importance of amino acid residues C118 and T120 in Sw-5b-mediated HR only for the NSM proteins from one cluster of tospoviruses within the American clade, and that the ability to support viral cell-to-cell movement is not required for effector functionalityWe thank Leonardo Boiteux (Embrapa Vegetable Crops - CNPH, Brasilia, DF, Brazil) for providing the isogenic line "CNPH LAM 147" essential to elaboration of this work. This research was financially supported by CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico), CAPES (Conselho de Aperfeicoamento de Pessoal de Nivel Superior), BIO2014-54862-R from the Spanish Direccion General de Investigacion Cientifica y Tecnica (DGICYT), the Prometeo Program GV2014/010 from the Generalitat Valenciana and Wageningen UniversityLeastro, MO.; De Oliveira, AS.; Pallás Benet, V.; Sanchez Navarro, JA.; Kormelink, R.; Resende, RO. (2017). The NSm proteins of phylogenetically related tospoviruses trigger Sw-5b-mediated resistance dissociated of their cell-to-cell movement function. Virus Research. 240:25-34. https://doi.org/10.1016/j.virusres.2017.07.019S253424