Making a better home: modulation of plant defensive response by brevipalpus mites.

False-spider mites of the genus Brevipalpus are highly polyphagous pests that attack hundreds of plant species of distinct families worldwide. Besides causing direct damage, these mites may also act as vectors of many plant viruses that threaten high-value ornamental plants like orchids and economically important crops such as citrus and coffee. To better understand the molecular mechanisms behind plant-mite interaction we used an RNA-Seq approach to assess the global response of Arabidopsis thaliana (Arabidopsis) plants along the course of the infestation with Brevipalpus yothersi, the main vector species within the genus. Mite infestation triggered a drastic transcriptome reprogramming soon at the beginning of the interaction and throughout the time course, deregulating 1755, 3069 and 2680 genes at 6 hours after infestation (hai), 2 days after infestation (dai), and 6 dai, respectively. Gene set enrichment analysis revealed a clear modulation of processes related to the plant immune system. Co-expressed genes correlated with specific classes of transcription factors regulating defense pathways and developmental processes. Up-regulation of defensive responses correlated with the down-regulation of growth-related processes, suggesting the triggering of the growthdefense crosstalk to optimize plant fitness. Biological processes (BPs) enriched at all time points were markedly related to defense against herbivores and other biotic stresses involving the defense hormones salicylic acid (SA) and jasmonic acid (JA). Levels of both hormones were higher in plants challenged with mites than in the noninfested ones, supporting the simultaneous induction of genes from both pathways. To further clarify the functional relevance of the plant hormonal pathways on the interaction, we evaluated the mite performance on Arabidopsis mutants impaired in SA- or JAmediated response. Mite oviposition was lower on mutants defective in SA biosynthesis (sid2) and signaling (npr1), showing a function for SA pathway in improving the mite reproduction, an unusual mechanism compared to closely-related spider mites. Here we provide the first report on the global and dynamic plant transcriptome triggered by Brevipalpus feeding, extending our knowledge on plant-mite interaction. Furthermore, our results suggest that Brevipalpus mites manipulate the plant defensive response to render the plant more susceptible to their colonization by inducing the SA-mediated pathway

Detección del virus de la leprosis de los cítricos tipo citoplasmático en los Llanos Orientales de Colombia.

Durante los años 2004 y 2005 se observaron síntomas de la leprosis de los cítricos en los departamentos de Meta y Casanare. Para confirmar la causa de esta sintomatología, se colectaron hojas de naranja 'Valencia' con lesiones típicas en municipios de Casanare (Yopal y Aguazul) y Meta (Guamal, Villavicencio y Cumaral). Las muestras fueron procesadas identificar el virus por medio de microscopía electrónica de transmisión (MET) y transcripción reversa - reacción en cadena de la polimerasa (RT-PCR). Diez de las 21 muestras analizadas por MET resultaron positivas para el virus de la leprosis (Citrus Leprosis Virus, CiLV-C). En algunas muestras se observaron partículas baciliformes características del CiLV-C en el retículo endoplasmático y formas irregularles de viroplasmas en el citoplasma. Catorce de las 32 muestras procesadas por RT-PCR resultaron positivas al CiLV-C. Los análisis RT-PCR amplificaron fragmentos de cADN del tamaño esperado para las muestras colectadas en el Meta. Una de las ampliaciones fue secuenciada (GenBank accessión No. DQ272491) y la secuencia obtenida guarda identidad del 98 por ciento con la secuencia brasilera aislada de nucleótidos para CiLV-C (GenBank accessión No. AY289190.1). La secuencia obtenida y los primers fueron entregados al Instituto Colombiano Agropecuario -ICA- para el diagnóstico del virus CiLV-C en Colombia. Entre los ácaros recolectados en las plantas afectadas se identificó a Brevipalpus phoenicis (Geijskes) reconocido como principal vector de CiLV-

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

Foliar and twigs lesions of sweet orange caused by citrus leprosis

O objetivo deste trabalho foi apresentar a descrição anatômica dos tecidos lesionados pelo Citrus leprosis virus do tipo citoplasmático (CiLV-C), em ramos e folhas de laranjeira-doce (Citrus sinensis (L.) Osbeck) 'Pêra', e fornecer dados para a compreensão das interações entre o vírus e a planta hospedeira. Tecidos que apresentavam lesões foram seccionados em micrótomo rotativo (5–7 µm de espessura) e, posteriormente, as lâminas foram coradas e montadas em resina sintética. Nas análises realizadas no microscópio eletrônico de varredura, as amostras, depois de fixadas, foram imersas em glicerina 30% e, em seguida, criofraturadas, desidratadas em série etílica, secadas ao ponto crítico de CO2, e cobertas com uma delgada camada de ouro. As lesões foliares se iniciaram como pontuações necróticas, envolvidas por halos cloróticos que limitam o crescimento da lesão. Nos ramos, ocorreram dois tipos de lesões com fendas. Na região da fenda, houve intensa hiperplasia do parênquima cortical e floemático, que promoveu a obliteração das células condutoras do floema. A formação de ductos gomosos traumáticos nas lesões caulinares foi descrita. As alterações verificadas no floema podem explicar porque o vírus torna-se não-sistêmico.The objective of this work was to present the anatomical description of tissues lesioned by Citrus leprosis virus cytoplasmatic type (CiLV-C), in leaves and twigs of 'Pêra' sweet orange (Citrus sinensis (L.) Osbeck) providing new data to understand the interactions between the virus and the plant host, in Brazil. Leprosis lesions were cut on a rotary microtome (5–7 µm thick), stained and mounted in synthetic resin. For scanning electron microscope analysis, fixed tissues were immersed in glycerin 30% and criofractured, dehydrated in a graded ethylic series, critical point drying and coated with gold. Foliar lesions began as necrotic points surrounded by a yellow halo that inhibited the lesion expansion. The twigs presented two different lesions: depressed ones and pustules with ruptures. There was hyperplasia of the cortical and phloematic parenchyma cells causing the obliteration of phloematic vessels. These histological alterations of the phloem tissue could explain why the virus does not become systemic even occasionally infecting phloem parenchyma cells. The development of gummosis traumatic ducts in twig lesions was described here

First Report of Orchid fleck virus in Costa Rica

copyright 2002 American Phytopathology Society JournalsOrchid fleck virus (OFV), a tentative member of the family Rhabdoviridae, infects orchids in several countries. The virus is vectored worldwide by the mite Brevipalpus californicus (Banks) (Acari: Tenuipalpidae). Eleven plants of Oncidium spp. and one plant each of the genera Cymbidium and Maxillaria exhibiting numerous yellow flecks and necrotic ringspot lesions on leaves were collected in two private orchid collections in Costa Rica. Presence of OFV was assessed by plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA) using an antiserum developed against an OFV isolate in Japan (2), analyses of ultrathin sections of the host cell with transmission electron microscopy (TEM), and reverse transcription polymerase chain reaction (RT-PCR) amplification using specific primers for the viral nucleocapsid gene (1). Eight of eleven Oncidium samples, and both Cymbidium and Maxillaria samples tested positive for OFV with PTA-ELISA having A values ranging from 3.9 to 14.6 times higher than negative controls. Thin sections from individual samples of Cymbidium, Oncidium, and Maxillaria revealed electronlucent intranuclear viroplasm and short, rodlike particles (40 to 50 × 100 nm) in the nucleus or cytoplasm typical of OFV-infected cells. RTPCR amplifications from one sample of each genera resulted in PCR product bands of approximately 800 bp. The Cymbidium RT-PCR product was cloned into a pGEM-T-Easy expression vector and sequenced using an ABI 3700 sequencer. The 619-bp nucleocapsid gene consensus sequence had 98% homology with the OFV isolate 0023 identified in Germany (GenBank Accession No. AF343870) (1). However, it had only approximately 85% nucleocapsid gene homology with other OFV isolates available through GenBank, including those from countries geographically closer to Costa Rica, such as Brazil (1).To our knowledge, this is the first report of OFV infecting orchids in Costa Rica.Universidad de Costa Rica/[801-A1-801]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Biología Celular y Molecular (CIBCM

Differential response of citrus species and hybrids to leprosis

O objetivo deste trabalho foi buscar novas fontes de resistência à leprose-dos-citros, no Banco Ativo de Germoplasma do Centro APTA Citros Sylvio Moreira, Instituto Agronômico, em Cordeirópolis, SP. Foram utilizadas plantas obtidas por sementes de 26 acessos, infectadas com o vírus da leprose-dos-citros (Citrus leprosis virus – CiLV), por meio do seu vetor Brevipalpus phoenicis. O aparecimento de lesões, a partir de 21 dias após a inoculação, foi observado em 11 dos genótipos testados (42,3%). Quinze espécies, entre elas Citrus pennivesiculata e C. celebica, comportaram-se como altamente resistentes, enquanto outras, como C. keraji, foram mais suscetíveis que o padrão C. sinensis. Os dados mostraram grande variação de respostas de Citrus spp. à leprose, com elevado número de espécies resistentes, que podem ser utilizadas como fonte de resistência à doença em programas de melhoramento.The objective of this work was to prospect for new sources of resistance to leprosis in the Citrus Germplasm Bank of Centro APTA Citros Sylvio Moreira, Instituto Agronômico, in Cordeirópolis, SP, Brazil. Plants obtained through seeds from 26 citrus accessions were mite-infected with Citrus leprosis virus (CiLV). Typical lesions started to appear in 11 genotypes (42.3% of the tested accessions) 21 days after inoculation. Fifteen species, among them Citrus pennivesiculata and C. celebica, behaved as highly resistant, while other genotypes, as C. keraji, were more susceptible than the standard C. sinensis. The data showed a wide variation in the response of Citrus spp. to leprosis, with a large number of resistant species, wich can be used as source of resistance to this disease, in breeding programs

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-transcription 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. ERE 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 vector7CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP573848/08-4; 375843/2012-42012/18771- 0; 2014/00366-8; 2008/57909-2; 2014/08458-