Reference gene selection for gene expression studies using RT-qPCR in virus-infected planthoppers

<p>Abstract</p> <p>Background</p> <p>Planthoppers not only severely affect crops by causing mechanical damage when feeding but are also vectors of several plant virus species. The analysis of gene expression in persistently infected planthoppers might unveil the molecular basis of viral transmission. Quantitative real-time RT-PCR (RT-qPCR) is currently the most accurate and sensitive method used for quantitative gene expression analysis. In order to normalize the resulting quantitative data, reference genes with constant expression during the experimental procedures are needed.</p> <p>Results</p> <p>Partial sequences of the commonly used reference genes actin (ACT), α1-tubulin (TUB), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), elongation factor 1 alpha (EF1A), ribosomal protein S18 (RPS18) and polyubiquitin C (UBI) from <it>Delphacodes kuscheli</it>, a planthopper capable of persistently transmitting the plant fijivirus <it>Mal de Río Cuarto virus </it>(MRCV), were isolated for the first time. Specific RT-qPCR primers were designed and the expression stability of these genes was assayed in MRCV-infective and naïve planthoppers using geNorm, Normfinder and BestKeeper tools. The overall analysis showed that UBI, followed by 18S and ACT, are the most suitable genes as internal controls for quantitative gene expression studies in MRCV-infective planthoppers, while TUB and EF1A are the most variable ones. Moreover, EF1A was upregulated by MRCV infection.</p> <p>Conclusions</p> <p>A RT-qPCR platform for gene expression analysis in the MRCV-infected planthopper vector <it>Delphacodes kuscheli </it>was developed. Our work is the first report on reference gene selection in virus-infected insects, and might serve as a precedent for future gene expression studies on MRCV and other virus-planthopper pathosystems.</p

In vivo subcellular localization of Mal de Río Cuarto virus (MRCV) non-structural proteins in insect cells reveals their putative functions

The in vivo subcellular localization of Mal de Río Cuarto virus (MRCV, Fijivirus, Reoviridae) non-structural proteins fused to GFP was analyzed by confocal microscopy. P5-1 showed a cytoplasmic vesicular-like distribution that was lost upon deleting its PDZ binding TKF motif, suggesting that P5-1 interacts with cellular PDZ proteins. P5-2 located at the nucleus and its nuclear import was affected by the deletion of its basic C-termini. P7-1 and P7-2 also entered the nucleus and therefore, along with P5-2, could function as regulators of host gene expression. P6 located in the cytoplasm and in perinuclear cloud-like inclusions, was driven to P9-1 viroplasm-like structures and co-localized with P7-2, P10 and α-tubulin, suggesting its involvement in viroplasm formation and viral intracellular movement. Finally, P9-2 was N-glycosylated and located at the plasma membrane in association with filopodia-like protrusions containing actin, suggesting a possible role in virus cell-to-cell movement and spread.Instituto de BiotecnologíaFil: Maroniche, Guillermo Andrés. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Mongelli, Vanesa Claudia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Llauger, Gabriela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Alfonso, Victoria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Taboga, Oscar Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Del Vas, Mariana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentin

AcMNPV Core Gene ac109 Is Required for Budded Virion Transport to the Nucleus and for Occlusion of Viral Progeny

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac109 core gene has been previously characterized as an essential late gene. Our results showed that budded virions could be detected in supernatants of infected Sf-9 cells, even when ac109 knockout viruses displayed a single-cell infection phenotype. Moreover, confocal microscopy analysis revealed that budded virions can enter the cytoplasm but are unable to enter the cell nucleus. This defect could be repaired by complementing ac109 in trans. In addition, polyhedra of normal size could be detected in Sf-9 nuclei infected with ac109 knockout viruses. However, electron microscopy demonstrated that these occlusion bodies were empty. Altogether, these results indicate that ac109 is required for infectivity of both phenotypes of virus.Instituto de BiotecnologíaFil: Alfonso, Victoria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Maroniche, Guillermo Andrés. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Reca, Sol Rita. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Lopez, Maria Gabriela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina.Fil: Del Vas, Mariana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Taboga, Oscar Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentin

A practical approach to the understanding and teaching of RNA silencing in plants

Gene silencing, also called RNA interference (RNAi) is a specific mechanism of RNA degradation involved in gene regulation, development and defense in eukaryotic organisms. It became an important subject in the teaching programs of molecular biology, genetics and biotechnology courses in the last years. The aim of this work is to provide simple and inexpensive assays to understand and teach gene silencing using plants as model systems. The use of transient and permanent transgenic plants for expressing reporter genes, like those derived from jellyfish green fluorescent protein (gfp) encoding gene, provides a nice, colorful and conclusive image of gene silencing. Three experimental approaches to evidence RNA silencing are depicted. In the first approach gene silencing is demonstrated after transient expression of reporter genes in non-transgenic plants. In the second, silencing is triggered against a reporter gene stably integrated into a transgenic plant. The third approach involves the triggering of RNA silencing against endogenous genes using viral vectors. In addition we illustrate systemic gene silencing showing how the silencing signal is spread over a plant and finally it is also demonstrated the suppression of gene silencing. The first group of experiments is recommended to be tough on undergraduate courses, the following two sections are recommended for graduate courses. Hopefully, it will help students to understand this important phenomenon and to unravel the importance of gene silencing as a key gene regulation mechanism and as a molecular and biotechnological tool

Occurrence of a closely-related isolate to Maize yellow striate virus in wheat plants

In Argentina, wheat fields have exhibited virus-like symptoms, such as chlorotic streaking, dwarfing, yellowing and empty ears since 2007. Symptomatic plants and leaves samples were collected in 2007 from Marcos Juarez and in 2008 and 2013 from Río Cuarto. The virus was experimentally transmitted from symptomatic wheat plants to wheat cv. Baguette 10 and cv. BioINTA 3005 using the vector Delphacodes kuscheli Fennah (Delphacidae), producing chlorotic streaking, dwarfing and yellowing in the inoculated cereals at 10?15 days post-inoculation. Virus presence was confirmed by electron microscopy and RT-PCR using degenerated primers, which amplified a conserved region of the plant rhabdovirus polymerase (L) gene. Sequence comparison showed 98% nucleotide identity with Maize yellow striate virus C. Caroya (JQ715419) isolated from corn in Argentina. To our knowledge, this is the first report of the occurrence of Maize yellow striate virus in wheat in Argentina.Fil: Dumón, Analía Delina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mattio, Maria Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; ArgentinaFil: Argüello Caro, Evangelina Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Alemandri, Vanina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; ArgentinaFil: Puyané, Valeria Aida. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; ArgentinaFil: del Vas, Mariana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: López Lambertini, Paola María. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; ArgentinaFil: Truol, Graciela Ana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; Argentin

Development of nanobodies against Mal de Río Cuarto virus major viroplasm protein P9‑1 for diagnostic sandwich ELISA and immunodetection

Mal de Río Cuarto virus (MRCV) is a member of the genus Fijivirus of the family Reoviridae that causes a devastating disease in maize and is persistently and propagatively transmitted by planthopper vectors. Virus replication and assembly occur within viroplasms formed by viral and host proteins. This work describes the isolation and characterization of llama-derived Nanobodies (Nbs) recognizing the major viral viroplasm component, P9-1. Specific Nbs were selected against recombinant P9-1, with affinities in the nanomolar range as measured by surface plasmon resonance. Three selected Nbs were fused to alkaline phosphatase and eGFP to develop a sandwich ELISA test which showed a high diagnostic sensitivity (99.12%, 95% CI 95.21–99.98) and specificity (100%, 95% CI 96.31–100) and a detection limit of 0.236 ng/ml. Interestingly, these Nanobodies recognized different P9-1 conformations and were successfully employed to detect P9-1 in pull-down assays of infected maize extracts. Finally, we demonstrated that fusions of the Nbs to eGFP and RFP allowed the immunodetection of virus present in phloem cells of leaf thin sections. The Nbs developed in this work will aid the study of MRCV epidemiology, assist maize breeding programs, and be valuable tools to boost fundamental research on viroplasm structure and maturation.Instituto de BiotecnologíaFil: Llauger, Gabriela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Llauger, Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Monti, Demian Esteban. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Monti, Demian Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Aduriz Guerrero, Matí­as. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología e Innovaciones Tecnológicas; ArgentinaFil: Aduriz Guerrero, Matí­as. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Romão, Ema. Vrije Universiteit Brussel. Lab of Cellular and Molecular Immunology; BélgicaFil: Dumon, Analia Delina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; ArgentinaFil: Dumon, Analia Delina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mattio, Maria Fernanda. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; ArgentinaFil: Mattio, Maria Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Wigdorovitz, Andres. Instituto Nacional de Tecnología Agropecuaria (INTA). INCUINTA. Instituto de Virología e Innovaciones Tecnológicas; ArgentinaFil: Wigdorovitz, Andres. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Muyldermans, Serge. Vrije Universiteit Brussel. Lab of Cellular and Molecular Immunology; BélgicaFil: Muyldermans, Serge. Dalian University of Technology. School of Bioengineering. Liaoning Key Laboratory of Molecular Recognition and Imaging; ChinaFil: Vincke, Cécile. Vrije Universiteit Brussel. Lab of Cellular and Molecular Immunology; BélgicaFil: Vincke, Cécile. VIB Center for Inflammation Research. Myeloid Cell Immunology Lab; BélgicaFil: Parreño, Viviana. Instituto Nacional de Tecnología Agropecuaria (INTA). INCUINTA. Instituto de Virología e Innovaciones Tecnológicas; ArgentinaFil: Parreño, Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Del Vas, Mariana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Del Vas, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Virus infection elevates transcriptional activity of miR164a promoter in plants

Background: Micro RNAs (miRs) constitute a large group of endogenous small RNAs that have crucial roles in many important plant functions. Virus infection and transgenic expression of viral proteins alter accumulation and activity of miRs and so far, most of the published evidence involves post-transcriptional regulations. Results: Using transgenic plants expressing a reporter gene under the promoter region of a characterized miR (P-miR164a), we monitored the reporter gene expression in different tissues and during Arabidopsis development. Strong expression was detected in both vascular tissues and hydathodes. P-miR164a activity was developmentally regulated in plants with a maximum expression at stages 1.12 to 5.1 (according to Boyes, 2001) along the transition from vegetative to reproductive growth. Upon quantification of P-miR164a-derived GUS activity after Tobacco mosaic virus Cg or Oilseed rape mosaic virus (ORMV) infection and after hormone treatments, we demonstrated that ORMV and gibberellic acid elevated P-miR164a activity. Accordingly, total mature miR164, precursor of miR164a and CUC1 mRNA (a miR164 target) levels increased after virus infection and interestingly the most severe virus (ORMV) produced the strongest promoter induction. Conclusion: This work shows for the first time that the alteration of miR pathways produced by viral infections possesses a transcriptional component. In addition, the degree of miR alteration correlates with virus severity since a more severe virus produces a stronger P-miR164a induction.Instituto de BiotecnologíaFil: Bazzini, Ariel Alejandro. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Almasia, Natalia Ines. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Manacorda, Carlos Augusto. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Mongelli, Vanesa Claudia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Conti, Gabriela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Maroniche, Guillermo Andrés. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rodriguez, Maria Cecilia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Distefano, Ana Julia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Hopp, Horacio Esteban. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Del Vas, Mariana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Asurmendi, Sebastian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

ICTV virus taxonomy profile : Sedoreoviridae 2022

Sedoreoviridae is a large family of icosahedral viruses that are usually regarded as non- enveloped with segmented (10–12 linear segments) dsRNA genomes of 18–26 kbp. Sedoreovirids have a broad host range, infecting mammals, birds, crustaceans, arthropods, algae and plants. Some of them have important pathogenic potential for humans (e.g. rotavirus A), livestock (e.g. bluetongue virus) and plants (e.g. rice dwarf virus).Instituto de BiotecnologíaFil: Matthijnssens, Jelle. University of Leuven; BélgicaFil: Attoui, Houssam. National Institute for Agricultural Research (INRA); FranciaFil: Bányai, Krisztián. Veterinary Medical Research Institute; HungríaFil: Brussaard, Corina P. D. NIOZ Royal Netherlands Institute for Sea Research; Países BajosFil: Brussaard, Corina P. D. University of Utrecht; Países BajosFil: Danthi, Pranav. Indiana University; Estados UnidosFil: Del Vas, Mariana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); ArgentinaFil: Dermody, Terence S. University of Pittsburgh. School of Medicine; Estados UnidosFil: Duncan, Roy. Dalhousie University; CanadáFil: Fāng, Qín. Wuhan Institute of Virology; ChinaFil: Johne, Reimar. German Federal Institute for Risk Assessment; AlemaniaFil: Mertens, Peter P. C. University of Nottingham; Reino UnidoFil: Jaafar, Fauziah Mohd. Ecole Nationale Vétérinaire d’Alfort; FranciaFil: Patton, John T. Indiana University; Estados UnidosFil: Sasaya, Takahide. National Agriculture and Food Research Organization; JapónFil: Suzuki, Nobuhiro. Okayama University. JapónFil: Wei, Taiyun. Fujian Agriculture and Forestry University; Chin

Identification and biological characterization of Barley yellow striate mosaic virus (BYSMV): a new wheat disease in Argentina

Wheat (Triticum aestivum L.) is the most important winter grain in Argentina. Its production is limited by several factors, including viral diseases. The aim of this study was to identify and characterize a recently detected disease of wheat crops in Argentina. Symptomatic plants were collected from different locations from the wheat production area, Miramar and Balcarce (Buenos Aires), Río Cuarto, Rosales and Marcos Juarez (Córdoba), Paraná (Entre Ríos) and General Pico (La Pampa). Samples were characterized by: electron microscopy (leaf-dip and ultrathin sections), serological tests (with antiserum reacting against different wheat viruses by DAS-ELISA and indirect ELISA), mechanical, seed and vector transmission assays, differential host range, and susceptibility of different wheat cultivars in natural infections. The results showed that this new disease is caused by Barley yellow striate mosaic virus (BYSMV) (Rhabdoviridae-Cytorhabdovirus), a widely distributed virus that can be transmitted to other crops such as barley, oat and triticale. This work representsthe first report of BYSMV in Argentina.El cultivo de trigo (Triticum aestivum L.), es el cereal de invierno de mayor importancia económica en Argentina. Su producción se halla limitada por diversos factores, entre ellos las enfermedades virales. El objetivo del presente trabajo fue identificar y caracterizar biológicamente una enfermedad viral detectada recientemente en el cultivo de trigo en Argentina, que ha presentado elevada incidencia en los últimos años. Para ello, se recolectaron plantas con síntomas en localidades de: Miramar y Balcarce (Buenos Aires), Río Cuarto, Rosales y Marcos Juárez (Córdoba), Paraná (Entre Ríos) y General Pico (La Pampa) de la región triguera Argentina. Las muestras fueron caracterizadas a través de: microscopia electrónica ("leaf-dip" y cortes ultrafinos), serología (con antisueros para diferentes virosis del trigo por DAS-ELISA y ELISA indirecto), transmisión mecánica, por semilla y por vectores (delfácidos), rango de hospedantes diferenciales y susceptibilidad diferencial de diferentes cultivares de trigo en infecciones naturales. Los resultados obtenidos evidenciaron que esta nueva enfermedad es causada por el Barley yellow striate mosaic virus (BYSMV) (Rhabdoviridae-Cytorhabdovirus), un virus ampliamente distribuido a nivel mundial y que representa un riesgo potencial para otros cultivos como cebada, avena y triticale. Este trabajo representa el primer reporte del BYSMV en Argentina.Fil: Dumón, Analía Delina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Unidad de Fitopatología y Modelización Agrícola - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Unidad de Fitopatología y Modelización Agrícola; ArgentinaFil: Argüello Caro, Evangelina Beatriz. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; ArgentinaFil: Alemandri, María V.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; ArgentinaFil: Bainotti, Carlos Tomas. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; ArgentinaFil: Mattio, María F.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; ArgentinaFil: Rodríguez, Sandra M.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; ArgentinaFil: del Vas, Mariana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Truol, Graciela Ana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; Argentin