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

Pyrophagus tigrinus Remes Lenicov & Varela (Hemiptera: Delphacidae), a new vector of the Mal de Río Cuarto virus (MRCV, Fijivirus) under experimental conditions

El Mal de Río Cuarto virus (MRCV), agente causal de la enfermedad más importante del maíz en la Argentina, es transmitido principalmente por Delphacodes kuscheli. La presencia de otros delfácidos en áreas con la virosis, permite suponer que éstos podrían tener un rol en su epidemiología. Pyrophagus tigrinus fue hallada por primera vez en 1997 en Jesús María (Córdoba) sobre cultivos de triticale afectados con el MRCV. El objetivo fue demostrar la capacidad de P. tigrinus para transmitir el MRCV a triticale en condiciones experimentales. Se inició la cría en condiciones controladas y se realizaron transmisiones a triticale, empleando el mismo cereal como fuente de inóculo. Los síntomas observados y el patrón electroforético del dsRNA viral se correspondieron con el del MRCV en transmisiones con D. kuscheli. Se observaron agregados de partículas virales en células floemáticas. La capacidad de P. tigrinus para transmitir el MRCV tiene importantes implicaciones en la epidemiología de esta enfermedad.Mal de Río Cuarto virus (MRCV), causal agent of the most important disease that affects maize in Argentina, is spread under natural conditions by Delphacodes kuscheli. The presence of other delphacid species in areas infected with this virus suggests that those species could have a function in the epidemiology of disease. Pyrophagus tigrinus was first found by chance in 1997 in Jesús María (Córdoba) in triticale crops affected by MRCV. The aim of this work was to demonstrate the capacity of P. tigrinus to transmit the MRCV to triticale under experimental conditions. The culture of P. tigrinus under controlled conditions was initiated and Mentransmissions to triticale using this cereal as source of inoculum were carried out. The symptoms observed and the electrophoretic pattern of viral dsRNA were corresponded with the MRCV using D. kuscheli as vector. Aggregates of MRCV-like particles in phloem tissue cells were also observed. The vector capacity of P. tigrinus has very important implications in the epidemiology of this disease.Facultad de Ciencias Naturales y Muse

Mal de Río Cuarto Virus infection triggers the production of distinctive viral-derived siRNA profiles in wheat and Its planthopper vector

Plant reoviruses are able to multiply in gramineae plants and delphacid vectors encountering different defense strategies with unique features. This study aims to comparatively assess alterations of small RNA (sRNA) populations in both hosts upon virus infection. For this purpose, we characterized the sRNA profiles of wheat and planthopper vectors infected by Mal de Río Cuarto virus (MRCV, Fijivirus, Reoviridae) and quantified virus genome segments by quantitative reverse transcription PCR We provide evidence that plant and insect silencing machineries differentially recognize the viral genome, thus giving rise to distinct profiles of virus-derived small interfering RNAs (vsiRNAs). In plants, most of the virus genome segments were targeted preferentially within their upstream sequences and vsiRNAs mapped with higher density to the smaller genome segments than to the medium or larger ones. This tendency, however, was not observed in insects. In both hosts, vsiRNAs were equally derived from sense and antisense RNA strands and the differences in vsiRNAs accumulation did not correlate with mRNAs accumulation. We also established that the piwi-interacting RNA (piRNA) pathway was active in the delphacid vector but, contrary to what is observed in virus infected mosquitoes, virus-specific piRNAs were not detected. This work contributes to the understanding of the silencing response in insect and plant hosts.Inst. de BiotecnologíaFil: De Haro, Luis Alejandro. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Dumon, Analia Delina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; ArgentinaFil: Mattio, Maria Fernanda. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; ArgentinaFil: Arguello Caro, Evangelina Beatriz. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; ArgentinaFil: Llauger, Gabriela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Zavallo, Diego. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Blanc, Hervé. Centre National de la Recherche Scientifique. Institut Pasteur, Viruses and RNA Interference Unit; FranciaFil: Truol, Graciela Ana Maria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; ArgentinaFil: Mongelli, Vanesa Claudia. Centre National de la Recherche Scientifique. Institut Pasteur, Viruses and RNA Interference Unit; FranciaFil: Saleh, María-Carla. Centre National de la Recherche Scientifique. Institut Pasteur, Viruses and RNA Interference Unit; FranciaFil: Asurmendi, Sebastian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Del Vas, Mariana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Wheat curl mite, Aceria tosichella, and transmitted viruses: an expanding pest complex affecting cereal crops

The wheat curl mite (WCM), Aceria tosichella, and the plant viruses it transmits represent an invasive mite-virus complex that has affected cereal crops worldwide. The main damage caused by WCM comes from its ability to transmit and spread multiple damaging viruses to cereal crops, with Wheat streak mosaic virus (WSMV) and Wheat mosaic virus (WMoV) being the most important. Although WCM and transmitted viruses have been of concern to cereal growers and researchers for at least six decades, they continue to represent a challenge. In older affected areas, for example in North America, this mite-virus complex still has significant economic impact. In Australia and South America, where this problem has only emerged in the last decade, it represents a new threat to winter cereal production. The difficulties encountered in making progress towards managing WCM and its transmitted viruses stem from the complexity of the pathosystem. The most effective methods for minimizing losses from WCM transmitted viruses in cereal crops have previously focused on cultural and plant resistance methods. This paper brings together information on biological and ecological aspects of WCM, including its taxonomic status, occurrence, host plant range, damage symptoms and economic impact. Information about the main viruses transmitted by WCM is also included and the epidemiological relationships involved in this vectored complex of viruses are also addressed. Management strategies that have been directed at this mite-virus complex are presented, including plant resistance, its history, difficulties and advances. Current research perspectives to address this invasive mite-virus complex and minimize cereal crop losses worldwide are also discussed.Instituto de Patología VegetalFil: Navia, Denise. Embrapa Recursos Genéticos e Biotecnologia; BrasilFil: Mendonça, Renata Santos de. Embrapa Recursos Genéticos e Biotecnologia; BrasilFil: Skoracka, Anna. Adam Mickiewicz University. Faculty of Biology. Institute of Environmental Biology. Department of Animal Taxonomy and Ecology; PoloniaFil: Szydło, Wiktoria. Adam Mickiewicz University. Faculty of Biology. Institute of Environmental Biology. Department of Animal Taxonomy and Ecology; PoloniaFil: Knihinicki, Danuta. Orange Agricultural Institute. Agricultural Scientific Collections Unit. NSW Department of Primary Industries; AustraliaFil: Hein, Gary L. University of Nebraska at Lincoln; Estados UnidosFil: Pereira, Paulo Roberto Valle da Silva. Embrapa Trigo; BrasilFil: Truol, Graciela Ana Maria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; ArgentinaFil: Lau, Douglas. Embrapa Trigo; Brasi