Abstract
BackgroundPlanthoppers 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.ResultsPartial 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 Delphacodes kuscheli, a planthopper capable of persistently transmitting the plant fijivirus Mal de Río Cuarto virus (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.ConclusionsA RT-qPCR platform for gene expression analysis in the MRCV-infected planthopper vector Delphacodes kuscheli 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.
Highlights
Planthoppers severely affect crops by causing mechanical damage when feeding but are vectors of several plant virus species
Our study aimed to examine the stability of potential reference genes expression upon Mal de Río Cuarto virus (MRCV) infection of single D. kuscheli planthoppers and to rank them according to their reliability as internal controls
Planthoppers able to transmit MRCV to wheat plants were kept for subsequent RT-qPCR studies along with control naïve insects subjected to the same procedure but always fed on uninfected plants
Summary
Planthoppers severely affect crops by causing mechanical damage when feeding but are vectors of several plant virus species. Gateway-based vectors applicable for the subcellular localization analysis of proteins in cultured Bombyx genus of the Reoviridae family) [13,14] and the planthopper Peregrinus maidis transmits Maize mosaic virus (MMV: Nucleorhabdovirus genus of the Rhabdoviridae family) [15]. Another planthopper species, Delphacodes kuscheli, is a natural vector that propagatively transmits Mal de Río Cuarto virus (MRCV, Fijivirus genus of the Reoviridae family). MRCV causes the most important maize disease in Argentina and its genome sequence has recently been completed [16,17,18,19]
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