Abstract
BackgroundCassava brown streak disease (CBSD) has a viral aetiology and is caused by viruses belonging to the genus Ipomovirus (family Potyviridae), Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Molecular and serological methods are available for detection, discrimination and quantification of cassava brown streak viruses (CBSVs) in infected plants. However, precise determination of the viral RNA localization in infected host tissues is still not possible pending appropriate methods.ResultsWe have developed an in situ hybridization (ISH) assay based on RNAscope® technology that allows the sensitive detection and localization of CBSV RNA in plant tissues. The method was initially developed in the experimental host Nicotiana rustica and was then further adapted to cassava. Highly sensitive and specific detection of CBSV RNA was achieved without background and hybridization signals in sections prepared from non-infected tissues. The tissue tropism of CBSV RNAs appeared different between N. rustica and cassava.ConclusionsThis study provides a robust method for CBSV detection in the experimental host and in cassava. The protocol will be used to study CBSV tropism in various cassava genotypes, as well as CBSVs/cassava interactions in single and mixed infections.
Highlights
Cassava brown streak disease (CBSD) has a viral aetiology and is caused by viruses belonging to the genus Ipomovirus, Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV)
We have developed an in situ hybridization (ISH) method which is based on RNAscope® technology, allowing detection and localization of RNA targets with high specificity and sensitivity [17, 18]
Because an in situ hybridization method for CBSV RNA has not been described, we developed an ISH protocol based on RNAscope®
Summary
Cassava brown streak disease (CBSD) has a viral aetiology and is caused by viruses belonging to the genus Ipomovirus (family Potyviridae), Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Enzyme-linked immunosorbent assays (ELISA) based on monoclonal antibodies [7], reverse transcription-polymerase chain reaction (RT-PCR) [8,9,10] and quantitative RT-PCR [11,12,13,14] are routinely used in diagnosis of the viruses While these methods are sensitive, reproducible and robust for virus detection in a given cassava sample, accurate. We are interested in following the movement of cassava brown streak viruses in cassava to study tissue invasion and the possible association of CBSV with specific plant tissues and organs. To address this aim, we have developed an in situ hybridization (ISH) method which is based on RNAscope® technology, allowing detection and localization of RNA targets with high specificity and sensitivity [17, 18]. Two studies have used RNAscope® in plant tissues: for the sensitive localization of messenger RNAs (mRNAs) coding for C4 photosynthetic enzymes in maize leaves [23] and for the simultaneous visualization of two isolates of Citrus tristeza virus (CTV) in the petioles and root tissues of citrus [24]
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