Centro de Edafología y Biología Aplicada del Segura (CEBAS-CSIC), Departamento de Biología del Estrés y Patología Vegetal, Murcia, Spain.
Centro de Edafología y Biología Aplicada del Segura (CEBAS-CSIC), Departamento de Biología del Estrés y Patología Vegetal, Murcia, Spain.
J Virol Methods. 2019 May;267:42-47. doi: 10.1016/j.jviromet.2019.02.003. Epub 2019 Feb 13.
In situ hybridization (ISH) is an informative and relatively accessible technique for the localization of viral genomes in plant tissue and cells. However, simultaneous visualization of related plant viruses in mixed infections may be limited by the nucleotide similarity in the genomes and the single chromogenic detection over the same sample preparation. To address this issue, we used two Pepino mosaic virus isolates and performed ISH over consecutive serial cross-sections of paraffin-embedded leaf samples of single and mixed infected Nicotiana benthamiana plants. Moreover, the probe design was optimized to reduce cross-hybridisation, and co-localization was based on the overlapping of consecutive cross-sections from mixed infected leaves; thus, our results showed that both Pepino mosaic virus isolates co-localized in the same leaf tissue. In turn, both isolates were localized in the cytoplasm of the same cells. These results provide valuable information for studying mixed infections in plants by using a simple ISH procedure that is accessible to any pathology laboratory.
原位杂交（ISH）是一种在植物组织和细胞中定位病毒基因组的有用且相对容易获得的技术。然而，在混合感染中同时可视化相关植物病毒可能会受到基因组中核苷酸相似性和同一样本制备中单色显色检测的限制。为了解决这个问题，我们使用了两种 Pepino mosaic 病毒分离株，并对单个和混合感染的 Nicotiana benthamiana 植物的石蜡包埋叶片样本进行了连续的串行切片的原位杂交。此外，优化了探针设计以减少交叉杂交，并且共定位基于混合感染叶片的连续切片的重叠；因此，我们的结果表明两种 Pepino mosaic 病毒分离株在同一叶片组织中共定位。反过来，两种分离株都定位于同一细胞的细胞质中。这些结果为使用任何病理实验室都可以获得的简单原位杂交程序研究植物中的混合感染提供了有价值的信息。
