Abstract
Translation is a key step in control of gene expression, yet most analyses of global responses to a stimulus focus on transcription and the transcriptome. For RNA viruses in particular, which have no DNA-templated transcriptional control, control of viral and host translation is crucial. Here, we describe the method of ribosome profiling (ribo-seq) in plants, applied to virus infection. Ribo-seq is a deep sequencing technique that reveals the translatome by presenting a snapshot of the positions and relative amounts of translating ribosomes on all mRNAs in the cell. In contrast to RNA-seq, a crude cell extract is first digested with ribonuclease to degrade all mRNA not protected by a translating 80S ribosome. The resulting ribosome-protected fragments (RPFs) are deep sequenced. The number of reads mapping to a specific mRNA compared to the standard RNA-seq reads reveals the translational efficiency of that mRNA. Moreover, the precise positions of ribosome pause sites, previously unknown translatable open reading frames, and noncanonical translation events can be characterized quantitatively using ribo-seq. As this technique requires meticulous technique, here we present detailed step-by-step instructions for cell lysate preparation by flash freezing of samples, nuclease digestion of cell lysate, monosome collection by sucrose cushion ultracentrifugation, size-selective RNA extraction and rRNA depletion, library preparation for sequencing and finally quality control of sequenced data. These experimental methods apply to many plant systems, with minor nuclease digestion modifications depending on the plant tissue and species. This protocol should be valuable for studies of plant virus gene expression, and the global translational response to virus infection, or any other biotic or abiotic stress, by the host plant.