Ribosome Profiling of Plants.

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.

Variational inference for detecting differential translation in ribosome profiling studies.

Translational efficiency change is an important mechanism for regulating protein synthesis. Experiments with paired ribosome profiling (Ribo-seq) and mRNA-sequencing (RNA-seq) allow the study of translational efficiency by simultaneously quantifying the abundances of total transcripts and those that are being actively translated. Existing methods for Ribo-seq data analysis either ignore the pairing structure in the experimental design or treat the paired samples as fixed effects instead of random effects. To address these issues, we propose a hierarchical Bayesian generalized linear mixed effects model which incorporates a random effect for the paired samples according to the experimental design. We provide an analytical software tool, "riboVI," that uses a novel variational Bayesian algorithm to fit our model in an efficient way. Simulation studies demonstrate that "riboVI" outperforms existing methods in terms of both ranking differentially translated genes and controlling false discovery rate. We also analyzed data from a real ribosome profiling experiment, which provided new biological insight into virus-host interactions by revealing changes in hormone signaling and regulation of signal transduction not detected by other Ribo-seq data analysis tools.

Effects of the Noncoding Subgenomic RNA of Red Clover Necrotic Mosaic Virus in Virus Infection.

In recent years, a new class of viral noncoding subgenomic RNA (ncsgRNA) has been identified. This RNA is generated as a stable degradation product via an exoribonuclease-resistant RNA (xrRNA) structure, which blocks the progression of 5'→3' exoribonuclease on viral RNAs in infected cells. Here, we assess the effects of the ncsgRNA of red clover necrotic mosaic virus (RCNMV), called SR1f, in infected plants. We demonstrate the following: (i) the absence of SR1f reduces symptoms and decreases viral RNA accumulation in Nicotiana benthamiana and Arabidopsis thaliana plants; (ii) SR1f has an essential function other than suppression of RNA silencing; and (iii) the cytoplasmic exoribonuclease involved in mRNA turnover, XRN4, is not required for SR1f production or virus infection. A comparative transcriptomic analysis in N. benthamiana infected with wild-type RCNMV or an SR1f-deficient mutant RCNMV revealed that wild-type RCNMV infection, which produces SR1f and much higher levels of virus, has a greater and more significant impact on cellular gene expression than the SR1f-deficient mutant. Upregulated pathways include plant hormone signaling, plant-pathogen interaction, MAPK signaling, and several metabolic pathways, while photosynthesis-related genes were downregulated. We compare this to host genes known to participate in infection by other tombusvirids. Viral reads revealed a 10- to 100-fold ratio of positive to negative strand, and the abundance of reads of both strands mapping to the 3' region of RCNMV RNA1 support the premature transcription termination mechanism of synthesis for the coding sgRNA. These results provide a framework for future studies of the interactions and functions of noncoding RNAs of plant viruses. IMPORTANCE Knowledge of how RNA viruses manipulate host and viral gene expression is crucial to our understanding of infection and disease. Unlike viral protein-host interactions, little is known about the control of gene expression by viral RNA. Here, we begin to address this question by investigating the noncoding subgenomic RNA (ncsgRNA) of red clover necrotic mosaic virus (RCNMV), called SR1f. Similar exoribonuclease-resistant RNAs of flaviviruses are well studied, but the roles of plant viral ncsgRNAs, and how they arise, are poorly understood. Surprisingly, we find the likely exonuclease candidate, XRN4, is not required to generate SR1f, and we assess the effects of SR1f on virus accumulation and symptom development. Finally, we compare the effects of infection by wild-type RCNMV versus an SR1f-deficient mutant on host gene expression in Nicotiana benthamiana, which reveals that ncsgRNAs such as SR1f are key players in virus-host interactions to facilitate productive infection.

Control of translation during the unfolded protein response in maize seedlings: Life without PERKs.

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) defines a condition called ER stress that induces the unfolded protein response (UPR). The UPR in mammalian cells attenuates protein synthesis initiation, which prevents the piling up of misfolded proteins in the ER. Mammalian cells rely on Protein Kinase RNA-Like Endoplasmic Reticulum Kinase (PERK) phosphorylation of eIF2α to arrest protein synthesis, however, plants do not have a PERK homolog, so the question is whether plants control translation in response to ER stress. We compared changes in RNA levels in the transcriptome to the RNA levels protected by ribosomes and found a decline in translation efficiency, including many UPR genes, in response to ER stress. The decline in translation efficiency is due to the fact that many mRNAs are not loaded onto polyribosomes (polysomes) in proportion to their increase in total RNA, instead some of the transcripts accumulate in stress granules (SGs). The RNAs that populate SGs are not derived from the disassembly of polysomes because protein synthesis remains steady during stress. Thus, the surge in transcription of UPR genes in response to ER stress is accompanied by the formation of SGs, and the sequestration of mRNAs in SGs may serve to temporarily relieve the translation load during ER stress.

A rapid and simple quantitative method for specific detection of smaller coterminal RNA by PCR (DeSCo-PCR): application to the detection of viral subgenomic RNAs.

RNAs that are 5'-truncated versions of a longer RNA but share the same 3' terminus can be generated by alternative promoters in transcription of cellular mRNAs or by replicating RNA viruses. These truncated RNAs cannot be distinguished from the longer RNA by a simple two-primer RT-PCR because primers that anneal to the cDNA from the smaller RNA also anneal to-and amplify-the longer RNA-derived cDNA. Thus, laborious methods, such as northern blot hybridization, are used to distinguish shorter from longer RNAs. For rapid, low-cost, and specific detection of these truncated RNAs, we report detection of smaller coterminal RNA by PCR (DeSCo-PCR). DeSCo-PCR uses a nonextendable blocking primer (BP), which outcompetes a forward primer (FP) for annealing to longer RNA-derived cDNA, while FP outcompetes BP for annealing to shorter RNA-derived cDNA. In the presence of BP, FP, and the reverse primer, only cDNA from the shorter RNA is amplified in a single-tube reaction containing both RNAs. Many positive strand RNA viruses generate 5'-truncated forms of the genomic RNA (gRNA) called subgenomic RNAs (sgRNA), which play key roles in viral gene expression and pathogenicity. We demonstrate that DeSCo-PCR is easily optimized to selectively detect relative quantities of sgRNAs of red clover necrotic mosaic virus from plants and Zika virus from human cells, each infected with viral strains that generate different amounts of sgRNA. This technique should be readily adaptable to other sgRNA-producing viruses, and for quantitative detection of any truncated or alternatively spliced RNA.

Noncoding RNAs of Plant Viruses and Viroids: Sponges of Host Translation and RNA Interference Machinery.

Noncoding sequences in plant viral genomes are well-known to control viral replication and gene expression in cis. However, plant viral and viroid noncoding (nc)RNA sequences can also regulate gene expression acting in trans, often acting like 'sponges' that bind and sequester host cellular machinery to favor viral infection. Noncoding sequences of small subgenomic (sg)RNAs of Barley yellow dwarf virus (BYDV) and Red clover necrotic mosaic virus (RCNMV) contain a cap-independent translation element that binds translation initiation factor eIF4G. We provide new evidence that a sgRNA of BYDV can globally attenuate host translation, probably by sponging eIF4G. Subgenomic ncRNA of RCNMV is generated via 5' to 3' degradation by a host exonuclease. The similar noncoding subgenomic flavivirus (sf)RNA, inhibits the innate immune response, enhancing viral pathogenesis. Cauliflower mosaic virus transcribes massive amounts of a 600-nt ncRNA, which is processed into small RNAs that overwhelm the host's RNA interference (RNAi) system. Viroids use the host RNAi machinery to generate viroid-derived ncRNAs that inhibit expression of host defense genes by mimicking a microRNA. More examples of plant viral and viroid ncRNAs are likely to be discovered, revealing fascinating new weaponry in the host-virus arms race.

Discovery of Novel Leaf Rust Responsive microRNAs in Wheat and Prediction of Their Target Genes.

MicroRNAs are endogenous small noncoding RNAs which play critical roles in gene regulation. Few wheat (Triticum aestivum L.) miRNA sequences are available in miRBase repertoire and knowledge of their biological functions related to biotic stress is limited. We identified 52 miRNAs, belonging to 19 families, from next-generation transcriptome sequence data based on homology search. One wheat specific novel miRNA was identified but could not be ascribed or assigned to any known miRNA family. Differentially expressed 22 miRNAs were found between susceptible and resistant wheat near-isogenic lines inoculated with leaf rust pathogen Puccinia triticina and compared with mock inoculated controls. Most miRNAs were more upregulated in susceptible NIL compared to resistant NIL. We identified 1306 potential target genes for these 52 miRNAs with vital roles in response to stimuli, signaling, and diverse metabolic and cellular processes. Gene ontology analysis showed 66, 20, and 35 target genes to be categorized into biological process, molecular function, and cellular component, respectively. A miRNA-mediated regulatory network revealed relationships among the components of the targetome. The present study provides insight into potential miRNAs with probable roles in leaf rust pathogenesis and their target genes in wheat which establish a foundation for future studies.