Plants are subject to a variety of abiotic and biotic stresses, including virus infection. This leads to enormous losses in crop yield and quality worldwide. Understanding how plants respond to these stresses can enable researchers to develop more healthy and robust plant varieties. The main objective of my research is to explore (i) the transcriptional and translational control of cellular gene expression in response to virus infection and (ii) the role(s) of viral noncoding subgenomic (ncsg)RNAs during infection. For this, I used red clover necrotic mosaic virus (RCNMV) as a model for economically important Tombusvirids. RCNMV generates a 3’ coterminal viral ncsgRNA, called SR1f, that belongs to the class of viral subgenomic (sg)RNAs that are functional in human flavivirus pathogenesis but which are still understudied in plant virus infection. Additionally, I also explored how translation is regulated in plants during unfolded protein response (UPR), which is elicited by many viruses and abiotic stresses.
A prerequisite for investigating viral sgRNAs is an RNA detection method that can distinguish between the coterminal genomic and sgRNAs. Using RCNMV SR1f and the analogous ncsgRNA from Zika virus (sfRNA), I developed a novel RT-PCR-based method, called DeSCo-PCR (Detection of smaller coterminal RNAs by PCR), for simple, quick, quantitative, and specific detection of viral sgRNAs. I demonstrate its advantages over the traditionally-used northern blot hybridization for detecting viral sgRNAs. This is the first RT-PCR method that distinguishes genomic from sgRNAs in most positive-sense RNA viruses.
Next, I wanted to assess the role(s) of RCNMV SR1f during infection. RCNMV SR1f belongs to the class of exoribonuclease-resistant (xr)RNA-derived viral ncsgRNAs. In plants, viral ncsgRNAs play a role in determining the severity of symptoms and the success of infection. Therefore, to explore the functions and effects of SR1f, I (i) used RNA sequencing (RNA-seq) to compare how infection with RCNMV constructs, which can or cannot produce SR1f, affect the transcriptomes of Nicotiana benthamiana and RCNMV, (ii) assessed the role of SR1f in counteracting the antiviral RNA silencing response in Arabidopsis thaliana, and (iii) determined the requirement of XRN4 for generating RCNMV SR1f in A. thaliana.
Next, I used ribosome profiling (Ribo-seq) to assess how host and viral genes are translationally regulated in RCNMV-infected plants. Most genome-wide host-virus interaction studies have used RNA-seq, which does not provide any information on translational control. Translational control is a tightly-regulated process that provides a more rapid change in gene expression than a transcriptional response. Furthermore, viruses rely completely on cellular translation machinery for viral protein synthesis. However, translational control during plant virus-host interaction has rarely been studied at the genome-wide level. Therefore, I used Ribo-seq to (i) assess the effects of RCNMV infection on the transcriptome and the translatome of A. thaliana at early and late stages of infection, (ii) identify cellular genes that are transcriptionally and translationally-regulated in response to virus infection, and (iii) assess the translational landscape of RCNMV mRNAs in infected cells.
Finally, I also used Ribo-seq to assess the translational control in roots of Zea mays seedlings during UPR. The PKR-like ER kinase (PERK)-mediated UPR pathway, which results in phosphorylation of eIF2α and subsequent inhibition of global translation in mammalian cells, is absent in the plant system. Therefore, I wanted to determine if translational control is as important in plants as it is in mammalian cells during UPR. I used Ribo-seq and other molecular assays to (i) determine if there is global inhibition of translation in plants during UPR, (ii) calculate the translational efficiencies of several UPR-responsive mRNAs, and (iii) determine the fate of the UPR-responsive mRNAs that were transcriptionally upregulated during UPR
