Identification of the putative plant-functional equivalent of double-stranded RNA-dependent protein kinase R.

Abstract

Viruses constantly challenge plants; in response, plants have evolved a number of defense systems that protect them or reduce the severity of the virus invasion. Translational inhibition, an innate antiviral defense mechanism that is utilised by, and most studied in, mammals, is proposed also to exist in plants. This mechanism is activated by the presence of double-stranded (ds) RNA, a necessary replication intermediate of RNA viruses, and relies on the phosphorylation of the α subunit of the eukaryotic initiation factor (eIF) 2 by the dsRNA-dependent protein kinase R (PKR). Phosphorylation of eIF2α results in the global inhibition of translational initiation in the host cell, and subsequently blocks the infection of the virus. Initial evidence supports the presence of a plant-functional homolog of PKR (pPKR), but no homologous sequence has so far been detected. The aim of this doctoral research was to identify pPKR via a proteomics approach using Arabidopsis thaliana, whereby the protein was first enriched as determined by the correlation of its activity, and then sequenced. The identification of pPKR would provide evidence for the presence of a novel antiviral defense mechanism in plants. The immobilised eIF2α peptide assay was developed, refined, and validated to allow the rapid detection and quantification of eIF2α phosphorylation activity in vitro. Flower buds from young Arabidopsis plants following light exposure (4 hr) had the highest eIF2α phosphorylation activity and ds but not single-stranded RNA activated this pPKR activity. Of the six different techniques used to enrich for pPKR from Arabidopsis, size exclusion chromatography was the most consistent. Mass spectrometry analysis of proteins associated with kinase activity identified a number of RNA binding proteins and protein kinases, including calcium-dependent protein kinases (CPKs). The assessment of ions and chelators on putative pPKR activity revealed that eIF2α phosphorylation was activated in the presence of both calcium and the chelator ethylene glycol tetraacetic acid (EGTA). Arabidopsis T-DNA insertion lines (15 of the 16 Arabidopsis dsRNA binding motif-containing genes, all 34 Arabidopsis CPKs, a kinase of unknown function, the eIF2α kinase GCN2, and the proposed plant-functional homolog of the mammalian PKR inhibitor p58IPK) were imported, and where possible, crossed to homozygosity and tested for eIF2α phosphorylation activity. Attempts were made to generate and test the eIF2α phosphorylation activity in nine transgenic Arabidopsis lines overexpressing either mammalian PKR, an inhibitor of PKR, or a disrupted eIF2α. Of the 20 transgenic plant lines that could be tested, a statistically significant decrease in eIF2α phosphorylation activity was seen in the CPK19 T-DNA insertion line and p58IPK overexpression line. These two plants also had similarly stunted phenotypes. These findings provide evidence for CPK19 as the putative plant-functional homolog of PKR