The response of eIF2α kinases to plant virus infection

Abstract

When the eukaryotic translation initiation factor 2 α (eIF2α) is phosphorylated in several eukaryotes, general mRNA translation is inhibited, but some mRNA species that contain an upstream open reading frame (uORF) are preferentially translated. In plants, two types of eIF2α kinases have been reported - general control non-derepressible-2 kinase (GCN2) and protein kinase RNA-activated (PKR). The aim of this research was to further elucidate the role of eIF2α phosphorylation in plants, with a particular focus on the response of eIF2α kinases, including a putative PKR, to virus infection. It is not clear whether in plants, as in other eukaryotes, if phosphorylation of eIF2 regulates translation of mRNAs with particular uORFs. To determine whether this type of regulation occurs in plants, a Luciferase/Renilla reporter system and yeast general control non-derepressible-4 (GCN4) uORFs were used. The uORFs did not regulate the translation of a main reporter ORF in Nicotiana benthamiana plants, regardless of the eIF2α phosphorylation status. In mammals, viral double-stranded RNA activates PKR. There is some evidence to suggest that plants also encode a virus-activated eIF2α kinase; however, this kinase has not been identified. Arabidopsis thaliana (At) calcium-dependent protein kinase-19 (CPK19) has been identified as a putative PKR-like kinase. This research demonstrated that although AtCPK19 phosphorylates eIF2, the phosphorylation event does not occur on the critical serine residue of eIF2, making it unlikely to be a plant PKR. Arabidopsis lines overexpressing AtGCN2 did not have viral resistance, nor was eIF2α phosphorylated in response to virus infection. In lines transformed to ectopically express human PKR, PKR could not be detected and had a similar phenotype. However, N. benthamiana plants that were treated with glyphosate to induce eIF2α phosphorylation did become infected less rapidly compared to plants that were left untreated. Finally, by undertaking research to repeat, connect and extend published work that indicated 58- kDa inhibitor of protein kinase (P58IPK) is an inhibitor of virus-activated plant eIF2α kinase activity; this research determined that the published results were not repeatable in a very similar pathosystem. Instead, there was no virus-activated eIF2α kinase activity in plants, with or without P58IPK. Overall, the conclusion from this thesis is that the only eIF2α kinase encoded by plants is GCN2 and that under the conditions tested, eIF2α is not phosphorylated in response to virus infection.