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Do Virus-Encoded Suppressors of Rna Silencing Reduce Plant eIF2a Kinase Activity and Thereby Increase Susceptibility to Infection

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Date

2014

Authors

Supervisor

Higgins, Colleen
MacDiarmid, Robin

Item type

Thesis

Degree name

Master of Applied Science

Journal Title

Journal ISSN

Volume Title

Publisher

Auckland University of Technology

Abstract

Plant cells inhibit viral infection using a process which targets double stranded RNA (dsRNA), a necessary intermediate in viral infection. This process is known as RNA silencing. Many plant viruses, and some animal viruses, encode a suppressor of RNA silencing (a VSR) which interferes with this process, thereby allowing infection to occur. In animals, cells also protect themselves against viral infection by shutdown of protein production in the invaded host cell. The production of viral dsRNA in vertebrate cells activates Protein Kinase R (PKR), which phosphorylates the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α). Phosphorylation of eIF2α interferes with protein production by the host cell, and consequently viral replication. It has been found that certain vertebrate-infecting viruses encode inhibitors of PKR activity which function in a number of ways, one of which is the activation of Inhibitor of Protein Kinase R (IPK), a host-encoded inhibitor of PKR. By inhibiting the ability of PKR to phosphorylate eIF2α, virus replication can proceed. Recently, some inhibitors of PKR were also shown to be VSRs. It is unknown if VSRs encoded by plant viruses can also affect phosphorylation of eIF2α. This research had two aims; firstly to determine whether or not eIF2α phosphorylation is triggered upon infection of Arabidopsis thaliana with the viruses Turnip vein clearing virus (TVCV) and Turnip yellow mosaic virus (TYMV), and if IPK activity alters this potential kinase activity. To determine this, wild-type A. thaliana and A. thaliana mutants lacking a functional IPK gene were infected with TVCV and TYMV and sampled over a time period of two weeks. Total plant protein was extracted from the sampled tissue and phosphorylated eIF2α detected via western blot. No eIF2α phosphorylation was seen in wild-type or mutant A. thaliana upon infection with either TVCV or TYMV. Secondly, to determine whether or not plant VSRs can also inhibit eIF2α phosphorylation by suppressing the function of plant eIF2α kinases. VSRs were transiently expressed in Nicotiana benthamiana plants, eIF2α phosphorylation by the eIF2α kinase GCN2 (general control nonrepressible 2) was triggered by amino acid starvation, and the quantity of phosphorylated eIF2α present measured via western blot to determine whether or not VSRs directly inhibit eIF2α kinase activity. No decrease in eIF2α phosphorylation levels was seen in plant tissue expressing any of the VSRs used, although expression of the p19 gene from Tomato bushy stunt virus (TBSV) appeared to cause an increase in eIF2α phosphorylation upon amino acid starvation. From this, it was concluded that neither infection with the viruses TVCV or TYMV or expression of the VSRs used in this study altered eIF2α kinase activity, with the exception of the VSR p19 from TBSV, which when expressed in N. benthamiana appears to increase eIF2α phosphorylation upon glyphosate treatment of the plant. The fact that no other VSR caused an increase in eIF2α phosphorylation upon glyphosate treatment suggests that the effect of p19 on eIF2α phosphorylation is not due to its ability to function as a VSR, and plant viruses have not developed a mechanism of inhibiting the phosphorylation of eIF2α. The lack of phosphorylation of eIF2α in A. thaliana upon infection with the viruses TVCV and TYMV suggests that eIF2α phosphorylation is not an antiviral mechanism utilised in plants, although further research in this area is required to determine if this is representative of all A. thaliana ecotypes.

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Keywords

Plant, Virus, RNA silencing, eIF2a, Supressor of silencing

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Permanent link

https://hdl.handle.net/10292/8006

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