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The plant-specific translation initiation complex eIFiso4F is encoded by three genes in Arabidopsis (Arabidopsis thaliana)-genes encoding the cap binding protein eIFiso4E (eifiso4e) and two isoforms of the large subunit scaffolding protein eIFiso4G (i4g1 and i4g2). To quantitate phenotypic changes, a phenomics platform was used to grow wild-type and mutant plants (i4g1, i4g2, i4e, i4g1 x i4g2, and i4g1 x i4g2 x i4e [i4f]) under various light conditions. Mutants lacking both eIFiso4G isoforms showed the most obvious phenotypic differences from the wild type. Two-dimensional differential gel electrophoresis and mass spectrometry were used to identify changes in protein levels in plants lacking eIFiso4G. Four of the proteins identified as measurably decreased and validated by immunoblot analysis were two light harvesting complex binding proteins 1 and 3, Rubisco activase, and carbonic anhydrase. The observed decreased levels for these proteins were not the direct result of decreased transcription or protein instability. Chlorophyll fluorescence induction experiments indicated altered quinone reduction kinetics for the double and triple mutant plants with significant differences observed for absorbance, trapping, and electron transport. Transmission electron microscopy analysis of the chloroplasts in mutant plants showed impaired grana stacking and increased accumulation of starch granules consistent with some chloroplast proteins being decreased. Rescue of the i4g1 x i4g2 plant growth phenotype and increased expression of the validated proteins to wild-type levels was obtained by overexpression of eIFiso4G1. These data suggest a direct and specialized role for eIFiso4G in the synthesis of a subset of plant proteins.

The Arabidopsis thaliana-potyvirus system was developed to identify compatibility and incompatibility factors involved during infection and disease caused by positive-strand RNA viruses [1, 2]. Several Arabidopsis mutants with increased susceptibility to Tobacco etch potyvirus (TEV) were isolated previously [1], revealing a virus-specific resistance system in the phloem [3–5]. In this study, Arabidopsis mutants with decreased susceptibility to Turnip mosaic potyvirus (TuMV) were isolated. Three independent mutants that conferred immunity to TuMV were isolated and assigned to the same complementation group. These mutants were also immune or near-immune to TEV but were susceptible to an unrelated virus. The locus associated with decreased susceptibility was named loss-of-susceptibility to potyviruses 1 (lsp1). The LSP1 locus was isolated by map-based cloning and was identified as the gene encoding translation factor eIF(iso)4E, one of several known Arabidopsis isoforms that has cap binding activity. eIF4E and eIF(iso)4E from different plant species were shown previously to interact with the genome-linked protein (VPg) of TEV and TuMV, respectively. Models to explain the roles of eIF(iso)4E during virus infection are presented.

The V20 cultivar of Nicotiana tabacum was shown previously to exhibit a strain-specific restriction of long-distance movement of tobacco etch potyvirus (TEV). In V20, both TEV-HAT and TEV-Oxnard strains are capable of genome amplification and cell-to-cell movement, but only TEV-Oxnard is capable of systemic infection by vasculature-dependent long-distance movement. To investigate the basis for host-specific movement of TEV, chimeric virus genomes were assembled from TEV-HAT and TEV-Oxnard. Viruses containing the TEV-Oxnard coding regions for HC-Pro and/or capsid protein (CP), two proteins that are known to be essential for TEV long-distance movement, failed to infect V20 systemically. In contrast, chimeric viruses encoding the TEV-Oxnard VPg domain of NIa were able to infect V20 systemically. The critical region controlling the infection phenotype in V20 was mapped to a 67-nucleotide segment containing 10-nucleotide differences, but only five amino acid differences, between TEV-HAT and TEV-Oxnard. In V20 coinfection experiments, a restricted strain had no effect on systemic infection by a long-distance movement-competent chimeric strain, suggesting that the restricted strain was not inducing a generalized systemic resistance response. These data suggest that the VPg domain, which is covalently attached to the 5' end of genomic RNA, interacts either directly or indirectly with host components to facilitate long-distance movement.

Arabidopsis thaliana knockout lines for the plant-specific eukaryotic translation initiation factors eIFiso4G1 (i4g1) and eIFiso4G2 (i4g2) genes have been obtained. To address the potential for functional redundancy of these genes, homozygous double mutant lines were generated by crossing individual knockout lines. Both single and double mutant plants were analyzed for changes in gross morphology, development, and responses to selected environmental stressors. Single gene knockouts appear to have minimal effect on morphology, germination rate, growth rate, flowering time, or fertility. However, double mutant i4g1/i4g2 knockout plants show reduced germination rates, slow growth rates, moderate chlorosis, impaired fertility and reduced long term seed viability. Double mutant plants also exhibit altered responses to dehydration, salinity, and heat stress. The i4g2 and i4g1/i4g2 double mutant has reduced amounts of chlorophyll a and b suggesting a role in the expression of chloroplast proteins. General protein synthesis did not appear to be affected as the levels of gross protein expression did not appear to change in the mutants. The lack of a phenotype for either of the single mutants suggests there is considerable functional overlap. However, the strong phenotypes observed for the double mutant indicates that the individual gene products may have specialized roles in the expression of proteins involved in plant growth and development. Electronic supplementary material The online version of this article (doi:10.1007/s11103-010-9670-z) contains supplementary material, which is available to authorized users.