eIFiso4G Augments the Synthesis of Specific Plant Proteins Involved in Normal Chloroplast Function.

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.

Deletion of the eIFiso4G subunit of the Arabidopsis eIFiso4F translation initiation complex impairs health and viability.

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.

Genetic and functional diversification of small RNA pathways in plants.

Multicellular eukaryotes produce small RNA molecules (approximately 21-24 nucleotides) of two general types, microRNA (miRNA) and short interfering RNA (siRNA). They collectively function as sequence-specific guides to silence or regulate genes, transposons, and viruses and to modify chromatin and genome structure. Formation or activity of small RNAs requires factors belonging to gene families that encode DICER (or DICER-LIKE [DCL]) and ARGONAUTE proteins and, in the case of some siRNAs, RNA-dependent RNA polymerase (RDR) proteins. Unlike many animals, plants encode multiple DCL and RDR proteins. Using a series of insertion mutants of Arabidopsis thaliana, unique functions for three DCL proteins in miRNA (DCL1), endogenous siRNA (DCL3), and viral siRNA (DCL2) biogenesis were identified. One RDR protein (RDR2) was required for all endogenous siRNAs analyzed. The loss of endogenous siRNA in dcl3 and rdr2 mutants was associated with loss of heterochromatic marks and increased transcript accumulation at some loci. Defects in siRNA-generation activity in response to turnip crinkle virus in dcl2 mutant plants correlated with increased virus susceptibility. We conclude that proliferation and diversification of DCL and RDR genes during evolution of plants contributed to specialization of small RNA-directed pathways for development, chromatin structure, and defense.

Loss-of-susceptibility mutants of Arabidopsis thaliana reveal an essential role for eIF(iso)4E during potyvirus infection.

The Arabidopsis thaliana-potyvirus system was developed to identify compatibility and incompatibility factors involved during infection and disease caused by positive-strand RNA viruses. Several Arabidopsis mutants with increased susceptibility to Tobacco etch potyvirus (TEV) were isolated previously, revealing a virus-specific resistance system in the phloem. 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.