Tandem affinity purification was used in Arabidopsis thaliana to identify cellular interactors of Turnip mosaic virus (TuMV) RNA-dependent RNA polymerase (RdRp). The heat shock cognate 70-3 (Hsc70-3) and poly(A)-binding (PABP) host proteins were recovered and shown to interact with the RdRp in vitro. As previously shown for PABP, Hsc70-3 was redistributed to nuclear and membranous fractions in infected plants and both RdRp interactors were co-immunoprecipitated from a membrane-enriched extract using RdRp-specific antibodies. Fluorescently tagged RdRp and Hsc70-3 localized to the cytoplasm and the nucleus when expressed alone or in combination in Nicotiana benthamiana. However, they were redistributed to large perinuclear ER-derived vesicles when co-expressed with the membrane binding 6K-VPg-Pro protein of TuMV. The association of Hsc70-3 with the RdRp could possibly take place in membrane-derived replication complexes. Thus, Hsc70-3 and PABP2 are potentially integral components of the replicase complex and could have important roles to play in the regulation of potyviral RdRp functions.

The poly(A)-binding protein (PABP) is an important translation initiation factor that binds to the polyadenylated 39 end of mRNA. We have previously shown that PABP2 interacts with the RNAdependent RNA polymerase (RdRp) and VPg-Pro of turnip mosaic virus (TuMV) within virusinduced vesicles. At least eight PABP isoforms are produced in Arabidopsis thaliana, three of which (PABP2, PABP4 and PABP8) are highly and broadly expressed and probably constitute the bulk of PABP required for cellular functions. Upon TuMV infection, an increase in protein and mRNA expression from PAB2, PAB4 and PAB8 genes was recorded. In vitro binding assays revealed that RdRp and the viral genome-linked protein (VPg-Pro) interact preferentially with PABP2 but are also capable of interaction with one or both of the other class II PABPs (i.e. PABP4 and PABP8). To assess whether PABP is required for potyvirus replication, A. thaliana single and double pab knockouts were isolated and inoculated with TuMV. All lines showed susceptibility to TuMV. However, when precise monitoring of viral RNA accumulation was performed, it was found to be reduced by 2.2-and 3.5-fold in pab2 pab4 and pab2 pab8 mutants, respectively, when compared with wild-type plants. PABP levels were most significantly reduced in the membrane-associated fraction in both of these mutants. TuMV mRNA levels thus correlated with cellular PABP concentrations in these A. thaliana knockout lines. These data provide further support for a role of PABP in potyvirus replication. INTRODUCTIONThe poly(A)-binding protein (PABP) is an abundant translation initiation factor in the cell that binds to the polyadenylated 39 end of mRNA. Its interaction with the eukaryotic translation initiation factor 4F complex (composed of the eIF4E, eIF4A and eIF4G proteins), which is bound to the 59 cap structure of the mRNA, results in the formation of a protein bridge that brings the 59 and 39 termini of the mRNA into proximity. This leads to a synergistic enhancement of translation (Sachs, 2000;Wells et al., 1998). PABP is also important for stability (BehmAnsmant et al., 2007;Parker & Song, 2004), biogenesis (Amrani et al., 1997;Brown & Sachs, 1998) and nuclear export of cellular mRNAs (Brune et al., 2005).Multiple PABP isoforms are normally found in animals and plants (Mangus et al., 2003). Eight PABP genes have been identified in Arabidopsis thaliana (Belostotsky, 2003).The corresponding proteins are divided into four classes based on gene expression and similarity. Class II genes (PAB2, PAB4 and PAB8) are highly and broadly expressed and probably encode the bulk of PABP required for cellular functions (Belostotsky, 2003).In animal cells, PABP is cleaved by proteinases of picornaviruses (Joachims et al., 1999;Kerekatte et al., 1999;Kuyumcu-Martinez et al., 2002, 2004bRodriguez Pulido et al., 2007;Zhang et al., 2007), caliciviruses (Kuyumcu-Martinez et al., 2004a) and retroviruses (Alvarez et al., 2006). The resultant cleavage leads to host translational shutdown (Joachims et al., 1999; KuyumcuMartinez ...

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Mountain ash (Sorbus decora and S. americana) is used by the Cree Nation of the James Bay region of Quebec (Eeyou Istchee) as traditional medicine. Its potential as an antidiabetic medicine is thought to vary across its geographical range, yet little is known about the factors that affect its antioxidant capacity. Here, we examined metabolite gene expression in relation to antioxidant activity, linking phytochemistry and medicinal potential. Samples of leaf and bark from S. decora and S. americana were collected from 20 populations at four different latitudes. Two genes known to produce antidiabetic substances, flavonol synthase and squalene synthase, were analyzed using quantitative real time PCR. Gene expression was significantly higher for flavonol synthase compared to squalene synthase and increased in the most Northern latitude. Corresponding differences observed in the antioxidant capacity of ethanolic extracts from the bark of Sorbus spp. confirm that plants at higher latitudes increase production of stress-induced secondary metabolites and support Aboriginal perceptions of their higher medicinal potential. Modern genetic techniques such as quantitative real time PCR offer unprecedented resolution to substantiate and scrutinise Aboriginal medicinal plant perception. Furthermore, it offers valuable insights into how environmental stress can trigger an adaptive response resulting in the accumulation of secondary metabolites with human medicinal properties.

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