Frequently, crop plants do not take up adequate amounts of iron from the soil, leading to chlorosis, poor yield and decreased nutritional quality. Extremely limited soil bioavailability of iron has led plants to evolve two distinct uptake strategies: chelation, which is used by the world's principal grain crops; and reduction, which is used by other plant groups. The chelation strategy involves extrusion of low-molecular-mass secondary amino acids (mugineic acids) known as 'phytosiderophores' which chelate sparingly soluble iron. The Fe(III)-phytosiderophore complex is then taken up by an unknown transporter at the root surface. The maize yellow stripe1 (ys1) mutant is deficient in Fe(III)-phytosiderophore uptake, therefore YS1 has been suggested to be the Fe(III)-phytosiderophore transporter. Here we show that ys1 is a membrane protein that mediates iron uptake. Expression of YS1 in a yeast iron uptake mutant restores growth specifically on Fe(III)-phytosiderophore media. Under iron-deficient conditions, ys1 messenger RNA levels increase in both roots and shoots. Cloning of ys1 is an important step in understanding iron uptake in grasses, and has implications for mechanisms controlling iron homeostasis in all plants.

Purified recombinant viral replicases are useful for studying the mechanism of viral RNA replication in vitro. In this work, we obtained a highly active template-dependent replicase complex for Cucumber necrosis tombusvirus (CNV), which is a plus-stranded RNA virus, from Saccharomyces cerevisiae. The recombinant CNV replicase showed properties similar to those of the plant-derived CNV replicase (P. D. Nagy and J. Pogany, Virology 276:279-288, 2000), including the ability (i) to initiate cRNA synthesis de novo on both plus-and minus-stranded templates, (ii) to generate replicase products that are shorter than full length by internal initiation, and (iii) to perform primer extension from the 3 end of the template. We also found that isolation of functional replicase required the coexpression of the CNV p92 RNA-dependent RNA polymerase and the auxiliary p33 protein in yeast. Moreover, coexpression of a viral RNA template with the replicase proteins in yeast increased the activity of the purified CNV replicase by 40-fold, suggesting that the viral RNA might promote the assembly of the replicase complex and/or that the RNA increases the stability of the replicase. In summary, this paper reports the first purified recombinant tombusvirus replicase showing high activity and template dependence, a finding that will greatly facilitate future studies on RNA replication in vitro.Plus-stranded RNA viruses, which constitute the largest group among plant and animal viruses, replicate in infected cells by using the viral replicase complex. The replicase complex consists of virus-coded proteins, such as the RNA-dependent RNA polymerase (RdRp), auxiliary proteins, and possibly host-derived proteins, and the RNA template (1,4,5,20,27). To study the mechanism of viral RNA replication, functional replicases are purified from virus-infected hosts (3,10,12,16,23,26,38,41,42,53,55) or from heterologous systems, including Escherichia coli (17,19,21,24,44,45), yeast (40), insect (22,24,58), Xenopus (13), and mammalian cells (14,24). The advantage of the heterologous systems is that expression of the replicase proteins can be achieved without dependence on virus replication, thus facilitating mutational analysis of the replicase genes. These studies have established that the RdRp of several viruses, including Turnip crinkle virus, Tobacco etch virus, Bamboo mosaic virus, Hepatitis C virus, Bovine viral diarrhea virus (17,[19][20][21][22]44,45), etc., are active when expressed without other virus-coded auxiliary proteins. On the contrary, RdRps for several other viruses, such as Brome mosaic virus (BMV) and Alfalfa mosaic virus (AMV), required the presence of several factors, such as the RdRp, a viral auxiliary protein, and the viral RNA, in order to be functional in vitro (40, 54). In summary, viral replicase systems, which are very useful to dissect the protein (trans-acting) and RNA (cis-acting) factors that control virus replication, have been developed only for a limited number of plus-stranded RNA viruses.Tombusviruses are small pl...

Replication of plus-stranded RNA viruses is performed by the viral replicase complex, which, together with the viral RNA, must be targeted to intracellular membranes, where replication takes place in membraneous vesicles/spherules. Tombusviruses code for two overlapping replication proteins, the p33 auxiliary protein and the p92 polymerase. Using replication-competent fluorescent protein-tagged p33 of Cucumber necrosis virus (CNV), we determined that two domains affected p33 targeting to peroxisomal membranes in yeast: an N-proximal hydrophobic trans-membrane sequence and the C-proximal p33:p33/p92 interaction domain. On the contrary, only the deletion of the p33:p33/p92 interaction domain, but not the trans-membrane sequence, altered the intracellular targeting of p92 protein in the presence of wt p33 and DI-72(+) RNA. Moreover, unlike p33, p92 lacking the trans-membrane sequence was still functional in supporting the replication of a replicon RNA in yeast, whereas the p33:p33/p92 interaction domain in both p33 and p92 was essential for replication. In addition, p33 was also shown to facilitate the recruitment of the viral RNA to peroxisomal membranes and that p33 is colocalized with (+) and (-)-stranded viral RNAs. Also, FRET and pull-down analyses confirmed that p33 interacts with other p33 molecules in yeast cells. Based on these data, we propose that p33 facilitates the formation of multimolecular complexes, including p33, p92, viral RNA, and unidentified host factors, which are then targeted to the peroxisomal membranes, the sites of CNV replication.

Plus-strand RNA virus replication requires the assembly of the viral replicase complexes on intracellular membranes in the host cells. The replicase of Cucumber necrosis virus (CNV), a tombusvirus, contains the viral p33 and p92 replication proteins and possible host factors. In addition, the assembly of CNV replicase is stimulated in the presence of plus-stranded viral RNA (Z. Panaviene et al., J. Virol. 78:8254-8263, 2004). To define cis-acting viral RNA sequences that stimulate replicase assembly, we performed a systematic deletion approach with a model tombusvirus replicon RNA in Saccharomyces cerevisiae, which also coexpressed p33 and p92 replication proteins. In vitro replicase assays performed with purified CNV replicase preparations from yeast revealed critical roles for three RNA elements in CNV replicase assembly: the internal p33 recognition element (p33RE), the replication silencer element (RSE), and the 3 -terminal minus-strand initiation promoter (gPR). Deletion or mutagenesis of these elements reduced the activity of the CNV replicase to a minimal level. In addition to the primary sequences of gPR, RSE, and p33RE, formation of two alternative structures among these elements may also play a role in replicase assembly. Altogether, the role of multiple RNA elements in tombusvirus replicase assembly could be an important factor to ensure fidelity of template selection during replication.The genomes of plus-stranded RNA viruses are replicated by viral replicase complexes assembled on intracellular membranes (1, 4). The replicase complex consists of virus-encoded proteins, such as the RNA-dependent RNA polymerase (RdRp), auxiliary viral proteins, and possibly host-derived proteins and the viral RNA template (2, 10). The viral RNA serves a more complex role than just being used as a template for replication. For example, the assembly of viral replicases of Brome mosaic virus (BMV) and Alfalfa mosaic virus requires the plus-stranded viral RNA in order to be functional (28, 37). In the case of Cucumber necrosis virus (CNV), the viral (ϩ)RNA, but not the (Ϫ)RNA, stimulated replicase assembly by 40-fold in yeast, a model host (22). All these works led to the model that the viral (ϩ)RNA likely serves as a platform to bring the viral replication proteins and host protein factors together, leading to efficient assembly of the viral replicase.Tombusviruses are nonsegmented plus-stranded viruses that code for five proteins. These include the p33 and p92 replication proteins (Fig. 1), a cell-to-cell movement protein (p22), a coat protein (p41), and a suppressor of gene silencing (p19). The overlapping p33 and p92 replicase proteins are essential for replication of the genomic RNA (gRNA) in plant cells (14,20,33,40). The p92 replication protein has the RdRp signature motifs in its unique C terminus, whereas the auxiliary p33 plays a role in template selection and the recruitment of the viral RNA into replication (12,26). In addition, mutagenesis of p33 within its RNA-binding site (an arginine-proline-rich motif, te...

Graminaceous monocots, including most of the world's staple grains (i.e. rice, corn, and wheat) use a chelation strategy (Strategy II) for primary acquisition of iron from the soil. Strategy II plants secrete phytosiderophores (PS), compounds of the mugineic acid family that form stable Fe(III) chelates in soil. Uptake of iron-PS chelates, which occurs through specific transporters at the root surface, thus represents the primary route of iron entry into Strategy II plants. The gene Yellow stripe1 (Ys1) encodes the Fe(III)-PS transporter of maize (Zea mays). Here the physiological functions performed by maize YS1 were further defined by examining the pattern of Ys1 mRNA and protein accumulation and by defining YS1 transport specificity in detail. YS1 is able to translocate iron that is bound either by PS or by the related compound, nicotianamine; thus, the role of YS1 may be to transport either of these complexes. Ys1 expression at both the mRNA and protein levels responds rapidly to changes in iron availability but is not strongly affected by limitation of copper or zinc. Our data provide no support for the idea that YS1 is a transporter of zinc-PS, based on YS1 biochemical activity and Ys1 mRNA expression patterns in response to zinc deficiency. YS1 is capable of transporting copper-PS, but expression data suggest that the copper-PS uptake has limited significance in primary uptake of copper.