While metal nanoparticles are being increasingly used in many sectors of the economy, there is growing interest in the biological and environmental safety of their production. The main methods for nanoparticle production are chemical and physical approaches that are often costly and potentially harmful to the environment. The present review is devoted to the possibility of metal nanoparticle synthesis using plant extracts. This approach has been actively pursued in recent years as an alternative, efficient, inexpensive, and environmentally safe method for producing nanoparticles with specified properties. This review provides a detailed analysis of the various factors affecting the morphology, size, and yield of metal nanoparticles. The main focus is on the role of the natural plant biomolecules involved in the bioreduction of metal salts during the nanoparticle synthesis. Examples of effective use of exogenous biomatrices (peptides, proteins, and viral particles) to obtain nanoparticles in plant extracts are discussed.

The RNA silencing suppressor activity of the 2b protein of Cucumber mosaic virus (CMV) has been variously attributed to its nuclear targeting, its interaction with and inhibition of Argonaute 1 (AGO1), or its ability to bind small RNAs in vitro. In addition, the 2b ortholog of Tomato aspermy virus forms aggregates and binds RNAs in vitro. We have further studied the relationships between CMV 2b protein silencing suppressor activity and its subcellular distribution, protein-protein interactions in vivo, and interactions with small interfering RNAs in vitro. To do this, we tagged the protein with fluorescent markers and showed that it retained suppressor activity. We showed that the 2b protein is present in the nucleolus and that it self-interacts and interacts with AGO1 and AGO4 in vivo. Using a battery of mutants, we showed that the putative nuclear localization signals and phosphorylation motif of the 2b protein are not required for self-interaction or for interaction with AGO proteins. The occurrence of neither of these interactions or of nucleolar targeting was sufficient to provide local silencing-suppression activity. In contrast, the ability of the 2b protein to bind small RNAs appears to be indispensable for silencing suppressor function.

The nucleolus and specific nucleolar proteins are involved in the life cycles of some plant and animal viruses, but the functions of these proteins and of nucleolar trafficking in virus infections are largely unknown. The ORF3 protein of the plant virus, groundnut rosette virus (an umbravirus), has been shown to cycle through the nucleus, passing through Cajal bodies to the nucleolus and then exiting back into the cytoplasm. This journey is absolutely required for the formation of viral ribonucleoprotein particles (RNPs) that, themselves, are essential for the spread of the virus to noninoculated leaves of the shoot tip. Here, we show that these processes rely on the interaction of the ORF3 protein with fibrillarin, a major nucleolar protein. Silencing of the fibrillarin gene prevents longdistance movement of groundnut rosette virus but does not affect viral replication or cell-to-cell movement. Repressing fibrillarin production also localizes the ORF3 protein to multiple Cajal bodylike aggregates that fail to fuse with the nucleolus. Umbraviral ORF3 protein and fibrillarin interact in vitro and, when mixed with umbravirus RNA, form an RNP complex. This complex has a filamentous structure with some regular helical features, resembling the RNP complex formed in vivo during umbravirus infection. The filaments formed in vitro are infectious when inoculated to plants, and their infectivity is resistant to RNase. These results demonstrate previously undescribed functions for fibrillarin as an essential component of translocatable viral RNPs and may have implications for other plant and animal viruses that interact with the nucleolus.Cajal bodies ͉ plant virus movement ͉ ribonucleoprotein particles T he nucleolus is a subnuclear domain and is the site of transcription and processing of rRNA and of ribosome biogenesis. In addition, the nucleolus also participates in other aspects of RNA metabolism and cell function (1, 2). The nucleolus is structurally and functionally associated with Cajal bodies (CBs), which are structures found in both animals and plants (3, 4). CBs contain different proteins including coilin, a protein essential for CB formation, and fibrillarin, a major nucleolar protein that is a core component of small nucleolar ribonucleoprotein particles (snoRNPs) and is required for rRNA processing (4-7). CBs are involved in the maturation of small nuclear RNPs (snRNPs) and snoRNPs, which traffic through CBs before accumulating in splicing speckles and the nucleolus, respectively (8, 9). Both the nucleolus and CBs have a role in RNA silencing in plants (10,11). Finally, a number of animal and plant viruses including the RNAcontaining tobacco etch virus and the DNA-containing tomato yellow leaf curl virus have a nucleolar phase in their life cycle (12, 13). Recently, we have shown that the ability of the umbravirus, groundnut rosette virus (GRV), to move long distances through the phloem, the specialized vascular system used by plants for the transport of assimilates and macromolecules, depends strictly on the...

The nucleolus and Cajal bodies (CBs) are prominent interacting subnuclear domains involved in a number of crucial aspects of cell function. Certain viruses interact with these compartments but the functions of such interactions are largely uncharacterized. Here, we show that the ability of the groundnut rosette virus open reading frame (ORF) 3 protein to move viral RNA long distances through the phloem strictly depends on its interaction with CBs and the nucleolus. The ORF3 protein targets and reorganizes CBs into multiple CB-like structures and then enters the nucleolus by causing fusion of these structures with the nucleolus. The nucleolar localization of the ORF3 protein is essential for subsequent formation of viral ribonucleoprotein (RNP) particles capable of virus long-distance movement and systemic infection. We provide a model whereby the ORF3 protein utilizes trafficking pathways involving CBs to enter the nucleolus and, along with fibrillarin, exit the nucleus to form viral 'transportcompetent' RNP particles in the cytoplasm.

To test the hypothesis that caspase-like proteases exist and are critically involved in the implementation of programmed cell death (PCD) in plants, a search was undertaken for plant caspases activated during the N gene-mediated hypersensitive response (HR; a form of pathogen-induced PCD in plants) in tobacco plants infected with Tobacco mosaic virus (TMV). For detection, characterization, and partial purification of a tobacco caspase, the Agrobacterium tumefaciens VirD2 protein, shown here to be cleaved specifically at two sites (TATD and GEQD) by human caspase-3, was used as a target. In tobacco leaves, specific proteolytic processing of the ectopically produced VirD2 derivatives at these sites was found to occur early in the course of the HR triggered by TMV. A proteolytic activity capable of specifically cleaving the model substrate at TATD was partially purified from these leaves. A tetrapeptide aldehyde designed and synthesized on the basis of the elucidated plant caspase cleavage site prevented fragmentation of the substrate protein by plant and human caspases in vitro and counteracted TMV-triggered HR in vivo. Therefore, our data provide a characterization of caspase-specific protein fragmentation in apoptotic plant cells, with implications for the importance of such activity in the implementation of plant PCD.

Previously, we found that silencing suppression by the 2b protein and six mutants correlated both with their ability to bind to double-stranded (ds) small RNAs (sRNAs) in vitro and with their nuclear/nucleolar localization. To further discern the contribution to suppression activity of sRNA binding and of nuclear localization, we have characterized the kinetics of in vitro binding to a ds sRNA, a single-stranded (ss) sRNA, and a micro RNA (miRNA) of the native 2b protein and eight mutant variants. We have also added a nuclear export signal (NES) to the 2b protein and assessed how it affected subcellular distribution and suppressor activity. We found that in solution native protein bound ds siRNA, miRNA, and ss sRNA with high affinity, at protein:RNA molar ratios~2:1. Of the four mutants that retained suppressor activity, three showed sRNA binding profiles similar to those of the native protein, whereas the remaining one bound ss sRNA at a 2:1 molar ratio, but both ds sRNAs with 1.5-2 times slightly lower affinity. Three of the four mutants lacking suppressor activity failed to bind to any sRNA, whereas the remaining one bound them at far higher ratios. NES-tagged 2b protein became cytoplasmic, but suppression activity in patch assays remained unaffected. These results support binding to sRNAs at molar ratios at or near 2:1 as critical to the suppressor activity of the 2b protein. They also show that cytoplasmically localized 2b protein retained suppressor activity, and that a sustained nuclear localization was not required for this function.

We report the synthesis and characterization of amorphous iron oxide nanoparticles from iron salts in aqueous extracts of monocotyledonous (Hordeum vulgare) and dicotyledonous (Rumex acetosa) plants. The nanoparticles were characterized by TEM, absorbance spectroscopy, SAED, EELS, XPS, and DLS methods and were shown to contain mainly iron oxide and iron oxohydroxide. H. vulgare extracts produced amorphous iron oxide nanoparticles with diameters of up to 30 nm. These iron nanoparticles are intrinsically unstable and prone to aggregation; however, we rendered them stable in the long term by addition of 40 mM citrate buffer pH 3.0. In contrast, amorphous iron oxide nanoparticles (diameters of 10-40 nm) produced using R. acetosa extracts are highly stable. The total protein content and antioxidant capacity are similar for both extracts, but pH values differ (H. vulgare pH 5.8 vs R. acetosa pH 3.7). We suggest that the presence of organic acids (such oxalic or citric acids) plays an important role in the stabilization of iron nanoparticles, and that plants containing such constituents may be more efficacious for the green synthesis of iron nanoparticles.

Cajal bodies (CBs) are distinct nuclear bodies physically and functionally associated with the nucleolus. In addition to their traditional function in coordinating maturation of certain nuclear RNAs, CBs participate in cell cycle regulation, development, and regulation of stress responses. A key "signature" component of CBs is coilin, the scaffolding protein essential for CB formation and function. Using an RNA silencing (loss-of-function) approach, we describe here new phenomena whereby coilin also affects, directly or indirectly, a variety of interactions between host plants and viruses that have RNA or DNA genomes. Moreover, the effects of coilin on these interactions are manifested differently: coilin contributes to plant defense against tobacco rattle virus (tobravirus), tomato black ring virus (nepovirus), barley stripe mosaic virus (hordeivirus), and tomato golden mosaic virus (begomovirus). In contrast, with potato virus Y (potyvirus) and turnip vein clearing virus (tobamovirus), coilin serves to increase virus pathogenicity. These findings show that interactions with coilin (or CBs) may involve diverse mechanisms with different viruses and that these mechanisms act at different phases of virus infection. Thus, coilin (CBs) has novel, unexpected natural functions that may be recruited or subverted by plant viruses for their own needs or, in contrast, are involved in plant defense mechanisms that suppress host susceptibility to the viruses.