Biological activities of human interferon and 2′–5′ oligoadenylates in plants

Exogenous human interferon 2 (IFN) and 2–5 oligoadenylates (2–5A) have been shown to cause at least a dual physiological effect in tobacco and wheat: (i) increased cytokinin activity and (ii) induced synthesis of numerous proteins, among which members of two groups of stress proteins have been identified, namely pathogenesis-related (PR) and heat shock (HS) proteins. These effects were observed only by low concentrations of these substances: IFN at 0.1–1 u/ml and 2–5A at 1–10 nM.     

An umbraviral protein,involved in long-distance RNA movement,binds viral RNA and forms unique,protective ribonucleoprotein complexes

Umbraviruses are different from most other viruses in that they do not encode a conventional capsid protein (CP); therefore, no recognizable virus particles are formed in infected plants. Their lack of a CP is compensated for by the ORF3 protein, which fulfils functions that are provided by the CPs of other viruses, such as protection and long-distance movement of viral RNA. When the Groundnut rosette virus (GRV) ORF3 protein was expressed from Tobacco mosaic virus (TMV) in place of the TMV CP [TMV(ORF3)], in infected cells it interacted with the TMV RNA to form filamentous ribonucleoprotein (RNP) particles that had elements of helical structure but were not as uniform as classical virions. These RNP particles were observed in amorphous inclusions in the cytoplasm, where they were embedded within an electron-dense matrix material. The inclusions were detected in all types of cells and were abundant in phloem-associated cells, in particular companion cells and immature sieve elements. RNP-containing complexes similar in appearance to the inclusions were isolated from plants infected with TMV(ORF3) or with GRV itself. In vitro, the ORF3 protein formed oligomers and bound RNA in a manner consistent with its role in the formation of RNP complexes. It is suggested that the cytoplasmic RNP complexes formed by the ORF3 protein serve to protect viral RNA and may be the form in which it moves through the phloem. Thus, the RNP particles detected here represent a novel structure which may be used by umbraviruses as an alternative to classical virions.     

Phytaspase,a relocalisable cell death promoting plant protease with caspase specificity

Caspases are cysteine‐dependent proteases and are important components of animal apoptosis. They introduce specific breaks after aspartate residues in a number of cellular proteins mediating programmed cell death (PCD). Plants encode only distant homologues of caspases, the metacaspases that are involved in PCD, but do not possess caspase‐specific proteolytic activity. Nevertheless, plants do display caspase‐like activities indicating that enzymes structurally distinct from classical caspases may operate as caspase‐like proteases. Here, we report the identification and characterisation of a novel PCD‐related subtilisin‐like protease from tobacco and rice named phytaspase (plant aspartate‐specific protease) that possesses caspase specificity distinct from that of other known caspase‐like proteases. We provide evidence that phytaspase is synthesised as a proenzyme, which is autocatalytically processed to generate the mature enzyme. Overexpression and silencing of the phytaspase gene showed that phytaspase is essential for PCD‐related responses to tobacco mosaic virus and abiotic stresses. Phytaspase is constitutively secreted into the apoplast before PCD, but unexpectedly is re‐imported into the cell during PCD providing insights into how phytaspase operates.     

Cajal bodies and the nucleolus are required for a plant virus systemic infection

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 'transport-competent' RNP particles in the cytoplasm.     

The extreme N-terminal domain of a hordeivirus TGB1 movement protein mediates its localization to the nucleolus and interaction with fibrillarin

The hordeiviral movement protein encoded by the first gene of the triple gene block (TGBp1) of Poa semilatent virus (PSLV), interacts with viral genomic RNAs to form RNP particles which are considered to be a form of viral genome capable of cell-to-cell and long-distance transport in infected plants. The PSLV TGBp1 contains a C-terminal NTPase/helicase domain (HELD) and an N-terminal extension region consisting of two structurally and functionally distinct domains: an extreme N-terminal domain (NTD) and an internal domain (ID). This study demonstrates that transient expression of TGBp1 fused to GFP in Nicotiana benthamiana leaves results in faint but obvious fluorescence in the nucleolus in addition to cytosolic distribution. Mutagenesis of the basic amino acids inside the NTD clusters A ¹¹⁶KSKRKKKNKK¹²⁵ and B ¹⁷⁵KKATKKESKKQTK¹⁸⁷ reveals that these clusters are indispensable for nuclear and nucleolar targeting of PSLV TGBp1 and may contain nuclear and nucleolar localization signals or their elements. The PSLV TGBp1 is able to bind to fibrillarin, the major nucleolar protein (AtFib2 from Arabidopsis thaliana) in vitro. This protein–protein interaction occurs between the glycine-arginine-rich (GAR) domain of fibrillarin and the first 82 amino acid residues of TGBp1. The interaction of TGBp1 with fibrillarin is also visualized in vivo by bimolecular fluorescence complementation (BiFC) during co-expression of TGBp1 or its deletion mutants, and fibrillarin as fusions to different halves of YFP in N. benthamiana plants. The sites responsible for nuclear/nucleolar localization and fibrillarin binding, have been located within the intrinsically disordered TGBp1 NTD. These data could suggest that specific functions of hordeivirus TGBp1 may depend on its interaction with nucleolar components.     

Plant phytaspases and animal caspases: structurally unrelated death proteases with a common role and specificity

Proteases with an aspartate cleavage specificity are known to contribute to programmed cell death (PCD) in animals and plants. In animal cells this proteolytic activity belongs to caspases, a well-characterized family of cysteine-dependent death proteases. Plants, however, lack caspase homologs and thus the origin of this type of proteolytic activity in planta was poorly understood. Here, we review recent data demonstrating that a plant serine-dependent protease, phytaspase, shares cleavage specificity and a role in PCD analogous to that of caspases. However, unlike caspases, regulation of phytaspase-mediated cleavage of intracellular target proteins appears to be attained not at the level of proenzyme processing/activation, which occurs, in the case of phytaspase, autocatalytically and constitutively. Rather, the mature phytaspase is excluded from healthy cells into the apoplast and is allowed to re-enter cells upon the induction of PCD. Thus, PCD-related proteases in animals and plants display both common features and important distinctions.     

RNA binding is more critical to the suppression of silencing function of Cucumber mosaic virus 2b protein than nuclear localization

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.     

In planta production of a candidate vaccine against bovine papillomavirus type 1

Bovine papillomavirus type 1 (BPV-1) is an economically important virus that induces tumourigenic pathologies in horses and cows. Given that the BPV-1 L1 major coat protein can self-assemble into highly immunogenic higher-order structures, we transiently expressed it in Nicotiana benthamiana as a prelude to producing a candidate vaccine. It was found that plant codon optimization of L1 gave higher levels of expression than its non-optimized counterpart. Following protein extraction, we obtained high yields (183 mg/kg fresh weight leaf tissue) of relatively pure L1, which had self-assembled into virus-like particles (VLPs). We found that these VLPs elicited a highly specific and strong immune response, and therefore they may have utility as a potential vaccine. This is the first report demonstrating the viable production of a candidate BPV vaccine protein in plants.