Production of Functionally Active and Immunogenic Non-Glycosylated Protective Antigen from Bacillus anthracis in Nicotiana benthamiana by Co-Expression with Peptide-N-Glycosidase F (PNGase F) of Flavobacterium meningosepticum.

Bacillus anthracis has long been considered a potential biological warfare agent, and therefore, there is a need for a safe, low-cost and highly efficient anthrax vaccine with demonstrated long-term stability for mass vaccination in case of an emergency. Many efforts have been made towards developing an anthrax vaccine based on recombinant protective antigen (rPA) of B. anthracis, a key component of the anthrax toxin, produced using different expression systems. Plants represent a promising recombinant protein production platform due to their relatively low cost, rapid scalability and favorable safety profile. Previous studies have shown that full-length rPA produced in Nicotiana benthamiana (pp-PA83) is immunogenic and can provide full protection against lethal spore challenge; however, further improvement in the potency and stability of the vaccine candidate is necessary. PA of B. anthracis is not a glycoprotein in its native host; however, this protein contains potential N-linked glycosylation sites, which can be aberrantly glycosylated during expression in eukaryotic systems including plants. This glycosylation could affect the availability of certain key epitopes either due to masking or misfolding of the protein. Therefore, a non-glycosylated form of pp-PA83 was engineered and produced in N. benthamiana using an in vivo deglycosylation approach based on co-expression of peptide-N-glycosidase F (PNGase F) from Flavobacterium meningosepticum. For comparison, versions of pp-PA83 containing point mutations in six potential N-glycosylation sites were also engineered and expressed in N. benthamiana. The in vivo deglycosylated pp-PA83 (pp-dPA83) was shown to have in vitro activity, in contrast to glycosylated pp-PA83, and to induce significantly higher levels of toxin-neutralizing antibody responses in mice compared with glycosylated pp-PA83, in vitro deglycosylated pp-PA83 or the mutated versions of pp-PA83. These results suggest that pp-dPA83 may offer advantages in terms of dose sparing and enhanced immunogenicity as a promising candidate for a safe, effective and low-cost subunit vaccine against anthrax.

Virus-derived gene expression and RNA interference vector for grapevine.

The improvement of the agricultural and wine-making qualities of the grapevine (Vitis vinifera) is hampered by adherence to traditional varieties, the recalcitrance of this plant to genetic modifications, and public resistance to genetically modified organism (GMO) technologies. To address these challenges, we developed an RNA virus-based vector for the introduction of desired traits into grapevine without heritable modifications to the genome. This vector expresses recombinant proteins in the phloem tissue that is involved in sugar transport throughout the plant, from leaves to roots to berries. Furthermore, the vector provides a powerful RNA interference (RNAi) capability of regulating the expression of endogenous genes via virus-induced gene-silencing (VIGS) technology. Additional advantages of this vector include superb genetic capacity and stability, as well as the swiftness of technology implementation. The most significant applications of the viral vector include functional genomics of the grapevine and disease control via RNAi-enabled vaccination against pathogens or invertebrate pests.

Class XI myosins are required for development, cell expansion, and F-Actin organization in Arabidopsis.

The actomyosin system is conserved throughout eukaryotes. Although F-actin is essential for cell growth and plant development, roles of the associated myosins are poorly understood. Using multiple gene knockouts in Arabidopsis thaliana, we investigated functional profiles of five class XI myosins, XI-K, XI-1, XI-2, XI-B, and XI-I. Plants lacking three myosins XI showed stunted growth and delayed flowering, whereas elimination of four myosins further exacerbated these defects. Loss of myosins led to decreased leaf cell expansion, with the most severe defects observed in the larger leaf cells. Root hair length in myosin-deficient plants was reduced approximately 10-fold, with quadruple knockouts showing morphological abnormalities. It was also found that trafficking of Golgi and peroxisomes was entirely myosin dependent. Surprisingly, myosins were required for proper organization of F-actin and the associated endoplasmic reticulum networks, revealing a novel, architectural function of the class XI myosins. These results establish critical roles of myosin-driven transport and F-actin organization during polarized and diffuse cell growth and indicate that myosins are key factors in plant growth and development.

Overlapping functions of the four class XI myosins in Arabidopsis growth, root hair elongation, and organelle motility.

Flowering plants have evolved multigene families of the class XI myosin motors, the functions of which remain poorly understood. Here, we investigated functional profiles of the Arabidopsis myosins that belong to two paralogous pairs, XI-K/XI-1 and XI-2/XI-B, using single and double gene-knockout mutants. It was found that the myosins XI-K, XI-2, and XI-B, but not XI-1 have overlapping and additive roles in the root hair elongation. A nonidentical set of the three myosins, XI-K, XI-1, and XI-2, exhibited partially redundant and additive roles in the transport of Golgi stacks, peroxisomes, and mitochondria. Conspicuously, the double xi-k/1 knockout plants that showed the largest cumulative reduction of the organelle velocities also exhibited a stunted plant growth and reduced fecundity phenotype. Collectively, these results suggest that the rapid, myosin-powered organelle trafficking is required for the optimal plant growth, whereas a distinct myosin function, presumably the vesicular transport, is involved in elongation of the root hairs. In addition, our data imply that the myosin gene duplication in plants has been followed by a gradual functional specialization of the resulting pairs of myosin paralogs.

Class VIII myosins are required for plasmodesmatal localization of a closterovirus Hsp70 homolog.

The Hsp70 homolog (Hsp70h) of Beet yellows virus (BYV) functions in virion assembly and cell-to-cell movement and is autonomously targeted to plasmodesmata in association with the actomyosin motility system (A. I. Prokhnevsky, V. V. Peremyslov, and V. V. Dolja, J. Virol. 79:14421-14428, 2005). Myosins are a diverse category of molecular motors that possess a motor domain and a tail domain involved in cargo binding. Plants have two classes of myosins, VIII and XI, whose specific functions are poorly understood. We used dominant negative inhibition to identify myosins required for Hsp70h localization to plasmodesmata. Six full-length myosin cDNAs from the BYV host plant Nicotiana benthamiana were sequenced and shown to encode apparent orthologs of the Arabidopsis thaliana myosins VIII-1, VIII-2, VIII-B, XI-2, XI-F, and XI-K. We found that the ectopic expression of the tail domains of each of the class VIII, but not the class XI, myosins inhibited the plasmodesmatal localization of Hsp70h. In contrast, the overexpression of the motor domains or the entire molecules of the class VIII myosins did not affect Hsp70h targeting. Further mapping revealed that the minimal cargo-binding part of the myosin VIII tails was both essential and sufficient for the inhibition of the proper Hsp70h localization. Interestingly, plasmodesmatal localization of the Tobacco mosaic virus movement protein and Arabidopsis protein RGP2 was not affected by myosin VIII tail overexpression. Collectively, our data implicate class VIII myosins in protein delivery to plasmodesmata and suggest that more than one mechanism of such delivery exist in plants.

Two class XI myosins function in organelle trafficking and root hair development in Arabidopsis.

Multigene families encoding class XI myosins are conserved in higher plants, however, little information is available on specific functions of these ubiquitous molecular motors. We isolated gene knockout mutants for all 13 class XI myosins present in Arabidopsis (Arabidopsis thaliana) genome. Inactivation of 11 myosin genes resulted in no discernible phenotypes under the normal growth conditions. In contrast, the knockouts of the remaining two myosin genes, XI-2 (formerly MYA2) and XI-K, exhibited similar defects in root hair elongation suggesting that the myosin-driven motility plays a significant role in a polar tip growth. Strikingly, inactivation of each of these myosins also reduced trafficking of Golgi stacks, peroxisomes, and mitochondria in root hairs and in leaf epidermal cells. These results indicate that myosins XI-K and XI-2 play major and overlapping roles in the cell dynamics in Arabidopsis and highlight the redundant nature of myosin function in plants.

Myosin XI-K Is required for rapid trafficking of Golgi stacks, peroxisomes, and mitochondria in leaf cells of Nicotiana benthamiana.

A prominent feature of plant cells is the rapid, incessant movement of the organelles traditionally defined as cytoplasmic streaming and attributed to actomyosin motility. We sequenced six complete Nicotiana benthamiana cDNAs that encode class XI and class VIII myosins. Phylogenetic analysis indicates that these two classes of myosins diverged prior to the radiation of green algae and land plants from a common ancestor and that the common ancestor of land plants likely possessed at least seven myosins. We further report here that movement of Golgi stacks, mitochondria, and peroxisomes in the leaf cells of N. benthamiana is mediated mainly by myosin XI-K. Suppression of myosin XI-K function using dominant negative inhibition or RNA interference dramatically reduced movement of each of these organelles. When similar approaches were used to inhibit functions of myosin XI-2 or XI-F, only moderate to marginal effects were observed. Organelle trafficking was virtually unaffected in response to inhibition of each of the three class VIII myosins. Interestingly, none of the tested six myosins appears to be involved in light-induced movements of chloroplasts. Taken together, these data strongly suggest that myosin XI-K has a major role in trafficking of Golgi stacks, mitochondria, and peroxisomes, whereas myosins XI-2 and XI-F might perform accessory functions in this process. In addition, our analysis of thousands of individual organelles revealed independent movement patterns for Golgi stacks, mitochondria, and peroxisomes, indicating that the notion of coordinated cytoplasmic streaming is not generally applicable to higher plants.

Intracellular targeting of a hordeiviral membrane-spanning movement protein: sequence requirements and involvement of an unconventional mechanism.

The membrane-spanning protein TGBp3 is one of the three movement proteins (MPs) of Poa semilatent virus. TGBp3 is thought to direct other viral MPs and genomic RNA to peripheral bodies located in close proximity to plasmodesmata. We used the ectopic expression of green fluorescent protein-fused TGBp3 in epidermal cells of Nicotiana benthamiana leaves to study the TGBp3 intracellular trafficking pathway. Treatment with inhibitors was used to reveal that the targeting of TGBp3 to plasmodesmata does not require a functional cytoskeleton or secretory system. In addition, the suppression of endoplasmic reticulum-derived vesicle formation by a dominant negative mutant of small GTPase Sar1 had no detectable effect on TGBp3 trafficking to peripheral bodies. Collectively, these results suggested the involvement of an unconventional pathway in the intracellular transport of TGBp3. The determinants of targeting to plasmodesmata were localized to the C-terminal region of TGBp3, including the conserved hydrophilic and terminal membrane-spanning domains.

Virion tails of Beet yellows virus: Coordinated assembly by three structural proteins.

Filamentous virions of Beet yellows virus contain a long body formed by a major capsid protein and a short tail that is assembled by a minor capsid protein (CPm), an Hsp70-homolog (Hsp70h), a 64-kDa protein (p64), and a 20-kDa protein (p20). Using mutation analysis and newly developed in planta assays, here we investigate the genetic requirements for the tail assembly. We show that the inactivation of CPm dramatically reduces incorporation of both Hsp70h and p64. Furthermore, inactivation of Hsp70h prevents incorporation of p64 into virions and vice versa. Hsp70h and p64 are each required for efficient incorporation of CPm. We also show that the tails possessing normal relative amounts of CPm, Hsp70h, and p64 can be formed in the absence of the major capsid protein and p20. Similar to the tails isolated from the wild-type virions, these mutant tails encapsidate the approximately 700 nt-long, 5'-terminal segments of the viral RNA. Taken together, our results imply that CPm, Hsp70h and p64 act cooperatively to encapsidate a defined region of the closterovirus genome.

Diverse suppressors of RNA silencing enhance agroinfection by a viral replicon.

Launching the Beet yellows virus (BYV) minireplicon by agrobacterial delivery resulted in an unexpectedly low number of infected cells per inoculated leaf. This effect was due to a strong RNA silencing response in the agroinfiltrated leaves. Strikingly, ectopic co-expression of p21, a BYV RNA silencing suppressor, increased minireplicon infectivity by three orders of magnitude. Mutational analysis demonstrated that this effect correlates with suppressor activity of p21. Five diverse, heterologous viral suppressors were also active in this system, providing a useful approach for a dramatic, up to 10,000-fold, increase of the efficiency of agroinfection. The minireplicon agroinfection assay was also used to identify a new suppressor, a homolog of BYV p21, derived from Grapevine leafroll-associated virus-2. In addition, we report preliminary data on the suppressor activity of the p10 protein of Grapevine virus A and show that this protein belongs to a family of Zn-ribbon-containing proteins encoded by filamentous plant RNA viruses from three genera. The members of this family are predicted to have RNA silencing suppressor activity.

Actin cytoskeleton is involved in targeting of a viral Hsp70 homolog to the cell periphery.

The cell-to-cell movement of plant viruses involves translocation of virus particles or nucleoproteins to and through the plasmodesmata (PDs). As we have shown previously, the movement of the Beet yellows virus requires the concerted action of five viral proteins including a homolog of cellular approximately 70-kDa heat shock proteins (Hsp70h). Hsp70h is an integral component of the virus particles and is also found in PDs of the infected cells. Here we investigate subcellular distribution of Hsp70h using transient expression of Hsp70h fused to three spectrally distinct fluorescent proteins. We found that fluorophore-tagged Hsp70h forms motile granules that are associated with actin microfilaments, but not with microtubules. In addition, immobile granules were observed at the cell periphery. A pairwise appearance of these granules at the opposite sides of cell walls and their colocalization with the movement protein of Tobacco mosaic virus indicated an association of Hsp70h with PDs. Treatment with various cytoskeleton-specific drugs revealed that the intact actomyosin motility system is required for trafficking of Hsp70h in cytosol and its targeting to PDs. In contrast, none of the drugs interfered with the PD localization of Tobacco mosaic virus movement protein. Collectively, these findings suggest that Hsp70h is translocated and anchored to PDs in association with the actin cytoskeleton.

Viral RNA silencing suppressors inhibit the microRNA pathway at an intermediate step.

RNA silencing suppressors from different plant viruses are structurally diverse. In addition to inhibiting the antiviral silencing response to condition susceptibility, many suppressors are pathogenicity factors that cause disease or developmental abnormalities. Here, unrelated suppressors from multiple viruses were shown to inhibit microRNA (miRNA) activities and trigger an overlapping series of severe developmental defects in transgenic Arabidopsis thaliana. This suggests that interference with miRNA-directed processes may be a general feature contributing to pathogenicity of many viruses. A normally labile intermediate in the miRNA biogenesis/RNA-induced silencing complex (RISC) assembly pathway, miRNA*, accumulated specifically in the presence of suppressors (P1/HC-Pro, p21, or p19) that inhibited miRNA-guided cleavage of target mRNAs. Both p21 and p19, but not P1/HC-Pro, interacted with miRNA/miRNA* complexes and hairpin RNA-derived short interfering RNAs (siRNAs) in vivo. In addition, p21 bound to synthetic miRNA/miRNA* and siRNA duplexes in vitro. We propose that several different suppressors act by distinct mechanisms to inhibit the incorporation of small RNAs into active RISCs.

Complex molecular architecture of beet yellows virus particles.

Closteroviruses possess exceptionally long filamentous virus particles that mediate protection and active transport of the genomic RNA within infected plants. These virions are composed of a long "body" and short "tail" whose principal components are the major and minor capsid proteins, respectively. Here we use biochemical, genetic, and ultrastructural analyses to dissect the molecular composition and architecture of particles of beet yellows virus, a closterovirus. We demonstrate that the virion tails encapsidate the 5'-terminal, approximately 650-nt-long, part of the viral RNA. In addition to the minor capsid protein, the viral Hsp70-homolog, 64-kDa protein, and 20-kDa protein are also incorporated into the virion tail. Atomic force microscopy of virions revealed that the tail possesses a striking, segmented morphology with the tip segment probably being built of 20-kDa protein. The unexpectedly complex structure of closterovirus virions has important mechanistic and functional implications that may also apply to other virus families.

Suppressor of RNA silencing encoded by Beet yellows virus.

Using an Agrobacterium-mediated transient assay, we screened the 15.5-kb genome of the Beet yellows virus for proteins with RNA silencing suppressor activity. Among eight proteins tested, only a 21-kDa protein (p21) was able to suppress double-stranded (ds) RNA-induced silencing of the green fluorescent protein (GFP) mRNA. Restoration of GFP expression by p21 under these conditions had no apparent effect on accumulation of the small interfering RNAs. In addition, p21 elevated the transient expression level of the GFP mRNA in the absence of dsRNA inducer. Similar activities were detected using homologs of p21 encoded by other members of the genus Closterovirus. Computer analysis indicated that p21-like proteins constitute a novel protein family that is unrelated to other recognized suppressors of RNA silencing. Examination of the subcellular distribution in BYV-infected plants revealed that p21 is partitioned between soluble cytoplasmic form and proteinaceous inclusion bodies at the cell periphery.

Interaction between long-distance transport factor and Hsp70-related movement protein of Beet yellows virus.

Systemic spread of viruses in plants involves local movement from cell to cell and long-distance transport through the vascular system. The cell-to-cell movement of the Beet yellows virus (BYV) is mediated by a movement protein that is an Hsp70 homolog (Hsp70h). This protein is required for the assembly of movement-competent virions that incorporate Hsp70h. By using the yeast two-hybrid system, in vitro coimmunoprecipitation, and in planta coexpression approaches, we show here that the Hsp70h interacts with a 20-kDa BYV protein (p20). We further demonstrate that p20 is associated with the virions presumably via binding to Hsp70h. Genetic and immunochemical analyses indicate that p20 is dispensable for assembly and cell-to-cell movement of BYV but is required for the long-distance transport of virus through the phloem. These results reveal a novel activity for the Hsp70h that provides a molecular link between the local and systemic spread of a plant virus by docking a long-distance transport factor to virions.