Tobacco calmodulin-like protein provides secondary defense by binding to and directing degradation of virus RNA silencing suppressors.

RNA silencing (RNAi) induced by virus-derived double-stranded RNA (dsRNA), which is in a sense regarded as a pathogen-associated molecular pattern (PAMP) of viruses, is a general plant defense mechanism. To counteract this defense, plant viruses express RNA silencing suppressors (RSSs), many of which bind to dsRNA and attenuate RNAi. We showed that the tobacco calmodulin-like protein, rgs-CaM, counterattacked viral RSSs by binding to their dsRNA-binding domains and sequestering them from inhibiting RNAi. Autophagy-like protein degradation seemed to operate to degrade RSSs with the sacrifice of rgs-CaM. These RSSs could thus be regarded as secondary viral PAMPs. This study uncovered a unique defense system in which an rgs-CaM-mediated countermeasure against viral RSSs enhanced host antiviral RNAi in tobacco.

Booster responses by oral vaccination with transgenic plants against chicken leucocytozoonosis.

We developed a transgenic potato (TrP/R7) expressing the recombinant R7 (rR7) antigen for use as an oral vaccine to protect against a chicken protozoan disease, chicken leucocytozoonosis. The TrP/R7 potato was produced by Agrobacterium tumefaciens-mediated transformation and regeneration, and the R7 gene insertion into potato chromosomes was confirmed by genomic polymerase chain reaction and Southern hybridization. rR7 antigen expression in TrP/R7 potato was also confirmed by sandwich enzyme-linked immunosorbent assay and western blotting using an antibody against the second-generation schizont of Leucocytozoon caulleryi. A transgenic potato clone with the highest rR7 antigen expression (3 µg rR7 antigen per gram of fresh-weight potato leaves) was selected, cultivated, and used in oral administration experiments to examine its ability to boost immunity. Chickens were immunized with chicken leucocytozoonosis vaccine "Hokken" by injection, and chickens that developed moderate levels of antibody titres were fed with TrP/R7 leaves. Chickens fed with TrP/R7 leaves showed increased antibody responses. In contrast, chickens fed with non-transgenic potato leaves showed a continuous decrease in antibody titres. Furthermore, chickens fed with TrP/R7 potato leaves showed strong resistance against experimental challenge with L. caulleryi infection. This study demonstrates the use of a plant-based oral vaccine to boost immunity against a protozoan disease.

Risk-managed production of bioactive recombinant proteins using a novel plant virus vector with a helper plant to complement viral systemic movement.

A plant viral vector has the potential to efficiently produce recombinant proteins at a low cost in a short period. Although recombinant proteins can be also produced by transgenic plants, a plant viral vector, if available, may be more convenient when urgent scale-up in production is needed. However, it is difficult to use a viral vector in open fields because of the risk of escape to the environment. In this study, we constructed a novel viral vector system using a movement-defective Cucumber mosaic virus (CMV) vector, which is theoretically localized in the inoculated cells but infects systemically only with the aid of the transgenic helper plant that complements viral movement, diminishing the risk of viral proliferation. Interestingly, the helper plant systemically infected with the vector gave strong cross-protection against challenge inoculation with wild-type CMVs. Using CMV strains belonging to two discrete CMV groups (subgroups I and II), we also improved the system to prevent recombination between the vector and the transgene transcript in the helper plant. We here demonstrate the expression of an anti-dioxin single chain variable fragment (DxscFv) and interleukin-1 receptor antagonist (IL1-Ra) in Nicotiana benthamiana by this viral vector confinement system, which is applicable for many useful high-quality recombinant proteins.

HC-Pro, a potyvirus RNA silencing suppressor, cancels cycling of Cucumber mosaic virus in Nicotiana benthamiana plants.

The mixed infection of Cucumber mosaic virus (CMV) and a potyvirus has been known to increase CMV titer in Nicotiana benthamiana plants, resulting in synergistic viral symptoms. We found that among three potyviruses--Potato virus Y (PVY), Turnip mosaic virus (TuMV), and Clover yellow vein virus (C1YVV)--synergistic effects on CMV (or a recombinant CMV vector) titers were most efficiently induced by a co-infection with PVY in N. benthamiana plants. In addition, the helper component-proteinase (HC-Pro) gene of PVY expressed by transgenic plants, which is a viral RNA silencing suppressor, was sufficient to cancel the cycling pattern of CMV titer, resulting in increased levels of overall CMV accumulation. Surprisingly, we found that the levels of CMV and the foreign protein expressed from the CMV vector were much higher in the HC-Pro-transgenic plants than the levels detected in the plants mixed-infected with CMV and PVY. The mechanism for canceling the cyclic infection of CMV by the HC-Pro protein alone is discussed in view of the interaction between RNA silencing and HC-Pro, as well as the possible involvement of the 3a protein.

Ability of orally administered IFN-alpha-containing transgenic potato extracts to inhibit Listeria monocytogenes infection.

Type I interferons (IFN-alpha/beta) were originally thought to be antiviral cytokines, but it has recently been reported that they also play an important role in potentiating innate and adaptive immune responses. Moreover, several studies have shown that the oral administration of type I IFN ameliorates various biologic activities. Here, we studied the ability of orally administered IFN-alpha to protect mice from systemic Listeria monocytogenes infection. Daily oral administration of purified natural IFN-alpha at a concentration of 1000 international units (IU)/20 microl reduced the bacterial burden in infected organs. We also examined the protective effect of IFN-alpha expressed in transgenic potato plants. A much lower concentration of IFN-alpha (20 IU/ 20 microl) in the plant extracts was almost as protective as much higher concentrations of purified natural IFN-alpha. Our observations indicate that transgenic cytokine-expressing plants can be used prophylactically as edible pharmaceuticals to enhance systemic defense responses in humans and animals.

Expression of biologically active human tumor necrosis factor-alpha in transgenic potato plant.

We report the successful insertion of the cDNA of human tumor necrosis factor-alpha (HuTNF-alpha) into the genome of potato plant species, Solanum tuberosum, using Agrobacterium tumefacience-mediated transformation. HuTNF-alpha is a known and essential cytokine mediating host defense against tumors and infectious diseases and an immunomodulating agent. To enhance the accumulation of foreign gene product expression in plant cells, the molecular design of the constructed HuTNF-alpha is presented. Transcription and translation of TNF-alpha in transformants were confirmed by Northern blot, RT-PCR, ELISA, and Western blot, respectively. Expression of the bioactive HuTNF-alpha in plant cells was confirmed by way of the cytotoxic effect of the extract obtained from the transformants against murine L929 cells. We think that the expression level of HuTNF-alpha (15 microg/g potato plant tissue) obtained in the present study may be sufficient to induce responses/effects similar to those generated by TNF-alpha in human milk administered orally. We believe that the TNF-alpha expressed in edible potato plants has tremendous potential for clinical use in the areas of medicine and veterinary science. Its usefulness and applicability, therefore, need to be fully explored.

Deletion of plant-specific sugar residues in plant N-glycans by repression of GDP-D-mannose 4,6-dehydratase and ß-1,2-xylosyltransferase genes.

Production of pharmaceutical glycoproteins, such as therapeutic antibodies and cytokines, in plants has many advantages in safety and reduced costs. However, plant-made glycoproteins have N-glycans with plant-specific sugar residues (core ß-1,2-xylose and α-1,3-fucose) and a Lewis a (Le(a)) epitope, Galß(1-3)[Fucα(1-4)]GlcNAc. Because it is likely that these sugar residues and glycan structures are immunogenic, many attempts have been made to delete them. Previously, we reported the simultaneous deletion of the plant-specific core α-1,3-fucose and α-1,4-fucose residues in Le(a) epitopes by repressing the GDP-D-mannose 4,6-dehydratase (GMD) gene, which is associated with GDP-L-fucose biosynthesis, in Nicotiana benthamiana plants (rGMD plants, renamed to ΔGMD plants) (Matsuo and Matsumura, Plant Biotechnol. J., 9, 264-281, 2011). In the present study, we generated a core ß-1,2-xylose residue-repressed transgenic N. benthamiana plant by co-suppression of ß-1,2-xylosyltransferase (ΔXylT plant). By crossing ΔGMD and ΔXylT plants, we successfully generated plants in which plant-specific sugar residues were repressed (ΔGMDΔXylT plants). The proportion of N-glycans with deleted plant-specific sugar residues found in total soluble protein from ΔGMDΔXylT plants increased by 82.41%. Recombinant mouse granulocyte/macrophage-colony stimulating factor (mGM-CSF) and human monoclonal immunoglobulin G (hIgG) harboring N-glycans with deleted plant-specific sugar residues were successfully produced in ΔGMDΔXylT plants. Simultaneous repression of the GMD and XylT genes in N. benthamiana is thus very useful for deleting plant-specific sugar residues.