Characterization of Virus-Inducible Orchid Argonaute 5b Promoter and Its Functional Characterization in Nicotiana benthamiana during Virus Infection.

Plant ARGONAUTES (AGOs) play a significant role in the defense against viral infection. Previously, we have demonstrated that AGO5s encoded in Phalaenopsis aphrodite subsp. formosana (PaAGO5s) took an indispensable part in defense against major viruses. To understand the underlying defense mechanism, we cloned PaAGO5s promoters (pPaAGO5s) and analyzed their activity in transgenic Nicotiana benthamiana using ß-glucuronidase (GUS) as a reporter gene. GUS activity analyses revealed that during Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) infections, pPaAGO5b activity was significantly increased compared to pPaAGO5a and pPaAGO5c. Analysis of pPaAGO5b 5'-deletion revealed that pPaAGO5b_941 has higher activity during virus infection. Further, yeast one-hybrid analysis showed that the transcription factor NbMYB30 physically interacted with pPaAGO5b_941 to enhance its activity. Overexpression and silencing of NbMYB30 resulted in up- and downregulation of GUS expression, respectively. Exogenous application and endogenous measurement of phytohormones have shown that methyl jasmonate and salicylic acid respond to viral infections. NbMYB30 overexpression and its closest related protein, PaMYB30, in P. aphrodite subsp. formosana reduced CymMV accumulation in P. aphrodite subsp. formosana. Based on these discoveries, this study uncovers the interaction between virus-responsive promoter and the corresponding transcription factor in plants.

A transgenic plant cell-suspension system for expression of epitopes on chimeric Bamboo mosaic virus particles.

We describe a novel strategy to produce vaccine antigens using a plant cell-suspension culture system in lieu of the conventional bacterial or animal cell-culture systems. We generated transgenic cell-suspension cultures from Nicotiana benthamiana leaves carrying wild-type or chimeric Bamboo mosaic virus (BaMV) expression constructs encoding the viral protein 1 (VP1) epitope of foot-and-mouth disease virus (FMDV). Antigens accumulated to high levels in BdT38 and BdT19 transgenic cell lines co-expressing silencing suppressor protein P38 or P19. BaMV chimeric virus particles (CVPs) were subsequently purified from the respective cell lines (1.5 and 2.1 mg CVPs/20 g fresh weight of suspended biomass, respectively), and the resulting CVPs displayed VP1 epitope on the surfaces. Guinea pigs vaccinated with purified CVPs produced humoral antibodies. This study represents an important advance in the large-scale production of immunopeptide vaccines in a cost-effective manner using a plant cell-suspension culture system.

Stable Display of Artificially Long Foreign Antigens on Chimeric Bamboo mosaic virus Particles.

Plant viruses can be genetically modified to generate chimeric virus particles (CVPs) carrying heterologous peptides fused on the surface of coat protein (CP) subunits as vaccine candidates. However, some factors may be especially significant in determining the properties of chimeras. In this study, peptides from various sources and of various lengths were inserted into the Bamboo mosaic virus-based (BaMV) vector CP N-terminus to examine the chimeras infecting and accumulating in plants. Interestingly, it was found that the two different strains Foot-and-mouth disease virus (FMDV) VP1 antigens with flexible linker peptides (77 or 82 amino acids) were directly expressed on the BaMV CP, and the chimeric particles self-assembled and continued to express FMDV antigens. The chimeric CP, when directly fused with a large foreign protein (117 amino acids), can self-fold into incomplete virus particles or disks. The physicochemical properties of heterologus peptides N-terminus, complex strand structures of heterologus peptides C-terminus and different flexible linker peptides, can affect the chimera accumulation. Based on these findings, using plant virus-based chimeras to express foreign proteins can increase their length limitations, and engineered plant-made CVP-based vaccines have increasing potential for further development as novel vaccines.

Argonaute 5 family proteins play crucial roles in the defence against Cymbidium mosaic virus and Odontoglossum ringspot virus in Phalaenopsis aphrodite subsp. formosana.

The orchid industry faces severe threats from diseases caused by viruses. Argonaute proteins (AGOs) have been shown to be the major components in the antiviral defence systems through RNA silencing in many model plants. However, the roles of AGOs in orchids against viral infections have not been analysed comprehensively. In this study, Phalaenopsis aphrodite subsp. formosana was chosen as the representative to analyse the AGOs (PaAGOs) involved in the defence against two major viruses of orchids, Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV). A total of 11 PaAGOs were identified from the expression profile analyses of these PaAGOs in P. aphrodite subsp. formosana singly or doubly infected with CymMV and/or ORSV. PaAGO5b was found to be the only one highly induced. Results from overexpression of individual PaAGO5 family genes revealed that PaAGO5a and PaAGO5b play central roles in the antiviral defence mechanisms of P. aphrodite subsp. formosana. Furthermore, a virus-induced gene silencing vector based on Foxtail mosaic virus was developed to corroborate the function of PaAGO5s. The results confirmed their importance in the defences against CymMV and ORSV. Our findings may provide useful information for the breeding of traits for resistance or tolerance to CymMV or ORSV infections in Phalaenopsis orchids.

Production of Japanese Encephalitis Virus Antigens in Plants Using Bamboo Mosaic Virus-Based Vector.

Japanese encephalitis virus (JEV) is among the major threats to public health in Asia. For disease control and prevention, the efficient production of safe and effective vaccines against JEV is in urgent need. In this study, we produced a plant-made JEV vaccine candidate using a chimeric virus particle (CVP) strategy based on bamboo mosaic virus (BaMV) for epitope presentation. The chimeric virus, designated BJ2A, was constructed by fusing JEV envelope protein domain III (EDIII) at the N-terminus of BaMV coat protein, with an insertion of the foot-and-mouth disease virus 2A peptide to facilitate the production of both unfused and epitope-presenting for efficient assembly of the CVP vaccine candidate. The strategy allowed stable maintenance of the fusion construct over long-term serial passages in plants. Immuno-electron microscopy examination and immunization assays revealed that BJ2A is able to present the EDIII epitope on the surface of the CVPs, which stimulated effective neutralizing antibodies against JEV infection in mice. This study demonstrates the efficient production of an effective CVP vaccine candidate against JEV in plants by the BaMV-based epitope presentation system.