The C2 protein of tomato leaf curl Taiwan virus is a pathogenicity determinant that interferes with expression of host genes encoding chromomethylases

Tomato (Solanum lycopersicum) is one of the most important crops worldwide and is severely affected by geminiviruses. Tomato leaf curl Taiwan virus (ToLCTWV), belonging to the geminiviruses, was isolated in Taiwan and causes tremendous crop loss. The geminivirus‐encoded C2 proteins are crucial for a successful interaction between the virus and host plants. However, the exact functions of the viral C2 protein of ToLCTWV have not been investigated. We analyzed the molecular function(s) of the C2 protein by transient or stable expression in tomato cv. Micro‐Tom and Nicotiana benthamiana. Severe stunting of tomato and N. benthamiana plants infected with ToLCTWV was observed. Expression of ToLCTWV C2‐green fluorescent protein (GFP) fusion protein was predominately located in the nucleus and contributed to activation of a coat protein promoter. Notably, the C2‐GFP fluorescence was distributed in nuclear aggregates. Tomato and N. benthamiana plants inoculated with potato virus X (PVX)‐C2 displayed chlorotic lesions and stunted growth. PVX‐C2 elicited hypersensitive responses accompanied by production of reactive oxygen species in N. benthamiana plants, which suggests that the viral C2 was a potential recognition target to induce host‐defense responses. In tomato and N. benthamiana, ToLCTWV C2 was found to interfere with expression of genes encoding chromomethylases. N. benthamiana plants with suppressed NbCMT3–2 expression were more susceptible to ToLCTWV infection. Transgenic N. benthamiana plants expressing the C2 protein showed decreased expression of the NbCMT3–2 gene and pNbCMT3–2::GUS (β‐glucuronidase) promoter activity. C2 protein is an important pathogenicity determinant of ToLCTWV and interferes with host components involved in DNA methylation.     

Transient expression of <Emphasis Type="Italic">Human papillomavirus</Emphasis> type 16 L2 epitope fused to N- and C-terminus of coat protein of <Emphasis Type="Italic">Potato virus X</Emphasis> in plants

Transient expression of foreign genes based on plant viral vectors is a suitable system for the production of relevant immunogens that can be used for the development of a new generation of vaccines against a variety of infectious diseases. In the present study the epitope derived from HPV-16 L2 minor capsid protein (amino acids 108–120) was expressed from Potato virus X (PVX)-based vector pGR106 as N- or C-terminal fusion with the PVX coat protein (PVX CP) in transgenic Nicotiana benthamiana plants. The fusion protein L2_108-120-PVX CP was successfully expressed in plants at a level of 170 mg/kg of fresh leaf tissue. The C-terminal fusion protein PVX CP- L2_108-120 was expressed using mutated vector sequence to avoid homologous recombination at a level of 8 mg/kg of fresh leaf tissue. Immunogenicity of L2_108-120-PVX CP virus-like particles was tested after immunization of mice by subcutaneous injection or tattoo administration. In animal sera the antibodies against the PVX CP and the L2_108-120 epitope were found after both methods of vaccine delivery.     

The influence of the N- and C- terminal modifications of Potato virus X coat protein on virus properties

The Potato virus X (PVX)-based vector was used for the construction of N- and C-terminally modified PVX coat protein (XCP) chimeras. N-terminal XCP modifications do not influence the viral life cycle, whereas the simple XCP C-terminal fusion impedes the viral replication. We designed several C-terminally modified XCP chimeras and tested their viabilities in various Nicotiana benthamiana genotypes. Our results showed the negative impact of 3′-terminal modification of XCP on the chimera’s life cycle. To ensure chimeric constructs stability, the second copy of the last 60 nucleotides of XCP followed by the 3′-untranslated region (UTR) was added downstream of the recombinant sequence. Simultaneously, the first copy of the last 60 nucleotides of XCP was mutated in order to prevent recombination between the two identical sequences. The movement protein of Tobacco mosaic virus expressed in transgenic N. benthamiana plants positively affected the cell-to-cell spread of C-terminally modified XCP chimeras.     

Influence of cytoplasmic heat shock protein 70 on viral infection of Nicotiana benthamiana

The accumulation of heat shock protein 70 (Hsp70) generally occurs in plants infected with viruses. However, the effect of Hsp70 accumulation on plant viral infection and pathogenesis remains elusive. In this study, the expression of six Hsp70 genes was found to be induced by the four diverse RNA viruses, Tobacco mosaic virus, Potato virus X (PVX), Cucumber mosaic virus and Watermelon mosaic virus, in Nicotiana benthamiana. Heat treatment enhanced the accumulation and systemic infection of these viruses. Similar results were obtained for viral infection in plants heterologously expressing an Arabidopsis cytoplasmic Hsp70 through either a PVX vector or Agrobacterium infiltration. In contrast, viral infection was compromised in cytoplasmic NbHsp70c‐1 gene‐silenced plants. These data demonstrate that the cytoplasmic Hsp70s can enhance the infection of N. benthamiana by diverse viruses.     

Stability of <Emphasis Type="Italic">Potato virus X</Emphasis> expression vectors is related to insert size: implications for replication models and risk assessment

We investigated the stability of expression constructs based on Potato virus X (PVX) as a function of insert length. Five different inserts ranging in length from 261 to 1,758 bp (human proinsulin, murine interleukin-10, HIV-1 nef, petunia expansin-1 and human gad65) were expressed using a PVX vector in Nicotiana benthamiana plants for three sequential passages. Using a competitive RT-PCR approach we demonstrated that insert–deletion could occur in the first infection cycle for all inserts, but that this was much more likely to be the case for longer ones. This suggested a negative correlation between insert length and vector stability. Sequence analysis of the deleted constructs suggested that recombination usually occurred at sites close to the duplicated sub-genomic promoter, but in a smaller number of cases the foreign gene itself was probably involved, resulting in partially deleted constructs containing transgene fragments. The implications of these results in the context of recombinant protein expression and its risks are discussed.     

Establishment of a novel virus-induced virulence effector assay for the identification of virulence effectors of plant pathogens using a PVX-based expression vector

Plant pathogens deliver virulence effectors into plant cells to modulate plant immunity and facilitate infection. Although species-specific virulence effector screening approaches have been developed for several pathogens, these assays do not apply to pathogens that cannot be cultured and/or transformed outside of their hosts. Here, we established a rapid and parallel screening assay, called the virus-induced virulence effector (VIVE) assay, to identify putative effectors in various plant pathogens, including unculturable pathogens, using a virus-based expression vector. The VIVE assay uses the potato virus X (PVX) vector to transiently express candidate effector genes of various bacterial and fungal pathogens into Nicotiana benthamiana leaves. Using the VIVE assay, we successfully identified Avh148 as a potential virulence effector of Phytophthora sojae. Plants infected with PVX carrying Avh148 showed strong viral symptoms and high-level Avh148 and viral RNA accumulation. Analysis of P. sojae Avh148 deletion mutants and soybean hairy roots overexpressing Avh148 revealed that Avh148 is required for full pathogen virulence. In addition, the VIVE assay was optimized in N. benthamiana plants at different developmental stages across a range of Agrobacterium cell densities. Overall, we identified six novel virulence effectors from seven pathogens, thus demonstrating the broad effectiveness of the VIVE assay in plant pathology research.     

Plant-produced recombinant influenza vaccine based on virus-like HBc particles carrying an extracellular domain of M2 protein

Conventional influenza vaccines are based on a virus obtained in chicken embryos or its components. The high variability of the surface proteins of influenza virus, hemagglutinin and neuraminidase, requires strain-specific vaccines matching the antigenic specificity of newly emerging virus strains to be developed. A recombinant vaccine based on a highly conservative influenza virus protein M2 fused to a nanosized carrier particle can be an attractive alternative to traditional vaccines. We have constructed a recombinant viral vector based on potato X virus that provides for expression in the Nicotiana benthamiana plants of a hybrid protein M2eHBc consisting of an extracellular domain of influenza virus M2 protein (M2e) fused to hepatitis B core antigen (HBc). This vector was introduced into plant cells by infiltrating leaves with agrobacteria carrying the viral vector. The hybrid protein M2eHBc was synthesized in the infected N. benthamiana plants in an amount reaching 1–2% of the total soluble protein and formed virus-like particles with the M2e peptide presented on the surface. Methods of isolation and purification of M2eHBc particles from plant producers were elaborated. Experiments on mice have shown a high immunogenicity of the plant-produced M2eHBc particles and their protective effect against lethal influenza challenge. The developed transient expression system can be used for production of M2e-based candidate influenza vaccine in plants.     

Analysis of Subcellular Localization and Pathogenicity of <i>Plum Bark Necrosis Stem-Pitting Associated Virus</i> Protein P6

Infection of plum bark necrosis stem pitting associated virus (PBNSPaV) has been reported in many Prunus species in several countries, causing significant economic losses. The very small proteins encoded by plant viruses are often overlooked due to their short sequences and uncertain significance. However, numerous studies have indicated that they might play important roles in the pathogenesis of virus infection. The role of small hydrophobic protein P6, encoded by the open reading frame 2 of PBNSPaV, has not been well explored. In this study, we amplified the P6 fragment from a PBNSPaV isolate by RT-PCR using specific primers and found that it is 174 bp long and encodes a protein of approximately 6.3 kD with a transmembrane domain. Subcellular localization analysis of P6 proteins in tobacco leaves showed that P6 localizes to the cytomembrane and nuclear membrane. To further clarify the pathogenicity of P6 proteins, we constructed a PVX-P6 expression vector by inserting the p6 fragment into a potato virus X (PVX)-based vector and transformed it into Agrobacterium tumefaciens GV3101. Infiltration of Nicotiana benthamiana (N. benthamiana) with the PVX vector-transformed A. tumefaciens led to slight mosaic symptoms at 14 days of post-inoculation. Meanwhile, infiltration with the PVX-P6 vector-transformed A. tumefaciens resulted in no significant symptoms. These results demonstrated that heterologous expression of P6 in N. benthamiana could not enhance the pathogenicity of PVX. Our study indicates that P6 may not be a potential pathogenic factor associate with the causing of symptoms, and the mode of action of PBNSPaV-P6 protein remains to be further studied.     

Differential contributions of plant Dicer‐like proteins to antiviral defences against potato virus X in leaves and roots

Members of the plant Dicer‐like (DCL) protein family are the critical components of the RNA‐silencing pathway that mediates innate antiviral defence. The distinct antiviral role of each individual DCL protein has been established with mostly based on observations of aerial parts of plants. Thus, although the roots are closely associated with the life cycle of many plant viruses, little is known about the antiviral activities of DCL proteins in roots. We observed that antiviral silencing strongly inhibits potato virus X (PVX) replication in roots of some susceptible Solanaceae species. Silencing of the DCL4 homolog in Nicotiana benthamiana partially elevated PVX replication levels in roots. In Arabidopsis thaliana, which was originally considered a non‐host plant of PVX, high levels of PVX accumulation in inoculated leaves were achieved by inactivation of DCL4, while in the upper leaves and roots, it required the additional inactivation of DCL2. In transgenic A. thaliana carrying the PVX amplicon with a green fluorescent protein (GFP) gene insertion in the chromosome (AMP243 line), absence of DCL4 enabled high levels of PVX‐GFP accumulation in various aerial organs but not in the roots, suggesting that DCL4 is critical for intracellular antiviral silencing in shoots but not in roots, where it can be functionally compensated by other DCL proteins. Together, the high level of functional redundancies among DCL proteins may contribute to the potent antiviral activities against PVX replication in roots.     

Improved in Planta Expression of the Human Islet Autoantigen Glutamic Acid Decarboxylase (GAD65)

The smaller isoform of the enzyme glutamic acid decarboxylase (GAD65) is a major islet autoantigen in autoimmune type 1 diabetes mellitus (T1DM). Transgenic plants expressing human GAD65 (hGAD65) are a potential means of direct oral administration of the islet autoantigen in order to induce tolerance and prevent clinical onset of disease. We have previously reported the successful generation of transgenic tobacco and carrot that express immunoreactive, full-length hGAD65. In the present study, we tested the hypothesis that the expression levels of recombinant hGAD65 in transgenic plants can be increased by targeting the enzyme to the plant cell cytosol and by mediating expression through the potato virus X (PVX) vector. By substituting the NH₂-terminal region of hGAD65 with a homologous region of rat GAD67, a chimeric GAD67_1-87/GAD65_88-585 molecule was expressed in transgenic tobacco plants. Immunolocalization analysis showed that immunoreactive GAD67/65 was found in the plant cell cytosol. By using a radio-immuno assay with human serum from a GAD65 autoantibody-positive T1DM patient, the highest expression level of the recombinant GAD67/65 protein was estimated to be 0.19% of the total soluble protein, compared to only 0.04% of wild-type hGAD65. Transient expression of wild-type, full-length hGAD65 in N. benthamiana mediated by PVX infection was associated with expression levels of immunoreactive protein as high as 2.2% of total soluble protein. This substantial improvement of the expression of hGAD65 in plants paves the way for immunoprevention studies of oral administration of GAD65-containing transgenic plant material in animal models of spontaneous autoimmune diabetes.