Disruption of prefoldin-2 protein synthesis in root-knot nematodes via host-mediated gene silencing efficiently reduces nematode numbers and thus protects plants.

MAIN CONCLUSION: The aim of this study is to demonstrate the feasibility of down-regulating endogeneous prefoldin-2 root-knot nematode transcripts by expressing dsRNA with sequence identity to the nematode gene in tobacco roots under the influence of strong Arabidopsis ubiquitin (UBQ1) promoter. Root-knot nematodes (RKNs) are sedentary endoparasites infecting a wide range of plant species. They parasitise the root system, thereby disrupting water and nutrient uptake and causing major reductions in crop yields. The most reliable means of controlling RKNs is via the use of soil fumigants such as methyl bromide. With the emergence of RNA interference (RNAi) technology, which permits host-mediated nematode gene silencing, a new strategy to control plant pathogens has become available. In the present study, we investigated host-induced RNAi gene silencing of prefoldin-2 in transgenic Nicotiana benthamiana. Reductions in prefoldin-2 mRNA transcript levels were observed when nematodes were soaked in a dsRNA solution in vitro. Furthermore, nematode reproduction was suppressed in RNAi transgenic lines, as evident by reductions in the numbers of root knots (by 34-60 % in independent RNAi lines) and egg masses (by 33-58 %). Endogenous expression of prefoldin-2, analysed via real-time polymerase chain reaction and Western blotting, revealed that the gene was strongly expressed in the pre-parasitic J2 stage. Our observations demonstrate the relevance and potential importance of targeting the prefoldin gene during the nematode life cycle. The work also suggests that further improvements in silencing efficiency in economically important crops can be accomplished using RNAi directed against plant-parasitic nematodes.

Enhanced expression of recombinant human cyclooxygenase 1 from stably-transfected Drosophila melanogaster S2 cells by dimethyl sulfoxide is mediated by up-regulation of nitric oxide synthase and transcription factor Kr-h1.

Recombinant human cyclooxygenase 1 (COX-1) was expressed from stably-transfected Drosophila melanogaster S2 (S2) cells. DMSO improved the expression of recombinant COX-1 by 180 %. DMSO increased the expression of nitric oxide synthase (NOS) at both the RNA and protein levels; NOS expression was closely correlated with the synthesis of recombinant COX-1 mRNA in stably-transfected S2 cells. DMSO also induced the gene encoding Kr-h1 which binds to the CACCC element of the metallothionein promoter to enhance the expression of recombinant COX-1. Therefore, DMSO improves the expression of recombinant COX-1 via NOS and/or the transcription factor Kr-h1.

Expression of a recombinant chimeric protein of hepatitis A virus VP1-Fc using a replicating vector based on Beet curly top virus in tobacco leaves and its immunogenicity in mice.

We describe the expression and immunogenicity of a recombinant chimeric protein (HAV VP1-Fc) consisting of human hepatitis A virus VP1 and an Fc antibody fragment using a replicating vector based on Beet curly top virus (BCTV) in Agrobacterium-infiltrated Nicotiana benthamiana leaves. Recombinant HAV VP1-Fc was expressed with a molecular mass of approximately 68 kDa. Recombinant HAV VP1-Fc, purified using Protein A Sepharose affinity chromatography, elicited production of specific IgG antibodies in the serum after intraperitoneal immunization. Following vaccination with recombinant HAV VP1-Fc protein, expressions of IFN-γ and IL-4 were increased in splenocytes at the time of sacrifice. Recombinant VP1-Fc from infiltrated tobacco plants can be used as an effective experimental immunogen for research into vaccine development.

Synthesis of double-layered rotavirus-like particles using internal ribosome entry site vector system in stably-transformed Drosophila melanogaster.

We established a bicistronic expression system using an encephalomyocarditis virus (EMCV)-derived internal ribosomal entry site (IRES) element to generate stably transformed Drosophila melanogaster Schneider 2 (S2) cells expressing human rotavirus Wa capsid proteins, VP2 and VP6, for the synthesis of VP2/6 double-layered virus-like particle (DVLP). The EMCV-derived IRES permitted bicistronic translation of recombinant VP6. Recombinant VP2 and VP6 were detected in extracellular fractions of stably transformed S2 cells. A wheel-like DVLP (diam ~ 50-55 nm) with short spikes was produced from the extracellular fraction of stably transformed S2 cells. A bicistronic expression system using an EMCV-derived IRES element can thus be used to express two proteins of interest in stably transformed S2 cells. The bi-or tri-cistronic expression of recombinant VP2/6/7 using stably transformed S2 cells can also be used to produce rotavirus VLPs.

Electrochemical microgap immunosensors for selective detection of pathogenic Aspergillus niger.

Aspergillus niger (A. niger) is a well-known allergenic, harmful fungus in the indoor environment that can cause asthmatic symptoms and atopy. Previous immunosensing approach suffers from an insufficient detection limit, mainly because there are no techniques for target amplification. We report an electrochemical immunosensor that selectively quantifies the A. niger based on the detection of extracellular proteins by using a specific interaction with antibody. The sensor was designed to show a decrease in redox current upon binding of the antigens secreted from A. niger onto an antibody-immobilized surface between the interdigitated electrodes. The extracellular proteins were profiled by LC-MS/MS to identify the antigens existing in the A. niger solution. Since the targets of the sensor are the proteins, its sensitivity and selectivity remain almost intact even after filtration of the spores. It was also found that the use of secretion promoter in the sampling stage greatly improved the sensor's limit of detection (LOD) for the spores. By this, the LOD was lowered by a few orders of magnitude so as to reach the value as low as ~101 spores/mL.

Expression and functional validation of heat-labile enterotoxin B (LTB) and cholera toxin B (CTB) subunits in transgenic rice (Oryza sativa).

We expressed the heat-labile enterotoxin B (LTB) subunit from enterotoxigenic Escherichia coli and the cholera toxin B (CTB) subunit from Vibrio cholerae under the control of the rice (Oryza sativa) globulin (Glb) promoter. Binding of recombinant LTB and CTB proteins was confirmed based on GM1-ganglioside binding enzyme-linked immunosorbent assays (GM1-ELISA). Real-time PCR of three generations (T3, T4, and T5) in homozygous lines (LCI-11) showed single copies of LTB, CTB, bar and Tnos. LTB and CTB proteins in rice transgenic lines were detected by Western blot analysis. Immunogenicity trials of rice-derived CTB and LTB antigens were evaluated through oral and intraperitoneal administration in mice, respectively. The results revealed that LTB- and CTB-specific IgG levels were enhanced in the sera of intraperitoneally immunized mice. Similarly, the toxin-neutralizing activity of CTB and LTB in serum of orally immunized mice was associated with elevated levels of both IgG and IgA. The results of the present study suggest that the combined expression of CTB and LTB proteins can be utilized to produce vaccines against enterotoxigenic strains of Escherichia coli and Vibrio cholera, for the prevention of diarrhea.