In planta expression of specific single chain fragment antibody (scFv) against nucleocapsid protein of fig mosaic virus (FMV).

Fig mosaic virus (FMV) is recognized as the main viral agent associated with the mosaic disease (MD) of fig trees (Ficus carica). Due to its worldwide occurrence, FMV represents the most significant global threat to the production of fig fruit. A disease management strategy against the MD in fig orchards has never been effective; and therefore, expression of recombinant antibody in plant cells could provide an alternative approach to suppress FMV infections. In this study we focused on expressing a specific recombinant antibody, a single-chain variable fragment (scFv), targeting the nucleocapsid protein (NP) of FMV in planta. To accomplish this objective, we inserted the scFv gene into a plant expression vector and conducted transient expression in leaves of Nicotiana tabacum cv. Samson plants. The construct was transiently expressed in tobacco plants by agroinfiltration, and antibody of the anticipated size was detected by immunoblotting. The produced plantibody was then assessed for specificity using ELISA and confirmed by Western blot analysis. In this study, the plantibody developed against FMV could be considered as a potential countermeasure to the infection by conferring resistance to MD.

Assessment of co-infection with BNYVV and BSCTV on resistance against Rhizomania disease in transgenic sugar beet plants.

Sugar beet is an economically important crop and one of the major sources of sucrose around the world. Beet necrotic yellow vein virus (BNYVV) and Beet severe curly top virus (BSCTV) are two widespread viruses in sugar beet that cause severe damage to its performance. Previously, we have successfully produced resistance to BNYVV based on RNA silencing in sugar beet by introducing constructs carrying the viral coat-protein-encoding DNA sequence, CP21, in sense and anti-sense orientations. Yet, the RNA silencing-mediated resistance to a specific virus could be affected by other ones as a part of synergistic interactions. In this study, we assayed the specificity of the induced resistance against BNYVV in two sets of transgenic events, S3 and S6 carrying 5'-UTR with or without CP21-coding sequences, respectively. These events were subjected to viral challenges with either BNYVV, an Iranian isolate of BSCTV (BSCTV-Ir) or both. All the plants inoculated with just BSCTV-Ir displayed curly-leaf symptoms. However, partial resistance was evident in S3 events as shown by mild symptoms and reduced PCR amplification of the BSCTV-Ir coat protein encoding sequence. Based on the presented data, resistance to BNYVV was stable in almost all the transgenic plants co-infected with BSCTV-Ir, except for one event, S3-229. In general, it seems that the co-infection does not affect the resistance to BNYVV in transgenic plants. These findings demonstrated that the introduced RNA silencing-mediated resistance against BNYVV in transgenic sugar beets is specific and is not suppressed after co-infection with a heterologous virus.

Developing of specific monoclonal recombinant antibody fused to alkaline phosphatase (AP) for one-step detection of fig mosaic virus.

Present study was performed to develop a fusion recombinant monoclonal antibody for one-step and accurate detection of FMV with a specific single-chain variable fragment (scFv) fused to alkaline phosphatase (AP) named as scFv(FMV-NP)-AP. The gene encoding-specific scFv recombinant antibody binding to nucleocapsid protein of Fig Mosaic Virus (FMV-NP) was fused to upstream of AP gene and integrated in pET26b bacterial expression vector. As vector contain pelB signal peptide, the expressed protein is secreted into periplasmic compartment. Recombinant fusion protein was produced in transformed E. coli following induction by IPTG. Extraction and purification of fusion protein was performed under denatured condition. The results of SDS-PAGE and western blot analysis indicated high integrity and purity with a single band protein with expected size of 72 kDa. The total yield of purified scFv(FMV-NP)-AP fusion protein estimated around 0.5-1 mg/l cultured medium. Subsequent colorimetric analysis confirmed presence of alkaline phosphatase activity in prepared scFv-AP fusion protein. Specificity of generated recombinant fusion antibody against cognate antigen and the native virus presented in infected plant extracts was assessed by ELISA, western blot and dot blot assays. Results revealed that scFv(FMV-NP)-AP is able to detect the presence of FMV in infected fig plants. The novel approach, implementing specific recombinant fusion antibody developed in this research, leads to one-step detection of FMV in plants by avoiding the use of chemical enzyme-labeled secondary antibodies.

Generation and molecular docking analysis of specific single-chain variable fragments selected by phage display against the recombinant nucleocapsid protein of fig mosaic virus.

The mosaic disease caused by fig mosaic virus (FMV) is considered the plague of fig worldwide. A naïve phage display library, raised against the recombinant nucleocapsid protein of FMV (FMV-Np) was screened to obtain specific monoclonal recombinant antibodies in the form of single chain variable fragments (scFvs). After three rounds of biopanning, the bacterially expressed FMV-Np was used as an antigen for selecting specific phages for the production of specific soluble scFvs to be used in immunological assays. The binding specificity of scFvs against FMV-infected fig samples was evaluated by immunoblotting and Plate trapped antigen-ELISA (PTA-ELISA), which revealed efficient of the resultant scFvs to the target antigen. Silico homology-modelling and molecular docking analysis confirmed the scFv and FMV-Np interactions with the anti-FMV-Np scFv through an estimated binding energy of -650 kj mol-1; considered to be generated from the interactions between 13 amino acids residues predicted as putative epitopes in the interface pocket of FMV-Np and scFv antibody. This high affinity was further confirmed in the specificity of ELISA and immunoblotting assays. This is the first report on the application of phage display technology to generate specific recombinant scFvs against FMV that can be applied in development of antibody-mediated protection strategy to control the fig mosaic disease.

Production of a polyclonal antiserum against recombinant nucleocapsid protein and its application for the detection of fig mosaic virus.

Mosaic disease (MD), caused by Fig mosaic emaravirus (FMV), is the most important and devastating virus disease of fig trees worldwide. The detection of FMV in infected plants is possible only through the use of molecular techniques, i.e. RT-PCR and LAMP, which both offer high sensitivity of detection, but are also considered laborious when dealing with a large number of samples. To cope with this restriction, a polyclonal antiserum through the immunization of a rabbit by injecting the recombinant nucleocapsid protein (NP) of FMV was raised and evaluated for its efficacy in Western Blot, Dot immuno-binding and DAS-ELISA. The results obtained showed that the raised antiserum was able to identify the nucleocapsid protein of FMV (p3) which was found to have an estimated molecular weight of ca. 35 KDa. In addition, the antiserum, when used in the three serological assays, was able to detect the p3 of FMV in protein extracts of infected plants with different levels of efficacy. Dot immuno-binding, using denatured plant protein extract, proved to be the most efficient serological assay for detecting FMV in samples collected from different fig orchards. This is the first report on an antiserum raised against FMV that could be used for immunological detection of the virus.

Effects of simultaneously elevated temperature and CO2 levels on Nicotiana benthamiana and its infection by different positive-sense RNA viruses are cumulative and virus type-specific.

We have studied how simultaneously elevated temperature and CO2 levels [climate change-related conditions (CCC) of 30°C, 970 parts-per-million (ppm) of CO2 vs. standard conditions (SC) of 25°C, ~ 405ppm CO2] affect physiochemical properties of Nicotiana benthamiana leaves, and also its infection by several positive-sense RNA viruses. In previous works we had studied effects of elevated temperature, CO2 levels separately. Under CCC, leaves of healthy plants almost doubled their area relative to SC but contained less protein/unit-of-area, similarly to what we had found under conditions of elevated CO2 alone. CCC also affected the sizes/numbers of different foliar cell types differently. Under CCC, infection outcomes in titers and symptoms were virus type-specific, broadly similar to those observed under elevated temperature alone. Under either condition, infections did not significantly alter the protein content of leaf discs. Therefore, effects of elevated temperature and CO2 combined on properties of the pathosystems studied were overall cumulative.

Silencing the tobacco gene for RNA-dependent RNA polymerase 1 and infection by potato virus Y cause remodeling of cellular organelles.

RNA-dependent RNA polymerase 1 (RDR1) has been shown to be involved in DNA methylation, RNA silencing and regulating expression of other genes. RDR1 gene expression is stimulated by infection with potato virus Y° (PVY). Transgenic Nicotiana tabacum plants silenced for RDR1 gene expression showed morphological changes in mesophyll cells, associated with remodeling of the nuclei, chloroplasts and mitochondria. RDR1 silencing led to decreased nuclear size, increased heterochromatin content and aggregation, decreased numbers of chloroplasts, plus changes in shape, internal structures and integrity of chloroplasts and mitochondria. RDR1-silenced transgenic plants showed increased PVY accumulation and ultrastructural remodeling was intensified in both chloroplasts and mitochondria of PVY-infected, RDR1-silenced plants. By contrast, heterochromatin condensation was reduced by PVY infection, and in non-transgenic plants the nuclei were translucent and lacked morphology after PVY infection. Thus, RDR1 regulates gene expression leading to remodeling of chromosomes, and PVY infection counteracts these effects on chromosomal remodeling.

Prevalence of Tobacco mosaic virus in Iran and Evolutionary Analyses of the Coat Protein Gene.

The incidence and distribution of Tobacco mosaic virus (TMV) and related tobamoviruses was determined using an enzyme-linked immunosorbent assay on 1,926 symptomatic horticultural crops and 107 asymptomatic weed samples collected from 78 highly infected fields in the major horticultural crop-producing areas in 17 provinces throughout Iran. The results were confirmed by host range studies and reverse transcription-polymerase chain reaction. The overall incidence of infection by these viruses in symptomatic plants was 11.3%. The coat protein (CP) gene sequences of a number of isolates were determined and disclosed to be a high identity (up to 100%) among the Iranian isolates. Phylogenetic analysis of all known TMV CP genes showed three clades on the basis of nucleotide sequences with all Iranian isolates distinctly clustered in clade II. Analysis using the complete CP amino acid sequence showed one clade with two subgroups, IA and IB, with Iranian isolates in both subgroups. The nucleotide diversity within each sub-group was very low, but higher between the two clades. No correlation was found between genetic distance and geographical origin or host species of isolation. Statistical analyses suggested a negative selection and demonstrated the occurrence of gene flow from the isolates in other clades to the Iranian population.

The influence of RNA-dependent RNA polymerase 1 on potato virus Y infection and on other antiviral response genes.

The gene encoding RNA-dependent RNA polymerase 1 (RDR1) is involved in basal resistance to several viruses. Expression of the RDR1 gene also is induced in resistance to Tobacco mosaic virus (TMV) mediated by the N gene in tobacco (Nicotiana tabacum cv. Samsun NN) in an incompatible hypersensitive response, as well as in a compatible response against Potato virus Y (PVY). Reducing the accumulation of NtRDR1 transcripts by RNA inhibition mediated by transgenic expression of a double-stranded RNA hairpin corresponding to part of the RDR1 gene resulted in little or no induction of accumulation of RDR1 transcripts after infection by PVY. Plants with lower accumulation of RDR1 transcripts showed much higher accumulation levels of PVY. Reduced accumulation of NtRDR1 transcripts also resulted in lower or no induced expression of three other antiviral, defense-related genes after infection by PVY. These genes encoded a mitochondrial alternative oxidase, an inhibitor of virus replication (IVR), and a transcription factor, ERF5, all involved in resistance to infection by TMV, as well as RDR6, involved in RNA silencing. The extent of the effect on the induced NtIVR and NtERF5 genes correlated with the extent of suppression of the NtRDR1 gene.