Infectious clone construction and pathogenicity confirmation of Cotton leaf curl Multan virus (CLCuMuV), Ramie mosaic virus (RamV) and Corchorus yellow vein Vietnam virus (CoYVV) by southern blot analysis.

Geminiviruses are insect-transmissible, economically vital group of plant viruses, which cause significant losses to crop production and ornamental plants across the world. During this study, infectious clones of three devastating begomoviruses, i.e., Cotton leaf curl Multan virus (CLCuMuV), Ramie mosaic virus (RamV) and Corchorus yellow vein Vietnam virus (CoYVV) were constructed by following novel protocol. All infectious clones were confirmed by cloning and sequencing. All of the infectious clones were agro-inoculated in Agrobacterium. After the agro-infiltrations, all clones were injected into Nicotiana benthamiana and jute plants under controlled condition. After 28 days of inoculation, plants exhibited typical symptoms of their corresponding viruses. All the symptomatic and asymptomatic leaves were collected from inoculated plants for further analysis. The southern blot analysis was used to confirm the infection of studied begomoviruses. At the end, all the products were sequenced and analyzed.

Agroinoculation of Grapevine Pinot Gris Virus in tobacco and grapevine provides insights on viral pathogenesis.

The Grapevine Pinot Gris disease (GPG-d) is a novel disease characterized by symptoms such as leaf mottling and deformation, which has been recently reported in grapevines, and mostly in Pinot gris. Plants show obvious symptoms at the beginning of the growing season, while during summer symptom recovery frequently occurs, manifesting as symptomless leaves. A new Trichovirus, named Grapevine Pinot gris virus (GPGV), which belongs to the family Betaflexiviridae was found in association with infected plants. The detection of the virus in asymptomatic grapevines raised doubts about disease aetiology. Therefore, the primary target of this work was to set up a reliable system for the study of the disease in controlled conditions, avoiding interfering factor(s) that could affect symptom development. To this end, two clones of the virus, pRI::GPGV-vir and pRI::GPGV-lat, were generated from total RNA collected from one symptomatic and one asymptomatic Pinot gris grapevine, respectively. The clones, which encompassed the entire genome of the virus, were used in Agrobacterium-mediated inoculation of Vitis vinifera and Nicotiana benthamiana plants. All inoculated plants developed symptoms regardless of their inoculum source, demonstrating a correlation between the presence of GPGV and symptomatic manifestations. Four months post inoculum, the grapevines inoculated with the pRI::GPGV-lat clone developed asymptomatic leaves that were still positive to GPGV detection. Three to four weeks later (i.e. ca. 5 months post inoculum), the same phenomenon was observed in the grapevines inoculated with pRI::GPGV-vir. This observation perfectly matches symptom progression in infected field-grown grapevines, suggesting a possible role for plant antiviral mechanisms, such as RNA silencing, in the recovery process.

Functional analysis of the N-terminal basic motif of a eukaryotic satellite RNA virus capsid protein in replication and packaging.

Efficient replication and assembly of virus particles are integral to the establishment of infection. In addition to the primary role of the capsid protein (CP) in encapsidating the RNA progeny, experimental evidence on positive sense single-stranded RNA viruses suggests that the CP also regulates RNA synthesis. Here, we demonstrate that replication of Satellite tobacco mosaic virus (STMV) is controlled by the cooperative interaction between STMV CP and the helper virus (HV) Tobacco mosaic virus (TMV) replicase. We identified that the STMV CP-HV replicase interaction requires a positively charged residue at the third position (3R) in the N-terminal 13 amino acid (aa) motif. Far-Northwestern blotting showed that STMV CP promotes binding between HV-replicase and STMV RNA. An STMV CP variant having an arginine to alanine substitution at position 3 in the N-terminal 13aa motif abolished replicase-CP binding. The N-terminal 13aa motif of the CP bearing alanine substitutions for positively charged residues located at positions 5, 7, 10 and 11 are defective in packaging full-length STMV, but can package a truncated STMV RNA lacking the 3' terminal 150 nt region. These findings provide insights into the mechanism underlying the regulation of STMV replication and packaging.

Virus-induced gene silencing as a scalable tool to study drought tolerance in plants.

Here we describe the methodology of using virus-induced gene silencing (VIGS) as a powerful and scalable tool to screen the function of genes that participate in adaptation to drought. Silencing of endogenous gene expression in Nicotiana benthamiana is achieved by systemic infection of the aerial parts of the plant with a virus engineered to contain homologous fragments of the target gene(s) of interest. Silenced plant material can be consistently produced with little optimization in less than 1 month without specialized equipment, using only simple cloning and transformation techniques. Although maximal silencing is localized to only a few leaves, when whole plants are subjected to water stress, the tissue from these silenced leaves can be characterized for physiological, biochemical, and transcriptional responses to determine the role of the candidate genes in drought tolerance.

Optimization and utilization of Agrobacterium-mediated transient protein production in Nicotiana.

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana × N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Sesbania mosaic virus (SeMV) infectious clone: possible mechanism of 3' and 5' end repair and role of polyprotein processing in viral replication.

Sesbania mosaic virus (SeMV) is a positive stranded RNA virus belonging to the genus Sobemovirus. Construction of an infectious clone is an essential step for deciphering the virus gene functions in vivo. Using Agrobacterium based transient expression system we show that SeMV icDNA is infectious on Sesbania grandiflora and Cyamopsis tetragonoloba plants. The efficiency of icDNA infection was found to be significantly high on Cyamopsis plants when compared to that on Sesbania grandiflora. The coat protein could be detected within 6 days post infiltration in the infiltrated leaves. Different species of viral RNA (double stranded and single stranded genomic and subgenomic RNA) could be detected upon northern analysis, suggesting that complete replication had taken place. Based on the analysis of the sequences at the genomic termini of progeny RNA from SeMV icDNA infiltrated leaves and those of its 3' and 5' terminal deletion mutants, we propose a possible mechanism for 3' and 5' end repair in vivo. Mutation of the cleavage sites in the polyproteins encoded by ORF 2 resulted in complete loss of infection by the icDNA, suggesting the importance of correct polyprotein processing at all the four cleavage sites for viral replication. Complementation analysis suggested that ORF 2 gene products can act in trans. However, the trans acting ability of ORF 2 gene products was abolished upon deletion of the N-terminal hydrophobic domain of polyprotein 2a and 2ab, suggesting that these products necessarily function at the replication site, where they are anchored to membranes.