RNA interference-based resistance against a legume mastrevirus

<p>Abstract</p> <p>Background</p> <p>RNA interference (RNAi) is a homology-dependant gene silencing mechanism and has been widely used to engineer resistance in plants against RNA viruses. However, its usefulness in delivering resistance against plant DNA viruses belonging to family <it>Geminiviridae </it>is still being debated. Although the RNAi approach has been shown, using a transient assay, to be useful in countering monocotyledonous plant-infecting geminiviruses of the genus <it>Mastrevirus</it>, it has yet to be investigated as a means of delivering resistance to dicot-infecting mastreviruses. <it>Chickpea chlorotic dwarf Pakistan virus </it>(CpCDPKV) is a legume-infecting mastrevirus that affects chickpea and other leguminous crops in Pakistan.</p> <p>Results</p> <p>Here a hairpin (hp)RNAi construct containing sequences encompassing part of replication-associated protein gene, intergenic region and part of the movement protein gene of CpCDPKV under the control of the <it>Cauliflower mosaic virus </it>35S promoter has been produced and stably transformed into <it>Nicotiana benthamiana</it>. Plants harboring the hairpin construct were challenged with CpCDPKV. All non-transgenic <it>N. benthamiana </it>plants developed symptoms of CpCDPKV infection within two weeks post-inoculation. In contrast, none of the inoculated transgenic plants showed symptoms of infection and no viral DNA could be detected by Southern hybridization. A real-time quantitative PCR analysis identified very low-level accumulation of viral DNA in the inoculated transgenic plants.</p> <p>Conclusions</p> <p>The results presented show that the RNAi-based resistance strategy is useful in protecting plants from a dicot-infecting mastrevirus. The very low levels of virus detected in plant tissue of transgenic plants distal to the inoculation site suggest that virus movement and/or viral replication was impaired leading to plants that showed no discernible signs of virus infection.</p

Interaction of eukaryotic proliferating cell nuclear antigen (PCNA) with the replication-associated protein (Rep) of cotton leaf curl Multan virus and pedilanthus leaf curl virus.

peer reviewedThe replication-associated (Rep) proteins of pathogenic begomoviruses, including cotton leaf curl Multan virus (CLCuMuV) and pedilanthus leaf curl virus (PeLCV), interact with the DNA replication machinery of their eukaryotic hosts. The analysis of Rep protein sequences showed that there is 13-28% sequence variation among CLCuMuV and PeLCV isolates, with phylogenetic clusters that can separated at least in part based on the country of origin of the respective viruses. To identify specific host factors involved in the virus replication cycle, we conducted yeast two-hybrid assays to detect possible interactions between the CLCuMuV and PeLCV Rep proteins and 30 protein components of the Saccharomyces cerevisiae DNA replication machinery. This showed that the proliferating cell nuclear antigen (PCNA) protein of S. cerevisiae interacts with Rep proteins from both CLCuMuV and PeLCV. We used the yeast PCNA sequence in BLAST comparisons to identify two PCNA orthologs each in Gossypium hirsutum (cotton), Arabidopsis thaliana (Arabidopsis), and Nicotiana benthamiana (tobacco). Sequence comparisons showed 38-40% identity between the yeast and plant PCNA proteins, and > 91% identity among the plant PCNA proteins, which clustered together in one phylogenetic group. The expression of the six plant PCNA proteins in the yeast two-hybrid system confirmed interactions with the CLCuMuV and PeLCV Rep proteins. Our results demonstrate that the interaction of begomovirus Rep proteins with eukaryotic PCNA proteins is strongly conserved, despite significant evolutionary variation in the protein sequences of both of the interacting partners

REPercussions: how geminiviruses recruit host factors for replication

Circular single-stranded DNA viruses of the family Geminiviridae encode replication-associated protein (Rep), which is a multifunctional protein involved in virus DNA replication, transcription of virus genes, and suppression of host defense responses. Geminivirus genomes are replicated through the interaction between virus Rep and several host proteins. The Rep also interacts with itself and the virus replication enhancer protein (REn), which is another essential component of the geminivirus replicase complex that interacts with host DNA polymerases α and δ. Recent studies revealed the structural and functional complexities of geminivirus Rep, which is believed to have evolved from plasmids containing a signature domain (HUH) for single-stranded DNA binding with nuclease activity. The Rep coding sequence encompasses the entire coding sequence for AC4, which is intricately embedded within it, and performs several overlapping functions like Rep, supporting virus infection. This review investigated the structural and functional diversity of the geminivirus Rep

Flame Retardancy, Physiological Comfort and Durability of Casein Treated Cotton Fabrics

The present work was aimed to develop the flame retardant textiles for prevention of second-degree burn injuries from low intensity heat flux accidents. The different concentration of casein suspension was applied on cotton fabrics and their thermo-oxidative properties, flame retardant behavior as well as physiological comfort were examined. From thermo gravimetric analysis, the percentage increase in char residue indicated improvement in thermo-oxidative properties. The maximum improvement in flame retardant behavior was found in case of 30 w/v % casein suspension due to higher production of thermally stable char. Their SEM micrographs also showed the formation of stronger and coherent char with presence of local intumescence. Furthermore, for estimating small differences in burn length and burn area, the flame propagation was studied in detail using the image analysis. The lower concentration of casein below 20 w/v % was found to provide adequate flame retardancy with acceptable physiological comfort and mechanical properties. Finally, the short term durability of casein treatment was verified under the effect of washing and ageing conditions

Rational design of multi epitope-based subunit vaccine by exploring MERS-COV proteome: Reverse vaccinology and molecular docking approach.

Middle East respiratory syndrome (MERS-COV), first identified in Saudi Arabia, was caused by a novel strain of coronavirus. Outbreaks were recorded from different regions of the world, especially South Korea and the Middle East, and were correlated with a 35% mortality rate. MERS-COV is a single-stranded, positive RNA virus that reaches the host by binding to the receptor of dipeptidyl-peptides. Because of the unavailability of the vaccine available for the protection from MERS-COV infection, the rapid case detection, isolation, infection prevention has been recommended to combat MERS-COV infection. So, vaccines for the treatment of MERS-COV infection need to be developed urgently. A possible antiviral mechanism for preventing MERS-CoV infection has been considered to be MERS-CoV vaccines that elicit unique T-cell responses. In the present study, we incorporated both molecular docking and immunoinformatic approach to introduce a multiepitope vaccine (MEP) against MERS-CoV by selecting 15 conserved epitopes from seven viral proteins such as three structural proteins (envelope, membrane, and nucleoprotein) and four non-structural proteins (ORF1a, ORF8, ORF3, ORF4a). The epitopes, which were examined for non-homologous to host and antigenicity, were selected on the basis of conservation between T-cell, B-cell, and IFN-γ epitopes. The selected epitopes were then connected to the adjuvant (β-defensin) at the N-terminal through an AAY linker to increase the immunogenic potential. Structural modelling and physiochemical characteristic were applied to the vaccine construct developed. Afterwards the structure has been successfully docked with antigenic receptor, Toll-like receptor 3 (TLR-3) and in-silico cloning ensures that its expression efficiency is legitimate. Nonetheless the MEP presented needs tests to verify its safety and immunogenic profile

Diversity, Mutation and Recombination Analysis of Cotton Leaf Curl Geminiviruses.

The spread of cotton leaf curl disease in China, India and Pakistan is a recent phenomenon. Analysis of available sequence data determined that there is a substantial diversity of cotton-infecting geminiviruses in Pakistan. Phylogenetic analyses indicated that recombination between two major groups of viruses, cotton leaf curl Multan virus (CLCuMuV) and cotton leaf curl Kokhran virus (CLCuKoV), led to the emergence of several new viruses. Recombination detection programs and phylogenetic analyses showed that CLCuMuV and CLCuKoV are highly recombinant viruses. Indeed, CLCuKoV appeared to be a major donor virus for the coat protein (CP) gene, while CLCuMuV donated the Rep gene in the majority of recombination events. Using recombination free nucleotide datasets the substitution rates for CP and Rep genes were determined. We inferred similar nucleotide substitution rates for the CLCuMuV-Rep gene (4.96X10-4) and CLCuKoV-CP gene (2.706X10-4), whereas relatively higher substitution rates were observed for CLCuMuV-CP and CLCuKoV-Rep genes. The combination of sequences with equal and relatively low substitution rates, seemed to result in the emergence of viral isolates that caused epidemics in Pakistan and India. Our findings also suggest that CLCuMuV is spreading at an alarming rate, which can potentially be a threat to cotton production in the Indian subcontinent

In Silico Analysis of Plant Flavonoids as Potential Inhibitors of Newcastle Disease Virus V Protein

Newcastle disease is a viral infection causing serious economic losses to the global poultry industry. The V protein of Newcastle disease virus (NDV) is a pathogenicity determinant having various functions such as the suppression of apoptosis and replication of the NDV. This study was designed to assess the resistance potential of plant flavonoids against the V protein of Newcastle disease virus. Sequence analysis was performed using EXPASY and ProtParam tools. To build the three-dimensional structure of V protein, a homology-modeling method was used. Plant flavonoids with formerly reported therapeutic benefits were collected from different databases to build a library for virtual screening. Docking analysis was performed using the modeled structure of V protein on MOE software. Interaction analysis was also performed by MOE to explain the results of docking. Sequence analysis and physicochemical properties showed that V protein is negatively charged, acidic in nature, and relatively unstable. The 3D structure of the V protein showed eight &beta;-pleated sheets, three helices, and ten coiled regions. Based on docking score, ten flavonoids were selected as potential inhibitors of V protein. Furthermore, a common configuration was obtained among these ten flavonoids. The interaction analysis also identified the atoms involved in every interaction of flavonoid and V protein. Molecular dynamics (MD) simulation confirmed the stability of two compounds, quercetin-7-O-[&alpha;-L-rhamnopyranosyl(1&rarr;6)-&beta;-D-galactopyranoside] and luteolin 7-O-neohesperidoside, at 100 ns with V protein. The identified compounds through molecular docking and MD simulation could have potential as NDV-V protein inhibitor after further validation. This study could be useful for the designing of anti-NDV drugs

Mitochondrial gene cytochrome c oxidase I (CO1) used for molecular identification of Bactrocera zonata in Pakistan

Bactrocera zonata is fruit pest mostly attacked on peach and cause heavy destruction in production of peach fruits by sucking their juice. For theirmanagement, we start to detect them on basis of their molecular characterization. As mitochondrial genome encodes a gene COI used as biomarker for identifcation of eukaryotes including insects. In present study, we amplifed COI gene and cloned into pTZ57R/T vector (Fermentas). Cloned gene was confrmed throughrestriction analysis and sequenced through its entirety on both strands from Macrogen (South Korea) by Sanger sequencing method. Different computational toolswere utilized for comparative analysis of sequence with other related sequences retrieved from databases. Related species were identifed through phylogeneticanalysis using Mega 7 tool. Pairwise sequence alignment showed the sequence identity about 96% with Bactrocera zonata. By identifying the pests with moreauthentic molecular biomarker may help the research to control them more effectively in future.</p