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Coat protein gene of potato virus X is a structural protein that plays a vital role in viral transmission and pathogenesis. RNA interference or post transcriptional gene silencing is a regulatory conserved mechanism that uses small interfering RNAs to control the expression of desired gene by inhibiting mRNA transcription. The present study aims to investigate the potential role of different siRNAs in down-regulating the mRNA expression of potato virus X in cell culture assays. Three specific siRNAs against CP-PVX gene were designed in which siRNA 3 significantly reduced the CP-PVX mRNA expression in HepG 2 cells upto 91.91% (12.5 fold) as compared to siRNA 1 and siRNA 2 where knockdown percentages were only 20 and 73.04%, respectively as revealed by realtime PCR. Maximum knockdown values were obtained at 100 nM concentration of siRNA 1 and 2, while for siRNA 3 promising knockdown was obtained at 50 nM concentration. For long lasting effect of siRNAs, short hairpin RNA corresponding to siRNA3 was designed and tested against CP-PVX which resulted in a similar pattern of inhibition on RNA expression of target gene as synthetic siRNA3. It was found that mRNA expression of targeted gene was reduced to 91% depicting strong RNAi response generated by shRNA. Our findings support the possibility of using consensus siRNA and shRNA-based approach as a promising strategy in effective inhibition of PVX in in-vitro assay. Down regulation of potato virus X through coat protein gene targeted siRNA is a potential way of inhibiting invading virus and to effectively stop its systemic spread. [ABSTRACT FROM AUTHOR]

RNA silencing is an important antiviral mechanism in plants. Small interfering RNAs (siRNA) or short hairpin RNAs (shRNA) are key stimulators for RNA silencing by acting as executors of viral restriction. Here, we have utilized RNAi technology to suppress potato virus X (PVX) in a transgenic potato cultivar. A stable shRNA of 107 bp directed against a conserved region in the coat protein (CP) gene of PVX was designed with stem and loop sequences derived from a microRNA (miR403; an active regulatory miRNA in potato). The shRNA transgene was directionally cloned in a plant binary vector under the influence of the cauliflower mosaic virus 35S (CaMV35S) constitutive promoter. The pre-shRNA construct was introduced into the potato cultivar Sante through Agrobacterium and transgene insertion was confirmed by testing using PCR (polymerase chain reaction). Upon artificial inoculation of transgenic and non-transgenic potato lines with PVX, variable resistance was revealed through qRT-PCR among the transgenic potato lines. Compared to non-transgenic potato plants, the transgenic potato lines—D5, P3, P9, P14, P21, S11 and S21—showed undetectable levels of CP-PVX mRNA. However, the transgenic lines D4 and P16 exhibited 70% and 75%, respectively, reducing mRNA expression of CP-PVX. The transgenic potato lines remained healthy, with no detectable disease symptoms as compared to the control plants which showed chlorosis and mosaic symptoms. Hence, the expression of virus specific shRNAs is a novel, effective and predictable approach to engineer resistance against PVX in transgenic plants. [ABSTRACT FROM AUTHOR]

The future of food security in Africa is being severely threatened due to an exponential increase in population, which is almost three times the increase of food production. Maize production is constrained by stem borers which cause significant yield losses. Yield losses can be further compounded by higher temperatures due to climate changes, which are expected to increase the population of maize stem borers. While several methods have been employed in stem borer management, there is still significant damage caused by maize stem borers. This necessitates better control methods including the adoption of recent biotechnological advancement in RNA interference (RNAi) technology. This review highlights evidence of an increase in the stem borer population as well as the foreseen decline in maize production worldwide due to the effects of climatic changes. Furthermore, we have drawn attention to improved methods that have been used to control stem borers in maize production as well as a reluctant acceptance of traditional biotechnology in Africa. Finally, we suggest the application of alternative RNA interference techniques to breed maize for efficient pest control in order to achieve food security, improve nutrition and promote sustainable maize production. [ABSTRACT FROM AUTHOR]

Cotton leaf curl virus disease (CLCuD) is caused by a suite of whitefly-transmitted begomovirus species and strains, resulting in extensive losses annually in India and Pakistan. RNA-interference (RNAi) is a proven technology used for knockdown of gene expression in higher organisms and viruses. In this study, a small interfering RNA (siRNA) construct was designed to target the AC1 gene of Cotton leaf curl Kokhran virus-Burewala (CLCuKoV-Bu) and the bC1 gene and satellite conserved region of the Cotton leaf curl Multan betasatellite (CLCuMB). The AC1 gene and CLCuMB coding and non-coding regions function in replication initiation and suppression of the plant host defense pathway, respectively. The construct, Vb, was transformed into cotton plants using the Agrobacterium-mediated embryo shoot apex cut method. Results from fluorescence in situ hybridization and karyotyping assays indicated that six of the 11 T1 plants harbored a single copy of the Vβ transgene. Transgenic cotton plants and non-transgenic (susceptible) test plants included as the positive control were challenge-inoculated using the viruliferous whitefly vector to transmit the CLCuKoV-Bu/CLCuMB complex. Among the test plants, plant Vβ-6 was asymptomatic, had the lowest amount of detectable virus, and harbored a single copy of the transgene on chromosome six. Absence of characteristic leaf curl symptom development in transgenic Vβ-6 cotton plants, and significantly reduced begomoviral-betasatellite accumulation based on real-time polymerase chain reaction, indicated the successful knockdown of CLCuKoV-Bu and CLCuMB expression, resulting in leaf curl resistant plants. [ABSTRACT FROM AUTHOR]

RNA interference is a defense response against virus multiplication. The goal of this paper was to test if expressing virus-specific shRNAs against potato virus Y (PVY) could confer resistance against PVY in potatoes. The recombinant binary vectors consisted of short hairpin RNA (shRNA) transgenes, expressed under the CaMV35S promoter, directed against the conserved portion of the potato virus Y (PVY) coat protein gene. To provide the shRNA a particular shape and stability in vivo , stem and loop sequences from a microRNA (miR403; an active regulatory microRNA in potato) were included with the siRNA sequences. The shRNA constructs were delivered into the “Cardinal” potato variety using Agrobacterium , and transgenic status was confirmed by polymerase chain reaction (PCR) and Southern blot. Compared to wild-type plants inoculated with PVY, expression of PVY coat protein mRNA, as assayed by reverse transcriptase (RT)-PCR, was reduced to 0.05% in shRNA1-transgenic potato lines and 22% in shRNA4-transgenic lines. In addition, via northern blots, the maximum accumulation of the transgene-specific siRNA was observed in shRNA1 transgenic plants, less was observed in shRNA4-transgenic lines, and the least amount was detected in control plants, which correlates with silencing of the PVY coat protein gene. Conclusively, by simultaneous RNA silencing, shRNA1-transgenic plants were found to be almost immune to PVY infection compared with shRNA4-potato plants. Therefore, the expression of virus-specific shRNAs is a novel, effective, and predictable approach to engineer resistance against PVY. [ABSTRACT FROM AUTHOR]