Comparison of Post-transcriptional Gene Silencing (PTGS) Strategies for Developing Transgenic Plants Resistant to Tomato Leaf Curl Virus (ToLCV)

Tomato (Solanum lycopersicum L.), an economically important crop is host to many whitefly transmitted geminiviruses including tomato leaf curl virus (ToLCV). Genetically engineering resistance of pathogen through Post-transcriptional gene silencing (PTGS/RNAi) is a powerful strategy that can provide alternative to existing methods of producing virus resistant plants. We cloned and characterized ToLCV-replicase (TRP) gene from a local Dharwad, Karnataka, India ToLCV isolate for development of transgenic tomato plants. Plant expression vectors carrying viral replicase (rep) gene in sense, antisense, ihp (intron spaced hairpin) and HUTR (inverted repeats of heterologous 3΄-untranslated region) were constructed. Transgenic tomato plants carrying rep gene in different strategies when challenged with whiteflies carrying ToLCV showed varied degrees of resistance. Such plants were confirmed through PCR, GUS, Dot blot, Southern blot and semiquantitative PCR analysis. High degree of resistance was observed in the construct carrying both sense and antisense strand interrupted by intron (ihp). Our results demonstrate that, transgenic plants with simultaneous expression of sense and antisense strands are more efficient in gene silencing of ToLCV than those expressing either sense or antisense strand alone.

Post-transcriptional gene silencing: Basic concepts and applications

Post-transcriptional gene silencing (PTGS)-mediated gene silencing exploits the cellular mechanism whereintranscripts having sequence similarity to the double-stranded RNA (dsRNA) molecules present in the cell will besubjected to degradation. PTGS is closely related to natural processes such as RNA-mediated virus resistance andcross-protection in plants. Gene silencing and the cellular machinery for affecting this phenomenon might haveevolved as a natural protective measure against viral infection in plants. In PTGS, small interfering RNA (siRNA)molecules of 21–23 nucleotides length act as homology guides for triggering the systemic degradation of transcriptshomologous to the siRNA molecules. PTGS phenomenon, first discovered in transgenic petunia plants harbouringchalcone synthase gene and termed co-suppression, has been subsequently exploited to target specific gene transcripts for degradation leading to manifestation of desirable traits in crop plants. Targeted gene silencing has beenachieved either through the introduction of DNA constructs encoding dsRNA or antisense RNA or by deploying cosuppression constructs producing siRNAs against the transcript of interest. Understanding the mechanism of genesilencing has led to the development of several alternative strategies for inducing gene silencing in a precise andcontrolled way. This has paved the way for using PTGS as one ofthe chief functional genomicstools in plants and hashelped in unraveling the mechanism of many cellular processes and identifying the focal points in pathways, besides,opening new vistas in genetic engineering of plants for human benefits. PTGS has shown great potential in silencingthe deleterious genes efficiently so that value-added plant products could be obtained. Thus, PTGS has ushered in anew era in the genetic manipulation of plants for both applied and basic studies. In this review, we have outlined thebasics of RNAi-mediated gene silencing and summarized the work carried out at our institute using this approach, ascase studies. In particular, adopting RNAi-mediated gene silencing (a) as a method to restore fertility in transgenicmale sterile lines developed based on orfH522 gene from sunflower PET1-CMS source, (b) as a tool to suppress theproduction of toxic proteins, ricin and RCA, in castor, and (c) as an approach to induce bud necrosis virus resistancein sunflower has been discussed. Examples from other plant systems also have been mentioned to exemplify theconcept and utility of gene silencing in crop plants.

Virus-induced gene silencing (VIGS) in cape gooseberry (Physalis peruviana L., Solanaceae) / Silenciamento de genes induzidos por vírus (VIGS) em physalis (Physalis peruviana L., Solanaceae) / Silenciamiento génico inducido por virus (VIGS) en uchuva (Physalis peruviana L., Solanaceae)

Abstract Cape gooseberry (Physalis peruviana, L.) is a herbaceous plant belonging to the Solanaceae family that produces an edible berry appreciated for its nutraceutical and pharmaceutical properties. Its production is often limited by diseases and reproducible fruit quality. Recent studies have reported genes associated with fruit quality and resistance response to the root-infecting fungus Fusarium oxysporum f. sp. physali (Foph,) which causes vascular wilt. In order to standardize a method to validate the biological function of candidate genes in the non-model species P. peruviana, we tested the robust approach in reverse genetics, virus-induced gene silencing (VIGS). In this study, we validated and optimized VIGS using an insert of the phytoene desaturase (PDS) gene in a silencing viral vector generated from tobacco rattle virus (TRV). Leaves infiltrated with Agrobacterium (GV3101 strain) showed photo-bleached segments, which were distinctive for PDS suppression at 7 days post-infection (dpi). More than half of the treated plants showed photo-bleaching, indicating an efficiency rate of 50 % of the VIGS protocol. The results of this study showed that VIGS can be used for future functional gene characterization implicated in the immune response, disease resistance and fruit quality in capegooseberry.

Resumo A physalis (Physalis peruviana, L.) é uma planta herbácea pertencente à família Solanaceae, que produz uma baga comestível apreciada por suas propriedades nutracêuticas e farmacêuticas. Sua produção com frequência se vê limitada devido a enfermidades e baixa reprodutibilidade na qualidade do fruto. Estudos recentes reportaram genes associados com a qualidade do fruto e com a resposta de resistência ao fungo radicular Fusarium oxysporum f. sp. physali (Foph.), que causa esmorecimento vascular. Com a finalidade de padronizar um método para validar a função biológica de genes candidatos na espécie não-modelo P. p ruviana, avaliamos uma aproximação robusta em genética invertida, o sil nciamento de genes induzidos por vírus (VIGS). Neste estudo, validamos e otimizamos o VIGS usando um inserto da fitoeno desaturase (PDS) em um vetor viral de silenciamento produzido a partir do vírus do chocalho do tabaco (TRV). As folhas infiltradas com Agrobacterium (cepa GV3101) mostraram segmentos fotobranqueados, que foram distintivos para a supressão de PDS a 7 dias pós-infecção (dpi). Mais da metade das plantas tratadas mostraram fotobranqueamento, o que indica uma taxa de eficiência de 50 % do procotolo VIGS. Os resultados de este estudo mostraram que o VIGS pode ser usado em caracterizações futuras de genes funcionais implicados na resposta imune, na resistência a enfermidades e na qualidade do fruto de physalis.

Resumen La uchuva (Physalis peruviana, L.) es una planta herbácea perteneciente a la familia de las solanáceas, que produce una baya comestible apreciada por sus propiedades nutracéuticas y farmacéuticas. Su producción con frecuencia se ve limitada debido a enfermedades y a falta de reproducibilidad en la calidad del fruto. Estudios recientes han reportado genes asociados con la calidad del fruto y con la respuesta de resistencia al hongo radicular Fusarium oxysporum f. sp. physali (Foph,), que causa marchitamiento vascular. Con el fin de estandarizar un método para validar la función biológica de genes candidatos en la especie no-modelo P. peruviana, evaluamos la aproximación robusta en genética inversa, el silenciamiento génico inducido por virus (VIGS). En este estudio, validamos y optimizamos el VIGS usando un inserto de la fitoeno desaturasa (PDS) en un vector viral de silenciamiento producido a partir del virus del cascabeleo del tabaco (TRV). Las hojas infiltradas con Agrobacterium (cepa GV3101) mostraron segmentos fotoblanqueados, que fueron distintivos para la supresión de PDS a 7 días pos-infeccion (dpi). Más de la mitad de las plantas tratadas mostraron fotoblanqueo, lo cual indica una tasa de eficiencia del 50 % del protocolo VIGS. Los resultados de este estudio mostraron que el VIGS se puede usar en futuras caracterizaciones de genes funcionales implicados en la respuesta inmune, la resistencia a enfermedad y la calidad del fruto en la uchuva.

The potential of virus-induced gene silencing for speeding up functional characterization of plant genes

Virus-induced gene silencing (VIGS) has been shown to be of great potential in plant reverse genetics. Advantages of VIGS over other approaches, such as T-DNA or transposon tagging, include the circumvention of plant transformation, methodological simplicity and robustness, and speedy results. These features make VIGS an attractive alternative instrument in functional genomics, even in a high throughput fashion. The system is already well established in Nicotiana benthamiana; however, efforts are being made to improve VIGS in other species, including monocots. Current research is focussed on unravelling the mechanisms of post-transcriptional gene silencing and VIGS, as well as on finding novel viral vectors in order to broaden the host species spectrum. We examined how VIGS has been used to assess gene functions in plants, including molecular mechanisms involved in the process, available methodological elements, such as vectors and inoculation procedures, and we looked for examples in which the system has been applied successfully to characterize gene function in plants.

RNA interference c-erbB-2 and c-raf-1 genes expression combined transfection in the Tca8113 cell lines / 重庆医科大学学报

Objective:To investigate the proliferating inhibition and the mechanisms of dsRNA interference c-erbB-2 and c-raf-1 genes expression combined transfection in the human tongue carcinoma Tca8113 cell lines.Methods:There were 3 groups in our study,control group,normal control group and RNAi experimental group.At different time after liposome-mediated transfection,the cell proliferation,apoptosis,mRNA level,protein expressing level and cell cycle were observed by MTT,RT-PCR and electronic microscope.Results:According to the results of RNAi experimental group,the OD-value were 0.073( P

RNA interference inhibits the secretion of IL-1? in mice spleen lymphocytes / 中国病理生理杂志

AIM: To decide the effect that selected siRNA degrades mRNA of IL-1? specifically and suppression of its expression after connected with target site with homology complementary sequence. METHODS: Synthesized DNA expression box aimed directly at target site through PCR reaction in vivo was purified, and transfected into lymphocytes stimulated by LPS. siRNA was transcribed by cellular endogenous RNA polymerase Ⅲ and then evoke the degradation of target mRNA. After 48 hours of transfection, the cell culture supernatant was collected and the concentration of IL-1? was assayed using ELISA. RESULTS: Compared with blank-control and negative-control, selected sequence decreased the expression of IL-1?. Rate of the suppression was about 15%. CONCLUSION: RNAi technology produces specific interference effect in mouse spleen lymphocytes in original culture and inhibits the excretion of IL-1?.