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Groundnut bud necrosis virus (GBNV), a member of the genus Orthotospovirus, is the most devastating pathogen causing bud blight of tomato and causes substantial crop losses in India. Current management strategies rely upon the use of virus tolerant cultivars, control of insect vectors, and other cultural practices. Under field conditions, these methods are ineffective in reducing the disease. Control can be achieved with RNA silencing, which regulates the homologous specific degradation of targeted genes, resulting in reduced virus multiplication. In the present study, virus infected tomato plant samples were collected from different parts of Tamil Nadu, and infection was confirmed through DAC-ELISA using a polyclonal antibody specific to GBNV. The virus inoculum was propagated on the local lesion host, cowpea (Vigna unguiculata), resulting in the production of chlorotic and necrotic spots on inoculated primary leaves. Our results demonstrated that the complete nucleotide sequence of the replicase gene identified using PCR shared an identity of 94.7 to 97.7% with other isolates of GBNV. To investigate the virus suppression mechanism, an effective RNAi construct was developed with the conserved sequence of the replicase gene for GBNV. A 3674 bp hpRNA cassette, comprising the sense and antisense fragments of 357 bp along with the flanking sequence, inserted in a pHANNIBAL vector, generated transgenic tomato plants using shoot apical meristem explants through Agrobacterium harboring the gene construct. The presence of the transgene in the developed putative transformants was assessed by PCR analysis using nptII and Rep genes and dot blot hybridization using a DIG luminescent detection kit. The expression of the replicase hpRNA construct revealed reduced symptom development upon artificial inoculation of GBNV. Further analysis of the transgenic tomato plants using DAC-ELISA confirmed the reduced level of virus titer. We propose that the RNAi construct, established with a conserved sequence of the replicase gene, showed a gene silencing mechanism as evidenced by reduced virus accumulation in putative transgenic lines, and this could be used as an effective strategy in the management of GNBV in tomato.

ToChLPV and PepGMV are Begomoviruses that have adapted to a wide host range and are able to cause major diseases in agronomic crops. We analyzed the efficacy of induced resistance to PepGMV in Nicotiana benthamiana plants with two constructs: one construct with homologous sequences derived from PepGMV, and the other construct with heterologous sequences derived from ToChLPV. Plants protected with the heterologous construct showed an efficacy to decrease the severity of symptoms of 45%, while plants protected with the homologous construct showed an efficacy of 80%. Plants protected with the heterologous construct showed a reduction of incidence of 42.86%, while the reduction of incidence in plants protected with the homologous construct was 57.15%. The efficacy to decrease viral load was 95.6% in plants protected with the heterologous construct, and 99.56% in plants protected with the homologous construct. We found, in both constructs, up-regulated key components of the RNAi pathway. This demonstrates that the efficacy of the constructs was due to the activation of the gene silencing mechanism, and is reflected in the decrease of viral genome copies, as well as in recovery phenotype. We present evidence that both constructs are functional and can efficiently induce transient resistance against PepGMV infections. This observation guarantees a further exploration as a strategy to control complex Begomovirus diseases in the field.

The development and testing in the field of genetically modified -so called- orphan crops like cassava in tropical countries is still in its infancy, despite the fact that cassava is not only used for food and feed but is also an important industrial crop. As traditional breeding of cassava is difficult (allodiploid, vegetatively propagated, outbreeding species) it is an ideal crop for improvement through genetic modification. We here report on the results of production and field testing of genetically modified low-amylose transformants of commercial cassava variety Adira4 in Indonesia. Twenty four transformants were produced and selected in the Netherlands based on phenotypic and molecular analyses. Nodal cuttings of these plants were sent to Indonesia where they were grown under biosafety conditions. After two screenhouse tests 15 transformants remained for a field trial. The tuberous root yield of 10 transformants was not significantly different from the control. Starch from transformants in which amylose was very low or absent showed all physical and rheological properties as expected from amylose-free cassava starch. The improved functionality of the starch was shown for an adipate acetate starch which was made into a tomato sauce. This is the first account of a field trial with transgenic cassava which shows that by using genetic modification it is possible to obtain low-amylose cassava plants with commercial potential with good root yield and starch quality.

Our goal was to target silencing of the Plum pox virus coat protein (PPV CP) gene independently expressed in plants. Clone C-2 is a transgenic plum expressing CP. We introduced and verified, in planta, the effects of the inverse repeat of CP sequence split by a hairpin (IRSH) that was characterized in the HoneySweet plum. The IRSH construct was driven by two CaMV35S promoter sequences flanking the CP sequence and had been introduced into C1738 plum. To determine if this structure was enough to induce silencing, cross-hybridization was made with the C1738 clone and the CP expressing but PPV-susceptible C2 clone. In total, 4 out of 63 clones were silenced. While introduction of the IRSH is reduced due to the heterozygous character in C1738 plum, the silencing induced by the IRSH PPV CP is robust. Extensive studies, in greenhouse containment, demonstrated that the genetic resource of C1738 clone can silence the CP production. In addition, these were verified through the virus transgene pyramiding in the BO70146 BlueByrd cv. plum that successfully produced resistant BlueByrd BO70146 × C1738 (HybC1738) hybrid plums.

Selective gene silencing by RNA interference (RNAi) involves double-stranded small interfering RNA (ds siRNA) composed of single-stranded (ss) guide and passenger RNAs. siRNA is recognized and processed by Ago2 and C3PO, endonucleases of the RNA-induced silencing complex (RISC). RISC cleaves passenger RNA, exposing the guide RNA for base-pairing with its homologous mRNA target. Remarkably, the 3′ end of passenger RNA can accommodate a DNA extension of 19-nucleotides without loss of RNAi function. This construct is termed passenger-3′-DNA/ds siRNA and includes a 3′-nuclease-resistant mini-hairpin structure. To test this novel modification further, we have now compared the following constructs: (I) guide-3′-DNA/ds siRNA, (II) passenger-3′-DNA/ds siRNA, (III) guide-3′-DNA/ss siRNA, and (IV) passenger-3′-DNA/ss siRNA. The RNAi target was SIRT1, a cancer-specific survival factor. Constructs I–III each induced selective knock-down of SIRT1 mRNA and protein in both noncancer and cancer cells, accompanied by apoptotic cell death in the cancer cells. Construct IV, which lacks the SIRT1 guide strand, had no effect. Importantly, the 3′-DNA mini-hairpin conferred nuclease resistance to constructs I and II. Resistance required the double-stranded RNA structure since single-stranded guide-3′-DNA/ss siRNA (construct III) was susceptible to serum nucleases with associated loss of RNAi activity. The potential applications of 3′-DNA/siRNA constructs are discussed.

White spot syndrome caused by white spot syndrome virus (WSSV) is one of the most threatening diseases of shrimp culture industry. Previous studies have successfully demonstrated the use of DNA- and RNA-based vaccines to protect WSSV infection in shrimp. In the present study, we have explored the protective efficacy of antisense constructs directed against WSSV proteins, VP24, and VP28, thymidylate synthase (TS), and ribonucleotide reductase-2 (RR2) under the control of endogenous shrimp histone-3 (H3) or penaedin (Pn) promoter. Several antisense constructs were generated by inserting VP24 (pH3–VP24, pPn–VP24), VP28 (pH3–VP28, pPn–VP28), TS (pH3–TS, pPn–TS), and RR2 (pH3–RR2) in antisense orientation. These constructs were tested for their protective potential in WSSV infected cell cultures, and their effect on reduction of the viral load was assessed. A robust reduction in WSSV copy number was observed upon transfection of antisense constructs in hemocyte cultures derived from Penaeus monodon and Scylla serrata . When tested in vivo, antisense constructs offered a strong protection in WSSV challenged P . monodon . Constructs expressing antisense VP24 and VP28 provided the best protection (up to 90 % survivability) with a corresponding decrease in the viral load. Our work demonstrates that shrimp treated with antisense constructs present an efficient control strategy for combating WSSV infection in shrimp aquaculture.

RNA interference-mediated silencing is an effective way of controlling white spot syndrome virus (WSSV). However, the effect of RNAi on the innate immune mechanism is not well understood. Prophenoloxidase (proPO) is an important component of the shrimp innate immunity. In the present study, nonspecific effect of two double-stranded (ds)RNA-expressing constructs, one targeting vp28 gene of WSSV (pCMV-VP28-LH) and another targeting green fluorescent protein (GFP) (pCMV-GFP-LH) on proPO2 gene expression, is investigated. mRNA expression levels of proPO2 in hemocytes of DNA construct-injected shrimp were estimated using real-time PCR with elongation factor 1-α as internal control. Empty vector (pcDNA)-injected shrimp were used as experimental control. In pCMV-VP28-LH-injected shrimp, proPO2 showed significant upregulation until 48 h post-injection (p.i.). Similarly, pCMV-GFP-LH-injected animals showed high levels of expression until 72 h p.i. WSSV-challenged animals, compared to pcDNA-injected control group, showed no significant change in expression of the gene until 24 h. However, an increased expression was noticed at 48 h p.i. Our results suggest that neither the plasmids nor the long hairpin RNA expressed by the constructs has any nonspecific silencing effect on the proPO2 expression. On the contrary, the consistent upregulation of proPO2 observed in shrimp injected with dsRNA at early time-points indicates the possibility of nonspecific protection against WSSV infection.

Fas/FasL is a member of Tumor Necrosis Factor (TNF) super family, and related to tumor cell apoptosis. It is hypothesis by forward study that activated lymphocytes is more sensitive with Fas/FasL due to up-regulation of FasL expression, so it can be inverse killedly and cleared by tumor cell. The aim of this investigate is to study the fuction of FasL gene and gene therapy of lung cancer by to down-regulationg the FasL gene expression with a siRNA expression plasmid in lung cancer cell A549 as well as its inverse killing effect between activated T lymphocytes and lung cancer cell A549. Potential RNAi oligonucleotides of FasL was designed and synthesized according to appropriate web site. Then a FasL siRNA plasmid was constructed using a pGCsi-U6 vector.The plasmid was sequenced to confirm the inserted sequence. Western blot analysis was used to assess the levels of FasL proteins after the constructed plasmids have been transfected into A549 cells. It was confirmed by sequencing that the plasmid was constructe

Background and objective Fas/FasL is a member of Tumor Necrosis Factor (TNF) super family and related to tumor cell apoptosis. It is hypothesis by forward study that activated lymphocytes is more sensitive with Fas/FasL due to up-regulation of FasL expression, so it can be inverse killedly and cleared by tumor cell. The aim of this investigate is to study the fuction of FasL gene and gene therapy of lung cancer by to down-regulationg the FasL gene expression with a siRNA expression plasmid in lung cancer cell A549 as well as its inverse killing effect between activated T lymphocytes and lung cancer cell A549. Methods Potential RNAi oligonucleotides of FasL was designed and synthesized according to appropriate web site. Then a FasL siRNA plasmid was constructed using a pGCsi-U6 vector.The plasmid was sequenced to confirm the inserted sequence. Western blot analysis was used to assess the levels of FasL proteins after the constructed plasmids have been transfected into A549 cells. Results It was confirmed by se