Insight from γC1 protein model for implication in cotton leaf curl disease

DNA γ is approximately half of the size of Begomovirus DNA. It encodes a γC1 gene that is conserved in position and size. Thisgene has the capacity to encode a 13 to 14 kDa protein comprising 118 amino acid residues. It has been shown earlier that γC1protein is necessary for inducing symptoms of cotton leaf curl disease. The structure for γC1 (CLCuDγ01-Pakistan) is stillunknown. Therefore, a model of γC1 (CLCuDγ01-Pakistan) was developed using DoBo and I-TASSER servers followed byvalidation by PROCHECK and VERIFY 3D servers. The developed model provides an insight in a role for this multifunctionalprotein in causing Cotton Leaf Curl Disease (CLCuD). A possible function of this protein might be the suppression of RNAsilencingin cotton plants.     

Tête à tête of tomato yellow leaf curl virus and tomato yellow leaf curl sardinia virus in single nuclei

Since 1997 two distinct geminivirus species, Tomato yellow leaf curl Sardinia virus (TYLCSV) and Tomato yellow leaf curl virus (TYLCV), have caused a similar yellow leaf curl disease in tomato, coexisted in the fields of southern Spain, and very frequently doubly infected single plants. Tomatoes as well as experimental test plants (e.g., Nicotiana benthamiana) showed enhanced symptoms upon mixed infections under greenhouse conditions. Viral DNA accumulated to a similar extent in singly and doubly infected plants. In situ tissue hybridization showed TYLCSV and TYLCV DNAs to be confined to the phloem in both hosts, irrespective of whether they were inoculated individually or in combination. The number of infected nuclei in singly or doubly infected plants was determined by in situ hybridization of purified nuclei. The percentage of nuclei containing viral DNA (i.e., 1.4% in tomato or 6% in N. benthamiana) was the same in plants infected with either TYLCSV, TYLCV, or both. In situ hybridization of doubly infected plants, with probes that discriminate between both DNAs, revealed that at least one-fifth of infected nuclei harbored DNAs from both virus species. Such a high number of coinfected nuclei may explain why recombination between different geminivirus DNAs occurs frequently. The impact of these findings for epidemiology and for resistance breeding concerning tomato yellow leaf curl diseases is discussed.     

Isolation of the promoter of a cotton β-galactosidase gene (GhGal1) and its expression in transgenic tobacco plants

β-galactosidases (GUS, EC 3.2.1.23) are character- ized by their ability to hydrolyze terminal, non-re- ducing β-D-galactosyl residues from β-D-galactosides and are widely distributed in microbes, plants and animals. To date, the primary structures of …     

The emergence of okra enation leaf curl virus—an important begomovirus,infecting okra in several states across India

Okra enation leaf curl virus (OELCuV), a begomovirus, is an emerging serious constraint for okra production in India. OELCuV was earlier reported in Haryana, Gujarat and Karnataka. In the current study, a survey was conducted on okra crop in the other predominant okra growing regions of India and identified for the first time, a widespread symptomology of OELCuV. The disease incidence was recorded between 5 and 74% in all the surveyed regions. The diseased samples collected from all the locations were amplified with coat protein specific new primer in PCR. The amplicons were sequenced and deposited to NCBI Gene Bank. The finding could be highly useful in okra breeding programs against OELCuV.     

Down regulation of gyrase A gene expression in E. coli by antisense ribozymes using RT-PCR

Nucleic acid-based gene interference technologies represent promising strategies for specific inhibition of mRNA sequences of choice. Recently, small interfering RNAs have been implicated in inducing endogenous RNase of the RNA-induced silencing complex in the RNA interference pathway to inhibit gene expression and growth of several human viruses. We report down regulation of gene expression of E. coli gyrase A, an essential gene for DNA supercoiling and antibiotic susceptibility in BL21 (DE3) strain of E. coli, using Ribonuclease P based external guide sequence (EGS) technique. EGS directed against gyrase A gene that was cloned into pUC vector, which contains the ampicillin (Amp) resistance gene. The recombinant plasmid pT7EGyrA was transformed into BL21 (DE3) and inductions were performed using IPTG. RT-PCR experiment was done to investigate the down regulation of gyrase A gene. RT-PCR results demonstrated a significant decrease of gyrase A gene after 18 h of induction of the transformants. These experiments showed that the down regulation of the gene was seen after 18 h of induction than earlier hours of induction with IPTG suggesting inhibition of gyrase A gene with profound effect on cell viability. These results demonstrate the utility of EGS RNAs in gene therapy applications, by inhibiting the expression of essential proteins.     

Age-dependent PPARα activation induces hepatic sulfatide accumulation in transgenic mice carrying the hepatitis C virus core gene

Sulfatides, a type of glycosphingolipid, are associated with carcinogenesis. Peroxisome proliferator-activated receptor α (PPARα) is involved in the regulation of sulfatide metabolism as well as in cancer development. We previously reported that transgenic (Tg) mice expressing hepatitis C virus core protein (HCVcp) exhibited age-dependent PPARα activation and carcinogenesis in liver. However, the metabolism of sulfatides in hepatocellular carcinoma is unknown. To examine the relationship between sulfatide metabolism, carcinogenesis, HCVcp, and PPARα, age-dependent changes of these factors were examined in HCVcpTg, PPARα inhibitor-treated HCVcpTg, and non-Tg mice. The sulfatide content in liver, the hepatic expression of two key enzymes catalyzing the initial and last reactions in sulfatide synthesis, the hepatic expression of known sulfatide-transferring protein, oxidative stress, and hepatic PPARα expression and its activation were age-dependently increased in HCVcpTg mice. The increased synthesis and accumulation of sulfatides and PPARα activation were significantly enhanced in liver cancer lesions. These changes were attenuated by PPARα inhibitor treatment and not observed in non-Tg mice. These results suggest that HCVcp-induced age-dependent PPARα activation increases synthesis of sulfatides and the resulting sulfatide accumulation affects HCV-related liver cancer. The monitoring of hepatic sulfatide content and the modulation of sulfatide generation by intervention using a PPARα inhibitor might be useful for the prediction and prevention of HCV-related hepatocarcinogenesis, respectively.     

Specific expression of a β-tubulin gene (GhTub1) in developing cotton fibers Specific expression of a b-tubulin gene (GhTub1) in developing cotton fibers

Eukaryotic microtubular cytoskeleton plays crucial roles in various cellular processes, in-cluding cell division, polar cell expansion and cell differentiation[1, 2]. The basic filament-forming unit of microtubules is a heterodimer protein composed of a-tubulin and b-tubulin polypeptides. In the higher plants studied, both a- and b-tubulin genes form multigene families. While some of these tubulin genes are expressed constitutively, many others exhibit tissue-, organ- or cell type-specific exp…     

Effects of the virus satellite gene βC1 on host plant defense signaling and volatile emission

Tomato Yellow Leaf Curl China virus spreads together with its invasive vector, the silverleaf whitefly B biotype, which exhibits higher growth rates on infected plants. Previous studies indicate that the virus satellite gene βC1 accounts for the visible symptoms of infection and inhibits the constitutive expression of jasmonic acid (JA)—a phytohormone involved in plant defense against whiteflies—and of some JA-regulated genes. Here we present new details of the effects of on plant signaling and defense, obtained with (non-host) transgenic Arabidopsis thaliana and Nicotiana benthamiana plants. We found that JA induction in response to wounding was reduced in plants expressing βC1. This result implies that βC1 acts on conserved plant regulation mechanisms and might impair the entire JA defense pathway. Furthermore, transformed N. benthamiana plants exhibited elevated emissions of the volatile compound linalool, suggesting that βC1 also influences plant-derived olfactory cues available to vector and non-vector insects.     

Characterization of transgenic rice plants over-expressing the stress-inducible β-glucanase gene Gns1

The Gns1 gene of rice (Oryza sativa L. japonica) encodes 1,3;1,4- glucanase (EC 3.2.1.73), which hydrolyzes 1,3;1,4--glucosidic linkages on 1,3;1,4--glucan, an important component of cell walls in the Poaceae family. RNA and protein gel blot analyses demonstrated that blast disease or dark treatment induced the expression of the Gns1 gene. To assess the function of the Gns1 gene in disease resistance, we characterized transgenic rice plants constitutively expressing the Gns1 gene. The introduced Gns1 gene was driven by the CaMV 35S promoter and its products were found in the apoplast and accumulated in up to 0.1% of total soluble protein in leaves. Although transgenic plants showed stunted growth and impaired root formation, fertility, germination, and coleoptile elongation appeared unaffected compared to non-transgenic control plants, indicating that Gns1 does not play a crucial role in rice germination and coleoptile elongation. When transgenic plants were inoculated with virulent blast fungus (Magnaporthe grisea), they developed many resistant-type lesions on the inoculated leaf accompanying earlier activation of defense-related genes PR-1 and PBZ1 than in control plants. Transgenic plants spontaneously produced brown specks, similar in appearance to those reported for an initiation type of disease-lesion-mimic mutants, on the third and fourth leaves and occasionally on older leaves without inoculation of pathogens. Expression of the two defense-related genes was drastically increased after the emergence of the lesion-mimic phenotype.     

Abomasal infusion of arginine stimulates SCD and C/EBPß gene expression,and decreases CPT1ß gene expression in bovine adipose tissue independent of conjugated linoleic acid

Based on previous research with bovine peadipocytes, we hypothesized that infusion of arginine into the abomasum of Angus steers stimulates stearoyl-CoA desaturase (SCD) gene expression in bovine subcutaneous (s.c.) adipose tissue, and that this would be attenuated by conjugated linoleic acid (CLA). Growing Angus steers were infused abomasally with l-arginine 50 g/day; n = 13; provided as l-arginine HCl) or l-alanine (isonitrogenous control, 100 g/day; n = 11) for 14 days. For the subsequent 14 days, half of the steers in each amino acid group were infused with CLA (100 g/day). Body weight gain and average daily gain were unaffected (P > 0.15) by infusion of arginine or CLA into the abomasum. The plasma concentrations of cis-9, trans-11 and trans-10, cis-12 CLA were increased CLA infusion (P = 0.001) and infusion of arginine increased plasma arginine (P = 0.01). Compared with day 0, fatty acid synthase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase enzyme activities in s.c. adipose tissue increased by day 14 in steers infused with either alanine or arginine (all P < 0.01). NADP-MDH activity was higher (P = 0.01) in steers infused with arginine than in steers infused with arginine plus CLA by day 28, but lipid synthesis in vitro from glucose and acetate was unaffected by infusion of either arginine or CLA (P > 0.40). By day 28, C/EBPβ and SCD gene expression was higher, and CPT1β gene expression was lower, in s.c. adipose tissue of steers infused with arginine than in steers infused with alanine (±CLA) (P = 0.05). CLA decreased adipose tissue oleic acid (18:1n-9) in alanine- or arginine-infused steers (P = 0.05), although CLA had no effect on SCD gene expression. The data indicate that supplemental arginine promotes adipogenic gene expression and may promote lipid accumulation in bovine adipose tissue. l-Arginine may beneficially improve beef quality for human consumption.     

Development and evaluation of a new detection tool—visual DNA microarray for simultaneous and specific detection of human immunodeficiency virus type-1 and hepatitis C virus

Human immunodeficiency virus type-1 (HIV-1) and hepatitis C virus (HCV) are transfusion-transmitted human pathogens that have a major impact on blood safety and public health. Based on multiplex asymmetrical PCR and coupled with gold labelled silver stain (GLSS), we developed the visual DNA microarray for sensitive and specific detection of these two viruses. Capturing probes of 5′-end-amino-modified oligonucleotides were immobilized on glass surface to bind the complement biotinylated target DNA. The Au–streptavidin probe was introduced to the microarray for specific binding to biotin. Black images of microarray spots which result from the precipitation of silver onto Au–streptavidin probes, were visualized by naked eyes. In order to improve the efficiency of microarray hybridization, triplex asymmetrical PCR of HIV-1, HCV and Human enterovirus 71 (EV-71, used as positive control) were performed to prepare abundant biotinylated single-stranded target DNA. The sensitivity of visual DNA microarray (10³ copies/ml) was higher than conventional PCR (10⁴ copies/ml) and was identical to FQ-PCR (10³ copies/ml). Total 152 blood samples containing the two viruses were tested using the DNA microarray and fluorescence quantitative real-time PCR (FQ-PCR). The results were identical (P > 0.05). So this system has high sensitivity and may have potential in clinical applications.     

Effect of antisense L-Δ1-pyrroline-5-carboxylate reductase transgenic soybean plants subjected to osmotic and drought stress

The potential value of proline accumulation during environmental stressreveals a collection of controversial statements. Some argue that prolineaccumulation is beneficial to the plant, while others suggest the oppositeto be true. It is thus still unknown whether or not a constitutive higherlevel of proline accumulation enhances plant tolerance to environmentalstress. Since proline in plants is synthesised from both glutamic acid andornithine, we generated antisense soybean plants with an L-¹-pyrroline-5-carboxylate reductase (P5CR)gene, as it controls thecommon step of both pathways. The gene expression and consequentlyproline production was manipulated, with the use of an inducible heat shockpromoter (IHSP). The activation of the IHSP resulted in the inactivation ofthe P5CR gene, which resulted in decreased proline synthesis. Theantisense plants have provided us with insight into the correlation betweenproline accumulation, drought and osmotic stress. A mannitol stress at 32and 42 °C enhanced the accumulation of proline in control plants, incontrast to a significant decrease observed in the transformants. Theproline accumulation documented in this paper provides additional evidencethat the increase in proline levels during osmotic stress constitute anadaptive response by the plant. It was confirmed that there is anassociation between P5CR translation and proline accumulation, as theproline accumulation was markedly decreased by the activation of the heatinducible promoter and thus the antisense construct in transformed plants.A woodenbox screening indicated that proline plays a definite role insurvival of soybean plants under a drought stress, the transformantsfailed to survive a 6 day drought stress at 37 °C. This was in contrastwith the control plants which experienced the treatment only as a mildstress.     

Effective control of yellow stem borer and rice leaf folder in transgenic rice indica varieties Basmati 370 and M 7 using the novel δ-endotoxin cry2A Bacillus thuringiensis gene

Transgenic rice indica varieties Basmati 370 and M 7 expressing the novel cry2A (Bt) insecticidal gene were generated by particle bombardment. Molecular and biochemical analyses in R0 and R1 populations confirmed stable integration and expression of this novel Bt transgene. We estimated that the gene product was expressed up to 5% of total leaf protein. Insect feeding bioassays demonstrated that the Cry2A protein was effective against the yellow stem borer and the rice leaf folder, two major rice pests in the Indian Subcontinent. This is the first report of the control of major rice pests using this specific Bt gene. The cry2A gene can now be used in combination with other insecticidal genes for pyramiding resistance against insect pests. This will delay, or perhaps in combination with integrated pest management practices, prevent evolution of insect populations resistant to single insecticidal genes.