Genetic transformation of garlic (Allium sativum L.) with tobacco chitinase and glucanase genes for tolerance to the fungus Sclerotium cepivorum

Garlic yield and quality have decreased due to white rot disease caused by Sclerotium cepivorum Berk. A transformation protocol to introduce tobacco chitinase and glucanase genes into garlic embryogenic calli using Agrobacterium tumefaciens has been established. LBA4404 strain having pC2301CHGLU plasmid with TaCh, glu, gus and nptII genes (coding for chitinase, glucanase, β-glucuronidase and neomycin phosphotransferase, respectively) was used. 30 putative transgenic clones were obtained from inoculated calli after six months. Histochemical assay revealed high gus activity in 43% of the clones. Molecular analysis of transgenic plants showed 92% of the clones carried TaCh gene. Eight culture media for plant regeneration from transgenic calli were evaluated; MTDZ-1 (thidiazuron 1 mg/l) medium induced the highest number of plants (38.4 plants). Transgenic plants were grown in the greenhouse and they developed normally. S. cepivorum in vitro bioassays showed 41 to 60% of mycelial invasion in the transgenic plants, and 80% in non-transgenic plants (control). Transformed plants were not completely resistant, but they showed a delay in fungal infection. This is the first report on the introduction of fungal resistance genes in garlic.Keywords: Allium sativum, Agrobacterium tumefaciens, Sclerotium cepivorum, glucanase, chitinaseAfrican Journal of Biotechnology Vol. 12(22), pp. 3482-349

Insulin-Regulated Trafficking of GLUT4 Requires Ubiquitination

A major consequence of insulin binding its receptor on fat and muscle cells is translocation of the facilitative glucose transporter GLUT4 from an intracellular store to the cell surface where it serves to clear glucose from the bloodstream. Sorting of GLUT4 into its insulin-sensitive store requires the GGA [Golgi-localized, γ-ear-containing, ADP ribosylation factor (ARF)-binding] adaptor proteins, but the signal on GLUT4 to direct this sorting step is unknown. Here, we have identified a role for ubiquitination of GLUT4 in this process. We demonstrate that GLUT4 is ubiquitinated in 3T3-L1 adipocytes, and that a ubiquitin-resistant version fails to translocate to the cell surface of these cells in response to insulin. Our data support a model in which ubiquitination acts as a signal for the trafficking of GLUT4 from the endosomal/trans-Golgi network (TGN) system into its intracellular storage compartment, from where it is mobilized to the cell surface in response to insulin

In vivo functional analysis of the Dicistroviridae intergenic region internal ribosome entry sites

Some viral and cellular messages use an alternative mechanism to initiate protein synthesis that involves internal recruitment of the ribosome to an internal ribosome entry site (IRES). The Dicistroviridae intergenic regions (IGR) have been studied as model IRESs to understand the mechanism of IRES-mediated translation. In this study, the in vivo activity of IGR IRESs were compared. Our analysis demonstrates that Class I and II IGR IRESs have comparable translation efficiency in yeast and that Class II is significantly more active in mammalian cells. Furthermore, while Class II IGR IRES activity was enhanced in yeast grown at a higher temperature, temperature did not affect IGR IRES activity in mammalian cells. This suggests that Class II IRESs may not function optimally with yeast ribosomes. Examination of chimeric IGR IRESs, established that the IRES strength and temperature sensitivity are mediated by the ribosome binding domain. In addition, the sequence of the first translated codon is also an important determinant of IRES activity. Our findings provide us with a comprehensive overview of IGR IRES activities and allow us to begin to understand the differences between Classes I and II IGR IRESs

Conversion of deoxynivalenol to 3-acetyldeoxynivalenol in barley-derived fuel ethanol co-products with yeast expressing trichothecene 3-O-acetyltransferases

<p>Abstract</p> <p>Background</p> <p>The trichothecene mycotoxin deoxynivalenol (DON) may be concentrated in distillers dried grains with solubles (DDGS; a co-product of fuel ethanol fermentation) when grain containing DON is used to produce fuel ethanol. Even low levels of DON (≤ 5 ppm) in DDGS sold as feed pose a significant threat to the health of monogastric animals. New and improved strategies to reduce DON in DDGS need to be developed and implemented to address this problem. Enzymes known as trichothecene 3-<it>O-</it>acetyltransferases convert DON to 3-acetyldeoxynivalenol (3ADON), and may reduce its toxicity in plants and animals.</p> <p>Results</p> <p>Two <it>Fusarium </it>trichothecene 3-<it>O-</it>acetyltransferases (FgTRI101 and FfTRI201) were cloned and expressed in yeast (<it>Saccharomyces cerevisiae</it>) during a series of small-scale ethanol fermentations using barley (<it>Hordeum vulgare</it>). DON was concentrated 1.6 to 8.2 times in DDGS compared with the starting ground grain. During the fermentation process, FgTRI101 converted 9.2% to 55.3% of the DON to 3ADON, resulting in DDGS with reductions in DON and increases in 3ADON in the Virginia winter barley cultivars Eve, Thoroughbred and Price, and the experimental line VA06H-25. Analysis of barley mashes prepared from the barley line VA04B-125 showed that yeast expressing FfTRI201 were more effective at acetylating DON than those expressing FgTRI101; DON conversion for FfTRI201 ranged from 26.1% to 28.3%, whereas DON conversion for FgTRI101 ranged from 18.3% to 21.8% in VA04B-125 mashes. Ethanol yields were highest with the industrial yeast strain Ethanol Red^®, which also consumed galactose when present in the mash.</p> <p>Conclusions</p> <p>This study demonstrates the potential of using yeast expressing a trichothecene 3-<it>O</it>-acetyltransferase to modify DON during commercial fuel ethanol fermentation.</p

The Arabidopsis Trehalose-6-P Synthase AtTPS1 Gene Is a Regulator of Glucose, Abscisic Acid, and Stress Signaling

In Arabidopsis (Arabidopsis thaliana), trehalose is present at almost undetectable levels, excluding its role as an osmoprotectant. Here, we report that overexpression of AtTPS1 in Arabidopsis using the 35S promoter led to a small increase in trehalose and trehalose-6-P levels. In spite of this, transgenic plants displayed a dehydration tolerance phenotype without any visible morphological alterations, except for delayed flowering. Moreover, seedlings overexpressing AtTPS1 exhibited glucose (Glc)- and abscisic acid (ABA)-insensitive phenotypes. Transgenic seedlings germinated on Glc were visibly larger with green well-expanded cotyledonary leaves and fully developed roots, in contrast with wild-type seedlings showing growth retardation and absence of photosynthetic tissue. An ABA dose-response experiment revealed a higher germination rate for transgenic plants overexpressing AtTPS1 showing insensitive germination kinetics at 2.5 μm ABA. Interestingly, germination in the presence of Glc did not trigger an increase in ABA content in plants overexpressing AtTPS1. Expression analysis by quantitative reverse transcription-PCR in transgenic plants showed up-regulation of the ABI4 and CAB1 genes. In the presence of Glc, CAB1 expression remained high, whereas ABI4, HXK1, and ApL3 levels were down-regulated in the AtTPS1-overexpressing lines. Analysis of AtTPS1 expression in HXK1-antisense or HXK1-sense transgenic lines suggests the possible involvement of AtTPS1 in the hexokinase-dependent Glc-signaling pathway. These data strongly suggest that AtTPS1 has a pivotal role in the regulation of Glc and ABA signaling during vegetative development

Truncation of Arabidopsis thaliana and Selaginella lepidophylla trehalose-6-phosphate synthase unlocks high catalytic activity and supports high trehalose levels on expression in yeast.

Plants, such as Arabidopsis thaliana and Selaginella lepidophylla, contain genes homologous with the trehalose-6-phosphate synthase (TPS) genes of bacteria and fungi. Most plants do not accumulate trehalose with the desert resurrection plant S. lepidophylla, being a notable exception. Overexpression of the plant genes in a Saccharomyces cerevisiae tps1 mutant results in very low TPS-catalytic activity and trehalose accumulation. We show that truncation of the plant-specific N-terminal extension in the A. thaliana AtTPS1 and S. lepidophylla SlTPS1 homologues results in 10-40-fold higher TPS activity and 20-40-fold higher trehalose accumulation on expression in yeast. These results show that the plant TPS enzymes possess a high-potential catalytic activity. The growth defect of the tps1 strain on glucose was restored, however, the proper homoeostasis of glycolytic flux was not restored, indicating that the plant enzymes were unable to substitute for the yeast enzyme in the regulation of hexokinase activity. Further analysis of the N-terminus led to the identification of two conserved residues, which after mutagenesis result in strongly enhanced trehalose accumulation upon expression in yeast. The plant-specific N-terminal region may act as an inhibitory domain allowing modulation of TPS activity

Identificación de líneas de tomates silvestres tolerantes a salinidad en germinación y plántula

Losrecursos genéticos de los tomates silvestres (Solanum spp.) han demostrado ser valiosos reservorios de genes de tolerancia al estrés biótico y abiótico. Dado el aumento de áreas agrícolas con incidencia de salinidad, es necesario identificar genes de tolerancia para ser transferidos a variedades comerciales. El objetivo de esta investigación fue identificar líneas con tolerancia a la salinidad en poblaciones silvestres nativas. Se evaluaron 96 líneas bajo dos concentraciones de NaCl (0 y 150 mM) durante las etapas de germinación y de plántula. Durante la germinación se evaluó el porcentaje de germinación y de plántulas normales, así como el índice de velocidad de germinación. Un total de 11 líneas mostraron tolerancia al estrés salino. En la etapa de plántula se evaluaron 41 líneas seleccionadas por su respuesta durante la germinación y se cuantificó el peso fresco y seco de la raíz y parte aérea, contenido relativo de agua, altura de la planta y longitud de la raíz. En esta etapa se identificaron seislíneas que mostraron mayor tolerancia a la salinidad. Las líneas identificadas con tolerancia a la salinidad se pueden utilizar como recursos en programas de mejoramiento genético