Enhanced fungal resistance in transgenic cotton expressing an endochitinase gene from Trichoderma virens

Mycoparasitic fungi are proving to be rich sources of antifungal genes that can be utilized to genetically engineer important crops for resistance against fungal pathogens. We have transformed cotton and tobacco plants with a cDNA clone encoding a 42 kDa endochitinase from the mycoparasitic fungus, Trichoderma virens. Plants from 82 independently transformed callus lines of cotton were regenerated and analysed for transgene expression. Several primary transformants were identified with endochitinase activities that were significantly higher than the control values. Transgene integration and expression was confirmed by Southern and Northern blot analyses, respectively. The transgenic endochitinase activities were examined in the leaves of transgenic tobacco as well as in the leaves, roots, hypocotyls and seeds of transgenic cotton. Transgenic plants with elevated endochitinase activities also showed the expected 42 kDa endochitinase band in fluorescence, gel-based assays performed with the leaf extracts in both species. Homozygous T2 plants of the high endochitinase-expressing cotton lines were tested for disease resistance against a soil-borne pathogen, Rhizoctonia solani and a foliar pathogen, Alternaria alternata. Transgenic cotton plants showed significant resistance to both pathogens.     

Expression of the baculovirus p35 protein in tobacco affects cell death progression and compromises N gene-mediated disease resistance response to Tobacco mosaic virus

The p35 protein from baculovirus is a broad-range caspase inhibitor and suppresses programmed cell death in animals. We report here the effects of transgenic expression in tobacco of the p35 protein during the hypersensitive response (HR). Expression of p35 causes partial inhibition of nonhost HR triggered by bacteria and gene-for-gene HR triggered by virus. Infection of p35-expressing tobacco plants with Tobacco mosaic virus (TMV) disrupts N-mediated disease resistance, causing systemic spreading of the virus within a resistant background. Mutant variants altered in aspartate residues within the loop region of p35 are inefficient substrates for caspases in vitro, and they do not suppress caspase proteolytic activity in animal systems. Tobacco plants expressing these mutant variants of the p35 protein do not show inhibition of HR cell death or enhanced virus systemic movement. Thus, HR inhibition and TMV systemic spreading phenotype in p35-expressing plants correlate with the ability of the p35 protein to suppress caspase activity in animal systems. In addition, a C-terminal truncated variant of p35 is unable to suppress cell death in animals as well as HR cell death in transgenic tobacco. Our results provide evidence for the participation of caspase-like proteases during the HR. In addition, they suggest that timely activation of cell death is necessary for effective TMV containment within the primary infection site.     

Insulin-like growth factor-binding protein-2 levels in pediatric patients with growth hormone deficiency, eating disorders and acute lymphoblastic leukemia

Insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2) is altered in different diseases and might be used as an indication of its severity. The aims of our study were to investigate: (1) the developmental pattern of the serum IGFBP-2 concentration at birth and during childhood and adolescence; (2) whether the serum IGFBP-2 level could be a marker for the diagnosis and evolution of diseases where the growth hormone (GH)-IGF axis is altered, and (3) whether this binding protein shows a relationship with IGF-I, its free fraction, IGFBP-1 and -3. We report reference values for 55 normal full-term newborns and 221 normal children who were divided into 5 groups according to their Tanner stage. Serum levels were higher in newborns when compared with Tanner stages I-V (p < 0.001, ANOVA), with no further changes throughout development. Furthermore, we studied IGFBP-2 levels in 24 children with congenital GH deficiency (GHD), 26 with acute lymphoblastic leukemia (ALL), 75 obese children, and 60 girls with anorexia nervosa (AN) at diagnosis and during a follow-up period. IGFBP-2 at diagnosis was increased in GHD, ALL and AN, and decreased in obesity (p < 0.05, ANOVA). During the follow-up, IGFBP-2 concentrations tended to normalize. IGFBP-2 correlated positively with IGFBP-1 and negatively with IGF-I and IGFBP-3 in normal subjects and at diagnosis of the pathologies studied. Although IGFBP-2 functions are not well understood, these results suggest a possible role for this protein in diseases where the GH-IGF axis is altered.     

Chitosanase and chitinase activities in tomato roots during interactions with arbuscular mycorrhizal fungi or Phytophthora parasitica

New chitosanase acidic isoforms have been shown in Glomus mosseae-colonized tomato roots and their induction, together with the previously described mycorrhiza-related chitinase isoform, has been further corroborated in plants colonized with another Glomus species (G. intraradices),as well as in tomato roots colonized in vitro by Giaspora rosea. The induction of these chitosanase isoforms appears as a specific response to the arbuscular mycorrhizal (AM) symbiosis, and does not correspond to unspecific defence mechanisms, since these isoforms were not induced by the pathogen Phytophthora parasitica. Analysis by isoelectrofocusing showed two closely migrating chitinase isoforms, specific to mycorrhizal plants colonized either with G. mosseae or G. intraradices, and their isoelectric points were estimated to be 4.5 and 4.7. The estimated molecular mass of chitosanases was 20 kDa, and after isoelectrofocusing, the chitosanase activities were detected along the acidic pH range (6.5-3.5). Constitutive and induced isoforms were also investigated during a time-course study. In some experiments, chitin and chitosan were embedded together as substrates in polyacrylamide gels with the aim of studying the capacity of some isoforms to display both chitinase and chitosanase activities. In extracts from plants colonized with either G. mosseae or G. intraradices, some constitutive chitinases and the previously described mycorrhiza-related chitinase isoform, appeared to display chitosanase activity, while this bifunctional character was not found for the chitinases from non-mycorrhizal tissue, nor in Phytophthora-infected plants. These results suggest some diversity in the chitinase activities concerning substrate specificity in mycorrhizal plants. The possible implications of these observations in the functioning of the symbiosis is discussed.Key words: Arbuscular mycorrhizas, chitinases, chitosanases, Phytophthora parasitica, tomato, Lycoperiscon esculentum.      

Keeping cell identity in Arabidopsis requires PRC1 RING-finger homologs that catalyze H2A monoubiquitination

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Highlights? AtBMI1A/B proteins prevent misexpression of embryonic traits in somatic cells ? PRC2 and AtBMI1A/B proteins maintain cells in their differentiated state ? AtBMI1A/B proteins mediate H2A monoubiquitination     

Fungal ribotoxins: molecular dissection of a family of natural killers

RNase T1 is the best known representative of a large family of ribonucleolytic proteins secreted by fungi, mostly Aspergillus and Penicillium species. Ribotoxins stand out among them by their cytotoxic character. They exert their toxic action by first entering the cells and then cleaving a single phosphodiester bond located within a universally conserved sequence of the large rRNA gene, known as the sarcin-ricin loop. This cleavage leads to inhibition of protein biosynthesis, followed by cellular death by apoptosis. Although no protein receptor has been found for ribotoxins, they preferentially kill cells showing altered membrane permeability, such as those that are infected with virus or transformed. Many steps of the cytotoxic process have been elucidated at the molecular level by means of a variety of methodological approaches and the construction and purification of different mutant versions of these ribotoxins. Ribotoxins have been used for the construction of immunotoxins, because of their cytotoxicity. Besides this activity, Aspf1, a ribotoxin produced by Aspergillus fumigatus, has been shown to be one of the major allergens involved in allergic aspergillosis-related pathologies. Protein engineering and peptide synthesis have been used in order to understand the basis of these pathogenic mechanisms as well as to produce hypoallergenic proteins with potential diagnostic and immunotherapeutic applications.     

Tuning immune tolerance with vasoactive intestinal peptide: a new therapeutic approach for immune disorders

The induction of immune tolerance is essential for the maintenance of immune homeostasis and to limit the occurrence of exacerbated inflammatory and autoimmune conditions. Multiple mechanisms act together to ensure self-tolerance, including central clonal deletion, cytokine deviation and induction of regulatory T cells. Identifying the factors that regulate these processes is crucial for the development of new therapies of autoimmune diseases and transplantation. The vasoactive intestinal peptide (VIP) is a well-characterized endogenous anti-inflammatory neuropeptide with therapeutic potential for a variety of immune disorders. Here, we examine the latest research findings, which indicate that VIP participates in maintaining immune tolerance in two distinct ways: by regulating the balance between pro-inflammatory and anti-inflammatory factors, and by inducing the emergence of regulatory T cells with suppressive activity against autoreactive T-cell effectors.     

Eco-efficiency

Goal, Scope and Background The eco-efficiency analysis and portfolio is a powerful decision support tool for various strategic and marketing issues. Since its original academic development, the approach has been refined during the last decade and applied to a multitude of projects. BASF, as possibly the most prominent company using and developing this tool, has applied the eco-efficiency approach to more than 300 projects in the last 7 years. One of the greatest difficulties is to cover both dimensions of eco-efficiency (costs or value added and environmental impact) in a comparable manner. This is particularly a challenge for the eco-efficiency analyses of products. Methods In this publication, an important approach and field of application dealing with product decisions based on the combination of Life Cycle Cost (LCC) and Life Cycle Assessment (LCA) is described in detail. Special emphasis is put on the quantitative assessment of the relation of costs and environmental impacts. In conventional LCA an assessment of environmental impact categories is often made by normalization with inhabitant equivalents. This is necessary to be able to compare the different environmental impact categories, because of each different unit. For the proposed eco-efficiency analysis, the costs of products or processes are also normalized with adapted gross domestic product figures. Results and Discussion The ratio between normalized environmental impact categories and normalized costs (R_E,C) is used for the graphical presentation of the results in an eco-efficiency portfolio. For the interpretation of the results of an eco-efficiency analysis, it is important to distinguish ratios R_E,C which are higher than one from ratios lower than one. In the first case, the environmental impact is higher than the cost impact, while the inverse is true in the second case. This is very important for defining which kind of improvement is needed and defining strategic management decisions. The paper shows a statistical evaluation of the R_E,C factor based on the results of different eco-efficiency analyses made by BASF. For industries based on large material flows (e.g. chemicals, steel, metals, agriculture), the R_E,C factor is typically higher than one. Conclusions and Recommendations This contribution shows that LCC and LCA may be combined in a way that they mirror the concept of eco-efficiency. LCAs that do not consider LCC may be of very limited use for company management. For that very reason, corporations should install a data management system that ensures equal information on both sides of the eco-efficiency coin.