Composite Phaseolus vulgaris plants with transgenic roots as research tool

Large seeded grain legumes such as the common bean (Phaseolus vulgaris) and cowpea (Vigna unguiculata) are very important crops with seeds that are major protein source for people in developing countries, but their yields and improvement lag behind the economically more important cereals. For research purposes, genetic transformation is a powerful tool to obtain valuable information on gene expression and putative gene functions. In addition, through genetic transformation, candidate genes can be evaluated for their potential in agricultural biotechnology applications, such as resistance against biotic stresses. However, it remains difficult to stable genetically transform large seeded grain legumes such as Phaseolus and cowpea using Agrobacterium tumefaciens. In this paper a system is described to obtain so-called transgenic composite plants from P. vulgaris. These have a transgenic root system, obtained through Agrobacterium rhizogenes transformation of de-rooted seedlings. Their potentials for studies on important processes in the root system will be discussed

Identification of PP2A complexes and pathways involved in cell transformation

The simian virus 40 small t (SV40ST) oncoprotein interacts with protein phosphatase 2A (PP2A), an abundantly expressed family of serine-threonine phosphatases. This interaction is essential for the transformation of human cells by SV40, and several PP2A subunits have been implicated as tumor suppressor genes. However, the pathways controlled by specific PP2A complexes involved in cell transformation remain incompletely understood. Using a comprehensive loss-of-function approach, we identified 4 PP2A regulatory subunits [B56α, B56γ, PR72/PR130, and PTPA (protein phosphatase 2A activator)], which when suppressed replaced the expression of SV40ST in human cell transformation. We found that manipulation of complexes containing PP2A B56α, B56γ, and PR72/PR130 activates the pathways regulated by c-Myc, Wnt, and PI3K (phosphoinositide 3-kinase)/Akt in a manner that depends on their specific phosphatase activity. In contrast, suppression of PTPA disrupts the assembly of PP2A heterotrimeric complexes, which leads to the activation of these same oncogenic pathways. These observations delineate the PP2A family members and pathways perturbed by SV40ST during human cell transformation.status: publishe

Sustainable management of Neo -Tropical Tree Genetic Resources: Combining molecular and modelling methods to understand the structure and dynamics of gene diversity. Final Scientific Report. February 2002 - January 2006

Original Objectives: • Examine the structure and dynamics of genetic variation for a range of economically and ecologically important Central and South American tree species within natural ecosystems and identify the main factors that are responsible for partitioning of variation within species • Examine the impact of identified extraction methods/habitat degradation (i.e. fragmentation, logging, forest clearance and domestication) on selected economically important species • Produce a model tailored to the individual study species that will integrate field observations and DNA-based technologies to provide realistic simulations of the impact of differing land-use strategies and extraction regimes on the genetic resource base of impacted species • Improve capacity to execute sound natural forest management by improving awareness of genetic implications of natural forest management and implementation of a modelling approach to setting sustainability strategies Results and Milestones: Development of molecular techniques Several new microsatellite primers were developed in a range of species; DNA extraction methods for target species were optimised; AFLP protocols were optimised and applied across the range of target species. Practical measures to maximise comparability of AFLP datasets between labs were designed, and standardised scoring and analysis techniques developed for the analysis stages. Several aspects of the methodological development were published in peer-reviewed journals. Identifying biological determinants of genetic diversity For a list of 50 target species collections and information on biological characteristics were prepared, following protocols from WP1. All target species were analysed using AFLPs and a meta-analysis conducted. Within the timescale of the project only preliminary analysis was possible but this indicated significant effects of pollination and seed dispersal mechanism on population differentiation and within-population genetic diversity respectively. Geographic distribution was not found to have any effect. Effect of human-mediated processes on genetic diversity Substantial case studies were carried out examining the effects of human-mediated processes on genetic diversity in Pinus oocarpa, Araucaria angustifolia, Swietenia macrophylla (Brazil and Belize), Vochysia ferruginea, Symphonia globulifera and Theobroma grandiflorum. Important outcomes of the case studies in general showed the impact of population modification on tree mating systems (and hence genetic diversity maintenance), the scale of real gene movement within tree populations, the significance of population densities for gene flow patterns and highlighted potential forest management strategies to mitigate the genetic diversity impacts of harvesting. Simulation modelling of population genetic dynamics The model, ECO-GENE, was adapted for use with the genetic datasets derived from case studies. In particular, a new module was designed for the model allowing sensitivity analysis to be performed. Simulations using empirical data from studies of Symphonia globulifera and Swietenia macrophylla were conducted. These studies highlighted the necessity for preparation of site-specific plans, and the importance of population density and the interaction between demography and growth and gene dispersal capability for capture and maintenance of genetic diversity in populations. Designing Management strategies to maximize diversity Both scientific and non-technical communications were achieved during the project lifetime. Amongst other individual peer-reviewed papers, special issues of the journals Heredity (Nature Publishing Group) and Silvae Genetica were produced. Further paper writing and publication is ongoing. Additional dissemination of project outputs was achieved through preparation of a board game for schools and colleges and a high-level public workshop for the biodiversity conservation sector (in San Jose, Costa Rica), including attendees from the government’s Biodiversity Conservation Commission as well as a range of other public and private bodies with interests in the field

Transcriptome analysis by cDNA-AFLP on Suillus luteus Cd-tolerant and Cd-sensitive isolates

The ectomycorrhizal basidiomycete Suillus luteus (L.Fr.), a typical pioneer species which associates with young pine trees colonizing disturbed sites, is a common root symbiont found at heavy metal contaminated sites. Three Cd-sensitive and three Cd-tolerant isolates of S. luteus, isolated respectively from non-polluted and a heavy metal-polluted site in Limburg (Belgium), were used for a transcriptomic analysis. We identified differentially expressed genes by cDNA-AFLP analysis. The possible roles of some of the encoded proteins in heavy metal (Cd) accumulation and tolerance are discussed. Despite the high conservation of coding sequences in S. luteus, a large intraspecific variation in the transcript profiles was observed. This variation was as large in Cd-tolerant as in sensitive isolates and may help this pioneer species to adapt to novel environments.info:eu-repo/semantics/publishe

Cd-tolerant Suillus luteus: a fungal insurance for pines exposed to Cd.

Soil metal pollution can trigger evolutionary adaptation in soil-borne organisms. An in vitro screening test showed cadmium adaptation in populations of Suillus luteus (L. Fr.) Roussel, an ectomycorrhizal fungus of pine trees. Cadmium stress was subsequently investigated in Scots pine (Pinus sylvestris L.) seedlings inoculated with a Cd-tolerant S. luteus, isolated from a heavy metal contaminated site, and compared to plants inoculated with a Cd-sensitive isolate from a non-polluted area. A dose-response experiment with mycorrhizal pines showed better plant protection by a Cd-adapted fungus: more fungal biomass and a higher nutrient uptake at high Cd exposure. In addition, less Cd was transferred to aboveground plant parts. Because of the key role of the ectomycorrhizal symbiosis for tree fitness, the evolution of Cd tolerance in an ectomycorrhizal partner such as S. luteus can be of major importance for the establishment of pine forests on Cd-contaminated soils.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Gene expression profiling of a Zn-tolerant and a Zn-sensitive Suillus luteus isolate exposed to increased external zinc concentrations

Complementary DNA (cDNA)-amplified fragment-length polymorphism (AFLP) was applied to analyze transcript profiles of a Zn-tolerant and a Zn-sensitive isolate of the ectomycorrhizal basidiomycete Suillus luteus, both cultured with and without increased external zinc concentrations. From the obtained transcript profiles that covered approximately 2% of the total expected complement of genes in S. luteus, 144 nonredundant, differentially expressed transcript-derived fragments (TDFs), falling in different classes of expression pattern, were isolated and sequenced. Thirty-six of the represented genes showed homology to function-known genes, whereas 6 matched unknown protein coding sequences, and 102 were possibly novel. Although relatively few TDFs were found to be responsive to the different zinc treatments, their modulated expression levels may suggest a different transcriptional response to zinc treatments in both isolates. Among the identified genes that could be related to heavy-metal detoxification or the tolerance trait were genes encoding for homologues of a heat-shock protein, a putative metal transporter, a hydrophobin, and several proteins involved in ubiquitin-dependent proteolysis. © 2007 Springer-Verlag.SCOPUS: ar.jinfo:eu-repo/semantics/publishe