Construction of plant transformation vectors carrying beet necrotic yellow vein virus coat protein gene (ii)- plant transformation

Fragments containing the coat protein gene of beet necrotic yellow vein virus were cloned in two plant transformation vectors: pCAMBIA3301M with the bar gene as selectable marker, and pCAMBIA1304M, with resistance to hygromycin. Three constructs were made of each vector: CPL, containing coat protein gene with leader sequence; CPS with coat protein gene, and CPSas with coat protein gene in antisense orientation. Vectors pC3301MCPL, pC3301MCPS. and pC3301MCPSas were used in Agrobacterium—mediated transformation of Nicotiana tabacum (tobacco), Nicotiana excelsior and Nicotiana benthamiana. Regenerants that developed roots on selective media were tested for the presence of CP fragments and the bar gene, but most regenerants were nontransformed (50-83% escapes). After all rooted plants had been selfed, and T1 seed germinated on selective media, only plants descending from one N. excelsior regenerant transformed with pC3301MCPS were positive for presence of bar gene and CPS fragment. Tobacco and Nicotiana benthamiana were transformed with constructs pC1304MCPS and pC1304MCPSas. Transformation efficiency was much higher and approximately 50% of regenerants that rooted on media with 20 mg l−1 hygromycin were positive for the presence of CP fragments. All T1 plants were positive for presence of CP fragments

PROTEOMICS

The denitrifying Aromatoleum aromaticum strain EbN1 utilizes a wide range of aromatic and nonaromatic compounds under anoxic and oxic conditions. The recently determined genome revealed corresponding degradation pathways and predicted a fine-tuned regulatory network. In this study, differential proteomics (2-D DIGE and MS) was used to define degradation pathway-specific subproteomes and to determine their growth condition dependent regulation. Differential protein profiles were determined for cultures adapted to growth under 22 different substrate and redox conditions. In total, 354 different proteins were identified, 199 of which displayed significantly changed abundances. These regulated proteins mainly represented enzymes of the different degradation pathways, and revealed different degrees of growth condition specific regulation. In case of three substrate conditions (e.g. phenylalanine, anoxic), proteins previously predicted to be involved in their degradation were apparently not involved (e.g. Pdh, phenylacetaldehyde dehydrogenase). Instead, previously not considered proteins were specifically increased in abundance (e.g. EbA5005, predicted aldehyde:ferredoxin oxidoreductase), shedding new light on the respective pathways. Moreover, strong evidence was obtained for thus far unpredicted degradation pathways of three hitherto unknown substrates (e.g. o-aminobenzoate, anoxic). Comparing all identified regulated and nonregulated proteins provided first insights into regulatory hierarchies of special degradation pathways versus general metabolism in strain EbN1

Functional proteomic view of metabolic regulation in "Aromatoleum aromaticum" strain EbN1

The denitrifying "Aromatoleum aromaticum" strain EbN1 utilizes a wide range of aromatic and nonaromatic compounds under anoxic and oxic conditions. The recently determined genome revealed corresponding degradation pathways and predicted a fine-tuned regulatory network. In this study, differential proteomics (2-D DIGE and MS) was used to define degradation pathway-specific subproteomes and to determine their growth condition dependent regulation. Differential protein profiles were determined for cultures adapted to growth under 22 different substrate and redox conditions. In total, 354 different proteins were identified, 199 of which displayed significantly changed abundances. These regulated proteins mainly represented enzymes of the different degradation pathways, and revealed different degrees of growth condition specific regulation. In case of three substrate conditions (e.g. phenylalanine, anoxic), proteins previously predicted to be involved in their degradation were apparently not involved (e.g. Pdh, phenylacetaldehyde dehydrogenase). Instead, previously not considered proteins were specifically increased in abundance (e.g. EbA5005, predicted aldehyde:ferredoxin oxidoreductase), shedding new light on the respective pathways. Moreover, strong evidence was obtained for thus far unpredicted degradation pathways of three hitherto unknown substrates (e.g. o-aminobenzoate, anoxic). Comparing all identified regulated and nonregulated proteins provided first insights into regulatory hierarchies of special degradation pathways versus general metabolism in strain EbN1

Characterization of protoplasts from hypocotyls of Helianthus annuus in relation to their tissue origin

International audienc

Antigenic analysis of the coat protein of beet necrotic yellow vein virus by means of monoclonal antibodies.

By means of monoclonal antibodies (MAbs), five (groups of) epitopes were identified on particles of beet necrotic yellow vein virus (BNYVV). Epitopes 1 and 2, which were located on the opposite extremities of virus particles, are discontinuous (SDS-labile) epitopes which were destroyed when the particles were treated with trypsin. Epitope 3 is a continuous (SDS-stable) epitope located at the same extremity as epitope 2. It was not destroyed when the particles were treated with trypsin and was present on an Escherichia coli-expressed fusion protein containing amino acids (aa) 1 to 103 of the BNYVV coat protein. The continuous epitope 4, which was located along the entire length of the particles, was found to be present on a fusion protein containing aa 104 to 188 of the BNYVV coat protein but not on trypsin-treated virus particles. In Western blots, these treated particles yielded two slightly smaller coat proteins which failed to react with MAbs specific for epitope 4 but did react with polyclonal antisera and MAbs specific for epitope 3. BNYVV coat protein has a trypsin cleavage site on the carboxyl side of arginine in position 182, so it is therefore suggested that epitope 4 is located on the exposed C terminus, which is composed of aa 183 to 188. Epitope 5 was also located along the entire length of the particles but in a more uneven distribution than epitope 4. This may be because it is a discontinuous epitope that is very sensitive to subtle changes in protein conformation.</p