Powdery mildew resistance in 155 Nordic bread wheat cultivars and landraces

The occurrence and distribution of seedling resistance genes and the presence of adult plant resistance to powdery mildew, was investigated in a collection of 155 Nordic bread wheat landraces and cultivars by inoculation with 11 powdery mildew isolates. Eighty-nine accessions were susceptible in the seedling stage, while 66 accessions showed some resistance. Comparisons of response patterns allowed postulation of combinations of genes Pm1a, Pm2, Pm4b, Pm5, Pm6, Pm8 and Pm9 in 21 lines. Seedling resistance was three times more frequent in spring wheat than in winter wheat. The most commonly postulated genes were Pm1a+Pm2+Pm9 in Sweden, Pm5 in Denmark and Norway, and Pm4b in Finland. Forty-five accessions were postulated to carry only unidentified genes or a combination of identified and unidentified genes that could not be resolved by the 11 isolates. Complete resistance to all 11 isolates was present in 18 cultivars. Adult plant resistance was assessed for 109 accessions after natural infection with a mixture of races. In all, 92% of the accessions developed less than 3-5% pathogen coverage while nine lines showed 10-15% infected leaf surface. The characterization of powdery mildew resistance in Nordic wheat germplasm could facilitate the combination of resistance genes in plant breeding programmes to promote durability of resistance and disease management.     

Production of recombinant and tagged proteins in the hyperthermophilic archaeon Sulfolobus solfataricus

Many systems are available for the production of recombinant proteins in bacterial and eukaryotic model organisms, which allow us to study proteins in their native hosts and to identify protein-protein interaction partners. In contrast, only a few transformation systems have been developed for archaea, and no system for high-level gene expression existed for hyperthermophilic organisms. Recently, a virus-based shuttle vector with a reporter gene was developed for the crenarchaeote Sulfolobus solfataricus, a model organism of hyperthermophilic archaea that grows optimally at 80 degrees C (M. Jonuscheit, E. Martusewitsch, K. M. Stedman, and C. Schleper, Mol. Microbiol. 48:1241-1252, 2003). Here we have refined this system for high-level gene expression in S. solfataricus with the help of two different promoters, the heat-inducible promoter of the major chaperonin, thermophilic factor 55, and the arabinose-inducible promoter of the arabinose-binding protein AraS. Functional expression of heterologous and homologous genes was demonstrated, including production of the cytoplasmic sulfur oxygenase reductase from Acidianus ambivalens, an Fe-S protein of the ABC class from S. solfataricus, and two membrane-associated ATPases potentially involved in the secretion of proteins. Single-step purification of the proteins was obtained via fused His or Strep tags. To our knowledge, these are the first examples of the application of an expression vector system to produce large amounts of recombinant and also tagged proteins in a hyperthermophilic archaeon.     

The Trophic Index of Macrophytes (TIM) – a New Tool for Indicating the Trophic State of Running Waters

In running waters, apart from structural degradation, nutrient input becomes increasingly important. To investigate the indicator values of as many species of submerged macrophytes as possible numerous samples of the sediment within macrophyte stands and the overlying water were taken in running waters throughout Bavaria, Germany. To develop the Trophic Index of Macrophytes (TIM), the concentrations of soluble reactive phosphorus of both the water body and the sediment pore water were used. Based on a weighted sum of the SRP‐concentrations of the water body and the sediment pore water, indicator values were determined for a total of 49 species of submerged macrophytes. A detailed method is described on how and depending on which preconditions the trophic state of running waters can be determined by the TIM. An example of the TIM in the stream Rotbach is given. It shows that the TIM is a useful means to detect differences in the phosphorus loading of running waters.     

Improved purification of the membrane-bound hydrogenase–sulfur-reductase complex from thermophilic archaea using ε-aminocaproic acid-containing chromatography buffers

A hydrogenase–sulfur reductase (SR) complex was purified from membrane preparations of the extremely thermophilic, acidophilic archaeon Acidianus ambivalens using a combination of sucrose density gradient centrifugation and column chromatography (FPLC). All chromatographic steps were performed in the presence of 0.5% ε-aminocaproic acid resulting in the elution of the SR complex as a sharp peak. In contrast, chromatography using buffers without ε-aminocaproic acid, or in the presence of detergents, were not successful. The purified A. ambivalens SR complex consisted of at least four subunits with relative molecular masses of 110 000, 66 000, 39 000 and 29 000, respectively. A similar procedure was applied to purify the membrane-bound hydrogenase from Thermoproteus neutrophilus, a non-related extremely thermophilic but neutrophilic archaeon, which consisted of only two subunits with relative molecular masses of 66 000 and 39 000, respectively.     

Common CFTR mutations are not likely to predispose to chronic bronchitis in Northern Germany

The frequency of six common mutations in the cystic fibrosis transmembrane conductance regulator gene was studied in 100 patients hospitalized with chronic bronchitis. Only one patient with chronic bronchitis and diffuse bronchiectasis was heterozygous for the common F508 mutation. R553X, G542X, G551D, N1303K and 621+1GT were not detected. This result is not significantly different from the frequency of cystic fibrosis carriers in Northern Europe. Predisposition of heterozygotes for chronic bronchitis is therefore unlikely.     

Formation of poly(3-hydroxyalkanoates) by phototrophic and chemolithotrophic bacteria

The formation of poly(3-hydroxyalkanoic acid), PHA, by various strains of chemolithotrophic and phototrophic bacteria has been examined. Chemolithotrophic bacteria were grown aerobically under nitrogen-limiting conditions on various aliphatic organic acids. Phototrophic bacteria were grown anaerobically in the light on a nitrogen-rich medium and were subsequently transferred to a nitrogen-free medium containing acetate, propionate, valerate, heptanoate or octanoate as carbon source. All 41 strains investigated in this study were able to synthesize and accumulate PHA. All 11 strains of chemolithotrophic bacteria and all 15 strains belonging to the non-sulfur purple bacteria synthesized a polymer, which contained 3-hydroxy-valerate (3HV) beside 3-hydroxybutyrate (3HB), if the cells were cultivated in the presence of propionate, valerate or heptanoate. Many non-sulfur purple bacteria synthesized copolyesters of 3HB and 3HV even with acetate as carbon source. In contrast, most sulfur purple bacteria did not incorporate 3HV at all. Among 15 strains tested, only Chromatium vinosum strain 1611, C. purpuratum strain BN5500 and Lamprocystis roseopersicina strain 3112 were able to synthesize polyesters containing 3HV with propionate, valerate or heptanoate as carbon source.     

The sulfur oxygenase reductase from Acidianus ambivalens is an icosatetramer as shown by crystallization and Patterson analysis

The sulfur oxygenase reductase (SOR) is the initial enzyme in the aerobic sulfur metabolism of the thermoacidophilic and chemolithoautotrophic crenarchaeote Acidianus ambivalens. Single colorless polyhedral crystals were obtained under two crystallization conditions from SOR preparations heterologously overproduced in Escherichia coli. They belonged to space-group I4 and diffraction data were collected up to 1.7 A resolution. Their Patterson symmetry shows additional 4-, 3- and 2-fold non-crystallographic symmetry rotation axes, characteristic of the point group 432. Taking into account the molecular mass of SOR, the crystal unit cell volume, the non-crystallographic symmetry operators and previous electron microscopy studies of the SOR, it was deduced that the quaternary structure of the functionally active enzyme is an icosatetramer with 871 kDa molecular mass.     

Screening and evaluation of resistance to downy mildew (Peronospora parasitica) and clubroot (Plasmodiophora brassicae) in genetic resources of Brassica oleracea

52 entries including landraces, old cultivars and wild accessions of B. oleracea and closely related Brassica species were screened for resistance against downy mildew and clubroot. Several accessions resistant to downy mildew and a few to clubroot were found. Genetic inheritance of the resistance in downy mildew was investigated by screening F1 and BC1F1 offspring from three resistant landrace accessions crossed with both a resistant and a susceptible father. The seedling resistance against downy mildew was found to be inherited recessively. This is a bit surprising as earlier papers mostly report of inheritance controlled by a single dominant gene. Previous screenings of B. oleracea resistance against downy mildew at the cotyledon stage have been done with P. parasitica isolated from B. oleracea as the original host plant. The recessive nature of the cotyledon resistance found in this screening might be due to the fact that the P. parasitica isolate was collected from B. napus fields. The clubroot seedling resistance was found to be controlled by recessive inheritance after screening the F1 offspring, this in agreement with earlier results/reports.     

Lessons to be learned from primary renal cell carcinomas: novel tumor antigens and HLA ligands for immunotherapy

The lack of sufficient well-defined tumor-associated antigens is still a drawback on the way to a cytotoxic T-lymphocyte-based immunotherapy of renal cell carcinoma (RCC). We are trying to define a larger number of such targets by a combined approach involving HLA ligand characterization by mass spectrometry and gene expression profiling by oligonucleotide microarrays. Here, we present the results of a large-scale analysis of 13 RCC specimens. We were able to identify more than 700 peptides, mostly from self-proteins without any evident tumor association. However, some HLA ligands derived from previously known tumor antigens in RCC. In addition, gene expression profiling of tumors and a set of healthy tissues revealed novel candidate RCC-associated antigens. For several of them, we were able to characterize HLA ligands after extraction from the tumor tissue. Apart from universal RCC antigens, some proteins seem to be appropriate candidates in individual patients only. This underlines the advantage of a personalized therapeutic approach. Further analyses will contribute additional HLA ligands to this repertoire of universal as well as patient-individual tumor antigens.Tobias Krüger and Oliver Schoor contributed equally to this work.