Tagging quantitative trait loci for maturity-corrected late blight resistance in tetraploid potato with PCR-based candidate gene markers

Late blight caused by the oomycete Phytophthora infestans is the economically most important and destructive disease in potato cultivation. Quantitative resistance to late blight available in tetraploid cultivars is correlated with late maturity in temperate climates, which is an undesirable characteristic. A total of 30 DNA-based markers known to be linked to loci for pathogen resistance in diploid potato were selected and tested as polymerase chain reaction-based markers for linkage with quantitative trait loci (QTL) for late blight resistance and plant maturity in two half-sib families of tetraploid potatoes. Most markers originated from within or were physically closely linked to candidate genes for quantitative resistance factors. The families were repeatedly evaluated in the field for quantitative resistance to late blight and maturity. Resistance was corrected for the maturity effect. Nine of eleven different map segments tagged by the markers harbored QTL affecting maturity-corrected resistance. Interactions were found between unlinked resistance QTL, providing testable strategies for marker-assisted selection in tetraploid potato. Based on the linkage observed between QTL for resistance and plant maturity and based on the genetic interactions observed between candidate genes tagging resistance QTL, we discuss models for the molecular basis of quantitative resistance and maturity.     

P signalling in unicellular cyanobacteria: analysis of redox-signals and energy charge

This communication presents a short outline of the current knowledge on the molecular basis of P_II signal transduction in unicellular cyanobacteria with respect to the perception of environmental stimuli. First, the general characteristics of the P_II signalling system in unicellular cyanobacteria are presented, the hallmark of which is modification by serine-phosphorylation, as compared to the paradigmatic P_II signal transduction system in proteobacteria, which is based on tyrosyl-uridylylation. Then, the focus is turned on the signals controlling P_II phosphorylation state. Recently, the cellular phosphatase (termed PphA), which specifically dephosphorylates phosphorylated P_II (P_II-P) was identified in Synechocystis sp. strain PCC 6803. With the availability of a PphA-deficient mutant and the purified components for in vitro assay of PphA mediated P_II-P dephosphorylation, novel insights into the signals, to which P_II-P dephosphorylation responds, can be obtained. Here we present an investigation of the response of P_II-P dephosphorylation towards treatments that affect the redox-balance of the cells. Furthermore, a possible role of varying ATP/ADP ratios on P_II-P dephosphorylation was examined. From these studies, together with previous investigations, we conclude that P_II-P dephosphorylation specifically responds to changes in the levels of central metabolites of carbon metabolism, in particular 2-oxoglutarate.     

Apoplastic binding of aluminum is involved in silicon-induced amelioration of aluminum toxicity in maize

The alleviating effect of silicon (Si) supply on aluminum (Al) toxicity was suggested to be based on ex or in planta mechanisms. In our experiments with the Al-sensitive maize (Zea mays) cultivar Lixis, Si treatment but not Si pretreatment ameliorated Al-induced root injury as revealed by less root-growth inhibition and callose formation. Si treatment did not affect monomeric Al concentrations in the nutrient solution, suggesting an in planta effect of Si on Al resistance. A fractionated analysis of Si and Al in the 1-cm root apices revealed that more than 85% of the root-tip Al was bound in the cell wall. Al contents in the apoplastic sap, the symplastic sap, and the cell wall did not differ between -Si and +Si plants. Si did not affect the Al-induced exudation of organic acid anions and phenols from the root apices. However, Al treatment greatly enhanced Si accumulation in the cell wall fraction, reducing the mobility of apoplastic Al. From our data we conclude that Si treatment leads to the formation of hydroxyaluminumsilicates in the apoplast of the root apex, thus detoxifying Al.     

Microarray and functional gene analyses of sulfate-reducing prokaryotes in low-sulfate, acidic fens reveal cooccurrence of recognized genera and novel lineages

Low-sulfate, acidic (approximately pH 4) fens in the Lehstenbach catchment in the Fichtelgebirge mountains in Germany are unusual habitats for sulfate-reducing prokaryotes (SRPs) that have been postulated to facilitate the retention of sulfur and protons in these ecosystems. Despite the low in situ availability of sulfate (concentration in the soil solution, 20 to 200 μM) and the acidic conditions (soil and soil solution pHs, approximately 4 and 5, respectively), the upper peat layers of the soils from two fens (Schlöppnerbrunnen I and II) of this catchment displayed significant sulfate-reducing capacities. 16S rRNA gene-based oligonucleotide microarray analyses revealed stable diversity patterns for recognized SRPs in the upper 30 cm of both fens. Members of the family “Syntrophobacteraceae” were detected in both fens, while signals specific for the genus Desulfomonile were observed only in soils from Schlöppnerbrunnen I. These results were confirmed and extended by comparative analyses of environmentally retrieved 16S rRNA and dissimilatory (bi)sulfite reductase (dsrAB) gene sequences; dsrAB sequences from Desulfobacca-like SRPs, which were not identified by microarray analysis, were obtained from both fens. Hypotheses concerning the ecophysiological role of these three SRP groups in the fens were formulated based on the known physiological properties of their cultured relatives. In addition to these recognized SRP lineages, six novel dsrAB types that were phylogenetically unrelated to all known SRPs were detected in the fens. These dsrAB sequences had no features indicative of pseudogenes and likely represent novel, deeply branching, sulfate- or sulfite-reducing prokaryotes that are specialized colonists of low-sulfate habitats.The dissimilatory reduction of sulfate is carried out exclusively by prokaryotic organisms and is one of the most important mineralization processes in anoxic aquatic environments, especially marine sediments (29, 30). In contrast to well-studied sulfate-reducing communities in marine (18, 19, 38, 41, 53, 56, 57, 72) and freshwater habitats (39, 40, 59, 60), relatively little is known about the distribution, diversity, and in situ activities of sulfate-reducing prokaryotes (SRPs) in terrestrial ecosystems. The contribution of terrestrial SRPs to the overall turnover of organic matter is likely of minor importance on a global scale. However, SRPs contribute to the biodegradation of pollutants in soils and subsurface environments (1, 15, 49, 71) and are important to the geomicrobiology of specialized terrestrial habitats that are subject to flooding, such as rice fields (68, 76, 77) and fens (3, 5).δ³⁴S values and ³⁵S-labeling patterns indicate that the dissimilatory reduction of sulfate is an ongoing process in the acidic fens of a forested catchment in northern Bavaria, Germany (Lehstenbach, Fichtelgebirge) (3, 5). The deposition of sulfur that originated from the combustion of soft coal in Eastern Europe (10) led to accumulation of sulfur in the soils of this catchment (4). Although pollution controls have lessened the deposition in recent years, desorption of sulfate in aerated upland soils causes sulfate to enter fens at lower elevations. It was hypothesized that the dissimilatory reduction of sulfate in these mainly anoxic, waterlogged acidic fen soils (the pH of the fen soils is approximately 4) contributes to the retention of sulfur in this ecosystem (3, 4, 50). The reduction of sulfate in these fens is also a sink for protons and thus decreases the acidity of the soil solution and groundwater of this habitat.The acidity and low sulfate content of some of the fens in the Lehstenbach catchment provide an unusual habitat for SRPs, and the occurrence and activity of these organisms in such habitats have received little attention. The main objectives of this study were (i) to assess the capacity of the fen soils to reduce sulfate along vertical soil profiles in the upper peat layers, (ii) to determine the vertical community profiles for all known SRP lineages that inhabit the fens by the use of a 16S rRNA-based oligonucleotide microarray (SRP-PhyloChip) (44), (iii) to resolve the possible existence of novel SRP lineages in the fens by retrieval of dsrAB, which are genes that encode the alpha and beta subunits of the siroheme dissimilatory (bi)sulfite reductase (EC 1.8.99.3) (34, 66, 74), and (iv) to deduce the possible in situ functional relationships that can be inferred from this collective information.     

Keratinocyte growth factor: effects on keratinocytes and mechanisms of action

Keratinocyte growth factor (KGF) is a potent and specific mitogen for different types of epithelial cells, and it can protect these cells from various insults. Due to these properties, it is of particular importance for the repair of injured epithelial tissues, and it is currently therapeutically explored for the treatment of radiation- and chemotherapy-induced mucosal epithelial damage in cancer patients. In this review we summarize the current knowledge on the role of KGF in tissue repair and cytoprotection, and we report on its mechanisms of action in keratinocytes.     

Disseminated lethal Encephalitozoon cuniculi (genotype III) infections in cotton-top tamarins (Oedipomidas oedipus)--a case report

For the first time, Encephalitozoon (E.) cuniculi genotype III ('dog strain') was verified in two cotton-top tamarins (Oedipomidas oedipus) by light microscopy, immunohistochemistry, electron microscopy, PCR and sequencing. The animals had a disseminated lethal infection with this protist. In earlier reports, genotype III had been found only in domestic dogs, man, emperor tamarins (Saguinus imperator) and golden lion tamarins (Leontopithecus rosalia). This investigation establishes now that the 'dog strain' can occur in cotton-top tamarins too. This is further evidence for the zoonotic potential of E. cuniculi. Furthermore, free E. cuniculi spores were identified also in blood vessels of several tissues. These findings indicate that during a disseminated infection E. cuniculi spores can occur in peripheral blood, too. We propose that blood should also be included in the investigations for the detection of microsporidia, so that a possible disseminated course of an infection can be detected.     

Protein expression profiling identifies molecular targets of quercetin as a major dietary flavonoid in human colon cancer cells

A high dietary intake of plant foods is thought to contribute to the prevention of colorectal cancers in humans and flavonoids as part of such a diet are considered to contribute to those protective effects. Quercetin is a major dietary flavonoid consumed with a diet rich in onions, tea, and apples. We used HT-29 human colon cancer cells and investigated the effects of quercetin on proliferation, apoptosis, and differentiation as processes shown to be disregulated during cancer development. To identify the cellular targets of quercetin action, two-dimensional gel electrophoresis was performed and proteins altered in expression level after quercetin exposure of cells were identified by mass spectrometry of peptide fragments generated by tryptic digestion. Quercetin inhibited the proliferation of HT-29 cells with an IC(50)-value of 81.2 +/- 6.6 microM. Cell differentiation based on surface expression of alkaline phosphatase was enhanced 4-fold and the activity of the pro-apoptotic effector caspase-3 increased 3-fold. Those effects were associated with the regulation of heat-shock proteins and annexins shown to both play a crucial role in the process of apoptosis. Cytoskeletal caspase substrates were found as regulated as well and various proteins involved in intermediary metabolism and in gene regulation showed altered steady-state expression levels upon quercetin treatment of cells. In conclusion, quercetin alters the levels of a variety of proteins involved in growth, differentiation, and apoptosis of colon cancer cells. Their identification as molecular targets of quercetin may explain the anti-cancer activities of this flavonoid.     

Setting up standards and a reference map for the alkaline proteome of the Gram-positive bacterium Lactococcus lactis

Despite the fact that almost 39% of the theoretical expressed proteins of Lactococcus lactis have a predicted isoelectric point above 7, these proteins have not been studied in previous proteome analyses. In the present study, we set up a reference map of alkaline lactococcal proteins by using immobilized pH gradients (IPG) spanning pH 6 to 12 and 9 to 12, and protein identification by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Different electrophoresis systems for isoelectric focusing were evaluated to optimize the first dimension. Best results were obtained by sample application using cup-loading at the anodic side and increasing the final voltage up to 8000 V for IPGs, using N,N-dimethylacrylamide as monomer. After two-dimensional gel electrophoresis of extracts obtained from exponentially growing cells, about 200 protein spots were selected for identification by peptide mass fingerprinting. With MALDI-TOF MS, 153 proteins were identified that were the products of 85 different genes. Their predicted isoelectric points range from as high as 11.31 to as low as 6.34. Ribosomal proteins, hypothetical proteins and proteins with unknown function represent the largest groups of identified proteins. For further classification, the codon adaptation index (CAI) and grand average of hydropathicity (GRAVY) for each lactococcal protein were calculated. The protein with the lowest CAI identified in this study is the manganese ABC transporter ATP-binding protein. Less than 10% of the alkaline lactococcal proteins have a smaller CAI. The highest GRAVY for an identified protein is 0.26. The complete in silico data of Lactococcus lactis as well as clickable reference maps are available at www.wzw.tum.de/proteomik/lactis.     

16S rRNA gene based analysis of <Emphasis Type="Italic">Enterobacter sakazakii</Emphasis> strains from different sources and development of a PCR assay for identification

Background  

E. sakazakii is considered to be an opportunistic pathogen, implicated in food borne diseases causing meningitis or enteritis especially in neonates and infants. Cultural standard identification procedures for E. sakazakii include the observation of yellow pigmentation of colonies and a positive glucosidase activity. Up to now, only one PCR system based on a single available 16S rRNA gene sequence has been published for E. sakazakii identification. However, in our hands a preliminary evaluation of this system to a number of target and non-target strains showed significant specificity problems of this system. In this study full-length 16S rRNA genes of thirteen E. sakazakii strains from food, environment and human origin as well as the type strain ATCC 51329 were sequenced. Based on this sequence data a new specific PCR system for E. sakazakii was developed and evaluated.     

A structural analysis of the interaction between ncd tail and tubulin protofilaments

ncd is a minus-end directed, kinesin-like motor, which binds to microtubules with its motor domain and its cargo domain as well. Typical of retrograde motors, the motor domain of ncd locates to the C-terminal end of the polypeptide chain, and hence, the cargo domain constitutes the N-terminal region. To date, several studies have investigated the interaction properties of the motor domain with microtubules, but very few structural data are available about the tail itself or its interaction with microtubules as cargo. Here, we applied cryo-electron microscopy and helical 3D image reconstruction to 15 protofilament microtubules decorated with an ncd tail fragment (N-terminal residues 83-187, named NT6). In our study, the ncd tail shows a behaviour resembling filamentous MAPs such as tau protein, exhibiting a highly flexible structure with no large globular domains. NT6 binds to four different sites on the outer side of microtubules within the proximity of the kinesin motor-binding site. Two of these sites locate within the groove between two neighbouring protofilaments, and appear as strong binding sites, while the other two sites, located at the outer rim, appear to play a secondary role. In addition, the ncd tail fragment induces the formation of large protofilament sheets, suggesting a tail-induced modification of lateral protofilament contacts.