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Fungi defend their ecological niche against antagonists by producing antibiosis molecules. Some of these molecules are only produced upon confrontation with the antagonist. The basidiomycete Coprinopsis cinerea induces the expression of the sesquiterpene synthase-encoding gene cop6 and its two neighboring genes coding for cytochrome P450 monooxygenases in response to bacteria. We further investigated this regulation of cop6 and examined if the gene product is involved in the production of antibacterials. Cell-free supernatants of axenic cultures of the Gram-positive bacterium Bacillus subtilis were sufficient to induce cop6 transcription assessed using a fluorescent reporter strain. Use of this strain in a microfluidic device revealed that the cop6 gene was induced in all hyphae directly exposed to the supernatant and that induction occurred within less than one hour. Targeted replacement of the cop6 gene demonstrated the requirement of the encoded synthase for the biosynthesis of the sesquiterpene lagopodin B, a previously reported antibacterial compound from related species. Accordingly, lagopodin B from C. cinerea inhibited the growth of several Gram-positive bacteria including B. subtilis but not Gram-negative bacteria. Our results demonstrate that the C. cinerea vegetative mycelium responds to soluble compounds of a bacterial culture supernatant by local production of an antibacterial secondary metabolite.

Chitin-binding lysin motif (LysM) effectors contribute to the virulence of various plant-pathogenic fungi that are causal agents of foliar diseases. Here, we report the LysM effectors of the soil-borne fungal vascular wilt pathogen Verticillium dahliae. Comparative genomics revealed three core LysM effectors that are conserved in a collection of V. dahliae strains. Remarkably, and in contrast with the previously studied LysM effectors of other plant pathogens, no expression of core LysM effectors was monitored in planta in a taxonomically diverse panel of host plants. Moreover, targeted deletion of the individual LysM effector genes in V. dahliae strain JR2 did not compromise virulence in infections on Arabidopsis, tomato or Nicotiana benthamiana. Interestingly, an additional lineage-specific LysM effector is encoded in the genome of V. dahliae strain VdLs17, but not in any other V. dahliae strain sequenced to date. Remarkably, this lineage-specific effector is expressed in planta and contributes to the virulence of V. dahliae strain VdLs17 on tomato, but not on Arabidopsis or N. benthamiana. Functional analysis revealed that this LysM effector binds chitin, is able to suppress chitin-induced immune responses and protects fungal hyphae against hydrolysis by plant hydrolytic enzymes. Thus, in contrast with the core LysM effectors of V. dahliae, this lineage-specific LysM effector of strain VdLs17 contributes to virulence in planta.

Molecular Microbiology, 112 (2), ISSN:0950-382x, ISSN:1365-2958

The clinical trial design including a test treatment, an active control and a placebo is called the gold standard design. In this paper, we develop a statistical method for planning and evaluating non-inferiority trials with gold standard design for right-censored time-to-event data. We consider both lost to follow-up and administrative censoring. We present a semiparametric approach that only assumes the proportionality of the hazard functions. In particular, we develop an algorithm for calculating the minimal total sample size and its optimal allocation to treatment groups such that a desired power can be attained for a specific parameter constellation under the alternative. For the purpose of sample size calculation, we assume the endpoints to be Weibull distributed. By means of simulations, we investigate the actual type I error rate, power and the accuracy of the calculated sample sizes. Finally, we compare our procedure with a previously proposed procedure assuming exponentially distributed event times. To illustrate our method, we consider a double-blinded, randomized, active and placebo controlled trial in major depression. Copyright © 2013 John Wiley & Sons, Ltd.

The large thickness of Upper Carboniferous strata found in the Netherlands suggests that the area was subject to long-term subsidence. However, the mechanisms responsible for subsidence are not quantified and are poorly known. In the area north of the London Brabant Massif, onshore United Kingdom, subsidence during the Namurian-Westphalian B has been explained by Dinantian rifting, followed by thermal subsidence. In contrast, south and east of the Netherlands, along the southern margin of the Northwest European Carboniferous Basin, flexural subsidence caused the development of a foreland basin. It has been proposed that foreland flexure due to Variscan orogenic loading was also responsible for Late Carboniferous subsidence in the Netherlands. In the first part of this paper, we present a series of modelling results in which the geometry and location of the Variscan foreland basin was calculated on the basis of kinematic reconstructions of the Variscan thrust system. Although several uncertainties exist, it is concluded that most subsidence calculated from well data in the Netherlands cannot be explained by flexural subsidence alone. Therefore, we investigated whether a Dinantian rifting event could adequately explain the observed subsidence by inverse modelling. The results show that if only a Dinantian rifting event is assumed, such as is found in the United Kingdom, a very high palaeowater depth at the end of the Dinantian is required to accommodate the Namurian-Westphalian B sedimentary sequence. To better explain the observed subsidence curves, we propose (1) an additional stretching event during the Namurian and (2) a model incorporating an extra dynamic component, which might well explain the very high wavelength of the observed subsidence compared with the wavelength of the predicted flexural foreland basin. © 2008 The Authors. Journal compilation © 2008 Blackwell Publishing Ltd.

Treatment of cultured parsley cells (Petroselinum crispum) with fungal elicitor caused large increases in the activities of chitinase and 1,3-beta-glucanase. Chitinase activity accumulated predominantly in the culture medium, whereas 1,3-beta-glucanase activity was located almost exclusively intracellularly. Extracellular chitinase activity was resolved into six different isoenzymes, all of which were purified and characterized. All six isoforms were acidic proteins (pI 3.8-5.3), with molecular mass 30-38 kDa. Four were exochitinases and two were endochitinases. The most abundant isoform also showed lysozyme activity. Three of the exochitinases were glycoproteins and two of these were reactive with an antiserum specific for xylose in complex glycosidic structures. The exochitinases constituted relatively small proportions of the total chitinase activity and may serve a different function in cellular metabolism compared to the more abundant endochitinases.

Summary Infection of potato (Solarium tuberosum L.) leaves with the fungal pathogen Phytophthora infestans caused a similar, strong and coordinated induction of 1, 3-β-glucanases and chitinases in compatible (plant susceptible) and incompatible (plant resistant) interactions of two selected plant cultivars with appropriate races of the fungus. The temporal and spatial patterns of 1, 3-β-glucanase induction were studied in further detail by immunohistochemical and in-situ hybridization methods. Accumulation of the protein was preceded by progressive activation of the corresponding gene, commencing near infection sites and spreading rapidly throughout the whole infected leaf as well as to adjacent, non-infected leaves. Protein and mRNA distribution patterns were nearly identical in compatible and incompatible interactions. In comparison with 1, 3-β-glucanase mRNA, phenyl-alanine ammonia-lyase mRNA accumulated more rapidly and remained restricted to the vicinity of fungal infection sites, in addition to its constitutive occurrence in the vascular bundles. Even more rapid than any detectable mRNA induction was the accumulation of auto fluorescing material in plant cells immediately surrounding fungal structures, particularly and invariably in incompatible interactions and less frequently in compatible interactions. It is concluded that cultivar-race-specific resistance is established early in the interaction of potato leaves with P. infestans and hence the observed massive accumulation of 1, 3-β-glucanase and chitinase is presumably not involved in determining this specificity.

The distribution of enzymes interconverting fructose 6-phosphate and fructose 1,6-bisphosphate has been studied in a range of tissues from castor bean seedlings. In each tissue the activity of PP i :fructose 6-phosphate phosphotransferase was greater than phosphofructokinase and substantial compared with fructose 1,6-bisphosphatase. PP i :fructose 6-phosphate phosphotransferase in endosperm is apparently confined to the cytoplasm. The role of this latter enzyme in vivo is discussed.

Zusammenfassung Die lytische Wirkung des polykationischen Makromolekuls DEAE-Dextran (Mol.-Gew. 500 000) auf Membranen wurde zur Isolierung von Chloroplasten aus der zellwandlosen, einzelligen Grunalge Dunaliella marina ausgenutzt. Derartig isolierte Chloroplasten waren morphologisch (Phasenkontrastmikroskopie) und biochemisch (Enzymausstattung) intakt. Es zeigte sich jedoch, das Kriterien funktioneller Intaktheit, wie photochemische Reduktion von 3-Phosphatglycerat oder CO2-abhangige O2-Entwicklung (CO2-Fixierung) nicht erfullt waren. Zur Klarung dieses scheinbaren Widerspruchs wurde der Einflus von DEAE-Dextran auf Chloroplasten untersucht; von anderen Polykationen (Histone, Polylysin) war bekannt, das sie mit Funktionen von biologischen Membranen interferieren und den Elektronentransport in Chloroplasten beeintrachtigen. DEAE-Dextran inhibierte in Konzentrationen, die zur Lysis der Zelle nicht ausreichten, die Photosyntheseaktivitat ganzer Zellen vollstandig. Die Inhibition wurde durch Magnesiumionen effektiv verhindert. Mit artifiziellen Elektronendonatoren und -akzeptoren lies sich die Stelle der Inhibition in der Elektronentransportkette lokalisieren. Wahrend die Photoreduktion von Ferri-cyanid an Photosystem II durch DEAE-Dextran nicht beeintrachtigt war, wurde der Elektronentransfer auf Methylviologen uber beide Photosysteme inhibiert. Nach Ausschalten von Photosystem II mit 3-(3′, 4′-Dichlorphenyl)-1, 1-dimethylharnstoff erfuhr die Photoreduktion von Methylviologen mit 2, 6-Dichlorphenol-indophenol als unspezifischem Elektronendonator fur Photosystem I zwar eine Steigerung durch DEAE-Dextran, aber die Reaktion mit dem spezifischen Elektronendonator N,N,N',N'-Tetramethyl-p-phenylendiamin wurde gehemmt. Die Ergebnisse zeigen, das DEAE-Dextran nicht nur eine offensichtliche Schadigung der auseren Membran der Chloroplasten hervorruft, sondern auch, wie andere Polykationen, selektiv die Aktivitat von Photosystem I im Bereich des Plastocyanins inhibiert. Die Untersuchungen wurden durch ein Stipendium des Deutschen Akademischen Austauschdienstes (DAAD) fur E. K., von der Adolf-Haeuser-Stiftung und von der Deutschen Forschungsgemeinschaft (SFB 103/A5) gefordert.