A possible role for Epstein-Barr virus in the pathogenesis of pleural effusion.

A high percentage of pleural effusions remain unexplained despite an intensive diagnostic workup. Epstein-Barr virus (EBV) infections occur worldwide and affect the majority of the population. The present study investigated the prevalence and clinical relevance of EBV in pleural effusions. A prospective study was performed in which 60 consecutive patients with pleural effusion were enrolled. Real-time quantitative EBV-PCR was performed on pleural fluid and serum. Pleural fluid was further evaluated using standard biochemical, cytological and microbiological procedures. Demographic data, medical history and medication were recorded. A total of 24 (40%), from 60 pleural fluids tested, were positive in the EBV-PCR. Median EBV-DNA levels for positive samples was 454 genome equivalents (geq).mL-1 (range 36-163,446 geq.mL-1). A total of 20 (59%) out of 34 unexplained pleural effusions were EBV-PCR positive. Serological analysis of all patients with a positive PCR revealed a previous infection. Patients with a positive EBV-PCR on pleural fluid were more likely to have a positive EBV-PCR on serum than patients with a negative PCR on pleural fluid. Epstein-Barr virus reactivation in pleural fluid is a frequent event and the absence of an alternative diagnosis to explain the nature of the effusion in the majority of cases suggests an aetiological role for Epstein-Barr virus in the development of pleural effusion.

The molecular basis of co-evolution between Cladosporium fulvum and tomato.

Cladosporiumfulvum is a semi-biotrophic pathogen, which causes leaf mold of tomato (Lycopersicon spp.). In our laboratory this pathosystem serves as a model to study gene-for-gene interactions between plants and pathogenic fungi (Joosten & De Wit 1999). Many avirulence (Avr) genes and matching resistance (CQ) genes have been cloned and we are now beginning to understand how their products can induce an array of plant defense responses, including the classic hypersensitive response (HR). Here, we will discuss the latest results of our molecular studies on this interaction. These include the isolation of: (i) two new Avr genes, Avr2 and Avr4E, (ii) the determination of the specificity determinants within the Cf-4 and Cf-9 genes by artificial domain swaps and introduction of point mutations, (iii) the analysis of polymorphism occurring in AVR9-responsive Cf genes occurring in natural populations of L. pimpinellifolium, and finally (iv) the description of an efficient method to identify early HR-related genes.

No evidence for binding between resistance gene product Cf-9 of tomato and avirulence gene product AVR9 of Cladosporium fulvum.

The gene-for-gene model postulates that for every gene determining resistance in the host plant, there is a corresponding gene conditioning avirulence in the pathogen. On the basis of this relationship, products of resistance (R) genes and matching avirulence (Avr) genes are predicted to interact. Here, we report on binding studies between the R gene product Cf-9 of tomato and the Avr gene product AVR9 of the pathogenic fungus Cladosporium fulvum. Because a high-affinity binding site (HABS) for AVR9 is present in tomato lines, with or without the Cf-9 resistance gene, as well as in other solanaceous plants, the Cf-9 protein was produced in COS and insect cells in order to perform binding studies in the absence of the HABS. Binding studies with radio-labeled AVR9 were performed with Cf-9-producing COS and insect cells and with membrane preparations of such cells. Furthermore, the Cf-9 gene was introduced in tobacco, which is known to be able to produce a functional Cf-9 protein. Binding of AVR9 to Cf-9 protein produced in tobacco was studied employing surface plasmon resonance and surface-enhanced laser desorption and ionization. Specific binding between Cf-9 and AVR9 was not detected with any of the procedures. The implications of this observation are discussed.

Microbial populations involved in cycling of dimethyl sulfide and methanethiol in freshwater sediments.

Although several microorganisms that produce and degrade methanethiol (MT) and dimethyl sulfide (DMS) have been isolated from various habitats, little is known about the numbers of these microorganisms in situ. This study reports on the identification and quantification of microorganisms involved in the cycling of MT and DMS in freshwater sediments. Sediment incubation studies revealed that the formation of MT and DMS is well balanced with their degradation. MT formation depends on the concentrations of both sulfide and methyl group-donating compounds. A most-probable number (MPN) dilution series with syringate as the growth substrate showed that methylation of sulfide with methyl groups derived from syringate is a commonly occurring process in situ. MT appeared to be primarily degraded by obligately methylotrophic methanogens, which were found in the highest positive dilutions on DMS and mixed substrates (methanol, trimethylamine [TMA], and DMS). Amplified ribosomal DNA restriction analysis (ARDRA) and 16S rRNA gene sequence analysis of the total DNA isolated from the sediments and of the DNA isolated from the highest positive dilutions of the MPN series (mixed substrates) revealed that the methanogens that are responsible for the degradation of MT, DMS, methanol, and TMA in situ are all phylogenetically closely related to Methanomethylovorans hollandica. This was confirmed by sequence analysis of the product obtained from a nested PCR developed for the selective amplification of the 16S rRNA gene from M. hollandica. The data from sediment incubation experiments, MPN series, and molecular-genetics detection correlated well and provide convincing evidence for the suggested mechanisms for MT and DMS cycling and the common presence of the DMS-degrading methanogen M. hollandica in freshwater sediments.

Avirulence proteins of plant pathogens: determinants of victory and defeat.

summary The simplest way to explain the biochemical basis of the gene-for-gene concept is by direct interaction between a pathogen-derived avirulence (Avr) gene product and a receptor protein, which is encoded by the matching resistance (R) gene of the host plant. The number of R genes for which the matching Avr gene has been cloned is increasing. The number of host-pathogen relationships, however, for which a direct interaction between R and Avr gene products could be proven is still very limited. This observation suggests that in various host-pathogen relationships no physical interaction between R and Avr proteins occurs, and that perception of AVR proteins by their matching R gene products is indirect. Indirect perception implies that at least a third component is required. The 'Guard hypothesis' proposes that this third component could be the virulence target of an AVR protein. Binding of the AVR protein to its virulence target is perceived by the matching R protein, which is 'guarding' the virulence target. An intriguing aspect of the 'Guard hypothesis' is that the Avr gene product causes avirulence of the pathogen through interaction with its virulence target in the plant. This would mean that, although AVR proteins are generally thought to be bifunctional (avirulence as well as virulence factors), this dual function might be based on a single biochemical event. This review focuses on the way AVR proteins are perceived by their matching R gene products. The various components that determine the outcome of the interaction will be discussed, with an emphasis on the dual function of AVR proteins.

A functional cloning strategy, based on a binary PVX-expression vector, to isolate HR-inducing cDNAs of plant pathogens.

We have devised a novel, high-throughput functional cloning method to isolate cDNAs from plant pathogens of which the products elicit a hypersensitive response (HR) in plants. Copy DNA, made from RNA isolated from the tomato pathogen Cladosporium fulvum grown under nutrient-limiting conditions in vitro, was cloned into a binary, potato virus X (PVX)-based expression vector and transformed to Agrobacterium tumefaciens. 9600 colonies were individually toothpick-inoculated onto leaflets of tomato plants resistant to C. fulvum. Four cDNAs were identified whose expression induced formation of a necrotic lesion around the inoculation site. One of these clones, specifically inducing HR on tomato plants carrying the Cf-4 resistance gene, encodes race-specific elicitor AVR4. The other three cDNAs, inducing a non-genotype-specific HR, encode a protein highly homologous to bZIP, basic transcription factors. To determine whether this approach has general applicability, part of the library was also inoculated onto Nicotiana tabacum var. Samsun NN, which is not a host for C. fulvum. Four independent HR-inducing cDNAs were identified which all encode ECP2, an extracellular protein of C. fulvum known to induce necrosis in certain Nicotiana species. These observations confirm that this functional screening method is a versatile strategy to identify cDNAs of pathogens that encode (race-specific) elicitors and other HR-inducing proteins.

Isolation and characterization of Methanomethylovorans hollandica gen. nov., sp. nov., isolated from freshwater sediment, a methylotrophic methanogen able to grow on dimethyl sulfide and methanethiol.

A newly isolated methanogen, strain DMS1(T), is the first obligately anaerobic archaeon which was directly enriched and isolated from a freshwater sediment in defined minimal medium containing dimethyl sulfide (DMS) as the sole carbon and energy source. The use of a chemostat with a continuous DMS-containing gas stream as a method of enrichment, followed by cultivation in deep agar tubes, resulted in a pure culture. Since the only substrates utilized by strain DMS1(T) are methanol, methylamines, methanethiol (MT), and DMS, this organism is considered an obligately methylotrophic methanogen like most other DMS-degrading methanogens. Strain DMS1(T) differs from all other DMS-degrading methanogens, since it was isolated from a freshwater pond and requires NaCl concentrations (0 to 0.04 M) typical of the NaCl concentrations required by freshwater microorganisms for growth. DMS was degraded effectively only in a chemostat culture in the presence of low hydrogen sulfide and MT concentrations. Addition of MT or sulfide to the chemostat significantly decreased degradation of DMS. Transient accumulation of DMS in MT-amended cultures indicated that transfer of the first methyl group during DMS degradation is a reversible process. On the basis of its low level of homology with the most closely related methanogen, Methanococcoides burtonii (94.5%), its position on the phylogenetic tree, its morphology (which is different from that of members of the genera Methanolobus, Methanococcoides, and Methanohalophilus), and its salt tolerance and optimum (which are characteristic of freshwater bacteria), we propose that strain DMS1(T) is a representative of a novel genus. This isolate was named Methanomethylovorans hollandica. Analysis of DMS-amended sediment slurries with a fluorescence microscope revealed the presence of methanogens which were morphologically identical to M. hollandica, as described in this study. Considering its physiological properties, M. hollandica DMS1(T) is probably responsible for degradation of MT and DMS in freshwater sediments in situ. Due to the reversibility of the DMS conversion, methanogens like strain DMS1(T) can also be involved in the formation of DMS through methylation of MT. This phenomenon, which previously has been shown to occur in sediment slurries of freshwater origin, might affect the steady-state concentrations and, consequently, the total flux of DMS and MT in these systems.