Recombination between diverged clusters of the tomato Cf-9 plant disease resistance gene family.
The tomato Cf-4 and Cf-9 genes are the founder members of a large gene family of homologues of Cladosporium fulvum resistance gene Cf-9 (Hcr9 genes), several of which confer resistance against C. fulvum through recognition of different pathogen-encoded avirulence determinants. Three loci of tandemly repeated Hcr9 genes-Southern Cross (SC), Milky Way (MW), and Northern Lights (NL)-are located on the short arm of tomato chromosome 1. Comparisons between 2 SC-Hcr9s, 11 from MW, and 5 from NL implicated sequence exchange between gene family members in their evolution. The extent to which novel variants can be generated by recombination depends on the degree of sequence polymorphism available within the gene family. Here we show that physical separation of Hcr9 genes can be associated with elevated sequence divergence. Two diverged subclasses of Hcr9s could be defined. These are physically separated from each other, with members of one class exclusively residing at Northern Lights. One exceptional Hcr9 at Northern Lights carried sequence features specific for Hcr9s at other loci, suggesting a recent transfer of this gene by an interlocus recombination event. As members of diverged subclasses are brought into physical vicinity within a tandem repeat, a larger spectrum of sequence variants can potentially be generated by subsequent interhomologue sequence exchange. (+info)
Folding and conformational analysis of AVR9 peptide elicitors of the fungal tomato pathogen Cladosporium fulvum.
The race-specific elicitor AVR9, produced by the phytopathogenic fungus Cladosporium fulvum, is a 28-residue beta-sheet peptide containing three disulfide bridges. The folding of this peptide to its native conformation was examined in the presence of oxidized (GSSG) and reduced (GSH) glutathione at concentrations resembling those present in the endoplasmic reticulum. The concentrations of GSH and GSSG, and the applied temperature strongly affected the folding efficiency. The effect of temperature appeared reversible. The conditions for in vitro folding were optimized and a maximum yield of 60-70% of correctly folded peptide was obtained. In vitro folded AVR9 is equally as active as native fungal AVR9. They both display similar NMR characteristics, indicating that they have the same 3D structure and identical disulfide bridges. Thus, AVR9 can be folded correctly in vitro. This folding can be described by disulfide bridge formation leading to scrambled three-disulfide species, followed by disulfide reshuffling to acquire the native structure. The presence of urea significantly affected the folding of AVR9, indicating that noncovalent interactions play a role in directing correct folding. Protein disulfide isomerase increased the folding rate at least 15-fold, but had no effect on the yield. The folding procedure has also been applied successfully to two mutant AVR9 peptides, (K23A)AVR9 and biotinylated AVR9. We conclude that the 28-residue sequence, without the preprosequence (as present in vivo), contains sufficient information to direct correct folding and disulfide bridge formation in vitro. (+info)
Sensitization to individual allergens and bronchial responsiveness in the ECRHS. European Community Respiratory Health Survey.
Little is known about the relation of bronchial responsiveness (BHR) to sensitization to individual allergens, or its variation between countries. Data were obtained for BHR, specific immunoglobulin E and confounding variables from 11,215 subjects, aged 20-44 yrs at the start of the European Community Respiratory Health Survey, in 34 centres in 15 countries. The relation of BHR to sensitization to cat, house dust mite, timothy grass and Cladosporium was estimated by means of multiple regression for each centre, and combined across centres by random effects meta-analysis, controlling for baseline lung function, height, sex, season of testing, age, smoking and age/sex and age/smoking interactions. BHR was greater, on average, in those sensitized to cat (p=0.023), house dust mite (p<0.001) and timothy grass (p=0.018), but not to Cladosporium (p=0.60), and increased with degree of sensitization (p<0.001). All relations showed heterogeneity between centres, although to a lesser extent in the relation to sensitization to house dust mite. More variation in bronchial responsiveness was explained by sensitization and degree of sensitization to the individual allergens than by atopy defined as any positive test in each centre, but the relative importance of each allergen varied. The use of atopy as a single variable in relation to bronchial hyperresponsiveness may be misleading. (+info)
Agroinfiltration is a versatile tool that facilitates comparative analyses of Avr9/Cf-9-induced and Avr4/Cf-4-induced necrosis.
The avirulence genes Avr9 and Avr4 from the fungal tomato pathogen Cladosporium fulvum encode extracellular proteins that elicit a hypersensitive response when injected into leaves of tomato plants carrying the matching resistance genes, Cf-9 and Cf-4, respectively. We successfully expressed both Avr9 and Avr4 genes in tobacco with the Agrobacterium tumefaciens transient transformation assay (agroinfiltration). In addition, we expressed the matching resistance genes, Cf-9 and Cf-4, through agroinfiltration. By combining transient Cf gene expression with either transgenic plants expressing one of the gene partners, Potato virus X (PVX)-mediated Avr gene expression, or elicitor injections, we demonstrated that agroinfiltration is a reliable and versatile tool to study Avr/Cf-mediated recognition. Significantly, agroinfiltration can be used to quantify and compare Avr/Cf-induced responses. Comparison of different Avr/Cf-interactions within one tobacco leaf showed that Avr9/Cf-9-induced necrosis developed slower than necrosis induced by Avr4/Cf-4. Quantitative analysis demonstrated that this temporal difference was due to a difference in Avr gene activities. Transient expression of matching Avr/Cf gene pairs in a number of plant families indicated that the signal transduction pathway required for Avr/Cf-induced responses is conserved within solanaceous species. Most non-solanaceous species did not develop specific Avr/Cf-induced responses. However, co-expression of the Avr4/Cf-4 gene pair in lettuce resulted in necrosis, providing the first proof that a resistance (R) gene can function in a different plant family. (+info)
Humoral immune response in chromoblastomycosis during and after therapy.
A longitudinal study was carried out in Madagascar, the most important focus of chromoblastomycosis (P. Esterre, A. Andriantsimahavandy, E. Ramarcel, and J. L. Pecarrere, Am. J. Trop. Med. Hyg. 55:45-47, 1996), to investigate natural immunity to this disease. Sequential blood samples were obtained before, during, and at the end of a successful therapeutic trial with terbinafine, a new antifungal drug. Using enzyme-linked immunosorbent assay and immunoblot methods, detailed analyses of antibody concentration and antigen mapping were conducted for 136 serum samples and tentatively correlated to epidemiological and pathobiological data. Two different cytoplasmic antigens, corresponding to the two fungal species involved (Fonsecaea pedrosoi and Cladophialophora carrionii), were used to analyze the distribution of different classes of immunoglobulins. This was done with respect to the origin of the isolates, clinical and pathobiological. Although strong individual variations were noticed, some major antigens (one of 18.5 kDa specific for F. pedrosoi and two of 23.5 and 33 kDa, respectively, specific for C. carrionii) corresponded to high antibody prevalence and concentration. As some antigenic components were also detected by immunoglobulin M (IgM) and IgA antibodies, the role that these specific antibodies could play in the immune response is discussed. (+info)
Biological screening of Brazilian medicinal plants.
In this study, we screened sixty medicinal plant species from the Brazilian savanna ("cerrado") that could contain useful compounds for the control of tropical diseases. The plant selection was based on existing ethnobotanic information and interviews with local healers. Plant extracts were screened for: (a) molluscicidal activity against Biomphalaria glabrata, (b) toxicity to brine shrimp (Artemia salina L.), (c) antifungal activity in the bioautographic assay with Cladosporium sphaerospermum and (d) antibacterial activity in the agar diffusion assay against Staphylococcus aureus, Escherichia coli, Bacillus cereus and Pseudomonas aeruginosa. Forty-two species afforded extracts that showed some degree of activity in one or more of these bioassays. (+info)
Growth and toxin production by Clostridium botulinum in moldy tomato juice.
Tomato juice inoculated with Cladosporium sp. or Penicillium sp. developed pH gradients with the upper portions near the mold mats having pH values near neutrality and the lower portions remaining more acid. Clostridium botulinum spores in these moldy tomato juices germinated, grew out, and produced toxin. (+info)
Identification of two highly divergent catalase genes in the fungal tomato pathogen, Cladosporium fulvum.
Catalases of pathogenic micro-organisms have attracted attention as potential virulence factors. Homology-based screens were performed to identify catalase genes in the fungal tomato pathogen Cladosporium fulvum. Two highly divergent genes, Cat1 and Cat2, were isolated and characterized. Cat1 codes for a putative 566-amino-acid catalase subunit and belongs to the gene family that also encodes the mainly peroxisome-localized catalases of animal and yeast species. Cat2 codes for a putative catalase subunit of 745 amino acids and belongs to a different gene family coding for the large-subunit catalases similar to ones found in bacteria and filamentous fungi. Neither catalase had an obvious secretory signal sequence. A search for an extracellular catalase was unproductive. The Cat1 and Cat2 genes showed differential expression, with the Cat1 mRNA preferentially accumulating in spores and the Cat2 mRNA preferentially accumulating in response to external H(2)O(2). With Cat2-deleted strains, activity of the Cat2 gene product (CAT2) was identified among four proteins with catalase activity separated on non-denaturing gels. The CAT2 activity represented a minor fraction of the catalase activity in spores and H(2)O(2)-stressed mycelium, and no phenotype was observed for Cat2-deleted strains, which showed a normal response to H(2)O(2) treatment. These results indicate the existence of a complex catalase system in C. fulvum, with regard to both the structure and regulation of the genes involved. In addition, efficient C. fulvum gene-replacement technology has been established. (+info)