Black Sigatoka or black leaf streak disease, caused by the Dothideomycete fungus Pseudocercospora fijiensis (previously: Mycosphaerella fijiensis), is the most significant foliar disease of banana worldwide. Due to the lack of effective host resistance, management of this disease requires frequent fungicide applications, which greatly increase the economic and environmental costs to produce banana. Weekly applications in most banana plantations lead to rapid evolution of fungicide-resistant strains within populations causing disease-control failures throughout the world. Given its extremely high economic importance, two strains of P. fijiensis were sequenced and assembled with the aid of a new genetic linkage map. The 74-Mb genome of P. fijiensis is massively expanded by LTR retrotransposons, making it the largest genome within the Dothideomycetes. Melting-curve assays suggest that the genomes of two closely related members of the Sigatoka disease complex, P. eumusae and P. musae, also are expanded. Electrophoretic karyotyping and analyses of molecular markers in P. fijiensis field populations showed chromosome-length polymorphisms and high genetic diversity. Genetic differentiation was also detected using neutral markers, suggesting strong selection with limited gene flow at the studied geographic scale. Frequencies of fungicide resistance in fungicide-treated plantations were much higher than those in untreated wild-type P. fijiensis populations. A homologue of the Cladosporium fulvum Avr4 effector, PfAvr4, was identified in the P. fijiensis genome. Infiltration of the purified PfAVR4 protein into leaves of the resistant banana variety Calcutta 4 resulted in a hypersensitive-like response. This result suggests that Calcutta 4 could carry an unknown resistance gene recognizing PfAVR4. Besides adding to our understanding of the overall Dothideomycete genome structures, the P. fijiensis genome will aid in developing fungicide treatment schedules to combat this pathogen and in improving the efficiency of banana breeding programs.

Several changes in the sequence of the gene encoding sterol 14alpha-demethylase were found that have been described in other fungi as being correlated with resistance to azole fungicides. No correlation was found between gene expression and propiconazole resistance.

Two perinaphthenone-type compounds (1 and 2) were isolated together with four known phenylphenalenones (3-6) from the rhizomes of Musa acuminata var. "Yangambi km 5". The structures of the new phenalenones were assigned as 2-hydroxy-1H-phenalen-1-one (1) and 2-methoxy-1H-phenalen-1-one (2) on the basis of their spectroscopic data and were confirmed by synthesis. Compounds 1 and 2 displayed significantly enhanced activity against Mycosphaerella fijiensis in comparison with other phenylphenalenones.

For proteins in solution the validity of certain crystallographic parameters can be ascertained by a combination of molecular-dynamics (MD) simulations and NMR spectroscopy. Using the laser photo-CIDNP (chemically induced dynamic nuclear polarization) technique as a measure for surface accessibility of histidine, tyrosine and tryptophan, the spectra of bovine galectin-I and Erythrina corallodendron lectin (EcorL) are readily reconcilable with the crystallographic data for these two proteins. The results emphasise the role of Trp68/Trp69 for carbohydrate binding in bovine galectin-l/chicken galectins and of Trp194 in murine galectin-3. This feature derived from the crystal structure of' bovine galectin-I is maintained in solution for the prototype human homologue, two avian galectins and the chimera-type murine galectin-3, as the spectra corroborate the CIDNP-inferable spatial parameters of the four calculated models for binding-site architecture. In EcorL, Tyr106/Tyr108 are constituents of the extended combining pocket, which can be shielded in solution by ligand presence. Discrepancies between results from modelling and CIDNP measurements concern primarily the lack of reactivity of histidine residues for human and avian prototype galectins and of Tyr82lTyr229 of the plant lectin. Site-directed mutagenesis of EcorL is assumed to provide information on the role of a certain residue for functional aspects. When singlesite mutants of EcorL ([Alal06]EcorL, [Alal08]EcorL, [Ala229]EcorL) were subjected to moleculardynamics (MD) simulations, the apparent surface accessibilities even of spatially separated amino acid side chains could non-uniformly be affected. This conclusion is supported by the assessment of the spectra for the mutant proteins. On the basis of these CIDNP-results modelling of the binding-site architecture of the lectin indicates the occurrence of notable alterations in the orientation of Tyr106/Tyr108 phenyl rings. The implied potential effect of single-site mutations on conformational features of a protein will deserve attention for the interpretation of studies comparing wild-type and mutant proteins.Keywords: galectin ; lectin ; Erythrina agglutinin ; NMR; molecular modeling.X-ray crystallography of proteins provides detailed information on three-dimensional structure. The precision of the derived spatial parameters of the protein in the crystal, however, should not be considered as proof for the solution structure. Among other factors, the often applied non-physiological conditions to favour crystal growth and the intermolecular interactions during packaging may lead to deviations from the solution structure(s) especially for surface group features (Wagner et al., 1992;MacArthur et al., 1994). Consequently, it is desirable to employ a sensitive spectroscopic method which in combination with mo- lecular-dynamics (MD) simulations can verify the applicability of distinct properties determined by crystallographic analysis for the solution structure. With a focus on surface epitopes, whose mobility can...

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Black leaf streak or "black Sigatoka" is one of the most important diseases affecting bananas and plantains worldwide. Very few studies have been published on the host-pathogen interaction of this pathosystem, particularly at the molecular level. The aim of this work was to analyze, under controlled conditions, the enzyme activity of peroxidase (POX), phenylalanine ammonia lyase (PAL), β-1, 3-glucanase (GLU) and chitinase (CHI) as well as the production of H 2 O 2 in banana plants infected with Mycosphaerella fijiensis. Defence responses were examined and compared in a resistant (Calcutta 4) and a susceptible (Williams) cultivar. Plants were inoculated and tested for enzyme activity at 0, 6, 12, 18, 24, 48 and 72 h after infection (HAI) and 6, 9, 12, 15 and 18 days after inoculation (DAI). A rapid induction of PAL, POX and GLU was observed in the resistant cultivar at 6-18 HAI as well as H 2 O 2 production at 72 HAI. In contrast, in the susceptible cultivar, induction of these enzymes was only observed from 6 DAI. These results suggest that the first 72 HAI are important in determining the response of the host to the disease. Further studies characterizing banana responses to M. fijiensis at the early stages of the infection are necessary in order to better understand this host-pathogen interaction.

The levels of native fungitoxic perinaphthenone phytoalexins in susceptible Musa varieties (banana), which are commercially grown in large plantations, are too low to provide plants with long-lasting protection against highly pathogenic fungi. Novel strategies for plant protection are necessary to reduce crop losses and to prevent the development of resistant fungal strains. The synthesis of novel fungicides based on the structures of perinaphthenone natural products is considered to be a promising strategy. Thirteen substituted perinaphthenones, among them two known natural products (1, 2) and 11 synthetics (3-13), were evaluated for their activity against Mycosphaerella fijiensis , and their half-maximal inhibitory concentrations (IC(50)) were calculated to establish structure-activity relationships (SAR). A SAR trend was hypothesized, leading to the design of a new compound, 4-methoxy-2-nitro-1H-phenalen-1-one (14); the new compound displayed significantly enhanced in vitro activity against M. fijiensis compared to other perinaphthenone derivatives. The activity of 14 was comparable to that of two commercial fungicides.

The cDNA of the Erythrina corallodendron lectin (ECorL) has been expressed in Escherichia coli. For this purpose, an NcoI site was inserted into the cDNA coding for the lectin precursor [Arango, R., Rozenblatt, S. & Sharon, N. (1990) FEBS Lett. 264, 109-1121 immediately before the codon GTG (103 -105) which codes for the N-terminal valine of the mature lectin. This introduced an ATG codon for a methionine preceding the valine. The mutated cDNA was ligated into pUC-8, then subcloned into the expression vector pET-3d, which carries a strong promoter derived from gene 10 of the phage T7. The recombinant plasmid was introduced into the E. coli lysogenic strain BL21(DE3).Recombinant ECorL was expressed by growing the bacteria in the presence of isopropyl a-Dthiogalactopyranoside. Most of the recombinant lectin was found in an insoluble aggregated form as inclusion bodies and only a small part was in the culture medium in a soluble active form. Functional recombinant lectin was recovered from the inclusion bodies by solubilization with 6 M urea in cyclohexylaminopropane sulfonate pH 10.5, renaturation by 1 0-fold dilution in the same buffer and further adjustment of the pH to 8.0. The recombinant lectin, obtained at a yield of 4-7 mg/l culture, had, by gel filtration, a slightly lower molecular mass (56 kDa) than the native lectin, and was devoid of covalently linked carbohydrate; it was, however, essentially indistinguishable from native ECorL by other criteria, including its dimeric structure, Western blot analysis with anti-ECorL polyclonal and monoclonal antibodies, and Ouchterlony double-diffusion analysis with polyclonal antibodies, as well as hemagglutinating activity and specificity for mono-or disaccharides.Lectins are carbohydrate-binding proteins present in a variety of organisms ranging from bacteria to higher vertebrates [I, 21. The most thoroughly studied lectins are those extracted from plants, especially from the seeds of the leguminosae [3, 41. They serve as invaluable carbohydratespecific tools in many areas of biological and medical research, and are also used clinically. Legume lectins comprise a large group of proteins that share extensive similarities in their primary amino acid sequences, and possess similar secondary and tertiary structures [4 -61. Despite these similarities, they display a wide range of carbohydrate specificities. The structural basis of these specificities is most likely due to differences in the architecture of the variable parts of their binding sites Erythrina is a family of over 100 species of deciduous leguminous trees and shrubs found in the tropics and subtropics. Since 1980, lectins from over 20 species of this family have been isolated in different laboratories, ten of these by us [3, 81. All Erythrina lectins are composed of two identical, or nearly identical, subunits with molecular masses close to 30 kDa. They are glycoproteins containing 3 -9% carbohydrate and are specific for galactose and N-acetylgalactosamine, with a preference for N-acetyllactosamine which ...