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. Black Sigatoka or black leaf streak disease, caused by the ascomycete fungus Pseudocercospora fijiensis, inflicts huge costs on banana producers, due to crop losses and expenses for disease control. The global banana export trade relies on Cavendish clones that are highly susceptible to P. fijiensis. Sustainable production of the world’s number one fruit requires a better understanding of host resistance and sophisticated management of fungicide resistance in the pathogen. In the P. fijiensis genome sequence we identified an effector that induced an HR-like necrosis on a resistant banana accession but not on a susceptible cultivar. If validated, this assay may be useful for identifying resistance in banana breeding programs. We also used the genomic sequence to develop highly polymorphic molecular markers for analyzing P. fijiensis field populations and identified a strong enrichment (nearly 100%) for fungicide resistance markers in fungicide-treated banana plantations compared to untreated wild-type populations. This rapid evolution of fungicide resistance poses an immediate threat to sustainable banana production.

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 photoCIDNP (chemically induced dynamic nuclear polarization) technique as a measure for surface accessibility of histidine, tyrosine and tryptophan, the spectra of bovine galectin‐1 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‐1/chicken galectins and of Trpl94 in murine galectin‐3. This feature derived from the crystal structure of bovine galectin‐1 is maintained in solution for the prototype human homologue, two avian galectins and the chimeratype 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 Tyr82/Tyr229 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 single‐site mutants of EcorL ([Ala106]EcorL, [Ala108]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.

Electrospray mass spectrometry was used to accurately measure the molecular masses of single chain lectins from legume seeds and also of three recombinant lectins, expressed in Escherichia coli. The five single chain lectins, Erythrina corallodendron lectin, soybean and peanut agglutinins, Dolichos biflorus lectin, and Phaseolus vulgaris hemagglutinin E, all showed evidence of C-terminal proteolytic processing, in some cases to “ragged” ends, when their masses were compared to those expected from their cDNA sequences and their known carbohydrate chains. Recombinant forms of the lectins from E. corallodendron, soybean, and peanut also showed C-terminal trimming, but not to the same points as the natural forms. Discrepancies between the protein and cDNA sequences of the E. corallodendron lectin were resolved by combined liquid chromatography-mass spectrometry peptide mapping and protein sequencing experiments, and the presence of a second glycosylation site was demonstrated. Our data show that all of these lectins undergo C-terminal proteolytic processing of a readily attacked peptide segment. This trimming is frequently imprecise, and the resulting heterogeneity may be a major contributor to the appearance of isolectin forms of these proteins.