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.

ABSTRACT. We searched the genome of Mycosphaerella fijiensis for molecular markers that would allow population genetics analysis of this plant pathogen. M. fijiensis, the causal agent of banana leaf streak disease, also known as black Sigatoka, is the most devastating pathogen attacking bananas (Musa spp). Recently, the entire genome sequence of M. fijiensis became available. We screened this database for VNTR markers. Forty-two primer pairs were selected for validation, based on repeat type and length and the number of repeat units. Five VNTR markers showing multiple alleles were validated with a reference set of isolates from different parts of the world and a population from a banana plantation in Costa Rica. Polymorphism information content values varied from 0.6414 to 0.7544 for the reference set and from 0.0400 and 0.7373 for the population set. Eighty percent of the polymorphism information content values were above 0.60, indicating that the markers are highly informative. These markers allowed robust scoring of agarose gels and proved to be useful for variability and population genetics studies. In conclusion, the strategy we developed to identify and validate VNTR markers is an efficient means to incorporate markers that can be used for fungicide resistance management and to develop breeding strategies to control banana black leaf streak disease. This is the first report of VNTR-minisatellites from the M. fijiensis genome sequence.

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The advent of high-throughput sequencing technology has generated abundant information on DNA sequences for the genomes of many plant species. Expressed Sequence Tags (ESTs), which are unique DNA sequences derived from a cDNA library and therefore representing genes transcribed in specific tissues or at some stage of development, are one type of DNA sequences highly available today for many important crop species. Molecular markers are used for bridging DNA sequence information with particular phenotypes and are useful tools for genotyping germplasm collections and also for tagging genes involved in desirable agronomic traits. In this sense, there is always a strong demand for suitable marker techniques to better utilise existing sequence information. A transcriptome database from banana (Musa spp.), DATAMusa, containing 42,724 ESTs from 11 different cDNA libraries and encompassing approximately 24 Mb of DNA sequence, was used in this study for the design of primers to PCR-amplify two types of EST-derived molecular markers, Variable Nucleotide Tandem Repeat (VNTR) and Target Region Amplification Polymorphism (TRAP). These primers were then validated against a panel of 14 diploid Musa genotypes and produced 32 (VNTR) and 119 (TRAP) alleles. Used separately or together, both types of markers were able to discriminate Musa genotypes from different genome background (A or B genomes). The TRAP alleles identified were derived from only one EST, while the VNTR alleles were derived from 12 unigenes. Based on the results of this study, EST-derived markers can be an important source of polymorphism to be used in genetic diversity and gene discovery studies in banana.

Plasma membrane proteins constitute a very important class of proteins. They are involved in the transmission of external signals to the interior of the cell and selective transport of water, nutrients and ions across the plasma membrane. However, the study of plasma membrane proteins is challenging because of their poor solubility in aqueous media and low relative abundance. In this work, we evaluated four different strategies for the characterization of plasma membrane proteins from banana roots: (i) the aqueous-polymer two-phase system technique (ATPS) coupled to gelelectrophoresis (gel-based), and (ii) ATPS coupled to LC-MS/MS (gel free), (iii) a microsomal fraction and (iv) a full proteome, both coupled to LC-MS/ MS. Our results show that the gel-based strategy is useful for protein visualization but has major limitations in terms of time reproducibility and efficiency. From the gel-free strategies, the microsomal-based strategy allowed the highest number of plasma membrane proteins to be identified, followed by the full proteome strategy and by the ATPS based strategy. The high yield of plasma membrane proteins provided by the microsomal fraction can be explained by the enrichment of membrane proteins in this fraction and the high throughput of the gel-free approach combined with the usage of a fast high-resolution mass spectrometer for the identification of proteins.Index terms: Aqueous-polymer two-phase system technique, microsomal and full proteome fractions, protein identification. RESUMOProteínas da membrana plasmática constituem uma importante classe de proteínas. Elas estão envolvidas na transmissão de sinais externos para o interior da célula e no transporte seletivo de nutrientes/ions, através da membrana plasmática. Porém, o estudo dessas proteinas é difícil, porque elas são poucoabundantes e apresentam baixa solubilidade em tampões aquosos. Neste trabalho, nós avaliamos quatro estratégias diferentes para extrair proteínas da membrana plasmática de raízes de banana: (i) a técnica de sistema aquoso de duas fases, constituída porpolímeros(ATPS) combinada com eletroforese em gel e (ii) ATPS sem gel usandoLC-MS/MS, (iii) uma fração microssomal e (iv) uma fração, contendo o proteoma total celular, ambas as frações avaliadas via LC-MS/MS. Nossos resultados mostram que a estratégia baseada em eletroforese em gel é útil para a visualização de proteínas, mas apresenta limitações em termos de reproducibilidade e eficiência. Dentre as estratégias sem o uso de gel, a fração microssomal permitiu a identificação do maior número de proteínas de membrana plasmática, seguida pela fração de proteoma total e pela técnica de sistema aquoso de duasfases. O alto rendimento de proteínas de membrana plasmática proporcionado pela fração microssomal pode ser explicado pelo enriquecimento de proteínas de membrana nessa fração e pela eficiência do espectrômetro de massa na identificação de proteínas.Termos para indexação: Sistema aquoso de duas fases, frações microssomal e de proteoma total, identificação de proteín...