101 Dothideomycetes genomes: A test case for predicting lifestyles and emergence of pathogens.

Dothideomycetes is the largest class of kingdom Fungi and comprises an incredible diversity of lifestyles, many of which have evolved multiple times. Plant pathogens represent a major ecological niche of the class Dothideomycetes and they are known to infect most major food crops and feedstocks for biomass and biofuel production. Studying the ecology and evolution of Dothideomycetes has significant implications for our fundamental understanding of fungal evolution, their adaptation to stress and host specificity, and practical implications with regard to the effects of climate change and on the food, feed, and livestock elements of the agro-economy. In this study, we present the first large-scale, whole-genome comparison of 101 Dothideomycetes introducing 55 newly sequenced species. The availability of whole-genome data produced a high-confidence phylogeny leading to reclassification of 25 organisms, provided a clearer picture of the relationships among the various families, and indicated that pathogenicity evolved multiple times within this class. We also identified gene family expansions and contractions across the Dothideomycetes phylogeny linked to ecological niches providing insights into genome evolution and adaptation across this group. Using machine-learning methods we classified fungi into lifestyle classes with >95 % accuracy and identified a small number of gene families that positively correlated with these distinctions. This can become a valuable tool for genome-based prediction of species lifestyle, especially for rarely seen and poorly studied species.

ABC transporters of the wheat pathogen Mycosphaerella graminicola function as protectants against biotic and xenobiotic toxic compounds.

We have studied the role of five ABC transporter genes (MgAtr to MgAtr5) from the wheat pathogen Mycosphaerella graminicola in multidrug resistance (MDR). Complementation of Saccharomyces cerevisiae mutants with the ABC transporter genes from M. graminicola showed that all the genes tested encode proteins that provide protection against chemically unrelated compounds, indicating that their products function as multidrug transporters with distinct but overlapping substrate specificities. Their substrate range in yeast includes fungicides, plant metabolites, antibiotics, and a mycotoxin derived from Fusarium graminearum (diacetoxyscirpenol). Transformants of M. graminicola in which individual ABC transporter genes were deleted or disrupted did not exhibit clear-cut phenotypes, probably due to the functional redundancy of transporters with overlapping substrate specificity. Independently generated MgAtr5 deletion mutants of M. graminicola showed an increase in sensitivity to the putative wheat defence compound resorcinol and to the grape phytoalexin resveratrol, suggesting a role for this transporter in protecting the fungus against plant defence compounds. Bioassays with antagonistic bacteria indicated that MgAtr2 provides protection against metabolites produced by Pseudomonas fluorescens and Burkholderia cepacia. In summary, our results show that ABC transporters from M. graminicola play a role in protection against toxic compounds of natural and artificial origin.

Efficient Agrobacterium tumefaciens-mediated gene disruption in the phytopathogen Mycosphaerella graminicola.

Agrobacterium tumefaciens-mediated transformation has been successfully applied to the wheat pathogen Mycosphaerella graminicola. Both protoplasts and intact cells have been transformed to hygromycin B resistance. Furthermore, A. tumefaciens-mediated transformation using homologous DNA originating from the M. graminicola ABC transporter gene MgAtr2 resulted in the efficient generation of disruption mutants. In 44% of the transformants, disruption of MgAtr2 was achieved and transformants resulted from the integration of a single copy of the transforming DNA. These results indicate that A. tumefaciens-mediated transformation is a useful tool to generate targeted gene disruption in the phytopathogen M. graminicola, where gene targeting by conventional methods is hardly possible.

Characterization of the ABC transporter genes MgAtr1 and MgAtr2 from the wheat pathogen Mycosphaerella graminicola.

ATP-binding cassette (ABC) transporters are membrane-bound transporters involved in various physiological processes. In this paper we describe the cloning of the ABC transporter encoding genes MgAtr1 and MgAtr2 from the wheat pathogen Mycosphaerella graminicola (anamorph Septoria tritici). Both deduced proteins MgAtr1 and MgAtr2 are highly homologous to other fungal ABC transporters. RT-PCR revealed that the MgAtr2 mRNA population consists of partially and fully spliced transcripts. Putative substrates of ABC transporters, modulators of ABC transporter activity, and inducers of ABC transporter gene transcription were analyzed for their potential to induce expression of MgAtr1 and MgAtr2 in m. graminicola. The genes are differently upregulated by compounds such as the plant secondary metabolites eugenol and reserpine. Similar results are obtained for several antibiotics and the azole fungicides cyproconazole and imazalil. Moreover, a different expression pattern between yeast-like cells and mycelium of this dimorphic fungus was observed. These results indicate that MgAtr1 and MgAtr2 play a role in protection of m. graminicola against natural toxic compounds and xenobiotics. A putative role in protection against plant defense compounds during pathogenesis is suggested.

Cloning, sequence and expression in Escherichia coli of the gene encoding phosphofructokinase from Bacillus macquariensis.

A chromosomal DNA fragment containing the Bacillus macquariensis (Bm) ATP-dependent phosphofructokinase-encoding gene (pfk) was cloned from a subgenomic library in pUC19 using a PCR-derived probe. The region containing pfk, including flanking sequences, was sequenced and the deduced amino acid sequence (aa) was found to be homologous to other PFK, but it contained two single-aa changes conserved in a range of other organisms from pro- and eukaryotic origins. Enzymatic studies with PFK purified from overproducing Escherichia coli (Ec) host cells showed that the Bm enzyme is similar to B. stearothermophilus (Bs) PFK in many respects and that it is relatively cold stable.

Simultaneous mutagenesis of multiple sites: application of the ligase chain reaction using PCR products instead of oligonucleotides.

A method is described for preparing mutants with multiple, site-directed mutations by ordered coupling of PCR-generated fragments catalyzed by a thermostable DNA ligase. Annealing of the sense strands of the fragments to a single-stranded (antisense) template created a full-length sense strand leaving only nicks that were closed by ligation. Mutations were introduced in the PCR primers. Following 40 cycles of denaturation and annealing, tags on the flanking primers of the ligase chain reaction product were used specifically to amplify the mutated product with specific primers that could not amplify the original template. The amplified ligation product was cloned and was found to contain the desired restriction sites introduced by way of the mutagenic primers.