Bcmfs1, a novel major facilitator superfamily transporter from Botrytis cinerea,provides tolerance towards the natural toxic compounds camptothecin and cercosporin and towards fungicides

Bcmfs1, a novel major facilitator superfamily gene from Botrytis cinerea, was cloned, and replacement and overexpression mutants were constructed to study its function. Replacement mutants showed increased sensitivity to the natural toxic compounds camptothecin and cercosporin, produced by the plant Camptotheca acuminata and the plant pathogenic fungus Cercospora kikuchii, respectively. Overexpression mutants displayed decreased sensitivity to these compounds and to structurally unrelated fungicides, such as sterol demethylation inhibitors (DMIs). A double-replacement mutant of Bcmfs1 and the ATP-binding cassette (ABC) transporter gene BcatrD was more sensitive to DMI fungicides than a single-replacement mutant of BcatrD, known to encode an important ABC transporter of DMIs. The sensitivity of the wild-type strain and mutants to DMI fungicides correlated with Bcmfs1 expression levels and with the initial accumulation of oxpoconazole by germlings of these isolates. The results indicate that Bcmfs1 is a major facilitator superfamily multidrug transporter involved in protection against natural toxins and fungicides and has a substrate specificity that overlaps with the ABC transporter BcatrD. Bcmfs1 may be involved in protection of B. cinerea against plant defense compounds during the pathogenic phase of growth on host plants and against fungitoxic antimicrobial metabolites during its saprophytic phase of growth.     

MgHog1 regulates dimorphism and pathogenicity in the fungal wheat pathogen Mycosphaerella graminicola

The dimorphic ascomycete pathogen Mycosphaerella graminicola switches from a yeastlike form to an infectious filamentous form that penetrates the host foliage through stomata. We examined the biological function of the mitogen-activated protein kinase-encoding gene MgHog1 in M. graminicola. Interestingly, MgHog1 mutants were unable to switch to filamentous growth on water agar that mimics the nutritionally poor conditions on the foliar surface and, hence, exclusively developed by a yeastlike budding process. Consequently, due to impaired initiation of infectious germ tubes, as revealed by detailed in planta cytological analyses, the MgHog1 mutants failed to infect wheat leaves. We, therefore, conclude that MgHog1 is a new pathogenicity factor involved in the regulation of dimorphism in M. graminicola. Furthermore, MgHog1 mutants are osmosensitive, resistant to phenylpyrrole and dicarboximide fungicides, and do not melanize.     

Origin and domestication of the fungal wheat pathogen Mycosphaerella graminicola via sympatric speciation

The Fertile Crescent represents the center of origin and earliest known place of domestication for many cereal crops. During the transition from wild grasses to domesticated cereals, many host-specialized pathogen species are thought to have emerged. A sister population of the wheat-adapted pathogen Mycosphaerella graminicola was identified on wild grasses collected in northwest Iran. Isolates of this wild grass pathogen from 5 locations in Iran were compared with 123 M. graminicola isolates from the Middle East, Europe, and North America. DNA sequencing revealed a close phylogenetic relationship between the pathogen populations. To reconstruct the evolutionary history of M. graminicola, we sequenced 6 nuclear loci encompassing 464 polymorphic sites. Coalescence analyses indicated a relatively recent origin of M. graminicola, coinciding with the known domestication of wheat in the Fertile Crescent around 8,000-9,000 BC. The sympatric divergence of populations was accompanied by strong genetic differentiation. At the present time, no genetic exchange occurs between pathogen populations on wheat and wild grasses although we found evidence that gene flow may have occurred since genetic differentiation of the populations.     

The landscape of transposable elements in the finished genome of the fungal wheat pathogen Mycosphaerella graminicola

Background

In addition to gene identification and annotation, repetitive sequence analysis has become an integral part of genome sequencing projects. Identification of repeats is important not only because it improves gene prediction, but also because of the role that repetitive sequences play in determining the structure and evolution of genes and genomes. Several methods using different repeat-finding strategies are available for whole-genome repeat sequence analysis. Four independent approaches were used to identify and characterize the repetitive fraction of the Mycosphaerella graminicola (synonym Zymoseptoria tritici) genome. This ascomycete fungus is a wheat pathogen and its finished genome comprises 21 chromosomes, eight of which can be lost with no obvious effects on fitness so are dispensable.

Results

Using a combination of four repeat-finding methods, at least 17% of the M. graminicola genome was estimated to be repetitive. Class I transposable elements, that amplify via an RNA intermediate, account for about 70% of the total repetitive content in the M. graminicola genome. The dispensable chromosomes had a higher percentage of repetitive elements as compared to the core chromosomes. Distribution of repeats across the chromosomes also varied, with at least six chromosomes showing a non-random distribution of repetitive elements. Repeat families showed transition mutations and a CpA → TpA dinucleotide bias, indicating the presence of a repeat-induced point mutation (RIP)-like mechanism in M. graminicola. One gene family and two repeat families specific to subtelomeres also were identified in the M. graminicola genome. A total of 78 putative clusters of nested elements was found in the M. graminicola genome. Several genes with putative roles in pathogenicity were found associated with these nested repeat clusters. This analysis of the transposable element content in the finished M. graminicola genome resulted in a thorough and highly curated database of repetitive sequences.

Conclusions

This comprehensive analysis will serve as a scaffold to address additional biological questions regarding the origin and fate of transposable elements in fungi. Future analyses of the distribution of repetitive sequences in M. graminicola also will be able to provide insights into the association of repeats with genes and their potential role in gene and genome evolution.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1132) contains supplementary material, which is available to authorized users.     

Finished genome of the fungal wheat pathogen Mycosphaerella graminicola reveals dispensome structure, chromosome plasticity, and stealth pathogenesis

The plant-pathogenic fungus Mycosphaerella graminicola (asexual stage: Septoria tritici) causes septoria tritici blotch, a disease that greatly reduces the yield and quality of wheat. This disease is economically important in most wheat-growing areas worldwide and threatens global food production. Control of the disease has been hampered by a limited understanding of the genetic and biochemical bases of pathogenicity, including mechanisms of infection and of resistance in the host. Unlike most other plant pathogens, M. graminicola has a long latent period during which it evades host defenses. Although this type of stealth pathogenicity occurs commonly in Mycosphaerella and other Dothideomycetes, the largest class of plant-pathogenic fungi, its genetic basis is not known. To address this problem, the genome of M. graminicola was sequenced completely. The finished genome contains 21 chromosomes, eight of which could be lost with no visible effect on the fungus and thus are dispensable. This eight-chromosome dispensome is dynamic in field and progeny isolates, is different from the core genome in gene and repeat content, and appears to have originated by ancient horizontal transfer from an unknown donor. Synteny plots of the M. graminicola chromosomes versus those of the only other sequenced Dothideomycete, Stagonospora nodorum, revealed conservation of gene content but not order or orientation, suggesting a high rate of intra-chromosomal rearrangement in one or both species. This observed "mesosynteny" is very different from synteny seen between other organisms. A surprising feature of the M. graminicola genome compared to other sequenced plant pathogens was that it contained very few genes for enzymes that break down plant cell walls, which was more similar to endophytes than to pathogens. The stealth pathogenesis of M. graminicola probably involves degradation of proteins rather than carbohydrates to evade host defenses during the biotrophic stage of infection and may have evolved from endophytic ancestors.     

A group of expressed cDNA sequences from the wheat fungal leaf blotch pathogen, Mycosphaerella graminicola (Septoria tritici)

A group of expressed sequence tags (ESTs) from the wheat fungal pathogen Mycosphaerella graminicola utilizing ammonium as a nitrogen source has been analyzed. Single pass sequences of complementary DNAs from 986 clones were determined. Contig analysis and sequence comparisons allowed 704 unique ESTs (unigenes) to be identified, of which 148 appeared as multiple copies. Searches of the nrdb95 protein database at EMBL using the BLAST2x algorithm revealed 407 (57.8%) sequences that generated high to moderate high scoring pairs with proteins of known and unknown function. The rest of the sequences (297) showed either weak or no similarities to database entries. Among the unigenes with assigned function, 26.7% were involved in primary metabolism and 17.9% were associated with protein and RNA metabolism. Fewer clones were ascribed roles in signal transduction (4.9%), transport and secretion (6.1%), cell structure (3.1%), and cell division (3.6%). Approximately 18.1% of the identities found were to hypothetical or unknown proteins mainly from the yeasts Saccharomyces cerevisiae and Schizosaccaromyces pombe. Comparison of the 297 sequences with no clear function to other fungal ESTs in the public domain revealed 12 sequences that had high to moderate similarity to Neurospora crassa, Emericella (Aspergillus) nidulans, or Magnaporthe grisea sequences.     

MgSlt2, a cellular integrity MAP kinase gene of the fungal wheat pathogen Mycosphaerella graminicola, is dispensable for penetration but essential for invasive growth

Among expressed sequence tag libraries of Mycosphaerella graminicola isolate IPO323, we identified a full-length cDNA clone with high homology to the mitogen-activated protein (MAP) kinase Slt2 in Saccharomyces cerevisiae. This MAP kinase consists of a 1242-bp open reading frame, and encodes a 414-amino-acid protein. We designated this homolog MgSlt2, generated MgSlt2 knockout strains in M. graminicola isolate IPO323, and found several altered phenotypes in vitro as well as in planta. In yeast glucose broth, MgSlt2 disruptants showed a defective polarized growth in the tip cells upon aging, causing substantial local enlargements culminating in large swollen cells containing two to four nuclei. The MgSlt2 disruptants showed a significantly increased sensitivity to several fungicides, including miconazole (2x), bifonazole (>4x), imazalil (5x), and cyproconazole (10x), and were hypersensitive to glucanase. Unlike the wild type, MgSlt2 disruptants did not produce aerial mycelia and did not melanize on potato dextrose agar. Although cytological analysis in planta showed normal penetration of wheat stomata by the germ tubes of the MgSlt2 disruptants, subsequently formed hyphal filaments frequently were unable to branch out and establish invasive growth resulting in highly reduced virulence, and prevented pycnidia formation. Therefore, we conclude that MgSlt2 is a new pathogenicity factor in M. graminicola.     

Mutagenesis and functional studies with succinate dehydrogenase inhibitors in the wheat pathogen Mycosphaerella graminicola

A range of novel carboxamide fungicides, inhibitors of the succinate dehydrogenase enzyme (SDH, EC 1.3.5.1) is currently being introduced to the crop protection market. The aim of this study was to explore the impact of structurally distinct carboxamides on target site resistance development and to assess possible impact on fitness. We used a UV mutagenesis approach in Mycosphaerella graminicola, a key pathogen of wheat to compare the nature, frequencies and impact of target mutations towards five subclasses of carboxamides. From this screen we identified 27 amino acid substitutions occurring at 18 different positions on the 3 subunits constituting the ubiquinone binding (Qp) site of the enzyme. The nature of substitutions and cross resistance profiles indicated significant differences in the binding interaction to the enzyme across the different inhibitors. Pharmacophore elucidation followed by docking studies in a tridimensional SDH model allowed us to propose rational hypotheses explaining some of the differential behaviors for the first time. Interestingly all the characterized substitutions had a negative impact on enzyme efficiency, however very low levels of enzyme activity appeared to be sufficient for cell survival. In order to explore the impact of mutations on pathogen fitness in vivo and in planta, homologous recombinants were generated for a selection of mutation types. In vivo, in contrast to previous studies performed in yeast and other organisms, SDH mutations did not result in a major increase of reactive oxygen species levels and did not display any significant fitness penalty. However, a number of Qp site mutations affecting enzyme efficiency were shown to have a biological impact in planta.Using the combined approaches described here, we have significantly improved our understanding of possible resistance mechanisms to carboxamides and performed preliminary fitness penalty assessment in an economically important plant pathogen years ahead of possible resistance development in the field.     

Whole-genome and chromosome evolution associated with host adaptation and speciation of the wheat pathogen Mycosphaerella graminicola

The fungus Mycosphaerella graminicola has been a pathogen of wheat since host domestication 10,000-12,000 years ago in the Fertile Crescent. The wheat-infecting lineage emerged from closely related Mycosphaerella pathogens infecting wild grasses. We use a comparative genomics approach to assess how the process of host specialization affected the genome structure of M. graminicola since divergence from the closest known progenitor species named M. graminicola S1. The genome of S1 was obtained by Illumina sequencing resulting in a 35 Mb draft genome sequence of 32X. Assembled contigs were aligned to the previously sequenced M. graminicola genome. The alignment covered >90% of the non-repetitive portion of the M. graminicola genome with an average divergence of 7%. The sequenced M. graminicola strain is known to harbor thirteen essential chromosomes plus eight dispensable chromosomes. We found evidence that structural rearrangements significantly affected the dispensable chromosomes while the essential chromosomes were syntenic. At the nucleotide level, the essential and dispensable chromosomes have evolved differently. The average synonymous substitution rate in dispensable chromosomes is considerably lower than in essential chromosomes, whereas the average non-synonymous substitution rate is three times higher. Differences in molecular evolution can be related to different transmission and recombination patterns, as well as to differences in effective population sizes of essential and dispensable chromosomes. In order to identify genes potentially involved in host specialization or speciation, we calculated ratios of synonymous and non-synonymous substitution rates in the >9,500 aligned protein coding genes. The genes are generally under strong purifying selection. We identified 43 candidate genes showing evidence of positive selection, one encoding a potential pathogen effector protein. We conclude that divergence of these pathogens was accompanied by structural rearrangements in the small dispensable chromosomes, while footprints of positive selection were present in only a small number of protein coding genes.     

Significant difference in pathogenicity between MAT1-1 and MAT1-2 isolates in the wheat pathogen Mycosphaerella graminicola

Five Mycosphaerella graminicola populations from four geographic regions (Australia, Israel, Switzerland, and the USA) were assayed for neutral RFLP markers and mating type idiomorphs. On average, 25-30 genetically distinct isolates were selected from each population and their pathogenicity was measured on two wheat cultivars in a common garden experiment conducted in a greenhouse. A significant difference in pathogenicity was found between MAT1-1 and MAT1-2 isolates. On average, MAT1-1 isolates had 14-22% greater pathogenicity than MAT1-2 isolates. The pattern of higher pathogenicity in MAT1-1 isolates was consistent across four geographical populations and on two wheat cultivars. A uniform and continuous variation in pathogenicity was found among isolates within each mating type, but no genetic differentiation in selectively neutral RFLP loci was found between mating types, consistent with the hypothesis that differences in pathogenicity were not due to the effects of specific pathogenicity genes or non-random genetic backgrounds.     

Functional and biochemical characterisation of the Escherichia coli major facilitator superfamily multidrug transporter MdtM

Multidrug resistance (MDR) occurs when bacteria simultaneously acquire resistance to a broad spectrum of structurally dissimilar compounds to which they have not previously been exposed. MDR is principally a consequence of the active transport of drugs out of the cell by proteins that are integral membrane transporters. We characterised and purified the putative Escherichia coli MDR transporter, MdtM, a 410 amino acid residue protein that belongs to the large and ubiquitous major facilitator superfamily. Functional characterisation of MdtM using growth inhibition and whole cell transport assays revealed its role in intrinsic resistance of E. coli cells to the antimicrobials ethidium bromide and chloramphenicol. Site-directed mutagenesis studies implied that the MdtM aspartate 22 residue and the highly conserved arginine at position 108 play a role in proton recognition. MdtM was homologously overexpressed and purified to homogeneity in dodecyl-β-D-maltopyranoside detergent solution and the oligomeric state and stability of the protein in a variety of detergent solutions was investigated using size-exclusion HPLC. Purified MdtM is monomeric and stable in dodecyl-β-D-maltopyranoside solution and binds chloramphenicol with nanomolar affinity in the same detergent. This work provides a firm foundation for structural studies on this class of multidrug transporter protein.     

A gene of the major facilitator superfamily encodes a transporter for enterobactin (Enb1p) in Saccharomyces cerevisiae

While in fungi iron transport via hydroxamate siderophores has been amply proven, iron transport via enterobactin is largely unknown. Enterobactin is a catecholate-type siderophore produced by several enterobacterial genera grown in severe iron deprivation. By using the KanMX disruption module in vector pUG6 in a fet3 background of Saccharomyces cerevisiae we were able to disrupt the gene YOL158c Sce of the major facilitator super family (MFS) which has been previously described as a gene encoding a membrane transporter of unknown function. Contrary to the parental strain, the disruptant was unable to utilize ferric enterobactin in growth promotion tests and in transport assays using ⁵⁵Fe-enterobactin. All other siderophore transport properties remained unaffected. The results are evidence that in S. cerevisiae the YOL158c Sce gene of the major facilitator super family, now designated ENB1, encodes a transporter protein (Enb1p), which specifically recognizes and transports enterobactin.     

Electrophoretic and cytological karyotyping of the foliar wheat pathogen Mycosphaerella graminicola reveals many chromosomes with a large size range.

The karyotypes of three isolates of Mycosphaerella graminicola, the septoria tritici blotch pathogen of wheat, were analyzed with both pulsed field gel electrophoresis (PFGE) and the cytological technique called germ tube burst method (GTBM). These analyses revealed a chromosome length polymorphism among these isolates. The estimated genome size was 31-40 Mb depending on the isolates, indicating 17-22% redundancy in the genome of the standard strain IP0323 because such differences do not affect development, pathogenicity and sexual reproduction of the other isolates. The chromosome numbers in the three isolates were 18-20 and the chromosome size was 0.3-6 Mb. These data show that M. graminicola has the highest chromosome number and the smallest autosomes (A chromosomes) in filamentous ascomycetes. Our data also confirmed a large (> or =6 Mb) chromosome that was assembled recently in the IPO323 genome sequence. GTBM analyses revealed the mitotic metaphase chromosomes, enabling chromosome quantification, which was fully congruent with the PFGE analyses. These data will be instrumental in the final assembly of the M. graminicola genome.     

The global genetic structure of the wheat pathogen Mycosphaerella graminicola is characterized by high nuclear diversity,low mitochondrial diversity,regular recombination,and gene flow

A total of 1673 Mycosphaerella graminicola strains were assayed for DNA fingerprints and restriction fragment length polymorphism (RFLP) markers in the nuclear and mitochondrial genomes. The isolates were collected from 17 wheat fields located in 11 countries on five continents over a six year period (1989-1995). Our results indicate that genetic diversity in the nuclear genome of this fungus was high for all but three of the field populations surveyed and that populations sampled from different continents had similar frequencies for the most common RFLP alleles. Hierarchical analysis revealed that more than 90% of global gene diversity was distributed within a wheat field, while approximately 5% of gene diversity was distributed among fields within regions and approximately 3% was distributed among regions on different continents. These findings suggest that gene flow has occurred on a global scale. On average, each leaf was colonized by a different nuclear genotype. In contrast, only seven mtDNA haplotypes were detected among the 1673 isolates and the two most common mtDNA haplotypes represented approximately 93% of the world population, consistent with a selective sweep. Analysis of multilocus associations indicated that all field populations were in gametic equilibrium, suggesting that sexual recombination is a regular occurrence globally.