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The role in virulence of the ATP-binding cassette (ABC) transporters MgAtr1, MgAtr2, MgAtr3, MgAtr4, and MgAtr5 from Mycosphaerella graminicola was analyzed by gene disruption or replacement on seedlings of the susceptible wheat cultivar Obelisk. Disruption strains of MgAtr1 and MgAtr2 and replacement strains of MgAtr3 and MgAtr5 displayed the same phenotype as control strains, while virulence of the MgAtr4 disruption strains was significantly reduced. This reduction in virulence was independent of the wheat cultivar used. Histopathological analysis of the infection process revealed that MgAtr4 disruption strains colonize substomatal cavities less efficiently and display reduced intercellular growth in the apoplast of wheat leaves. In vitro growth experiments in different media showed no fitness penalty associated with the disruption of MgAtr4. Expression analysis demonstrated that transcripts of the constitutively expressed gene CYP51 encoding the fungal-specific cytochrome P450 sterol 14α-demethylase from M. graminicola were not detectable in interaction RNA from wheat infected with MgAtr4 disruption strains, thus confirming the reduced intercellular growth of these strains. The results indicate that MgAtr4 is a virulence factor of M. graminicola during pathogenesis on wheat and may function in protection against fungitoxic compounds present around the substomatal cavities of wheat leaves. MgAtr4 is the first virulence factor cloned from this important plant pathogen.

This review provides an overview of members of the ATP-binding cassette (ABC) and major facilitator superfamily (MFS) of transporters identified in filamentous fungi. The most common function of these membrane proteins is to provide protection against natural toxic compounds present in the environment of fungi, such as antibiotics produced by other microorganisms. In plant pathogenic fungi, these transporters can also be an important determinant of virulence on host plants by providing protection against plant defence compounds or mediating the secretion of host-specific toxins. Furthermore, they play a critical role in determining base-line sensitivity to fungicides and other antimycotic agents. Overexpression of some of these transporters can lead to the development of resistance to chemically-unrelated compounds, a phenomenon described as multidrug resistance (MDR). This has been observed in a variety of organisms and can impose a serious threat to the effective control of pathogenic fungi.

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.

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 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.

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.

ATP-binding cassette (ABC) transporters belong to one of the largest protein families known. They play a role in numerous vital processes in the cell and are characterised by their capacity to transport a broad variety of substrates, ranging from simple ions to complex polypeptides. Many human diseases are correlated to malfunctioning of ABC transporters. Historically, ABC transporters became known because of their role in the development of multidrug resistance (MDR) during cancer therapy. MDR is the simultaneous development of resistance against chemically unrelated compounds and can be the consequence of overproduction of ABC transporters. MDR is not limited to human tumour cells but also occurs during chemical treatment of parasites, bacteria, and fungi. In plant pathogenic fungi ABC transporters can play a role in resistance against azole fungicides. Chapter 1 gives an overview of common functions and characteristics of ABC transporters. This thesis describes the functional analysis of ABC transporters in Mycosphaerella graminicola, one of the most important pathogens on wheat. This fungus is the causal agent of septoria tritici leaf blotch. When not properly controlled, yield losses caused by this disease can be as high as 50%. Despite its importance, not much is known about the molecular biology of this fungus. Chapter 2 describes the cloning of the first two ABC transporter genes of this fungus, MgAtrl and MgAtrl. Both genes were isolated by means of heterologous hybridisation using a probe derived from the Saccharomyces cerevisiae ABC transporter PDR5. Exposure of M. graminicola to a broad variety of chemicals showed that several compounds enhanced expression of one or both genes. Compounds capable of increasing the expression include antibiotics, plant secondary metabolites, and fungicides used to control M. graminicola, suggesting a role for MgAtrl and MgAtr2 in protection against these compounds. Another striking result is the differential expression of the two genes in yeast-like cells and mycelium, suggesting a morphology dependent regulation of expression. Chapter 3 describes the development of an Agrobacterium tumefaciens-mediated transformation protocol. In contrast to several other methods, A. tumefaciens-mediated transformation is highly effective and results in the efficient generation of disruption mutants. The establishment of this transformation protocol enabled the functional analysis of ABC transporters of M. graminicola by gene disruption. Independent knockout mutants were generated for five ABC transporter genes from M, graminicola and the virulence of these mutants was tested on wheat. As described in Chapter 4, AMgAtr4 mutants were less virulent, while all the other transformants showed unaltered virulence. However, it is still unclear how MgAtr4 exerts its action. MgAtr4 is the first described virulence factor of M. graminicola and the fourth ABC transporter described as a virulence factor in plant pathogenic fungi. The substrate specificity of the ABC transporters was studied by complementation of S. cerevisiae mutants with the M. graminicola ABC transporter genes and by analysis of the M. graminicola knockout mutants for sensitivity to a broad variety of compounds. The results of the yeast complementation assay presented in Chapter 5 clearly show that the ABC transporters tested function as multidrug transporters with overlap in substrate specificity. The substrate range in yeast includes antibiotics, fungicides, plant secondary metabolites, and a mycotoxin. The M. graminicola knockout mutants did not show altered sensitivity to any of these potential substrates. This can be explained by the fact that multiple ABC transporters exist with overlap in substrate specificity, which can take over the function of the distorted transporter. MgAtr5 deletion mutants showed a small increase in sensitivity to the putative wheat defence compound resorcinol, suggesting a role for MgAtr5 in the M. graminicolawheat pathosystem. Bioassays to test the effect of antagonistic bacteria on the growth of the ABC transporter knockout mutants, indicate that MgAtr2 can provide protection against antibiotics produced by these bacteria. Thus, the data presented in Chapter 5 indicate that ABC transporters from M. graminicola play a role in the protection against toxic compounds. Both the expression analysis (Chapter 2) and the yeast complementation data (Chapter 5) suggest that azole fungicides can act as substrates for ABC transporters from M. graminicola. In Chapter 6 the potential role of ABC transporters in fungicide sensitivity is studied in more detail. M. graminicola mutants with decreased sensitivity to the azole fungicide cyproconazole were generated and shown to exhibit a MDR phenotype. Decreased azole sensitivity correlated with altered accumulation of cyproconazole, indicative for the involvement of ABC transporters. Expression of one or more of the ABC transporter genes studied was altered in all mutants. However, changes in fungicide sensitivity did not correlate with alterations in expression of a specific ABC transporter gene. Disruption of MgAtrl in two mutants showing constitutive MgAtrl overexpression restored cyproconazole sensitivity to wild-type levels in only one of these mutants. These results show that overexpression of ABC transporters is one of the mechanisms leading to azole resistance in M. graminicola. In conclusion, the data presented in this thesis show that M. graminicola possesses ABC transporters with overlapping substrate specificity. Substrates include xenobiotics and natural toxicants. This is confirmed by the findings that ABC transporters from M. graminicola act as virulence factor and can provide protection against mycotoxins, bacterial antibiotics, and azole fungicides. Therefore, ABC transporters of M. graminicola contribute to the success of this fungus as a pathogen.