ABC transporters of the wheat pathogen Mycosphaerella graminicola

<strong> A</strong> TP- <strong>b</strong> inding <strong>c</strong> assette (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, MgAtr1 and MgAtr2 . 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 MgAtr1 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,<FONT FACE="Symbol">D</font>MgAtr4 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. graminicola -wheat 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 correlatewith alterations in expression of a specific ABC transporter gene. Disruption of MgAtr1 in two mutants showing constitutive MgAtr1 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