ABC and MFS transporters from Botrytis cinerea involved in sensitivity to fungicides and natural toxic compounds
ATP-binding cassette (ABC) and major facilitator superfamily (MFS) transporters are two major classes of proteins involved in drug resistance. ABC transporter proteins are primary transporters that use the energy generated by ATP hydrolysis to transport drugs over membranes, while MFS transport proteins are secondary transporters that use the proton motive force as an energy source. The substrate range of both transporters is very broad and may include ions, amino acids, peptides, sugars, secondary metabolites, and drugs. The goal of this thesis was to identify ABC and MFS transporter genes from Botrytis cinerea, which are involved in transport of sterol demethylation inhibitor (DMI) fungicides, and to discover compounds, which can modulate the activity of such transporters. Such modulators may be useful in practice to counteract resistance development to DMIs.An overview of characteristics of B. cinerea , commercial fungicides used in control of the pathogen, modes of action of botryticides, and resistance development to fungicides that inhibit sterol biosynthesis is described in Chapter 1. This chapter also describes the relevance of ABC and MFS transporters in fungicide resistance and the importance of fungicide mixtures to delay resistance development.The DMI fungicide oxpoconazole, developed by Ube industries, Ltd., is introduced in Chapter 2. This fungicide is effective against diseases that are commonly controlled by DMIs, such as rusts and scabs. In addition, it is effective against grey mould caused by B. cinerea under field condition. B. cinerea is known for its ability to acquire resistance to fungicides easily. ABC and MFS transporters may play a role in such a resistance development. The physiological functions of these transporters may be to cope with natural plant toxins since the pathogen has an extraordinary wide host range.Chapter 3 describes fourteen ABC and three MFS transporter genes from B. cinerea . Two of the ABC transporters were described previously and the others were identified in an EST library of the fungus. Their role in DMI resistance was investigated by studying basal and induced expression in wild-type and DMI-resistant strains of B. cinerea. From this screen, it appeared that BcatrD was the most probable ABC transporter gene encoding a putative DMI transporter since basal expression in three isolates correlated with sensitivity to DMIs. Induced expression after treatment with DMIs also correlated with sensitivityto DMIs. The MFS transporter gene Bcmfs1 might encode another DMI transporter since its basal expression in DMI resistant strains was also higher than that in the wild-type strain.The functional analysis of BcatrD and Bcmfs1 is described in Chapters 4 and 5, respectively. This was achieved by phenotyping of gene replacement and overexpression mutants generated from the haploid wild-type strain B05.10. Replacement and overexpression mutants of BcatrD displayed an increased and decreased sensitivity to DMIs, respectively. Overexpression mutants of Bcmfs1 also exhibited a decreased sensitivity to these fungicides while Bcmfs1 replacement mutants showed similar sensitivity to DMIs as compared to the wild-type. To clarify the role of Bcmfs1 in DMI-sensitivity in more detail, we constructed a double replacement mutant of BcatrD and Bcmfs1 . The double replacement mutant was more sensitive to DMIs than the single replacement mutant of BcatrD. These results suggest that BcatrD functions as a major DMI transporter and that Bcmfs1 is of minor importance. Accumulation of oxpoconazole by germlings of the mutants is also described in Chapter 4 and 5. Accumulation of oxpoconazole was transient in time as observed before for other DMIs and other filamentous fungi. The transient accumulation pattern is the result of passive influx and inducible active efflux of fungicides by transporters. The initial accumulation (20 min after the addition of oxpoconazole) level of oxpoconazole in the strains tested correlated with sensitivity to oxpoconazole. The steady state levels (more than 60 min after the addition of oxpoconazole) of accumulation by BcatrD and Bcmfs1 replacement mutants were higher than that by the wild-type isolate. These results indicate that BcatrD and Bcmfs1 mediate the sensitivity of B. cinerea to oxpoconazole by reducing the accumulation of the fungicide in mycelial cells.
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| Format: | Doctoral thesis biblioteca |
| Language: | English |
| Subjects: | atp, botrytis cinerea, fungicide tolerance, fungicides, multiple drug resistance, pesticide resistance, plant pathogenic fungi, plant pathogens, fungiciden, fungicidentolerantie, meervoudige resistentie tegen geneesmiddelen, plantenziekteverwekkende schimmels, plantenziekteverwekkers, resistentie tegen pesticiden, |
| Online Access: | https://research.wur.nl/en/publications/abc-and-mfs-transporters-from-botrytis-cinerea-involved-in-sensit |
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| Summary: | ATP-binding cassette (ABC) and major facilitator superfamily (MFS) transporters are two major classes of proteins involved in drug resistance. ABC transporter proteins are primary transporters that use the energy generated by ATP hydrolysis to transport drugs over membranes, while MFS transport proteins are secondary transporters that use the proton motive force as an energy source. The substrate range of both transporters is very broad and may include ions, amino acids, peptides, sugars, secondary metabolites, and drugs. The goal of this thesis was to identify ABC and MFS transporter genes from Botrytis cinerea, which are involved in transport of sterol demethylation inhibitor (DMI) fungicides, and to discover compounds, which can modulate the activity of such transporters. Such modulators may be useful in practice to counteract resistance development to DMIs.An overview of characteristics of B. cinerea , commercial fungicides used in control of the pathogen, modes of action of botryticides, and resistance development to fungicides that inhibit sterol biosynthesis is described in Chapter 1. This chapter also describes the relevance of ABC and MFS transporters in fungicide resistance and the importance of fungicide mixtures to delay resistance development.The DMI fungicide oxpoconazole, developed by Ube industries, Ltd., is introduced in Chapter 2. This fungicide is effective against diseases that are commonly controlled by DMIs, such as rusts and scabs. In addition, it is effective against grey mould caused by B. cinerea under field condition. B. cinerea is known for its ability to acquire resistance to fungicides easily. ABC and MFS transporters may play a role in such a resistance development. The physiological functions of these transporters may be to cope with natural plant toxins since the pathogen has an extraordinary wide host range.Chapter 3 describes fourteen ABC and three MFS transporter genes from B. cinerea . Two of the ABC transporters were described previously and the others were identified in an EST library of the fungus. Their role in DMI resistance was investigated by studying basal and induced expression in wild-type and DMI-resistant strains of B. cinerea. From this screen, it appeared that BcatrD was the most probable ABC transporter gene encoding a putative DMI transporter since basal expression in three isolates correlated with sensitivity to DMIs. Induced expression after treatment with DMIs also correlated with sensitivityto DMIs. The MFS transporter gene Bcmfs1 might encode another DMI transporter since its basal expression in DMI resistant strains was also higher than that in the wild-type strain.The functional analysis of BcatrD and Bcmfs1 is described in Chapters 4 and 5, respectively. This was achieved by phenotyping of gene replacement and overexpression mutants generated from the haploid wild-type strain B05.10. Replacement and overexpression mutants of BcatrD displayed an increased and decreased sensitivity to DMIs, respectively. Overexpression mutants of Bcmfs1 also exhibited a decreased sensitivity to these fungicides while Bcmfs1 replacement mutants showed similar sensitivity to DMIs as compared to the wild-type. To clarify the role of Bcmfs1 in DMI-sensitivity in more detail, we constructed a double replacement mutant of BcatrD and Bcmfs1 . The double replacement mutant was more sensitive to DMIs than the single replacement mutant of BcatrD. These results suggest that BcatrD functions as a major DMI transporter and that Bcmfs1 is of minor importance. Accumulation of oxpoconazole by germlings of the mutants is also described in Chapter 4 and 5. Accumulation of oxpoconazole was transient in time as observed before for other DMIs and other filamentous fungi. The transient accumulation pattern is the result of passive influx and inducible active efflux of fungicides by transporters. The initial accumulation (20 min after the addition of oxpoconazole) level of oxpoconazole in the strains tested correlated with sensitivity to oxpoconazole. The steady state levels (more than 60 min after the addition of oxpoconazole) of accumulation by BcatrD and Bcmfs1 replacement mutants were higher than that by the wild-type isolate. These results indicate that BcatrD and Bcmfs1 mediate the sensitivity of B. cinerea to oxpoconazole by reducing the accumulation of the fungicide in mycelial cells. |
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