Fungicide-driven evolution and molecular basis of multidrug resistance in field populations of the grey mould fungus Botrytis cinerea
Kretschmer, M. ; Leroch, M. ; Mosbach, A. ; Walker, A.S. ; Fillinger, S. ; Mernke, D. ; Schoonbeek, H.J. ; Pradier, J.M. ; Leroux, P. ; Waard, M.A. de; Hahn, M. - \ 2009
PLoS Pathogens 5 (2009)12. - ISSN 1553-7366 - 13 p.
abc transporter bcatrb - natural toxic compounds - botryotinia-fuckeliana - penicillium-digitatum - candida-albicans - functional-characterization - virulence factor - drug-resistance - gene - sensitivity
The grey mould fungus Botrytis cinerea causes losses of commercially important fruits, vegetables and ornamentals worldwide. Fungicide treatments are effective for disease control, but bear the risk of resistance development. The major resistance mechanism in fungi is target protein modification resulting in reduced drug binding. Multiple drug resistance (MDR) caused by increased efflux activity is common in human pathogenic microbes, but rarely described for plant pathogens. Annual monitoring for fungicide resistance in field isolates from fungicide-treated vineyards in France and Germany revealed a rapidly increasing appearance of B. cinerea field populations with three distinct MDR phenotypes. All MDR strains showed increased fungicide efflux activity and overexpression of efflux transporter genes. Similar to clinical MDR isolates of Candida yeasts that are due to transcription factor mutations, all MDR1 strains were shown to harbor activating mutations in a transcription factor (Mrr1) that controls the gene encoding ABC transporter AtrB. MDR2 strains had undergone a unique rearrangement in the promoter region of the major facilitator superfamily transporter gene mfsM2, induced by insertion of a retrotransposon-derived sequence. MDR2 strains carrying the same rearranged mfsM2 allele have probably migrated from French to German wine-growing regions. The roles of atrB, mrr1 and mfsM2 were proven by the phenotypes of knock-out and overexpression mutants. As confirmed by sexual crosses, combinations of mrr1 and mfsM2 mutations lead to MDR3 strains with higher broad-spectrum resistance. An MDR3 strain was shown in field experiments to be selected against sensitive strains by fungicide treatments. Our data document for the first time the rising prevalence, spread and molecular basis of MDR populations in a major plant pathogen in agricultural environments. These populations will increase the risk of grey mould rot and hamper the effectiveness of current strategies for fungicide resistance management.
The drug transporter MgMfs1 can modulate sensitivity of field strains of the fungal wheat pathogen Mycosphaerella graminicola to the strobilurin fungicide trifloxystrobin
Roohparvar, R. ; Mehrabi, R. ; Nistelrooy, J.G.M. van; Zwiers, L.H. ; Waard, M.A. de - \ 2008
Pest Management Science 64 (2008)7. - ISSN 1526-498X - p. 685 - 693.
qo inhibitor fungicides - natural toxic compounds - mycosphaerella-graminicola - multidrug-resistance - genetic-variation - candida-albicans - abc transporters - mechanisms - respiration - pathosystem
BACKGROUND: The major facilitator superfamily (MFS) drug transporter MgMfs1 of the wheat pathogen Mycosphaerella graminicola (Fuckel) J Schroeter is a potent multidrug transporter with high capacity to transport strobilurin fungicides in vitro. The data presented in this paper indicate that, in addition to the predominant cause of strobilurin resistance, cytochrome b G143A subsititution, MgMfs1 can play a role in sensitivity of field strains of this pathogen to trifloxystrobin. RESULTS: In a major part of field strains of M. graminicola (collected in the Netherlands in 2004) containing the cytochrome b G143A substitution, the basal level of expression of MgMfs1 was elevated as compared with sensitive strains lacking the G143A substitution. Induction of MgMfs1 expression in wild-type isolates upon treatment with trifloxystrobin at sublethal concentrations proceeded rapidly. Furthermore, in disease control experiments on wheat seedlings, disruption mutants of MgMfs1 displayed an increased sensitivity to trifloxystrobin. CONCLUSION: It is concluded that the drug transporter MgMfs1 is a determinant of strobilurin sensitivity of field strains of M. graminicola. (c) 2008 Society of Chemical Industry
Cloning and functional characterization of BcatrA, a gene encoding an ABC transporter of the plant pathogenic fungus Botryotinia fuckeliana (Botrytis cinerea)
Sorbo, G. Del; Ruocco, M. ; Schoonbeek, H. ; Scala, F. ; Pane, C. ; Vinale, F. ; Waard, M.A. de - \ 2008
Mycological Research 112 (2008)6. - ISSN 0953-7562 - p. 737 - 746.
binding-cassette transporter - natural toxic compounds - saccharomyces-cerevisiae - multidrug-resistance - mycosphaerella-graminicola - drosophila-melanogaster - aspergillus-nidulans - efflux pump - cutinase-a - yeast
BcatrA was cloned from the plant pathogenic fungus Botryotinia fuckeliana (Botrytis cinerea) and sequenced. Sequence analysis revealed that BcatrA encodes a protein composed of 1562 amino acid residues displaying high similarity with various fungal ATP-binding cassette (ABC) transporters having the (NBF-TM6)2 topology. Expression of BcatrA is barely detectable during normal vegetative growth in liquid substrates. Transcript levels of BcatrA are enhanced in a dose- and time-dependent manner after treatment with cycloheximide or catechol, but not by a number of other drugs or fungicides, including fludioxonil, fenarimol, imazalil, and the plant defense compounds pisatin and resveratrol. Quantitative analysis of BcatrA during the synchronized infection of bean leaves revealed an overaccumulation of the gene transcript at 6, 12 and 24 h post-inoculation, suggesting an involvement of the gene in the first steps of pathogenesis. Functional analysis of BcatrA was performed by targeted gene replacement in a wild-type strain of the fungus, and by overexpression in a mutant of Saccharomyces cerevisiae carrying multiple non-functional multidrug-resistance genes. BcatrA replacement mutants did not show any significant increase in sensitivity to drugs, including inducers of BcatrA transcription, and displayed an unaltered virulence on several common host plants of B. cinerea. However, when expressed in the heterologous system, BcatrA reduced sensitivity to cycloheximide and catechol, thus indicating the ability of the BcatrA product to function as a multidrug transporter.
Control of Mycosphaerella graminicola on wheat seedlings by medical drugs known to modulate the activity of ATP-binding cassette transporters
Roohparvar, R. ; Huser, A. ; Zwiers, L.H. ; Waard, M.A. de - \ 2007
Applied and Environmental Microbiology 73 (2007)15. - ISSN 0099-2240 - p. 5011 - 5019.
major facilitator superfamily - natural toxic compounds - multidrug-resistance - abc transporters - botrytis-cinerea - efflux pump - fungicide sensitivity - virulence - plant - reversal
Medical drugs known to modulate the activity of human ATP-binding cassette (ABC) transporter proteins (modulators) were tested for the ability to potentiate the activity of the azole fungicide cyproconazole against in vitro growth of Mycosphaerella graminicola and to control disease development due to this pathogen on wheat seedlings. In vitro modulation of cyproconazole activity could be demonstrated in paper disk bioassays. Some of the active modulators (amitriptyline, flavanone, and phenothiazines) increased the accumulation of cyproconazole in M. graminicola, suggesting that they reversed cyproconazole efflux. However, synergism between cyproconazole and modulators against M. graminicola on wheat seedlings could not be shown. Despite their low in vitro toxicity to M. graminicola, some modulators (amitriptyline, loperamide, and promazine) did show significant intrinsic disease control activity in preventive and curative foliar spray tests with wheat seedlings. The results suggest that these compounds have indirect disease control activity based on modulation of fungal ABC transporters essential for virulence and constitute a new class of disease control agents.
Impact of fungal drug transporters on fungicide sensitivity, multidrug resistance and virulence
Waard, M.A. de; Andrade, A.C. ; Hayashi, K. ; Schoonbeek, H. ; Stergiopoulos, I. ; Zwiers, L.H. - \ 2006
Pest Management Science 62 (2006)3. - ISSN 1526-498X - p. 195 - 207.
atp-binding cassette - pathogen mycosphaerella-graminicola - azole antifungal agents - natural toxic compounds - yeast abc proteins - candida-albicans - botrytis-cinerea - aspergillus-nidulans - penicillium-digitatum - venturia-inaequalis
Drug transporters are membrane proteins that provide protection for organisms against natural toxic products and fungicides. In plant pathogens, drug transporters function in baseline sensitivity to fungicides, multidrug resistance (MDR) and virulence on host plants. This paper describes drug transporters of the filamentous fungi Aspergillus nidulans (Eidam) Winter, Botrytis cinerea Pers and Mycosphaerella graminicola (Fu¿ckel) Schroter that function in fungicide sensitivity and resistance. The fungi possess ATP-binding cassette (ABC) drug transporters that mediate MDR to fungicides in laboratory mutants. Similar mutants are not pronounced in field resistance to most classes of fungicide but may play a role in resistance to azoles. MDR may also explain historical cases of resistance to aromatic hydrocarbon fungicides and dodine. In clinical situations, MDR development in Candida albicans (Robin) Berkhout mediated by ABC transporters in patients suffering from candidiasis is common after prolonged treatment with azoles. Factors that can explain this striking difference between agricultural and clinical situations are discussed. Attention is also paid to the risk of MDR development in plant pathogens in the future. Finally, the paper describes the impact of fungal drug transporters on drug discovery
Modulators of membrane drug transporters potentiate the activity of the DMI fungicide oxpoconazole against Botrytis cinerea
Hayashi, K. ; Schoonbeek, H. ; Waard, M.A. de - \ 2003
Pest Management Science 59 (2003)3. - ISSN 1526-498X - p. 294 - 302.
sterol demethylation inhibitors - natural toxic compounds - multidrug-resistance - efflux pump - wild-type - botryotinia-fuckeliana - penicillium-italicum - candida-albicans - accumulation - fenarimol
Modulators known to reduce multidrug resistance in tumour cells were tested for their potency to synergize the fungitoxic activity of the fungicide oxpoconazole, a sterol demethylation inhibitor (DMI), against Botrytis cinerea Pers. Chlorpromazine, a phenothiazine compound known as a calmodulin antagonist, appeared the most potent compound. Tacrolimus, a macrolide compound with immunosuppressive activity, was also active. The synergism of chlorpromazine negatively correlated with the sensitivity of the parent strain and mutants of B cinerea. The synergism was highest in a mutant that overexpressed the ATP-binding cassette transporter BcatrD, known to transport DMI fungicides such as oxpoconazole. The synergism of chlorpromazine positively correlated with its potency to enhance the accumulation of oxpoconazole in BcatrD mutants. These results indicate that chlorpromazine is a modulator of BcatrD activity in B cinerea and suggest that mixtures of DMI fungicides with modulators may represent a perspective for the development of new resistance management strategies.