Báo cáo khoa học: Modeling the Qo site of crop pathogens in Saccharomyces cerevisiae cytochrome b ppt

The G143A mutation caused a high level of resistance to QoI compounds such as myxothiazol, axoxystrobin and pyraclostrobin, but not to stigmatellin.. Two target site mutations in cytochr

Modeling the Qo site of crop pathogens in Saccharomyces cerevisiae

Nicholas Fisher1, Amanda C Brown1, Graham Sexton2, Alison Cook2, John Windass2and Brigitte Meunier1

1

The Wolfson Institute for Biomedical Research, UCL, London, UK;2Syngenta, Jealott’s Hill International Research Centre, Bracknell, Berkshire, UK

Saccharomyces cerevisiaehas been used as a model system

to characterize the effect of cytochrome b mutations found

in fungal and oomycete plant pathogens resistant to Qo

inhibitors (QoIs), including the strobilurins, now widely

employed in agriculture to control such diseases Specific

residues in the Qosite of yeast cytochrome b were modified

to obtain four new forms mimicking the Qobinding site of

Erysiphe graminis, Venturia inaequalis, Sphaerotheca

fuligi-neaand Phytophthora megasperma These modified versions

of cytochrome b were then used to study the impact of the

introduction of the G143A mutation on bc1complex

activ-ity In addition, the effects of two other mutations F129L

and L275F, which also confer levels of QoI insensitivity,

were also studied The G143A mutation caused a high

level of resistance to QoI compounds such as myxothiazol,

axoxystrobin and pyraclostrobin, but not to stigmatellin The pattern of resistance conferred by F129L and L275F was different Interestingly G143A had a slightly deleterious effect on the bc1 function in V inaequalis, S fuliginea and P megasperma Qo site mimics but not in that for

E graminis.Thus small variations in the Qo site seem to affect the impact of the G143A mutation on bc1 activity Based on this observation in the yeast model, it might be anticipated that the G143A mutation might affect the fitness

of pathogens differentially If so, this could contribute to observed differences in the rates of evolution of QoI resist-ance in fungal and oomycete pathogens

Keywords: Qo inhibitors; bc1 complex; cytochrome b; resistance; plant pathogens

The mitochondrial bc1 complex is a membrane-bound

enzyme that catalyzes the transfer of electrons from

ubiquinol to cytochrome c and couples this electron

transfer to the vectorial translocation of protons across

the inner mitochondrial membrane In eukaryotes it is

comprised of 10 or 11 different polypeptides, and

addi-tionally operates as a structural and functional dimer

Cytochrome b, cytochrome c1and the Rieske iron–sulfur

protein (ISP) form the catalytic core of the enzyme The

catalytic mechanism, called the Q-cycle, requires two

distinct quinone-binding sites (Qo, quinol oxidation site,

and Qi, quinone reduction site), which are located on

opposite sides of the membrane and linked by a

trans-membrane electron-transfer pathway The mitochondrially

encoded cytochrome b subunit provides both the quinol

and quinone binding pockets and the transmembrane

electron pathway (via hemes bland bh)

A number of quinol antagonists are known that inhibit

bc1activity These are either specific for the Qisite, such

as antimycin, or for the Qo site, such as myxothiazol,

stigmatellin, natural and synthetic strobilurins Some of

the latter Qoinhibitor compounds (QoIs) are now widely

used in agriculture to control fungal and oomycete plant pathogens Resistance to these inhibitors has, however, emerged in field populations of some such plant patho-gens Two target site mutations in cytochrome b in particular appear to play a central role in the mechanism

of resistance: G143A which has been reported in resistant isolates from various important pathogens ([1] and references within) and F129L which has been found in pathogens of turf grass, vines and potatoes A143 is also found in the strobilurin-producing basidiomycete Mycena galopoda [2] In E graminis, the mutation G143A has spread widely and is without any apparent fitness penalty

In other pathogens, such as V inaequalis, G143A has thus far been detected only in a localized geographical area Still other pathogens have, however, not yet shown QoI resistance despite their exposure to QoI fungicides ([1]) Several mechanisms might explain differences in the emergence of such resistance One factor may be subtle variations in the structure and function of the Qobinding domain of the pathogens

In this work, the resistance mutations, in particular G143A were investigated in the context of yeast bc1 structures Yeast was used as a model system to construct several forms of the Qo domain, mimicking distinctive plant pathogen derived forms of this region based on both primary and tertiary structure comparisons, and to study the effect of the introduction of the QoI resistance mutation G143A on enzyme activity Some of these distinctive changes in the Qodomain have been found to affect the impact of the resistance mutation on enzyme activity

Correspondence to B Meunier, the Wolfson Institute for Biomedical

Research, UCL, Gower Street, London, WC1E 6BT, UK.

E-mail: b.meunier@ucl.ac.uk

Abbreviations: ISP, iron–sulfur protein; PMSF, phenylmethylsulfonyl

fluoride; QoI, Q o inhibitor.

(Received 27 February 2004, revised 5 April 2004,

accepted 16 April 2004)

Experimental procedures

Media and chemicals

The following media were used for the growth of yeast:

YPD [1% (w/v) yeast extract, 2% (w/v) peptone, 3% (w/v)

glucose], YPG [1% (w/v) yeast extract, 2% (w/v) peptone,

3% (w/v) glycerol], transformation medium [0.7% (w/v)

yeast nitrogen base, 3% (w/v) glucose, 2% (w/v) agar, 1M

sorbitol, and 0.8 gÆL)1of a complete supplement mixture

minus uracil; Anachem] Decyl ubiquinone and

myxo-thiazol were purchased from Sigma Stigmatellin was

purchased from Fluka

Generation of the yeast mutant strains

Plasmid pBM5, carrying the wild-type intron-free version of

the CYTB gene, was constructed by blunt end cloning of a

PCR product of CYTB into the pCRscript vector

(Strata-gene) Site directed mutageneses were performed using the

Quickchange Site-Directed Mutagenesis Kit (Stratagene)

according to the manufacturer’s recommendations After

verification of the sequence, plasmids carrying the intended

mutant genes were used for microprojectile bombardment

mediated mitochondrial transformation of yeast as

des-cribed in [3]

Preparation of decylubiquinol

Ten milligrams of 2,3-dimethoxy-5-methyl

n-decyl-1,4-ben-zoquinone (decylubiquinone, Sigma), an analogue of

ubi-quinone was dissolved in 0.4 mL nitrogen-saturated hexane

An equal volume of aqueous 1.15Msodium dithionite was

added, and the mixture shaken vigorously until colorless

The upper, organic phase was collected, and the

decyl-ubiquinol recovered by evaporating off the hexane under

nitrogen The decylubiquinol was dissolved in 100 lL 96%

(v/v) EtOH (acidified with 10 mM HCl) and stored in

aliquots at )80 °C The concentration of decylubiquinol

was determined spectrophotometrically from absolute

spectra, using e288)320¼ 4.14 mM )1Æcm)1

Preparation of crude mitochondrial membranes and

measurement of cytochromec reductase activity

Wild-type and mutant yeast strains were grown to stationary

phase (48 h) in 200 mL YPD cultures at 28°C The cells

(approximately 2 g wet weight per culture) were then

harvested by centrifugation at 4000 g for 10 min Cell pellets

were then washed by resuspension in 40 mL 50 mM

potas-sium phosphate, 2 mMEDTA (pH 7.5) and centrifuged as

before The harvested cells were resuspended in 10 mL

50 mM potassium phosphate, 2 mMEDTA (pH 7.5)

sup-plemented with 0.2 mM phenylmethylsulfonyl fluoride

(PMSF) and 0.05% (w/v) bovine serum albumin prior to

disruption in a Retsch MM300 glass bead mill operating at

30 Hz for 10 min at 4°C Membranes were separated from

cell debris by centrifugation at 10 000 g for 20 min The

supernatant was centrifuged at 100 000 g for 90 min and the

pelleted membranes resuspended in 1 mL of 50 mM

potas-sium phosphate (pH 7.5), 2 mM EDTA containing 10%

(v/v) glycerol Resuspended membranes were stored in

0.1 mL aliquots at)80 °C Cytochrome c reductase activity measurements were made in 50 mMpotassium phosphate,

pH 7.5, 2 mM EDTA, 10 mM KCN, 0.025% (w/v) lauryl maltoside and 30 lM equine cytochrome c at room tem-perature Membranes were diluted to 2.5 nMcytochrome bc1 complex (determined from the reduced minus oxidized difference spectra, using e¼ 28.5 mM )1Æcm)1at 562–575 nm [4] Cytochrome c reductase activity was initiated by the addition of decylubiquinol (5–100 lM) Reduction of cyto-chrome c was monitored in a Cary 4000 spectrophotometer

at 550 vs 542 nm over a 4 min time-course Initial rates (computer-fitted as zero-order kinetics) were measured as a function of decylubiquinol concentration, and Vmand Km values derived from Eadie–Hofstee (v vs v/[S]) plots [5] All rate measurements were performed in triplicate

Spectroscopic analysis of cytochromes in whole cells Spectra were generated by scanning cell suspensions with a single beam spectrophotometer built in-house and operating

at room temperature The cells, grown on YPD plates for

48 h, were resuspended at a concentration of 200 mg cells per milliliter and reduced by dithionite The cytochrome concentration was estimated from the reduced spectra as described in [3]

Results and discussion

Construction of yeast mutants with modified cytochromeb Qosites

The sequence of cytochrome b is highly conserved between species, especially in catalytic domains such as the Qo

region This site is actually a relatively large domain formed from components encompassing amino acid residues 120–150 and 260–280 of cytochrome b The cavity consists

of two lobes, a heme blproximal lobe and a distal lobe The distal lobe is close to the surface region of cytochrome b and is involved in interactions with the peripheral domain of the iron–sulphur protein The stigmatellin head-group binds

in this distal lobe of the Qosite and is positioned in a pocket formed by amino acid tracts 122–131 (transmembrane helix C), 142–152 (helix cd1 and the cd1-cd2 linker), 268–280 (helix ef) The methoxyacrylamide moiety of myxothiazol, and methoxyacrylate moiety of strobilurin-related inhibi-tors, occupy the proximal domain, and are closely associ-ated (< 5 A˚ separation) with the sidechains of residues F129 (transmembrane helix C), Y132 (ibid), G143 (helix cd1) and F275 (helix ef) [1,6]

Comparison of cytochrome b sequences around residues

129 and 143, involved in QoI resistance, showed some variations between pathogen species (Fig 1) Firstly,

S cerevisiae, used as a model system in this work, has

a unique feature: the CCV(133–135) sequence which, although also found in related yeast (Fig 1A), is replaced

by the sequence VLP(133–135) in most other organisms, including all plant pathogens we have analyzed and more distantly related species including mammals To address the question whether the CCV sequence is essential to yeast bc1 complex function or assembly, this sequence was replaced

by the more common VLP sequence and the respiratory growth competence, the cytochrome b level and bc activity

were monitored (Tables 1 and 2) No effect was observed,

suggesting that the yeast enzyme can accommodate the VLP

sequence without loss of function This new form of Qo

domain, with the common VLP(133–135) sequence, has

therefore been used throughout the other studies reported

here

The effect of other variations in the Qobinding domain

on bc1 function and inhibitor resistance was then

investi-gated Four plant pathogens were chosen for this study,

E graminis(Ascomycete, pathogen of wheat), V inaequalis

(Ascomycete, pathogen of apple), S fuliginea (Ascomycete,

pathogen of cucumber) and P megasperma (Oomycete,

causing root rot disease) based on comparison of their

primary sequences The cytochrome b sequences of these

plant pathogens, either obtained from public databases or

by targeted PCR amplification and sequencing of field isolates, showed only small but distinctive changes in the Qo site (Fig 1) Three permutations at position 136: tyrosine, phenylalanine and tryptophan, and three permutations at position 141: histidine, leucine and phenylalanine were observed in the four pathogens In addition, a change of residue 275 from leucine to phenylalanine is seen P mega-sperma cytochrome b This latter change has been also reported in Pneumocystis carinii resistant to atovaquone treatment [7] and is naturally present in the corresponding mammalian enzyme [8] Appropriate changes in the yeast cytochrome b sequence were introduced in order to obtain four new forms of cytochrome b: E graminis-like (AB1),

Fig 1 Comparison of cytochrome b sequences in a region comprising the Q o domain (A) Aligned sequences from yeasts and, as a representative mammal, humans (residues 121–155, S cerevisiae numbering) (B) Corresponding sequence comparison of S cerevisiae with the four plant pathogens employed in this study (C) The sequence of the 15 yeast variants constructed and analyzed in this work The mutated residues are in bold The sequences of E graminis, V inaequalis and P megasperma are available from the EMBL database The sequence of S fuliginea was determined by targeted RT-PCR amplification as described in [11].

V inaequalis-like (AB4), S fuliginea-like (AB7) and

P megasperma-like (AB9) mutants (Fig 1C) These new

forms of cytochrome b were also used to compare the

impact of the introduction of the mutations G143A, F129L and L275F on bc1 complex activity To this end, we introduced these additional mutations into the

Table 1 Respiratory growth competence, cytochrome b content and resistance to Q o inhibitors To determine the doubling time, cells were inoculated

in respiratory medium (YPG) and the optical density was monitored periodically at 600 nm The cytochrome b (cyt b) content was determined in whole cells by spectrophotometry as described in experimental procedures, using e ¼ 25 m M-1 cm -1 at 562–575 nm The cyt b concentration in the wild type cells was 5.7 nmol per gram of cells The respiratory growth in presence of inhibitor was monitored on respiratory media (YPG) plus 1 or

10 l M inhibitor as described in Fig 3 +++ indicates vigorous growth; ++ and +, weaker growth; – , no growth.

Strains Mutations

Doubling Time (hrs)

Cyt b content (%)

Growth on Myxothiazol Stigmatellin Azoxystrobin Pyraclostrobin

Erysiphe graminis-like

Venturia inaequalis-like

Sphaerotheca fuliginea -like

Phytophthora megasperma-like

Table 2 QH 2 cytochrome c reductase activities QH 2 cytochrome c reductase activity was assayed as described in experimental procedures.

Strains Mutations

bc 1 Complex activity Rates (s)1)

at 50 l M QH 2 V m (s)1)

K m (QH 2 l M )

Erysiphe graminis-like

Venturia inaequalis-like

Sphaerotheca fuliginea-like

Phytophthora megasperma-like

pathogen-like mutants In total, 15 variants were constructed

(Figs 1C and 2) These were generated by a biolistic

trans-formation procedure, which produces homoplasmic yeast

strains carrying only the variant cytochrome b sequence [3],

andthenusedtomonitorrespiratoryfunctioninvariousways

Effects of mutations on respiratory growth and

cytochromeb content

All the variant cytochrome b yeast strains constructed were

respiration competent Their doubling times in

nonferment-able medium (YPG) were 4–5 h, with the exception of

strains AB5 and AB13 which showed doubling times of

6 and 10 h, respectively This phenotype was not

investi-gated further In order to assess the effect of mutations on

the assembly of the bc1 complex, we also monitored the

concentration of cytochromes in whole cells, as described in

experimental procedures: changes introduced in the Qo

domain had little effect on cytochrome b assembly

Cyto-chrome b content was between 90 and 100% of that of the

wild-type, in the E graminis-, V inaequalis- and S

fuligi-nea-like constructs; though the changes introduced in the

P megasperma-like constructs seemed to hinder enzyme

assembly slightly as judged by the decrease in cytochrome b

content (Table 1) Lowest cytochrome b levels were

observed in the strain harboring the three mutations

Y136W, H141F and L275F (60% of the wild type)

Interestingly, these three changes are naturally present in mammals The introduction of a fourth mutation, F129L restored the cytochrome b content to near wild-type level (Table 1) It seems likely that the introduction of three bulky residues, Y136W, H141F and L275F, sterically hinders the folding of cytochrome b and the assembly of the complex The replacement of phenylalanine at position 129 by a smaller residue leucine may then alleviate the hindrance and restore the proper folding of cytochrome b

Resistance to Qoinhibitors

As mutations G143A and F129L had been found in plant pathogen isolates resistant to QoIs, we monitored the respiratory growth competence of the different constructs in the presence of stigmatellin, which binds in the distal lobe

of the Qo site, and myxothiazol, azoxystrobin and pyra-clostrobin, which bind at the proximal lobe of the Qosite (Fig 3 and Table 1)

The control strains, AB1, AB4, AB7 and AB9 were all sensitive to myxothiazol, stigmatellin, azoxystrobin and pyraclostrobin Introduction of G143A in all four Qoforms led to strong resistance to myxothiazol, azoxystrobin and pyraclostrobin: strains AB2, AB13, AB8 and AB10 grew on nonfermentable medium in presence of 10 lM of each of these compounds but were still sensitive to stigmatellin Interestingly structural studies suggest that the Ca hydrogen

Fig 2 Structure of the Q o site The cyto-chrome b a-carbon backbone is shown in orange The location of residues altered to model the Q o -sites from the pathogenic fungi discussed in the text are shown in green The VLP(133-135) region of cytochrome b is indi-cated in white Q o -bound stigmatellin and hemes b l /b h are represented in cyan and red, respectively This figure was prepared from the yeast bc 1 crystal structure coordinates 1KYO.pdb [12] using VISUAL MOLECULAR

software [13].

atom of G143 approaches within 3.5 A˚ of the

methoxy-acrylamide moiety of myxothiazol and hence mutation to

the bulkier residue alanine is likely to abolish the binding of

this class of Qoantagonist [1,6] A similarly close interaction

with the benzene ring linker region of azoxystrobin and

pyraclostrobin could explain resistance to these compounds

The pattern of resistance induced by F129L was different

Strains AB3 and AB16 were resistant to myxothiazol and

stigmatellin They also show limited cross-resistance to

azoxystrobin as growth was observed at 1 lMazoxystrobin

but not at 10 lM Yeast cells carrying this mutation were

rather more sensitive to pyraclostrobin: no growth was

observed at 1 lM pyraclostrobin The sidechain of F129

approaches within 3 A˚ of the myxothiazol

methoxyacryl-amide moiety By contrast, F129 has a closest approach of

4 A˚ with the hydrophobic tail of stigmatellin The likely

mechanism of F129L stigmatellin resistance is therefore not

clear, but it could be due to a subtle alteration of the

backbone fold at Qo, or a change in accessibility for the

antagonist to the Qosite The slight variance in sensitivity

to azoxystrobin and pyraclostrobin is likely to be due to the

difference in pharmacophore structure between these two

compounds, as discussed in more detail below As

men-tioned above, strain AB5 showed a weaker growth that

could explain the apparent sensitivity

Interestingly AB12, which combined F129L with L275F,

was sensitive to azoxystrobin but resistant to

pyraclostro-bin In this case it is likely that the two changes have slightly

modified the structure of the Qo site, which can now

accommodate azoxystrobin but not pyraclostrobin The

sidechain of F275 in chicken bc1complex is involved in a

stabilizing ring–stacking hydrophobic interaction with the

phenyl group of MOA-stilbene [6], a Qoinhibitor closely

related to strobilurin This may explain why strain AB11

(Y136W, H141F, L275F) retains sensitivity to the

strobilu-rin-related inhibitor azoxystrobin Strain AB12 (Y136W,

H141F, L275F + F129L) demonstrated resistance to both

myxothiazol and pyraclostrobin, but remained sensitive to

azoxystrobin Pyraclostrobin and Azoxystrobin differ in

pharmacophore structure; the former contains an

alkoxy-amino moiety, whereas the latter is methoxyacrylate based

(Fig 4) Significantly, the pharmacophore of pyraclostrobin occupies a smaller volume than that of azoxystrobin, and might have a greater degree of rotational freedom due to the

Fig 4 Structure of Q o inhibitors azoxystrobin and pyraclostrobin [1] Pharmacophore groups are indicated by boxes.

Fig 3 Sensitivity to Q o inhibitor exposure.

The name and position of the strains are

shown in the right-hand panel A drop of each

strain was inoculated on a nonfermentable

medium plate (YPG) with or without 10 l M

inhibitor and incubated for 3–4 days.

lack of methoxyacrylate p-bonded structure Mutation of

both F129 and L275 to leucine and phenylalanine,

respect-ively, are required to inhibit pyraclostrobin binding

As expected, strains AB17 and AB18 harboring both

G143A and F129L combined resistance to myxothiazol,

azoxystrobin and pyraclostrobin with resistance to

stig-matellin

In order to quantify the level of resistance induced by

G143A, bc1 complex sensitivity to myxothiazol and

stig-matellin was monitored in membranes from strain AB2 and

its control AB1 QH2cytochrome c reductase activity (using

2.5 nM bc1 complex), as in Table 2, was measured in

presence of increasing concentration of inhibitors The

concentration of stigmatellin required for 50% decrease of

activity (I50) was around 2.5 nMfor AB1 and AB2, whereas

the I50for myxothiazol was 2.5 nMfor AB1 and 18 lMfor

AB2: a 7500-fold increase This is in good agreement with

previous results The G143A mutation was first reported in

mammalian cells after selection in presence of myxothiazol,

conferring > 7000-fold resistance to the inhibitor [9]

Effect of mutations on bc1complex activity

In order to study possible effects of the mutations on bc1

function, mitochondrial membranes were prepared from the

different strains and cytochrome c reductase activity was

monitored spectrophotometrically as described in

experi-mental procedures As shown in Table 1, the replacement

of the yeast sequence CCV(133-135) by the much more

common sequence VLP, in the E graminis-like strain had

no effect on enzyme activity In the V inaequalis-like strain

(AB4), histidine 141 was replaced by leucine The activity of

the resultant enzyme was then decreased by 30% compared

to the wild-type yeast Activity was however, restored to

near wild-type levels by the introduction of a second change,

Y136F, in the S fuliginea-like strain (AB7) The P

mega-sperma-like enzyme (in strain AB9), which harbored

Y136W and H141F also showed a 30% decrease in bc1

activity

The introduction of G143A, F129L or both changes

together (though this has not been seen in any natural

isolate to our knowledge) in the E graminis-like Qosite had

little effect on bc1activity (80–87% of wild-type rate) Thus

this Qo site can accommodate the G143A and F129L

mutations without loss of function This is consistent with

previous observations with E graminis itself, which showed

that the isolates carrying the G143A mutation did not suffer

any fitness penalty [10] In the V inaequalis-like strains, the

situation was different As mentioned above, the control

strain (AB4) harboring the change H141L showed a lower

activity than the wild-type yeast strain (turnover number

28 s)1 vs 40 s)1) Interestingly the introduction of the

G143A mutation in this Qosite further decreased the bc1

activity to 14 s)1(50% of the control AB4) In contrast,

F129L had no effect In AB18, which combined G143A and

F129F, the enzyme activity was 43% of the control It seems

therefore that the V inaequalis-like enzyme cannot

accom-modate the G143A mutation without reduction of function

Similar results were obtained with the P megasperma- and

the S fuliginea-like Qo sites The introduction of G143A

caused, respectively, a 60% and 33% decrease of the bc1

activity compared to the controls We have also used the

P megasperma-like form to monitor the effect of F129L and L275F The mutation L275F is naturally occurring in Phytophothora sp The introduction of these mutations decreased the bc1activity to 44% of the control AB9 Their combination in AB12 restored the activity to 67% of the control AB9 Thus the introduction of L275F in the

P megasperma-like Qo caused a decrease both in bc1 content and activity, while F129L partially compensated the defect

To gain further information on the effect of the mutation G143A, steady-state cytochrome c reductase activity was monitored as a function of decylubiquinol (QH2) concen-tration The apparent Vmand Kmfor QH2were calculated from initial rate measurements using derived Eadie–Hofstee plots (Table 1) The mutation G143A appeared to decrease both the Vm and the Kmfor quinol in AB13, AB8 and AB10 It might therefore be that this mutation slightly affects the structure of the Qo site which, as a result, becomes saturated with substrate more rapidly than the control due to lower electron transfer, or alternatively it may reflect a decreased on rate for quinol binding The replacement of glycine by alanine is a relatively conservative structural change, and unlikely to disrupt the fold of the cd1 helix The introduced methyl group may sterically hinder interactions with the quinol headgroup, or unfavorably alter the conformation of bound quinol such that electron transfer or deprotonation rates are decreased

Thus variations in the Qo domain seem to affect the impact of the QoI resistance mutation G143A on cyto-chrome bc1 activity In some cytochrome b forms, the introduction of G143A decreases the QH2 cytochrome c activity of the complex Under standard laboratory condi-tions in S cerevisiae, this decrease has no effect on cell growth as little as 20% of bc1complex activity is enough to support respiratory growth Therefore a decline in respir-atory growth will only be seen when the complex is severely inhibited However in other organisms, such as plant pathogens, when the energetic demands are higher, this decrease might affect the fitness of the cells In combination with other factors, this could explain the differences in the evolution of QoI resistance in fungal and oomycete pathogens Interestingly the characteristic Qo site features

of E graminis, one of the pathogens which showed field resistance to QoI fungicides particularly quickly, seem to be most functionally accommodating of the resistance-associ-ated G143A mutation

Acknowledgements

This work was supported by Syngenta The authors acknowledge the contributions made by our colleagues, Ms Carole Stanger and Ms Judith Burbidge, to the analysis of cytochrome b gene and/or mRNA sequences from plant pathogen isolates We would also particularly wish to recognize the interest, enthusiasm and insight in initiating these studies shown by our late colleague Steve Heaney, and this paper is dedicated to his memory.

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