High resistance of Isaria fumosorosea to carbendazim arises from the overexpression of an ATP-binding cassette transporter (ifT1) rather than tubulin mutation

Authors


Ming-Guang Feng, Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China.
E-mail:mgfeng@zju.edu.cn

Abstract

Aims:  Probing possible mechanisms involved in the resistance of entomopathogenic fungus Isaria fumosorosea to carbendazim fungicide.

Methods and Results:  A carbendazim-sensitive strain (If116) selected from 15 wild-type strains was subjected to NaNO2-induced mutagenesis, yielding nine mutants with carbendazim resistance increased by 82- to 830-fold and thermotolerance decreased by 15–51%. Comparing the protein sequences deduced from the α- and β-tubulin genes of If116 and its mutants revealed no traceable site mutation relating to the enhanced resistance although the transcripts levels of β-tubulin gene in all mutants were 0·87- to 7·16-fold of that in If116. Three examined mutants showed multidrug resistance because they were significantly more resistant to glufosinate, imidacloprid and other six fungicides than If116 during growth. Further examination of rhodamine-stained blastospores revealed existence of drug efflux pump protein(s) in all carbendazim-resistant mutants. Thus, the sequences of an ATP-binding cassette (ABC) transporter gene (ifT1) and its promoter region cloned from the wild-type and mutant strains were analysed. Three common point mutations were located, respectively, at the binding sites of Gal4, Abf1 and Raf, which are crucial transcription factors in the regulative network of numerous protein loci. Such point mutations elevated the ifT1 expression by 17 to 137-fold in all the mutants.

Conclusions:  The overexpression of the ABC transporter caused by the point mutations at the binding sites was responsible for the fungal resistance to various pesticides including carbendazim.

Significance and Impact of Study:  The transporter-mediated multidrug resistance found for the first time in entomopathogenic fungi is potential for use in improving mycoinsecticide compatibility with chemical pesticides.

Introduction

Site mutations of β-tubulin are usually responsible for the resistance of phytopathogenic and entomopathogenic fungi to benzimidazole fungicides, such as carbendazim (methyl 2-benzimidazole carbamate, abbreviated as MBC in this report), benomyl and nocodazole (Ma et al. 2003, 2005; Zou et al. 2006) and also associated with fungal sensitivity to heat stress (Yan and Dickman 1996; Ma et al. 2003; Kiso et al. 2004; Zou et al. 2006). Fungal resistance to the fungicides is a very useful phenotype for the control of insect pests by mycoinsecticides, which include fungal cells (e.g., conidia) as active ingredients and are not compatible with common fungicides sprayed for plant disease control.

Pleiotropic drug resistance (PDR) exists in eukaryotes, which resist various compounds unrelated in structure and function. Such multidrug resistance (MDR) is associated with ATP-binding cassette (ABC) transporters embedded in cell membrane for energy transport (Nakaune et al. 1998; Andrade et al. 2000a,b; Michalkova-Papajova et al. 2000; Gupta and Chattoo 2008). ABC transporters are known for their vital effects on the MDR of human tumours and pathogens and thus on the curative effect of anticancer drugs and antibiotics (Sanglard et al. 1997; Skatrud 2002). Saccharomyces cerevisiae possesses ∼30 ABC transporter genes, of which some mediate PDR/MDR (Taglicht and Michaelis 1998). Similar transporter-mediated PDR/MDR also exists in the fungal pathogens Candida glabrata (Parkinson et al. 1995), Aspergillus nidulans (Andrade et al. 2000a,b) and Penicillium digitatum (Nakaune et al. 1998, 2002). However, no report has dealt with the transporter-mediated PDR/MDR in entomopathogenic fungi used for mycoinsecticide development.

The initial goal of this study was to determine a variability in the MBC resistance and thermotolerance of Isaria fumosorosea (formerly Paecilomyces fumosoroseus) strains as fungal biocontrol agents of insect pests (Faria and Wraight 2001) and to find possible MBC-sensitive sites in the fungal tubulin genes for genetic manipulation. A wild-type strain (If116) sensitive to the fungicide was selected from 15 strains with different geographical and host origins and subjected to chemical mutagenesis. Mutants from a single round of mutagenesis showed extremely high MBC resistance and reduced thermotolerance. However, sequence analysis of their tubulin genes failed to reveal β-tubulin site mutations that were supposed to confer the enhanced MBC resistance, as reported previously in other fungi (Butters et al. 2003; Ma et al. 2003; Zou et al. 2006). This implies that a mechanism other than tubulin mutation is involved in the I. fumosorosea resistance to MBC. The different mechanism was thus probed by detecting possible PDR/MDR in wild-type and mutant cells stained with rhodamine 123, which is often used to reflect the cellular efflux activity of an ABC transporter pump (Ludescher et al. 1992; Clark et al. 1996; Nakaune et al. 2002), and by comparing the sequences of an ABC transporter gene and its promoter region cloned from the wild-type and mutant strains. The overexpression of the target gene caused by point mutations at the binding sites of three transcription factors in the promoter region was found responsible not only for the high MBC resistance of I. fumosorosea mutants but also for their resistance to other pesticides of different types.

Materials and Methods

Fungal isolates and conidial preparations

Twelve I. fumosorosea (If) isolates were from the ARS Collection of Entomopathogenic Fungal Cultures (RW Holley Center for Agriculture and Health, Ithaca, NY, USA; see ARSEF accession numbers in Table 1 and host and geographic origins at http://arsef.fpsnl. cornell.edu), and other three whitefly-derived isolates were from the USDA-ARS Biological Control of Pests Research Unit, Weslaco, Texas. All strains were stored at −76°C and recovered on the slants of Sabouraud dextrose agar plus 1% yeast extract (SDAY) at 25°C, followed by 7- to 9-day incubation for conidiation on SDAY plates at 25°C and 12 : 12 h (light/dark cycle). Aerial conidia scraped from the cultures of each isolate were suspended in 0·02% Tween 80 and standardized to ∼2 × 104 conidia ml−1 for the following use.

Table 1.   Comparative MBC sensitivities of different wild-type strains of Isaria fumosorosea
Fungal strains*MBC range† (μg ml−1)Fitted parameters‡Fitness (r2)EC50 (μg ml−1)MIC§ (μg ml−1)Resistance ranking¶
α ± SEβ ± SE
  1. *ARSEF accession numbers for all strains except asterisked ones, which were from the USDA-ARS Biological Control of Pests Research Unit, Weslaco, Texas.

  2. †Each parenthesized value denotes the number of MBC concentrations used in the assay.

  3. ‡Fitted to the equation Is = 1/[1 + exp(α βC)] at the significance level of < 0·001 in all fitness F tests.

  4. §Estimated as EC99, i.e., an MBC concentration to inhibit 99% CFU growth on SDAY plates.

  5. ¶Resistance ranking: MIC ≤ 1·0 μg ml−1 for super sensitivity (SS); 1·0 < MIC ≤ 5·0 for sensitivity (S); 5·0 < MIC ≤ 20·0 for low resistance (LR); 20·0 < MIC ≤ 100·0 for medium resistance (MR); and MIC > 100·0 for high resistance (HR) (URL: http://www.fao.org/ag/AGP/AGPP/Pesticid/JMPR/).

116*1–32 (6)−1·579 ± 0·5800·946 ± 0·3350·9101·676·52LR
153*1–128 (8)−1·310 ± 0·3690·278 ± 0·0830·9124·7221·25MR
612*1–128 (8)−1·251 ± 0·3440·305 ± 0·0860·9264·1019·14LR
21751–32 (6)−1·592 ± 0·4430·251 ± 0·0800·9096·3524·70MR
23754–128 (6)−1·870 ± 0·7450·367 ± 0·1340·9105·0917·61LR
26582–64 (6)−1·823 ± 0·4720·525 ± 0·1360·9583·4712·23LR
30762–128 (7)−2·112 ± 0·4550·866 ± 0·1710·9742·447·74LR
35192–128 (7)−1·801 ± 0·4390·419 ± 0·1030·9514·3015·26LR
35772–128 (7)−1·755 ± 0·4490·335 ± 0·0890·9435·2318·94LR
38432–128 (7)−1·951 ± 0·3930·539 ± 0·1110·9733·6212·15LR
38782–128 (7)−1·483 ± 0·4130·256 ± 0·0740·9305·7923·71MR
42051–32 (6)−3·084 ± 0·3750·547 ± 0·0700·9895·6414·04LR
50831–128 (8)−1·545 ± 0·4070·569 ± 0·1550·9342·7110·79LR
60322–64 (6)−1·739 ± 0·4220·350 ± 0·0870·9564·9618·08LR
62064–128 (6)−1·701 ± 0·4010·075 ± 0·0200·92222·7684·28MR

Assays for MBC sensitivities of fungal isolates

Three 50-μl aliquots (replicates) of the conidial suspension of each strain were spread evenly on 9-cm-diameter SDAY plates containing 0 (control) to 128 μg MBC ml−1 at the interval of 0·2, 0·25 or 0·5 μg ml−1 in terms of their MBC sensitivities in preliminary assays. To distribute the fungicide as evenly in SDAY as possible, MBC was dissolved in 0·01 mol l−1 HCl prior to inclusion into the autoclaved medium. All spread plates were incubated for up to 6 days at 25°C and 12 : 12 h. Counts of colony-forming units (CFU) were then made from the plates at the gradient MBC concentrations. An MBC concentration to cause 100% inhibitory effect on fungal growth was used as a maximum in the assay of each strain. The ratio of a CFU count at a given MBC concentration over that in the control was defined as survival index (Is) for each of the tested strains.

Preparation of Isaria fumosorosea mutants

An MBC-sensitive isolate, If116, selected from the above assays was subjected to chemical mutagenesis as described previously (Zou et al. 2006). Briefly, conidial suspension (5 × 107 conidia ml−1) in 0·02% Tween 80 and 0·01 mol l−1 phosphate-buffered saline (PBS; pH 7·4) was mixed with 2% (v/v) of 0·2 mol l−1 NaNO2 (as mutagen) for 5-min reaction at 25°C to cause an expected loss of ∼95% viability. The reaction was terminated immediately by adding ten-fold volume of 0·02% Tween 80 to the mixture. Subsequently, 50-μl aliquots of the reacted suspension were spread on SDAY plates including 100 μg MBC ml−1, a concentration much higher than the maximum (30 μg ml−1) used in the previous assay of the same strain. After 7-day incubation at 25°C and 12 : 12 h, vigorous colonies were subjected to three rounds of subculture on MBC-free SDAY at the same regime. Nine mutants showing normal growth and conidiation were chosen for the following experiments.

Assays for MBC resistance and thermotolerance of mutant strains

The nine mutants were assayed for their MBC resistance as described earlier but the MBC concentration added to SDAY ranged from 0 (control) to 1000 μg ml−1 at an interval of 100 or 200. Conidial thermotolerance of each mutant was assessed using the method of wet-heat stress (Ying and Feng 2004). Briefly, 1-ml aliquots of conidial suspension (106 conidia ml−1 0·02% Tween 80) in 1·5-ml glass vials were exposed to water bath at 45°C for up to 25 min. During the exposure, a 100-μl aliquot was pipetted from each vial (three vials per mutant) at an interval of 1 to 5 min, varying with conidial sensitivity to the heat stress in preliminary assays and spread on an SDAY plate (6 cm diameter). After 24-h incubation at 25°C and 12 : 12 h, conidial viability was determined using microscopic counts of germinated and ungerminated conidia on each plate triangularly covered with three glass coverslips (three counts per slip, ∼100 conidia per count). The sampling continued until no viable conidia were found in the sample. The ratio of the per cent germination of each mutant at a given sampling time over that in blank control (not stressed) was defined as survival index (Is) under the heat stress. The wild-type strain was included in the assays for comparison.

MDR assays

To assay multidrug sensitivities of three selected mutants (M1-14, M1-47, and M1-80) in parallel with their parental strain (If116), three 200-μl aliquots (replicates) of 108 conidia ml−1 suspension were evenly spread onto the plates of a medium consisting of 1% glucose, 0·6% NaNO2, 0·152% KH2PO4, 0·052% KCl and 0·02% Hutner’s trace element and incubated for 24-h germination at 25°C. Filter paper discs (5 mm diameter) dripped with 50 μl dimethyl sulfoxide (DMSO) containing a chemical drug were placed on the centre of the plates (9 cm diameter). Dissolved in 50 μl DMSO were six fungicides (5 mg iprodione, 5 mg thiophanate-methyl, 1 mg shenqinmycin, 5 mg cymoxanil, 5 mg azoxystrobin and 5 mg tricyclazole), one herbicide (0·5 mg glufosinate), and one insecticide (5 mg imidacloprid), respectively. All plates were incubated at 25°C for 7–10 days, followed by cross-measuring the fungal growth-inhibiting zones around the discs for comparison among the tested drugs. The drug solvent showed no effect on the fungal growth in a preliminary assay.

Assays for the presence of drug efflux pump

Conidial suspension (108 conidia ml−1) of the wild-type strain If116 was shaken in Sabouraud dextrose broth (SDB) for 48 h at 25°C. Blastospores were harvested by filtering the SDB culture through four-layer lens papers, washed three times with PBS and then resuspended in 1 ml PBS in 1·5-ml microfuge tube. Adding rhodamine 123 (Sigma, St Louis, MO) to the final concentration of 10 μmol l−1, the suspension was incubated for 30 min in water bath at 37°C. The stained blastospores were collected by centrifugation and washed twice with PBS. To induce the production of possible PDR-mediating ABC transporter protein(s) for uptake and efflux by the blastospores, MBC was added to the SDB culture at the final concentrations of 0–2 μg ml−1 after the first 24-h incubation, followed by additional 24-h incubation. To induce possible MBC competition for the protein-binding site of the fluorescent stain, MBC was added to the blastospore suspension at the final concentrations of 0–30 μg ml−1 during the 30-min period of staining at 37°C. The fluorescence intensity (FI) of the stained blastospores at a given MBC concentration was assessed on BD-LSR Cytofluorimeter with CellQuest software (BD Biosciences, San Jose, CA, USA) at the excitation and emission wavelengths of 488 and 525 nm, respectively.

Finally, the blastospores of all mutants and If116 were produced in MBC-free SDB cultures grown for 48 h at 25°C and harvested for the same period of staining at 37°C. The FI values of their stained blastospores were assessed using the same protocol. The FI ratio of each mutant over If116 was defined as relative FI (RFI). All assays were repeated three times.

Cloning and analysis of target genes associated with drug resistance

Total RNAs were extracted from the cultures of If116 and its nine mutants using a method described elsewhere (Zou et al. 2006). Each RNA sample (1 μg) was reversely transcribed with AMV reverse transcriptase and oligo(dT) primer (Takara, Tokyo, Japan) and the cDNA was PCR amplified to clone α- and β-tubulin genes with paired primers αtub-F/R (5′-ATG CGTGAGGTTATCAGTATC-3′; 5′-ATACTCAATCTCGCCCTCAT-3′) and βtub1-F/R (5′-AT GCGTGAGATTGTTCACCTC-3′; 5′-TTACATGGGCTCCTCAGCCTCA-3′), respectively. The PCR was run in a 50-μl reaction volume consisting of 50 ng cDNA, 0·2 μmol l−1 each primer, 0·2 mmol l−1 each dNTP, 2·5 mmol l−1 MgCl2, 1 × Taq polymerase buffer and 2·5 U Taq polymerase. The reaction began from denaturation at 94°C for 3 min, followed by 35 cycles of 30-s denaturation at 94°C, 30-s annealing at 50 (α-tubulin) or 55°C (β-tubulin) and 90-s extension at 72°C and the final extension of 7 min at 72°C.

To clone an ABC transporter gene (ifT1) from the wild-type and mutant strains via PCR, degenerate primers ifT1-F/R (5′-TCCGGNGCCCGHAARAC-3′; 5′-CCSGAGGTNGGYTCRTC-3′) were designed based on the conserved C-terminal regions of glycine-rich Walker A and hydrophobic Walker B motifs of the genes encoding other fungal ABC transporter proteins in GenBank database. The PCR began from 5-min pre-activation at 94°C, followed by 40 cycles of 30 s at 94°C, 30 s at 50°C and 1 min at 72°C. An amplified product (404 bp) was cloned into pGEM®-T Easy (Promega, Madison, WI, USA.) for sequencing at Invitrogen (Shanghai, China). Based on the sequenced fragment, six pairs of primers were designed for six rounds of DNA walking with SpeedUpTM Premix Kit II (Neuro-Hemin Biotec, Hangzhou, China) to amplify the full-length ifT1 gene and upstream promoter region (PifT1) from If116 genome. Based on the PifT1 and ifT1 sequences, paired primers PifT1-F/R (5′-TG GGCTATATTGGTTCAGGC-3′; 5′-ATACCTGAGACCAATCCTGCG-3′) and ifT1-F/R (5′-T TATTTGGTCGCCTTTTCC-3′; 5′-TCTATTGCTTATGGAATGACGG-3′) were designed to amplify the PifT1 and ifT1 sequences from all mutants via PCR.

All DNA fragments of the target genes cloned from If116 and its MBC-resistant mutants were gel-purified, cloned into pGEM-T-Easy and sequenced at Invitrogen. The protein sequences deduced from the cloned genes were analysed online (http://www.dnastar.com/ products/lasergene.php; http://www.ncbi.nlm.nih.gov/BLAST/) for locating possible site mutations. All putative PifT1 regions from the strains were analysed for locating possible mutations in transcription factors (TF) as well as initial transcription site, TATA box and CAAT box by motif search (http://www.cbil.upenn.edu/cgi-bin/tess/tess) and TF search (http://www.Cbrc.jp/research/db/tfsearch.html).

Assays for ifT1 expression levels in wild-type and mutant strains

Aliquots of 2 μg total RNA extracted from the SDB cultures of If116 and its mutants were reversely transcribed using PrimeScriptTM RT reagent kit (Takara, Japan). Synthesized cDNA (diluted to 10 ng μl−1) was used as template for quantitative real-time PCR (qRT-PCR) to assess the transcript levels of the β-tubulin and ifT1 genes and the fungal 18S rRNA (as an internal standard) with paired primers βtub2-F/R (5′-CGCCGTCCTCGTCGATCTTGAG-3′; 5′-GCACCCTCAGTGTAGTGACCCTTG-3′), ifT3-F/R (5′-TCGCCAGTGCATTGGAGAT T-3′; 5′-ATAGAGGAGATGGCTTCGGCA-3′) and 18S-F/R (5′-CGGCTACCACATCCAAGGAA-3′; 5′-GCTGGAATTACCGCGGCT-3′), respectively. The reaction began from 5-min pre-heating at 95°C, followed by 40 cycles of 20 s at 95°C, 20 s at 62°C and 20 s at 72°C. The relative transcript level of each target gene was estimated as the transcript ratio of each mutant over the wild-type strain using the inline image method (Livak and Schmittgen 2001). Each qRT-PCR was repeated three times.

Results

Variability in MBC resistance of wild-type strains

For all 15 I. fumosorosea wild-type strains tested in this study, the inverted sigmoid trends of conidial survival indices (Is) over the gradient MBC concentrations (C) fit well the survival equation Is = 1/[1 + exp(α βC)], resulting in the minimal inhibiting concentration (MIC) estimates of 6·5 (If116) to 84·3 (If6206) μg ml−1 (Table 1). These data indicated low to medium levels of MBC resistance in the tested strains, contrasting with the sensitivity of most B. bassiana wild-type strains to the same fungicide (Zou et al. 2006).

Changes in MBC resistance and thermotolerance of If116 mutants

The wild-type strain If116 showing the least MBC resistance in the previous experiment was exposed to the selective pressure of NaNO2 for chemical mutagenesis, yielding nine mutants with mitosis stability, which was confirmed after three rounds of culturing on MBC-free SDAY. Surprisingly, all the mutants became extremely resistant to MBC because 20–83% of their conidia survived (i.e. formed CFUs) even at 1000 μg MBC ml−1 maximally dissolvable in the medium. Their Is-C trends were not in the typically inverted sigmoid type, thereby fitting the density equation Is = (α βC)−1/λ with very high coefficients of determination (r 0·99). With the fitted equations, median effective concentration (EC50) for MBC to inhibit 50% CFU formation was estimated as 99·4 to more than 1000 μg ml−1 (Table 2), but no meaningful MIC values (far beyond maximal MBC concentration tested) were computable. The EC50 values indicated that MBC resistance in all mutants was enhanced by 82- to 830-fold.

Table 2.   Conidial MBC resistance (EC50) and thermotolerance (LT50) of Isaria fumosorosea wild-type strain (If116) and nine MBC-resistant mutants
Fungal strainMBC resistance*Thermotolerance†
Fitted parametersFitness (r2)EC50 (μg ml−1)Fitted parametersFitness (r2)LT50 (min)
αβλαβ
  1. *Fitted to the density equation Is = (α βC)−1/λ, where C was the MBC concentration. The EC50 estimate denotes an effective MBC concentration to inhibit 50% CFU growth on SDAY.

  2. †Fitted to the equation Is = 1/[1 + exp(α βt)], where t was the time length (min) of wet-heat stress at 45°C. The LT50 estimate denotes the media lethal time of fungal conidia under the heat stress.

If1161·0040·52180·7110·9911·2−8·6070·6250·99913·8
M1-31·0020·10863·5600·99599·4−2·7400·2810·9839·7
M1-140·9980·00060·4460·993605·8−6·6430·9890·9686·7
M1-331·0030·019216·2980·994>1000−4·3210·4740·9989·1
M1-471·053−0·0010−3·9010·997985·6−8·4410·8050·99510·5
M1-670·9890·00130·9690·991745·2−6·9840·5910·99911·8
M1-801·0010·02894·5820·998794·1−2·6270·4160·9906·3
M1-811·0010·01581·8590·997166·2−3·4850·5050·9986·9
M1-931·033−0·0010−4·0550·998973·2−4·3520·5610·9917·8
M1-940·999−0·0001−0·0770·995509·4−4·4120·5680·9987·8

Accompanied by the greatly enhanced MBC resistance, all mutants were less tolerant to the wet-heat stress at 45°C. Under this stress, median lethal time (LT50) estimates of their conidia fell in the range 6·7–11·8 min (Table 2), which were computed by fitting the time-declining Is trends to the survival equation (Ying and Feng 2004). This indicates that the conidial thermotolerance of the mutants was reduced by 14·5–51·4% compared with the LT50 estimate of 13·8 min from the wild-type strain. However, no correlation was found between increased EC50 values and decreased LT50 values.

Features of α- and β-tubulin sequences in MBC-resistant mutants

The sequences of both α- and β-tubulin genes (GenBank codes: JN170092 to JN170111) amplified from the genomes of the wild-type strain and its MBC-resistant mutants with paired primers αtub-F/R and βtub-F/R were characteristic with 1883- and 1727-bp open reading frames (ORF) with six and three introns, respectively. Online alignments of all deduced amino acid sequences revealed that no site mutation occurred in any amino acid residues of α-tubulin from the mutants. The deduced β-tubulin sequences of three mutants (M1-3, M1-67 and M1-93) were identical to that of the wild-type strain while the same sequences of other six mutants suffered from one to three random site mutations (M1-14: N227Y; M1-33: L44F and S365P; M1-47: R380C and E412G; M1-80: G13S, F90L and S386F; M1-81: F266L; and M1-94: F305L). However, none of such mutations occurred at the amino acid residues known to confer fungal MBC resistance (Butters et al. 2003; Ma et al. 2003, 2005; Zou et al. 2006).

Detected multidrug resistance

The three MBC-resistant mutants M1-14, M1-47 and M1-80 displayed significantly greater resistance to almost all tested pesticides of different types than the wild-type strain, as indicated by smaller growth-inhibiting zones around the 5-mm-diameter paper discs with the pesticides (Table 3). These data clue that multidrug resistance exists in the tested mutants.

Table 3.   Sensitivities of Isaria fumosorosea wild-type strain (If116) and three MBC-resistant mutants to different types of chemical pesticides
DrugMean (±SD) diameter (cm) of growth-inhibiting zone*F value†
If116M1-14M1-47M1-80
  1. *Table entries were estimated from three replicates. Those followed by different letters in each line differed significantly (Fisher’s LSD, < 0·05).

  2. < 0·01 (A) or 0·05 (a) in F3,8 tests (one-way anova); m: marginal effect at = 0·05.

Iprodione1·27 ± 0·12a0·93 ± 0·06b0·70 ± 0·10c1·03 ± 0·06b22·1A
Tricycleazole2·63 ± 0·32a1·50 ± 0·44b1·53 ± 0·42ab1·43 ± 0·49b5·6a
Shenqinmycin1·20 ± 0·10a0·73 ± 0·06c0·93 ± 0·06bc1·10 ± 0·20ab8·8A
Thiophanate-methyl1·53 ± 0·25a0·77 ± 0·12b0·60 ± 0·06b0·60 ± 0·10b25·7A
Azoxysyrobin1·93 ± 0·06a1·53 ± 0·15b1·17 ± 0·06c0·87 ± 0·15d47·9A
Cymoxanil1·07 ± 0·15a0·73 ± 0·12b0·63 ± 0·06b0·90 ± 0·26ab4·0m
Glufosinate3·97 ± 0·15a0·87 ± 0·06c3·43 ± 0·21b1·07 ± 0·12c366·4A
Imidacloprid2·03 ± 0·25a1·27 ± 0·06b0·57 ± 0·06c0·60 ± 0·00c81·8A

Detected drug efflux pump

The blastospores harvested from the If116 SDB cultures were well stained with rhodamine after 30-min incubation at 37°C. Their FI values were drastically decreased by adding 0·25–0·5 μg MBC ml−1 to the SDB cultures for 24-h induction prior to staining but not reduced further by adding more MBC (Fig. 1a). The decreased FI trend indicated that the added MBC induced the production of possible PDR-associated ABC transporter protein(s), thus causing more rhodamine efflux from the stained spores. Moreover, the blastospores from the MBC-free cultures showed an FI trend increased over the range of 0–30 μg ml−1 MBC during the staining period (Fig. 1b), suggesting that the stain and MBC compete for the same transporter protein(s). Interestingly, such protein(s) were overexpressed in the blastospores of all MBC-resistant mutants produced in normal SDB cultures, as indicated by the significant RFI decreases in their stained blastospores (Fig. 1c).

Figure 1.

 Fluorescence intensity (FI) change in I. fumosorosea blastospores stained with rhodamine 123 (Rh123) for 30 min at 37°C. (a) Effect of increased Rh123 efflux on the FI values of If116 (wild-type) blastospores stained after the SDB culture was supplemented with 0–5 μg MBC ml−1 for the last 24-h incubation at 25°C to induce the production of possible PDR/MDR protein in the blastospores. (b) Effect of decreased Rh123 efflux on the FI values of If116 blastospores stained together with 0–30 μg MBC ml−1 to induce possible MBC competition for the protein-binding site of Rh123. (c) Relative FI (RFI) values of the stained blastospores of MBC-resistant mutants over If116 counterparts. All blastospores were produced in normal SDB cultures prior to standard staining. Different letters on the bars denote significant difference (Fisher’s LSD, < 0.05). Error bars: SD of the mean from three replicates.

Features of cloned ifT1 and PifT1 sequences

All DNA sequences of the ifT1 gene cloned from wild-type and mutant strains (GenBank codes: JN170112 to JN170121) were found sharing a 4584-bp ORF with a 214-bp intron and encoding for the sequences of 1527 amino acids with no site mutation found in sequence alignments. In hydropathy analysis (http://www.cbs.dtu.dk/services/TMHMM/), the deduced protein was predicted as a typical ABC transporter with two membrane-anchored hydrophobic domains at each terminus, each domain including six transmembrane-spanning helices and a pair of conserved motifs, i.e., Walker A (GPPGSGCST) and Walker B (CWDNSTRGLD) in the N-terminal domain and motif A (GVSGAGKTT) and motif B (LFVDEPTSGLD) in the C-terminal domain. Online BLASTP analysis and sequence alignments showed only 35–64% sequence identity of the deduced ifT1 protein to the ABC transporters known from other fungi (Prasad et al. 1995; Urban et al. 1999; Andrade et al. 2000a; Andrade et al. 2000b; Nakaune et al. 2002) and yeasts (Balzi et al. 1994; Turi and Rose 1995). These data indicate that the ifT1-coding protein is a new member in the family of ABC transporter proteins as PDR/MDR regulators.

Although all ifT1 ORF sequences from the wild-type and mutant strains were identical, the 2400-bp promoter (PifT1) regions upstream of their ORFs were found harbouring three common point mutations, i.e., −2202G→A, −836C→T and −302C→T (Table 4). Such point mutations identified by online search occurred respectively at the binding sites of Gal4 as transcription activator (Brand and Perrimon 1993; Davison et al. 2007), Abf1 as autonomously replicating sequence-binding factor 1 (Miyake et al. 2002; Schlecht et al. 2008), and Raf as part of a protein kinase cascade in the MAPK/ERK signal transduction pathway (Li et al. 1991; Sridhar et al. 2005; Terai and Matsuda 2005). Apart from the common point mutations, one to four other random point mutations were also found in the PifT1 regions of the mutant strains, but such mutations were not always associated with meaningful factors to regulate gene transcripts.

Table 4.   Point mutations in the promoter regions (PifT1) upstream of the ifT1 gene cloned from the nine MBC-resistant mutants of the wild-type Isaria fumosorosea strain (If116)
StrainsCommon point mutations*Other random point mutations*
Gal4Abf1Raf
  1. *Found by online Motif Search (http://motif.genome.jp) and TFSEARCH (http://www.Cbrc.jp/research/db/TFSEARCH.html). Gal4, Abf1 and Raf are located at the sites from −2206 to −2201 bp, −838 to −833 bp and −303 to −300 bp, respectively. The common point mutation of each factor is underlined.

If116TGGGGGCTCGTCCCGA 
M1-3TGGGAGCTTGTCCTGA−893A→T
M1-14TGGGAGCTTGTCCTGA−1617C→T, −1435C→T
M1-33TGGGAGCTTGTCCTGA−1455C→T, −1008T→C
M1-47TGGGAGCTTGTCCTGA−2270G→C, −1680C→T
M1-67TGGGAGCTTGTCCTGA−1181C→T, −343C→T
M1-80TGGGAGCTTGTCCTGA−2079A→G, −549C→T, −355C→T
M1-81TGGGAGCTTGTCCTGA−946T→C
M1-93TGGGAGCTTGTCCTGA−1617C→T, −1435C→T, −1313A→G, −212C→T
M1-94TGGGAGCTTGTCCTGA−1617C→T, −1435C→T, −244C→T

Transcript levels of β-tubulin and ifT1 genes in MBC-resistant mutants

The β-tubulin and ifT1 genes were transcribed in the SDB cultures of the wild-type and mutant strains grown under normal conditions. The transcript levels of β-tubulin gene in all mutants were only 0·87- to 7·16-fold of that in the wild type (Fig. 2a). In contrast, the ifT1 transcript levels in all MBC-resistant mutants were 17- to 137-fold of that in the wild type (Fig. 2b). These data indicate that the enhanced multidrug resistance in the mutant strains was likely attributed to the ifT1 overexpression.

Figure 2.

 The transcript levels of the β-tubulin (a) and ifT1 (b) genes in the normal SDB cultures of MBC-resistant mutants relative to the wild-type strain If116, determined by quantitative real-time PCR. Error bars: SD of the mean from three replicates.

Discussion

All tested wild-type strains of I. fumosorosea were generally more resistant to MBC than the wild-type strains of B. bassiana assayed previously (Zou et al. 2006). The extraordinarily high MBC resistance of the fungal mutants derived from a single round of NaNO2-induced mutagenesis was not associated with any β-tubulin site mutations that confer MBC resistance in other fungi (Butters et al. 2003; Ma et al. 2003; Zou et al. 2006). The relative small changes, including up to six-fold increase and 13% decrease, in the β-tubulin gene transcripts of all mutants were also unlikely to increase the fungal MBC resistance by 82- to 830-fold. A drug efflux pumping mechanism revealed in the assays of rhodamine-stained blastospores was confirmed by the drastic increases of ifT1 transcripts in all the MBC-resistant mutants. Thus, we consider that the common point mutations at the binding sites of Gal4, Abf1 and Raf factors in the PifT1 regions induced the ifT1 overexpression, which resulted in the fungal resistance to not only MBC but also other different pesticides tested in the study. This is the first report on PDR/MDR mediated by an ABC transporter in entomopathogenic fungi. Nonetheless, some more MBC-resistant mutants, e.g., M1-93 and M1-94, displayed lower levels of ifT1 expression than M1-3, a mutant much less resistant to MBC. This implies that the ifT1 is not the only transporter responsible for the high resistance. Some other transporter proteins in the fungal genome are also likely to take parts in the fungal resistance to MBC and other pesticides.

Interestingly, the enhancements of the ifT1 expression in all MBC-resistant mutants were consistently associated with three common point mutations in the PifT1 regions rather than any change in the coding regions of the target gene. Such mutations occurred at the biding sites of the crucial factors Gal4, Abf1 and Raf, which are well known involved in the regulative network of numerous protein loci (Miyake et al. 2002; Davison et al. 2007; Schlecht et al. 2008). Several studies have shown that the expression levels of drug efflux pumping proteins increased in yeast response to different xenobiotics and that Pdr1p/Pdr3p was required for the response (Mamnun et al. 2004; Lucau-Danila et al. 2005; Alenquer et al. 2006; Fardeau et al. 2007). A Pdr1p orthologue of C. glabrata (CgPdr1p) was also found regulating the drug efflux pumps and thus responsible for the fungal MDR (Vermitsky et al. 2006). Treatments with antifungal drugs may activate similar drug efflux pumps in yeast (Lucau-Danila et al. 2005). The activation of the pump genes in fungi was proven to result from binding a drug or xenobiotic to a hydrophobic domain in Pdr1p/Pdr3p (Thakur et al. 2008). The increased knowledge about the PDR/MDR regulation helps to infer that the overexpression of the ABC transporter ifT1 is responsible for the high resistance of all I. fumosorosea mutants to MBC and other different pesticides. However, it is unclear at present how the recognized transcription factors and/or other unrecognized factors regulate the ifT1 expression for drug efflux, warranting further studies.

Acknowledgements

We thank R.A. Humber (RW Holley Center for Agriculture and Health, Ithaca, NY, USA) and T.J. Poprawski (Biological Control of Pests Research Unit, Weslaco, Texas, USA) for providing fungal strains. Funding of this study was provided by the Natural Science Foundation (grant nos. 30930018 and 31021033) and the Ministry of Science and Technology of China (grant nos. 2009CB118904 and 2011AA10A204).

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