MBC Resistance Research Articles

Abstract PS5-10: Esr1 mutation as a potential predictor of abemaciclib benefit following prior cdk4/6 inhibitor (cdk4/6i) progression in hormone receptor-positive (hr+) metastatic breast cancer (mbc): A translational investigation

Abstract Background: CDK4/6 inhibitors have emerged as the standard of care for HR+ MBC. However, there is limited insight into the potential benefit of abemaciclib following prior progression on palbociclib or ribociclib. Based on a multi-center cohort of patients with HR+ MBC who had received abemaciclib after prior palbociclib progression (Wander SA et al ASCO 2019), we have previously reported that abemaciclib after prior CDK4/6i progression was well tolerated and that a subset of patients derived durable clinical benefit. Identifying molecular predictors of sensitivity to abemaciclib after prior CDK4/6i progression constitutes an important area of research. Given the high frequency of ESR1 mutations in HR+ MBC with antiestrogen resistance, we evaluated the translational impact of ESR1 mutations in mediating response to abemaciclib in this setting. Methods: To evaluate abemaciclib sensitivity in ESR1 mutant cell lines, T47D HR+ breast cancer cells were modified to over-express multiple mutant ESR1 isoforms via lentiviral infection and antibiotic selection. These isoforms included ESR1 Y537S, Y537N, and D538G. In an additional T47D cell line, RB1 expression was knocked down via CRISPR. The resulting derivative cell lines were grown in the absence of estrogen (via charcoal-stripped serum, CSS) or in escalating doses of abemaciclib. Cell viability was measured via cell-titer-glo assay. For clinical validation, we identified patients with MBC who had ESR1 mutations detected by targeted sequencing of cell-free DNA (cfDNA), via CLIA certified Guardant assay, and had abemaciclib exposure following prior progression on palbociclib or ribociclib in the existing multi-center cohort from six US institutions. Results: All ESR1 mutant derivative cells demonstrated enhanced growth in estrogen deprivation compared to GFP controls, as expected, and were similarly sensitive to escalating doses of abemaciclib monotherapy in vitro, suggesting that ESR1 mutations do not confer resistance to abemaciclib. Interestingly, two patients with ESR1 mutations (in the absence of concurrent driver alterations in RB1, FGFR, CCNE2, and ERBB2) demonstrated progression on palbociclib and sensitivity to abemaciclib. In one patient, cfDNA obtained prior to palbociclib and fulvestrant exposure failed to reveal any ESR1 alteration. Following progression on palbociclib, and prior to sequential exposure to abemaciclib, an ESR1 Y537N alteration was identified. The patient went on to receive 16 months of abemaciclib monotherapy. In a second patient, an ESR1 D538G alteration was identified following progression on palbociclib and fulvestrant. The patient had several intervening regimens, and subsequently went on to receive abemaciclib and fulvestrant for 16 months. RB1-null T47D cells were resistant to abemaciclib monotherapy in vitro, as expected and, in the clinical dataset, the presence of alterations in previously identified genomic mediators of CDK4/6i resistance, such as RB1, were associated with progression on both palbociclib and abemaciclib. Conclusions: HR+ breast cancer cells expressing mutant ESR1 isoforms were resistant to estrogen deprivation but retained sensitivity to abemaciclib in vitro. Furthermore, patients harboring ESR1 mutations via targeted sequencing of cfDNA, in the absence of other known mediators of CDK4/6i resistance, were shown to derive clinical benefit from abemaciclib following prior progression on palbociclib. These results suggest that patients with HR+ MBC, ESR1 mutation, and clinical resistance to anti-estrogen treatment and palbociclib may be candidates for abemaciclib treatment. Further research is warranted to confirm these novel translational observations. Citation Format: Seth A. Wander, Hyo S. Han, Gabriela N. Johnson, Maxwell R. Lloyd, Pingping Mao, Utthara Nayar, Kailey Kowalski, Casey R. Stein, Veronica Mariotti, Leslie SL Kim, Maren Levin, Jing Xi, Apurva Pandey, Siobhan Dunne, Azadeh Nasrazadani, Adam Brufsky, Kevin Kalinsky, Cynthia X Ma, Joyce O’Shaughnessy, Nikhil Wagle, Aditya Bardia. Esr1 mutation as a potential predictor of abemaciclib benefit following prior cdk4/6 inhibitor (cdk4/6i) progression in hormone receptor-positive (hr+) metastatic breast cancer (mbc): A translational investigation [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-10.

Carbendazim-resistance of Gibberella zeae associated with fusarium head blight and its management in Jiangsu Province, China

Fusarium head blight (FHB) caused by Gibberella zeae occurs in wheat-producing areas worldwide. FHB results in substantial economic losses and produces various mycotoxins, notably the type B trichothecene (B-Trich) deoxynivalenol and its acetylated derivatives and nivalenol, as well as the sterol zearalenone, which are toxic to humans and animals, respectively. Here, 1313 (667 from conventional fields [CFs] and 646 from systematic observation fields [SOFs]), 1915 (1246 from CFs, 669 from SOFs) and 1703 (1118 from CFs, 585 from SOFs) isolates of G. zeae were collected from 37, 43 and 35 counties (cities), respectively, in Jiangsu Province in 2016–2018. The sensitivity to carbendazim (MBC) was determined, and 2-year field trials on the management of FHB severity and B-Trich contamination were conducted using 10 representative fungicides registered by the Institute for the Control of Agrochemicals, Ministry of Agriculture of China. The frequencies of MBC-resistant (R) G. zeae collected from CFs in 2016–2018 were 61.32%, 58.03% and 56.80%, respectively, and those from SOFs were 38.24%, 43.80% and 41.37%, respectively. The frequency of MBC-R G. zeae in Jiangsu Province was found to be the highest in the world, and this development of MBC resistance requires the application of alternative chemicals to fight FHB. In field trials in 2017–2018, tebuconazole and phenamacril showed >85% efficacy in the control of FHB severity and B-Trich contamination. A mixture of pyraclostrobin and tebuconazole showed >82% and <58% efficacy in the control of FHB severity and B-Trich contamination, respectively. Azoxystrobin showed <61% efficacy in FHB severity control, and increased B-Trich accumulation. Prochloraz and epoxiconazole showed ∼67–78% efficacy in the control of FHB severity and B-Trich contamination. A mixture of MBC and triadimefon, MBC, thiophanate-methyl and thiram, which represented ∼70% of the relevant registered fungicides, showed <60% efficacy in the control of FHB severity and B-Trich contamination. The information obtained in this study will be useful for the management of MBC-R G. zeae and control of FHB and B-Trich.

Molecular and Biochemical Characterization of Carbendazim-Resistant Botryodiplodia theobromae Field Isolates.

Stem-end rot caused by Botryodiplodia theobromae is a destructive disease of mango. B. theobromae field isolates resistant to carbendazim (MBC) were collected in Hainan Province, China. In this study, the characteristics of these field isolates with resistance to MBC were investigated. The resistance of B. theobromae isolates to MBC was stably inherited. Both the MBC-resistant and MBC-sensitive isolates had similar mycelial growth rates, pathogenicity, sensitivity to high glucose, glycerol content, and peroxidase activity. Compared with MBC-sensitive isolates, MBC-resistant isolates were more sensitive to low temperature and had a significant decrease in sensitivity to high NaCl and a significant increase in catalase (CAT) and glutathione S-transferase (GST) activities. After MBC treatment, the cell membrane permeability of the sensitive isolates was markedly increased compared with that of the resistant isolates. Analysis of the β-tubulin gene sequence revealed point mutations resulting in substitutions at codon 198 from glutamic acid (GAG) to alanine (GCG) in moderately resistant isolates, and at codon 200 from phenylalanine (TTC) to tyrosine (TAC) in highly resistant isolates. These β-tubulin gene mutations were consistently associated with MBC resistance. Overall, we infer that the altered cell membrane permeability and the increase in CAT and GST activities of the resistant isolates are linked to MBC resistance.

Sunitinib Treatment Enhances Metastasis of Innately Drug-Resistant Breast Tumors.

Antiangiogenic therapies have failed to confer survival benefits in patients with metastatic breast cancer (mBC). However, to date, there has not been an inquiry into the roles for acquired versus innate drug resistance in this setting. In this study, we report roles for these distinct phenotypes in determining therapeutic response in a murine model of mBC resistance to the antiangiogenic tyrosine kinase inhibitor sunitinib. Using tumor measurement and vascular patterning approaches, we differentiated tumors displaying innate versus acquired resistance. Bioluminescent imaging of tumor metastases to the liver, lungs, and spleen revealed that sunitinib administration enhances metastasis, but only in tumors displaying innate resistance to therapy. Transcriptomic analysis of tumors displaying acquired versus innate resistance allowed the identification of specific biomarkers, many of which have a role in angiogenesis. In particular, aquaporin-1 upregulation occurred in acquired resistance, mTOR in innate resistance, and pleiotrophin in both settings, suggesting their utility as candidate diagnostics to predict drug response or to design tactics to circumvent resistance. Our results unravel specific features of antiangiogenic resistance, with potential therapeutic implications. Cancer Res; 77(4); 1008-20. ©2016 AACR.

Detection and characterization of carbendazim resistance in Sclerotinia sclerotiorum isolates from oilseed rape in Anhui Province of China.

Stem rot caused by Sclerotinia sclerotiorum is a devastating disease of oilseed rape (Brassica napus) in Anhui Province of China. The fungicide carbendazim (methyl benzimidazole-2-yl carbamate; MBC) has been used to control this fungal disease since the 1980s. In the present study, 74 isolates of S. sclerotiorum from 13 regions of Anhui were collected, and the sensitivities of these isolates to MBC were examined to monitor fungicide resistance. We found that 22 of the 74 isolates showed resistance to MBC, indicating that S. sclerotiorum has developed resistance in parts of Anhui Province. PCR assays and DNA sequence analysis showed that isolates with high MBC resistance had a point mutation at position 198 in the β-tubulin gene that caused a glutamic acid-to-alanine change in the protein. The β-tubulin gene in low-resistance isolates did not have the mutation. These results indicate that the mutation in β-tubulin gene may be associated with high MBC resistance in S. sclerotiorum. The present study also found no correlation between MBC resistance and pathogenicity of S. sclerotiorum isolates, suggesting that the pathogenicity of S. sclerotiorum isolates on oilseed rape did not vary with MBC resistance status.

Molecular mechanism of resistance of Fusarium fujikuroi to benzimidazole fungicides.

Although carbendazim (MBC) and other benzimidazole fungicides have effectively controlled bakanae disease of rice (which is caused by Fusarium fujikuroi, F. proliferatum, and F. verticillioides) in the past, MBC resistance has become common. Previous research has shown that MBC resistance results from a mutation in the β1 -tubulin (β1 tub) gene in F. verticillioides. However, MBC resistance in F. fujikuroi, a predominant species in China, does not result from a mutation in the β1 tub. The molecular mechanism of F. fujikuroi resistance against benzimidazole fungicides is poorly understood. In this study, we determined that although β1 tub and β2 -tubulin (β2 tub) in F. fujikuroi have high homology with β1 tub and β2 tub in F. verticillioides, MBC resistance in F. fujikuroi results from mutations in β2 tub [GAG(Glu)→GTG(Val) at codon 198, TTC(Phe)→TAC(Tyr) at codon 200, and GGC(Gly)→GGT(Gly) at codon 235] but not in β1 tub. Δβ2 tub (β2 tub deletion) mutants were highly sensitive to MBC, produced fewer conidia and were less virulent than parental strains. Complementation of the Δβ2 tub mutants with a copy of the whole β2 tub locus from their parental strains restored the level of MBC resistance (or sensitivity) to that of the parental strain.

Characterization of carbendazim sensitivity and trichothecene chemotypes of Fusarium graminearum in Jiangsu Province of China

Fusarium head blight, one of the most damaging plant diseases in Jiangsu province of China, is a leading cause of economic loss and toxin accumulation in the crop, including nivalenol, deoxynivalenol and its acetylated derivatives. Disease control by carbendazim (MBC) has been applicated for many years, and the resistance frequency increased steadily. Furthermore, resistance may trigger toxin growth. Here, a total of 7261 isolates were collected throughout Jiangsu province from 2010 to 2012 to determine their sensitivity to MBC and trichothecene chemotypes. We studied the relevance between trichothecene chemotype and MBC-sensitivity, and found that the MBC-sensitive isolates occupied more NIV chemotype proportion up to date; 15-AcDON chemotype only existed in MBC-sensitive isolates; and most MBC-resistant isolates secreted 3-AcDON in chemotype. Besides, trichothecene production analyses indicated that MBC resistance increased 3-AcDON yield and percentage, especially site-directed mutants at codon 167 in the β2-tubulin gene.

Integrated Use of Pyraclostrobin and Epoxiconazole for the Control of Fusarium Head Blight of Wheat in Anhui Province of China.

Fusarium asiaticum and F. graminearum are the primary causal agents of Fusarium head blight (FHB) of wheat in China. Carbendazim (a benzimadazole fungicide, MBC), has been extensively used for the control of FHB, resulting in severe MBC resistance in China. This article presents the baseline sensitivity of F. asiaticum and F. graminearum isolates from Anhui Province of China to fungicides pyraclostrobin (a quinone outside inhibitor) and epoxiconazole (a sterol demethylation inhibitor). In the presence of salicylhydroxamic acid, the 50% effective concentration (EC50) values for pyraclostrobin in inhibiting mycelial growth of the 126 F. asiaticum isolates and 63 F. graminearum isolates were 0.012 to 0.135 μg/ml and 0.010 to 0.105 μg/ml, and the EC50 values for pyraclostrobin in inhibiting conidium germination of the F. asiaticum and F. graminearum populations were 0.047 to 0.291 and 0.042 to 0.255 μg/ml, respectively. The EC50 values for epoxiconazole in inhibiting mycelial growth of the F. asiaticum and F. graminearum populations were 0.12 to 0.95 and 0.16 to 0.93 μg/ml, respectively. All of the baseline sensitivity curves were unimodal. This study also suggested that there was no cross-resistance between MBC and pyraclostrobin or epoxiconazole. In the protective and curative tests, pyraclostrobin and epoxiconazole applied at 200 and 300 μg/ml exhibited over 75% protective and curative control efficacy in all treatments. In field trials, both pyraclostrobin and epoxiconazole at 225 g a.i./ha provided over 80% efficacy in 2010 and 2011 at both sites where MBC resistance occurred, suggesting excellent activity against FHB. Interestingly, integrated use of pyraclostrobin + epoxiconazole applied at 150 + 150 g a.i./ha provided over 85% efficacy at both sites in 2010 and 2011. Pyraclostrobin and epoxiconazole should be good alternatives to MBC for the control of FHB, and integrated use of these two fungicides might achieve greater efficacy.

Activity of carbendazim, dimethachlon, iprodione, procymidone and boscalid against Sclerotinia stem rot in Jiangsu Province of China

Carbendazim (MBC) was widely used to control Sclerotinia stem rot routinely during the 1980s in China, but development of MBC resistance in the causal agent Sclerotinia sclerotiorum led to control failures of this disease. In this study it was found that the MBC resistance in S. sclerotiorum populations was widespread throughout Jiangsu Province with a resistance frequency of 29.54% in the 1786 collected isolates during the growing seasons of 2006 to 2008. The resistance frequencies differed among sampled cities, ranging from 3.1% to 54.9%. The field MBC-resistant isolates showed comparable mycelial growth, sclerotia production and pathogenicity to the wild-type sensitive isolates, which suggested that the field MBC-resistant isolates might have sufficient parasitic fitness to compete with the field MBC-sensitive isolates in the field. In the in vitro sensitivity test, boscalid showed greater activity against S. sclerotiorum than dicarboximide fungicides (dimethachlon, iprodione and procymidone). The treatment 50% boscalid (WG) 125 g a.i. ha−1 was comparable in efficacy to the treatment 50% iprodione (WP) 600 g a.i. ha−1, and better than other treatments of 6% dimethachlon (WP) 690 g a.i. ha−1 and 50% procymidone (WP) 337.5 g a.i. ha−1, whereas MBC failed to control Sclerotinia stem rot (control efficacy only 16.0%). The most active agent for controlling Sclerotinia stem rot was boscalid in our study.

Effect of Carbendazim Resistance on Trichothecene Production and Aggressiveness of Fusarium graminearum

Fusarium graminearum (teleomorph, Gibberella zeae) causes head blight of cereals and contaminates grains with trichothecene mycotoxins that are harmful to humans and domesticated animals. Control of Fusarium head blight relies on carbendazim (MBC) in China, but resistance to MBC in F. graminearum is now widespread. Sixty-seven strains were evaluated for trichothecene production in shake culture or in the field. The strains included 60 wild-type strains (30 MBC-resistant and 30 MBC-sensitive), three MBC-resistant site-directed mutants at codon 167 in beta(2)-tubulin, three MBC-sensitive site-directed mutants at codon 240 in beta(2)-tubulin, and their MBC-sensitive wild-type progenitor strain ZF21. The incidence of infected spikelets and the amount of F. graminearum DNA in field grain (AFgDNA) also were evaluated for all strains. MBC resistance increased trichothecene production in shake culture or in the field. Although MBC resistance did not change the incidence of infected spikelets, it did increase AFgDNA. Tri5 gene expression increased in MBC-resistant strains grown in shake culture. We found a significant exponential relationship between trichothecene production and Tri5 gene expression in shake culture and a linear relationship between the incidence of infected spikelets or AFgDNA and trichothecene production in field grain.

Detection and Characterization of Benzimidazole Resistance in California Populations of Colletotrichum cereale.

Colletotrichum cereale is the causal agent of turfgrass anthracnose, which has become a serious problem on annual bluegrass (Poa annua) and creeping bentgrass (Agrostis palustris) golf course putting greens. Thiophanate-methyl is a benzimidazole (methyl benzimidazole carbamate [MBC]) fungicide used for the management of anthracnose. In this study, we examined 481 isolates from 10 California populations to determine the presence and frequency of MBC resistance. An in vitro methodology was developed to construct a baseline sensitivity distribution using 60 isolates from an unexposed population (TCGC). The 50% effective dose (ED50) values for the baseline sensitivity distribution for thiophanate-methyl ranged from 0.14 to 2.3 μg/ml with a mean of 0.75 μg/ml. For 60 isolates assayed from an exposed population (AHCC), 57 isolates were not responsive to in vitro concentrations of thiophanate-methyl of up to 30 μg/ml. Isolates nonresponsive to thiophanate-methyl were not responsive to benomyl in vitro. Two isolates nonresponsive in vitro to thiophanate-methyl or benomyl were not controlled in vivo on annual bluegrass plants treated preventively with either fungicide at 11 mg/ml, confirming the results of the in vitro testing. The remaining 361 isolates from eight populations were tested using the single discriminatory dose of thiophanate-methyl at 10 μg/ml. A high proportion (>90%) of isolates from six of the populations were resistant to thiophanate-methyl, indicating the presence of practical resistance at these locations. To determine the molecular mechanism of MBC resistance, the two β-tubulin genes, TUB1 and TUB2, of 12 resistant and 6 sensitive isolates were amplified and sequenced, revealing a glutamic acid to lysine substitution at position 198 of TUB2 that was present in all resistant isolates. This work confirms the presence of MBC resistance in C. cereale populations from California and presents methods and information that can be used to manage resistance to the MBC fungicides and improve anthracnose management programs.

Characterization and fitness of carbendazim‐resistant strains of Fusarium graminearum (wheat scab)

Carbendazim (MBC) has failed to control wheat scab, caused by Fusarium graminearum Schwabe, on the eastern coast of China in recent years after about 30 years of application. MBC resistance was found to be common in pathogen populations on the eastern coast and along areas of the Yangtze River. EC(50) and minimum inhibitive concentration (MIC) values of MBC inhibiting mycelium growth of wild-type isolates were less than 0.9 and 1.4 microg mL(-1) respectively, while EC(50) values of resistant collections averaged 7.02 +/- 11.86 microg mL(-1). The slope of the MBC dosage-response curve (DRC) for resistant isolates of F. graminearum was flat: 1 < b < 2.8 for resistant isolates and 3.5 < b < 11 for sensitive isolates). Both field resistant and sensitive MBC strains shared similar temperature sensitivity, fitness and virulence on ears. Field resistant strains and UV-induced mutants showed positive cross-resistance to other benzimidazole derivatives and were mainly at intermediate MBC resistance level. Highly resistant field MBC strains rarely appeared, but only some of the highly resistant MBC UV mutants were insensitive to N-phenylaminecarbamates. No mutation in beta-tubulin was found in F. graminearum, in contrast to mutation in this tubulin which has led to MBC resistance in other plant pathogens. MBC(R) isolates have high fitness and competition in field, conferred by a novel molecular mechanism.

Tetrad analysis of mating type, mutations, esterase and aggressiveness in Phaeosphaeria nodorum

Ascospore progenies from two crosses between a wild single-ascospore strain and two mutants of Phaeosphaeria nodorum were obtained in vitro. Cultural characters, sensitivity to carbendazim (MBC), nitrate utilization, mating type, esterase zymograms and aggressiveness of these progenies and parents were compared and differences were detected between the ascospores. The single-ascospore strains from each single ascus could always be grouped into four pairs using a combination of markers. Recombinations were observed between nitrate non-utilizing phenotype and mating type or esterase patterns for one cross. For the other cross, recombinations between MBC resistance and esterase patterns were detected. The values of aggressiveness in each progeny were distributed along a wide range confirming the polygenic character of aggressiveness in P. nodorum. No close relationship was observed between in vitro conidiogenesis, the intensity of leaf necrosis and the fertile pycnidia production on leaf lesions.

Sensitivity of Neurospora crassa to benzimidazoles and N-phenylcarbamates: Effect of amino acid substitutions at position 198 in β-tubulin

Diethofencarb-resistant revertants have been isolated from the Neurospora crassa mutant F914 which shows both carbendazim (MBC) resistance and diethofencarb sensitivity. Revertant strains FR422 and FR424 were true revertants showing high resistance to N-phenylcarbamates and sensitivity to benzimidazoles, the same as wild-type. However, other revertant strains, FR511, FR513, FR521, FR421, and FRB3, were more resistant to diethofencarb than the strain F914 but less resistant to diethofencarb than the wild-type strain. These moderately resistant strains were also more resistant to benzimidazoles than the parent strain F914, Strains FR421 and FR521 were also resistant to griseofulvin. All the diethofencarb-resistant mutations were linked to the Bml locus that encodes β-tubulin. The β-tubulin gene of FRB3 strain was cloned and sequenced, and an amino acid substitution at the position 198 was found. In the strains FRB3, F914, and wild-type, the amino acids at position 198 were Lys, Gly, and Glu, respectively. A new β-tubulin gene with Ala at position 198 was constructed by site-directed mutagenesis. This gene transformed the wild-type strain to MBC resistance, while transformation of the F914 strain with this gene resulted in growth on the medium containing 0.5 μg/ml diethofencarb but not on that containing 75 μg/ml diethofencarb. The F914 strain transformed by β-tubulin genes of the wild-type strain and the FRB3 strain grew even on medium containing 75 μg/ml diethofencarb. These data suggest that the presence of Glu at position 198 in β-tubulin is responsible for MBC sensitivity. Different amino acid substitutions at position 198 confer different sensitivity to diethofencarb and the order of amino acids which result in increasing sensitivity are Glu, Lys, Ala, and Gly (most sensitive). These approaches should work in clarifying the function of β-tubulin and also aid in the design of new potent fungicides.

A single amino-acid substitution in the beta-tubulin gene of Neurospora confers both carbendazim resistance and diethofencarb sensitivity.

Two MBC-resistant mutants of Neurospora crassa, F914 and F939, were sensitive to diethofencarb at a concentration of 0.1 micrograms/ml, while the wild-type strain and other MBC-resistant mutants showed resistance to diethofencarb at a concentration of 100 micrograms/ml. Genetic analysis suggested that the mutations in these two strain were closely linked to the Bml locus which codes for beta-tubulin. When the wild-type strain was transformed by the cloned beta-tubulin gene of the F914 strain, the transformants showed both MBC resistance and diethofencarb sensitivity. On the other hand, the diethofencarb sensitivity of the F914 strain was cancelled by transformation with the wild-type beta-tubulin gene. DNA sequencing of F914 beta-tubulin revealed that glycine was substituted for glutamic acid at position 198 in the F914 strain. Therefore, a single base change in the beta-tubulin gene was proved to confer both MBC resistance and diethofencarb sensitivity.

Isolation of a benomyl-resistant allele of the -tubulin gene from Septoria nodorum and its use as a dominant selectable marker

Summary: We have developed a homologous transformation system for the wheat-pathogenic fungus Septoria nodorum based on a benomyl-(MBC-) resistant allele of the β-tubulin gene. The β-tubulin gene was isolated by heterologous hybridization from a cosmid library prepared from an MBC-resistant mutant. Cosmids carrying the gene conferred MBC resistance when introduced into a sensitive strain, demonstrating that resistance to MBC fungicides in S. nodorum may be determined by the β-tubulin gene. This MBC resistant allele of the β-tubulin gene (tubAR) was subcioned into pUC18 and used as a dominant selectable marker for transformation of wild-type sensitive strains. Transformants arose at frequencies of approximately 5 per μg of DNA, were integrative in nature and were mitotically stable. Some transformants showed a marked reduction in vigour, both in the presence and absence of MBC; this is thought to arise from overproduction of β-tubulin. The S. nodorum tubARgene also conferred MBC resistance on the related species Leptosphaeria maculans, a pathogen of Brassica, following its introduction by cotransformation. Probing digested S. nodorum DNA with tubARat low stringency revealed only a single β-tubulin gene. We anticipate that tubARwill prove a useful tool for the investigation of the pathogenicity of S. nodorum and other fungi.

Isolation and sequence analysis of a beta-tubulin gene from Aspergillus flavus and its use as a selectable marker

An altered beta-tubulin gene that confers resistance to benomyl [whose active ingredient is 2-(methoxycarbonylamino)benzimidazole (MBC)] was isolated from a DNA library of Aspergillus flavus and used as a selectable marker for transformation. The beta-tubulin gene was cloned into a plasmid vector containing the pyr-4 gene of Neurospora crassa, and transformants were selected either for uracil prototrophy or MBC resistance. Transformants selected for uracil prototrophy were of three phenotypic classes: sensitive, intermediate, and resistant to MBC. Transforming DNA appeared to integrate at several sites in the genome, with the more resistant phenotypes having more copies of the altered beta-tubulin gene than the sensitive and intermediate phenotypes. Transformants were also selected on medium containing MBC. The average frequency of transformation (1 to 3 transformants per micrograms of transforming DNA) was lower than that obtained by selection for uracil prototrophy, presumably because of failure to select transformants that contained few copies of the altered beta-tubulin gene. The sequence of the beta-tubulin gene was determined and compared with the published sequence of the benA gene of A. nidulans; the beta-tubulin gene was found to be highly conserved between the two Aspergillus species. Notable differences were that the beta-tubulin gene of A. flavus lacks intron 6 present in benA and has an additional leucine at position 148. This is the first gene sequence reported from an aflatoxin-producing fungus and adds to the growing body of knowledge of the beta-tubulin genes and their use as selectable markers for transformation of filamentous fungi.