Additive effect involving a new locus of benomyl resistance in Aspergillus nidulans

Most of the fungicides based on the benzimidazole nucleus, including benomyl, thiabendazole and thiophanate, are systemic and because they control many important fungal diseases. This regular paper is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol36/iss1/9 Martinez-Rossi, N.M.^1 and J.L. Azevedo^2 Most of the fungicides based on the benzimidazole nucleus, including benomyl, thiabendazole Additive effect involving a new and thiophanate, are systemic and because they have a broad action spectrum they can be used to locus of benomyl resistance in control many important fungal diseases. Even though the chemical structures of benomyl and Aspergillus nidulans thiophanate may appear not to be similar, the two substances are metabolized to a common compound which is probably the cause of cross-resistance between them. Benomyl-resistant mutants are of interest from at least two points of view: the understanding of the genetic bases that govern this resistance and the study of microtubulins using a genetical-biochemical approach, since benomyl is an antimicrotubular drug. Three loci of benomyl resistance have been described in A. nidulans: benA (Hastie and Georgopoulos 1971 J. Gen. Microbiol. 67:371-373, benB and benC (van Tuyl 1977 Ph.D. Thesis, Agricultural University, Wageningen, The Netherlands). The benA locus maps on linkage group VIII, confers high resistance to benomyl even though it does not permit conidiation of resistant mutants and also codes for ß-1 and ß-2 tubulin (Sheir-Neiss, G. et al. 1978 Cell 15:639-647). The other two loci map elsewhere and make the fungus resistant to low fungicide levels. To determine the possible existence and interrelationship of other loci responsible for this resistance, conidia of the proA1 pabaA6 yA2 strain were irradiated with UV light and inoculated at 37°C in complete medium dishes containing thiophanate-methyl fungicide (40 ug/ml). This concentration inhibits the growth of sensitive strains. A mutant obtained under these conditions (BEN-35) proved to be resistant to benomyl and showed normal conidiation in complete medium containing up to 10 ug/ml benomyl or 50 ug/ml thiophanate-methyl. In contrast FGSC A524 (benAl0 biAl fwA1) did not conidiate at a concentration exceeding 5 ug/ml benomyl or 10 ug/ml thiophanate-methyl. Genetic analyses of this new mutant (BEN-35) carried out by cross with the Master Strain E (MSE) showed that a single gene mutation which also mapped on linkage group VIII was responsible for the benomyl and thiophanate-methyl resistance. About 300 segregants obtained from a cross between BEN-35 and FGSC A524 were tested for allelism between these resistant genes by incubation in several concentrations of benomyl. Two of the segregants were as sensitive as the wild type strain and another one showed high resistance to the fungicide. Thus, it seems that a new locus (benD), located about 1.0 unit from benA and probably centromere-proximal to it, is responsible for the resistance. The ED50 values of the BEN-35, FGSC A524, sensitive strains, heterozygous diploids and double mutant (Table 1) obtained from dose-response curves for benomyl and thiophanate-methyl showed: a) cross resistance between benomyl and thiophanate-methyl, b) a higher level of resistance to both fungicides in the BEN-35 mutant as compared to the FGSC A524 strain, c) the resistance of the BEN-35 strain to thiophanate-methyl was about 60 times higher than to benomyl. For FGSC A524 and sensitive strains the ratio between the two fungicides was only 10, d) intermediate resistance of the diploid heterozygous for benDl/+ indicating a semidominant trait, e) the double mutant obtained by crossing the BEN-35 strain (benD1) with FGSC A524 (benA10) has a high level of resistance to benomyl. This interaction has also been detected among the benA, benB and benC loci of A. nidulans (van Tuyl 1977 Ph.D. Thesis, Agricultural University, Wageningen, The Netherlands). The double mutant, benA benB, showed an intermediate type of resistance to benomyl and somewhat higher resistance to thiabendazole. When benB and benC were recombined into one strain, only a slight increase in resistance was observed. Thus, it seems that benomyl (or benzimidazole) resistance is governed by a multigenic system. Furthermore, the additive effect and the physical closeness of the benA and benD loci suggests that the latter might be responsible for the synthesis of another tubulin polypeptide. Table 1. ED50 values from dose-response curves of benomyl and thiophanate-methyl for various strains ED50^a(ug/ml) Strains Relevant genotype benomyl^b thiophanate-methyl^c M S E 0 . 7 7 . 0 proA1 pabaA6 yA2 0.7 benA10 biA1 fwA1 (A524) benA10 BEN-35//MSE benDl Double resistant benA10 benD1 >100.0 7 . 1 BEN-35 benD1 31.3 19.4 7 . 0 80.0 >2000.0 794.3 a Concentration reducing the colony radial growth by 50% b Methyl-1-butylcarbamoyl-2-benzimidazole carbamate c 1,2-bis (3 methoxy-carbonyl-2-thioureido)-benzene ^1 Dept. de Genetica, Universidade de Sao Paula, 14049 Ribeirao Preto, SP, Brazil; 2Inst. de Genetica, ESALQ-USP, Caixa Postal 83, Sao Paulo, Brazil Metzenberg, R.L. and J. Grotelueschen When a gene or other fragment of DNA is cloned. it is often useful to identify the Restriction polymorphism maps of chromosomal region from which it arose. This is conveniently done with a set of progeny from one Neurospora crassa: update. or more reference crosses in which many polymorphic differences are segregating. Data which allow the mapping of cloned genes have been published (Metzenberg et al. 1984, Neurospora Newsl. 31:35-39; ibid. Proc. Nat. Acad. Sci. U.S. 1985, 82:2067-2071; Metzenberg and Grotelueshen, 1988, Fungal Genetics Newsl. 35:30-35). The following is an update of the 1988 article. As noted previously, 38 segregants from the first cross were taken from ordered asci, and provide somewhat more information than can be obtained from the 18 segregants which represent random spores from the second cross. Both crosses, however, have been used in a number of laboratories, and data from both are presented. The scoring of segregants is coded in the same way as before: "M" or "0" indicate segregants that are like the Mauriceville parent or like the Oak-Ridge-derived parent, respectively: "-" indicates that the scoring was not done or was equivocal for technical reasons; and (0) in Isolate 1 and (M) in Isolate 6 for all lanes of the second cross means that these are not progeny but are the parental strains of the cross, and are 0 and M by definition. Loci which were previously identified in the 1988 article, or are in the Compendium (Perkins et al. 1982, Microbiol. Reviews 46:426-570) are not further identified. All loci corresponding to 5S RNA-coding regions, previously given only as Arabic numerals starting with 1, are now identified as Fsr-1, etc. All of thetelomeres (Tel) which have been included are due to the efforts of Michael Schechtman, whose article in this issue should be consulted. A number of loci whose identities were being kept confidential are now shown with informative names, instead of coded numbers beginning with a string of zeroes. COXVIII is subunit VIII of cytochrome oxidase, and is coded by a cloned, but unnamed, gene (M. Suarez/U.L. RajBhandary, in preparation). The following are newly named or unpublished loci, with the name(s) of the person or persons who should be consulted about them: cyt-21 (M. Kuiper/A. Lambowitz) ; sod-1 superoxide dismutase (D. Natvig); pma-l, plasma membrane ATPase (K. Hager/C.W. Slayman/B. Bowman); lys-6, (Ming Chow/U.L. RajBhandary); cyt-2, (A. Kubelik/A. Lambowitz); cyt-8 and cya-2, (M. David/U.L. RajBhandary); cat-3, (D. Natvig); ccg-1 and ccg-2, (J. Lores/J. Dunlap); SPIAE, an anonymous DNA fragment, (M. Schechtman); frq and for, (J. Dunlap). pho-4 was formerly called van (B. Bowman). Finally, the scoring of rDNA (LG V) of isolate C4 has been changed from M to 0. The previous result may have been in error, but inspection of the original blots suggests it may not have been. Initially, C4 was an exceptional strain having both M and 0 types of rDNA, with a large preponderance of M, but subsequent preparations have been pure 0. The preponderant rDNA form of the strain may thus have changed during mitotic events, and should be considered questionable. The other genes of this strain seem to behave conventionally. If you have found these data useful, please pass on the favor by pencilling in any results of your own onto a copy of this table and sending it to RLM. You may ask that it be assigned a number which preserves confidentiality. Supported by NIH Grant GM 08995. Dept. of Physiological Chemistry, University of Wisconsin, Madison, WI 53706.


Martinez
Most of the fungicides based on the benzimidazole nucleus, including benomyl, thiabendazole Additive effect involving a new and thiophanate, are systemic and because they have a broad action spectrum they can be used to locus of benomyl resistance in control many important fungal diseases. Even though the chemical structures of benomyl and Aspergillus nidulans thiophanate may appear not to be similar, the two substances are metabolized to a common compound which is probably the cause of cross-resistance between them. Benomyl-resistant mutants are of interest from at least two points of view: the understanding of the genetic bases that govern this resistance and the study of microtubulins using a genetical-biochemical approach, since benomyl is an antimicrotubular drug.
The benA locus maps on linkage group VIII, confers high resistance to benomyl even though it does not permit conidiation of resistant mutants and also codes for ß-1 and ß-2 tubulin (Sheir-Neiss, G. et al. 1978 Cell 15:639-647). The other two loci map elsewhere and make the fungus resistant to low fungicide levels.
To determine the possible existence and interrelationship of other loci responsible for this resistance, conidia of the proA1 pabaA6 yA2 strain were irradiated with UV light and inoculated at 37°C in complete medium dishes containing thiophanate-methyl fungicide (40 ug/ml). This concentration inhibits the growth of sensitive strains. A mutant obtained under these conditions (BEN-35) proved to be resistant to benomyl and showed normal conidiation in complete medium containing up to 10 ug/ml benomyl or 50 ug/ml thiophanate-methyl.
Genetic analyses of this new mutant (BEN-35) carried out by cross with the Master Strain E (MSE) showed that a single gene mutation which also mapped on linkage group VIII was responsible for the benomyl and thiophanate-methyl resistance. About 300 segregants obtained from a cross between BEN-35 and FGSC A524 were tested for allelism between these resistant genes by incubation in several concentrations of benomyl. Two of the segregants were as sensitive as the wild type strain and another one showed high resistance to the fungicide. Thus, it seems that a new locus (benD), located about 1.0 unit from benA and probably centromere-proximal to it, is responsible for the resistance.
The ED50 values of the BEN-35, FGSC A524, sensitive strains, heterozygous diploids and double mutant (Table 1) obtained from dose-response curves for benomyl and thiophanate-methyl showed: a) cross resistance between benomyl and thiophanate-methyl, b) a higher level of resistance to both fungicides in the BEN-35 mutant as compared to the FGSC A524 strain, c) the resistance of the BEN-35 strain to thiophanate-methyl was about 60 times higher than to benomyl. For FGSC A524 and sensitive strains the ratio between the two fungicides was only 10, d) intermediate resistance of the diploid heterozygous for benDl/+ indicating a semidominant trait, e) the double mutant obtained by crossing the BEN-35 strain (benD1) with FGSC A524 (benA10) has a high level of resistance to benomyl. This interaction has also been detected among the benA, benB and benC loci of A. nidulans (van Tuyl 1977 Ph.D. Thesis, Agricultural University, Wageningen, The Netherlands).
The double mutant, benA benB, showed an intermediate type of resistance to benomyl and somewhat higher resistance to thiabendazole. When benB and benC were recombined into one strain, only a slight increase in resistance was observed. Thus, it seems that benomyl (or benzimidazole) resistance is governed by a multigenic system. Furthermore, the additive effect and the physical closeness of the benA and benD loci suggests that the latter might be responsible for the synthesis of another tubulin polypeptide. a -Concentration reducing the colony radial growth by 50% b -Methyl-1-butylcarbamoyl-2-benzimidazole carbamate c -1,2-bis (3 methoxy-carbonyl-2-thioureido)-benzenê