Tubulins in Aspergillus nidulans

The discovery and characterization of the tubulin superfamily in Aspergillus nidulans is described. Remarkably, the genes that encode alpha-, beta-, and gamma-tubulins were all identified first in A. nidulans. There are two alpha-tubulin genes, tubA and tubB, two beta-tubulin genes, benA and tubC, and one gamma-tubulin gene, mipA. Hyphal tubulin is encoded mainly by the essential genes tubA and benA. TubC is expressed during conidiation and tubB is required for the sexual cycle. Promoter swapping experiments indicate that the alpha-tubulins encoded by tubA and tubB are functionally interchangeable as are the beta-tubulins encoded by benA and tubC. BenA mutations that alter resistance to benzimidazole antimicrotubule agents are clustered and define a putative binding region for these compounds. gamma-Tubulin localizes to the spindle pole body and is essential for mitotic spindle formation. The phenotypes of mipA mutants suggest, moreover, that gamma-tubulin has essential functions in addition to microtubule nucleation.     

Isolation of mip (microtubule-interacting protein) mutations of Aspergillus nidulans.

We identified four mutations in two previously undescribed loci involved in microtubule function in Aspergillus nidulans as extragenic suppressors of benA33, a heat-sensitive beta-tubulin mutation. Three of the four mutations map to a locus closely linked to riboB on linkage group VIII; we designated this locus mipA (for microtubule-interacting protein). We were not able to map the remaining suppressor because of chromosomal rearrangements. However, since it recombines with riboB at a significantly higher frequency than the mipA alleles, it is unlikely to be in mipA; thus, we designated it mipB1. The mip mutations are not allelic to the previously identified loci that encode alpha- and beta-tubulin, and it is likely that mipA and mipB encode previously unidentified nontubulin proteins involved in microtubule function. Each of the mip mutations suppresses the heat sensitivity conferred by benA33 and suppresses the blockage of nuclear division and movement conferred by this mutation at high temperatures. Interactions between mipA and benA are allele specific. All of the mipA mutations are cryptic in a wild-type benA background but cause cold sensitivity in combination with benA33. These mutations also confer cold sensitivity in combination with benA31 and benA32 and reduce the resistance conferred by these mutations to the antimicrotubule agent benomyl but do not suppress the heat sensitivity conferred by these alleles. Finally, the mipA alleles suppress the heat sensitivity conferred by benA11, benA17, and benA21 but do not confer cold sensitivity in combination with these alleles.     

The highly divergent beta-tubulins of Aspergillus nidulans are functionally interchangeable

An internal 1.4-kb Bst EII fragment was used to disrupt the benA gene and establish heterokaryons. The heterokaryons demonstrated that the molecular disruption of benA results in a recessive benA null mutation. Conidia from a heterokaryon swell and germinate but cannot undergo nuclear division and are thus inviable. A chimeric beta-tubulin gene was constructed with the benA promoter driving the tubC structural gene. This chimeric gene construction was placed on a plasmid containing a selectable marker for Aspergillus transformation and the gene disrupting fragment of benA. Integration of this plasmid at benA by the internal gene disrupting fragment of benA simultaneously disrupts the benA gene and replaces it with the chimeric beta-tubulin gene, rescuing the benA null generated by the integration. Strains generated by this procedure contain only tubC beta-tubulin for all beta-tubulin functions. Strains having only tubC beta-tubulin are viable and exhibit no detectable microtubule dysfunction though they are more sensitive than wild-type strains to the antimicrotubule drug benomyl. It is concluded that the two beta-tubulin genes of Aspergillus nidulans, though highly divergent, are interchangeable.     

Interaction of thiabendazole with fungal tubulin.

Thiabendazole, 2-(4'-thiazolyl)benzimidazole, at 80 micrometer completely inhibits mitosis in hyphae of Aspergillus nidulans, growing in liquid culture. DNA and RNA synthesis and mycelial growth are only partially inhibited at this concentration. Binding studies with cell-free mycelial extracts from Penicillium expansum showed that thiabendazole competitively inhibits [14C]carbendazim binding to tubulin, which suggests that the antimitotic activity of thiabendazole is based on interference with microtubule assembly. Tubulin from a thiabendazole-resistant and carbendazim-highly sensitive mutant of P. expansum has a lower affinity to thiabendazole and a higher affinity to carbendazim than tubulin from a wide-type strain. This indicates that in this mutant the structure of the binding site is affected. The data presented suggest that several sites of both the tubulin and ligand molecule are involved in the binding of benzimidazole compounds to fungal tubulin.     

Increasing tubC beta-tubulin synthesis by placing it under the control of a benA beta-tubulin upstream sequence causes a reduction in benA beta-tubulin level but has no effect on microtubule function

We have constructed a chimeric beta-tubulin gene that places the structural gene for the tubC beta-tubulin of Aspergillus nidulans under the control of the benA beta-tubulin promoter. Introduction of either this chimeric gene or a second wild-type benA gene into a benomyl-resistant benA22 strain causes it to become benomyl sensitive, indicating that the introduced genes are functional. Analysis of the tubulin proteins synthesized in benA22 strains into which a second wild-type benA beta-tubulin gene was transformed showed that the total amount of beta-tubulin protein was the same as in the parental strain with a single benA gene. Thus the level of beta-tubulin must be regulated. This was also true of transformants carrying an extra copy of the chimeric beta-tubulin gene. The total amount of beta-tubulin was the same as in the parental strain. Two-dimensional gel analysis showed that the endogenous benA22 and the introduced chimeric tubC gene contributed equally to the total beta-tubulin pool. The fact that one-half of the benA beta-tubulin could be replaced by tubC beta-tubulin with no effect on the growth of the cells suggests that the benA and tubC beta-tubulins are functionally interchangeable.     

Aspergillus contains multiple tubulin genes

Previous work with benomyl-resistant mutants of Aspergillus nidulans has demonstrated that the benA locus is a structural gene for beta-tubulin. Two of the benA mutants, benA22 and benA85, show altered electrophoretic mobilities on two-dimensional gels for two beta-tubulins (designated beta 1 and beta 2). These shifts of the beta 1- and beta 2-tubulins uncover a spot in the region where wild-type beta-tubulins migrate that is occluded on gels of wild-type extracts by the beta 1- and beta 2-tubulins. Evidence has now been obtained indicating that this spot represents an additional beta-tubulin (designated beta 3). Tubulin was partially purified from Aspergillus and run on one- and two-dimensional gels and the band or spot uncovered by the shift of the beta 1- and beta 2-tubulins was identified as a beta-tubulin by immunoblotting with monoclonal and affinity-purified polyclonal anti-tubulin antibodies and by one-dimensional peptide mapping. These observations show that Aspergillus contains at least two structural genes for beta-tubulins. Similar techniques have also been applied to a mutant showing altered alpha-tubulins to confirm and modify earlier observations suggesting that at least two structural genes for alpha-tubulins are also present.     

Identification and functional analysis of beta-tubulin genes by site specific integrative transformation in Aspergillus nidulans

We have cloned two different beta-tubulin sequences from the filamentous fungus Aspergillus nidulans. Each was used in the construction of transforming plasmids that carry the pyr4 gene of Neurospora crassa. We used these plasmids to transform a pyrG-strain of Aspergillus to uridine prototrophy. Both plasmids were shown to integrate site specifically into the homologous chromosomal sequences. We then used transformant strains in genetic crosses to demonstrate that one of the cloned beta-tubulin sequences was the benA beta-tubulin gene, which codes for the beta 1-and beta 2-tubulins. The other cloned beta-tubulin sequence was shown to be the structural gene for beta 3-tubulin by gene disruption and to participate in conidial development. This is the first report of a gene disruption by site specific, integrative recombination in Aspergillus nidulans.     

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.     

Interaction of thiabendazole with fungal tubulin

Thiabendazole, 2-(4′-thiazolyl)benzimidazole, at 80 μM completely inhibits mitosis in hyphae of Aspergillus nidulans, growing in liquid culture. DNA and RNA synthesis and mycelial growth are only partially inhibited at this concentration.Binding studies with cell-free mycelial extracts from Penicillium expansum showed that thiabendazole competitively inhibits [¹⁴C]carbendazim binding to tubulin, which suggests that the antimitotic activity of thiabendazole is based on interference with microtubule assembly.Tubulin from a thiabendazole-resistant and carbendazim-highly sensitive mutant of P. expansum has a lower affinity to thiabendazole and a higher affinity to carbendazim than tubulin from a wild-type strain. This indicates that in this mutant the structure of the binding site is affected.The data presented suggest that several sites of both the tubulin and ligand molecule are involved in the binding of benzimidazole compounds to fungal tubulin.     

禾谷镰孢菌β2微管蛋白与苯并咪唑类杀菌剂互作研究

小麦赤霉病是小麦上的主要病害之一,在全世界范围内引起该病害的致病菌主要是禾谷镰孢菌Fusarium graminearum。目前,使用杀菌剂是生产上防治小麦赤霉病发生和危害的主要手段,常用的杀菌剂主要有苯并咪唑类杀菌剂(benzimidazoles)等,苯并咪唑类杀菌剂的作用靶标是β2微管蛋白。本研究旨在探究小麦赤霉病菌中β2微管蛋白与苯并咪唑类杀菌剂的互作机制,通过同源建模的方法获得了禾谷镰孢菌β2微管蛋白的三维结构,并在此基础上将β2微管蛋白与4种苯并咪唑类杀菌剂(多菌灵、苯菌灵、噻菌灵、甲基硫菌灵)进行分子对接。分子对接结果显示β2微管蛋白第198位苯丙氨酸和第236位缬氨酸与4种苯并咪唑类杀菌剂直接互作形成氢键,第50、134、165、167、198、200、236、237、239、240、250、253、257、314位氨基酸形成药剂结合口袋。通过比较β2微管蛋白与4种苯并咪唑类杀菌剂结合自由能,发现与其他3种杀菌剂相比,β2微管蛋白与多菌灵的结合自由能最小(-5.72 kcal/mol),说明其与多菌灵互作亲和力更强。采用菌丝生长速率法测定了禾谷镰孢菌对4种苯并咪唑类杀菌剂的EC₅₀值,禾谷镰孢菌对多菌灵、苯菌灵、噻菌灵、甲基硫菌灵的EC₅₀值分别为0.772、0.862、1.088、13.266 mg/L,该结果表明禾谷镰孢菌对多菌灵的敏感性强于其他3种杀菌剂,与分子对接结果相吻合。     

Sequence variation in the Trichuris trichiura beta-tubulin locus: implications for the development of benzimidazole resistance

Benzimidazole resistance has evolved in a variety of organisms and typically results from mutations in the beta-tubulin locus at specific amino acid sites. Despite widespread treatment of human intestinal nematodes with benzimidazole drugs, there have been no unambiguous reports of resistance. However, since beta-tubulin mutations conferring resistance are generally recessive, frequencies of resistance alleles less than 30% would be difficult to detect on the basis of drug treatment failures. Here we investigate sequence variation in a 1079 bp segment of the beta-tubulin locus in the human whipworm Trichuris trichiura from 72 individual nematodes from seven countries. We did not observe any alleles with amino acid mutations indicative of resistance, and of 40 point mutations there were only four non-synonymous mutations all of which were singletons. Estimated effective population sizes are an order of magnitude lower than those from another nematode species in which benzimidazole resistance has developed (Haemonchus contortus). Both the lower diversity and reduced population sizes suggest that benzimidazole resistance is likely to evolve less rapidly in Trichuris than in trichostrongyle parasites of livestock. We observed moderate levels of population subdivision (Phi(ST)=0.26) comparable with that previously observed in Ascaris lumbricoides, and identical alleles were frequently found in parasites from different continents, suggestive of recent admixture. A particularly interesting feature of the data is the high nucleotide diversities observed in nematodes from the Caribbean. This genetic complexity may be a direct result of extensive admixture and complex history of human populations in this region of the world. These data should encourage (but not make complacent) those involved in large-scale benzimidazole treatment of human intestinal nematodes.     

Effects of mitotic and tubulin mutations on microtubule architecture in actively growing protoplasts of Aspergillus nidulans

We used immunofluorescent microscopy to characterize microtubule (MT) architecture in wild-type and mutant protoplasts of Aspergillus nidulans at interphase and at mitosis. Because the visualization of MTs by immunofluorescence is technically difficult in intact hyphae of A. nidulans, we developed a method for removing the cell wall under conditions that do not perturb cell physiology, as evidenced by the fact that the resulting protoplasts undergo nuclear division at a normal rate and that cell cycle mutant phenotypes are expressed at restrictive temperature. Interphase cells exhibited an extensive network of cytoplasmic MTs. During mitosis the cytoplasmic MTs mostly disappeared and an intranuclear mitotic spindle appeared. We have previously shown that the benA 33 beta-tubulin mutation causes hyperstabilization of the mitotic spindle, and we have presented additional indirect evidence that suggested that the tubA1 and tubA4 alpha-tubulin mutations destabilize spindle MTs. In this paper, we show that the benA33 mutation increases the stability of cytoplasmic MTs as well as spindle MTs and that the tubA1 and tubA4 mutations destabilize both spindle and cytoplasmic MTs.     

Multi-sited mutations of beta-tubulin are involved in benzimidazole resistance and thermotolerance of fungal biocontrol agent Beauveria bassiana

Fungicide resistance and thermotolerance of biocontrol agents in mitosporic fungi are of merits for enhancing fungal formulations against insect pests in the field. Among 20 wild strains of Beauveria bassiana (a well-known fungal biocontrol agent) tested in this study, 19 were sensitive or highly sensitive to carbendazim (methyl 2-benzimidazole carbamate), a typical benzimidazole fungicide, despite low resistance found in one strain. Sequential mutagenesis of a carbendazim-sensitive wild strain [minimal inhibitory concentration (MIC) = 1.32 microg ml(-1)] under artificial selection pressure generated 11 mutants sharing a common MIC of > 1000 microg ml(-1) without visible variation in colony growth and conidiation capacity. This represents at least 758-fold enhancement of the resistance among the mutants. However, accompanied with the enhanced resistance, all the mutants became less thermotolerable. Stressed at 48 degrees C, conidial LT(50)s of the mutants varied from 1.8 to 9.6 min and were lower than the parental LT(50) (36 min). Moreover, the contents of hydrophobin-like proteins in conidial walls declined significantly among the mutants compared with that of the wild parent. Mutations commonly relating to benzimidazole resistance in fungi were located at Q134, F167 and/or E198 around the taxol-binding site of beta-tubulin by sequencing the beta-tubulin of the mutants. Also, mutations of other 37 amino acid residues in the sequences (each having one to five residues mutated) were found for the first time and they were diverse in spatial structure. All mutations restricted to the half of beta-tubulin close to alpha-tubulin were likely involved in variation in each of the traits concerned but their interactions were complicated.     

Alanine-scanning Mutagenesis of Aspergillus γ-Tubulin Yields Diverse and Novel Phenotypes

We have created 41 clustered charged-to-alanine scanning mutations of the mipA, gamma-tubulin, gene of Aspergillus nidulans and have created strains carrying these mutations by two-step gene replacement and by a new procedure, heterokaryon gene replacement. Most mutant alleles confer a wild-type phenotype, but others are lethal or conditionally lethal. The conditionally lethal alleles exhibit a variety of phenotypes under restrictive conditions. Most have robust but highly abnormal mitotic spindles and some have abnormal cytoplasmic microtubule arrays. Two alleles appear to have reduced amounts of gamma-tubulin at the spindle pole bodies and nucleation of spindle microtubule assembly may be partially inhibited. One allele inhibits germ tube formation. The cold sensitivity of two alleles is strongly suppressed by the antimicrotubule agents benomyl and nocodazole and a third allele is essentially dependent on these compounds for growth. Together our data indicate that gamma-tubulin probably carries out functions essential to mitosis and organization of cytoplasmic microtubules in addition to its well-documented role in microtubule nucleation. We have also placed our mutations on a model of the structure of gamma-tubulin and these data give a good initial indication of the functionally important regions of the molecule.     

禾谷镰孢菌β-微管蛋白基因克隆及其与多菌灵抗药性关系的分析

应用3对引物,从禾谷镰孢菌(Gibberella zeae)对多菌灵(MBC)的敏感菌株(MBC^R)和田间及室内诱导抗药性菌株(MBC^R)中扩增β-微管蛋白基因。该基因全长1631bp,包含3个内含子,编码447aa,与其他常见植物病原丝状真菌β-微管蛋白基因的氨基酸同源性达95．12％～99．30％。MBC^R和MBC^R菌株核苷酸序列分析表明,MBCR菌株未发生任何位点的突变,说明G．zeae对MBC的抗药性机制并非像其他丝状真菌一样由β-微管蛋白198位氨基酸突变所致。     

Genetics of the Tubulin Gene Families of Physarum

The organization of the alpha- and beta-tubulin gene families in Physarum was investigated by Mendelian analysis. Restriction endonuclease-generated DNA fragments homologous to alpha- and beta-tubulin show length polymorphisms that can be used as markers for genetic mapping. Analysis of meiotic assortment among progeny of heterozygotes allowed alpha- and beta-tubulin sequence loci to be defined. There are four unlinked alpha-tubulin sequence loci (altA, altB, altC and altD) and at least three unlinked beta-tubulin sequence loci (betA, betB and betC). The alpha-tubulin loci are not linked to the beta-tubulin loci. --Segregation of tubulin sequence loci with respect to ben mutations that confer resistance to antitubulin benzimidazole drugs was used to investigate whether any members of the alpha- or beta-tubulin gene families are allelic to ben loci. The beta-tubulin sequence locus betB is allelic to the resistance locus benD, the betA locus is probably allelic to benA and the alpha-tubulin sequence locus altC may be allelic to benC. The molecular implications of benzimidazole resistance phenotypes when only one of the expressed beta-tubulin gene family members mutates to drug resistance are discussed in relation to tubulin function.     

Characterization of an inducible expression system in Aspergillus nidulans using alcA and tubulin-coding genes

Plasmids have been constructed in which expression of a gene can be placed under the control of the inducible promoter of the alcA gene encoding alcohol dehydrogenase I in Aspergillus nidulans. Simplified shuttle vectors carrying pyr4 which complements pyrG89 mutations have also been constructed. These are based on pUC19 and retain alpha-peptide expression. The beta-tubulin genes, tubC and benA, have been placed under the control of alcA and their expression studied. Levels of expression can be assayed phenotypically because increased synthesis of beta-tubulin inhibits vegetative growth. Sensitivity of asexual spore formation to the anti-microtubule drug benomyl provides a means of detecting very low levels of expression of the chimeric genes. Glucose almost completely represses the chimeric genes. Induction is rapid and is maximal within an hour. When a strain carrying seven copies of an alcA::tubC gene fusion was grown under inducing conditions, 6.5% of total sulfate labelled protein consisted of tubC product. Cyclopentanone was the most potent inducer of the chimeric genes on solid media but it also partially inhibited growth. Chimeric alcA::tubC and alcA::benA genes were expressed to very similar levels despite the fact that tubC utilizes many rare codons.