Α-tubulin Genes Research Articles

3'UTR of mRNA Encoding CPEB Protein Orb2 Plays an Essential Role in Intracellular Transport in Neurons.

Intracellular trafficking plays a critical role in the functioning of highly polarized cells, such as neurons. Transport of mRNAs, proteins, and other molecules to synaptic terminals maintains contact between neurons and ensures the transmission of nerve impulses. Cytoplasmic polyadenylation element binding (CPEB) proteins play an essential role in long-term memory (LTM) formation by regulating local translation in synapses. Here, we show that the 3'UTR of the Drosophila CPEB gene orb2 is required for targeting the orb2 mRNA and protein to synapses and that this localization is important for LTM formation. When the orb2 3'UTR is deleted, the orb2 mRNAs and proteins fail to localize in synaptic fractions, and pronounced LTM deficits arise. We found that the phenotypic effects of the orb2 3'UTR deletion were rescued by introducing the 3'UTR from the orb, another Drosophila CPEB gene. In contrast, the phenotypic effects of the 3'UTR deletion were not rescued by the 3'UTR from one of the Drosophila α-tubulin genes. Our results show that the orb2 mRNAs must be targeted to the correct locations in neurons and that proper targeting depends upon sequences in the 3'UTR.

Toxicity and tissue accumulation characteristics of the herbicide pendimethalin under silicon application in ginger (Zingiber officinale Roscoe).

Environmental health and food safety issues potentially caused by the dinitroaniline herbicide pendimethalin (PM) are a worldwide concern. The toxicity response of ginger and tissue accumulation effects of PM on ginger biomass were studied by utilizing PM (CK (clean water), PM1 (0.4%), PM2 (0.67%), PM3 (1.0%), and PM4 (1.67%)) in a dose-response study. It significantly reduced the biomass of ginger under PM4, which is attributed to root damage. The net photosynthetic rate of ginger under PM4 was 11.37% lower than that of CK, which is mainly caused by stomatal limitation. In addition, the ultrastructure of chloroplasts has changed. PM4 caused the accumulation of reactive oxygen species (ROS) in ginger. The activity of superoxide dismutase (SOD) and peroxidase (POD) increased accordingly, maintaining the dynamic balance of ROS content. PM had no significant effect on the expression of ginger α-tubulin genes. PM was significantly accumulated in ginger roots, but not rhizomes. Si increased the productivity of ginger under PM4, which is mainly related to the increase of root development (root application of silicon) and photosynthetic efficiency (foliar application of silicon). Si reduced the ROS content due to the increase in SOD, POD, and catalase (CAT) activity and photosynthetic efficiency.

Dinitroaniline Herbicide Resistance and Mechanisms in Weeds.

Dinitroanilines are microtubule inhibitors, targeting tubulin proteins in plants and protists. Dinitroaniline herbicides, such as trifluralin, pendimethalin and oryzalin, have been used as pre-emergence herbicides for weed control for decades. With widespread resistance to post-emergence herbicides in weeds, the use of pre-emergence herbicides such as dinitroanilines has increased, in part, due to relatively slow evolution of resistance in weeds to these herbicides. Target-site resistance (TSR) to dinitroaniline herbicides due to point mutations in α-tubulin genes has been confirmed in a few weedy plant species (e.g., Eleusine indica, Setaria viridis, and recently in Lolium rigidum). Of particular interest is the resistance mutation Arg-243-Met identified from dinitroaniline-resistant L. rigidum that causes helical growth when plants are homozygous for the mutation. The recessive nature of the TSR, plus possible fitness cost for some resistance mutations, likely slows resistance evolution. Furthermore, non-target-site resistance (NTSR) to dinitroanilines has been rarely reported and only confirmed in Lolium rigidum due to enhanced herbicide metabolism (metabolic resistance). A cytochrome P450 gene (CYP81A10) has been recently identified in L. rigidum that confers resistance to trifluralin. Moreover, TSR and NTSR have been shown to co-exist in the same weedy species, population, and plant. The implication of knowledge and information on TSR and NTSR in management of dinitroaniline resistance is discussed.

Chlamydomonas reinhardtii tubulin-gene disruptants for efficient isolation of strains bearing tubulin mutations.

The single-cell green alga Chlamydomonas reinhardtii possesses two α-tubulin genes (tua1 and tua2) and two β-tubulin genes (tub1 and tub2), with the two genes in each pair encoding identical amino acid sequences. Here, we screened an insertional library to establish eight disruptants with defective tua2, tub1, or tub2 expression. Most of the disruptants did not exhibit major defects in cell growth, flagellar length, or flagellar regeneration after amputation. Because few tubulin mutants of C. reinhardtii have been reported to date, we then used our disruptants, together with a tua1 disruptant obtained from the Chlamydomonas Library Project (CLiP), to isolate tubulin-mutants resistant to the anti-tubulin agents propyzamide (pronamide) or oryzalin. As a result of several trials, we obtained 8 strains bearing 7 different α-tubulin mutations and 12 strains bearing 7 different β-tubulin mutations. One of the mutations is at a residue similar to that of a mutation site known to confer drug resistance in human cancer cells. Some strains had the same amino acid substitutions as those reported previously in C. reinhardtii; however, the mutants with single tubulin genes showed slightly stronger drug-resistance than the previous mutants that express the mutated tubulin in addition to the wild-type tubulin. Such increased drug-resistance may have facilitated sensitive detection of tubulin mutation. Single-tubulin-gene disruptants are thus an efficient background of generating tubulin mutants for the study of the structure–function relationship of tubulin.

Reference Gene and Protein Expression Levels in Two Different NAFLD Mouse Models.

The expression levels of some reference genes and proteins are used for data normalization and quantification. However, these levels can change in response to experimental conditions or treatments. Aim. The aim of this work was to evaluate reference gene and protein expression in models of nonalcoholic fatty liver disease, using mice fed with a high-fat diet (HFD) and mice that are genetically obese (ob/ob). Main Methods. Histological staining techniques were used to verify the morphology and quantify the amount of lipid droplets present in the liver. Real-time polymerase chain reaction and immunoblotting were employed for monitoring protein expression and gene expression levels, respectively. Key Finding. The results showed that there was a substantial increase in the amount of lipid droplets in the livers of HFD and ob/ob animals when compared to the standard diet (SD) group. There was an observed reduction in the expression of β-actin (10%), α-tubulin (6%), GAPDH (19%), and RPL3 (15%) genes when comparing the ob/ob group to the HFD group. Additionally, the ob/ob mice displayed GAPDH protein levels that were substantially, but not significantly, reduced when compared to SD. Significance. It was concluded that there are slight differences in the expression levels of reference genes and proteins in these two NAFLD animal models, and researchers should consider these alterations when working with these models.

Stage-specific functional relationships between Tub1 and Tub2 beta-tubulins in the wheat scab fungus Fusarium graminearum

The filamentous ascomycete Fusarium graminearum contains two β-tubulin genes TUB1 and TUB2 that differ in functions during vegetative growth and sexual reproduction. To further characterize their functional relationship, in this study we determined the co-localization of Tub1 and Tub2 and assayed their expression levels in different mutants and roles in DON production. Tub1 co-localized with Tub2 to the same regions of microtubules in conidia, hyphae, and ascospores. Whereas deletion of TUB1 had no obvious effect on the transcription of TUB2 and two α-tubulin genes (TUB4 and TUB5), the tub2 mutant was up-regulated in TUB1 transcription. To assay their protein expression levels, polyclonal antibodies that could specifically detect four α- and β-tubulin proteins were generated. Western blot analyses showed that the abundance of Tub1 proteins was increased in tub2 but reduced in tub4 and tub5 mutants. Interestingly, protein expression of Tub4 and Tub5 was decreased in the tub1 mutant in comparison with the wild type, despite a lack of obvious changes in their transcription. In contrast, deletion of TUB2 had no effect on translation of TUB4 and TUB5. Ectopic expression of Tub2-mCherry partially recovered the growth defect of the tub1 mutant but did not rescue its defect in sexual reproduction. Expression of Tub1-GFP in the tub2 mutant also partially rescued its defects in vegetative growth, suggesting that disturbance in the balance of α- and β-tubulins contributes to mutant defects. The tub2 but not tub1 mutant was almost blocked in DON biosynthesis. Expression of TRI genes, toxisome formation, and DON-related cellular differentiation were significantly reduced in the tub2 mutant. Overall, our results showed that Tub1 and Tub2 share similar subcellular localization and have overlapping functions during vegetative growth but they differ in functions in DON production and ascosporogenesis in F. graminearum.

Вивчення поліморфізму довжини інтронів генів α-тубуліну як метод аналізу генетичної диференціації рослин

Вивчення поліморфізму довжини інтронів генів α-тубуліну як метод аналізу генетичної диференціації рослин

Analysis of α-Tubulin Gene Expression During Cold Acclimation of Winter and Spring Soft Wheat

The expression profiles of 15 α-tubulin genes in spring and winter wheat varieties during cold acclimation were studied. Among the studied genes, two subfamilies (three genes in each) were identified with elevated expression levels detected at the initial stages of cold acclimation. The Tuba-2-3 gene, which, within its subfamily, is characterized by the most significant amplitude of the increase in the initial expression level, deserves special attention. In addition, the greatest differences in expression levels between varieties were found for this gene within its subfamily. For the winter variety, higher expression levels of this tubulin gene remaining for a long time (up to the seventh day of acclimation) were detected. A significant initial increase in expression levels for all α-tubulin genes of the fourth subfamily reaching maximum values during further acclimation was revealed. The high initial values of the expression levels of genes from this subfamily may also indicate their important role in the resistance of wheat microtubules to low temperatures in the early stages of cold acclimation.

A paradox: rapid evolution rates of germline-limited sequences are associated with conserved patterns of rearrangements in cryptic species of Chilodonella uncinata (Protista, Ciliophora).

Some of the most extreme genome wide rearrangements are found in ciliates, which are unique in possessing both germline micronucleus and somatic macronucleus in every cell/organism. A series of DNA rearrangement events, including DNA elimination, chromosomal fragmentation, gene unscrambling and alternative processing, happen during macronuclear development. To assess the molecular evolution of macronuclear and germline-limited sequences in different cryptic species of Chilodonella uncinata, we characterized the actin, α-tubulin and β-tubulin genes in the micronucleus and macronucleus genomes of USA-SC2 strain and compared them with other strains (i.e. cryptic species). Three main results are: (i) rearrangement patterns between germline and soma are conserved for each gene among the cryptic species of C. uncinata; (ii) in contrast, the germline-limited regions are highly divergent in sequence and length among the cryptic species; (iii) pointer shifting is frequent among the cryptic species. We speculate that pointer sequences may serve as the buffer between the conserved macronuclear destined sequences and rapidly-evolving internal eliminated sequences. The data combined with previous studies demonstrate the plasticity of gene rearrangement among different groups of ciliates and add to the growing data for the role of genome rearrangements in species differentiation.

Genome-wide identification, phylogenetic classification, and exon-intron structure characterization of the tubulin and actin genes in flax (Linum usitatissimum).

Flax (Linum usitatissimum L.) is a valuable food and fiber crop cultivated for its quality fiber and seed oil. α-, β-, γ-tubulins and actins are the main structural proteins of the cytoskeleton. α- and γ-tubulin and actin genes have not been characterized yet in the flax genome. In this study, we have identified 6 α-tubulin genes, 13 β-tubulin genes, 2 γ-tubulin genes, and 15 actin genes in the flax genome and analyzed the phylogenetic relationships between flax and Arabidopsis thaliana tubulin and actin genes. Six α-tubulin genes are represented by three paralogous pairs, among 13 β-tubulin genes 7 different isotypes can be distinguished, 6 of which are encoded by two paralogous genes each. γ-tubulin is represented by a paralogous pair of genes one of which may be not functional. Fifteen actin genes represent seven paralogous pairs-seven actin isotypes and a sequentially duplicated copy of one of the genes of one of the isotypes. Exon-intron structure analysis has shown intron length polymorphism within the β-tubulin genes and intron number variation among the α-tubulin gene: three or four introns are found in two or four genes, respectively. Intron positioning occurs at conservative sites, as observed in numerous other plant species. Flax actin genes show both intron length polymorphisms and variation in the number of intron that may be two or three. These data will be useful to support further studies on the specificity, functioning, regulation, and evolution of the flax cytoskeleton proteins.

Influence of sucrose starvation, osmotic and salt stresses on expression profiles of genes involved in the development of autophagy by means of microtubules

Aim. The aim of this work was to investigate changes in expression profiles of key genes involved in the development of autophagy by means of microtubules under the influence of sucrose starvation, osmotic and salt stresses. Methods. Arabidopsis thaliana seeds were sown aseptically on Murashige and Skoog solid medium. Salt and osmotic stresses were simulated by seed germination and seedlings cultivation on the media containing 150 mM NaCl and 10 mM mannitol, respectively. For investigation of starvation-induced autophagy plants were germinated and grown on sucrose-free medium. Results. Changes in expression of α-tubulin and atg8 genes had clearly defined stressdependent nature. Overexpression of tua1 and atg8e under starvation; tua3 and atg8f under osmotic stress; tua3 and atg8f, atg8e during salt stress indirectly testifies interaction between the structural units of autophagosomes and microtubules. It was shown that influence of investigated abiotic stimuli results in overexpression of elp3 and hda6 genes. Small increase in expression levels of hexokinase 2 and 3 was demonstrated. Conclusions. Transcriptome analysis of key genes involved in realization of autophagy induced by sucrose starvation, osmotic and salt stresses in Arabidopsis thaliana cells was conducted. Received data indirectly testifies interaction between the structural units of autophagosomes and microtubules and enables to point α-tubulin and atg8 genes, which are specific for the realization of autophagy induced by a certain abiotic stimuli. Expression profiles of elp3/deacetylases as well as hexokinases indicate the critical role of α-tubulin acetylation for autophagic response, that is involved in the development of programmed cell death.Keywords: autophagy, sucrose starvation, osmotic stress, salt stress, transcriptome analysis, α-tubulin, atg8.

Novel α-Tubulin Mutations Conferring Resistance to Dinitroaniline Herbicides in Lolium rigidum.

The dinitroaniline herbicides (particularly trifluralin) have been globally used in many crops for selective grass weed control. Consequently, trifluralin resistance has been documented in several important crop weed species and has recently reached a level of concern in Australian Lolium rigidum populations. Here, we report novel mutations in the L. rigidum α-tubulin gene which confer resistance to trifluralin and other dinitroaniline herbicides. Nucleotide mutations at the highly conserved codon Arg-243 resulted in amino acid substitutions of Met or Lys. Rice calli transformed with the mutant 243-Met or 243-Lys α-tubulin genes were 4- to 8-fold more resistant to trifluralin and other dinitroaniline herbicides (e.g., ethalfluralin and pendimethalin) compared to calli transformed with the wild type α-tubulin gene from L. rigidum. Comprehensive modeling of molecular docking predicts that Arg-243 is close to the trifluralin binding site on the α-tubulin surface and that replacement of Arg-243 by Met/Lys-243 results in a spatial shift of the trifluralin binding domain, reduction of trifluralin-tubulin contacts, and unfavorable interactions. The major effect of these substitutions is a significant rise of free interaction energy between α-tubulin and trifluralin, as well as between trifluralin and its whole molecular environment. These results demonstrate that the Arg-243 residue in α-tubulin is a determinant for trifluralin sensitivity, and the novel Arg-243-Met/Lys mutations may confer trifluralin resistance in L. rigidum.

Expression analysis of cellulose synthase and main cytoskeletal protein genes in flax (Linum usitatissimum L.).

Fiber flax is an important source of natural fiber and a comprehensive model for the plant fiber biogenesis studies. Cellulose-synthase (CesA) and cytoskeletal genes are known to be important for the cell wall biogenesis in general and for the biogenesis of flax fibers in particular. Currently, knowledge about activity of these genes during the plant growth is limited. In this study, we have investigated flax fiber biogenesis by measuring expression of CesA and cytoskeletal genes at two stages of the flax development (seedlings and stems at the rapid growth stage) in several flax subspecies (elongatum, mediterraneum, crepitans). RT-qPCR has been used to quantify the expression of LusСesA1, LusСesA4, LusСesA7, LusСesA6, Actin, and α-Tubulin genes in plant samples. We report that CesA genes responsible for the secondary cell wall synthesis (LusCesA4, LusCesA7) have different expression pattern compared with CesA genes responsible for the primary cell wall synthesis (LusCesA1, LusCesA6): an average expression of LusCesA4 and LusCesA7 genes is relatively high in seedlings and further increases in stems at the rapid growth stage, whereas an average expression of LusCesA1 and LusCesA6 genes decreases. Interestingly, LusCesA1 is the only studied gene with different expression dynamics between the flax subspecies: its expression decreases by 5.2-10.7 folds in elongatum and mediterraneum but does not change in crepitans subspecies when the rapid growth stage and seedlings are compared. The expression of cytoskeleton genes (coding actin and α-tubulin) is relatively stable and significantly higher than the expression of cellulose-synthase genes in all the studied samples.

Reference gene selection for quantitative real-time polymerase chain reaction analysis in Bombyx mori nucleopolyhedrovirus-infected silkworms

Bombyx mori nucleopolyhedrovirus (BmNPV) is the most serious viral disease in silkworms. To investigate the mechanisms of the immune responses of B. mori to a BmNPV infection, a suitable reference gene (RG) is necessary for normalizing data when studying the expression of genes in BmNPV-infected silkworms or cells. Thus, quantitative real-time PCR polymerase chain reaction was used to compare the stability of expression of nine potential RGs, including the actin A3, translation initiation factor 3 (TIF-3), TIF-A4, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S RNA, 28S RNA, TATA-binding protein (TBP), ribosomal protein L3 (Rpl3), and α-tubulin genes, in silkworms infected with BmNPV. The results were analyzed by BestKeeper, geNorm, and NormFinder software. Overall, α-tubulin exhibited the most stable gene expression in BmNPV-infected silkworms, and this was verified by western blotting of the α-tubulin protein. Moreover, we detected the expression of some genes involved in the immune signaling pathways of silkworms after BmNPV infection using α-tubulin as an internal RG.

Cadmium affects microtubule organization and post-translational modifications of tubulin in seedlings of soybean (Glycine max L.).

Cadmium (Cd) is a non-essential heavy metal, toxic to all living organisms. The microtubule (MT) cytoskeleton appears to be one of the main targets of Cd action. In this study we present, with the use of various immunological approaches, the effect of Cd at moderate (85 μM) and high (170 μM) concentrations on the structure and functioning of the MT cytoskeleton in the root cells of soybean seedlings. As the result of heavy metal action, root growth was significantly diminished and was accompanied by a reduction in mitotic activity and disturbance in the structure of the MT arrays, including randomization of the cortical MT arrangement, distorted mitotic arrays and complete depolymerization of the MTs. Biochemical analysis revealed decreased levels of various α- and β-tubulin isoforms with a parallel down-regulation of most examined α-tubulin genes. Simultaneously, Cd treatment led to differentiated changes in the level of tubulin post-translational modifications, including tyrosination, detyrosination, acetylation, and polyglutamylation. Decreased tyrosination and polyglutamylation of particular tubulin isoforms accompanied by increase in the level of specific detyrosinated and acetylated isoforms implies augmented stability and reduced turnover of the MTs during stress conditions. Taken together, the obtained results indicate the significant impact of Cd on gene expression levels and subsequent post-translational processing of tubulin, which may be related to the impairment of MT cytoskeleton functioning in root cells.

Optimal Reference Genes for Gene Expression Normalization in Trichomonas vaginalis.

Trichomonas vaginalis is the etiologic agent of trichomonosis, the most common non-viral sexually transmitted disease worldwide. This infection is associated with several health consequences, including cervical and prostate cancers and HIV acquisition. Gene expression analysis has been facilitated because of available genome sequences and large-scale transcriptomes in T. vaginalis, particularly using quantitative real-time polymerase chain reaction (qRT-PCR), one of the most used methods for molecular studies. Reference genes for normalization are crucial to ensure the accuracy of this method. However, to the best of our knowledge, a systematic validation of reference genes has not been performed for T. vaginalis. In this study, the transcripts of nine candidate reference genes were quantified using qRT-PCR under different cultivation conditions, and the stability of these genes was compared using the geNorm and NormFinder algorithms. The most stable reference genes were α-tubulin, actin and DNATopII, and, conversely, the widely used T. vaginalis reference genes GAPDH and β-tubulin were less stable. The PFOR gene was used to validate the reliability of the use of these candidate reference genes. As expected, the PFOR gene was upregulated when the trophozoites were cultivated with ferrous ammonium sulfate when the DNATopII, α-tubulin and actin genes were used as normalizing gene. By contrast, the PFOR gene was downregulated when the GAPDH gene was used as an internal control, leading to misinterpretation of the data. These results provide an important starting point for reference gene selection and gene expression analysis with qRT-PCR studies of T. vaginalis.

Physiological and molecular changes in barley and wheat under salinity.

In this study, it was aimed to compare salinity-induced changes in barley (Hordeum vulgare L. cv. Bornova-92) and bread wheat (Triticum aestivum L. cv. Gerek-79). Seeds were germinated under saline conditions (0, 50, 100, 250, and 500mM NaCl) for 2days and recovered under non-saline conditions for 2days. At the end of the salt treatment, germination, water content (WC), total soluble protein content, and catalase (CAT, EC 1.11.1.6) activity were affected in both species, while superoxide dismutase (SOD, EC 1.15.1.1) activity was affected in barley. Salinity affected WC, protein content, and CAT activity in both species, while it affected germination in barley and affected fresh weight and SOD activity in wheat after recovery. Physiological responses of both species were correlated. Expression of α-tubulin, Atls1, and Lls1 genes was down-regulated in barley after 250mM NaCl treatment. HVA1 gene was highly (more than 50-fold) stimulated by salinity in barley. However, α-tubulin and Atls1 genes were down-regulated, and Lls1 gene was up-regulated in wheat after recovery from 250-mM NaCl treatment. Increase in HVA1 expression was not significant in wheat. The expression profiles of barley and wheat under salinity are different, and barley tended to regulate gene expression faster than wheat.

Salinity-induced Physiological and Molecular Changes in Barley and Wheat

In this study, it was aimed to compare salinity-induced changes in barley (Hordeum vulgare L. cv. Bornova-92) and bread wheat (Triticum aestivum L. cv. Gerek-79). Seeds were germinated under saline conditions (0, 50, 100, 250 and 500mM NaCl) for 2 days and recovered under non-saline conditions for 2 days. At the end of the salt-treatment, germination, water content (WC), total soluble protein content, catalase (CAT, EC 1.11.1.6) activity were affected in both species while superoxide dismutase (SOD, EC 1.15.1.1) activity was affected in barley. Salinity affected WC, protein content and CAT activity in both species while affected germination in barley and affected fresh weight and SOD activity in wheat after recovery. Physiological responses of both species were correlated. Expression of α-tubulin, Atls1 and Lls1 genes were down- regulated in barley after 250mM NaCl treatment. HVA1 gene was highly (more than 50-fold) stimulated by salinity in barley. However, α-tubulin, Atls1 genes were down-; Lls1 gene was up-regulated in wheat after recovery from 250mM NaCl treatment. Increase in HVA1 expression was not significant in wheat. Expression profiles of barley and wheat under salinity are different and barley tended to regulate gene expression faster than wheat.

Semi-dwarfism and lodging tolerance in tef (Eragrostis tef) is linked to a mutation in the α-Tubulin 1 gene.

Genetic improvement of native crops is a new and promising strategy to combat hunger in the developing world. Tef is the major staple food crop for approximately 50 million people in Ethiopia. As an indigenous cereal, it is well adapted to diverse climatic and soil conditions; however, its productivity is extremely low mainly due to susceptibility to lodging. Tef has a tall and weak stem, liable to lodge (or fall over), which is aggravated by wind, rain, or application of nitrogen fertilizer. To circumvent this problem, the first semi-dwarf lodging-tolerant tef line, called kegne, was developed from an ethyl methanesulphonate (EMS)-mutagenized population. The response of kegne to microtubule-depolymerizing and -stabilizing drugs, as well as subsequent gene sequencing and segregation analysis, suggests that a defect in the α-Tubulin gene is functionally and genetically tightly linked to the kegne phenotype. In diploid species such as rice, homozygous mutations in α-Tubulin genes result in extreme dwarfism and weak stems. In the allotetraploid tef, only one homeologue is mutated, and the presence of the second intact α-Tubulin gene copy confers the agriculturally beneficial semi-dwarf and lodging-tolerant phenotype. Introgression of kegne into locally adapted and popular tef cultivars in Ethiopia will increase the lodging tolerance in the tef germplasm and, as a result, will improve the productivity of this valuable crop.

Molecular evolution and functional divergence of tubulin superfamily in the fungal tree of life.

Microtubules are essential for various cellular activities and β-tubulins are the target of benzimidazole fungicides. However, the evolution and molecular mechanisms driving functional diversification in fungal tubulins are not clear. In this study, we systematically identified tubulin genes from 59 representative fungi across the fungal kingdom. Phylogenetic analysis showed that α-/β-tubulin genes underwent multiple independent duplications and losses in different fungal lineages and formed distinct paralogous/orthologous clades. The last common ancestor of basidiomycetes and ascomycetes likely possessed two paralogs of α-tubulin (α1/α2) and β-tubulin (β1/β2) genes but α2-tubulin genes were lost in basidiomycetes and β2-tubulin genes were lost in most ascomycetes. Molecular evolutionary analysis indicated that α1, α2, and β2-tubulins have been under strong divergent selection and adaptive positive selection. Many positively selected sites are at or adjacent to important functional sites and likely contribute to functional diversification. We further experimentally confirmed functional divergence of two β-tubulins in Fusarium and identified type II variations in FgTub2 responsible for function shifts. In this study, we also identified δ-/ε-/η-tubulins in Chytridiomycetes. Overall, our results illustrated that different evolutionary mechanisms drive functional diversification of α-/β-tubulin genes in different fungal lineages, and residues under positive selection could provide targets for further experimental study.