Intron-mediated regulation of β-tubulin genes expression affects the sensitivity to carbendazim in Fusarium graminearum

  • Yanjun Li
  • Dongming Chen
  • Shunwen Luo
  • Yuanye Zhu
  • Xiaojing Jia
  • Yabing Duan
  • Mingguo ZhouEmail author
Original Article


The plant pathogenic fungus, Fusarium graminearum, is known to have two β-tubulin genes (named Fg-β1tub and Fg-β2tub). Mutations in Fg-β2tub rather than in Fg-β1tub have been shown to confer resistance to carbendazim (MBC), even though Fg-β1tub has higher homology than Fg-β2tub to the β-tubulin isotypes related to benzimidazole resistance in other fungi. However, sequence alignment of β-tubulin isotypes related to benzimidazole resistance showed that the number and position of introns in Fg-β2tub are more consistent than Fg-β1tub to those in other β-tubulin genes. In detail, Fg-β1tub lacks three introns, i.e., intron i3, i4, and i6 corresponding to positions in Fg-β2tub of F. graminearum. To investigate the effects of the divergence introns on the function of β-tubulins in F. graminearum, a strategy of intron deletion and insertion was used. Our results showed that deletion of the second intron from Fg-β1tub gene increased Fg-β1tub expression levels leading to increased sensitivity to MBC. Besides, inserting the divergence introns into Fg-β1tub can increase Fg-β1tub expression leading to increased sensitivity to MBC. In addition, intron-mediated Fg-β1tub gene expression requires a splicing-competent intron within the body of the host gene. Furthermore, the insertion and deletion of introns in Fg-β1tub gene have no significant effect on hyphal growth, conidiation and virulence in F. graminearum. Thus, we proposed that introns may be among the factors contributing to the evolution and functional divergence of two β-tubulin genes and also significantly regulate the expression of β-tubulin genes, which, in turn, affects sensitivity to MBC fungicides in F. graminearum.


Fusarium graminearum β-tubulin Intron Evolution Gene expression Fungicide sensitivity 



This research was supported by The National Natural Science Foundation of China (31730072; 31572025) and The National Key Research and Development Program of China (2016YFD0200503-04, 2016YFD0300706).

Supplementary material

294_2019_960_MOESM1_ESM.docx (1.2 mb)
Supplementary file1 (DOCX 1250 kb)
294_2019_960_MOESM2_ESM.docx (27 kb)
Supplementary file2 (DOCX 27 kb)


  1. Akua T, Berezin I, Shaul O (2010) The leader intron of AtMHX can elicit, in the absence of splicing, low-level intron-mediated enhancement that depends on the internal intron sequence. BMC Plant Biol 10:93–93CrossRefGoogle Scholar
  2. Barrett LW, Fletcher S, Wilton SD (2012) Regulation of eukaryotic gene expression by the untranslated gene regions and other non-coding elements. Cell Mol Life Sci 69:3613–3634CrossRefGoogle Scholar
  3. Callis J, Fromm M, Walbot V (1987) Introns increase gene expression in cultured maize cells. Genes Dev 1:1183–1200CrossRefGoogle Scholar
  4. Chatterjee S, Min L, Karuturi RKM, Lufkin T (2010) The role of post-transcriptional RNA processing and plasmid vector sequences on transient transgene expression in zebrafish. Transgenic Res 19:299–304CrossRefGoogle Scholar
  5. Chen C, Yu J, Bi C, Zhang Y, Xu J, Wang J, Zhou M (2009) Mutations in a β-tubulin confer resistance of Gibberella zeae to benzimidazole fungicides. Phytopathology 99:1403–1411CrossRefGoogle Scholar
  6. Choi T, Huang M, Gorman C, Jaenisch R (1991) A generic intron increases gene expression in transgenic mice. Mol Cell Biol 11:3070–3074CrossRefGoogle Scholar
  7. Clancy M, Hannah LC (2002) Splicing of the maize Sh1 first intron is essential for enhancement of gene expression, and a T-rich motif increases expression without affecting splicing. Plant Physiol 130:918–929CrossRefGoogle Scholar
  8. Croll D, Mcdonald BA (2012) Intron gains and losses in the evolution of Fusarium and Cryptococcus fungi. Genome Biol Evol 4:1148–1161CrossRefGoogle Scholar
  9. Curi GC, Chan RL, Gonzalez DH (2005) The leader intron of Arabidopsis thaliana genes encoding cytochrome c oxidase subunit 5c promotes high-level expression by increasing transcript abundance and translation efficiency. J Exp Bot 56:2563–2571CrossRefGoogle Scholar
  10. Ding M, Li J, Fan X, He F, Yu X, Chen L, Zou S, Liang Y, Yu J (2018) Aquaporin1 regulates development, secondary metabolism and stress responses in Fusarium graminearum. Curr Genet 64:1057–1069CrossRefGoogle Scholar
  11. Faghihi MA, Wahlestedt C (2009) Regulatory roles of natural antisense transcripts. Nat Rev Mol Cell Biol 10:637–643CrossRefGoogle Scholar
  12. Frugoli JA, Mcpeek MA, Thomas TL, Mcclung CR (1998) Intron loss and gain during evolution of the catalase gene family in angiosperms. Genetics 149:355Google Scholar
  13. Gao X, Zhang J, Song C, Yuan K, Wang J, Jin Q, Xu J (2018) Phosphorylation by Prp4 kinase releases the self-inhibition of FgPrp31 in Fusarium graminearum. Curr Genet 64:1261–1274CrossRefGoogle Scholar
  14. Gomez-Gil L, Almiron J, Carrillo P, Medina C, Ruiz G, Rodriguez P, Escobosa A, Corona F, Roncero M (2018) Nitrate assimilation pathway (NAP): role of structural (nit) and transporter (ntr1) genes in Fusarium oxysporum f. sp. lycopersici growth and pathogenicity. Curr Genet 64:493–507CrossRefGoogle Scholar
  15. Graveley BR (2001) Alternative splicing: increasing diversity in the proteomic world. Trends Genet 17:100–107CrossRefGoogle Scholar
  16. Juneau K, Miranda M, Hillenmeyer ME, Nislow C, Davis RW (2006) Introns regulate RNA and protein abundance in yeast. Genetics 174:511–518CrossRefGoogle Scholar
  17. Koonin EV (2009) Intron-dominated genomes of early ancestors of eukaryotes. J Hered 100:618–623CrossRefGoogle Scholar
  18. Li Y, Luo S, Jia X, Zhu Y, Chen D, Duan Y, Hou Y, Zhou M (2017) Regulatory roles of introns in fungicide sensitivity of Fusarium graminearum. Environ Microbiol 19:4140–4153CrossRefGoogle Scholar
  19. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔ C t method. Methods 25:402–408CrossRefGoogle Scholar
  20. Long M, de Souza SJ, Gilbert W (1995) Evolution of the intron-exon structure of eukaryotic genes. Curr Opin Genet Dev 5:774–778CrossRefGoogle Scholar
  21. Luo MJ, Reed R (1999) Splicing is required for rapid and efficient mRNA export in metazoans. Proc Natl Acad Sci 96:14937–14942CrossRefGoogle Scholar
  22. Maniatis T, Tasic B (2002) Alternative pre-mRNA splicing and proteome expansion in metazoans. Nature 418:236CrossRefGoogle Scholar
  23. Mascarenhas D, Mettler IJ, Pierce DA, Lowe HW (1990) Intron-mediated enhancement of heterologous gene expression in maize. Plant Mol Biol 15:913–920CrossRefGoogle Scholar
  24. Moabbi AM, Agarwal N, El-Kaderi B, Ansari A (2012) Role for gene looping in intron-mediated enhancement of transcription. Proc Natl Acad Sci 109:8505–8510CrossRefGoogle Scholar
  25. Nielsen CB, Friedman B, Birren B, Burge CB, Galagan JE (2004) Patterns of intron gain and loss in fungi. PLoS Biol 2:e422CrossRefGoogle Scholar
  26. Okkema PG, Harrison SW, Plunger V, Aryana A, Fire A (1993) Sequence requirements for myosin gene expression and regulation in Caenorhabditis elegans. Genetics 135:385–404Google Scholar
  27. Proctor RH, Hohn TM, McCormick SP (1995) Reduced virulence of Gibberella zeae caused by disruption of a trichothecene toxin biosynthetic gene. Mol Plant Microbe Interact 8:593–601CrossRefGoogle Scholar
  28. Qiu J, Xu J, Yu J, Bi C, Chen C, Zhou M (2011) Localisation of the benzimidazole fungicide binding site of Gibberella zeae β 2-tubulin studied by site-directed mutagenesis. Pest Manag Sci 67:191–198CrossRefGoogle Scholar
  29. Qiu J, Huang T, Xu J, Bi C, Chen C, Zhou M (2012) β-tubulins in Gibberella zeae: their characterization and contribution to carbendazim resistance. Pest Manag Sci 68:1191–1198CrossRefGoogle Scholar
  30. Rodrã-Guez-Trelles F, Tarrã-O R, Ayala FJ (2006) Origins and evolution of spliceosomal introns. Annu Rev Genet 40:47–76CrossRefGoogle Scholar
  31. Rose AB (2004) The effect of intron location on intron-mediated enhancement of gene expression in Arabidopsis. Plant J 40:744–751CrossRefGoogle Scholar
  32. Rose AB (2008) Intron-mediated regulation of gene expression. In: Reddy ASN, Golovkin M (eds) Nuclear pre-mRNA processing in plants. Springer, Berlin, pp 277–290CrossRefGoogle Scholar
  33. Rose AB, Beliakoff JA (2000) Intron-mediated enhancement of gene expression independent of unique intron sequences and splicing. Plant Physiol 122:535–542CrossRefGoogle Scholar
  34. Roy SW, Gilbert W (2005) Rates of intron loss and gain: implications for early eukaryotic evolution. Proc Natl Acad Sci USA 102:5773–5778CrossRefGoogle Scholar
  35. Roy SW, Gilbert W (2006) The evolution of spliceosomal introns: patterns, puzzles and progress. Nat Rev Genet 7:211–221Google Scholar
  36. Roy SW, Irimia M (2009) Mystery of intron gain: new data and new models. Trends Genet 25:67–73CrossRefGoogle Scholar
  37. Sambrook J (1977) Adenovirus amazes at Cold Spring Harbor. Nature 268:101–104CrossRefGoogle Scholar
  38. Sinibaldi RM, Mettler IJ (1992) Intron splicing and intron-mediated enhanced expression in monocots. Prog Nucleic Acid Res Mol Biol 42:229–257CrossRefGoogle Scholar
  39. Ueki J, Komari T, Imaseki H (2004) Enhancement of reporter-gene expression by insertions of two introns in maize and tobacco protoplasts. Plant Biotechnol 21:15–24CrossRefGoogle Scholar
  40. Vasil V, Clancy M, Ferl RJ, Vasil IK, Hannah LC (1989) Increased gene expression by the first intron of maize shrunken-1 locus in grass species. Plant Physiol 91:1575–1579CrossRefGoogle Scholar
  41. Yang P, Chen Y, Wu H, Fang W, Liang Q, Zheng Y, Olsson S, Zhang D, Zhou J, Wang Z, Zheng W (2018) The 5-oxoprolinase is required for conidiation, sexual reproduction, virulence and deoxynivalenol production of Fusarium graminearum. Curr Genet 64:285–301CrossRefGoogle Scholar
  42. Ying SY, Lin SL (2004) Intron-derived microRNA—fine tuning of gene functions. Gene 342:25–28CrossRefGoogle Scholar
  43. Yu JH, Hamari Z, Han KH, Seo JA, Reyes-Domínguez Y, Scazzocchio C (2004) Double-joint PCR: a PCR-based molecular tool for gene manipulations in filamentous fungi. Fungal Genet Biol 41:973–981CrossRefGoogle Scholar
  44. Zhang X, Jia L, Zhang Y, Jiang G, Li X, Zhang D, Tang WH (2012) In planta stage-specific fungal gene profiling elucidates the molecular strategies of Fusarium graminearum growing inside wheat coleoptiles. Plant Cell 24:5159–5176CrossRefGoogle Scholar
  45. Zhao Z, Liu H, Luo Y, Zhou S, An L, Wang C, Zhou M, Xu J (2014) Molecular evolution and functional divergence of tubulin superfamily in the fungal tree of life. Sci Rep 4:6746CrossRefGoogle Scholar
  46. Zheng Z, Gao T, Hou Y, Zhou M (2013) Involvement of the anucleate primary sterigmata protein FgApsB in vegetative differentiation, asexual development, nuclear migration, and virulence in Fusarium graminearum. FEMS Microbiol Lett 349:88–98CrossRefGoogle Scholar
  47. Zheng Z, Gao T, Zhang Y, Hou Y, Wang J, Zhou M (2014) FgFim, a key protein regulating resistance to the fungicide JS399-19, asexual and sexual development, stress responses and virulence in Fusarium graminearum. Mol Plant Pathol 15:488–499CrossRefGoogle Scholar
  48. Zhu Y, Liang X, Li Y, Duan Y, Zheng Z, Wang J, Zhou M (2018) F240 of β 2-tubulin explains why Fusarium graminearum is less sensitive to carbendazim than Botrytis cinerea. Phytopathology 108:352–361CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Yanjun Li
    • 1
  • Dongming Chen
    • 1
  • Shunwen Luo
    • 1
  • Yuanye Zhu
    • 1
  • Xiaojing Jia
    • 1
  • Yabing Duan
    • 1
  • Mingguo Zhou
    • 1
    Email author
  1. 1.College of Plant ProtectionNanjing Agricultural UniversityNanjingChina

Personalised recommendations