AoRim15 is involved in conidial stress tolerance, conidiation and sclerotia formation in the filamentous fungus Aspergillus oryzae

https://doi.org/10.1016/j.jbiosc.2015.08.011Get rights and content

Highlights

  • AoRim15 is involved in the stress tolerance of conidia in A. oryzae.

  • Both deletion and overexpression of Aorim15 cause a decrease in conidiation.

  • AoRim15 plays a positive regulatory role in sclerotia formation.

  • AoRim15 differentially regulates between conidia and sclerotia formation.

The serine–threonine kinase Rim15p is a master regulator of stress signaling and is required for stress tolerance and sexual sporulation in the yeast Saccharomyces cerevisiae. However, in filamentous fungi that reproduce asexually via conidiation, the physiological function of Rim15p homologs has not been extensively analyzed. Here, we functionally characterized the protein homolog of Rim15p in the filamentous fungus Aspergillus oryzae, by deleting and overexpressing the corresponding Aorim15 gene and examining the role of this protein in stress tolerance and development. Deletion of Aorim15 resulted in an increase in the sensitivity of conidia to oxidative and heat stresses, whereas conidia of the Aorim15 overexpressing strain were more resistant to these stresses. These results indicated that AoRim15 functions in stress tolerance, similar to S. cerevisiae Rim15p. Phenotypic analysis revealed that conidiation was markedly reduced by overexpression of Aorim15 in A. oryzae, and was completely abolished in the deletion strain. In addition, the formation of sclerotia, which is another type of developmental structure in filamentous fungi, was decreased by the deletion of Aorim15, whereas Aorim15 overexpression increased the number of sclerotia. These results indicated that AoRim15 is a positive regulator of sclerotia formation and that overexpression of AoRim15 shifts the developmental balance from conidiation towards sclerotia formation. Collectively, we demonstrated that AoRim15 is involved in the stress tolerance of conidia and differentially regulates between the two developmental fates of conidiation and sclerotia formation.

Section snippets

Strains, media and transformation

The strains used in this study are listed in Table 1. A. oryzae wild-type strain RIB40 (30) and strain NSPlD1, which has a highly efficient gene-targeting background (niaD- sC- ΔpyrG ΔligD) (31), were used as a DNA donor and transformation host, respectively. M + Met medium (0.2% NH4Cl, 0.1% (NH4)2SO4, 0.05% KCl, 0.05% NaCl, 0.1% KH2PO4, 0.05% MgSO4·7H2O, 0.002% FeSO4·7H2O, 2% glucose, and 0.15% methionine, pH 5.5) was used as a selective medium for deleting the Aorim15 gene. CD + Met medium

Deletion and overexpression of the Aorim15 gene in A. oryzae

We searched for gene(s) homologous to S. cerevisiae RIM15 in the A. oryzae genome database (http://www.bio.nite.go.jp/dogan/project/view/AO) using the BLAST algorithm and found AO090012000420, which was named Aorim15. Based on rapid amplification of 5′ and 3′ cDNA ends (5′ and 3′ RACE) analysis (data not shown), it was concluded that the identified Aorim15 gene contains two exons and one intron, encoding a polypeptide of 2055 amino acids, which shares 21% identity with S. cerevisiae Rim15p (

Discussion

S. cerevisiae Rim15p functions as a central regulator of stress signaling and is required for stress tolerance; however, the physiological functions of the corresponding proteins have been poorly characterized in filamentous fungi. In the present study, by deleting and overexpressing the Aorim15 gene in A. oryzae, we clearly demonstrated that AoRim15 is involved in conidial stress tolerance, conidiation, and sclerotia formation.

Genomic deletion of Aorim15 significantly reduced the number of

Acknowledgments

This work was supported by a Grant-in-Aid for Young Scientists from the Japan Society for the Promotion of Science (grant 25712007). Funding for this study was also provided by Research and Development Projects for Application in Promoting New Policy of Agriculture, Forestry and Fisheries from National Agriculture and Food Research Organization, Japan (grant 25027A), and by the Institute for Fermentation, Osaka (IFO), Japan.

References (46)

  • K. Wuichet et al.

    Evolution and phyletic distribution of two-component signal transduction systems

    Curr. Opin. Microbiol.

    (2010)
  • C.L. Wang et al.

    Aspergillus nidulans striatin (StrA) mediates sexual development and localizes to the endoplasmic reticulum

    Fungal Genet. Biol.

    (2010)
  • P. Lee et al.

    Rim15-dependent activation of Hsf1 and Msn2/4 transcription factors by direct phosphorylation in Saccharomyces cerevisiae

    FEBS Lett.

    (2013)
  • I. Skromne et al.

    Starvation stress modulates the expression of the Aspergillus nidulans brlA regulatory gene

    Microbiology

    (1995)
  • L. Kawasaki et al.

    SakA MAP kinase is involved in stress signal transduction, sexual development and spore viability in Aspergillus nidulans

    Mol. Microbiol.

    (2002)
  • F. Lara-Rojas et al.

    Aspergillus nidulans transcription factor AtfA interacts with the MAPK SakA to regulate general stress responses, development and spore functions

    Mol. Microbiol.

    (2011)
  • J.R. Coley-Smith et al.

    Survival and germination of fungal sclerotia

    Annu. Rev. Phytopathol.

    (1971)
  • B.W. Horn et al.

    Sexual reproduction in Aspergillus flavus

    Mycologia

    (2009)
  • B.W. Horn et al.

    The sexual state of Aspergillus parasiticus

    Mycologia

    (2009)
  • M. Sideri et al.

    Differentiation and hydrogen peroxide production in Sclerotium rolfsii are induced by the oxidizing growth factors, light and iron

    Mycologia

    (2000)
  • K. Grintzalis et al.

    Role of oxidative stress in sclerotial differentiation and aflatoxin B1 biosynthesis in Aspergillus flavus

    Appl. Environ. Microbiol.

    (2014)
  • E. Swinnen et al.

    Rim15 and the crossroads of nutrient signalling pathways in Saccharomyces cerevisiae

    Cell Div.

    (2006)
  • M. Wei et al.

    Life span extension by calorie restriction depends on Rim15 and transcription factors downstream of Ras/PKA, Tor, and Sch9

    PLoS Genet.

    (2008)
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    Present address: College of Biology and the Environment, Nanjing Forestry University, 159 Longpan Road, Nanjing 210037, China.

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