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MrArk1, an actin-regulating kinase gene, is required for endocytosis and involved in sustaining conidiation capacity and virulence in Metarhizium robertsii

  • Applied genetics and molecular biotechnology
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Abstract

Actin-regulating kinase (Ark) plays an important role in controlling endocytosis, which has been shown to be involved in the development and virulence of several fungal pathogens. However, it remains unclear whether Ark1 is required for the development and pathogenicity of an entomopathogenic fungus. Here, MrArk1 (MAA_03415), a homologue of yeast Ark1, was characterized in the insect pathogenic fungus, Metarhizium robertsii. Disruption of MrArk1 led to defects in endocytosis and a marked reduction (58%) in conidiation capacity. The reduced conidiation level was accompanied by repression of several key conidiation-related genes, including brlA, abaA, and wetA. Additionally, the deletion mutant showed a significant decrease in its tolerance to heat shock, but not to UV-B irradiation. Bioassays demonstrated attenuated virulence for the deletion mutant against Galleria mellonella via normal cuticle infection, accompanied by suppressed appressorium formation and reduced transcript levels of several genes involved in cuticle penetration. Taken together, our results indicate that MrArk1 is involved in the heat tolerance, sporulation, and virulence of M. robertsii, and thus is an important factor for sustaining the fungal potential against insect pests.

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References

  • Albataineh MT, Kadosh D (2016) Regulatory roles of phosphorylation in model and pathogenic fungi. Med Mycol 54(4):333–352

    Article  PubMed  Google Scholar 

  • Atkinson HA, Daniels A, Read ND (2002) Live-cell imaging of endocytosis during conidial germination in the rice blast fungus, Magnaporthe grisea. Fungal Genet Biol 37(3):233–244

    Article  PubMed  Google Scholar 

  • Berndt P, Lanver D, Kahmann R (2010) The AGC Ser/Thr kinase Aga1 is essential for appressorium formation and maintenance of the actin cytoskeleton in the smut fungus Ustilago maydis. Mol Microbiol 78(6):1484–1499

    Article  CAS  PubMed  Google Scholar 

  • Bruno KS, Tenjo F, Li L, Hamer JE, Xu JR (2004) Cellular localization and role of kinase activity of PMK1 in Magnaporthe grisea. Eukaryot Cell 3(6):1525–1532

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chen Y, Zhu J, Ying SH, Feng MG (2014) Three mitogen-activated protein kinases required for cell wall integrity contribute greatly to biocontrol potential of a fungal entomopathogen. PLoS One 9(2):e87948

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chen XX, Xu C, Qian Y, Liu R, Zhang QQ, Zeng GH, Zhang X, Zhao H, Fang WG (2016) MAPK cascade-mediated regulation of pathogenicity, conidiation and tolerance to abiotic stresses in the entomopathogenic fungus Metarhizium robertsii. Environ Microbiol 18(3):1048–1062

    Article  CAS  PubMed  Google Scholar 

  • Chen YX, Li B, Cen K, Lu YZ, Zhang SW, Wang CS (2018) Diverse effect of phosphatidylcholine biosynthetic genes on phospholipid homeostasis, cell autophagy and fungal developments in Metarhizium robertsii. Environ Microbiol 20(1):293–304

    Article  CAS  PubMed  Google Scholar 

  • de Vries RP, Riley R, Wiebenga A, Aguilar-Osorio G, Amillis S, Uchima CA, Anderluh G, Asadollahi M, Askin M, Barry K, Battaglia E, Bayram O, Benocci T, Braus-Stromeyer SA, Caldana C, Canovas D, Cerqueira GC, Chen FS, Chen WP, Choi C, Clum A, dos Santos RAC, Damasio ARD, Diallinas G, Emri T, Fekete E, Flipphi M, Freyberg S, Gallo A, Gournas C, Habgood R, Hainaut M, Harispe ML, Henrissat B, Hilden KS, Hope R, Hossain A, Karabika E, Karaffa L, Karanyi Z, Krasevec N, Kuo A, Kusch H, LaButti K, Lagendijk EL, Lapidus A, Levasseur A, Lindquist E, Lipzen A, Logrieco AF, MacCabe A, Makela MR, Malavazi I, Melin P, Meyer V, Mielnichuk N, Miskei M, Molnar AP, Mule G, Ngan CY, Orejas M, Orosz E, Ouedraogo JP, Overkamp KM, Park HS, Perrone G, Piumi F, Punt PJ, Ram AFJ, Ramon A, Rauscher S, Record E, Riano-Pachon DM, Robert V, Rohrig J, Ruller R, Salamov A, Salih NS, Samson RA, Sandor E, Sanguinetti M, Schutze T, Sepcic K, Shelest E, Sherlock G, Sophianopoulou V, Squina F, Sun H, Susca A, Todd RB, Tsang A, Unkles SE, van de Wiele N, van Rossen-Uffink D, Oliveira JVD, Vesth TC, Visser J, Yu JH, Zhou MM, Andersen MR, Archer DB, Baker SE, Benoit I, Brakhage AA, Braus GH, Fischer R, Frisvad JC, Goldman GH, Houbraken J, Oakley B, Pocsi I, Scazzocchio C, Seiboth B, vanKuyk PA, Wortman J, Dyer PS, Grigoriev IV (2017) Comparative genomics reveals high biological diversity and specific adaptations in the industrially and medically important fungal genus Aspergillus. Genome Biol 18(1):28

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dou XY, Wang Q, Qi ZQ, Song WW, Wang W, Guo M, Zhang HF, Zhang ZG, Wang P, Zheng XB (2011) MoVam7, a conserved SNARE involved in vacuole assembly, is required for growth, endocytosis, ROS accumulation, and pathogenesis of Magnaporthe oryzae. PLoS One 6(1):e16439

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Du YR, Jin K, Xia YX (2018) Involvement of MaSom1, a downstream transcriptional factor of cAMP/PKA pathway, in conidial yield, stress tolerances, and virulence in Metarhizium acridum. Appl Microbiol Biotechnol 102(13):5611–5623

    Article  CAS  PubMed  Google Scholar 

  • Fang WG, Bidochka MJ (2006) Expression of genes involved in germination, conidiogenesis and pathogenesis in Metarhizium anisopliae using quantitative real-time RT-PCR. Mycol Res 110:1165–1171

    Article  CAS  PubMed  Google Scholar 

  • Fang WG, Pei Y, Bidochka MJ (2006) Transformation of Metarhizium anisopliae mediated by Agrobacterium tumefaciens. Can J Microbiol 52(7):623–626

    Article  CAS  PubMed  Google Scholar 

  • Fang WG, Pava-Ripoll M, Wang SB, Leger RS (2009) Protein kinase A regulates production of virulence determinants by the entomopathogenic fungus, Metarhizium anisopliae. Fungal Genet Biol 46(3):277–285

    Article  CAS  PubMed  Google Scholar 

  • Fang WG, Azimzadeh P, St Leger RJ (2012) Strain improvement of fungal insecticides for controlling insect pests and vector-borne diseases. Curr Opin Microbiol 15(3):232–238

    Article  PubMed  Google Scholar 

  • Fischer-Parton S, Parton RM, Hickey PC, Dijksterhuis J, Atkinson HA, Read ND (2000) Confocal microscopy of FM4-64 as a tool for analysing endocytosis and vesicle trafficking in living fungal hyphae. J Microsc 198:246–259

    Article  CAS  PubMed  Google Scholar 

  • Fuchs U, Hause G, Schuchardt I, Steinberg G (2006) Endocytosis is essential for pathogenic development in the corn smut fungus Ustilago maydis. Plant Cell 18(8):2066–2081

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gao Q, Jin K, Ying SH, Zhang YJ, Xiao GH, Shang YF, Duan ZB, Hu XA, Xie XQ, Zhou G, Peng GX, Luo ZB, Huang W, Wang B, Fang WG, Wang SB, Zhong Y, Ma LJ, St Leger RJ, Zhao GP, Pei Y, Feng MG, Xia YX, Wang CS (2011) Genome sequencing and comparative transcriptomics of the model entomopathogenic fungi Metarhizium anisopliae and M. acridum. PLoS Genet 7(1):e1001264

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gao Q, Shang YF, Huang W, Wang CS (2013) Glycerol-3-phosphate acyltransferase contributes to triacylglycerol biosynthesis, lipid droplet formation, and host invasion in Metarhizium robertsii. Appl Environ Microbiol 79(24):7646–7653

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gao Q, Lu YZ, Yao HY, Xu YJ, Huang W, Wang CS (2016) Phospholipid homeostasis maintains cell polarity, development and virulence in Metarhizium robertsii. Environ Microbiol 18(11):3976–3990

    Article  CAS  PubMed  Google Scholar 

  • Goode BL, Eskin JA, Wendland B (2015) Actin and endocytosis in budding yeast. Genetics 199(2):315–358

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Higuchi Y, Shoji JY, Arioka M, Kitamoto K (2009) Endocytosis is crucial for cell polarity and apical membrane recycling in the filamentous fungus Aspergillus oryzae. Eukaryot Cell 8(1):37–46

    Article  CAS  PubMed  Google Scholar 

  • Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33(7):1870–1874

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li L, Wang JY, Chen HL, Chai RY, Zhang Z, Mao XQ, Qiu HP, Jiang H, Wang YL, Sun GC (2017a) Pex14/17, a filamentous fungus-specific peroxin, is required for the import of peroxisomal matrix proteins and full virulence of Magnaporthe oryzae. Mol Plant Pathol 18(9):1238–1252

    Article  CAS  PubMed  Google Scholar 

  • Li LW, Chen XL, Zhang SP, Yang J, Chen D, Liu MX, Zhang HF, Zheng XB, Wang P, Peng YL, Zhang ZG (2017b) MoCAP proteins regulated by MoArk1-mediated phosphorylation coordinate endocytosis and actin dynamics to govern development and virulence of Magnaporthe oryzae. PLoS Genet 13(5):e1006814

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Liu J, Wang ZK, Sun HH, Ying SH, Feng MG (2017) Characterization of the Hog1 MAPK pathway in the entomopathogenic fungus Beauveria bassiana. Environ Microbiol 19(5):1808–1821

    Article  CAS  PubMed  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2 -∆∆CT method. Methods 25(4):402–408

    Article  CAS  PubMed  Google Scholar 

  • Lovett B, St Leger RJ (2015) Stress is the rule rather than the exception for Metarhizium. Curr Genet 61(3):253–261

    Article  CAS  PubMed  Google Scholar 

  • Meng HM, Wang ZX, Wang YL, Zhu H, Huang B (2017) Dicer and argonaute genes involved in RNA interference in the entomopathogenic fungus Metarhizium robertsii. Appl Environ Microbiol 83(7):e03230–e03216

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Miaczynska M, Stenmark H (2008) Mechanisms and functions of endocytosis. J Cell Biol 180(1):7–11

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ming Y, Wei QL, Jin K, Xia YX (2014) MaSnf1, a sucrose non-fermenting protein kinase gene, is involved in carbon source utilization, stress tolerance, and virulence in Metarhizium acridum. Appl Microbiol Biotechnol 98(24):10153–10164

    Article  CAS  PubMed  Google Scholar 

  • Miranda-Saavedra D, Barton GJ (2007) Classification and functional annotation of eukaryotic protein kinases. Proteins 68(4):893–914

    Article  CAS  PubMed  Google Scholar 

  • Muniz-Paredes F, Miranda-Hernandez F, Loera O (2017) Production of conidia by entomopathogenic fungi: from inoculants to final quality tests. World J Microbiol Biotechnol 33(3):57

    Article  CAS  PubMed  Google Scholar 

  • Ortiz-Urquiza A, Keyhani NO (2015) Stress response signaling and virulence: insights from entomopathogenic fungi. Curr Genet 61(3):239–249

    Article  CAS  PubMed  Google Scholar 

  • Peng YJ, Ding JL, Feng MG, Ying SH (2019) Glc8, a regulator of protein phosphatase type 1, mediates oxidation tolerance, asexual development and virulence in Beauveria bassiana, a filamentous entomopathogenic fungus. Curr Genet 65(1):283–291

    Article  CAS  PubMed  Google Scholar 

  • Rangel DEN, Alston DG, Roberts DW (2008) Effects of physical and nutritional stress conditions during mycelial growth on conidial germination speed, adhesion to host cuticle, and virulence of Metarhizium anisopliae, an entomopathogenic fungus. Mycol Res 112:1355–1361

    Article  PubMed  Google Scholar 

  • Shen G, Whittington A, Song KJ, Wang P (2010) Pleiotropic function of intersectin homologue Cin1 in Cryptococcus neoformans. Mol Microbiol 76(3):662–676

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Smythe E, Ayscough KR (2003) The Ark1/Prk1 family of protein kinases—regulators of endocytosis and the actin cytoskeleton. EMBO Rep 4(3):246–251

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tong SM, Feng MG (2019) Insights into regulatory roles of MAPK-cascaded pathways in multiple stress responses and life cycles of insect and nematode mycopathogens. Appl Microbiol Biotechnol 103(2):577–587

    Article  CAS  PubMed  Google Scholar 

  • Turra D, Segorbe D, Di Pietro A (2014) Protein kinases in plant-pathogenic fungi: conserved regulators of infection. Annu Rev Phytopathol 52:267–288

    Article  CAS  PubMed  Google Scholar 

  • Wang CS, Leger RJS (2007) The Metarhizium anisopliae perilipin homolog MPL1 regulates lipid metabolism, appressorial turgor pressure, and virulence. J Biol Chem 282(29):21110–21115

    Article  CAS  PubMed  Google Scholar 

  • Wang CS, Wang SB (2017) Insect pathogenic fungi: genomics, molecular interactions, and genetic improvements. Annu Rev Entomol 62:73–90

    Article  CAS  PubMed  Google Scholar 

  • Wang CF, Zhang SJ, Hou R, Zhao ZT, Zheng Q, Xu QJ, Zheng DW, Wang GH, Liu HQ, Gao XL, Ma JW, Kistler HC, Kang ZS, Xu JR (2011) Functional analysis of the kinome of the wheat scab fungus Fusarium graminearum. PLoS Pathog 7(12):e1002460

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang JM, Du Y, Zhang HF, Zhou C, Qi ZQ, Zheng XB, Wang P, Zhang ZG (2013a) The actin-regulating kinase homologue MoArk1 plays a pleiotropic function in Magnaporthe oryzae. Mol Plant Pathol 14(5):470–482

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang ZX, Meng HM, Zhuang ZL, Chen MJ, Xie L, Huang B (2013b) Molecular cloning of a novel subtilisin-like protease (Pr1A) gene from the biocontrol fungus Isaria farinosa. Appl Entomol Zool 48(4):477–487

    Article  CAS  Google Scholar 

  • Wang ZX, Zhou XZ, Meng HM, Liu YJ, Zhou Q, Huang B (2014) Comparative transcriptomic analysis of the heat stress response in the filamentous fungus Metarhizium anisopliae using RNA-Seq. Appl Microbiol Biotechnol 98(12):5589–5597

    Article  CAS  PubMed  Google Scholar 

  • Wang YL, Wang TT, Qiao LT, Zhu JY, Fan JR, Zhang TT, Wang ZX, Li WZ, Chen AH, Huang B (2017) DNA methyltransferases contribute to the fungal development, stress tolerance and virulence of the entomopathogenic fungus Metarhizium robertsii. Appl Microbiol Biotechnol 101(10):4215–4226

    Article  CAS  PubMed  Google Scholar 

  • Wang ZK, Cai Q, Tong SM, Ying SH, Feng MG (2018a) C-terminal Ser/Thr residues are vital for the regulatory role of Ste7 in the asexual cycle and virulence of Beauveria bassiana. Appl Microbiol Biotechnol 102(16):6973–6986

    Article  CAS  PubMed  Google Scholar 

  • Wang ZX, Zhou Q, Li YD, Qiao LT, Pang Q, Huang B (2018b) iTRAQ-based quantitative proteomic analysis of conidia and mycelium in the filamentous fungus Metarhizium robertsii. Fungal Biol 122(7):651–658

    Article  PubMed  Google Scholar 

  • Watson HA, Cope M, Groen AC, Drubin DG, Wendland B (2001) In vivo role for actin-regulating kinases in endocytosis and yeast epsin phosphorylation. Mol Biol Cell 12(11):3668–3679

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Xu JR, Hamer JE (1996) MAP kinase and cAMP signaling regulate infection structure formation and pathogenic growth in the rice blast fungus Magnaporthe grisea. Genes Dev 10(21):2696–2706

    Article  CAS  PubMed  Google Scholar 

  • Yang WJ, Wu H, Wang ZX, Sun Q, Qiao LT, Huang B (2018) The APSES gene MrStuA regulates sporulation in Metarhizium robertsii. Front Microbiol 9:1208

    Article  PubMed  PubMed Central  Google Scholar 

  • Zhang LB, Feng MG (2018) Antioxidant enzymes and their contributions to biological control potential of fungal insect pathogens. Appl Microbiol Biotechnol 102(12):4995–5004

    Article  CAS  PubMed  Google Scholar 

  • Zhang HF, Li B, Fang Q, Li Y, Zheng XB, Zhang ZG (2016) SNARE protein FgVam7 controls growth, asexual and sexual development, and plant infection in Fusarium graminearum. Mol Plant Pathol 17(1):108–119

    Article  CAS  PubMed  Google Scholar 

  • Zhong K, Li X, Le X, Kong X, Zhang H, Zheng X, Wang P, Zhang Z (2016) MoDnm1 dynamin mediating peroxisomal and mitochondrial fission in complex with MoFis1 and MoMdv1 is important for development of functional appressorium in Magnaporthe oryzae. PLoS Pathog 12(8):e1005823

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhou R, Zhou XZ, Fan AL, Wang ZX, Huang B (2018) Differential functions of two metalloproteases, Mrmep1 and Mrmep2, in growth, sporulation, cell wall integrity, and virulence in the filamentous fungus Metarhizium robertsii. Front Microbiol 9:1528

    Article  PubMed  PubMed Central  Google Scholar 

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Funding

The research was supported by the National Natural Science Foundation of China (grant numbers 31572060, 31471821, and 31772226) and the National Key R&D Program of China (grant numbers 2017YFD0200400).

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  1. Zhangxun Wang and Yuanyuan Jiang contributed equally to this work.

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  1. Anhui Provincial Key Laboratory of Microbial Pest Control, Anhui Agricultural University, Hefei, 230036, China

    Zhangxun Wang, Yuanyuan Jiang, Yuandong Li, Jianyu Feng & Bo Huang

  2. School of Plant Protection, Anhui Agricultural University, Hefei, 230036, China

    Zhangxun Wang, Yuanyuan Jiang, Yuandong Li, Jianyu Feng & Bo Huang

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  1. Zhangxun Wang
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Wang, Z., Jiang, Y., Li, Y. et al. MrArk1, an actin-regulating kinase gene, is required for endocytosis and involved in sustaining conidiation capacity and virulence in Metarhizium robertsii. Appl Microbiol Biotechnol 103, 4859–4868 (2019). https://doi.org/10.1007/s00253-019-09836-6

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