Abstract
In eukaryotic cells, protein O-glycosylation is an essential protein modification. Analysis of the Metarhizium acridum genome database revealed a total of three O-glycoside mannosyltransferase homologs (Pmt1, Pmt2 and Pmt4), closely related to Saccharomyces cerevisiae Pmt1, Pmt2, and Pmt4. In this study, the functions of MaPmt4, encoding a protein O-mannosyltransferase in M. acridum, were characterized using disruption and complementation strategies. Disruption of MaPmt4 delayed the conidial germination and reduced the fungal tolerances to heat shock and UV-B irradiation, but did not affect conidial yield. Inactivation of MaPmt4 displayed increased sensitivity to cell wall-perturbing agents, formed thinner cell walls, and changed composition of fungal cell wall, demonstrating that MaPmt4 was also important to maintain fungal cell wall integrity. Bioassays by topical inoculation and intrahemocoel injection showed that the MaPmt4 deletion mutant exhibited greatly reduced virulence. The subsequent examination revealed that the inactivation of MaPmt4 impaired appressorium formation, decreased fungal growth in locust hemolymph in vitro, and boosted insect immune responses, the latter in part potentially owing to the changes in conidial surface structures, and thus attenuated the virulence of MaPmt4 deletion mutant. Furthermore, the results of comparative proteomics showed that MaPmt4 played important roles in fungal cell wall integrity, stress tolerances, and virulence via broad genetic pathways.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bai Z, Harvey LM, Mcneil B (2003) Elevated temperature effects on the oxidant/antioxidant balance in submerged batch cultures of the filamentous fungus Aspergillus niger B1-D. Biotechnol Bioeng 83(7):772–779. https://doi.org/10.1002/bit.10726
Beck MH, Strand MR (2007) A novel polydnavirus protein inhibits the insect prophenoloxidase activation pathway. Proc Natl Acad Sci USA 104(49):19267–19272. https://doi.org/10.1073/pnas.0708056104
Bourmaud A, Gallien S, Domon B (2016) Parallel reaction monitoring using quadrupole-orbitrap mass spectrometer: principle and applications. Proteomics 16(15–16):2146–2159. https://doi.org/10.1002/pmic.201500543
Bowman SM, Free SJ (2006) The structure and synthesis of the fungal cell wall. Bioessays 28(8):799–808. https://doi.org/10.1002/bies.20441
Cao Y, Du M, Luo S, Xia Y (2014) Calcineurin modulates growth, stress tolerance, and virulence in Metarhizium acridum and its regulatory network. Appl Microbiol Biotechnol 98(19):8253–8265. https://doi.org/10.1007/s00253-014-5876-3
Chung D, Dellaire G (2015) The role of the COP9 signalosome and neddylation in DNA damage signaling and repair. Biomolecules 5(4):2388–2416. https://doi.org/10.3390/biom5042388
Clarkson JM, Charnley AK (1996) New insights into the mechanisms of fungal pathogenesis in insects. Trends Microbiol 4(5):197–203. https://doi.org/10.1016/0966-842x(96)10022-6
Condit CM, Meagher RB (1986) A gene encoding a novel glycine-rich structural protein of Petunia. Nature 323(6084):178–181. https://doi.org/10.1038/323178a0
Dijkgraaf GJP, Brown JL, Bussey H (1996) The KNHI gene of Saccharomyces cerevisiae is a functional homolog of KRE9. Yeast 12(7):683–692. https://doi.org/10.1002/(SICI)1097-0061(19960615)12:7%3C683::AID-YEA959%3E3.0.CO;2-8
dos Reis MC, Pelegrinelli Fungaro MH, Delgado Duarte RT, Furlaneto L, Furlaneto MC (2004) Agrobacterium tumefaciens-mediated genetic transformation of the entomopathogenic fungus Beauveria bassiana. J Microbiol Methods 58(2):197–202. https://doi.org/10.1016/j.mimet.2004.03.012
Du Y, Jin K, Xia Y (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. https://doi.org/10.1007/s00253-018-9020-7
Fernandes ÉK, Rangel DE, Braga GU, Roberts DW (2015) Tolerance of entomopathogenic fungi to ultraviolet radiation: a review on screening of strains and their formulation. Curr Genet 61(3):427–440. https://doi.org/10.1007/s00294-015-0492-z
Fernández-Álvarez A, Elías-Villalobos A, Ibeas JI (2009) The O-mannosyltransferase PMT4 is essential for normal appressorium formation and penetration in Ustilago maydis. Plant Cell 21(10):3397–3412. https://doi.org/10.1105/tpc.109.065839
Fernández-Álvarez A, Marín-Menguiano M, Lanver D, Jiménez-Martín A, Elías-Villalobos A, Pérez-Pulido AJ, Kahmann R, Ibeas JI (2012) Identification of O-mannosylated virulence factors in Ustilago maydis. PLoS Pathog 8(3):e1002563. https://doi.org/10.1371/journal.ppat.1002563
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. https://doi.org/10.1371/journal.pgen.1001264
Gentzsch M, Tanner W (1997) Protein-O-glycosylation in yeast: Protein-specific mannosyltransferases. Glycobiology 7(4):481–486. https://doi.org/10.1093/glycob/7.4.481
Girrbach V, Zeller T, Priesmeier M, Strahl-Bolsinger S (2000) Structure-function analysis of the dolichyl phosphate-mannose: protein O-mannosyltransferase ScPmt1p. J Biol Chem 275(25):19288–19296. https://doi.org/10.1074/jbc.M001771200
Goldsworthy G, Chandrakant S, Opoku-Ware K (2003) Adipokinetic hormone enhances nodule formation and phenoloxidase activation in adult locusts injected with bacterial lipopolysaccharide. J Insect Physiol 49(8):795–803. https://doi.org/10.1016/s0022-1910(03)00118-5
González M, Brito N, Frías M, González C (2013) Botrytis cinerea protein O-mannosyltransferases play critical roles in morphogenesis, growth, and virulence. PLoS One 8(6):e65924. https://doi.org/10.1371/journal.pone.0065924
Goto M (2007) Protein O-glycosylation in fungi: diverse structures and multiple functions. Biosci Biotech Bioch 71(6):1415–1427. https://doi.org/10.1271/bbb.70080
Goto M, Harada Y, Oka T, Matsumoto S, Takegawa K, Furukawa K (2009) Protein O-mannosyltransferase B and C support hyphal development and differentiation in Aspergillus nidulans. Eukaryot Cell 8(10):1465–1474. https://doi.org/10.1128/EC.00371-08
Guerreiro ACL, Benevento M, Lehmann R, van Breukelen B, Post H, Giansanti P, Maarten Altelaar AF, Axmann IM, Heck AJ (2014) Daily rhythms in the cyanobacterium synechococcus elongatus probed by high-resolution mass spectrometry-based proteomics reveals a small defined set of cyclic proteins. Mol Cell Proteom 13(8):2042–2055. https://doi.org/10.1074/mcp.M113.035840
Guo M, Tan LY, Nie X, Zhu XL, Pan YM, Gao ZM (2016) The Pmt2p-mediated protein O-mannosylation is required for morphogenesis, adhesive properties, cell wall integrity and full virulence of Magnaporthe oryzae. Front Microbiol 7:630. https://doi.org/10.3389/fmicb.2016.00630
Harries E, Gandía M, Carmona L, Marcos JF (2015) The Penicillium digitatum protein O-mannosyltransferase Pmt2 is required for cell wall integrity, conidiogenesis, virulence and sensitivity to the antifungal peptide PAF26. Mol Plant Pathol 16(7):748–761. https://doi.org/10.1111/mpp.12232
He M, Xia Y (2009) Construction and analysis of a normalized cDNA library from Metarhizium anisopliae var. acridum germinating and differentiating on Locusta migratoria wings. FEMS Microbiol Lett 291(1):127–135. https://doi.org/10.1111/j.1574-6968.2008.01447.x
He PH, Dong WX, Chu XL, Feng MG, Ying SH (2016a) The cellular proteome is affected by a gelsolin (BbGEL1) during morphological transitions in aerobic surface versus liquid growth in the entomopathogenic fungus Beauveria bassiana. Environ Microbiol 18(11):4153–4169. https://doi.org/10.1111/1462-2920.13500
He Z, Luo L, Keyhani NO, Yu X, Ying S, Zhang Y (2016b) The C-terminal MIR-containing region in the Pmt1 O-mannosyltransferase restrains sporulation and is dispensable for virulence in Beauveria bassiana. Appl Microbiol Biot 101(3):1143–1161. https://doi.org/10.1007/s00253-016-7894-9
Hunter DM, Milner RJ, Spurgin PA (2001) Aerial treatment of the Australian plague locust, Chortoicetes terminifera (Orthoptera: Acrididae) with Metarhizium anisopliae (Deuteromycotina: Hyphomycetes). Bull Entomol Res 91(2):93–99. https://doi.org/10.1079/BER200080
Islam KT, Bond JP, Fakhoury AM (2017) FvSNF1, the sucrose non-fermenting protein kinase gene of Fusarium virguliforme, is required for cell-wall-degrading enzymes expression and sudden death syndrome development in soybean. Curr Genet 63(4):723–738. https://doi.org/10.1007/s00294-017-0676-9
Jin K, Ming Y, Xia YX (2012) MaHog1, a Hog1-type mitogen-activated protein kinase gene, contributes to stress tolerance and virulence of the entomopathogenic fungus Metarhizium acridum. Microbiology 158(12):2987–2996. https://doi.org/10.1099/mic.0.059469-0
Kapteyn JC, van Egmond P, Sievi E, van den Ende H, Makarow M, Klis FM (1999) The contribution of the O-glycosylated protein Pir2p/Hsp150 to the construction of the yeast cell wall in wild-type cells and beta 1,6-glucan-deficient mutants. Mol Microbiol 31(6):1835–1844. https://doi.org/10.1046/j.1365-2958.1999.01320.x
Katja B, Klara H, Petra S, Reinau LM, Michael DM, Peter S (2017) Lipoprotein glycosylation by protein-O-mannosyltransferase (MAB_1122c) contributes to low cell envelope permeability and antibiotic resistance of Mycobacterium abscessus. Front Microbiol 8:2123. https://doi.org/10.3389/fmicb.2017.02123
Keshishian H, Burgess MW, Gillette MA, Mertins P, Clauser KR, Mani DR, Kuhn EW, Farrell LA, Gerszten RE, Carr SA (2015) Multiplexed, quantitative workflow for sensitive biomarker discovery in plasma yields novel candidates for early myocardial injury. Mol Cell Proteomics 14(9):2375–2393. https://doi.org/10.1074/mcp.M114.046813
Lazo GR, Stein PA, Ludwig RA (1991) A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Biotechnology 9(10):963–967. https://doi.org/10.1038/nbt1091-963
Le THT, Oki A, Goto M, Shimizu K (2018) Protein O-mannosyltransferases are required for sterigmatocystin production and developmental processes in Aspergillus nidulans. Curr Genet 64(5):1043–1056. https://doi.org/10.1007/s00294-018-0816-x
Lee CG, Silva CAD, Lee JY, Hartl D, Elias JA (2008) Chitin regulation of immune responses: an old molecule with new roles. Curr Opin Immunol 20(6):684–689. https://doi.org/10.1016/j.coi.2008.10.002
Levitz SM, Madhani HD (2010) Innate recognition of fungal cell walls. PLoS Pathog 6(4):e1000758. https://doi.org/10.1371/journal.ppat.1000758
Liu CF, Tonini L, Malaga W, Beau M, Stella A, Bouyssié D, Jackson MC, Nigou J, Puzo G, Guilhot C (2013) Bacterial protein-O-mannosylating enzyme is crucial for virulence of Mycobacterium tuberculosis. Proc Natl Acad Sci USA 110(16):6560–6565. https://doi.org/10.1073/pnas.1219704110
Loibl M, Strahl S (2013) Protein O-mannosylation: what we have learned from baker’s yeast. Biochim Biophys Acta 1833(11):2438–2446. https://doi.org/10.1016/j.bbamcr.2013.02.008
Lommel M, Strahl S (2009) Protein O-mannosylation: conserved from bacteria to humans. Glycobiology 19(8):816–828. https://doi.org/10.1093/glycob/cwp066
Matar KAO, Chen X, Chen D, Anjago WM, Norvienyeku J, Lin Y, Chen M, Wang Z, Ebbole DJ, Lu GD (2017) WD40-repeat protein MoCreC is essential for carbon repression and is involved in conidiation, growth and pathogenicity of Magnaporthe oryzae. Curr Genet 63(4):685–696. https://doi.org/10.1007/s00294-016-0668-1
Merret R, Carpentier MC, Favory JJ, Picart C, Descombin J, Bousquet-Antonelli C, Tillard P, Lejay L, Deragon JM, Charng YY (2017) Heat shock protein HSP101 affects the release of ribosomal protein mRNAs for recovery after heat shock. Plant Physiol 174(2):1216–1225. https://doi.org/10.1104/pp.17.00269
Mouyna I, Kniemeyer O, Jank T, Loussert C, Mellado E, Aimanianda V, Beauvais A, Wartenberg D, Sarfati J, Bayry J (2010) Members of protein O-mannosyltransferase family in Aspergillus fumigatus differentially affect growth, morphogenesis and viability. Mol Microbiol 76(5):1205–1221. https://doi.org/10.1111/j.1365-2958.2010.07164.x
Oka T, Hamaguchi T, Sameshima Y, Goto M, Furukawa K (2004) Molecular characterization of protein O-mannosyltransferase and its involvement in cell-wall synthesis in Aspergillus nidulans. Microbiol 150(6):1973–1982. https://doi.org/10.1099/mic.0.27005-0
Olson GM, Fox DS, Wang P, Alspaugh JA, Buchanan KL (2007) Role of protein O-mannosyltransferase Pmt4 in the morphogenesis and virulence of Cryptococcus neoformans. Eukaryot Cell 6(2):222–234. https://doi.org/10.1128/EC.00182-06
Ortiz-Urquiza A, Keyhani NO (2015) Stress response signaling and virulence: insights from entomopathogenic fungi. Curr Genet 61(3):239–249. https://doi.org/10.1007/s00294-014-0439-9
Pan X, Heitman J (2002) Protein kinase A operates a molecular switch that governs yeast pseudohyphal differentiation. Mol Cell Biol 22(12):3981–3993. https://doi.org/10.1128/MCB.22.12.3981-3993.2002
Pan YM, Pan R, Tan LY, Zhang ZG, Guo M (2018) Pleiotropic roles of O-mannosyltransferase MoPmt4 in development and pathogenicity of Magnaporthe oryzae. Curr Genet 65(1):223–239. https://doi.org/10.1007/s00294-018-0864-2
Peng G, Wang Z, Yin Y, Zeng DY, Xia YX (2008) Field trials of Metarhizium anisopliae var. acridum (Ascomycota: Hypocreales) against oriental migratory locusts, Locusta migratoria manilensis (Meyen) in Northern China. Crop Prot 27(9):1244–1250. https://doi.org/10.1016/j.cropro.2008.03.007
Phaff HJ (1963) Cell wall of yeasts. Annu Rev Microbiol 17(1):15–30. https://doi.org/10.1146/annurev.mi.17.100163.000311
Prill SK, Klinkert B, Timpel C, Gale CA, Schröppel K, Ernst JF (2004) PMT family of Candida albicans: five protein mannosyltransferase isoforms affect growth, morphogenesis and antifungal resistance. Mol Microbiol 55(2):546–560. https://doi.org/10.1111/j.1365-2958.2004.04401.x
Raeder U, Broda P (1985) Rapid preparation of DNA from filamentous fungi. Lett Appl Microbiol 1(1):17–20. https://doi.org/10.1111/j.1472-765X.1985.tb01479.x
Schiestl RH, Prakash S (1989) Interactions of the RAD7 and RAD23 excision repair genes of Saccharomyces cerevisiae with DNA repair genes in different epistasis groups. Curr Genet 16(4):219–223. https://doi.org/10.1007/BF00422107
Screen S, Bailey A, Charnley K, Cooper R, Clarkson J (1998) Isolation of a nitrogen response regulator gene (nrr1) from Metarhizium anisopliae. Gene 221(1):17–24. https://doi.org/10.1016/S0378-1119(98)00430-2
Shimizu K, Imanishi Y, Toh-E A, Uno J, Chibana H, Hull CM, Kawamoto S (2014) Functional characterization of Pmt2, encoding a protein-O-mannosyltransferase, in the human pathogen Cryptococcus neoformans. Fungal Genet Biol 69:13–22. https://doi.org/10.1016/j.fgb.2014.05.007
Strahl-Bolsinger S, Gentzsch M, Tanner W (1999) Protein O-mannosylation. Biochim Biophys Acta 1426(2):297–307. https://doi.org/10.1016/S0304-4165(98)00131-7
Sukhithasri V, Nisha N, Biswas L, Anil KV, Biswas R (2013) Innate immune recognition of microbial cell wall components and microbial strategies to evade such recognitions. Microbiol Res 168(7):396–406. https://doi.org/10.1016/j.micres.2013.02.005
van Vu B, Itoh K, Nguyen QB, Tosa Y, Nakayashiki H (2012) Cellulases belonging to glycoside hydrolase families 6 and 7 contribute to the virulence of Magnaporthe oryzae. Mol Plant Microbe Interact 25(9):1135–1141. https://doi.org/10.1094/MPMI-02-12-0043-R
van den Toorn HW, Muñoz J, Mohammed S, Raijmakers R, Heck AJ, van Breukelen B (2011) RockerBox: analysis and filtering of massive proteomics search results. J Proteome Res 10(3):1420–1424. https://doi.org/10.1021/pr1010185
Wanchoo A, Lewis MW, Keyhani NO (2009) Lectin mapping reveals stage-specific display of surface carbohydrates in in vitro and haemolymph-derived cells of the entomopathogenic fungus Beauveria bassiana. Microbiology 155(9):3121–3133. https://doi.org/10.1099/mic.0.029157-0
Wang C, St Leger RJ (2007) The Metarhizium anisopliae perilipin homolog MPL1 regulates lipid metabolism, appressorial turgor pressure, and virulence. J Biol Chem 282(29):21110–21115. https://doi.org/10.1074/jbc.M609592200
Wang N, Yoshida Y, Hasunuma K (2007) Catalase-1 (CAT-1) and nucleoside diphosphate kinase-1 (NDK-1) play an important role in protecting conidial viability under light stress in Neurospora crassa. Mol Genet Genom 278(3):235–242. https://doi.org/10.1007/s00438-007-0244-y
Wang JJ, Qiu L, Chu ZJ, Ying SH, Feng MG (2014) The connection of protein O-mannosyltransferase family to the biocontrol potential of Beauveria bassiana, a fungal entomopathogen. Glycobiology 24(7):638–648. https://doi.org/10.1093/glycob/cwu028
Willer T, Brandl M, Sipiczki M, Strahl S (2005) Protein O-mannosylation is crucial for cell wall integrity, septation and viability in fission yeast. Mol Microbiol 57(1):156–170. https://doi.org/10.1111/j.1365-2958.2005.04692.x
Xie XQ, Li F, Ying SH, Feng MG (2012) Additive contributions of two manganese-cored superoxide dismutases (MnSODs) to antioxidation, UV tolerance and virulence of Beauveria bassiana. PLoS One 7(1):e30298. https://doi.org/10.1371/journal.pone.0030298
Zhang Q, Peng G, Wang Z, Yin Y, Xia Y (2005) Detection of Metarhizium anisopliae rDNA in haemalymph of infected adult locust by real-time fluorescence quantitative PCR. Prog Biotech 25(11):71–75. https://doi.org/10.1016/j.cyto.2004.11.006
Zhang S, Peng G, Xia Y (2010) Microcycle conidiation and the conidial properties in the entomopathogenic fungus Metarhizium acridum on agar medium. Biocontrol Sci Technol 20(8):809–819. https://doi.org/10.1080/09583157.2010.482201
Zhou H, Hu H, Zhang L, Li R, Ouyang H, Ming J, Jin C (2007) O-mannosyltransferase 1 in Aspergillus fumigatus (AfPmt1p) is crucial for cell wall integrity and conidium morphology, especially at an elevated temperature. Eukaryot Cell 6(12):2260–2268. https://doi.org/10.1128/EC.00261-07
Zimmermann G (1993) The entomopathogenic fungus Metarhizium anisopliae and its potential as a biocontrol agent. Pest Manag Sci 37(4):375–379. https://doi.org/10.1002/ps.2780370410
Acknowledgements
This work is supported by the Natural Science Foundation of China (31270040 and 31471820), the Fundamental Research Funds for the Central Universities (106112017CDJQJ298831), and the Natural Science Foundation Project of Chongqing (cstc2018jcyjAX0554).
Author information
Authors and Affiliations
Corresponding authors
Additional information
Communicated by M. Kupiec.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Zhao, T., Tian, H., Xia, Y. et al. MaPmt4, a protein O-mannosyltransferase, contributes to cell wall integrity, stress tolerance and virulence in Metarhizium acridum. Curr Genet 65, 1025–1040 (2019). https://doi.org/10.1007/s00294-019-00957-z
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00294-019-00957-z