Abstract
Intracellular trehalose accumulation is relevant to fungal life and pathogenicity. Trehalose-6-phosphate synthase (TPS) is known to control the first step of trehalose synthesis, but functions of multiple TPS genes in some filamentous fungi are variable. Here, we examined the functions of two TPS genes (tpsA and tpsB) in Beauveria bassiana, a fungal insect pathogen widely applied in arthropod pest control. Intracellular TPS activity and trehalose content decreased by 71–75 and 72–80% in ΔtpsA, and 21–30 and 15–45% in ΔtpsB, respectively, and to undetectable levels in ΔtpsAΔtpsB, under normal and stressful conditions. The three mutants lost 33, 50, and 98% of conidiation capacity in standard cultures. Conidial quality indicated by viability, density, intracellular trehalose content, cell wall integrity, and hydrophobicity was more impaired in ΔtpsA than in ΔtpsB and mostly in ΔtpsAΔtpsB, which was also most sensitive to nutritional, chemical, and environmental stresses and least virulent to Galleria mellonella larvae. Almost all of phenotypic defects in ΔtpsAΔtpsB approached to the sums of those observed in ΔtpsA and ΔtpsB and were restored by targeted gene complementation. Altogether, TpsA and TpsB play complementary roles in sustaining trehalose synthesis, conidiation capacity, conidial quality, multiple stress tolerance, and virulence, highlighting a significance of both for the fungal adaptation to environment and host.
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References
Al-Bader N, Vanier G, Liu H, Gravelat FN, Urb M, Hoareau CMQ, Campoli P, Chabot J, Filler SG, Sheppard DC (2010) Role of trehalose biosynthesis in Aspergillus fumigatus development, stress response, and virulence. Infect Immun 78:3007–3018
Avonce N, Mendoza-Vargas A, Morett E, Iturriaga G (2006) Insights on the evolution of trehalose biosynthesis. BMC Evol Biol 6:109
Bell W, Sun WN, Hohmann S, Wera S, Reinders A, De Virgilio C, Wiemken A, Thevelein JM (1998) Composition and functional analysis of the Saccharomyces cerevisiae trehalose synthase complex. J Biol Chem 273:33311–33319
Benaroudj N, Lee DH, Goldberg AL (2001) Trehalose accumulation during cellular stress protects cells and cellular proteins from damage by oxygen radicals. J Biol Chem 276:24261–24267
Boudreau BA, Larson TM, Brown DW, Busman M, Roberts ES, Kendra DF, McQuade KL (2013) Impact of temperature stress and validamycin a on compatible solutes and fumonisin production in F. verticillioides role of trehalose-6-phosphate synthase. Fungal Genet Biol 57:1–10
Crowe JH, Hoekstra FA, Crowe LM (1992) Anhydrobiosis. Annu Rev Physiol 54:579–599
De Virgilio C, Bürckert N, Bell W, Jenö P, Boller T, Wiemken A (1993) Disruption of TPS2, the gene encoding the 100-kDa subunit of the trehalose-6-phosphate synthase phosphatase complex in Saccharomyces cerevisiae, causes accumulation of trehalose-6-phosphate and loss of trehalose-6-phosphate phosphatase-activity. Eur J Biochem 212:315–323
Doehlemann G, Berndt P, Hahn M (2006) Trehalose metabolism is important for heat stress tolerance and spore germination of Botrytis cinerea. Microbiol-SGM 152:2625–2634
Elbein AD, Pan YT, Pastuszak I, Carroll D (2003) New insights on trehalose: a multifunctional molecule. Glycobiology 13:17R–27R
Fang WX, Yu XY, Wang B, Zhou H, Ouyang HM, Ming J, Jin C (2009) Characterization of the Aspergillus fumigatus phosphomannose isomerase Pmi1 and its impact on cell wall synthesis and morphogenesis. Microbiol-SGM 155:3281–3293
Fernandez J, Wright JD, Hartline D, Quispe CF, Madayiputhiya N, Wilson RA (2012) Principles of carbon catabolite repression in the rice blast fungus: Tps1, Nmr1-3, and a mate-family pump regulate glucose metabolism during infection. PLoS Genet 8:e1002673
Fillinger S, Chaveroche MK, van Dijck P, de Vries R, Ruijter G, Thevelein J, d'Enfert C (2001) Trehalose is required for the acquisition of tolerance to a variety of stresses in the filamentous fungus Aspergillus nidulans. Microbiol-SGM 147:1851–1862
Flores CL, Gancedo C, Petit T (2011) Disruption of Yarrowia lipolytica TPS1 gene encoding trehalose-6P synthase does not affect growth in glucose but impairs growth at high temperature. PLoS One 6:e23695
Foster AJ, Jenkinson JM, Talbot NJ (2003) Trehalose synthesis and metabolism are required at different stages of plant infection by Magnaporthe grisea. EMBO J 22:225–235
Gancedo C, Flores CL (2004) The importance of a functional trehalose biosynthetic pathway for the life of yeasts and fungi. FEMS Yeast Res 4:351–359
Hottiger T, Schmutz P, Wiemken A (1987) Heat-induced accumulation and futile cycling of trehalose in Saccharomyces cerevisiae. J Bacteriol 169:5518–5522
Lee JI, Yu YM, Rho YM, Park BC, Choi JH, Park HM, Maeng PJ (2005) Differential expression of the chsE gene encoding a chitin synthase of Aspergillus nidulans in response to developmental status and growth conditions. FEMS Microbiol Lett 249:121–129
Liu Q, Ying SH, Feng MG, Jiang XH (2009) Physiological implication of intracellular trehalose and mannitol changes in response of entomopathogenic fungus Beauveria bassiana to thermal stress. Antonie Van Leeuwenhoek 95:65–75
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2 -ΔΔCT method. Methods 25:402–408
Lowe RGT, Lord M, Rybak K, Trengove RD, Oliver RP, Solomon PS (2009) Trehalose biosynthesis is involved in sporulation of Stagonospora nodorum. Fungal Genet Biol 46:381–389
Martínez-Esparza M, Aguinaga A, González-Párraga P, García-Peñarrubia P, Jouault T, Argüelles JC (2007) Role of trehalose in resistance to macrophage killing: study with a tps1/tps1 trehalose-deficient mutant of Candida albicans. Clin Microbiol Infect 13:384–394
Ngamskulrungroj P, Himmelreich U, Breger JA, Wilson C, Chayakulkeeree M, Krockenberger MB, Malik R, Daniel HM, Toffaletti D, Djordjevic JT, Mylonakis E, Meyer W, Perfect JR (2009) The trehalose synthesis pathway is an integral part of the virulence composite for Cryptococcus gattii. Infect Immun 77:4584–4596
Noubhani A, Bunoust O, Rigoulet M, Thevelein JM (2000) Reconstitution of ethanolic fermentation in permeabilized spheroplasts of wild-type and trehalose-6-phosphate synthase mutants of the yeast Saccharomyces cerevisiae. Eur J Biochem 267:4566–4576
Nwaka S, Mechler B, Destruelle M, Holzer H (1995) Phenotypic features of trehalase mutants in Saccharomyces cerevisiae. FEBS Lett 360:286–290
Ocón A, Hampp R, Requena N (2007) Trehalose turnover during abiotic stress in arbuscular mycorrhizal fungi. New Phytol 174:879–891
Ram AF, Klis FM (2006) Identification of fungal cell wall mutants using susceptibility assays based on calcofluor white and Congo red. Nat Protoc 1:2253–2256
Rangel DEN, Anderson AJ, Roberts DW (2006) Growth of Metarhizium anisopliae on non-preferred carbon sources yields conidia with increased UV-B tolerance. J Invertebr Pathol 93:127–134
Rangel DEN, Anderson AJ, Roberts DW (2008) Evaluating physical and nutritional stress during mycelial growth as inducers of tolerance to heat and UV-B radiation in Metarhizium anisopliae conidia. Mycol Res 112:1362–1372
Rangel DEN, Braga GUL, Fernandes ÉKK, Keyser CA, Hallsworth JE, Roberts DW (2015) Stress tolerance and virulence of insect-pathogenic fungi are determined by environmental conditions during conidial formation. Curr Genet 61:383–404
Ratnakumar S, Tunnacliffe A (2006) Intracellular trehalose is neither necessary nor sufficient for desiccation tolerance in yeast. FEMS Yeast Res 6:902–913
Reinders A, Burckert N, Hohmann S, Thevelein JM, Boller T, Wiemken A, DeVirgilio C (1997) Structural analysis of the subunits of the trehalose-6-phosphate synthase/phosphatase complex in Saccharomyces cerevisiae and their function during heat shock. Mol Microbiol 24:687–695
Ribeiro MJS, Reinders A, Boller T, Wiemken A, DeVirgilio C (1997) Trehalose synthesis is important for the acquisition of thermotolerance in Schizosaccharomyces pombe. Mol Microbiol 25:571–581
Serneels J, Tournu H, Van Dijck P (2012) Tight control of trehalose content is required for efficient heat-induced cell elongation in Candida albicans. J Biol Chem 287:36873–36882
Singer MA, Lindquist S (1998) Thermotolerance in Saccharomyces cerevisiae: the Yin and Yang of trehalose. Trends Biotechnol 16:460–468
Song XS, Li HP, Zhang JB, Song B, Huang T, Du XM, Gong AD, Liu YK, Feng YN, Agboola RS, Liao YC (2014) Trehalose 6-phosphate phosphatase is required for development, virulence and mycotoxin biosynthesis apart from trehalose biosynthesis in Fusarium graminearum. Fungal Genet Biol 63:24–41
Svanström A, Van Leeuwen MR, Dijksterhuis J, Melin P (2014) Trehalose synthesis in Aspergillus niger: characterization of six homologous genes, all with conserved orthologs in related species. BMC Microbiol 14:90
Thevelein JM (1984) Regulation of trehalose mobilization in fungi. Microbiol Rev 48:42–59
Tournu H, Fiori A, Van Dijck P (2013) Relevance of trehalose in pathogenicity: some general rules, yet many exceptions. PLoS Pathog 9:e1003447
Trevisol ETV, Panek AD, De Mesquita JF, Eleutherio ECA (2014) Regulation of the yeast trehalose-synthase complex by cyclic AMP-dependent phosphorylation. BBA-Gen Subj 1840:1646–1650
Vuorio OE, Kalkkinen N, Londesborough J (1993) Cloning of 2 related genes encoding the 56-kDa and 123-kDa subunits of trehalose synthase from the yeast Saccharomyces cerevisiae. Eur J Biochem 216:849–861
Wang ZL, Lu JD, Feng MG (2012) Primary roles of two dehydrogenases in the mannitol metabolism and multi-stress tolerance of entomopathogenic fungus Beauveria bassiana. Environ Microbiol 14:2139–2150
Wang JJ, Qiu L, Cai Q, Ying SH, Feng MG (2014) Three α-1, 2-mannosyltransferases contribute differentially to conidiation, cell wall integrity, multistress tolerance and virulence of Beauveria bassiana. Fungal Genet Biol 70:1–10
Wilson RA, Jenkinson JM, Gibson RP, Littlechild JA, Wang ZY, Talbot NJ (2007) Tps1 regulates the pentose phosphate pathway, nitrogen metabolism and fungal virulence. EMBO J 26:3673–3685
Wilson RA, Gibson RP, Quispe CF, Littlechild JA, Talbot NJ (2010) An NADPH-dependent genetic switch regulates plant infection by the rice blast fungus. Proc Natl Acad Sci U S A 107:21902–21907
Winkler K, Kienle I, Burgert M, Wagner JC, Holzer H (1991) Metabolic-regulation of the trehalose content of vegetative yeast. FEBS Lett 291:269–272
Wolschek MF, Kubicek CP (1997) The filamentous fungus Aspergillus niger contains two “differentially regulated” trehalose-6-phosphate synthase-encoding genes, tpsA and tpsB. J Biol Chem 272:2729–2735
Xiao GH, Ying SH, Zheng P, Wang ZL, Zhang SW, Xie XQ, Shang YF, St Leger RJ, Zhao GP, Wang CS, Feng MG (2012) Genomic perspectives on the evolution of fungal entomopathogenicity in Beauveria bassiana. Sci Rep 2:483
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:e30298
Zaragoza O, González-Párraga P, Pedreño Y, Alvarez-Peral FJ, Argüelles JC (2003) Trehalose accumulation induced during the oxidative stress response is independent of TPS1 mRNA levels in Candida albicans. Int Microbiol 6:121–125
Acknowledgements
We thank Jun-Ying Li (Analysis Center of Agrobiology and Environmental Sciences, Zhejiang University) for technical assistance with TEM. This work was financially supported by the National Natural Science Foundation of China (Grant nos. 31572054 and 31270537).
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Juan-Juan Wang and Qing Cai contribute equally to this study.
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Wang, JJ., Cai, Q., Qiu, L. et al. Additive roles of two TPS genes in trehalose synthesis, conidiation, multiple stress responses and host infection of a fungal insect pathogen. Appl Microbiol Biotechnol 101, 3637–3651 (2017). https://doi.org/10.1007/s00253-017-8155-2
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DOI: https://doi.org/10.1007/s00253-017-8155-2