Abstract
PacC is a pH-responsive transcription factor gene highly expressed at alkaline pH and plays distinct roles in environmental fitness, conidiation and virulence of different fungi. Here, we show biological functions of orthologous MaPacC in the locust-specific fungal pathogen Metarhizium acridum. Disruption of MapacC slowed down the fungal growth only under alkaline conditions. Intriguingly, the fungal thermotolerance was enhanced by the MapacC deletion, accompanied by transcriptional upregulation of some heat shock-responsive genes. The disruptant suffered a reduction in conidial yield and a change in conidial surface structure, but showed little change in cell wall integrity. The virulence of the disruptant against a locust species was markedly attenuated due to delayed appressorium formation, repressed expression of some insect cuticle hydrolases and slowed growth in locust hemolymph. The phenoloxidase activity and nodules of the locusts infected by the disruptant were also boosted. All of these phenotypic changes were restored by targeted gene complementation. Our results indicate that MaPacC acts a negative regulator of thermotolerance and contributes to the virulence of M. acridum by an involvement in hyphal penetration through insect cuticle and evasion from insect immunity.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Aréchiga-Carvajal ET, Ruiz-Herrera J (2005) The RIM101/pacC homologue from the basidiomycete Ustilago maydis is functional in multiple pH-sensitive phenomena. Eukaryot Cell 4:999–1008. https://doi.org/10.1128/EC.4.6.999-1008.2005
Arst HN Jr, Peñalva MA (2003) pH regulation in Aspergillus and parallels with higher eukaryotic regulatory systems. Trends Genet 19(4):224–231. https://doi.org/10.1016/S0168-9525(03)00052-0
Bansal R, Mukherjee M, Horwitz BA, Mukherjee PK (2019) Regulation of conidiation and antagonistic properties of the soil-borne plant beneficial fungus Trichoderma virens by a novel proline-, glycine-, tyrosine-rich protein and a GPI-anchored cell wall protein. Curr Genet 65(4):953–964. https://doi.org/10.1007/s00294-019-00948-0
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
Bignell E, Negrete-Urtasun S, Calcagno AM, Haynes K, Arst HN Jr, Rogers T (2005) The Aspergillus pH-responsive transcription factor pacC regulates virulence. Mol Microbiol 55(4):1072–1084. https://doi.org/10.1111/j.1365-2958.2004.04472.x
Braga GUL, Rangel DEN, Fernandes ÉKK, Flint SD, Roberts DW (2015) Molecular and physiological effects of environmental UV radiation on fungal conidia. Curr Genet 61(3):405–425. https://doi.org/10.1007/s00294-015-0483-0
Brune T, Kunze-Schumacher H, Kölling R (2019) Interactions in the ESCRT-III network of the yeast Saccharomyces cerevisiae. Curr Genet 65(2):607–619. https://doi.org/10.1007/s00294-018-0915-8
Caracuel Z, Roncero MI, Espeso EA, González-Verdejo CI, García-Maceira FI, Di Pietro A (2003) The pH response transcription factor pacC controls virulence in the plant pathogen Fusarium oxysporum. Mol Microbiol 48(3):765–779. https://doi.org/10.1046/j.1365-2958.2003.03465.x
Cerenius L, Söderhäll K (2004) The prophenoloxidase-activating system in invertebrates. Immunol Rev 198(1):116–126. https://doi.org/10.1111/j.0105-2896.2004.00116.x
Chen Y, Li BQ, Xu XD, Zhang ZQ, Tian SP (2018) The pH-responsive PacC transcription factor plays pivotal roles in virulence and patulin biosynthesis in Penicillium expansum. Environ Microbiol 20(11):4063–4078. https://doi.org/10.1111/1462-2920.14453
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
Cornet M, Gaillardin C (2014) pH signaling in human fungal pathogens: a new target for antifungal strategies. Eukaryot Cell 13(3):342–352. https://doi.org/10.1128/EC.00313-13
Davis D, Edwards JE, Mitchell AP, Ibrahim AS (2000) Candida albicans Rim101 pH response pathway is required for host-pathogen interactions. Infect Immun 68(10):5953–5959. https://doi.org/10.1128/iai.68.10.5953-5959.2000
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. https://doi.org/10.1007/s00253-018-9020-7
Espeso EA, Tilburn J, Sánchez-Pulido L, Brown CV, Valencia A, Arst HN Jr, Peñalva MA (1997) Specific DNA recognition by the Aspergillus nidulans three zinc finger transcription factor PacC. J Mol Biol 274(4):466–480. https://doi.org/10.1006/jmbi.1997.1428
Fang WG, Leng B, Xiao YH, Jin K, Ma JC, Fan YH, Feng J, Yang XY, Zhang YJ, Pei Y (2005) Cloning of Beauveria bassiana chitinase gene Bbchit1 and its application to improve fungal strain virulence. Appl Environ Microbiol 71(1):363–370. https://doi.org/10.1128/aem.71.1.363-370.2005
Fang WG, Pei Y, Bidochka MJ (2006) Transformation of Metarhizium anisopliae mediated by Agrobacterium tumefaciens. Can J Microbiol 52(7):623–626. https://doi.org/10.1139/w06-014
Fernandes ÉKK, Bittencourt VREP, Roberts DW (2012) Perspectives on the potential of entomopathogenic fungi in biological control of ticks. Exp Parasitol 130:300–305. https://doi.org/10.1016/j.exppara.2011.11.004
Fernandes ÉKK, Rangel DEN, Braga GUL, 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-Martínez J, Brown CV, Díez E, Tilburn J, Arst HN Jr, Peñalva MA, Espeso EA (2003) Overlap of nuclear localisation signal and specific DNA binding residues within the zinc finger domain of PacC. J Mol Biol 334:667–684. https://doi.org/10.1016/j.jmb.2003.09.072
Gao Q, Jin K, Ying SH, Zhang YJ, Xiao GH, Shang YF, Duan ZB, Hu X, 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
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
Gow NA, van de Veerdonk FL, Brown AJ, Netea MG (2012) Candida albicans morphogenesis and host defence: discriminating invasion from colonization. Nat Rev Microbiol 10:112–122. https://doi.org/10.1038/nrmicro2711
He M, Xia YX (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
Hervás-Aguilar A, Rodríguez JM, Tilburn J, Arst HN Jr, Peñalva MA (2007) Evidence for the direct involvement of the proteasome in the proteolytic processing of the Aspergillus nidulans zinc finger transcription factor PacC. J Biol Chem 282(48):34735–34747. https://doi.org/10.1074/jbc.M706723200
Holder DJ, Kirkland BH, Lewis MW, Keyhani NO (2007) Surface characteristics of the entomopathogenic fungus Beauveria (Cordyceps) bassiana. Microbiology 153(Pt 10):3448–3457. https://doi.org/10.1099/mic.0.2007/008524-0
Huang W, Shang YF, Chen PL, Gao Q, Wang CS (2015) MrpacC regulates sporulation, insect cuticle penetration and immune evasion in Metarhizium robertsii. Environ Microbiol 17(4):994–1008. https://doi.org/10.1111/1462-2920.12451
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(Pt 12):2987–2996. https://doi.org/10.1099/mic.0.059469-0
Landraud P, Chuzeville S, Billon-Grande G, Poussereau N, Bruel C (2013) Adaptation to pH and role of PacC in the rice blast fungus Magnaporthe oryzae. PLoS One 8:e69236. https://doi.org/10.1371/journal.pone.0069236
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25(4):402–408. https://doi.org/10.1006/meth.2001.1262
Lord JC (2001) Response of the wasp Cephalonomia tarsalis (Hymenoptera: Bethylidae) to Beauveria bassiana (Hyphomycetes: Moniliales) as free conidia or infection in its host, the sawtoothed grain beetle, Oryzaephilus surinamensis (Coleoptera: Silvanidae). Biol Control 21:300–304. https://doi.org/10.1006/bcon.2001.0942
Lord JC (2005) From Metchnikoff to Monsanto and beyond: the path of microbial control. J Invertebr Pathol 89:19–29. https://doi.org/10.1016/j.jip.2005.04.006
Loser K, Weltring K (1998) Induction of a polyubiquitin gene (ubi1) by potato phytoalexins and heat shock in Gibberella pulicaris. Curr Genet 34(5):404–409. https://doi.org/10.1007/s002940050414
Lovett B, St Leger RJ (2018) Genetically engineering better fungal biopesticides. Pest Manag Sci 74(4):781–789. https://doi.org/10.1002/ps.4734
Luo ZB, Ren H, Mousa JJ, Rangel DE, Zhang YJ, Bruner SD, Keyhani NO (2017) The PacC transcription factor regulates secondary metabolite production and stress response, but has only minor effects on virulence in the insect pathogenic fungus, Beauveria bassiana. Environ Microbiol 19(2):788–802. https://doi.org/10.1111/1462-2920.13648
Merhej J, Richard-Forget F, Barreau C (2011) The pH regulatory factor Pac1 regulates Tri gene expression and trichothecene production in Fusarium graminearum. Fungal Genet Biol 48(3):275–284. https://doi.org/10.1016/j.fgb.2010.11.008
Nielsen C, Skovgard H, Steenberg T (2005) Effect of Metarhizium anisopliae (Deuteromycotina: Hyphomycetes) on survival and reproduction of the filth fly parasitoid, Spalangia cameroni (Hymenoptera: Pteromalidae). Environ Entomol 34:133–139. https://doi.org/10.1603/0046-225X-34.1.133
Noventa-Jordão MA, Couto RM, Goldman MH, Aguirre J, Iyer S, Caplan A, Terenzi HF, Goldman GH (1999) Catalase activity is necessary for heat-shock recovery in Aspergillus nidulans germlings. Microbiology 145(Pt 11):3229–3234. https://doi.org/10.1099/00221287-145-11-3229
Ochiai M, Ashida M (2000) A pattern-recognition protein for β-1,3-glucan. The binding domain and the cDNA cloning of β-1,3-glucan recognition protein from the silkworm. Bombyx mori. J Biol Chem 275(7):4995–5002. https://doi.org/10.1074/jbc.275.7.4995
Parsell DA, Lindquist S (1993) The function of heat-shock proteins in stress tolerance: degradation and reactivation of damaged proteins. Annu Rev Genet 27(1):437–496. https://doi.org/10.1146/annurev.ge.27.120193.002253
Peñalva MA, Arst HN Jr (2002) Regulation of gene expression by ambient pH in filamentous fungi and yeast. Microbiol Mol Biol Rev 66:426–446. https://doi.org/10.1128/mmbr.66.3.426-446.2002
Peñalva MA, Tilburn J, Bignell E, Herbert N (2008) Ambient pH gene regulation in fungi: making connections. Trends Microbiol 16(6):291–300. https://doi.org/10.1016/j.tim.2008.03.006
Peñalva MA, Lucena-Agell D, Arst HN Jr (2014) Liaison alcaline: Pals entice non-endosomal ESCRTs to the plasma membrane for pH signaling. Curr Opin Microbiol 22(8):49–59. https://doi.org/10.1016/j.mib.2014.09.005
Peng GX, Wang ZK, Yin YP, 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
Rangel DEN, Braga GUL, Anderson AJ, Roberts DW (2005) Influence of growth environment on tolerance to UV-B radiation, germination speed, and morphology of Metarhizium anisopliae var. acridum conidia. J Invertebr Pathol 90:55–58. https://doi.org/10.1016/j.jip.2005.05.005
Rangel DEN, Braga GUL, Fernandes ÉKK, Keyser CA, Hallsworth JE, Roberts DW (2015) Stress tolerance and virulence of insectpathogenic fungi are determined by environmental conditions during conidial formation. Curr Genet 61:383–404. https://doi.org/10.1007/s00294-015-0477-y
Rollins JA (2003) The Sclerotinia sclerotiorum pac1 gene is required for sclerotial development and virulence. Mol Plant Microbes Interact 16(9):785–795. https://doi.org/10.1094/MPMI.2003.16.9.785
St Leger R, Joshi L, Bidochka MJ, Roberts DW (1996) Construction of an improved mycoinsecticide overexpressing a toxic protease. Proc Natl Acad Sci USA 93(13):6349–6354. https://doi.org/10.1073/pnas.93.13.6349
Tang QY, Zhang CX (2013) Data processing system (DPS) software with experimental design, statistical analysis and data mining developed for use in entomological research. Insect Sci 20(2):254–260. https://doi.org/10.1111/j.1744-7917.2012.01519.x
Tilburn J, Sarkar S, Widdick D, Espeso E, Arst H (1995) The Aspergillus PacC zinc finger transcription factor mediates regulation of both acid- and alkaline-expressed genes by ambient pH. EMBO J 14(4):779–790. https://doi.org/10.1002/j.1460-2075.1995.tb07056.x
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. https://doi.org/10.1007/s00253-018-9516-1
Virgilio S, Bertolini MC (2018) Functional diversity in the pH signaling pathway: an overview of the pathway regulation in Neurospora crassa. Curr Genet 64(3):529–534. https://doi.org/10.1007/s00294-017-0772-x
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 CS, St Leger RJ (2006) A collagenous protective coat enables Metarhizium anisopliae to evade insect immune responses. Proc Natl Acad Sci USA 103(17):6647–6652. https://doi.org/10.1073/pnas.0601951103
Wang CS, 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 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. https://doi.org/10.1007/s00253-014-5763-y
Wei QL, Du YR, Jin K, Xia YX (2017) The Ste12-like transcription factor MaSte12 is involved in pathogenicity by regulating the appressorium formation in the entomopathogenic fungus, Metarhizium acridum. Appl Microbiol Biotechnol 101(23–24):8571–8584. https://doi.org/10.1007/s00253-017-8569-x
Wu YX, Yin ZY, Xu LS, Feng H, Huang LL (2018) VmPacC is required for acidification and virulence in Valsa mali. Front Microbiol 9:1981. https://doi.org/10.3389/fmicb.2018.01981
You BJ, Choquer M, Chung KR (2007) The Colletotrichum acutatum gene encoding a putative pH-responsive transcription regulator is a key virulence determinant during fungal pathogenesis on citrus. Mol Plant Microbes Interact 20(9):1149–1160. https://doi.org/10.1094/MPMI-20-9-1149
Zhang LB, Feng MG (2018) Antioxidant enzymes and their contributions to biological control potential of fungal insect pathogens. Appl Microbiol Biotechnol 102:4995–5004. https://doi.org/10.1007/s00253-018-9033-2
Zhang QP, Peng GX, Wang ZK, Yin YP, Xia YX (2005) Detection of Metarhizium anisopliae rDNA in haemalymph of infected adult locust by real-time fluorescence quantitative PCR. Chin Prog Biotech 25(11):71–75. https://doi.org/10.1016/j.cyto.2004.11.006
Zhang SZ, Peng GX, Xia YX (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
Zhao TT, Tian HT, Xia YX, Jin K (2019) MaPmt4, a protein O-mannosyltransferase, contributes to cell wall integrity, stress tolerance and virulence in Metarhizium acridum. Curr Genet 65(4):1025–1040. https://doi.org/10.1007/s00294-019-00957-z
Zhu J, Ying SH, Feng MG (2016) The Pal pathway required for ambient pH adaptation regulates growth, conidiation, and osmotolerance of Beauveria bassiana in a pH dependent manner. Appl Microbiol Biotechnol 100:4423–4433. https://doi.org/10.1007/s00253-016-7282-5
Zou CG, Tu HH, Liu XY, Tao N, Zhang KQ (2010) PacC in the nematophagous fungus Clonostachys rosea controls virulence to nematodes. Environ Microbiol 12(7):1868–1877. https://doi.org/10.1099/mic.0.043653-0
Acknowledgements
This work was supported by the Natural Science Foundation of China (31471820), the Natural Science Foundation of Chongqing (cstc2015jcyja80037 and 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
Zhang, M., Wei, Q., Xia, Y. et al. MaPacC, a pH-responsive transcription factor, negatively regulates thermotolerance and contributes to conidiation and virulence in Metarhizium acridum. Curr Genet 66, 397–408 (2020). https://doi.org/10.1007/s00294-019-01032-3
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00294-019-01032-3