Abstract
We investigated the cell functions of the Ca2+ signaling genes phospholipase C-1 (plc-1), Ca2+/H+ exchanger (cpe-1), and secretory phospholipase A2 (splA2) for stress responses and cellulose utilization in Neurospora crassa. The Δplc-1, Δcpe-1, and ΔsplA2 mutants displayed increased sensitivity to the alkaline pH and reduced survival during induced thermotolerance. The ΔsplA2 mutant also exhibited hypersensitivity to the DTT-induced endoplasmic reticulum (ER) stress, increased microcrystalline cellulose utilization, increased protein secretion, and glucose accumulation in the culture supernatants. Moreover, the ΔsplA2 mutant could not grow on microcrystalline cellulose during ER stress. Furthermore, plc-1, cpe-1, and splA2 synthetically regulate the acquisition of thermotolerance induced by heat shock, responses to alkaline pH and ER stress, and utilization of cellulose and other alternate carbon sources in N. crassa. In addition, expression of the alkaline pH regulator, pac-3, and heat shock proteins, hsp60, and hsp80 was reduced in the Δplc-1, Δcpe-1, and ΔsplA2 single and double mutants. The expression of the unfolded protein response (UPR) markers grp-78 and pdi-1 was also significantly reduced in the mutants showing growth defect during ER stress. The increased cellulolytic activities of the ΔsplA2 and Δcpe-1; ΔsplA2 mutants were due to increased cbh-1, cbh-2, and endo-2 expression in N. crassa. Therefore, plc-1, cpe-1, and splA2 are involved in stress responses and cellulose utilization in N. crassa.
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References
Andoh T, Yoko-O T, Matsui Y, Toh-E A (1995) Molecular cloning of the plc1+ gene of Schizosaccharomyces pombe, which encodes a putative phosphoinositide-specific phospholipase C. Yeast 11:179–185. https://doi.org/10.1002/yea.320110209
Andoh T, Kato T Jr, Matsui Y, Toh-e A (1998) Phosphoinositide-specific phospholipase C forms a complex with 14–3-3 proteins and is involved in expression of UV resistance in fission yeast. Mol Gen Genet 258:139–147. https://doi.org/10.1007/s004380050716
Arst HN Jr, Peñalva MA (2003) pH regulation in Aspergillus and parallels with higher eukaryotic regulatory systems. Trends Genet 19:224–231. https://doi.org/10.1016/S0168-9525(03)00052-0
Barman A (2017) Studies on calcium signaling genes phospholipase C-1, secretory phospholipase A2, and calcium proton exchanger-1 in Neurospora crassa. Doctoral dissertation
Barman A, Tamuli R (2015) Multiple cellular roles of Neurospora crassa plc-1, splA2, and cpe-1 in regulation of cytosolic free calcium, carotenoid accumulation, stress responses, and acquisition of thermotolerance. J Microbiol 53:226–235. https://doi.org/10.1007/s12275-015-4465-1
Barman A, Tamuli R (2017) The pleiotropic vegetative and sexual development phenotypes of Neurospora crassa arise from double mutants of the calcium signaling genes plc-1, splA2, and cpe-1. Curr Genet 63:861–875. https://doi.org/10.1007/s00294-017-0682-y
Berridge MJ (1987) Inositol trisphosphate and diacylglycerol: two interacting second messengers. Ann Rev Biochem 56:159–193. https://doi.org/10.1146/annurev.bi.56.070187.001111
Berridge MJ (1993) Inositol trisphosphate and calcium signaling. Nature 361:315–325. https://doi.org/10.1038/361315a0
Berridge MJ, Irvine RF (1984) Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature 312:315–321. https://doi.org/10.1038/312315a0
Berridge MJ, Bootman MD, Lipp P (1998) Calcium-a life and death signal. Nature 395:645–648. https://doi.org/10.1038/27094
Boilard E, Lai Y, Larabee K, Balestrieri B, Ghomashchi F, Fujioka D, Gobezie R, Coblyn JS, Weinblatt ME, Massarotti EM, Thornhill TS (2010) A novel anti-inflammatory role for secretory phospholipase A2 in immune complex-mediated arthritis. EMBO Mol Med 2:172–187. https://doi.org/10.1002/emmm.201000072
Borkovich KA, Alex LA, Yarden O et al (2004) Lessons from the genome sequence of Neurospora crassa: tracing the path from genomic blueprint to multicellular organism. Microbiol Mol Biol Rev 68:1–108. https://doi.org/10.1128/mmbr.68.1.1-108.2004
Bowman BJ, Abreu S, Margolles-Clark E, Draskovic M, Bowman EJ (2011) Role of four calcium transport proteins, encoded by nca-1, nca-2, nca-3, and cax, in maintaining intracellular calcium levels in Neurospora crassa. Eukaryot Cell 10:654–661. https://doi.org/10.1128/ec.00239-10
Bravo R, Gutierrez T, Paredes F, Gatica D, Rodriguez AE, Pedrozo Z, Chiong M, Parra V, Quest AF, Rothermel BA, Lavandero S (2012) Endoplasmic reticulum: ER stress regulates mitochondrial bioenergetics. Int J Biochem Cell Biol 44:16–20. https://doi.org/10.1016/j.biocel.2011.10.012
Cavazzini D, Meschi F, Corsini R, Bolchi A, Rossi GL, Einsle O, Ottonello S (2013) Autoproteolytic activation of a symbiosis-regulated truffle phospholipase A2. J Biol Chem 288:1533–1547. https://doi.org/10.1074/jbc.M112.384156
Cerella C, Diederich M, Ghibelli L (2010) The dual role of calcium as messenger and stressor in cell damage, death, and survival. Int J Cell Biol 2010:1–14. https://doi.org/10.1155/2010/546163
Chen Y, Fan X, Zhao X, Shen Y, Xu X, Wei L, Wang W, Wei D (2021) cAMP activates calcium signaling via phospholipase C to regulate cellulase production in the filamentous fungus Trichoderma reesei. Biotechnol Biofuels 14:1–13. https://doi.org/10.1186/s13068-021-01914-0
Chung HJ, Kim MJ, Lim JY, Park SM, Cha BJ, Kim YH, Yang MS, Kim DH (2006) A gene encoding phosphatidyl inositol-specific phospholipase C from Cryphonectria parasitica modulates the lac1 expression. Fungal Genet Biol 43:326–336. https://doi.org/10.1016/j.fgb.2005.12.009
Clapham DE (2007) Calcium signaling. Cell 131:1047–1058. https://doi.org/10.1016/j.cell.2007.11.028
Cornelius G, Gebauer G, Techel D (1989) Inositol trisphosphate induces calcium release from Neurospora crassa vacuoles. Biochem Biophys Res Commun 162:852–856. https://doi.org/10.1016/0006-291X(89)92388-7
Cunningham KW, Fink GR (1996) Calcineurin inhibits VCX1-dependent H+/Ca2+ exchange and induces Ca2+ ATPases in Saccharomyces cerevisiae. Mol Cell Biol 16:2226–2237. https://doi.org/10.1128/MCB.16.5.2226
Cupertino FB, Freitas FZ, de Paula RM, Bertolini MC (2012) Ambient pH controls glycogen levels by regulating glycogen synthase gene expression in Neurospora crassa. New insights into the pH signaling pathway. Plos one 7:e44258. https://doi.org/10.1371/journal.pone.0044258
Dennis EA, Cao J, Hsu YH, Magrioti V, Kokotos G (2011) Phospholipase A2 enzymes: physical structure, biological function, disease implication, chemical inhibition, and therapeutic intervention. Chem Rev 111:6130–6185. https://doi.org/10.1021/cr200085w
Fan F, Ma G, Li J, Liu Q, Benz JP, Tian C, Ma Y (2015) Genome-wide analysis of the endoplasmic reticulum stress response during lignocellulase production in Neurospora crassa. Biotechnol Biofuels 8:1–17. https://doi.org/10.1186/s13068-015-0248-5
Fankhauser H, Schweingruber AM, Edenharter E, Schweingruber ME (1995) Growth of a mutant defective in a putative phosphoinositide-specific phospholipase C of Schizosaccharomyces pombe is restored by low concentrations of phosphate and inositol. Curr Genet 28:199–203. https://doi.org/10.1007/BF00315789
Flick JS, Thorner J (1993) Genetic and biochemical characterization of a phosphatidylinositol-specific phospholipase C in Saccharomyces cerevisiae. Mol Cell Biol 13:5861–5876. https://doi.org/10.1128/mcb.13.9.5861-5876.1993
Galagan JE, Calvo SE, Borkovich KA, Selker EU, Read ND, Jaffe D, FitzHugh W, Ma LJ, Smirnov S, Purcell S, Rehman B (2003) The genome sequence of the filamentous fungus Neurospora crassa. Nature 422:859–868. https://doi.org/10.1038/nature01554
Gavric O, dos Santos DB, Griffiths A (2007) Mutation and divergence of the phospholipase C gene in Neurospora crassa. Fungal Genet Biol 44:242–249. https://doi.org/10.1016/j.fgb.2006.09.010
Hetz C (2012) The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nat Rev Mol Cell Biol 13:89–102. https://doi.org/10.1038/nrm3270
Hu Y, Wang J, Ying SH, Feng MG (2014) Five vacuolar Ca2+ exchangers play different roles in calcineurin-dependent Ca2+/Mn2+ tolerance, multistress responses, and virulence of a filamentous entomopathogen. Fungal Genet Biol 73:12–19. https://doi.org/10.1016/j.fgb.2014.09.005
Huang Y, Li Y, Li D, Bi Y, Prusky DB, Dong Y, Wang T, Zhang M, Zhang X, Liu Y (2020) Phospholipase C from Alternaria alternata is induced by physiochemical cues on the pear fruit surface that dictate infection structure differentiation and pathogenicity. Front Microbiol 11:1279. https://doi.org/10.3389/fmicb.2020.01279
Kapoor M, Lewis J (1987) Heat shock induces peroxidase activity in Neurospora crassa and confers tolerance toward oxidative stress. Biochem Biophys Res Commun 147:904–910. https://doi.org/10.1016/S0006-291X(87)80156-0
Kapoor M, Sreenivasan GM, Goel N, Lewis J (1990) Development of thermotolerance in Neurospora crassa by heat shock and other stresses eliciting peroxidase induction. J Bacteriol 172:2798–2801. https://doi.org/10.1128/jb.172.5.2798-2801.1990
Kapoor M, Curle CA, Runham C (1995) The hsp70 gene family of Neurospora crassa: cloning, sequence analysis, expression, and genetic mapping of the major stress-inducible member. J Bacteriol 177:212–221. https://doi.org/10.1128/jb.177.1.212-221.1995
Kmetzsch L, Staats CC, Simon E, Fonseca FL, de Oliveira DL, Sobrino L, Rodrigues J, Leal AL, Nimrichter L, Rodrigues ML, Schrank A (2010) The vacuolar Ca2+ exchanger Vcx1 is involved in calcineurin-dependent Ca2+ tolerance and virulence in Cryptococcus neoformans. Eukaryot Cell 9:1798–1805. https://doi.org/10.1128/ec.00114-10
Kmetzsch L, Staats CC, Cupertino JB, Fonseca FL, Rodrigues ML, Schrank A, Vainstein MH (2013) The calcium transporter Pmc1 provides Ca2+ tolerance and influences the progression of murine cryptococcal infection. FEBS J 280:4853–4864. https://doi.org/10.1111/febs.12458
Kumar A, Roy A, Deshmukh MV, Tamuli R (2020) Dominant mutants of the calcineurin catalytic subunit (CNA-1) showed developmental defects, increased sensitivity to stress conditions, and CNA-1 interacts with CaM and CRZ-1 in Neurospora crassa. Arch Microbiol 202:921–934. https://doi.org/10.1007/s00203-019-01768-z
Kunze D, Melzer I, Bennett D, Sanglard D, MacCallum D, Nörskau J, Coleman DC, Odds FC, Schäfer W, Hube B (2005) Functional analysis of the phospholipase C gene CaPLC1 and two unusual phospholipase C genes, CaPLC2 and CaPLC3, of Candida albicans. Microbiol 151:3381–3394. https://doi.org/10.1099/mic.0.28353-0
Lev S, Desmarini D, Li C, Chayakulkeeree M, Traven A, Sorrell TC, Djordjevic JT (2013) Phospholipase C of Cryptococcus neoformans regulates homeostasis and virulence by providing inositol trisphosphate as a substrate for Arg1 kinase. Infect Immun 81:1245–1255. https://doi.org/10.1128/iai.01421-12
Lew RR, Giblon RE, Lorenti MS (2015) The phenotype of a phospholipase C (plc-1) mutant in a filamentous fungus, Neurospora crassa. Fungal Genet Biol 82:158–167. https://doi.org/10.1016/j.fgb.2015.07.007
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. https://doi.org/10.1006/meth.2001.1262
Ma Y, Hendershot LM (2004) ER chaperone functions during normal and stress conditions. J Chem Neuroanat 28:51–65. https://doi.org/10.1016/j.jchemneu.2003.08.007
Maddi A, Fu C, Free SJ (2012) The Neurospora crassa dfg5 and dcw1 genes encode α-1, 6-mannanases that function in the incorporation of glycoproteins into the cell wall. PLoS one 7:e38872. https://doi.org/10.1371/journal.pone.0038872
Maeda T (2012) The signaling mechanism of ambient pH sensing and adaptation in yeast and fungi. FEBS J 279:1407–1413. https://doi.org/10.1111/j.1742-4658.2012.08548.x
McCluskey K, Wiest A, Plamann M (2010) The Fungal Genetics Stock Center: a repository for 50 years of fungal genetics research. J Biosci 35:119–126. https://doi.org/10.1007/s12038-010-0014-6
Miseta A, Kellermayer R, Aiello DP, Fu L, Bedwell DM (1999) The vacuolar Ca2+/H+ exchanger Vcx1p/Hum1p tightly controls cytosolic Ca2+ levels in S. cerevisiae. FEBS Lett 451:132–136. https://doi.org/10.1016/S0014-5793(99)00519-0
Murakami M, Kudo I (2004) Secretory phospholipase A2. Biol Pharma Bullet 27:1158–1164. https://doi.org/10.1248/bpb.27.1158
Nakahama T, Nakanishi Y, Viscomi AR, Takaya K, Kitamoto K, Ottonello S, Arioka M (2010) Distinct enzymatic and cellular characteristics of two secretory phospholipases A2 in the filamentous fungus Aspergillus oryzae. Fungal Genet Biol 47:318–331. https://doi.org/10.1016/j.fgb.2009.12.011
Nguyen QB, Kadotani N, Kasahara S, Tosa Y, Mayama S, Nakayashiki H (2008) Systematic functional analysis of calcium-signaling proteins in the genome of the rice-blast fungus, Magnaporthe oryzae, using a high-throughput RNA-silencing system. Mol Microbiol 68:1348–1365. https://doi.org/10.1111/j.1365-2958.2008.06242.x
Nishizuka Y (1984) The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature 308:693–698
Peñalva MA, Tilburn J, Bignell E, Arst HN Jr (2008) Ambient pH gene regulation in fungi: making connections. Trends Microbiol 16:291–300. https://doi.org/10.1016/j.tim.2008.03.006
Pittman JK, Cheng NH, Shigaki T, Kunta M, Hirschi KD (2004) Functional dependence on calcineurin by variants of the Saccharomyces cerevisiae vacuolar Ca2+/H+ exchanger Vcx1p. Mol Microbiol 54:1104–1116. https://doi.org/10.1111/j.1365-2958.2004.04332.x
Quandt K, Frech K, Karas H, Wingender E, Werner T (1995) Matlnd and Matlnspector: new fast and versatile tools for detection of consensus matches in nucleotide sequence data. Nucleic Acids Res 23:4878–4884. https://doi.org/10.1093/nar/23.23.4878
Rhee SG, Bae YS (1997) Regulation of phosphoinositide-specific phospholipase C isozymes. J Biol Chem 272:15045–15048. https://doi.org/10.1074/jbc.272.24.15045
Rho HS, Jeon J, Lee YH (2009) Phospholipase C-mediated calcium signaling is required for fungal development and pathogenicity in Magnaporthe oryzae. Mol Plant Pathol 10:337–346. https://doi.org/10.1111/j.1364-3703.2009.00536.x
Rizzuto R, Duchen MR, Pozzan T (2004) Flirting in little space: the ER/mitochondria Ca2+ liaison. Sci STKE. https://doi.org/10.1126/stke.2152004re1
Roy A, Tamuli R (2022) Heat shock proteins and the calcineurin-crz1 signaling regulate stress responses in fungi. Arch Microbiol 204:1–3. https://doi.org/10.1007/s00203-022-02833-w
Roy A, Kumar A, Baruah D, Tamuli R (2021) Calcium signaling is involved in diverse cellular processes in fungi. Mycol 12:10–24. https://doi.org/10.1080/21501203.2020.1785962
Schaloske RH, Dennis EA (2006) The phospholipase A2 superfamily and its group numbering system. Biochim Biophys Acta-Mol Cell Biol Lipids. 1761:1246–1259. https://doi.org/10.1016/j.bbalip.2006.07.011
Schumacher J, Viaud M, Simon A, Tudzynski B (2008) The Gα subunit BCG1, the phospholipase C (BcPLC1) and the calcineurin phosphatase co-ordinately regulate gene expression in the grey mould fungus Botrytis cinerea. Mol Microbiol 67:1027–1050. https://doi.org/10.1111/j.1365-2958.2008.06105.x
Selker EU, Garrett PW (1988) DNA sequence duplications trigger gene inactivation in Neurospora crassa. Pro Natl Acad Sci 85:6870–6874. https://doi.org/10.1073/pnas.85.18.6870
Serrano R, Ruiz A, Bernal D, Chambers JR, Ariño J (2002) The transcriptional response to alkaline pH in Saccharomyces cerevisiae: evidence for calcium-mediated signaling. Mol Microbiol 46:1319–1333. https://doi.org/10.1046/j.1365-2958.2002.03246.x
Soragni E, Bolchi A, Balestrini R, Gambaretto C, Percudani R, Bonfante P, Ottonello S (2001) A nutrient-regulated, dual localization phospholipase A2 in the symbiotic fungus Tuber borchii. EMBO J 20:5079–5090. https://doi.org/10.1093/emboj/20.18.5079
Sun J, Glass NL (2011) Identification of the CRE-1 cellulolytic regulon in Neurospora crassa. PloS one 6:e25654. https://doi.org/10.1371/journal.pone.0025654
Takayanagi A, Miyakawa T, Asano A, Ohtsuka J, Tanokura M, Arioka M (2015) Expression, purification, refolding, and enzymatic characterization of two secretory phospholipases A2 from Neurospora crassa. Protein Exp Purif 115:69–75. https://doi.org/10.1016/j.pep.2015.08.007
Tamuli R, Deka R, Borkovich KA (2016) Calcineurin subunits A and B interact to regulate growth and asexual and sexual development in Neurospora crassa. PloS one 11:e0151867. https://doi.org/10.1371/journal.pone.0151867
Tian C, Beeson WT, Iavarone AT, Sun J, Marletta MA, Cate JH, Glass NL (2009) Systems analysis of plant cell wall degradation by the model filamentous fungus Neurospora crassa. Proc Natl Acad Sci 106:22157–22162. https://doi.org/10.1073/pnas.0906810106
Tsai HC, Chung KR (2014) Calcineurin phosphatase and phospholipase C are required for developmental and pathological functions in the citrus fungal pathogen Alternaria alternata. Microbiol 160:1453–1465. https://doi.org/10.1099/mic.0.077818-0
Verghese J, Abrams J, Wang Y, Morano KA (2012) Biology of the heat shock response and protein chaperones: budding yeast (Saccharomyces cerevisiae) as a model system. Microbiol Mol Biol Rev 76:115–158. https://doi.org/10.1128/mmbr.05018-11
Viladevall L, Serrano R, Ruiz A, Domenech G, Giraldo J, Barceló A, Ariño J (2004) Characterization of the calcium-mediated response to alkaline stress in Saccharomyces cerevisiae. J Biol Chem 279:43614–43624. https://doi.org/10.1074/jbc.M403606200
Virgilio S, Cupertino FB, Bernardes NE, Freitas FZ, Takeda AA, Fontes MR, Bertolini MC (2016) Molecular components of the Neurospora crassa pH signaling pathway and their regulation by pH and the PAC-3 transcription factor. PloS one 11:e0161659. https://doi.org/10.1371/journal.pone.0161659
Virgilio S, Cupertino FB, Ambrosio DL, Bertolini MC (2017) Regulation of the reserve carbohydrate metabolism by alkaline pH and calcium in Neurospora crassa reveals a possible cross-regulation of both signaling pathways. BMC Genomics 18:1–5. https://doi.org/10.1186/s12864-017-3832-1
Vogel HJ (1964) Distribution of lysine pathways among fungi: evolutionary implications. Am Nat 98:435–446
Westergaard M, Mitchell HK (1947) Neurospora V.A synthetic medium favouring sexual reproduction. Am J Bot 34:573–577. https://doi.org/10.2307/2437339
Yang Q, Borkovich KA (1999) Mutational activation of a Gαi causes uncontrolled proliferation of aerial hyphae and increased sensitivity to heat and oxidative stress in Neurospora crassa. Genet 151:107–117. https://doi.org/10.1093/genetics/151.1.107
Zelter A, Bencina M, Bowman BJ, Yarden O, Read ND (2004) A comparative genomic analysis of the calcium signaling machinery in Neurospora crassa, Magnaporthe grisea, and Saccharomyces cerevisiae. Fungal Genet Biol 41:827–841. https://doi.org/10.1016/j.fgb.2004.05.001
Zhang X, Ren A, Li MJ, Cao PF, Chen TX, Zhang G, Shi L, Jiang AL, Zhao MW (2016) Heat stress modulates mycelium growth, heat shock protein expression, ganoderic acid biosynthesis, and hyphal branching of Ganoderma lucidum via cytosolic Ca2+. Appl Environ Microbiol 82:4112–4125. https://doi.org/10.1128/AEM.01036-16
Zhu Q, Zhou B, Gao Z, Liang Y (2015) Effects of phospholipase C on Fusarium graminearum growth and development. Curr Microbiol 71:632–637. https://doi.org/10.1007/s00284-015-0901-z
Zhu Q, Sun L, Lian J, Gao X, Zhao L, Ding M, Li J, Liang Y (2016) The phospholipase C (FgPLC1) is involved in regulation of development, pathogenicity, and stress responses in Fusarium graminearum. Fungal Genet Biol 97:1–9. https://doi.org/10.1016/j.fgb.2016.10.004
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We thank Kevin McCluskey and Aric Wiest at the Fungal Genetics Stock Center for generously waiving charges for strains. DB was supported by a Research Fellowship from the Ministry of Human Resource Development (MHRD), Government of India. We thank MHRD and IITG for partial financial support.
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We thank the MHRD, IIT Guwahati, and DBT-NER twinning grant BT/PR24473/NER/95/737/2017, Govt. of India for partial financial support.
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Baruah, D., Tamuli, R. The cell functions of phospholipase C-1, Ca2+/H+ exchanger-1, and secretory phospholipase A2 in tolerance to stress conditions and cellulose degradation in Neurospora crassa. Arch Microbiol 205, 327 (2023). https://doi.org/10.1007/s00203-023-03662-1
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DOI: https://doi.org/10.1007/s00203-023-03662-1