The Transcriptomic Signature of RacA Activation and Inactivation Provides New Insights into the Morphogenetic Network of Aspergillus niger

RacA is the main Rho GTPase in Aspergillus niger regulating polarity maintenance via controlling actin dynamics. Both deletion and dominant activation of RacA (RacG18V) provoke an actin localization defect and thereby loss of polarized tip extension, resulting in frequent dichotomous branching in the ΔracA strain and an apolar growing phenotype for RacG18V. In the current study the transcriptomics and physiological consequences of these morphological changes were investigated and compared with the data of the morphogenetic network model for the dichotomous branching mutant ramosa-1. This integrated approach revealed that polar tip growth is most likely orchestrated by the concerted activities of phospholipid signaling, sphingolipid signaling, TORC2 signaling, calcium signaling and CWI signaling pathways. The transcriptomic signatures and the reconstructed network model for all three morphology mutants (ΔracA, RacG18V, ramosa-1) imply that these pathways become integrated to bring about different physiological adaptations including changes in sterol, zinc and amino acid metabolism and changes in ion transport and protein trafficking. Finally, the fate of exocytotic (SncA) and endocytotic (AbpA, SlaB) markers in the dichotomous branching mutant ΔracA was followed, demonstrating that hyperbranching does not per se result in increased protein secretion.

[1]  J. A. Roubos,et al.  Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88 , 2007, Nature Biotechnology.

[2]  X. Ye,et al.  Induction of Apoptosis by Sphingoid Long-Chain Bases in Aspergillus nidulans , 2003, Molecular and Cellular Biology.

[3]  Katsuhiko Kitamoto,et al.  Systematic analysis of SNARE localization in the filamentous fungus Aspergillus oryzae. , 2007, Fungal genetics and biology : FG & B.

[4]  Y. Hannun,et al.  Molecular Systems Biology 6; Article number 349; doi:10.1038/msb.2010.3 Citation: Molecular Systems Biology 6:349 , 2022 .

[5]  L. Du,et al.  Distinct ceramide synthases regulate polarized growth in the filamentous fungus Aspergillus nidulans. , 2005, Molecular biology of the cell.

[6]  A. Nienow,et al.  Dependence of morphology on agitation intensity in fed-batch cultures of Aspergillus oryzae and its implications for recombinant protein production. , 2002, Biotechnology and bioengineering.

[7]  G. Robson,et al.  Effect of branch frequency in Aspergillus oryzae on protein secretion and culture viscosity. , 1999, Biotechnology and bioengineering.

[8]  Nobumichi Furuta,et al.  Endocytic recycling in yeast is regulated by putative phospholipid translocases and the Ypt31p/32p-Rcy1p pathway. , 2006, Molecular biology of the cell.

[9]  S. Morales,et al.  Analysis of cell-cycle specific localization of the Rdi1p RhoGDI and the structural determinants required for Cdc42p membrane localization and clustering at sites of polarized growth , 2004, Current Genetics.

[10]  S. Moukha,et al.  Localization of growth and secretion of proteins in Aspergillus niger. , 1991, Journal of general microbiology.

[11]  M. Kaksonen,et al.  Harnessing actin dynamics for clathrin-mediated endocytosis , 2006, Nature Reviews Molecular Cell Biology.

[12]  Y. Benjamini,et al.  Controlling the false discovery rate: a practical and powerful approach to multiple testing , 1995 .

[13]  O. Merkel,et al.  Regulation of activity in vitro and in vivo of three phospholipases B from Saccharomyces cerevisiae. , 2005, The Biochemical journal.

[14]  J. Nielsen,et al.  Metabolic Engineering of the Morphology of Aspergillus oryzae by Altering Chitin Synthesis , 2002, Applied and Environmental Microbiology.

[15]  J. Bennett,et al.  More gene manipulations in fungi , 1991 .

[16]  G. Robson,et al.  Glucoamylase::green fluorescent protein fusions to monitor protein secretion in Aspergillus niger. , 2000, Microbiology.

[17]  L. H. Grimm,et al.  Morphology and productivity of filamentous fungi , 2005, Applied Microbiology and Biotechnology.

[18]  P. Pouwels,et al.  Development of a homologous transformation system for Aspergillus niger based on the pyrG gene , 2004, Molecular and General Genetics MGG.

[19]  Alexander Lichius,et al.  Form follows function -- the versatile fungal cytoskeleton. , 2011, Fungal biology.

[20]  A. Fleissner,et al.  Expression and export: recombinant protein production systems for Aspergillus , 2010, Applied Microbiology and Biotechnology.

[21]  Johannes A Roubos,et al.  Effective lead selection for improved protein production in Aspergillus niger based on integrated genomics. , 2009, Fungal genetics and biology : FG & B.

[22]  R. P. Cox,et al.  On-line growth measurements in bioreactors by titrating metabolic proton exchange , 1994, Applied Microbiology and Biotechnology.

[23]  J. Kunz,et al.  The pleckstrin homology domain proteins Slm1 and Slm2 are required for actin cytoskeleton organization in yeast and bind phosphatidylinositol-4,5-bisphosphate and TORC2. , 2005, Molecular biology of the cell.

[24]  R. Duncan,et al.  Expression of the Cameleon calcium biosensor in fungi reveals distinct Ca(2+) signatures associated with polarized growth, development, and pathogenesis. , 2012, Fungal genetics and biology : FG & B.

[25]  Deborah A. Brown,et al.  Lipid-dependent Targeting of G Proteins into Rafts* , 2000, The Journal of Biological Chemistry.

[26]  J. Ariño,et al.  Copper and Iron Are the Limiting Factors for Growth of the Yeast Saccharomyces cerevisiae in an Alkaline Environment* , 2004, Journal of Biological Chemistry.

[27]  Vera Meyer,et al.  The use of open source bioinformatics tools to dissect transcriptomic data. , 2012, Methods in molecular biology.

[28]  J. Qin,et al.  The Yeast PH Domain Proteins Slm1 and Slm2 Are Targets of Sphingolipid Signaling during the Response to Heat Stress , 2006, Molecular and Cellular Biology.

[29]  C. G. Reynaga-Peña,et al.  Apical branching in a temperature sensitive mutant of Aspergillus niger. , 1997, Fungal genetics and biology : FG & B.

[30]  A. Ram,et al.  Genetics, Genetic Manipulation, and Approaches to Strain Improvement of Filamentous Fungi , 2010 .

[31]  K. Takegawa,et al.  Mannosylinositol phosphorylceramide is a major sphingolipid component and is required for proper localization of plasma-membrane proteins in Schizosaccharomyces pombe , 2010, Journal of Cell Science.

[32]  Vera Meyer,et al.  Aspergillus as a multi-purpose cell factory: current status and perspectives , 2010, Biotechnology Letters.

[33]  L. Harvey,et al.  Effect of oxygen enrichment on morphology, growth, and heterologous protein production in chemostat cultures of Aspergillus niger B1-D. , 1999, Biotechnology and bioengineering.

[34]  E. Bi,et al.  Regulation of Cell Polarity by Interactions of Msb3 and Msb4 with Cdc42 and Polarisome Components , 2005, Molecular and Cellular Biology.

[35]  S. Winder,et al.  The WASP homologue Las17 activates the novel actin-regulatory activity of Ysc84 to promote endocytosis in yeast. , 2009, Molecular biology of the cell.

[36]  C. Nombela,et al.  The sequential activation of the yeast HOG and SLT2 pathways is required for cell survival to cell wall stress. , 2007, Molecular biology of the cell.

[37]  Junya Hasegawa,et al.  SH3YL1 regulates dorsal ruffle formation by a novel phosphoinositide-binding domain , 2011, The Journal of cell biology.

[38]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[39]  Hua Wei,et al.  The important role of actinin-like protein (AcnA) in cytokinesis and apical dominance of hyphal cells in Aspergillus nidulans. , 2009, Microbiology.

[40]  E. Espeso,et al.  Preferential localization of the endocytic internalization machinery to hyphal tips underlies polarization of the actin cytoskeleton in Aspergillus nidulans , 2008, Molecular microbiology.

[41]  Gordon K Smyth,et al.  Linear Models and Empirical Bayes Methods for Assessing Differential Expression in Microarray Experiments , 2004, Statistical applications in genetics and molecular biology.

[42]  C. J. Bos,et al.  Genetic analysis and the construction of master strains for assignment of genes to six linkage groups in Aspergillus niger , 1988, Current Genetics.

[43]  A. Rodal,et al.  Structural and functional dissection of the Abp 1 ADFH actin-binding domain reveals versatile in vivo adapter functions , 2005 .

[44]  T. Pakula,et al.  The cargo and the transport system: secreted proteins and protein secretion in Trichoderma reesei (Hypocrea jecorina). , 2012, Microbiology.

[45]  R. Fisher 019: On the Interpretation of x2 from Contingency Tables, and the Calculation of P. , 1922 .

[46]  Vera Meyer,et al.  Expanding the ku70 toolbox for filamentous fungi: establishment of complementation vectors and recipient strains for advanced gene analyses , 2010, Applied Microbiology and Biotechnology.

[47]  M. Balasubramanian,et al.  Yeast lipid rafts?--an emerging view. , 2006, Trends in cell biology.

[48]  G. Carman,et al.  Regulation of phospholipid synthesis in Saccharomyces cerevisiae by zinc depletion. , 2007, Biochimica et biophysica acta.

[49]  T. Höfken,et al.  The Rho GDI Rdi1 regulates Rho GTPases by distinct mechanisms. , 2008, Molecular biology of the cell.

[50]  K. Mikoshiba,et al.  Role of synaptotagmin, a Ca2+ and inositol polyphosphate binding protein, in neurotransmitter release and neurite outgrowth. , 1999, Chemistry and physics of lipids.

[51]  Alexander Lichius,et al.  Actin organization and dynamics in filamentous fungi , 2011, Nature Reviews Microbiology.

[52]  Brenda J. Andrews,et al.  Dissecting BAR Domain Function in the Yeast Amphiphysins Rvs161 and Rvs167 during Endocytosis , 2010, Molecular biology of the cell.

[53]  Richard H. Scheller,et al.  SNARE-mediated membrane fusion , 2001, Nature Reviews Molecular Cell Biology.

[54]  B. Oakley,et al.  The Functions of Myosin II and Myosin V Homologs in Tip Growth and Septation in Aspergillus nidulans , 2012, PloS one.

[55]  N. Zhang,et al.  Nonapoptotic Death of Saccharomyces cerevisiae Cells That Is Stimulated by Hsp90 and Inhibited by Calcineurin and Cmk2 in Response to Endoplasmic Reticulum Stresses , 2008, Eukaryotic Cell.

[56]  Y. Ho,et al.  Characterization of the yeast amphiphysins Rvs161p and Rvs167p reveals roles for the Rvs heterodimer in vivo. , 2005, Molecular biology of the cell.

[57]  M. Penttilä,et al.  Spatially Segregated SNARE Protein Interactions in Living Fungal Cells* , 2007, Journal of Biological Chemistry.

[58]  M. Akeroyd,et al.  The carbon starvation response of Aspergillus niger during submerged cultivation: Insights from the transcriptome and secretome , 2012, BMC Genomics.

[59]  C. Yu,et al.  Molecular level interaction of inositol hexaphosphate with the C2B domain of human synaptotagmin I. , 2012, Biochemistry.

[60]  de Winde,et al.  University of Groningen Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88 Pel, , 2006 .

[61]  Arnold L. Demain,et al.  Manual of Industrial Microbiology and Biotechnology , 1986 .

[62]  J. Hamer,et al.  Identification and characterization of Aspergillus nidulans mutants defective in cytokinesis. , 1994, Genetics.

[63]  Hans Ulrich Bergmeyer,et al.  Methods of Enzymatic Analysis , 2019 .

[64]  A. Ram,et al.  Survival in the Presence of Antifungals , 2007, Journal of Biological Chemistry.

[65]  Francesc Posas,et al.  Yeast HOG1 MAP Kinase Cascade Is Regulated by a Multistep Phosphorelay Mechanism in the SLN1–YPD1–SSK1 “Two-Component” Osmosensor , 1996, Cell.

[66]  Manabu Ishitani,et al.  Regulation of Osmotic Stress-responsive Gene Expression by theLOS6/ABA1 Locus inArabidopsis * , 2002, The Journal of Biological Chemistry.

[67]  C. G. Reynaga-Peña,et al.  Reconstruction of Signaling Networks Regulating Fungal Morphogenesis by Transcriptomics , 2009, Eukaryotic Cell.

[68]  M. Del Poeta,et al.  Lipid signalling in pathogenic fungi , 2011, Cellular microbiology.

[69]  G. Cerqueira,et al.  New resources for functional analysis of omics data for the genus Aspergillus , 2011, BMC Genomics.

[70]  K. Kitamoto,et al.  Septum‐directed secretion in the filamentous fungus Aspergillus oryzae , 2011, Molecular microbiology.

[71]  Jijun Cheng,et al.  Cell Cycle Progression and Cell Polarity Require Sphingolipid Biosynthesis in Aspergillus nidulans , 2001, Molecular and Cellular Biology.

[72]  E. Espeso,et al.  The tip growth apparatus of Aspergillus nidulans. , 2008, Molecular biology of the cell.

[73]  E. Record,et al.  Branching mutants of Aspergillus oryzae with improved amylase and protease production on solid substrates , 2005, Applied Microbiology and Biotechnology.

[74]  Vera Meyer,et al.  Genetic engineering of filamentous fungi--progress, obstacles and future trends. , 2008, Biotechnology advances.

[75]  K. Kuchler,et al.  Yeast ATP-binding cassette transporters: cellular cleaning pumps. , 2005, Methods in enzymology.

[76]  L. Lanzetti,et al.  Actin in membrane trafficking. , 2007, Current opinion in cell biology.

[77]  Maria Papagianni,et al.  Fungal morphology and metabolite production in submerged mycelial processes. , 2004, Biotechnology advances.

[78]  C. G. Reynaga-Peña,et al.  Cytoplasmic contractions in growing fungal hyphae and their morphogenetic consequences , 2005, Archives of Microbiology.

[79]  S. Harris,et al.  Functional Characterization of Aspergillus nidulans Homologues of Saccharomyces cerevisiae Spa2 and Bud6 , 2006, Eukaryotic Cell.

[80]  S. Henry,et al.  Cell Wall Integrity MAPK Pathway Is Essential for Lipid Homeostasis* , 2008, Journal of Biological Chemistry.

[81]  J. Cooper,et al.  Interactions with PIP2, ADP-actin monomers, and capping protein regulate the activity and localization of yeast twinfilin , 2001, The Journal of cell biology.

[82]  Vera Meyer,et al.  Highly efficient gene targeting in the Aspergillus niger kusA mutant. , 2007, Journal of biotechnology.

[83]  A. Griffiths,et al.  A mutation in the Neurospora crassa actin gene results in multiple defects in tip growth and branching. , 2004, Fungal genetics and biology : FG & B.

[84]  D. Eide,et al.  Combinatorial Control of Yeast FET4 Gene Expression by Iron, Zinc, and Oxygen* , 2002, The Journal of Biological Chemistry.

[85]  Benjamin M. Nitsche,et al.  Transcriptomic Insights into the Physiology of Aspergillus niger Approaching a Specific Growth Rate of Zero , 2010, Applied and Environmental Microbiology.

[86]  Jared L. Johnson,et al.  New Insights into How the Rho Guanine Nucleotide Dissociation Inhibitor Regulates the Interaction of Cdc42 with Membranes* , 2009, The Journal of Biological Chemistry.

[87]  A. Kihara,et al.  The Sjögren-Larsson syndrome gene encodes a hexadecenal dehydrogenase of the sphingosine 1-phosphate degradation pathway. , 2012, Molecular cell.

[88]  J. Nielsen,et al.  Metabolic engineering of the morphology of Aspergillus. , 2001, Advances in biochemical engineering/biotechnology.

[89]  J. W. Bennett,et al.  Appendix B – Growth Media , 1991 .

[90]  R. Fisher On the Interpretation of χ2 from Contingency Tables, and the Calculation of P , 2010 .

[91]  A. Ram,et al.  The polarisome component SpaA localises to hyphal tips of Aspergillus niger and is important for polar growth. , 2008, Fungal genetics and biology : FG & B.

[92]  A. Ram,et al.  Functional characterization of Rho GTPases in Aspergillus niger uncovers conserved and diverged roles of Rho proteins within filamentous fungi , 2011, Molecular microbiology.