Lessons from the genome sequence of Neurospora crassa: tracing the path from genomic blueprint to multicellular organism

Abstract

We present an analysis of over 1,100 of the approximately 10,000 predicted proteins encoded by the genome sequence of the filamentous fungus Neurospora crassa. Seven major areas of Neurospora genomics and biology are covered. First, the basic features of the genome, including the automated assembly, gene calls, and global gene analyses are summarized. The second section covers components of the centromere and kinetochore complexes, chromatin assembly and modification, and transcription and translation initiation factors. The third area discusses genome defense mechanisms, including repeat induced point mutation, quelling and meiotic silencing, and DNA repair and recombination. In the fourth section, topics relevant to metabolism and transport include extracellular digestion; membrane transporters; aspects of carbon, sulfur, nitrogen, and lipid metabolism; the mitochondrion and energy metabolism; the proteasome; and protein glycosylation, secretion, and endocytosis. Environmental sensing is the focus of the fifth section with a treatment of two-component systems; GTP-binding proteins; mitogen-activated protein, p21-activated, and germinal center kinases; calcium signaling; protein phosphatases; photobiology; circadian rhythms; and heat shock and stress responses. The sixth area of analysis is growth and development; it encompasses cell wall synthesis, proteins important for hyphal polarity, cytoskeletal components, the cyclin/cyclin-dependent kinase machinery, macroconidiation, meiosis, and the sexual cycle. The seventh section covers topics relevant to animal and plant pathogenesis and human disease. The results demonstrate that a large proportion of Neurospora genes do not have homologues in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. The group of unshared genes includes potential new targets for antifungals as well as loci implicated in human and plant physiology and disease.

FIG. 2.

Life cycle of Neurospora. “Depending on surrounding conditions, the vegetative mycelium can undergo the asexual sporulation processes of macroconidiation and microconidiation. It can enter the sexual cycle by forming protoperithecia; which, upon fertilization, can initiate development leading to the production of meiotically-derived ascospores.” Reprinted from reference with permission from the publisher.

FIG. 3.

Transcription factor distribution. (Left) proportion of analyzed Neurospora transcription factors in each of the indicated classes. (Right) Venn diagram showing the distribution of Zn₂Cys₆, C2H2, BZIP, and bHLH transcription factors in bacteria, fungi, and animals.

FIG. 4.

Neurospora silencing pathways. The stages of the life cycle of Neurospora are presented, indicating points where the various gene silencing pathways are active. Quelling is a post-transcriptional gene silencing (PTGS) pathway that is active in the vegetative phase of the life cycle, from germination of ascospores or conidia to formation of the mycelium and differentiation of conidiophores and conidia. RIP and meiotic silencing are silencing pathways that are specific to the sexual cycle but differ in their molecular mechanisms. RIP scans for the presence of duplicated copies of DNA fragments present in the genomes destined to participate in meiosis. Duplicated regions are inundated with a series of transition mutations, and most of the remaining nonmutated cytosine bases are methylated. This process occurs in the heterokaryotic ascogeneous tissue formed following fertilization but prior to karyogamy. Meiotic silencing, like quelling, is a PTGS-like mechanism that is activated when a discrete region of DNA fails to sense (i.e., trans-sense) an equivalent region in the opposite chromosome. This failure of trans-sensing in turn triggers the silencing of all genes contained in the loop of unpaired DNA.

FIG. 5.

Relative numbers of Neurospora, S. cerevisiae, and S. pombe transporters in various families. Neurospora was compared to S. cerevisiae (605) and S. pombe (http://www.membranetransport.org) with respect to transporters in the major facilitator superfamily (MFS), ATP binding cassette (ABC) superfamily, amino acid/polyamine/choline (APC) superfamily, mitochondrial carrier (MC) family, P-type ATPase family, and other families.

FIG. 6.

Glycolysis and alcoholic fermentation. Neurospora contains genes encoding enzymes of the EM (blue arrows) and HM (red arrows) pathways of glycolysis. Enzymes required for hexose phosphorylation are indicated above the black arrows, while those involved in the fermentation pathway are indicated by green arrows. Abbreviations for enzyme names are presented in Table 29. NCU numbers for predicted Neurospora proteins corresponding to each enzyme are indicated alongside each arrow.

FIG. 7.

Pathway of sulfur acquisition leading to sulfur assimilation and cysteine biosynthesis in Neurospora. Potential sulfur sources from the environment or internal stores are indicated. The alkylsulfonate and cysteic acid conversions are predicted from the genome analysis. EC designations are shown; in some cases, putative multiple forms are indicated from the analysis (see Tables 32 and 35). Corresponding NCU numbers are as follows: EC 3.1.6.1 (arylsulfatase, NCU06041.1), EC 3.1.6.7 (choline sulfatase, NCU08364.1), EC 2.7.7.4 (ATP sulfurylase, NCU01985.1), EC 2.7.1.25 (adenylyl sulfate kinase, NCU0896.1), EC 1.8.99.4 (PAPS reductase, NCU02005.1), EC 4.1.1.15 (cysteic acid decarboxylase, NCU06112.1), EC 1.14.11.17 (taurine dioxygenase, NCU07610.1, NCU07819.1, NCU09738.1, NCU09800.1), EC 1.14.14.5 (alkanesulfonate monooxygenase, NCU05340.1, NCU10015.1), EC 1.8.1.2 (sulfite reductase, NCU04077.1, NCU05238.1), and EC 2.5.1.47 (cysteine synthase, NCU02564.1, NCU03788.1, NCU06452.1). Abbreviations: APS, adenosine-5′-phosphosulfate; PAPS, 3′-phosphoadenosine-5′-phosphosulfate.

FIG. 8.

Neurospora dolichol pathway. The mechanism for sequential glycosylation of proteins in the ER is shown. S. cerevisiae homologues are presented in parentheses. GlcNAc, N-acetylglucosamine. Modified with permission from Markus Aebi (personal communication, 2003)

FIG. 9.

Proteins of the secretory pathway. Neurospora predicted proteins homologous to those involved in the various steps of protein sorting to membranous organelles are shown. Abbreviations: EGA, early Golgi apparatus; LGA, late Golgi apparatus; PM, plasma membrane; V, vacuole; LE, late endosome, and EE, early endosome. NCU numbers for putative Rab proteins are indicated using boldface type.

FIG. 10.

Neurospora endocytosis proteins. The percentage of Neurospora proteins with the greatest homology to proteins in S. cerevisiae, S. pombe, animals, and plants is shown.

FIG. 11.

Domain organization of Neurospora HKs. Abbreviations: An, A. nidulans; Af, A. fumigatus; Ca, C. albicans; Sc, S. cerevisiae; Gc, Glomerella cingulata. Note that assignment of the total number of PAS/PAC domains in the relevant proteins is somewhat subjective, since it depends on the threshold values used during BLAST analyses.

FIG. 12.

Known and predicted heterotrimeric G-protein, Ras, cAMP, and PAK/MAPK signaling pathways in Neurospora. The number of images for each signaling protein class in the cartoon represents the number of Neurospora predicted gene products in each group. Arrows depict interactions that are supported by evidence from other systems but have not yet been demonstrated in Neurospora. The MAPK cascade(s) may also receive input from two-component signaling pathways (Fig. 11). Various Rho GTPase superfamily members (Fig. 18) may regulate certain signaling events downstream of Ras and upstream of PAK proteins. Abbreviations: GPCR, G-protein-coupled receptor; RGS, regulator of G-protein signaling; PAK, p21-activated kinase; GCK, germinal-center kinase; AC, adenylyl cyclase; CAP, cyclase-associated protein; PKA-R, regulatory subunit of cAMP-dependent protein kinase; PKA-C, catalytic subunit of cAMP-dependent protein kinase; MAPK, MAPKK, and MAPKKK, mitogen-activated protein kinase, kinase kinase, and kinase kinase kinase, respectively.

FIG. 13.

Calcium signaling proteins in Neurospora. The numbers of each gene in a particular class are in parentheses. An asterisk indicates that the location in the plasma membrane and/or intracellular calcium store membrane has not been determined. CPC, Ca²⁺-permeable channel; CT, Ca²⁺-transporter (Ca²⁺-ATPases, cation-ATPases, Ca²⁺/H⁺ exchangers, and Ca²⁺/Na⁺ exchangers); CaM, calmodulin; reg, regulated.

FIG. 14.

Calcium signaling proteins. The percentage of Neurospora proteins with the greatest homology to proteins in S. cerevisiae, S. pombe, animals, and plants is shown.

FIG. 15.

Known molecular components in the coupled feedback loops of the Neurospora circadian system. The WC-1 and WC-2 proteins form a White Collar Complex (WCC) that activates frq gene expression and also clock-controlled gene (ccg, output) and vvd expression in the dark. The WCC also mediates light-induced transcription from frq, ccg genes, vvd, and wc-1 (gold arrows). VVD expression is strongly light induced, and VVD in turn is a photoreceptor that mediates light adaptation responses, transiently turning down the WCC activity. In the circadian cycle in the dark, frq mRNA is translated to make FRQ proteins which dimerize and play two roles: (i) FRQ feeds back into the nucleus to rapidly block the activity of the WCC in driving frq transcription, and (ii) FRQ acts to promote the synthesis of new WC-1 and wc-2 mRNA, thus making more WCC, which is held inactive by FRQ. Phosphorylation of FRQ by several kinases, including casein kinases 1 and 2 and CAMK-1, triggers its turnover mediated by an interaction with the ubiquitin ligase encoded by fwd-1; the kinetics of phosphorylation-mediated turnover is a major determinant of period length in the clock. When FRQ is degraded in the proteasome, the pool of WCC is released to reinitiate the cycle. See the text for details. Adapted from reference

FIG. 16.

Real and potential Neurospora photoreceptors. The approximate sizes and locations of pertinent protein functional domains are shown for this series of proteins having known or plausible roles in photobiology. WC-1 and WC-2 work together as the White Collar Complex they are known to comprise a photoreceptor that appears to be the circadian photoreceptor and a major blue light photoreceptor in Neurospora. VVD is also a blue light photoreceptor that is responsible for modulating the WCC and contributing to photoadaptation. NOP-1 binds retinal and undergoes a photocycle, but the associated photobiology has not been elucidated (see also Fig. 12). Likewise, CRY, PHY-1, and PHY-2 all show strong sequence homology to known photoreceptors from other organisms (Table 49; Fig. 11) but do not yet have any demonstrated role in photobiology. aa, amino acids.

FIG. 17.

Metabolic pathways leading to the synthesis of cell wall precursors. The various pathways, including the glyoxylate shunt (isocitrate lyase and malate synthase), are shown. Abbreviations: glc-6-P; glucose-6-phosphate: glc-1-P; glucose-1-phosphate: UDP-gluc; uridine diphosphoglucose: fru-6-P; fructose-6-phosphase: glcN-6-P; glucosamine-6-phosphase; glcN-1-P; glucoseamine-1-phosphate: glcNAc-6-P; N-acetylglucosamine-6-phosphate: glcNAc-1-P; N-acetylglucosamine-1-phosphase; UDP-glcNAc; uridine-diphospho-N-acetylglusoamine: PG mutase; phosphoglucomutase: PG isomerase; phosphoglucoisomerase; PAG mutase, phospho-N-acetylglucosamine mutase.

FIG. 18.

Phylogenetic tree of Neurospora Rho proteins. Rho proteins were manually annotated and analyzed using Clustal W. The numbers adjacent to nodes indicate the percentages of 1,000 additional bootstrap trials in which the indicated protein groups were found.

FIG. 1.

Plate from the first published scientific study of Neurospora. Plate 1 from Payen (607). The following translated legend for the portions of the figure labeled a, a′, b′, c, g", i, and k′ is taken from reference . “a. Colonies of the red-orange fungus Oidium aurantiacum as they appear to the naked eye in the cavities of infected bread. a′. A similar colony cut in two, showing in the red area a thick layer composed of innumerable small spores formed at the end of radiating filaments. The latter are yellowish white. b. Similar colonies that have grown up completely in the dark, with the result that the red color has not developed. b′. One of the colonies in b seen after exposure to light for one hour. Color begins to appear and then pigmentation progresses rapidly. c. Branching filament, about 150×. g". Spore treated successively, under the microscope, with a dilute solution of potassium hydroxide, and aqueous alcoholic solution of iodine, then with gradually more concentrated solutions of sulfuric acids. This acid, which separates parts of the cellulose envelope that contains less nitrogenous substance, results in a blue color turning to purple, which is characteristic of the state intermediate between cellulose and dextrin. i. Normal vegetative growth as seen with the naked eye; well-developed, especially under conditions of high humidity. k′. Termini of well developed filaments, showing spores and young cells.”