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Sustainable biofuel production from renewable biomass will require the efficient and complete use of all abundant sugars in the plant cell wall. Using the cellulolytic fungus Neurospora crassa as a model, we identified a xylodextrin transport and consumption pathway required for its growth on hemicellulose. Reconstitution of this xylodextrin utilization pathway in Saccharomyces cerevisiae revealed that fungal xylose reductases act as xylodextrin reductases, producing xylosyl-xylitol oligomers as metabolic intermediates. These xylosyl-xylitol intermediates are generated by diverse fungi and bacteria, indicating that xylodextrin reduction is widespread in nature. Xylodextrins and xylosyl-xylitol oligomers are then hydrolyzed by two hydrolases to generate intracellular xylose and xylitol. Xylodextrin consumption using a xylodextrin transporter, xylodextrin reductases and tandem intracellular hydrolases in cofermentations with sucrose and glucose greatly expands the capacity of yeast to use plant cell wall-derived sugars and has the potential to increase the efficiency of both first-generation and next-generation biofuel production.

• RHO-3 is essential for normal development of N. crassa. • RHO-3 displays a dynamic ring-shaped structure surrounding the inner core of the Spk. • The Δ rho-3 strain is unable to produce ascospores. • The Δ rho-3 strain showed a disaggregated and unstable Spk. The Rho family of monomeric GTPases act as signaling proteins to establish and maintain cell polarity and other essential cellular processes. Rho3 is a GTPase of the Rho family that is exclusive of fungi that regulate cell polarity in yeast. However, studies have yet to explore its function in filamentous fungi. In this work, we investigated the role of RHO-3 in the model organism Neurospora crassa. Confocal microscopy analysis revealed that RHO-3 localizes in the outer region of the Spitzenkörper (Spk), in the plasma membrane from region II to the beginning of region III, and in the septa of mature hyphae. The phenotypic effect of the rho-3 deletion was analyzed. The results revealed that the rho-3 null strain showed severe defects in growth rate, aerial hyphae length, and conidia production. The organization of the Spk is also affected in the absence of RHO-3. Co-expression analysis of GFP-RHO-3 with glucan synthase 1 (GS-1-mChFP) and chitin synthase 1 (CHS-1-mChFP) revealed that RHO-3 localizes in the external region of the Spk in the macrovesicles zone. In summary, our results suggest that RHO-3 is not essential for the polarized growth of hyphae but plays a significant role in hyphal extension rate, conidiation, sexual reproduction and the integrity of the Spk, possibly regulating the delivery of macrovesicles to the apical dome. [ABSTRACT FROM AUTHOR]

Circadian clocks are composed of transcription-translation negative feedback loops that pace rhythms of gene expression to the diurnal cycle. In the filamentous fungus Neurospora crassa, the proteins F requency ( F RQ), the F RQ-interacting RNA helicase (FRH), and C asein-Kinase I (CK1) form the FFC complex that represses expression of genes activated by the white-collar complex (WCC). FRQ orchestrates key molecular interactions of the clock despite containing little predicted tertiary structure. Spin labeling and pulse-dipolar electron spin resonance spectroscopy provide domain-specific structural insights into the 989-residue intrinsically disordered FRQ and the FFC. FRQ contains a compact core that associates and organizes FRH and CK1 to coordinate their roles in WCC repression. FRQ phosphorylation increases conformational flexibility and alters oligomeric state, but the changes in structure and dynamics are non-uniform. Full-length FRQ undergoes liquid-liquid phase separation (LLPS) to sequester FRH and CK1 and influence CK1 enzymatic activity. Although FRQ phosphorylation favors LLPS, LLPS feeds back to reduce FRQ phosphorylation by CK1 at higher temperatures. Live imaging of Neurospora hyphae reveals FRQ foci characteristic of condensates near the nuclear periphery. Analogous clock repressor proteins in higher organisms share little position-specific sequence identity with FRQ; yet, they contain amino acid compositions that promote LLPS. Hence, condensate formation may be a conserved feature of eukaryotic clocks., Competing Interests: DT, NM, BB, SC, JD, AB, BC No competing interests declared, (© 2023, Tariq, Maurici et al.)

• The bud-1 gene is essential for normal development of N. crassa. • The Δ bud-1 strain presents slow growth rate, delayed germination, scarce aerial hyphae, decreased conidiation and hyphae display a zigzag trajectory. • The Δ bud-1 strain showed a disaggregated and unstable Spk. In filamentous fungi, the hypha orientation is essential for polarized growth and morphogenesis. The ability to re-orient tip growth in response to environmental cues is critical for the colony survival. Therefore, hyphal tip orientation and tip extension are distinct mechanisms that operate in parallel during filamentous growth. In yeast, the axial growth orientation requires a pathway regulated by Rsr1p/Bud1p, a Ras-like GTPase protein, which determines the axial budding pattern. However, in filamentous fungi the function of the Rsr1/Bud1p gene (krev-1 homolog) has not been completely characterized. In this work, we characterized the phenotype of a homokaryon mutant Bud1p orthologous in Neurospora crassa (△bud-1) and tagged BUD-1 with the green fluorescent protein (GFP) to determine its localization and cell dynamics under confocal microscopy. During spore germination BUD-1 was localized at specific points along the plasma membrane and during germ tube emergence it was located at the tip of the germ tubes. In mature hyphae BUD-1 continued to be located at the cell tip and was also present at sites of branch emergence and at the time of septum formation. The △bud-1 mutant showed a delayed germination, and the orientation of hyphae was somewhat disrupted. Also, the hypha diameter was reduced approximately 37 % with respect to the wild type. The lack of BUD-1 affected the Spitzenkörper (Spk) formation, trajectory, the localization of polarisome components BNI-1 and SPA-2, and the actin cytoskeleton polarization. The results presented here suggest that BUD-1 participates in the establishment of a new polarity axis. It may also mediate the delivery of secretory vesicles for the efficient construction of new plasma membrane and cell wall. [ABSTRACT FROM AUTHOR]

Circadian clocks are evolved to adapt to the daily environmental changes under different conditions. The ability to maintain circadian clock functions in response to various stresses and perturbations is important for organismal fitness. Here, we show that the nutrient-sensing GCN2 signaling pathway is required for robust circadian clock function under amino acid starvation in Neurospora . The deletion of GCN2 pathway components disrupts rhythmic transcription of clock gene frq by suppressing WC complex binding at the frq promoter due to its reduced histone H3 acetylation levels. Under amino acid starvation, the activation of GCN2 kinase and its downstream transcription factor CPC-1 establish a proper chromatin state at the frq promoter by recruiting the histone acetyltransferase GCN-5. The arrhythmic phenotype of the GCN2 kinase mutants under amino acid starvation can be rescued by inhibiting histone deacetylation. Finally, genome-wide transcriptional analysis indicates that the GCN2 signaling pathway maintains robust rhythmic expression of metabolic genes under amino acid starvation. Together, these results uncover an essential role of the GCN2 signaling pathway in maintaining the robust circadian clock function in response to amino acid starvation, and demonstrate the importance of histone acetylation at the frq locus in rhythmic gene expression., Competing Interests: XL, YY, YH, JW, CH, QL, XG, SQ, JG, QH, YL, XL No competing interests declared, (© 2023, Liu, Yang et al.)

The circadian clock governs rhythmic cellular functions by driving the expression of a substantial fraction of the genome and thereby significantly contributes to the adaptation to changing environmental conditions. Using the circadian model organism Neurospora crassa, we show that molecular timekeeping is robust even under severe limitation of carbon sources, however, stoichiometry, phosphorylation and subcellular distribution of the key clock components display drastic alterations. Protein kinase A, protein phosphatase 2 A and glycogen synthase kinase are involved in the molecular reorganization of the clock. RNA-seq analysis reveals that the transcriptomic response of metabolism to starvation is highly dependent on the positive clock component WC-1. Moreover, our molecular and phenotypic data indicate that a functional clock facilitates recovery from starvation. We suggest that the molecular clock is a flexible network that allows the organism to maintain rhythmic physiology and preserve fitness even under long-term nutritional stress., Competing Interests: AS, OS, GS, OK, AD, MB, NG, KK No competing interests declared, (© 2023, Szőke et al.)

Abstract: Transcriptional analysis using qRT-PCR of 62 metal ion transporters during conidial germination of Neurospora crassa showed a significant up regulation of a hypothetical copper transporter gene, tcu-1, that belongs to the Ctr family. Herein we characterised the Ctr family genes (tcu-1, tcu-2 and tcu-3) and deciphered their role in various developmental phases of the N. crassa life cycle. Cross complementation assays in copper uptake mutant of Saccharomyces cerevisiae revealed that tcu-1, tcu-2 and tcu-3 are functional homologs of S. cerevisiae copper transporters. Expression studies of Ctr family members in various developmental phases of N. crassa showed differential expression pattern for high-affinity copper transporter, TCU1. Functional analysis of their gene knockout mutants showed that tcu-1 is essential for saprophytic conidial germination, vegetative growth and perithecia development under copper limited conditions while conidiation remained unaffected. [Copyright &y& Elsevier]

Abstract: FK506 binding proteins (FKBPs) belong to the family of peptidyl prolyl cis-trans isomerases (PPIases) catalyzing the cis/trans isomerisation of Xaa-Pro bonds in oligopeptides and proteins. FKBPs are involved in folding, assembly and trafficking of proteins. However, only limited knowledge is available about the roles of FKBPs in the endoplasmic reticulum (ER) and their interaction with other proteins. Here we show the ER located Neurospora crassa FKBP22 to be a dimeric protein with PPIase and a novel chaperone activity. While the homodimerization of FKBP22 is mediated by its carboxy-terminal domain, the amino-terminal domain is a functional FKBP domain. The chaperone activity is mediated by the FKBP domain but is exhibited only by the full-length protein. We further demonstrate a direct interaction between FKBP22 and BiP, the major Hsp70 chaperone in the ER. The binding to BiP is mediated by the FKBP domain of FKBP22. Interestingly BiP enhances the chaperone activity of FKBP22. Both proteins form a stable complex with an unfolded substrate protein and thereby prevent its aggregation. These results suggest that BiP and FKBP22 form a folding helper complex with a high chaperoning capacity in the ER of Neurospora crassa. [Copyright &y& Elsevier]

Cyclophilins belong to the family of peptidyl-prolyl cis/trans isomerases (PPIases), which are ubiquitous and highly conserved enzymes capable of cis/trans isomerizing Xaa-Pro peptide bonds. Members of the CyP40-type cyclophilins have originally been described as components of hormone receptor complexes. Here, we describe NcCyP41, a CyP40 ortholog from Neurospora crassa, its expression in Escherichia coli and subsequent purification. Characterization of NcCyP41 reveals that it is a heat shock protein, which is active as a cyclosporin A-sensitive PPIase. Affinity chromatography using immobilized recombinant NcCyP41 yielded two major NcCyP41-binding proteins: Hsp80 (a Hsp90 ortholog from N. crassa) and CyPBP37. CyPBP37 has not been described. In addition, this is the first record describing an interaction between a member of Cyp40-type cyclophilins and of CyPBP37-type proteins, respectively. CyPBP37 expression is repressed by thiamine and in the stationary phase in N. crassa. CyPBP37 is present in different isoforms. The expression of a CyPBP37 ortholog in yeast, Thi4p, is diminished in a mutant lacking one of the two CyP40 orthologs (Cpr7p). In addition, the ΔCpr7p deletion mutant shows a thiamine-dependent growth defect. We conclude that, in yeast, Cpr7p and Thi4p interact functionally. [Copyright &y& Elsevier]

Establishing and maintaining appropriate gene repression is critical for the health and development of multicellular organisms. Histone H3 lysine 27 (H3K27) methylation is a chromatin modification associated with repressed facultative heterochromatin, but the mechanism of this repression remains unclear. We used a forward genetic approach to identify genes involved in transcriptional silencing of H3K27-methylated chromatin in the filamentous fungus Neurospora crassa . We found that the N. crassa homologs of ISWI (NCU03875) and ACF1 (NCU00164) are required for repression of a subset of H3K27-methylated genes and that they form an ACF chromatin-remodeling complex. This ACF complex interacts with chromatin throughout the genome, yet association with facultative heterochromatin is specifically promoted by the H3K27 methyltransferase, SET-7. H3K27-methylated genes that are upregulated when iswi or acf1 are deleted show a downstream shift of the +1 nucleosome, suggesting that proper nucleosome positioning is critical for repression of facultative heterochromatin. Our findings support a direct role of the ACF complex in Polycomb repression., Competing Interests: EW, CM, KM, ES No competing interests declared, HT Kevin J. McNaught is affiliated with Genapsys, Inc The author has no financial interests to declare, (© 2022, Wiles et al.)

A cobalt-resistant strain of Neurospora crassa (cor) is 20-fold more resistant to Co when compared with the wild type. DEAE-cellulose and metal-chelate affinity chromatography of cell-free extracts separated cobalt into protein-bound and free ionic fractions. In N. crassa cor about 80% cobalt of cell-free extracts was protein bound while the same in wild type was only 25%. Cobalt content of the protein-bound fraction increased with time and cobalt concentration in the growth medium, and was not influenced by related metal ions. A cobaltoprotein (CBP) which is overproduced in N. crassa cor and constitutes up to 12% of total protein of extracts was purified. CPB is a brown coloured (absorption peaks at 275, 350 and 440nm), small molecular weight glycoprotein (Mr 8200 daltons) with 28 - 30% carbohydrate (mannose). CBP has 70 μg cobalt mg-1 protein. Cysteine, glycine, glutamic acid and aspartic acid are the major amino acid constituents. The role of CBP in cobalt resistance is discussed in relation to other known metalloproteins involved in resistance. © Rapid Science 1998. [ABSTRACT FROM AUTHOR]

A cobalt-resistant strain of Neurospora crassa (cor) was obtained by repeated subculturing of the wild type on cobalt-containing agar medium. N. crassa cor is twentyfold more resistant to cobalt ions compared with the wild type. Resistance was stable on repeated subculturing of cor on cobalt-free media. N. crassa cor is also cross-resistant to nickel (fourfold), but not to zinc or copper. Higher concentrations of iron and magnesium ions are required to reverse growth inhibition due to cobalt toxicity in N. crassa cor, compared with the wild type. Germinating conidia and mycelia of the cor strain accumulated lower levels of cobalt ions compared with the parent N. crassa. The partial transport block for cobalt uptake is shown to be primarily due to decreased surface binding of cobalt to mycelia and cell walls. Efflux of mycelial cobalt was also observed in wild type and cobalt-resistant N. crassa. The characteristics of cor in comparison with wild type N. crassa are discussed in relation to the mechanisms of cobalt resistance. [ABSTRACT FROM AUTHOR]

A Neurospora crassa library, constructed in a derivative of the plasmid pBR322 (pRK9), was used to transform two E. coli ch1D molybdenum cofactor mutants ( ch1D, ch1D::Mu). Subsequently, one transformant from each of three independent transformation experiments was restriction mapped. All three transformants had an identical N. crassa DNA insert (4.2 kb). Southern Blot analysis with one of the plasmids (pMoCo, 1:4) showed hybridization to a single band of N. crassa genomic DNA. When pMoCo plasmid (1:4) was used to transform various E. coli nitrate reductase mutants ( ch1A, ch1B, ch1C, ch1D, ch1E, ch1G and ch1M), the pMoCo plasmid was capable of restoring E. coli nitrate reductase activity to only the ch1D mutant. In vitro reconstitution experiments using wild-type, ch1D and ch1D; pMoCo cell-free extracts as a source of molybdenum cofactor (MoCo) were performed with the N. crassa MoCo mutants nit-1, nit-7 and nit-8. MoCo from wild-type E. coli cell-free extracts was capable of reconstituting NADPH : nitrate reductase activity to all three N. crassa mutants. MoCo from ch1D; pMoCo cell-free extracts was capable of reconstituting more NADPH : nitrate reductase activity to the N. crassa mutants than cell-free extracts from the original ch1D mutant. [ABSTRACT FROM AUTHOR]

The process of repeat- induced point mutation (RIP) was used to disrupt hsps-1, the gene encoding the major heat-inducible member of the HSP70 family of Neurospora crassa. A plasmid DNA, containing an incomplete copy of hsps-1 and the selectable marker qa-2, was introduced into germinated conidia. The sexual progeny of transformants with ectopically integrated hsps-1 DNA was examined for RIP by Southern-blot analysis of MboI- and Sau3A-digested genomic DNA. Progeny strains, showing RIP, were tested for heat shock-responsive expression of hsps-1, by RNA-blot hybridization and Western-blot analysis, as well as for thermotolerance. Isolates with RIP showed low levels of hsps-1 mRNA and a lack of induction of HSP70 protein by heat shock, accompanied by only a marginal decrease in the acquisition of thermotolerance. [ABSTRACT FROM AUTHOR]

Wild-type Neurospora crassa, strain Singapore, was transformed with a N. crassa metallothionein promoter/protyrosinase fusion gene. Transformants produced tyrosinase during vegetative growth, as determined by Western analyses and activity assays. This is in sharp contrast to wild-type strains, where this enzyme is only expressed in situations of starvation or sexual differentiation. Complete integration of a 400 bp metallothionein promoter-fragment leads to constitutive expression of protyrosinase, whereas a 3.6 kb promoter-fragment conferred copper inducibility on the reporter gene in four transformants. A transformant with high constitutive tyrosinase levels was able to produce melanin on complete medium agar plates supplemented with 1 mg/ml L-tyrosine. [ABSTRACT FROM AUTHOR]

Circadian clocks in fungi and animals are driven by a functionally conserved transcription-translation feedback loop. In Neurospora crassa , negative feedback is executed by a complex of Frequency (FRQ), FRQ-interacting RNA helicase (FRH), and casein kinase I (CKI), which inhibits the activity of the clock's positive arm, the White Collar Complex (WCC). Here, we show that the prd-2 ( period-2 ) gene, whose mutation is characterized by recessive inheritance of a long 26 hr period phenotype, encodes an RNA-binding protein that stabilizes the ck-1a transcript, resulting in CKI protein levels sufficient for normal rhythmicity. Moreover, by examining the molecular basis for the short circadian period of upf-1 prd-6 mutants, we uncovered a strong influence of the Nonsense Mediated Decay pathway on CKI levels. The finding that circadian period defects in two classically derived Neurospora clock mutants each arise from disruption of ck-1a regulation is consistent with circadian period being exquisitely sensitive to levels of casein kinase I ., Competing Interests: CK, RL, QX, CB, JL, JD No competing interests declared, (© 2020, Kelliher et al.)

• Agmatinase (AGM-1) is first reported in fungi. • The agm-1 gene is essential for the development of N. crassa. • The Δ agm-1 het strain showed defects in colony development. • The Δ agm-1 het strain showed a disaggregated and unstable Spk. • AGM-1 can hydrolyze agmatine then is an agmatinase like protein. • The AGM-1 protein closely resembles the F-actin population. • Apparently AGM-1 is in association with F-actin via its substrate agmatine. Agmatinase is known as a metalloenzyme which hydrolyzes agmatine to produce urea and putrescine, being crucial in the alternative pathway to produce polyamines. In this study, an agmatinase-like protein (AGM-1) (NCU 01348) in the filamentous fungus Neurospora crassa is reported. Purified AGM-1 from N. crassa displays enzymatic activity hydrolyzing agmatine; therefore, it can be considered as an agmatinase-like protein. However, its role in the alternative pathway to produce polyamines apparently is not its main function since only a slight reduction of polyamines concentration was detected in the Δ agm-1 het strain. Moreover, the null mutant Δ agm-1 (homokaryon strain) was unable to grow and the deficiency of agm-1 in the heterokaryon strain provoked a decrease in elongation rate, conidia and biomass production, despite of having de constitutive pathway via the ornithine decarboxylase (ODC). Additionally, mature hyphae of the Δ agm-1 het strain presented unusual apical branching and a disorganized Spitzenkörper (Spk). Trying to reveal the role of AGM-1in N. crassa , the protein was tagged with GFP and interestingly the dynamics and intracellular localization of AGM-1 closely resembles the F-actin population. This finding was further examined in order to elucidate if AGM-1is in a close association with F-actin. Since polyamines, among them agmatine, have been reported to act as stabilizers of actin filaments, we evaluated in vitro G-actin polymerization in the presence of agmatine and the effect of purified AGM-1 from N. crassa on these polymerized actin filaments. It was found that polymerization of actin filaments increases in the presence of agmatine and the addition of purified AGM-1 from N. crassa depolymerizes these actin filaments. Also, it was determined that an intact substrate binding site of the enzyme is necessary for the localization pattern of the native AGM-1. These results suggest that in N. crassa AGM-1 has a close association with the F-actin population via its substrate agmatine, playing an essential role during cell development. [ABSTRACT FROM AUTHOR]

Methylation of histone H3 at lysine 36 (H3K36me), a widely-distributed chromatin mark, largely results from association of the lysine methyltransferase (KMT) SET-2 with RNA polymerase II (RNAPII), but most eukaryotes also have additional H3K36me KMTs that act independently of RNAPII. These include the orthologs of ASH1, which are conserved in animals, plants, and fungi but whose function and control are poorly understood. We found that Neurospora crassa has just two H3K36 KMTs, ASH1 and SET-2, and were able to explore the function and distribution of each enzyme independently. While H3K36me deposited by SET-2 marks active genes, inactive genes are modified by ASH1 and its activity is critical for their repression. ASH1-marked chromatin can be further modified by methylation of H3K27, and ASH1 catalytic activity modulates the accumulation of H3K27me2/3 both positively and negatively. These findings provide new insight into ASH1 function, H3K27me2/3 establishment, and repression in facultative heterochromatin., Competing Interests: VB, TO, KA, SH, ES No competing interests declared, (© 2018, Bicocca et al.)

Development in higher organisms requires selective gene silencing, directed in part by di-/trimethylation of lysine 27 on histone H3 (H3K27me2/3). Knowledge of the cues that control formation of such repressive Polycomb domains is extremely limited. We exploited natural and engineered chromosomal rearrangements in the fungus Neurospora crassa to elucidate the control of H3K27me2/3. Analyses of H3K27me2/3 in strains bearing chromosomal rearrangements revealed both position-dependent and position-independent facultative heterochromatin. We found that proximity to chromosome ends is necessary to maintain, and sufficient to induce, transcriptionally repressive, subtelomeric H3K27me2/3. We ascertained that such telomere-proximal facultative heterochromatin requires native telomere repeats and found that a short array of ectopic telomere repeats, (TTAGGG) 17, can induce a large domain (~225 kb) of H3K27me2/3. This provides an example of a cis -acting sequence that directs H3K27 methylation. Our findings provide new insight into the relationship between genome organization and control of heterochromatin formation.