RHO-3 plays a significant role in hyphal extension rate, conidiation, and the integrity of the Spitzenkörper in Neurospora crassa.

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.

The small Ras-like GTPase BUD-1 modulates conidial germination and hyphal growth guidance in the filamentous fungus Neurospora crassa.

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.

Role and dynamics of an agmatinase-like protein (AGM-1) in Neurospora crassa.

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 Δagm-1 het strain. Moreover, the null mutant Δagm-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 Δagm-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.

Phosphorylation, disorder, and phase separation govern the behavior of Frequency in the fungal circadian clock.

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 Frequency (FRQ), the FRQ-interacting RNA helicase (FRH), and Casein-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.

Natural oscillations known as circadian rhythms influence many processes in humans and other animals including sleep, eating, brain activity and body temperature. These rhythms allow us to anticipate and prepare for regular changes in our environment including day-night cycles and the temperature of our surroundings. Circadian clocks in animals, fungi and other 'eukaryotic' organisms rely on networks of components that repress their own production to generate oscillations in their levels in cells over the course of a 24-hour period. The components in animal and fungus circadian clocks are different but there are strong similarities in their properties and how the networks operate. As a result, a type of fungus known as Neurospora crassa is often used as a model to study how circadian rhythms work in animals. A central component in the N. crassa circadian clock is a protein known as Frequency (FRQ). It is a large protein that, unlike most proteins, lacks a well-defined, three-dimensional structure. Despite this, it is able to bind to and regulate other proteins to repress its own production. One of its protein partners known as CK1 attaches small tags known as phosphate groups to FRQ to set the length of the circadian rhythm. However, it remains unclear how FRQ interacts with its protein partners or what effect the phosphate groups have on its activity. To address this question, Tariq, Maurici et al. used biochemical approaches to study the structure of FRQ. The experiments revealed that it contains a compact core that is able to bind to CK1 and other protein partners. The way FRQ regulates its protein partners is unusual: it undergoes a chemical process known as liquid-liquid phase separation to sequester other circadian clock proteins and modulate their enzymatic activities. In this process, a solution containing molecules of FRQ separates into two distinct components (known as phases), one of which contains FRQ and its partners in a concentrated liquid-like mixture. Evidence for such mixtures has also been found in living fungal cells. Further experiments suggest that liquid-liquid phase separation of FRQ may allow the clock to compensate for changes in temperature to maintain a regular rhythm. The circadian clocks of animals and other organisms all have proteins that perform similar roles as FRQ and maintain sequence properties that promote liquid-liquid phase separation. Therefore, it is possible that liquid-liquid phase separation may be a common feature of circadian rhythms in nature.

Adaptation to glucose starvation is associated with molecular reorganization of the circadian clock in Neurospora crassa.

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.

The nutrient-sensing GCN2 signaling pathway is essential for circadian clock function by regulating histone acetylation under amino acid starvation.

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.

The ACF chromatin-remodeling complex is essential for Polycomb repression.

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.

All the cells in an organism contain the exact same DNA, yet each type of cell performs a different role. They achieve this by turning specific genes on or off. To do this, cells wind their genetic code into structures called nucleosomes, which work a bit like spools of thread. Chemical modifications on these nucleosomes can determine whether a cell will use the genes spooled around it or not. In many organisms, cells can turn genes off using a modification called H3K27 methylation. This mark attracts a protein complex called PRC1 that packs the genes away, making them inaccessible to the proteins that would activate them. But the filamentous fungus Neurospora crassa does not produce PRC1. This suggests that this organism must keep genes with the H3K27 mark switched off in a different way. One possibility is that H3K27 methylation somehow leads to changes in the position of nucleosomes on the genome, since having nucleosomes near the beginning of gene sequences can stop the cell from reading the code. One protein complex responsible for positioning nucleosomes is known as the ATP-utilizing chromatin assembly and remodeling factor (ACF) complex, but it remained unknown whether it interacted with H3K27 methylation marks. To investigate further, Wiles et al. generated strains of Neurospora crassa that did not synthesize ACF and discovered that many of their genes, including ones marked with H3K27, were turned on. This was probably because the nucleosomes had shifted out of position, allowing the proteins responsible for activating the genes to gain access to the start of the genes' sequences. Turning genes on and off at the right time is crucial for development, cell survival, and is key in tissues and organs working properly. Understanding the role of ACF adds to what we know about this complex process, which is involved in many diseases, including cancer.

PRD-2 directly regulates casein kinase I and counteracts nonsense-mediated decay in the Neurospora circadian clock.

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-1prd-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.

Telomere repeats induce domains of H3K27 methylation in Neurospora.

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.

ASH1-catalyzed H3K36 methylation drives gene repression and marks H3K27me2/3-competent chromatin.

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.

Expanding xylose metabolism in yeast for plant cell wall conversion to biofuels.

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.