The C-terminal domain of SEC-10 is fundamental for exocyst function, Spitzenkorper organization and cell morphogenesis in Neurospora crassa.

The exocyst is a conserved multimeric complex that participates in the final steps of the secretion of vesicles. In the filamentous fungus Neurospora crassa, the exocyst is crucial for polar growth, morphology, and the organization of the Spitzenkorper (Spk), the apical body where secretory vesicles accumulate before being delivered to the plasma membrane. In the highly polarized cells of N. crassa, the exocyst subunits SEC-3, SEC-5, SEC-6, SEC-8, and SEC-15 were previously found localized at the plasma membrane of the apices of the cells, while EXO-70 and EXO-84 occupied the frontal outer layer of the Spk, occupied by vesicles. The localization of SEC-10 had remained so far elusive. In this work, SEC-10 was tagged with the green fluorescent protein (GFP) either at its N- or C-terminus and found localized at the plasma membrane of growing hyphal tips, similar to what was previously observed for some exocyst subunits. While expression of an N-terminally tagged version of SEC-10 at its native locus was fully viable, expression of a C-terminally tagged version at its native locus resulted in severe hyphal growth and polarity defects. Additionally, a sec-10 knockout mutant in a heterokaryotic state (with genetically different nuclei) was viable but showed a strongly aberrant phenotype, confirming that this subunit is essential to maintain hyphal morphogenesis. Transmission electron microscopy analysis revealed the lack of a Spk in the SEC-10-GFP strain, suggesting a critical role of the exocyst in the vesicular organization at the Spk. Mass spectrometry analysis revealed fewer peptides of exocyst subunits interacting with SEC-10-GFP than with GFP-SEC-10, suggesting an essential role of the C-terminus of SEC-10 in exocyst assembly and/or stability. Altogether, our data suggest that an unobstructed C-terminus of SEC-10 is indispensable for the exocyst complex function and that a GFP tag could be blocking important subunit-subunit interactions.

The Resonance and Adaptation of Neurospora crassa Circadian and Conidiation Rhyth ms to Short Light-Dark Cycles

Circadian clocks control the physiological and behavioral rhythms to adapt to the environment with a period of ~24 h. However, the influences and mechanisms of the extreme light/dark cycles on the circadian clock remain unclear. We showed that, in Neurospora crassa, both the growth and the microconidia production contribute to adaptation in LD12:12 (12 h light/12 h dark, periodically). Mathematical modeling and experiments demonstrate that in short LD cycles, the expression of the core clock protein FREQUENCY was entrained to the LD cycles when LD > 3:3 while it free ran when T ≤ LD3:3. The conidial rhythmicity can resonate with a series of different LD conditions. Moreover, we demonstrate that the existence of unknown blue light photoreceptor(s) and the circadian clock might promote the conidiation rhythms that resonate with the environment. The ubiquitin E3 ligase FWD-1 and the previously described CRY-dependent oscillator system were implicated in regulating conidiation under short LD conditions. These findings shed new light on the resonance of Neurospora circadian clock and conidiation rhythms to short LD cycles, which may benefit the understandings of both the basic regulatory aspects of circadian clock and the adaptation of physiological rhythms to the extreme conditions.

Coordinated Regulation of Protoperithecium Development by MAP Kinases MAK-1 and MAK-2 in Neurospora crassa

Mitogen-activated protein (MAP) kinase pathways function as signaling hubs that are integral for many essential cellular processes, including sexual development. The molecular mechanisms and cross-talk between PR and CWI MAP kinase pathways have been extensively studied during asexual development. However, if these can be extended to sexual development remains elusive. By analyzing genome-wide transcriptional responses to deletion of each of two MAP kinase coding genes mak-2 (PR-MAP kinase pathway) and mak-1 (CWI-MAP kinase pathway) in Neurospora crassa during protoperithecium formation, 430 genes co-regulated by the MAK-1 and MAK-2 proteins were found, functionally enriched at integral components of membrane and oxidoreductase. These genes include 13 functionally known genes participating in sexual development (app, poi-2, stk-17, fsd-1, vsd-8, and NCU03863) and melanin synthesis (per-1, pkh-1, pkh-2, mld-1, scy-1, trn-2, and trn-1), as well as a set of functionally unknown genes. Phenotypic analysis of deletion mutants for the functionally unknown genes revealed that 12 genes were essential for female fertility. Among them, single-gene deletion mutants for NCU07743 (named as pfd-1), NCU02250 (oli), and NCU05948 (named as pfd-2) displayed similar protoperithecium development defects as the Δmak-1 and Δmak-2 mutants, failing to form protoperithecium. Western blotting analysis showed that both phosphorylated and total MAK-1 proteins were virtually abolished in the Δnrc-1, Δmek-2, and Δmak-2 mutants, suggesting that the posttranscriptional regulation of MAK-1 is dependent on the PR-MAP kinase pathway during the protoperithecium development. Taken together, this study revealed the regulatory roles and cross-talk between PR and CWI-MAP kinase pathways during protoperithecium development.

Analysis of Codon Usage Pattern and Predicted Gene Expression in Neurospora Crassa: A Novel in Silico Approach

The codon usage pattern of genes has a key role in the gene expression and adaptive evolution of an organism. It is very significant in understanding the role of complex genomic structure in defining cell fates and regulating diverse biological functions. In this paper, we discussed that the codon usage index (CAIg) based on all protein-coding genes is a promising alternative to the Codon Adaptation Index (CAI). CAIg which measures the extent that a gene uses a subset of preferred codons relies exclusively on sequence features and is used as a good indicator of the strength of codon bias. A critical analysis of predicted highly expressed (PHE) genes in Neurospora crassa has been performed using codon usage index (CAIg) as a numerical estimator of gene expression level. Analyzing compositional properties and codon usage pattern of genes in Neurospora crassa, our study indicates that codon composition plays an important role in the regulation of gene expression. We found a systematic strong correlation between CAIg and CBI (codon bias index) or other expression-measures. Here, we show that codon usage index CAIg correlates well with both protein and mRNA levels; suggesting that codon usage is an important determinant of gene expression. Our study highlights the relationship between gene expression and compositional signature in relation to codon usage bias in Neurospora crassa and sets the ground for future investigation in eukaryotic biology.

Courtship Ritual of Male and Female Nuclei during Fertilization in Neurospora crassa

Using live-cell fluorescence imaging, for the first time we have observed live male and female nuclei during sexual reproduction in the model fungus Neurospora crassa . This study reveals the specific behavior of resident female nuclei within the trichogyne (the female organ) after fertilization and the extraordinary manner in which male nuclei migrate across the trichogyne toward their final destination, the protoperithecium, where karyogamy takes place.

Quantifying oxidation of cellulose-associated glucuronoxylan by two lytic polysaccharide monooxygenases from Neurospora crassa

Family AA9 lytic polysaccharide monooxygenases (LPMOs) are abundant in fungi where they catalyze oxidative depolymerization of recalcitrant plant biomass. These AA9 LPMOs cleave cellulose, and some also act on hemicelluloses, primarily other (substituted) β-(1→4)-glucans. Oxidative cleavage of xylan has been shown for only a handful AA9 LPMOs, and it remains unclear whether this activity is a minor side reaction or primary function. Here, we show that Nc LPMO9F and the phylogenetically related, hitherto uncharacterized Nc LPMO9L from Neurospora crassa are active on both cellulose and cellulose-associated glucuronoxylan, but not on glucuronoxylan alone. A newly developed method for simultaneous quantification of xylan-derived and cellulose-derived oxidized products showed that Nc LPMO9F preferentially cleaves xylan when acting on a cellulose–beechwood glucuronoxylan mixture, yielding about three times more xylan-derived than cellulose-derived oxidized products. Interestingly, under similar conditions, Nc LPMO9L and previously characterized Mc LPMO9H from Malbranchea cinnamomea showed different xylan-to-cellulose preferences, giving oxidized product ratios of about 0.5:1 and 1:1, respectively, indicative of functional variation among xylan-active LPMOs. Phylogenetic and structural analysis of xylan-active AA9 LPMOs led to the identification of characteristic structural features, including unique features that do not occur in phylogenetically remote AA9 LPMOs, such as four AA9 LPMOs whose lack of activity towards glucuronoxylan was demonstrated in the present study. Taken together, the results provide a path towards discovery of additional xylan-active LPMOs and show that the huge family of AA9 LPMOs has members that preferentially act on xylan. These findings shed new light on the biological role and industrial potential of these fascinating enzymes. Importance Plant cell wall polysaccharides are highly resilient to depolymerization by hydrolytic enzymes, partly due to cellulose chains being tightly packed in microfibrils that are covered by hemicelluloses. Lytic polysaccharide monooxygenases (LPMOs) seem well suited to attack these resilient co-polymeric structures, but the occurrence and importance of hemicellulolytic activity among LPMOs remains unclear. Here we show that certain AA9 LPMOs preferentially cleave xylan when acting on a cellulose–glucuronoxylan mixture, and that this ability is the result of protein evolution that has resulted in a clade of AA9 LPMOs with specific structural features. Our findings strengthen the notion that the vast arsenal of AA9 LPMOs in certain fungal species provides functional versatility, and that AA9 LPMOs may have evolved to promote oxidative depolymerization of a wide variety of recalcitrant, co-polymeric plant polysaccharide structures. These findings have implications for understanding the biological roles and industrial potential of LPMOs.