Biology subjects

Glass, N. L.

Publications and source records attributed to Glass, N. L..

6 recordsLinked to original sources

bioRxiv2025

Identification of regions required for allelic specificity at the cell wall remodeling allorecognition checkpoint in Neurospora crassa

Allorecognition is the ability of organisms/cells to differentiate self from non-self. In the fungus Neurospora crassa, allorecognition systems serve as checkpoints to restrict germling/hyphal fusion between genetically incompatible strains. The cell wall remodeling (cwr) checkpoint functions after chemotrophic interactions and is triggered upon cell/hyphal contact, regulating cell wall dissolution and subsequent cell fusion. The cwr region consists of two linked loci, cwr-1 and cwr-2, that are under severe linkage disequilibrium. Phylogenetic analysis of wild N. crassa populations showed that cwr-1/cwr-2 alleles fall into six different haplogroups (HGs). Strains containing deletions of cwr-1 and cwr-2 will fuse with previously HG incompatible cells, indicating cwr negatively regulates cell fusion. CWR-1 encodes a polysaccharide monooxygenase (PMO) domain that oxidatively degrades chitin; the PMO domain is sufficient to cause fusion arrest and confers allelic specificity by interacting in trans with CWR-2, a predicted transmembrane protein. However, the catalytic activity of CWR-1 is not required for triggering a block in cell fusion. The L2 and LC regions of the CWR-1 PMO domain show high levels of structural variability between different HGs. CWR-1 chimeras containing a LC region from a different HG were sufficient to trigger a cell fusion block, but not quite at wild type levels, suggesting that the complete PMO structure is necessary for allorecognition. Modeling of the transmembrane protein CWR-2 revealed allelic variability in the two major extracellular domains (ED2/ED4). Chimeras of CWR-2 with swapped ED2 or ED4 or ED2/ED4 domains from different cwr-2 haplogroups also altered allelic specificity. SummaryAllorecognition or nonself recognition enables fungi to distinguish genetically different individuals, thereby regulating cooperation to form mycelial networks. This study focused on the cell wall remodeling checkpoint (cwr), where genetic differences in two genes, cwr-1 and cwr-2, triggers allorecognition. Upon cell contact, CWR-1 in one cell functions in trans with CWR-2 in a second cell to confer a block in cell fusion. Chimeric proteins were created and tested to pinpoint domains involved in allelic specificity. For CWR-1, the L2 and LC domains are critical, while for CWR-2, the ED2 and ED4 domains have an important role in regulating cell fusion. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/633681v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@7e150org.highwire.dtl.DTLVardef@1596b00org.highwire.dtl.DTLVardef@1800a37org.highwire.dtl.DTLVardef@11ea8d4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

genetics↗

bioRxiv2022

A moonlighting function of a chitin polysaccharide monooxygenase, CWR-1, in allorecognition in Neurospora crassa

Organisms require the ability to differentiate themselves from organisms of different or even the same species. Allorecognition processes in filamentous fungi are essential to ensure identity of an interconnected syncytial colony to protect it from exploitation and disease. Neurospora crassa has three cell fusion checkpoints controlling formation of an interconnected mycelial network. The locus that controls the second checkpoint, which allows for cell wall dissolution and subsequent fusion between cells/hyphae, cwr, encodes two linked genes, cwr-1 and cwr-2. Previously, it was shown that cwr-1 and cwr-2 show severe linkage disequilibrium with six different haplogroups present in N. crassa populations. Isolates from an identical cwr haplogroup show robust fusion, while somatic cell fusion between isolates of different haplogroups is significantly blocked in cell wall dissolution. The cwr-1 gene encodes a putative polysaccharide monooxygenase (PMO). Herein we confirm that CWR-1 is a C1-oxidizing chitin PMO. We show that the PMO domain of CWR-1 was sufficient for checkpoint function and cell fusion blockage; however, through analysis of active-site, histidine-brace mutants, the catalytic activity of CWR-1 was ruled out as a major factor for allorecognition. Swapping a portion of the PMO domain (V86 to T130) did not switch cwr haplogroup specificity, but rather cells containing this chimera exhibited a novel haplogroup specificity. Allorecognition to mediate cell fusion blockage is likely occurring through a protein-protein interaction between CWR-1 with CWR-2. These data highlight a moonlighting role in allorecognition of the CWR1 PMO domain.

genetics↗

bioRxiv2021

Aspects of the Neurospora crassa sulfur starvation response are revealed by transcriptional profiling and DNA affinity purification sequencing

Accurate nutrient sensing is important for rapid fungal growth and exploitation of available resources. Sulfur is an important nutrient source found in a number of biological macromolecules, including proteins and lipids. The model filamentous fungus Neurospora crassa is capable of utilizing sulfur found in a variety of sources from amino acids to sulfate. During sulfur starvation, the transcription factor CYS-3 is responsible for upregulation of genes involved in sulfur uptake and assimilation. Using a combination of RNA sequencing and DNA affinity purification sequencing, we performed a global survey of the N. crassa sulfur starvation response and the role of CYS-3 in regulating sulfur responsive genes. Along with genes known to be involved in sulfur metabolism, the CYS-3 transcription factor also directly activated the expression of a number of uncharacterized transporter genes, suggesting that regulating sulfur import is an important aspect of regulation by CYS-3. Additionally, CYS-3 directly regulated the expression of genes involved in mitochondrial electron transfer. During sulfur starvation, genes involved in nitrogen metabolism, such as amino acid and nucleic acid metabolic pathways, along with genes encoding proteases and nucleases that are necessary for scavenging nitrogen, were activated. Sulfur starvation also caused changes in the expression of genes involved in carbohydrate metabolism, such as those encoding glycosyl hydrolases. Thus, our data suggest a connection between sulfur metabolism and other aspects of cellular metabolism.

genetics↗

bioRxiv2021

Spray-induced gene silencing for disease control is dependent on the efficiency of pathogen RNA uptake

Recent discoveries show that fungi can take up environmental RNA, which can then silence fungal genes through environmental RNA interference. This discovery prompted the development of Spray-Induced Gene Silencing (SIGS) for plant disease management. In this study, we aimed to determine the efficacy of SIGS across a variety of eukaryotic microbes. We first examined the efficiency of RNA uptake in multiple pathogenic and non-pathogenic fungi, and an oomycete pathogen. We observed efficient double-stranded RNA (dsRNA) uptake in the fungal plant pathogens Botrytis cinerea, Sclerotinia sclerotiorum, Rhizoctonia solani, Aspergillus niger, and Verticillium dahliae, but no uptake in Colletotrichum gloeosporioides, and weak uptake in a beneficial fungus, Trichoderma virens. For the oomycete plant pathogen, Phytophthora infestans, RNA uptake was limited, and varied across different cell types and developmental stages. Topical application of dsRNA targeting virulence-related genes in the pathogens with high RNA uptake efficiency significantly inhibited plant disease symptoms, whereas the application of dsRNA in pathogens with low RNA uptake efficiency did not suppress infection. Our results have revealed that dsRNA uptake efficiencies vary across eukaryotic microbe species and cell types. The success of SIGS for plant disease management can largely be determined by the pathogen RNA uptake efficiency.

microbiology↗

bioRxiv2018

A network of MAP-Kinase pathways and transcription factors regulates cell-to-cell communication and cell wall integrity in Neurospora crassa

Maintenance of cell integrity and cell-to-cell communication are fundamental biological processes. Filamentous fungi, such as Neurospora crassa, depend on communication to locate compatible cells, coordinate cell fusion, and establish a robust hyphal network. Two MAP-Kinase pathways are essential for communication and cell fusion in N. crassa; the Cell Wall Integrity/MAK-1 pathway and the MAK-2 (signal response) pathway. Previous studies have demonstrated several points of cross talk between the MAK-1 and MAK-2 pathways, which is likely necessary for oordinating chemotropic growth toward an extracellular signal, and then mediating cell fusion. Canonical MAP-Kinase pathways begin with signal reception and end with a transcriptional response. Two transcription factors, ADV-1 and PP-1, are essential for communication and cell fusion. PP-1 is the conserved target of MAK-2, while it is unclear what targets ADV-1. We did RNAseq on{Delta} adv-1, {Delta}pp-1, and wild-type cells and found that ADV-1 and PP-1 have a shared regulon including many genes required for communication, cell fusion, growth, development, and stress response. We identified ADV-1 and PP-1 binding sites across the genome by adapting the in vitro method of DNA-Affinity Purification sequencing (DAP-seq) for N. crassa. To elucidate the regulatory network, we misexpressed each transcription factor in each upstream MAPK deletion mutant. Misexpression of adv-1 was sufficient to fully suppress the phenotype of the{Delta} pp-1 mutant and partially suppress the phenotype of the{Delta} mak-1 mutant. Collectively, our data demonstrate that the MAK-1-ADV-1 and MAK-2- PP-1 pathways form a tight regulatory network that maintains cell integrity and mediates communication and cell fusion.

genetics↗

bioRxiv2017

Network Reconstruction And Systems Analysis Of Plant Cell Wall Deconstruction By Neurospora crassa

Plant biomass degradation by fungal derived enzymes is rapidly expanding in economic importance as a clean and efficient source for biofuels. The ability to rationally engineer filamentous fungi would facilitate biotechnological applications for degradation of plant cell wall polysaccharides. However, incomplete knowledge of biomolecular networks responsible for plant cell wall deconstruction impedes experimental efforts in this direction. To expand this knowledge base, a detailed network of reactions important for deconstruction of plant cell wall polysaccharides into simple sugars was constructed for the filamentous fungus Neurospora crassa. To reconstruct this network, information was integrated from five heterogeneous data types: functional genomics, transcriptomics, proteomics, genetics, and biochemical characterizations. The combined information was encapsulated into a feature matrix and the evidence weighed to assign annotation confidence scores for each gene within the network. Comparative analyses of RNA-seq and ChIP-seq data shed light on the regulation of the plant cell wall degradation network (PCWDN), leading to a novel hypothesis for degradation of the hemicellulose mannan. The transcription factor CLR-2 was subsequently experimentally shown to play a key role in the mannan degradation pathway of Neurospora crassa. Our network serves as a scaffold for integration of diverse experimental data, leading to elucidation of regulatory design principles for plant cell wall deconstruction by filamentous fungi, and guiding efforts to rationally engineer industrially relevant hyper-production strains.

systems biology↗