Endocytic Recycling Pathways in Aspergillus nidulans

ungi, which dominate many ecosystems as major decomposers and pathogens, generally colonize through the formation of long, tubular cells called hyphae. Understanding hyphal growth has been of interest to cell biologists for a century, and holds the potential to provide insights for many eukaryotic systems. Hyphal growth and shape are intimately connected with membrane trafficking, which is divided into exocytosis (membrane fusion) and endocytosis (membrane fission). Both of these processes are, in turn, essential for hyphal growth. However, endocytosis works to remove membrane, and why it should be important for cell expansion is not known. Here, using genetic manipulation combined with live cell imaging, the role of endocytosis in hyphal growth was scrutinized in the fungus Aspergillus nidulans. First, I examined the localization and function of the canonical endocytic coat protein, clathrin, and discovered that it is involved in budding at the Golgi apparatus, but does not appear to play a significant role in endocytosis. Second, I looked for proteins that may traffic through an endocytic recycling pathway within the hyphal tip, which resulted in identification and characterization of the phospholipid flippases DnfA-D in A. nidulans. These proteins regulate phospholipid asymmetry in the plasma membrane and the endocytic pathway, and are predicted to be involved in linking endocytosis and exocytosis. I discovered that DnfA and DnfB are stratified within the hyphal tip, likely on different vesicles, and require endocytosis for their steady-state localization. Loss of either DnfA or DnfB function has a minor effect on hyphal shape and growth, but loss of both is lethal. Additionally, the phospholipid phosphatidylserine is normally concentrated strictly on the outside of secretory vesicles, but the localization of this phospholipid, as well as several secretory proteins, was disrupted and diffused through the cytoplasm in the absence of DnfA. These results highlight the importance of endocytic recycling in the maintenance of polarized hyphal growth, as well the complexity of the homeostatic mechanisms at work in an actively expanding hyphal cell, which involve proteins and a variety of lipids for normal function

A Gin4-Like Protein Kinase GIL1 Involvement in Hyphal Growth, Asexual Development, and Pathogenesis in Fusarium graminearum

Fusarium graminearum is the main causal agent of Fusarium head blight (FHB) on wheat and barley. In a previous study, a GIN4-like protein kinase gene, GIL1, was found to be important for plant infection and sexual reproduction. In this study we further characterized the functions of GIL1 kinase in different developmental processes. The Δgil1 mutants were reduced in growth, conidiation, and virulence, and formed whitish and compact colonies. Although phialide formation was rarely observed in the mutants, deletion of GIL1 resulted in increased hyphal branching and increased tolerance to cell wall and cell membrane stresses. The Δgil1mutants produced straight, elongated conidia lacking of distinct foot cells and being delayed in germination. Compared with the wild type, some compartments in the vegetative hyphae of Δgil1 mutants had longer septal distances and increased number of nuclei, suggesting GIL1 is related to cytokinesis and septation. Localization of the GIL1-GFP fusion proteins to the septum and hyphal branching and fusion sites further supported its roles in septation and branching. Overall, our results indicate that GIL1 plays a role in vegetative growth and plant infection in F. graminearum, and is involved in septation and hyphal branching

Proteomics Reveals Distinct Changes Associated with Increased Gamma Radiation Resistance in the Black Yeast Exophiala dermatitidis

The yeast Exophiala dermatitidis exhibits high resistance to γ-radiation in comparison to many other fungi. Several aspects of this phenotype have been characterized, including its dependence on homologous recombination for the repair of radiation-induced DNA damage, and the transcriptomic response invoked by acute γ-radiation exposure in this organism. However, these findings have yet to identify unique γ-radiation exposure survival strategies—many genes that are induced by γ-radiation exposure do not appear to be important for recovery, and the homologous recombination machinery of this organism is not unique compared to more sensitive species. To identify features associated with γ-radiation resistance, here we characterized the proteomes of two E. dermatitidis strains—the wild type and a hyper-resistant strain developed through adaptive laboratory evolution—before and after γ-radiation exposure. The results demonstrate that protein intensities do not change substantially in response to this stress. Rather, the increased resistance exhibited by the evolved strain may be due in part to increased basal levels of single-stranded binding proteins and a large increase in ribosomal content, possibly allowing for a more robust, induced response during recovery. This experiment provides evidence enabling us to focus on DNA replication, protein production, and ribosome levels for further studies into the mechanism of γ-radiation resistance in E. dermatitidis and other fungi

Oxylipin biosynthetic gene families of Cannabis sativa

Cannabis sativa is a global multi-billion-dollar cash crop with numerous industrial uses, including in medicine and recreation where its value is largely owed to the production of pharmacological and psychoactive metabolites known as cannabinoids. Often underappreciated in this role, the lipoxygenase (LOX)-derived green leaf volatiles (GLVs), also known as the scent of cut grass, are the hypothetical origin of hexanoic acid, the initial substrate for cannabinoid biosynthesis. The LOX pathway is best known as the primary source of plant oxylipins, molecules analogous to the eicosanoids from mammalian systems. These molecules are a group of chemically and functionally diverse fatty acid-derived signals that govern nearly all biological processes including plant defense and development. The interaction between oxylipin and cannabinoid biosynthetic pathways remains to be explored. Despite their unique importance in this crop, there has not been a comprehensive investigation focusing on the genes responsible for oxylipin biosynthesis in any Cannabis species. This study documents the first genome-wide catalogue of the Cannabis sativa oxylipin biosynthetic genes and identified 21 LOX, five allene oxide synthases (AOS), three allene oxide cyclases (AOC), one hydroperoxide lyase (HPL), and five 12-oxo-phytodienoic acid reductases (OPR). Gene collinearity analysis found chromosomal regions containing several isoforms maintained across Cannabis, Arabidopsis, and tomato. Promoter, expression, weighted co-expression genetic network, and functional enrichment analysis provide evidence of tissue- and cultivar-specific transcription and roles for distinct isoforms in oxylipin and cannabinoid biosynthesis. This knowledge facilitates future targeted approaches towards Cannabis crop improvement and for the manipulation of cannabinoid metabolism

Oxylipin biosynthetic gene families of Cannabis sativa.

Cannabis sativa is a global multi-billion-dollar cash crop with numerous industrial uses, including in medicine and recreation where its value is largely owed to the production of pharmacological and psychoactive metabolites known as cannabinoids. Often underappreciated in this role, the lipoxygenase (LOX)-derived green leaf volatiles (GLVs), also known as the scent of cut grass, are the hypothetical origin of hexanoic acid, the initial substrate for cannabinoid biosynthesis. The LOX pathway is best known as the primary source of plant oxylipins, molecules analogous to the eicosanoids from mammalian systems. These molecules are a group of chemically and functionally diverse fatty acid-derived signals that govern nearly all biological processes including plant defense and development. The interaction between oxylipin and cannabinoid biosynthetic pathways remains to be explored. Despite their unique importance in this crop, there has not been a comprehensive investigation focusing on the genes responsible for oxylipin biosynthesis in any Cannabis species. This study documents the first genome-wide catalogue of the Cannabis sativa oxylipin biosynthetic genes and identified 21 LOX, five allene oxide synthases (AOS), three allene oxide cyclases (AOC), one hydroperoxide lyase (HPL), and five 12-oxo-phytodienoic acid reductases (OPR). Gene collinearity analysis found chromosomal regions containing several isoforms maintained across Cannabis, Arabidopsis, and tomato. Promoter, expression, weighted co-expression genetic network, and functional enrichment analysis provide evidence of tissue- and cultivar-specific transcription and roles for distinct isoforms in oxylipin and cannabinoid biosynthesis. This knowledge facilitates future targeted approaches towards Cannabis crop improvement and for the manipulation of cannabinoid metabolism

Phylogenetic, genetic, motif, and domain analysis of the CsAOC gene family.

(A) Cladogram of peptide sequences and conserved domains. (B) Distribution of conserved peptide sequence motifs. Colors are described in legend; x-axis represents length of peptides in amino acids. (C) Diagram of genetic structure. Blue bars, orange bars, and gray lines represent untranslated regions, exons, and introns, respectively; x-axis represents length of gene in nucleotides. (D) Polar cladogram depicting evolutionary relationship with gene families of selected species. Node labels show confidence values from 1000 bootstrap replications.</p

Phylogenetic, genetic, motif, and domain analysis of the CsLOX gene family.

(A) Cladogram of peptide sequences and conserved domains. (B) Distribution of conserved peptide sequence motifs. Colors are described in legend; x-axis represents length of peptides in amino acids. (C) Diagram of genetic structure. Blue bars, orange bars, and gray lines represent untranslated regions, exons, and introns, respectively; x-axis represents length of gene in nucleotides. (D) Polar cladogram depicting evolutionary relationship with gene families of selected species. Node labels show confidence values from 1000 bootstrap replications.</p

Regions of duplicated <i>OPR</i>, <i>AOS</i>, and <i>AOC</i> genes on <i>C</i>. <i>sativa</i> Chromosomes 1, 8, and X.

Regions of duplicated OPR, AOS, and AOC genes on C. sativa Chromosomes 1, 8, and X.</p