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Maximal polar growth potential depends on the polarisome component AgSpa2 in the filamentous fungus Ashbya gossypii.

ABSTRACT: We used actin staining and videomicroscopy to analyze the development from a spore to a young mycelium in the filamentous ascomycete Ashbya gossypii. The development starts with an initial isotropic growth phase followed by the emergence of germ tubes. The initial tip growth speed of 6-10 microm/h increases during early stages of development. This increase is transiently interrupted in response to the establishment of lateral branches or septa. The hyphal tip growth speed finally reaches a maximum of up to 200 micro/h, and the tips of these mature hyphae have the ability to split into two equally fast-growing hyphae. A search for A. gossypii homologs of polarisome components of the yeast Saccharomyces cerevisiae revealed a remarkable size difference between Spa2p of both organisms, with AgSpa2p being double as long as ScSpa2p due to an extended internal domain. AgSpa2 colocalizes with sites of polarized actin. Using time-lapse videomicroscopy, we show that AgSpa2p-GFP polarization is established at sites of branch initiation and then permanently maintained at hyphal tips. Polarization at sites of septation is transient. During apical branching the existing AgSpa2p-GFP polarization is symmetrically divided. To investigate the function of AgSpa2p, we generated two AgSPA2 mutants, a partial deletion of the internal domain alone, and a complete deletion. The mutations had an impact on the maximal hyphal tip growth speed, on the hyphal diameter, and on the branching pattern. We suggest that AgSpa2p is required for the determination of the area of growth at the hyphal tip and that the extended internal domain plays an important role in this process.

SUBMITTER: Knechtle P

PROVIDER: S-EPMC207006 | biostudies-literature |

REPOSITORIES: biostudies-literature

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A Ras-like GTPase is involved in hyphal growth guidance in the filamentous fungus Ashbya gossypii.

Project description:Characteristic features of morphogenesis in filamentous fungi are sustained polar growth at tips of hyphae and frequent initiation of novel growth sites (branches) along the extending hyphae. We have begun to study regulation of this process on the molecular level by using the model fungus Ashbya gossypii. We found that the A. gossypii Ras-like GTPase Rsr1p/Bud1p localizes to the tip region and that it is involved in apical polarization of the actin cytoskeleton, a determinant of growth direction. In the absence of RSR1/BUD1, hyphal growth was severely slowed down due to frequent phases of pausing of growth at the hyphal tip. During pausing events a hyphal tip marker, encoded by the polarisome component AgSPA2, disappeared from the tip as was shown by in vivo time-lapse fluorescence microscopy of green fluorescent protein-labeled AgSpa2p. Reoccurrence of AgSpa2p was required for the resumption of hyphal growth. In the Agrsr1/bud1Delta deletion mutant, resumption of growth occurred at the hyphal tip in a frequently uncoordinated manner to the previous axis of polarity. Additionally, hyphal filaments in the mutant developed aberrant branching sites by mislocalizing AgSpa2p thus distorting hyphal morphology. These results define AgRsr1p/Bud1p as a key regulator of hyphal growth guidance.

| S-EPMC519154 | biostudies-literature

Differential selection on gene translation efficiency between the filamentous fungus Ashbya gossypii and yeasts.

Project description:BackgroundThe filamentous fungus Ashbya gossypii grows into a multicellular mycelium that is distinct from the unicellular morphology of its closely related yeast species. It has been proposed that genes important for cell cycle regulation play central roles for such phenotypic differences. Because A. gossypii shares an almost identical set of cell cycle genes with the typical yeast Saccharomyces cerevisiae, the differences might occur at the level of orthologous gene regulation. Codon usage patterns were compared to identify orthologous genes with different gene regulation between A. gossypii and nine closely related yeast species.ResultsHere we identified 3,151 orthologous genes between A. gossypii and nine yeast species. Two groups of genes with significant differences in codon usage (gene translation efficiency) were identified between A. gossypii and yeasts. 333 genes (Group I) and 552 genes (Group II) have significantly higher translation efficiency in A. gossypii and yeasts, respectively. Functional enrichment and pathway analysis show that Group I genes are significantly enriched with cell cycle functions whereas Group II genes are biased toward metabolic functions.ConclusionBecause translation efficiency of a gene is closely related to its functional importance, the observed functional distributions of orthologous genes with different translation efficiency might account for phenotypic differentiation between A. gossypii and yeast species. The results shed light on the mechanisms for pseudohyphal growth in pathogenic yeast species.

| S-EPMC2632675 | biostudies-literature

Axl2 integrates polarity establishment, maintenance, and environmental stress response in the filamentous fungus Ashbya gossypii.

Project description:In budding yeast, new sites of polarity are chosen with each cell cycle and polarization is transient. In filamentous fungi, sites of polarity persist for extended periods of growth and new polarity sites can be established while existing sites are maintained. How the polarity establishment machinery functions in these distinct growth forms found in fungi is still not well understood. We have examined the function of Axl2, a transmembrane bud site selection protein discovered in Saccharomyces cerevisiae, in the filamentous fungus Ashbya gossypii. A. gossypii does not divide by budding and instead exhibits persistent highly polarized growth, and multiple axes of polarity coexist in one cell. A. gossypii axl2Δ (Agaxl2Δ) cells have wavy hyphae, bulbous tips, and a high frequency of branch initiations that fail to elongate, indicative of a polarity maintenance defect. Mutant colonies also have significantly lower radial growth and hyphal tip elongation speeds than wild-type colonies, and Agaxl2Δ hyphae have depolarized actin patches. Consistent with a function in polarity, AgAxl2 localizes to hyphal tips, branches, and septin rings. Unlike S. cerevisiae Axl2, AgAxl2 contains a Mid2 homology domain and may function to sense or respond to environmental stress. In support of this idea, hyphae lacking AgAxl2 also display hypersensitivity to heat, osmotic, and cell wall stresses. Axl2 serves to integrate polarity establishment, polarity maintenance, and environmental stress response for optimal polarized growth in A. gossypii.

| S-EPMC3232731 | biostudies-literature

Septin phosphorylation and coiled-coil domains function in cell and septin ring morphology in the filamentous fungus Ashbya gossypii.

Project description:Septins are a class of GTP-binding proteins conserved throughout many eukaryotes. Individual septin subunits associate with one another and assemble into heteromeric complexes that form filaments and higher-order structures in vivo. The mechanisms underlying the assembly and maintenance of higher-order structures in cells remain poorly understood. Septins in several organisms have been shown to be phosphorylated, although precisely how septin phosphorylation may be contributing to the formation of high-order septin structures is unknown. Four of the five septins expressed in the filamentous fungus, Ashbya gossypii, are phosphorylated, and we demonstrate here the diverse roles of these phosphorylation sites in septin ring formation and septin dynamics, as well as cell morphology and viability. Intriguingly, the alteration of specific sites in Cdc3p and Cdc11p leads to a complete loss of higher-order septin structures, implicating septin phosphorylation as a regulator of septin structure formation. Introducing phosphomimetic point mutations to specific sites in Cdc12p and Shs1p causes cell lethality, highlighting the importance of normal septin modification in overall cell function and health. In addition to discovering roles for phosphorylation, we also present diverse functions for conserved septin domains in the formation of septin higher-order structure. We previously showed the requirement for the Shs1p coiled-coil domain in limiting septin ring size and reveal here that, in contrast to Shs1p, the coiled-coil domains of Cdc11p and Cdc12p are required for septin ring formation. Our results as a whole reveal novel roles for septin phosphorylation and coiled-coil domains in regulating septin structure and function.

| S-EPMC3571309 | biostudies-literature

The small GTP-binding proteins AgRho2 and AgRho5 regulate tip-branching, maintenance of the growth axis and actin-ring-integrity in the filamentous fungus Ashbya gossypii.

Project description:GTPases of the Rho family are important molecular switches that regulate many basic cellular processes. The function of the Rho2 and Rho5 proteins from Saccharomyces cerevisiae and of their homologs in other species is poorly understood. Here, we report on the analysis of the AgRho2 and AgRho5 proteins of the filamentous fungus Ashbya gossypii. In contrast to S. cerevisiae mutants of both encoding genes displayed a strong morphological phenotype. The Agrho2 mutants showed defects in tip-branching, while Agrho5 mutants had a significantly decreased growth rate and failed to maintain their growth axis. In addition, the Agrho5 mutants had highly defective actin rings at septation sites. We also found that a deletion mutant of a putative GDP-GTP-exchange factor (GEF) that was homologous to a Rac-GEF from other species phenocopied the Agrho5 mutant, suggesting that both proteins act in the same pathway, but the AgRho5 protein has acquired functions that are fulfilled by Rac-proteins in other species.

| S-EPMC4149541 | biostudies-literature

One-vector CRISPR/Cas9 genome engineering of the industrial fungus Ashbya gossypii.

Project description:The filamentous fungus Ashbya gossypii is currently used for the industrial production of vitamin B2. Furthermore, the ability of A. gossypii to grow using low-cost substrates together with the inexpensive downstream processing makes this fungus an attractive biotechnological chassis. Indeed, the production in A. gossypii of other high-added value compounds such as folic acid, nucleosides and biolipids has been described. Hence, the development of new methods to expand the molecular toolkit for A. gossypii genomic manipulation constitutes an important issue for the biotechnology of this fungus. In this work, we present a one-vector CRISPR/Cas9 system for genomic engineering of A. gossypii. We demonstrate the efficiency of the system as a marker-less approach for nucleotide deletions and substitutions both with visible and invisible phenotypes. Particularly, the system has been validated for three types of genomic editions: gene inactivation, the genomic erasure of loxP scars and the introduction of point mutations. We anticipate that the use of the CRISPR/Cas9 system for A. gossypii will largely contribute to facilitate the genomic manipulations of this industrial fungus in a marker-less manner.

| S-EPMC6801137 | biostudies-literature

Phosphoribosyl pyrophosphate synthetase activity affects growth and riboflavin production in Ashbya gossypii.

Project description:BACKGROUND: Phosphoribosyl pyrophosphate (PRPP) is a central compound for cellular metabolism and may be considered as a link between carbon and nitrogen metabolism. PRPP is directly involved in the de novo and salvage biosynthesis of GTP, which is the immediate precursor of riboflavin. The industrial production of this vitamin using the fungus Ashbya gossypii is an important biotechnological process that is strongly influenced by substrate availability. RESULTS: Here we describe the characterization and manipulation of two genes of A. gossypii encoding PRPP synthetase (AGR371C and AGL080C). We show that the AGR371C and AGL080C gene products participate in PRPP synthesis and exhibit inhibition by ADP. We also observed a major contribution of AGL080C to total PRPP synthetase activity, which was confirmed by an evident growth defect of the Deltaagl080c strain. Moreover, we report the overexpression of wild-type and mutant deregulated isoforms of Agr371cp and Agl080cp that significantly enhanced the production of riboflavin in the engineered A. gossypii strains. CONCLUSION: It is shown that alterations in PRPP synthetase activity have pleiotropic effects on the fungal growth pattern and that an increase in PRPP synthetase enzymatic activity can be used to enhance riboflavin production in A. gossypii.

| S-EPMC2551608 | biostudies-literature

Developmental Growth Control Exerted via the Protein A Kinase Tpk2 in Ashbya gossypii.

Project description:Sporulation in Ashbya gossypii is induced by nutrient-limited conditions and leads to the formation of haploid spores. Using RNA-seq, we have determined a gene set induced upon sporulation, which bears considerable overlap with that of Saccharomyces cerevisiae but also contains A. gossypii-specific genes. Addition of cyclic AMP (cAMP) to nutrient-limited media blocks sporulation and represses the induction of sporulation specific genes. Deletion of the protein kinase A (PKA) catalytic subunits encoded by TPK1 and TPK2 showed reduced growth in tpk1 but enhanced growth in the tpk2 strain; however, both mutants sporulated well. Sporulation can be blocked by cAMP in tpk1 but not in tpk2 strains. Similarly, TPK2 acts at a second developmental switch promoting the break in spore dormancy. In S. cerevisiae, PKA phosphorylates and inhibits Msn2/4. The transcript profiles of the tpk1 and msn2/4 mutants were very similar to that of the wild type under sporulation conditions. However, deletion of the single A. gossypii MSN2/4 homolog generated a specific sporulation defect. We identified a set of genes involved in spore wall assembly that was downregulated in the msn2/4 mutant, particularly DIT2, suggesting that poor spore viability may be due to lysis of spores. Our results reveal specific functional differences between the two catalytic PKA subunits in A. gossypii and identified Tpk2 as the key A kinase that transduces developmental decisions of growth. Our data also suggest that Msn2/4 is involved only at a late step of sporulation in A. gossypii and is not a major regulator of IME1.

| S-EPMC4452567 | biostudies-literature

From function to shape: a novel role of a formin in morphogenesis of the fungus Ashbya gossypii.

Project description:Morphogenesis of filamentous ascomycetes includes continuously elongating hyphae, frequently emerging lateral branches, and, under certain circumstances, symmetrically dividing hyphal tips. We identified the formin AgBni1p of the model fungus Ashbya gossypii as an essential factor in these processes. AgBni1p is an essential protein apparently lacking functional overlaps with the two additional A. gossypii formins that are nonessential. Agbni1 null mutants fail to develop hyphae and instead expand to potato-shaped giant cells, which lack actin cables and thus tip-directed transport of secretory vesicles. Consistent with the essential role in hyphal development, AgBni1p locates to tips, but not to septa. The presence of a diaphanous autoregulatory domain (DAD) indicates that the activation of AgBni1p depends on Rho-type GTPases. Deletion of this domain, which should render AgBni1p constitutively active, completely changes the branching pattern of young hyphae. New axes of polarity are no longer established subapically (lateral branching) but by symmetric divisions of hyphal tips (tip splitting). In wild-type hyphae, tip splitting is induced much later and only at much higher elongation speed. When GTP-locked Rho-type GTPases were tested, only the young hyphae with mutated AgCdc42p split at their tips, similar to the DAD deletion mutant. Two-hybrid experiments confirmed that AgBni1p interacts with GTP-bound AgCdc42p. These data suggest a pathway for transforming one axis into two new axes of polar growth, in which an increased activation of AgBni1p by a pulse of activated AgCdc42p stimulates additional actin cable formation and tip-directed vesicle transport, thus enlarging and ultimately splitting the polarity site.

| S-EPMC1345653 | biostudies-literature

Cell polarity and hyphal morphogenesis are controlled by multiple rho-protein modules in the filamentous ascomycete Ashbya gossypii.

Project description:Polarized cell growth requires a polarized organization of the actin cytoskeleton. Small GTP-binding proteins of the Rho-family have been shown to be involved in the regulation of actin polarization as well as other processes. Hyphal growth in filamentous fungi represents an ideal model to investigate mechanisms involved in generating cell polarity and establishing polarized cell growth. Since a potential role of Rho-proteins has not been studied so far in filamentous fungi we isolated and characterized the Ashbya gossypii homologs of the Saccharomyces cerevisiae CDC42, CDC24, RHO1, and RHO3 genes. The AgCDC42 and AgCDC24 genes can both complement conditional mutations in the S. cerevisiae CDC42 and CDC24 genes and both proteins are required for the establishment of actin polarization in A. gossypii germ cells. Agrho1 mutants show a cell lysis phenotype. Null mutant strains of Agrho3 show periodic swelling of hyphal tips that is overcome by repolarization and polar hyphal growth in a manner resembling the germination pattern of spores. Thus different Rho-protein modules are required for distinct steps during polarized hyphal growth of A. gossypii.

| S-EPMC1461536 | biostudies-other

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