45 research outputs found

    VIB1 is not required for cellobiose sensing or signaling.

    No full text
    <p>(A) Expression levels of two cellulase genes (<i>cbh-1</i> and <i>gh5-1</i>) were assessed in the Δ3βG; <i>Δvib-1</i> strain versus WT, and the Δ3βG and <i>Δvib-1</i> strains after a shift from sucrose VMM to 0.2% cellobiose versus 2% Avicel. Gene expression levels were measured by relative quantitative-PCR using actin as a control and normalized to expression level when cultures were shifted to VMM with no carbon source. (B) CMCase activity after 24 hrs of growth on 2% cellobiose of the Δ3βG; <i>Δvib-1</i> strain relative to the WT, and the Δ3βG and <i>Δvib-1</i> strains. Measured enzyme activity in arbitrary unit (AU) was normalized against to the mycelial biomass of each culture.</p

    Constitutive expression of <i>clr-2</i> rescued the cellulase production defect of the Δ<i>vib-1</i> mutant.

    No full text
    <p>(A) Protein concentration and cellulase activity in a Δ<i>vib-1</i> mutant versus a Δ<i>vib-1</i> strain constitutively expressing <i>clr-2</i> (Pc <i>clr-2</i>; Δ<i>vib-1</i>) and WT and a Pc <i>clr-2</i> strain under Avicel conditions. (B) Expression levels from RNA-seq data of genes encoding major classes of CAZy proteins from WT and Δ<i>vib-1</i> shifted to Avicel versus the Pc <i>clr-2</i> and Pc <i>clr-2</i>; Δ<i>vib-</i>1 strains shifted to minimal media with no carbon source. FPKM (Fragment Per Kilobase per exon per Megabase mapped) for individual genes were averaged between three biological replicates and pooled by CAZy class. (C) Hierarchical clustering of FPKM for 91 Avicel-regulon genes in the Δ<i>vib-1</i> mutant and WT on Avicel (Av) and the Δ<i>vib-1</i>, WT, Pc <i>clr-2</i> and the Pc <i>clr-2</i>; Δ<i>vib-</i>1 strains switched to no carbon conditions (Nc). Results are displayed as heat maps with log (FPKM) from minimum (bright blue) to maximum (bright yellow).</p

    Deletion of <i>vib-1</i> abolishes production of cellulases and utilization of cellulosic material.

    No full text
    <p>(A) Growth of WT and Δ<i>vib-1</i> on Avicel after 4 days; growth of WT is indicated by formation of orange mycelia, versus no growth of the Δ<i>vib-1</i> mutant. (B) Cellulase activity from 4-day old culture supernatants from Avicel-grown cultures of WT, the Δ<i>vib-1</i> mutant, the P<i>vib-1</i> strain (constitutive expression of <i>vib-1</i> in a Δ<i>vib-1</i> strain) and the P<i>Trvib1</i> strain (constitutive expression of <i>T. reesei vib1</i> in a Δ<i>vib-1</i> strain). Cellulase activity was measured using Avicel as a substrate and represented by the amount of glucose and cellobiose released. The equivalent of glucose from cellobiose was calculated and represented by the light gray bar. (C) The secretomes of strains analyzed in panel B are shown.</p

    Deletion of <i>col-26</i> causes defects in glucose sensing/metabolism.

    No full text
    <p>(A) Mycelial biomass of the Δ<i>col-26</i> mutant relative to WT and the Δ<i>cre-1</i> strains on glucose, fructose, sucrose, or cellobiose. Mycelial biomass was measured at 24 hrs after inoculation. (B) Glucose uptake of WT, Δ<i>vib-1</i>, Δ<i>cre-1</i>, Δ<i>col-26</i>; Δ<i>vib-1</i> and the Δ<i>cre-1</i>; Δ<i>col-26</i>; Δ<i>vib-1</i> mutants was assayed by monitoring glucose remaining in the medium at 5 min, 20 min, and 60 min from cultures of identical biomass.</p

    VIB1, a Link between Glucose Signaling and Carbon Catabolite Repression, Is Essential for Plant Cell Wall Degradation by <i>Neurospora crassa</i>

    No full text
    <div><p>Filamentous fungi that thrive on plant biomass are the major producers of hydrolytic enzymes used to decompose lignocellulose for biofuel production. Although induction of cellulases is regulated at the transcriptional level, how filamentous fungi sense and signal carbon-limited conditions to coordinate cell metabolism and regulate cellulolytic enzyme production is not well characterized. By screening a transcription factor deletion set in the filamentous fungus <i>Neurospora crassa</i> for mutants unable to grow on cellulosic materials, we identified a role for the transcription factor, VIB1, as essential for cellulose utilization. VIB1 does not directly regulate hydrolytic enzyme gene expression or function in cellulosic inducer signaling/processing, but affects the expression level of an essential regulator of hydrolytic enzyme genes, CLR2. Transcriptional profiling of a Δ<i>vib-1</i> mutant suggests that it has an improper expression of genes functioning in metabolism and energy and a deregulation of carbon catabolite repression (CCR). By characterizing new genes, we demonstrate that the transcription factor, COL26, is critical for intracellular glucose sensing/metabolism and plays a role in CCR by negatively regulating <i>cre-1</i> expression. Deletion of the major player in CCR, <i>cre-1</i>, or a deletion of <i>col-26</i>, did not rescue the growth of Δ<i>vib-1</i> on cellulose. However, the synergistic effect of the Δ<i>cre-1</i>; Δ<i>col-26</i> mutations circumvented the requirement of VIB1 for cellulase gene expression, enzyme secretion and cellulose deconstruction. Our findings support a function of VIB1 in repressing both glucose signaling and CCR under carbon-limited conditions, thus enabling a proper cellular response for plant biomass deconstruction and utilization.</p></div

    Cellulase production in <i>N. crassa</i> is regulated by cellobiose induction and CCR.

    No full text
    <p>CCR is decreased in absence of glucose, allowing scouting enzymes to liberate cellobiose from cellulose. Cellobiose (or a derivative) results in activation of the transcription factor CLR1, which induces expression of transporters for cellodextrins, β-glucosidases, and <i>clr-2</i>. Production of the transcription factor CLR2 drives cellulase gene expression. Both intracellular and extracellular β-glucosidase enzymes catalyze conversion of cellobiose to glucose, which can trigger carbon catabolite repression via glucose sensing mechanisms and transcriptional repression by CRE1.</p

    Simultaneous deletion of <i>cre-1</i> and <i>col-26</i> rescues the phenotype of Δ<i>vib-1</i> on cellulose.

    No full text
    <p>(A) Cellulase activity of culture supernatants after 4-days of growth on Avicel from WT versus Δ<i>vib-1</i>, Δ<i>cre-1</i>, Δ<i>col-26</i>, Δ<i>cre-1</i>; Δ<i>vib-1</i>, Δ<i>col-26</i>; Δ<i>vib-1</i> and Δ<i>cre-1</i>; Δ<i>col-26</i>; Δ<i>vib-1</i> strains. (B) RT-PCR measurements of <i>clr-2</i> and <i>cbh-1</i> expression in the WT versus the Δ<i>col-26</i>, Δ<i>cre-1</i>, and the Δ<i>cre-1</i>; Δ<i>col-26</i>; Δ<i>vib-1</i> cultures after 5-days of growth on Avicel. Expression levels were normalized to WT. (C) The CMCase activity of Avicel cultures of WT versus Δ<i>col-26</i>, Δ<i>cre-1</i>, and the Δ<i>cre-1</i>; Δ<i>col-26</i>; Δ<i>vib-1</i> mutants during a time course of growth on Avicel.</p

    Screen for function of new proteins involved in CCR.

    No full text
    <p>(A) Growth assays of the WT, <i>Δvib-1</i>, Δ<i>cre-1</i>, Δ<i>col-26</i>, Δ<i>creB</i>, and Δ<i>creD</i> strains on 2-deoxy-glucose (2-DG) when grown on 2% cellobiose VMM for 2 days or on 2% Avicel VMM for 4 days. (B) Effects of <i>col-26</i> and <i>cre-1</i> deletions on sensitivity to 2-DG and allyl alcohol. Strains were inoculated and grown in 2% cellobiose VMM with 100 mM allyl alcohol for 40 hrs. For 2-DG sensitivity tests, the strains were inoculated and grown in 2% Avicel with either 0.2% 2-DG or 0.5% 2-DG for 5 days.</p

    Suppression of <i>cre-1</i> and <i>col-26</i> expression by VIB1 plays a role in early inductive and utilization phase during growth on cellulose.

    No full text
    <p>The transcriptional expression of <i>cre-1</i>, <i>vib-1</i>, <i>col-26</i>, <i>clr-2</i>, and <i>cbh-1</i> were measured by RT-PCR at 4 hrs (A) and 24 hrs (B) after 16 hr sucrose growth cultures were transferred to Avicel conditions. Expression levels were normalized to WT.</p

    DOC-1-GFP co-oscillates with MAK-2 during chemotropic interactions.

    No full text
    <p>(A) Germlings expressing DOC-1-GFP (green) were paired with germlings expressing MAK-2-mCherry (red). When MAK-2-mCherry accumulated at one CAT tip (arrows left and right panel), DOC-1-GFP was absent from the tip of the interacting germling. When DOC-1-GFP accumulated at the second CAT tip (arrow middle panel), MAK-2-mCherry was absent from the first CAT tip, indicating that DOC-1-GFP showed identical oscillation dynamics to MAK-2-mCherry. (B) Germlings expressing DOC-1-GFP (green) were paired with germlings expressing SOFT-mCherry (red). When SOFT-mCherry accumulated at one CAT tip, DOC-1-GFP accumulated at the CAT tip of its interacting partner (arrows left and right panel). When SOFT-mCherry was absent from the first CAT tip, DOC-1-GFP was absent from the second CAT tip (middle panel), indicating that DOC-1-GFP showed opposite oscillation dynamics to SOFT-mCherry. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002431#pbio.1002431.s008" target="_blank">S5B Fig</a> for oscillation intervals. (C) Co-localization and co-oscillation of DOC-1-GFP (left panel) with MAK-2-mCherry (middle panel) in heterokaryotic germlings undergoing chemotropic interactions (overlay, right panel). Bottom panel: Graphic representation of DOC-1-GFP and MAK-2-mCherry signals at the tips of conidial anastomosis tubes in germlings undergoing chemotropic interactions. <i>y</i>-axis shows the ratio of relative fluorescence intensity (RFI) in the interacting zone as compared to background. <i>x</i>-axis shows time (min). Note the co-oscillation of both DOC-1-GFP and MAK-2-mCherry in both germlings following fusion, as shown previously for MAK-2 and SOFT [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002431#pbio.1002431.ref045" target="_blank">45</a>].</p
    corecore