Program to compute protein abundance in mass spectrometry

The program is to compute protein abundance from spectral counts from mass spectrometry. It computes protein abundance from unique peptides and hared peptides among proteins considered. It is also available at http://kiharalab.org/RACalculator

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

<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

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

<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

MyoE localization.

<p>A–F are images of the same field and are maximum intensity projections of a Z-series stack. A–C show the co-localization of MyoE-GFP and mCherry-SynA at the Spitzenkörper (arrows). SynA localizes to the Spitzenkörper and to the plasma membrane near the apex (B). In D–F, the thresholds are chosen to reveal the punctate staining in the hypha while overexposing the MyoE-GFP and mCherry-SynA at the hyphal tip. MyoE-GFP localizes to numerous small puncta and some larger structures that may be endosomes (e.g. arrow). G. Faint localization of MyoE-GFP at forming septa (arrows). H. A three-dimensional projection of a hyphal tip showing MyoE-GFP and mCherry-SynA. Although MyoE and SynA co-localize at the Spitzenkörper, many puncta behind the tip show GFP fluorescence or mCherry fluorescence, but it was not clear that there was any obligate co-localization.</p

Deletion of myoB inhibits septum formation.

<p>All panels are images of living cells. In A and B, nuclei are shown with histone H1-mRFP and chitin is stained with calcofluor (10 µg/ml). A. a <i>myoB</i>⁺ strain (LO1516). Multiple septa are visible (arrows). B. a <i>myoB</i>Δ hypha. The <i>myoB</i> gene was deleted in LO1516 and nuclei carrying the deletion were maintained in a heterokaryon. No septa are present but there are thickened regions containing chitin (e.g. arrow) and chitin is highly concentrated near the hyphal tip. C. Shows a hyhal tip region in a <i>myoB</i>Δ strain stained with calcofluor but nuclei are not imaged. Note the absence of septa and side branches. The circular objects are ungerminated conidia resulting from the heterokaryon rescue technique.</p

VIB1 is not required for cellobiose sensing or signaling.

<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

Screen for function of new proteins involved in CCR.

<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.

<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

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

<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

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

<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