The <i>Δdfg5, Δdcw1</i> cell wall is deficient in cell wall protein.

<p>Coomassie brilliant blue dye assay of cell wall protein. Increasing amounts of wild type, <i>Δdfg5</i>, <i>Δdcw1</i>, and <i>Δdfg5, Δdcw1</i> cell walls were incubated in a solution of Coomassie Blue, and the amounts of dye absorbed by the cell walls were determined.</p

Linkage analysis of the carbohydrates present in wild type and mutant cell walls.

<p>The amounts of the different sugar linkages found in the analysis are given as a percentile of the total carbohydrate. The total percentage of mannose, galactose and glucose in the analysis was determined by the addition of all of the different linked mannose, galactose and glucose residues.</p

SDS PAGE analysis of secreted protein shows that <i>Δdfg5, Δdcw1</i> secretes large amounts of protein.

<p>Samples of secreted protein representing the amount of protein secreted from cells containing 300 µg of cytosolic protein were subjected to SDS PAGE and stained with silver stain. Lane 1, proteins secreted by wild type cells. Lane 2, proteins secreted by <i>Δdcw1</i>. Lane 3, proteins secreted by <i>Δdfg5</i>. Lane 4, proteins secreted by <i>Δdfg5, Δdcw1</i>.</p

RIP mutation of <i>dcw1</i> in the <i>Δdfg5</i> background recreates the <i>Δdfg5, Δdcw1</i> phenotype.

<p>Slants containing Vogel’s sucrose medium were inoculated with mutant and wild type isolates and grown for 48 hours at room temperature. The isolates shown include: 1) wild type (WT), 2) <i>Δdfg5</i> mutant, 3) <i>Δdcw1</i> mutant, 4) <i>Δdfg5, Δdcw1</i> double mutant, 5) <i>Δdfg5, dcw1RIP</i> mutant, 6) <i>Δoch-1</i> mutant.</p

The wild type copy of <i>dfg5</i> complements the <i>Δdfg5</i> mutation.

<p>Colonies of wild type, <i>Δdfg5</i>, and a <i>Δdfg5</i> mutant that has been transformed with a wild type copy of the <i>dfg5</i> gene (labeled as transformant) are shown. The colonies were inoculated in the middle of Petri dishes containing Vogel’s sucrose medium and grown for 48 hours at room temperature.</p

WSC-1 and HAM-7 Are MAK-1 MAP Kinase Pathway Sensors Required for Cell Wall Integrity and Hyphal Fusion in <em>Neurospora crassa</em>

<div><p>A large number of cell wall proteins are encoded in the <em>Neurospora crassa</em> genome. Strains carrying gene deletions of 65 predicted cell wall proteins were characterized. Deletion mutations in two of these genes (<em>wsc-1</em> and <em>ham-7</em>) have easily identified morphological and inhibitor-based defects. Their phenotypic characterization indicates that HAM-7 and WSC-1 function during cell-to-cell hyphal fusion and in cell wall integrity maintenance, respectively. <em>wsc-1</em> encodes a transmembrane protein with extensive homology to the yeast Wsc family of sensor proteins. In <em>N. crassa</em>, WSC-1 (and its homolog WSC-2) activates the cell wall integrity MAK-1 MAP kinase pathway. The GPI-anchored cell wall protein HAM-7 is required for cell-to-cell fusion and the sexual stages of the <em>N. crassa</em> life cycle. Like WSC-1, HAM-7 is required for activating MAK-1. A Δ<em>wsc-1;</em>Δ<em>ham-7</em> double mutant fully phenocopies mutants lacking components of the MAK-1 MAP kinase cascade. The data identify WSC-1 and HAM-7 as the major cell wall sensors that regulate two distinct MAK-1-dependent cellular activities, cell wall integrity and hyphal anastomosis, respectively.</p> </div

Δ<i>wsc-1</i>, Δ<i>ham-7</i>, and Δ<i>wsc-1;</i>

<p><b>Δ</b><b><i>ham-7</i></b><b> are deficient in MAK-1 activation.</b> (<b>A</b>) In the upper panel, extracts of non-stressed and oxidatively stressed wild type, Δ<i>wsc-1</i>, Δ<i>ham-7</i>, and Δ<i>wsc-1;</i> Δ<i>ham-7</i> cells were prepared and assayed for the presence of phosphorylated MAK-1 and MAK-2 by a Western blot assay using antibody that specifically recognizes the phosphorylated proteins. Extracts from non-stressed and from stressed cells are denoted by – and +. The sizes of the MAK-1 and MAK-2 proteins are shown at the side of the Western blot. A Western blot against tubulin was used as a control and to calibrate the amounts of protein in each of the samples. The amounts of activated MAK-1 (middle panel) and MAK-2 (lower panel) in each of the samples relative to the amount of activated MAK-1 or MAK-2 in the non-stressed wild type cell are shown (n = 5). (<b>B</b>) The colony extension rates for wild type, Δ<i>wsc-1</i>, Δ<i>ham-7</i>, Δ<i>wsc-1;</i> Δ<i>ham-7</i>, and Δ<i>mak-1</i> are shown. (<b>C</b>) The colony morphology (upper panel), the lack of protoperithecia production (middle panel), and lack of CAT fusion (lower panel) are shown for Δ<i>wsc-1;</i> Δ<i>ham-7</i>, and Δ<i>mak-1</i>.</p

Mutant Phenotypes.

<p>Mutant Phenotypes.</p

The Δ<i>wsc-1</i> mutant is deficient in MAK-1 activation.

<p>In the upper panel, extracts of non-stressed and oxidatively stressed wild type, Δ<i>wsc-1</i>, Δ<i>wsc-2</i>, and Δ<i>wsc-1;</i> Δ<i>wsc-2</i> cells were prepared and assayed for the presence of phosphorylated MAK-1 and MAK-2 by a Western blot assay using antibody that specifically recognizes the phosphorylated proteins. Extracts from non-stressed and from stressed cells are denoted by – and +. The sizes of the MAK-1 and MAK-2 proteins are shown at the side of the Western blot. A Western blot against tubulin was used as a control and to calibrate the amounts of protein in each of the samples. The amounts of activated MAK-1 (middle panel) and MAK-2 (lower panel) in each of the samples relative to the amount of activated MAK-1 or MAK-2 in the non-stressed wild type cell are shown (n = 5).</p

Complementation and RIP analysis of <i>wsc-1</i>.

<p>Colony morphologies are shown for wild type, Δ<i>wsc-1</i>, a transformant of Δ<i>wsc-1</i> containing a wild type copy of the <i>wsc-1</i> gene inserted at the <i>his-3</i> locus, and the <i>wsc-1^RIP1</i> mutant grown for 24 hours on a Vogel’s sucrose agar medium.</p