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

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

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

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

Δ<i>ham-7</i> morphologies.

<p>In the upper panels, the colonial morphology of Δ<i>ham-7</i> and wild type are shown for cells grown on a Vogel’s sucrose agar medium for 48 hours. The morphology of the hyphae at the edge of a 24 hour colony growing between two sheets of cellophane is shown for wild type and mutant cells in the lower panels. The black bar represents a distance of 20 µm.</p