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

Complementation of CAT fusion activities by different HA, GFP and RFP tagged proteins.

<p>A) The levels of CAT fusion activity for the gene deletion mutants and for transformants expressing a tagged version of the deleted gene are shown as a percentile of the cell fusion activity for wild type CATs. B) Photograph of CAT fusion activities in wild type (WT), <i>Δham-8</i>, and <i>Δham-8</i> transformed with <i>HA-ham-8</i>. Arrows point to examples of CAT fusion in the wild-type and <i>Δham-8</i> transformed with <i>HA-ham-8</i> panels.</p

Characterization of the <i>Neurospora crassa</i> Cell Fusion Proteins, HAM-6, HAM-7, HAM-8, HAM-9, HAM-10, AMPH-1 and WHI-2

<div><p>Intercellular communication of vegetative cells and their subsequent cell fusion is vital for different aspects of growth, fitness, and differentiation of filamentous fungi. Cell fusion between germinating spores is important for early colony establishment, while hyphal fusion in the mature colony facilitates the movement of resources and organelles throughout an established colony. Approximately 50 proteins have been shown to be important for somatic cell-cell communication and fusion in the model filamentous fungus <i>Neurospora crassa</i>. Genetic, biochemical, and microscopic techniques were used to characterize the functions of seven previously poorly characterized cell fusion proteins. HAM-6, HAM-7 and HAM-8 share functional characteristics and are proposed to function in the same signaling network. Our data suggest that these proteins may form a sensor complex at the cell wall/plasma membrane for the MAK-1 cell wall integrity mitogen-activated protein kinase (MAPK) pathway. We also demonstrate that HAM-9, HAM-10, AMPH-1 and WHI-2 have more general functions and are required for normal growth and development. The activation status of the MAK-1 and MAK-2 MAPK pathways are altered in mutants lacking these proteins. We propose that these proteins may function to coordinate the activities of the two MAPK modules with other signaling pathways during cell fusion.</p></div

MAK-2-GFP localization in wild type and mutant germ tubes/CATs.

<p>MAK-2-GFP expressing wild type (WT) and mutant conidia were grown under CAT induction conditions for 4 hours. DIC images (left column), GFP fluorescent images (middle column), and merged images (right column) are shown. The images show germ tubes/CATs for wild type (WT) (row 1), <i>Δham-6</i> (row 2), <i>Δham-7</i> (row 3), <i>Δham-8</i> (row 4), <i>Δham-9</i> (row 5), <i>Δham-10</i> (row 6), <i>Δamph-1</i> (row 7), and <i>Δwhi-2</i> (row 8). The arrows in the WT GFP fluorescent image point to the localization of MAK-2-GFP at the sites of cell fusion.</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

Fusion between MAK-2-GFP-expressing cell fusion mutants and RFP-expressing wild type cells.

<p>Conidia samples containing equal number of RFP-expressing wild type conidia and MAK-2-GFP-expressing wild type or mutant conidia were grown under CAT induction conditions for 4 hours. DIC images, GFP fluorescent images, RFP fluorescent images, and merged images for each combination of conidia types are shown in the columns from left to right respectively. Each row shows the images for RFP-expressing wild type conidia mixed with MAK-2-GFP-expressing wild type (WT) (row 1), <i>Δham-6</i> (row 2), <i>Δham-7</i> (row 3), <i>Δham-8</i> (row 4), <i>Δham-9</i> (row 5), <i>Δham-10</i> (row 6), <i>Δamph-1</i> (row 7), and <i>Δwhi-2</i> (row 8) conidia. Wild type conidia frequently engaged in cell fusion, while <i>Δamph-1</i> and <i>Δwhi-2</i> conidia engaged in cell fusion with w conidia at a low frequency.</p