Figure 1.
Morphology of Δdcw1, Δdfg5, and Δdfg5, Δdcw1 mutants.
Colonies of wild type, Δdcw1, Δdfg5, and Δdfg5, Δdcw1 mutants growing for 48 hours on Vogel’s sucrose medium in Petri dishes are shown in the upper panels. The lower panels show the morphology of hyphae growing at the edge of the colony growing between sheets of cellophane on a Vogel’s sucrose agar medium. The small black bar in the lower panel pictures is 5 µm in length.
Figure 2.
The wild type copy of dfg5 complements the Δdfg5 mutation.
Colonies of wild type, Δdfg5, and a Δdfg5 mutant that has been transformed with a wild type copy of the dfg5 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.
Figure 3.
RIP mutation of dcw1 in the Δdfg5 background recreates the Δdfg5, Δdcw1 phenotype.
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) Δdfg5 mutant, 3) Δdcw1 mutant, 4) Δdfg5, Δdcw1 double mutant, 5) Δdfg5, dcw1RIP mutant, 6) Δoch-1 mutant.
Figure 4.
The Δdfg5, Δdcw1 cell wall is deficient in cell wall protein.
Coomassie brilliant blue dye assay of cell wall protein. Increasing amounts of wild type, Δdfg5, Δdcw1, and Δdfg5, Δdcw1 cell walls were incubated in a solution of Coomassie Blue, and the amounts of dye absorbed by the cell walls were determined.
Figure 5.
SDS PAGE analysis of secreted protein shows that Δdfg5, Δdcw1 secretes large amounts of protein.
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 Δdcw1. Lane 3, proteins secreted by Δdfg5. Lane 4, proteins secreted by Δdfg5, Δdcw1.
Figure 6.
Western blot analysis of ACW-1 and GEL-1 secretion in wild type and mutant cells.
Western blot analyses using antibodies directed against ACW-1 (top panel) and GEL-1 (bottom panel) were carried out on the secreted protein from cells having 300 µg of cytosolic protein. ACW-1 and GEL-1 are two well-characterized N. crassa cell wall proteins. Lane 1, wild type secreted protein. Lane 2, Δdcw1 secreted protein. Lane 3, Δdfg5 secreted protein. Lane 4, Δdfg5, Δdcw1 secreted protein.
Table 1.
Linkage analysis of the carbohydrates present in wild type and mutant cell walls.