Coexpression of DN CtCdc42 (DN Cdc42-1 and DN Cdc42-2) or antisense CtCdc42 (As Cdc42) in a DA Ras mutant leads to phenotypic restoration and appressorial formation under noninducible conditions. The double mutants were grown on minimal medium for 6 days, and the morphologies of hyphae were observed with light microscopy. To detect appressorial development, spores of two double mutants (DA Ras As Cdc42 and DA Ras DN Cdc42-1) were inoculated onto soft water agar medium (1.6%) and cell morphologies were observed with light microscopy after 24 h postinoculation. (A to D) Hyphal phenotypes after 6 days of incubation. A, wild type; B, DA Ras; C, DA Ras DN Cdc42-1; D, DA Ras DN Cdc42-2. (E and F) Development of appressoria on noninducible, soft water agar medium following 24 h of incubation. E, DA Ras As Cdc42 (arrows show the generation of appressoria); F, DA Ras DN Cdc42-1.