## 2 Results  
### Mechanical and Genetic Determinants of Substrate Invasion  
We developed a model for _C. albicans_ invasion of noncellular substrates that employed polyacrylamide hydrogel discs. Cells were initially inoculated on the surface and incubated at 37 \({}^{\circ}\)C in YPD+serum medium. At 48 h there was confluent biofilm growth, and the hypothesis that we explore below is that biofilm formation is necessary for invasion. We quantified the depth of cell invasion into the substrate through microscopy (Figure 1A). The gel surface was marked with fluorescent microparticles, and fungal chitin was stained with Calcofluor White. We then measured the maximum distance over which chitin was detected beneath the surface through fluorescence microscopy of a slice through the hydrogel. Figure 1: _Candida albicans_ invasion into an elastic substrate. (**A**) Schematic diagram of invasion assay. Fungal cells grow as a biofilm on the surface, and invading hyphae penetrate into the polyacrylamide substrate. Our observations argue that turgor is required for invasion; (**B**) Depth of hyphal invasion _vs._ acrylamide concentration in polymerized gel substrate. Hyphal invasion depths were measured from collected images. Each point on the graph indicates an independent measurement. Error-bars correspond to standard deviation around the mean, which is marked by the horizontal line; (**C**) Confocal micrographs of _C. albicans_ invasive growth into elastic hydrogels of increasing modulus of elasticity (Gpolyacryl.). Circular polyacrylamide discs containing _C. albicans_ biofilms were fixed, stained with Calcofluor white, and incubated with FluoSpheres to mark the surface. A 1–2 mm strip was then cut across the disc diameter, and rotated 90 degrees onto a cover-glass. Images were taken with 405 and 488 nm laser lines, then compiled and pseudo-colored green (Calcofluor white) and red (FluoSpheres). Numbers at bottom indicate Gpolyacryl for each hydrogel; (**D**) Relationship between _RHR2_ and invasion. Biofilms of the indicated strains were grown on polyacrylamide (18%/0.5% acryl./bis. ), and specimens were prepared for imaging as described above. We note that _rhr2 Al/A_ mutants are not defective in formation of hyphae [20]. The abundant yeast form cells in images of the mutant invasion assays represent yeast cells that sloughed off of the hydrogel surface and accumulated on the cover slip when the gel slice was rotated for imaging. We observed that substrate invasion was accomplished by hyphal cells, in keeping with observations from many other _C. albicans_ invasion models [24; 25; 26]. In addition, the invasion depth was inversely proportional to the acrylamide concentration in the substrate polymerization mixture (Figure 1B). Increases in the acrylamide concentration ultimately increase the rigidity of the substrate, an expectation that we verified by determination of the Young's modulus of each hydrogel (Figure S1A,B). Our data are consistent with a model (see Discussion and Supplemental Information) in which invasive growth of the hydrogel depends on the effective hyphal turgor. It seemed reasonable that biofilm formation on the surface of the substrate may be necessary for subsequent invasive growth. Our reasoning was that adhesion between the biofilm and the substrate would be necessary for mechanical penetration, much as has been found for the apprescoria of plant pathogens [27]. If that is the case, then biofilm-defective mutants should be defective in invasive growth. We tested this prediction through assays of invasive growth capacities among a panel of 150 _C. albicans_ transcription factor mutant strains [28]. Six mutants were defective in invasion (Table 1). Included in this group were five mutants known to have defects in both biofilm formation and hyphal morphogenesis. These mutants had individual deletions of the genes _BRG1_, _NDT80_, _EFG1_, _TEC1_, and _ROB1_[29; 30; 31; 32]. We discovered an additional mutant, _dbp4A/A_, that was defective in invasion.