## 3 Discussion  
The ability to invade tissues is a critical element in the arsenal of virulence traits of many microbial pathogens. Two invasion strategies are known to be employed by _C. albicans_. One is considered a passive mechanism in which _C. albicans_ binds to host cell receptors to induce its endocytosis. The second is an active mechanism in which _C. albicans_ can penetrate tissues and, rarely, implanted-abiotic substrates as well [11, 15, 35, 36]. Here, we have focused on the active mechanism, with the specific goal of understanding the biological function of glycerol biosynthetic gene _RHR2_. Our study was motivated by two perplexing observations: that _RHR2_ is very highly expressed during biofilm growth, and that _RHR2_ is required for expression of several adhesin genes. Although _RHR2_ is required for biofilm formation _in vitro_ and _in vivo_, our studies indicated that this requirement reflected mainly the role of _RHR2_ in adhesin gene expression [21]. One biological rationale for the coupling of _RHR2_ and biofilm formation is that biofilm growth may generally precede substrate invasion. In that context, biofilm cells may express _RHR2_ and accumulate glycerol in order to generate the turgor necessary for active invasion. The results in this study support that idea with two lines of evidence. First, we show that biofilm formation ability correlates with active invasion in a simple _in vitro_ model. Second, we show that _RHR2_ is required for invasion in some capacity beyond the control of adhesin gene expression. In addition, we find that _RHR2_ is necessary for pathogenicity in an infection model, and that _RHR2_ has two separable functions in that context as well. Our observations argue that the two functions of _RHR2_ in invasion _in vitro_ are the stimulation of adhesin gene expression, and the generation of turgor. Adhesin expression enables biofilm formation, which occurs prior to invasion in our system. Our data suggest that turgor is required for invasion as well, based on a model in which hyphal invasion is represented as an expansion of a borehole in an infinite elastic medium (see Supplemental Information). According to the model, the extent of hyphal invasion into the substrate depends on the difference between hyphal turgor and the elastic modulus, or stiffness, of the substrate. The elastic modulus of polyacrylamide varies with the acrylamide concentration in the polymerization mixture, as confirmed by our direct measurements (Figure S1). The important observation is that hyphal invasion depth decreases linearly with increasing substrate elastic modulus. This relationship is expected if hyphal invasion is driven by hyphal turgor. How much turgor is required for invasion? Theoretical extrapolation from hyphal invasion depth _vs._ polyacrylamide elastic moduli data (Figure 1B) suggests that hyphae can accumulate effective turgor of 0.7-1.0 MPa (7-10 atm), comparable to what has been reported for single cells pressing against medical-grade silicone rubber, approximately \(\sim\)1.2 MPa [37]. The fact that biofilm-derived hyphae have greater turgor than planktonic hyphae is consistent with the higher glycerol content of biofilm cells compared to planktonic cells [21; 22]. Our studies also reveal that _RHR2_ is required for infection in an IAC model. We observed previously that _RHR2_ is dispensable for oropharyngeal candidiasis [21], and [PERSON] _et al._ found that _RHR2_ is required for a pathogenic interaction--specifically, damage--of oral epithelial cells [23]. The fact that these three infection models have different genetic requirements is consistent with our observation that gene expression profiles differ between the various infection environments [34; 38]. Our results argue that one function of _RHR2_ is to promote adhesin expression in this infection environment, because overexpression of _ALS1_ suppresses one aspect of the _rhr2A/A_ pathogenicity defect and enables organ infection.