## 2 Results
### RHR2 Function in Invasive Infection  
We sought to determine whether the _in vitro_ findings that point to two functions for _RHR2_ might be relevant to infection biology. Prior studies indicate that biofilm formation in a rat catheter infection model depends mainly upon the adhesin expression function of _RHR2_. We turned to an intraabdominal candidiasis (IAC) model of infection because gene expression data suggested that glycerol synthesis is up-regulated in this model [34]. Indeed, the _rhr24/l_ mutant was severely defective in infecting the peritoneum and intra-abdominal organs (Figure 3A-D). For example, at three days post-infection, mutant titers were severely reduced in the peritoneum, liver, spleen and pancreas compared to the wild-type and complemented strains. In addition, the _rhr24/l_ mutant had reduced persistence within intra-abdominal abscesses compared to wild-type and complemented strains (Figures 3E and S2). These results show that _RHR2_ is required for infection of organs and persistence in abscesses in the IAC infection model.  
Figure 2: Functional requirement for _RHR2_ in invasion. (**A**) Biofilm formation on polyacrylamide discs. Biofilms for the indicated strains were grown on polyacrylamide gel discs (18%/0.5% acryl./bis.) for 48 h, then photographed; (**B**) Gene expression analysis. The indicated strains were grown in YPD with 10% fetal bovine serum for the indicated strains for 8 h, and RNA was used for RT-qPCR measurements. _TDH3_ expression was used as the normalization standard. The normalized data were then used to compute expression changes relative to the wild-type strain; (**C**) Confocal micrographs of invading hyphae. Biofilms of the indicated strains were grown on polyacrylamide gel discs (18%/0.5% acryl./bis.) and prepared for imaging as described in the Figure 1 legend. The strains used were: wild type (DAY185), JVD006 (_rhr24/l_ + _pRHR2_), JVD005 (_rhr24/l_), JVD018 (_rhr24/l_ + _ALS1-OE_), JVD039 (_rhr24/l_ + _BCR1-OE_) and JVD051 (_rhr24/l_ + _UME6-OE_), and JVD065 (_rhr24/l_ + _BRG1-OE_).  
Figure 3: Association between _RHR2_ function and abdominal infection. (**A**–**D**) Fungal burdens in peritoneal fluid and abdominal organs. Three days after infection, the peritoneum was lavaged twice with 1 mL PBS each, and the peritoneal fluid was obtained for CFU enumeration. The abdominal organs (liver, spleen and pancreas) were also removed, homogenized and enumerated for fungal burden. The plot shows median log\({}_{10}\)CFU /L of peritoneal fluid or log\({}_{10}\)CFU/g of tissue for the indicated _C. albicans_ strains; (**E**) Fungal burden in intra-abdominal abscesses. Abscesses \(>\) 1 mm in size were excised and homogenized for CFU determination. The plot shows median log\({}_{10}\)CFU/g of abscesses for the indicated _C. albicans_ strains. Note that very few abscesses were present at day 14 in the animals infected with the _rhr2 Al/_ and _rhr2 Al/_ + _ALS1-OE_ strains. The strains used were wild type (DAY185), JVD006 (_rhr2 Al/_ + _pRHR2_), JVD005 (_rhr2 Al/_), and JVD018 (_rhr2 Al/_ + _ALS1-OE_). The capped lines on each plot show values from Mann-Whitney tests.  
These defects may arise from the _RHR2_ function in adhesin expression. To test this hypothesis, we examined the _rhr2 Al/A_ mutant that overexpresses _ALS1_ in the IAC infection model (Figure 3A-E). _ALS1_ overexpression restored the mutant's ability to infect the peritoneum and organs. However, _ALS1_ overexpression had no significant effect on persistence of the mutant within abscesses. These results argue that _RHR2_ has two functions in the IAC infection model: it is required for adhesin expression in order to infect organs, and it is required in some capacity beyond the expression of adhesin genes that enables persistence in abscesses.