Table S1 presents information on the strains, and Table S2 shows the primers used in this study. For general growth and propagation, C. albicans strains were cultured at 30°C in yeast-extract–peptone–dextrose (YPD) medium (1% yeast extract, 2% peptone, 2% dextrose, and 2% agar). First, C. albicans mutants and complement strains were created following previous studies (Noble and Johnson, 2005; Nobile et al., 2009; Noble et al., 2010), and the overexpression strain was examined as previously described (Gerami-Nejad et al., 2013). In brief, the gene-knockout strategy of the present study involved gene knockout using polymerase chain reaction (PCR)-based homologous recombination. This strategy uses C. albicans SN152 with defective histidine (HIS), leucine (LEU), and arginine (ARG) genes as the parent strain and HIS1 (Cd. HIS1) from C. dubliniensis, LEU2 (C. maltose LEU2, Cm.LEU2) from C. maltose, and ARG4 (C. dubliniensis ARG4, Cd. ARG4) from C. dubliniensis as the target genes to replace nutritional-marker genes for selection. This method typically knocks out the target genes one after the other, and two screening markers can be arbitrarily selected to knock out the target gene. In this study, the LEU2 cassette from plasmid pSN40 was amplified, and the strain SN152 was transformed with the fusion PCR products of LEU2 flanked by QCR7 5′ and 3′ fragments. The plasmid pSN52 was used as a PCR template to amplify the HIS1 cassette, and the second QCR7 allele was deleted using fusion PCR products of the HIS1 marker flanked by QCR7 5′- and 3′-fragments ( Figure S1 ). A copy of QCR7 was returned to the QCR7 locus to construct the gene-reconstituted strain. The entire QCR7 open-reading frame, including 743 bp of the 5′end, was amplified. Next, the entire QCR7 open-reading frame with upstream and the ARG4 cassette from plasmid pSN69 and downstream flanks (approximately 400 bp) were amplified using fusion PCR assays. The PCR products were transformed into the qcr7Δ/Δ mutant to construct the qcr7/QCR7 gene-reconstituted strain (Noble and Johnson, 2005) ( Figure S2 ). The qcr7Δ/Δ mutant and reconstituted strain (qcr7/QCR7) were confirmed through PCR with the primer pairs shown in Supplementary Figure S3 . However, the gene-overexpression strategy of the present study involved the amplification of the coding region of the corresponding genes by PCR and downstream cloning of the S. cerevisiae ADH1 promoter to integrate inserts into a large intergenic region, NEUT5L which facilitates the integration and expression of ectopic genes (Gerami-Nejad et al., 2013) ( Figure S4 ).

To evaluate the growth of C. albicans strains cultured with different carbon sources, strains were cultured overnight in YPD. Strains diluted to varying concentrations (5 μl of 10⁷ cells/ml to 10³ cells/ml) were spotted on yeast-extract–peptone solid medium (YEP; 1% yeast extract, 2% peptone). Next, washing and serial dilution with phosphate-buffered saline (PBS) were performed. This medium contained 2% glucose, 2% glycerol, 2% citrate, 2% acetate, 2% maltose, 2% ethanol, 2% lactate, and 50 mM GlcNAc. Subsequently, the sensitivity of C. albicans to cell-wall stress was assessed by spotting on YPD containing cell-wall stressors, such as Congo red (CR, 300 µg/ml), calcofluor white (CFW, 50 µg/ml), 0.04%SDS and caspofungin (0.25 µg/ml).

We evaluated the biofilm-formation mass of C. albicans using crystal-violet staining as described previously (Silva et al., 2009; Abbes et al., 2017; Neji et al., 2017). First, strains were cultured overnight in liquid YPD at 30°C and 220 rpm and then harvested via centrifugation at 3,000 rpm after washing twice with sterile PBS. Subsequently, the mixture was resuspended in Spider liquid medium. Next, supplementation was performed using appropriate carbon sources and 2-mililiter C. albicans-strain suspensions (1 ml containing 5 × 10⁶ cells) and the suspensions were incubated overnight in 12-well flat-bottomed plates containing fetal bovine serum (FBS) before the final incubation for 90 min at 37°C. Next, the nonattached cells were removed from the wells by washing once with 2 ml PBS, after which 2 ml of fresh corresponding induction medium was added to each well and incubated at 37°C for 48 h. Following the removal of medium, each well was washed once with PBS and treated with equivalent concentrations of methanol for 30 min. Next, equivalent concentrations of 1% crystal violet (Solarbio, China) were added to the wells for drying, and staining was performed for 1 h. Washing was performed under a gentle water flow until a colorless mixture was obtained, followed by incubation with equivalent amounts of acetic acid for 30 min to decolorize the mixture. Measurements were performed at 620 nm using a microplate reader.

The wild-type (WT) strain was cultured overnight in YPD, followed by washing and dilution with PBS to achieve a suspension with OD₆₀₀ = 0.1Abs. Next, the mixture was aliquoted in Spider liquid medium using appropriate carbon sources such as fermentable sugars (e.g., 2% glucose, 2% maltose, and 2% sucrose) or alternative carbon sources (e.g., 2% mannitol and 50mM GlcNAc) for hyphal induction at 37°C (Huang et al., 2017). The cells were harvested, and hyphal morphologies were visualized under a fluorescence microscope (Olympus, Japan) at indicated time points. Subsequently, each cell type (1 × 10⁵ cells in 5 µl of PBS) was spotted on Spider solid medium with appropriate carbon sources. The cultures were then incubated at 37°C, and photographs were taken after 7 days.

Kidney tissues were paraffin-embedded sectioned, Each slide was dewaxed by xylene, ethanol treatment, and antigen repair with citric acid as the repair solution, after which it was closed with a serum of the same source as the secondary antibody (usually 10% goat serum was used) and incubated for 30 min at 37°C (Henkels et al., 2016; Sasaki et al., 2021). Then treated slides with anti-Ly-6G (Servicebio; GB11229), anti-F4/80 antibodies (28463-1-AP, Proteintech, Wuhan, China) overnight at 4°C. All slides were washed with PBS before incubation with FITC-labeled goat anti-mouse antibody and CY3-labeled goat anti-mouse antibody (Pinuofei Biotechnology Co, China). After PBS rinsing, the slides were exposed to DAPI for 5 min to stain cell nuclei. Then, the slides were analyzed under a fluorescence microscope.

To determine the mitochondrial membrane potential (MMP), the MMP assay kit with JC-1 (Beyotime Institute of Biotechnology) was used. MMP decreases can be easily detected through JC-1’s transition from red to green fluorescence (She et al., 2015). Cells were collected when strains subcultured in fresh medium reached the log phase of growth and incubated with equal volumes of JC-1 dyeing solution at 37°C for 15 min in the dark. Cells were washed twice and resuspended in JC-1 dyeing solution. Next, we explored the emission spectra at 488 nm and excitation spectra at 595 nm using the Cytomics FC500 flow cytometer (Beckman Coulter); the red/green mean fluorescence intensities (FIs) were recorded for each sample.

All strains were cultured overnight in YPD at 30°C and 220 rpm, followed by washing and resuspension in PBS. Subsequently, 2 × 10⁶ cells were stained using fluorescent dichlorodihydro fluorescein diacetate dye (20 µg/ml) (Guo et al., 2014). Next, these cells were incubated at 37°C for 20 min in the dark. Subsequently, FIs were recorded using a multifunctional enzyme-mark instrument (SpectraMax Paradigm) with an excitation wavelength of 480 nm and emission wavelength of 530 nm. Finally, the levels of reactive oxygen species (ROS) were determined by evaluating each strain thrice.

All strains were cultured overnight in YPD at 30°C. Cells were then subcultured in fresh medium until the log phase of growth was reached. Next, 2 × 10⁶ cells of each strain were mixed completely with equal volumes of the BacTiter-Glo reagent (Promega Corporation, Madison, WI) (Lin et al., 2019; Li et al., 2021), followed by incubation at room temperature for 15 min in the dark. Finally, luminescent signals were detected using the full-wavelength multifunctional enzyme-mark instrument (SpectraMax Paradigm).

Secreted aspartyl protease (Sap) activity assays were performed using 0.17% yeast-nitrogen-base (YNB) medium (without aa or AS) + 0.1% BSA, containing 2% glucose as the carbon source and bovine serum albumin (BSA])as the sole nitrogen source (Crandall and Edwards, 1987). Water was used to suspend 1 × 10⁵ cells, after which they were spotted on YNB–BSA agar plates and incubated at 37°C for 5 days. The size of the white halo rings indicated the activity level. This experiment was repeated thrice.

Through transcriptome sequencing, we evaluated the global transcriptional response of C. albicans in RPMI 1640. All strains were cultured overnight in YPD at 30°C and subcultured in fresh YPD (buffered) at 30°C. Subsequently, C. albicans cells at the log phase of growth were resuspended in RPMI 1640, followed by incubation at 37°C for 4h according to the manufacturer’s instructions. Next, RNA extraction was performed using TRIzol (TIANGEN). In brief, 5 × 10⁶ cells were isolated and added to a precooled mortar. The cells were then grounded to powder with liquid nitrogen, after which the sample was stirred in 1 ml TRIzol for 10 min. Subsequently, chloroform (200 µl) was added to separate the aqueous RNA-containing solution from the intermediate and organic layers, which was followed by RNA precipitation using isopropyl alcohol (equal volume) and the addition of 1 ml ethanol (75%) to each RNA pellet. Finally, the samples were centrifuged (12,000 rpm/5 min/4°C), and the RNA was resuspended in 20 μL nuclease-free water and then stored at −80°C. Furthermore, the RNA samples were assessed using Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and NanoPhotometer spectrophotometer (Implen). Triplicate samples used in all assays were used to construct an independent library, and the sequencing and analysis were performed. The libraries for sequencing were constructed using the NEB Next Ultra RNA Library Prep Kit for Illumina (NEB). The poly (A)-tailed mRNA was enriched using the NEB Next Poly (A) mRNA Magnetic Isolation Module kit. The mRNA was fragmented into segments of nearly 200 bp. First-strand cDNA was synthesized from the mRNA fragments through reverse transcription using random hexamer primers. Next, second-strand cDNA was synthesized using DNA polymerase I and RNaseH. The end of the cDNA fragment was subjected to an end repair that included adding a single “A” base, followed by adapter ligation. PCR was used to purify and enrich the products to construct a DNA library. The final libraries were quantified using Qubit 2.0 Fluorometer (Invitrogen) and Agilent 2100 Bioanalyzer. After performing validation through quantitative reverse transcription-PCR, libraries were subjected to paired-end sequencing with a reading length of 150 bp using Illumina Novaseq 6000 (Illumina).

All the strains were cultured overnight in YPD at 30°C and subcultured in fresh YPD (buffered) at 30°C until growth at the log phase. Subsequently, cells were resuspended in RPMI 1640, followed by incubation at 37°C for 4h. The methods for RNA extraction were those described above. Extracted RNA was treated with DNase I (Takara) to remove contaminating DNA, and cDNA was synthesized with a PrimeScript™ RT Reagent Kit (Takara) following the manufacturer’s protocol. The cDNA was used as template for RT–qPCR using a SYBR Green mix kit (Mei5 Biotechnology Co., Ltd.) on a CFX Connect Real-Time PCR Detection System. The 18S rRNA genes were used as endogenous controls as specified. The results were analyzed using the 2^-ΔΔCT method. The primers for the genes used in the RT-qPCR are shown in Supplementary Table S2 .

All experiments were biologically replicated thrice. Data were presented as means ± standard errors of the means. Statistical analyses were performed using GraphPad Prism 8 software. Significant differences were calculated using two-way analyses of variance and Student’s t-tests.

Previous studies identified that disabling Complex III can prevent fungal adaptation to nutrient deprivation, sensitize C. albicans to attack by macrophages, and curtail virulence in mice (Vincent et al., 2016). However, in these studies, the catalytic subunit RIP1 was chosen as a representative and used in the remaining experiments. It is unclear whether other subunits are equally important for the functional performance of mitochondrial Complex III (CIII) and the maintenance of C. albicans virulence. In this study, we identified ten genes (COB, RIP1, CYR1, COR1, QCR2, QCR6, QCR7, QCR8, QCR9, and QCR10) encoding the subunit of mitochondrial Complex III using the Candida Genome Database (CGD, http://www.candidagenome.org/). Among them, apart from cobΔ/Δ and cyr1Δ/Δ mutants, which were inviable, we successfully knocked out the other eight subunits and tested the utilization of several carbon sources as energy sources for the pathogens. As shown in Figure 1A , in the right panel, in media with glucose, sucrose, and mannitol as carbon sources, rip1Δ/Δ, cor1Δ/Δ, qcr2Δ/Δ, qcr7Δ/Δ, and qcr8Δ/Δ showed a similar lag phase. However, the above mutant displayed remarkable growth defects compared to that of the WT in media with other host-relevant carbohydrates (e.g., maltose, citrate, acetate) as carbon sources. To examine the effect of Complex III mutants on the major virulence factors associated with C. albicans, the panel of mutants was examined for their biofilm-formation ability, hyphal growth, etc. Consistent with an alteration in carbon assimilation, the corresponding mutants also displayed defects in hyphal growth on solid media and were unable to form the dense matrix of the biofilm ( Figures 1B, C ). The remaining subunit formed Complex III; these coding gene mutants showed a similar phenotype to the wild-type strain. Surprisingly, we found that the absence of QCR7 had a more evident phenotypic impact on the ability of C. albicans to maintain hyphal growth on solid media and biofilm formation than the other subunit knockout strains. The results indicate that Qcr7 is critical to virulence in C. albicans. Thus, a series of experimental studies were conducted on QCR7, as described below.

Based on the above findings, it is reasonable to assume that QCR7 mutant could influence the fatal infections of C. albicans. Previous study showed the inhibition of Complex III was effective in attenuating the virulence of C. albicans in mice (Vincent et al., 2016). Therefore, we performed relevant experiments and analyzed in a mouse model of systemic candidiasis to investigate the effect of Qcr7 on C.albicans-infected hosts. The survival rate of the mice injected with the qcr7Δ/Δ mutant was much higher than that of those injected with the WT and QCR7 complement strains (QCR7 AB). Furthermore, no mice died within 3 weeks of infection with the qcr7Δ/Δ mutant ( Figure 2A ). During pathological characterization, the kidneys, the main target organ, of the infected mice served as the key indicators of host infection (Leunk and Moon, 1979). Regarding the kidney anatomy, the mice injected with the qcr7Δ/Δ mutant had smaller kidneys than those injected with the WT strain ( Figure 2B ). To quantitatively evaluate the invasive effects of C. albicans on the kidney, we investigated the fungal loads that correlated with the severity of kidney failure. By calculating the total number of colony-forming units (CFU) in the kidneys on day 2, we observed that the fungal burden in the kidneys injected with the qcr7Δ/Δ mutant significantly reduced ( Figure 2C ). Single kidney weight and kidney weigh/body weigh were also significantly reduce compared to wild type ( Figures 2D, E ). A histological analysis of the kidney-tissue samples obtained from the mice infected with the WT strain also revealed large areas with inflammatory cell infiltration ( Figure 2F ).

The kidney tissues obtained from the qcr7Δ/Δ mutant-infected mice showed significantly less inflammatory cells. Periodic acid-Schiff-base staining consistently revealed more tissue damage in the kidney, which differed between the WT-strain-infected mice with several C. albicans hyphae (red arrow), pseudo-hyphae(yellow arrow), and spores(black arrow), and the qcr7Δ/Δ mutant-infected mice ( Figure 2F ). Therefore, these results suggest that the absence of lethal injury in mice by the qcr7Δ/Δ mutant results from defective tissue invasion and colonization ability. To further clarify the spatial and temporal distribution of immune cells infiltrating the kidneys of mice during systemic infection, we observed the recruitment of neutrophils and macrophages in the kidney tissue of mice infected with the wild-type, mutant and complement strains for two days. Immunofluorescence staining was performed using Ly6G antibody for neutrophils and F4/80 antibody for macrophages. It can be visually seen that the number of both immune cells recruited in the kidneys of qcr7Δ/Δ infected mice was significantly reduced (p<0.0001), while their immune cells infiltration increased significantly after qcr7 was reconstituted (p<0.0001) ( Figures 2G , S5 ). Once again, it was well demonstrated that qcr7 is important for C. albicans to invade the attack of host immune system.

Fungi can use carbon sources to obtain energy for anabolic metabolism through glycolysis and aerobic respiration. The energy generated through the electronic transport chain is the primary source of energy for fungal growth. Therefore, if mitochondrial dysfunction affects aerobic respiration, the glycolytic pathway becomes the main metabolic pathway for energy metabolism. In addition, glycolysis is crucial for carbon assimilation, and this pathway also influences the invasiveness and virulence of C. albicans (Barelle et al., 2006). C. albicans uses multiple carbon sources in vitro, including glucose, GlcNAc, carboxylic acids (e.g., lactate), and amino acids (Williams and Lorenz, 2020). Compared with the WT strain, the qcr7Δ/Δ mutant grew slightly slower in the glucose-, sucrose-, and mannitol-only media. However, the growth of the mutant in the media containing GlcNAc or other non-glucose carbon sources was more significant than that of the WT strain ( Figure 3A ). The qcr7Δ/Δ mutant did not use amino acids as carbon sources ( Figure S6 ). The results indicate that the qcr7Δ/Δ mutant has a defective respiratory metabolism, which may affect ATP production during carbon metabolism. Furthermore, this implies that qcr7Δ/Δ does not substantially regulate the in vitro growth of C. albicans in the presence of alternative carbon sources.

Because Qcr7 is a component of the mitochondrial respiratory chain, we also examined the intracellular ATP content, endogenous ROS production, and mitochondrial membrane potential (MMP) of qcr7Δ/Δ mutants during logarithmic growth phase to determine their effect on mitochondrial functions. As expected, a significant decrease in intracellular ATP contents was observed in the qcr7Δ/Δ mutant among logarithmic growth phase ( Figure 3B ). Moreover, after evaluating the MMP using the JC-1 assay kit, we observed that the MMP of the mutant was significantly decreased compared with that of the WT strain ( Figure 3D ). In addition, the qcr7Δ/Δ mutant produced a higher level of ROS ( Figure 3C ). These data suggest the important function of Qcr7 in maintaining mitochondrial homeostasis.

Biofilms are defined as an association of microorganisms attached to a biotic or abiotic surface, the formation of which is considered a potential cause of antifungal drug failure and is associated with 65%-80% of microbial infections (Jamal et al., 2018). C.albicans biofilms, which often colonizes implanted medical devices and mucosal surfaces, and aggressive systemic infections of tissues and organs caused by dissemination into the bloodstream as a reservoir of pathogenic cells (Nobile and Johnson, 2015), which leads to inflammation and lethality. Thus, to understand the importance of QCR7 in biofilm formation, we performed an additional biofilm analysis under multiple conditions. Defective biofilm formation was evident in the qcr7Δ/Δ mutant when incubated in the Spider medium supplemented with glucose, mannitol, GlcNAc, maltose, and sucrose ( Figure 4A ). Therefore, these results indicate the importance of QCR7 in appropriate carbon-induced biofilm formation by C. albicans.

C. albicans can form hyphal cells both in planktonic cultures and during the maturation step of biofilm formation (Sudbery, 2011). In this study, we assessed the influence of QCR7 on the hyphal induction of C. albicans cultured in media containing various carbon sources. As expected, the results showed that the differences in hyphal morphology were more evident on the solid media. After culturing at 37°C, although the WT formed significant wrinkled colonies and hyphal extensions, the mutant formed only smooth colonies and slight hyphae. Particularly under GlcNAc, the colony derived from the qcr7Δ/Δ mutant was smooth ( Figure 4B ). Accordingly, the qcr7Δ/Δ mutant showed shorter and sparser hyphae when incubated in a liquid-induced medium with GlcNAc as the sole source of carbon ( Figure 4C ). However, no significant differences were observed in the hyphal length of the fungi cultured in liquid media with other carbon sources except maltose media ( Figures S7 , Figure S8 ) compared with the colony formed by the qcr7Δ/Δ mutant, where the WT strain developed wrinkled macro-colonies and extended the hyphae on solid media more evidently. In addition, this was accompanied by the loss of hyphal-related hydrolase activity ( Figure S9 ). Therefore, the qcr7Δ/Δ mutant might have affected the hyphal growth because of its impaired use of carbon sources.

The transcriptome of the qcr7Δ/Δ mutant was compared with that of the WT strain in media mimicking the in vivo conditions (RPMI 1640 medium). The results indicated that 307 genes were downregulated and 54 genes were upregulated with expression-fold changes of >2.0 and <−2.0 (P ≤ 0.05), respectively, in the qcr7Δ/Δ mutant compared with the WT strain. Therefore, we subjected these gene sets to pathway analysis using Gene Ontology (GO). Among the 307 downregulated genes, the most enriched GO term was transport (22.5%). The enriched terms also included processes such as translation (21.8%), ribosome biogenesis (20.5%), RNA metabolic processes (20.2%), the regulation of biological processes (18.9%), organelle organization (15.6%), response to stress (13.0%), and lipid metabolic processes (12.1%), followed by those related to filamentous growth (10.4%), cellular protein modification processes, response to chemicals, cellular homeostasis, biofilm formation (4.6%), carbohydrate metabolic processes (4.2%), and cell adhesion (2%) ( Figure 5A ). Furthermore, the list of the 54 upregulated genes list comprised 26 genes with unknown functions. The eight highest gene categories that were downregulated included those related to transport (33.3%), the regulation of biological processes (22.2%), cellular homeostasis (11.1%), protein catabolic process (9.3%), and responses to chemicals, including those related to filamentous growth, carbohydrate metabolic process, and cell adhesion (1.9%) ( Figure 5B ). An analysis of the GO category “cellular components” also found GO terms associated with the membrane and cell wall to be significantly (P ≤ 0.05) enriched in the downregulated gene sets ( Figure S10 ). Regarding the process related to sugar transportation through the plasma membrane, it can be proposed that membrane downregulation by some genes can affect the use of sugar. Previous evidence showed that mitochondrial function directly influences the cell wall (Duvenage et al., 2019). In addition, the absence of the Complex I regulator Goa1 (She et al., 2016) or the mitochondrial sorting and assembly machinery subunit Sam37 (Qu et al., 2012) results in defective cell-wall integrity (CWI), suggesting that the mitochondrial gene defects affecting the cell wall also apply to the Complex III regulator Qcr7. Intriguingly, an analysis of the downregulated gene sets related to filamentous growth and biofilm formation revealed that significantly downregulated genes were involved in regulating biofilm formation (SAP6, HWP1, XOG1, HYR1, YWP1, ALS3, CSH1, PGA10, and SUN41) and filamentous growth (ENO1, SSB1, DDR48, ALS3, and SUN41); these genes were also identified as cell-wall-related genes, indicating that impaired filamentous growth and biofilm formation were partly affected by the downregulation of cell-wall-associated genes. Moreover, several genes encoding glycosylphosphatidylinositol-anchored cell-wall proteins that influenced the cross-linking of cell-wall components were also downregulated (PGA3, PGA6, PGA7, PGA10, PGA34, PGA35, PGA53, PGA54, PGA56, PGA59, and PGA63) ( Figure S10 ). To verify whether QCR7 deletion negatively affects CWI, we spotted it on a medium containing cell-wall stressors, such as CFW, CR, caspofungin, and SDS. We observed that disrupting Qcr7 led to damaged cell-wall biosynthesis ( Figure S10 ).

Furthermore, our RNA-sequencing data revealed the most significant downregulation of several genes of biofilm formation, and we confirmed the reliability of the results by RT-qPCR ( Figures 5C, D ). In C. albicans, biofilm development requires six master transcriptional regulators (Bcr1, Brg1, Ndt80, Rob1, Tec1, Efg1) (Nobile et al., 2012). We further investigated whether six core biofilm regulatory factors in C. albicans regulate QCR7 expression. As shown in Figure S11 , the deletion of these master regulator significantly affects QCR7 expression. In addition, we overexpressed QCR7 in these transcription-factor-encoding knockout strains and performed biofilm formation assays, showing that QCR7 could partially restore the biofilm deficiency caused by NDT80 deletion ( Figure S12 ). These data can demonstrate that Qcr7 is regulated by Ndt80 to influence biofilm formation.

To further identify downstream biofilm-formation-related genes regulated by QCR7 and the probable mechanisms involved, we selected five genes (HWP1, YWP1, XOG1, HYR1, and SAP6) known to play an important role in biofilm formation and that were downregulated with an expression-fold change of < -5.0 (P ≤ 0.05) in the qcr7Δ/Δ mutant compared with the WT strain based on the transcriptome and overexpression of theses gene in the qcr7Δ/Δ background. We found that the overexpression of four genes (HWP1, YWP1, XOG1, and SAP6) partly but significantly rescued the qcr7Δ/Δ biofilm phenotype, but that HYR1 overexpression in this genetic background suppressed the biofilm phenotype of the qcr7Δ/Δ mutant even more ( Figure 6A ). Furthermore, the partial restoration of the phenotype also occurred in hyphal growth in solid medium. The increased expression levels of HWP1, YWP1, XOG1, and SAP6 in the qcr7Δ/Δ mutant formed a halo of more abundant and longer agar-invading hyphae around the colony center than those seen with the qcr7Δ/Δ mutant, whereas the overexpression of HYR1 showed no formation of a halo of agar-invading hyphae ( Figure 6B ). Considering whether the same mechanism regulates the reduction of each virulence phenotype, and restricted carbon source utilization, we tested for growth on several carbon sources to examine the utilization of the carbon sources by these strains. The results showed that carbon use was consistent with the virulence phenotype, with four genes (HWP1, YWP1, XOG1, SAP6) overexpressed in the qcr7Δ/Δ background showing a partial reversal of the use of these carbon sources, while the overexpression of HYR1 inhibited the use of the carbon source even more ( Figure 6C ). The mechanism behind this is not yet clear.

Altogether, the above results confirm that the restricted carbon utilization caused by the deletion of QCR7 is related to the impairment of the virulence phenotype and cell-surface changes.