The Claudin Family Protein FigA Mediates Ca2+ Homeostasis in Response to Extracellular Stimuli in Aspergillus nidulans and Aspergillus fumigatus

The claudin family protein Fig1 is a unique fungal protein that is involved in pheromone-induced calcium influx and membrane fusion during the mating of Saccharomyces cerevisiae and Candida albicans. Whether and how Fig1 regulates Ca2+ homeostasis in response to extracellular stimuli is poorly understood. Previously, we found Aspergillus nidulans FigA, a homolog of Fig1 in S. cerevisiae, similar to the high-affinity calcium uptake system, is required for normal growth under low-Ca2+ minimal medium. In this study, using the calcium-sensitive photoprotein aequorin to monitor cytosolic free calcium concentration ([Ca2+]c) in living cells, we found that the FigA dysfunction decreases the transient [Ca2+]c induced by a high extracellular calcium stress. Furthermore, FigA acts synergistically with CchA (a high-affinity Ca2+ channel) to coordinate cytoplasmic Ca2+ influx in response to an extracellular Ca2+ stimulus. Moreover, FigA mediates ER stress-induced transient [Ca2+]c in the presence or absence of extracellular calcium. Most importantly, these [Ca2+]c responses mediated by FigA are closely related to its conserved claudin superfamily motif, which is also required for hyphal growth and asexual development in A. nidulans. Finally, the function of FigA in Aspergillus fumigatus, the most common airborne human fungal pathogen was studied. The result showed that the two FigA homologous in A. nidulans and A. fumigatus have a large degree of functional homology not only in asexual development but also in regulating transient [Ca2+]c. Our study expands the knowledge of claudin family protein FigA in Ca2+ homeostasis in response to extracellular stimuli.


INTRODUCTION
Calcium is a highly versatile intracellular signal (second messenger) in eukaryotes. At resting state, the cytosolic Ca 2+ concentration ([Ca 2+ ] c ) in fungal cells is very low, ranging from 50 to 100 nM (Cui et al., 2009;. In response to various external stresses, a Ca 2+mediated signaling pathway is employed to regulate a wide variety of cellular processes through a transient increase in cytosolic Ca 2+ , which is elevated by activating the plasma membrane Ca 2+ influx system and/or secreting Ca 2+ from internal compartments (Berridge et al., 2000;Harren and Tudzynski, 2013;Munoz et al., 2015;Wang et al., 2016). In Saccharomyces cerevisiae, at least two different Ca 2+ influx systems, the high-affinity Ca 2+ influx system (HACS) and the low-affinity Ca 2+ influx system (LACS) have been identified. The HACS was activated in response to mating pheromones, alkaline pH, oxidative stress and compounds such as azole-class antifungal agents or ER-stress agents (Muller et al., 2001;Popa et al., 2010;Zhang et al., 2016). The HACS is primarily composed of two subunits: the voltage-gated Ca 2+ channel Cch1 (calcium channel) and the stretch-activated calcium channel/regulatory protein Mid1 (mating-induced death) (Iida et al., 1994;Fischer et al., 1997). Deletion of homologs of mid1 and cch1 results in calcium accumulation and growth defects under low-calcium conditions (Liu et al., 2006;Hallen and Trail, 2008;Wang et al., 2012;Harren and Tudzynski, 2013). Besides, Ecm7, as a new regulator of the HACS, directly or indirectly interacts with subunits of the HACS and may regulate the HACS through some unknown mechanisms in S. cerevisiae and Candida albicans (Martin et al., 2011;Ding et al., 2013).
The HACS is responsible primarily for the calcium response in the low external Ca 2+ concentration but suppressed under high external Ca 2+ concentration, so that the LACS becomes essential for this response (Muller et al., 2001(Muller et al., , 2003. Fig1 is the main component or regulator of LACS and was first identified as a pheromone-regulated protein involved in membrane fusion during yeast mating differentiation (Erdman et al., 1998). Fig1 was shown to be required for LACS activity, but not required for activation of Mpk1 mitogen-activated protein kinase in response to pheromones (Muller et al., 2001). LACS activity is insensitive to calcineurin activity, independent of Cch1p and Mid1p, and sufficient to elevate [Ca 2+ ] c in spite of its 16-fold lower affinity for Ca 2+ (Muller et al., 2001). Consistent with the reports in S. cerevisiae, CaFig1 facilitates calcium influx during mating in C. albicans (Yang et al., 2011). CaFig1 is also involved in regulating the fungal hyphal thigmotropic orientation. However, deletion of Cafig1 did not affect Ca 2+ accumulation in low-Ca 2+ conditions, suggesting the calcium independent roles of Fig1 (Brand et al., 2007). The function of Fig1 homologs has also been characterized in several filamentous fungi. In Neurospora crassa, loss of Fig1 decreased female fertility and arrested perithecium development in a mating type α background (Cavinder and Trail, 2012). In the plant pathogen Fusarium graminearum, loss of Fig1 resulted in phenotypes with reduced hyphal growth, failed perithecia and reduced virulence (Cavinder and Trail, 2012). Most recently, it was further showed that FigA in F. graminearum plays distinct roles from that of Mid1 and Cch1 in the formation of Ca 2+ signature in hyphal cells (Kim et al., 2018).
Genus Aspergillus contains important species including the premier model filamentous fungus Aspergillus nidulans and the human pathogenic fungus Aspergillus fumigatus. The functions of Cch1 and Mid1 homologs (CchA and MidA) have been characterized in A. nidulans. Consistent with the function of Cch1 and Mid1 as a member of the HACS, CchA and MidA are required for conidial development, hyphal polarity establishment, and cell wall integrations in low-calcium environmental conditions (Wang et al., 2012). However, no Ecm7 homolog was reported in Aspergillus. Our previous work has demonstrated that FigA, the homolog of Fig1 in A. nidulans, is involved in hyphal growth, asexual and sexual development (Zhang et al., 2014). There are no clear homologs of Fig1 outside of fungi and it lacks the homology to any known ion influx channel. Instead, Fig1 shows similar secondary structure and topology to the claudin/PMP22/EMP superfamily. In mammals, claudin superfamily members are involved in vesicle trafficking and membrane-membrane interactions (Van Itallie and Anderson, 2006;Guenzel and Fromm, 2012;Overgaard et al., 2012). Although it has been proven that S. cerevisiae and C. albicans Fig1 is required for the low-affinity calcium transport during cell fusion (Muller et al., 2003;Yang et al., 2011) Our previous data has shown that loss of CchA/MidA or FigA caused a reduced hyphal growth under calcium-limited minimal medium (MM) in A. nidulans. In contrast with cchA and midA mutants, there were no obvious differences in polarized growth and the sensitivity to the cell wall stressor Congo Red between the figA mutant and wild-type in MM (Wang et al., 2012;Zhang et al., 2014) (Supplementary Figure S1). To further characterize the relationship between figA and midA/cchA in mediating calcium uptake, figA midA and figA cchA double deletion mutants were grown on MM and the hyphal radial growth were observed. The phenotypic defect in hyphal radial growth was exacerbated in the figA cchA and figA midA double deletion mutants compared to their respective parental single mutants figA, cchA, or midA ( Figure 1A   real-time monitoring of [Ca 2+ ] c in living hyphal cells was performed. The pre-stimulatory resting [Ca 2+ ] c in A. nidulans is typically between 0.05 and 0.1 µM (Berridge et al., 2000;Munoz et al., 2015). As expected, the basal [Ca 2+ ] c resting level was approximately 0.09 µM in these experiments. Upon application of a 0.1 M CaCl 2 stimulus (high external calcium), the highest [Ca 2+ ] c in wild-type cells increased to approximately 0.9 µM. Interestingly, it was the cchA mutant (p = 0.0062) but not the midA mutant (p = 0.1241) that showed a significant reduction of the transient [Ca 2+ ] c compared to its wild-type strain (100%) (Figures 1B,C). Surprisingly, the transient [Ca 2+ ] c in the figA mutant showed a similar decrease as the cchA mutant under the same stimulus. Moreover, the figA cchA mutant, but not the figA midA mutant exhibited a dramatic further reduction in transient [Ca 2+ ] c compared to the figA mutant under the same stimulus (Figures 1B,C). Collectively, these data suggest that FigA and CchA synergistically coordinate cytoplasmic Ca 2+ influx in response to an extracellular calcium stimulus.

FigA Is Required for [Ca 2+ ] c Transient in Response to the ER Stress
Endoplasmic reticulum (ER) is a multifunctional organelle required for calcium storage, protein folding and processing. Our previous data indicate that CchA, but not MidA influences ER stress-induced calcium influx in A. nidulans . To explore the role of FigA in mediating transient [Ca 2+ ] c in response to the ER stress, we measured the transient [Ca 2+ ] c in hyphal cells following treatment with the ER-stress agent tunicamycin (TM). When the parental wild-type strain was treated with 5 µg/mL TM, an immediate transient increase in [Ca 2+ ] c to 0.6 µM was observed. Consistent with our previous result, the transient [Ca 2+ ] c in the cchA mutant but not in midA mutant showed a significant 27% reduction compared to the parental wild-type strain in response to TM (Figures 2A,B). Notably, the transient [Ca 2+ ] c in the figA mutant decreased by approximately 24% compared to the parental wild-type strain under the same stimulus (Figures 2A,B). The results suggest that both CchA and FigA are involved in mediating the ER stress-induced calcium influx in A. nidulans.
Furthermore, we tested whether the transient [Ca 2+ ] c resulting from the TM treatment is dependent on external calcium or internal calcium stores. Exposure of the figA mutant cells to the calcium chelator EGTA (1 mM) prior to the TM treatment showed a significant 22% reduction in the transient [Ca 2+ ] c compared to wild-type. However, there was no significant difference between midA or cchA and the wild-type strain (Figures 3A,B). Collectively, these data showed that both CchA and FigA mediate ER stress-induced calcium influx in A. nidulans. Moreover, FigA also contributes to ER stress-induced transient [Ca 2+ ] c in the absence of extracellular calcium.

The Claudin Motif Is Essential for FigA Function
The conserved motif in FigA, G GxC(n)C, is a feature of the claudin superfamily. The glycine and cysteine residues in the conserved motif are located near the end of the first predicted extracellular loop region, between TM1 and TM2 (Yang et al., 2011;Zhang et al., 2014) (Figure 4A). Our previous studies have demonstrated that FigA is essential for hyphal growth, asexual, and sexual development in A. nidulans (Zhang et al., 2014). However, the molecular characteristics of the key FigA motifs have not yet been identified. Accordingly, we constructed individual site-directed FigA mutants at two conserved glycine residues (G97A and G100A), two cysteine residues (C102A and C112A), and a combined mutant at all four residues (figA mt -4G/CA). Plate assays revealed that G97A and G100A mutants displayed a wild-type like phenotype in hyphal growth and asexual conidiation either on MM or MM plus calcium media ( Figure 4B). The C112A mutant displayed reduced conidia production on MM while exogenous calcium addition was able to almost completely restore the asexual conidiation defect in the C112A mutant. In comparison, the C102A mutant exhibited both hyphal and conidiation defects while the addition of calcium could completely restore the hyphal growth defect but only partially restore the conidiation defect in the C102A mutant. The figA mt -4G/CA mutant showed identical defects to a figA mutant both in reduced colony size and lack of asexual conidia on MM. In addition, calcium supplementation completely rescued the hyphal growth retardation but did not restore the asexual conidiation defect in the figA mt -4G/CA and figA mutants ( Figure 4B)

FigA Homologs Display Functional Homology in A. fumigatus
Aspergillus fumigatus is the most common airborne fungal pathogen for human (Dagenais and Keller, 2009). FigA in A. fumigatus (KEGG Accession No. Afu3g09060) has 82% identity with its homolog in A. nidulans. Both proteins have conserved topology and contain the claudin motif. To explore the function of the FigA in A. fumigatus, an AffigA deletion strain was constructed by homologous gene replacement employing the N. crassa pyr4 gene as a selectable marker (Supplementary Figure S3A). The resulting strain, AffigA, was confirmed by diagnostic PCR and Southern blot (Supplementary  Figures S3B,C). As is shown in Figure 5A, the loss of figA in A. fumigatus caused reduced conidia production on MM. Consistent with our observations in A. nidulans, the conidiation defects in the AffigA mutant could not be rescued by the addition of extracellular calcium ( Figure 5A). However, it seems that FigA plays a more important role in conidiation in A. nidulans than that in A. fumigatus. Conidia production in the AffigA mutant was 20% of the reference strain (100%). In comparison, conidiation was almost completely abolished in the A. nidulans figA deletion strain. In addition, loss of figA in A. fumigatus did not affect the hyphal radial and polarized growth ( Figure 5A and Supplementary Figure S4). To verify the functional homology between the two FigA homologs in A. nidulans and A. fumigatus, two wild-type figA genes were used to cross complement each figA deletion strain. Functional assays showed that the A. fumigatus FigA could completely restore the asexual conidiation and hyphal growth defects seen in the A. nidulans figA deletion strain and vice versa under all test conditions including MM, MM plus EGTA or MM plus calcium (Figures 5A,B). The results indicated that the two FigA homologs in A. fumigatus and A. nidulans have a large degree of functional homology.

FigA Mediates Transient [Ca 2+ ] c in A. fumigatus
The above results suggested that loss of figA affects calcium influx in A. nidulans. In order to explore whether A. fumigatus FigA regulates calcium influx, we monitored the transient [Ca 2+ ] c in response to extracellular calcium and TM by expressing codon-optimized aequorin in living cells of A. fumigatus wild-type A1160 and the AffigA mutant. As expected, the [Ca 2+ ] c in the AffigA mutant exposed to the 0.1 M CaCl 2 stimulus significantly decreased by 26% compared to that of the parental wild-type strain (Figures 6A,B). When treated with the ER stressor TM, the [Ca 2+ ] c in the AffigA mutant significantly decreased by 16% compared to that of the parental wild-type strain (Figures 6C,D). In summary, the above data showed that FigA possess a conserved role in [Ca 2+ ] c regulation in A. fumigatus.

DISCUSSION
The Ca 2+ -mediated signaling pathway plays a crucial role in fungal growth and survival under various stresses. In yeast, the HACS components Cch1 and Mid1 are the primary channels involved in Ca 2+ homeostasis under low-Ca 2+ conditions. The function of LACS component or regulator Fig1 seems to be confined to the pheromone-induced calcium influx in the rich medium which the function of the HACS is inhibited  by calcineurin (Muller et al., 2001;Yang et al., 2011 (Locke et al., 2000;Hong et al., 2010;Zhang et al., 2016). Interestingly, we found that in addition to the HACS component CchA, FigA is also involved in ER stress-induced calcium influx. However, unlike CchA, which is mainly responsible for calcium uptake from the extracellular environment, FigA  In mammals, the functions of claudin superfamily members are involved in epithelial tight-junction formation, which allow ions and other solutes to pass between cells (Van Itallie and Anderson, 2006;Guenzel and Fromm, 2012;Overgaard et al., 2012). The site-directed mutation experiments showed that the glycine and cysteine residues in the conserved motif [G GxC(n)C] of the claudin superfamily is important both for fungal low calcium adaptation and transient [Ca 2+ ] c in response to extracellular stimuli. In S. cerevisiae, Fig1 is localized predominantly to plasma membrane of shmoos and deletion of ScFig1 results in incomplete fusions between tips of mating shmoos, which might be due to loss of a calciumdependent membrane repair. However, such fusion defects were not observed in fig1 null mutants of C. albicans. Instead, Cafig1 presumably has an ability in maintaining the membrane stability during morphological transitions (Muller et al., 2003;Aguilar et al., 2007;Yang et al., 2011). However, FigA is located at the center of the septa of mature hyphae (probably around the pore) in Aspergillus (Zhang et al., 2014). The specific location of FigA at the center of hyphal septa indicates that FigA may also selectively allow solutes (calcium, for instance) to flow between cells (Harris, 2001;Zhong et al., 2012). Otherwise, FigA may work as a regulator of calcium channels.
Further studies will be required to identify and characterize the potential partners that interact with FigA. Overall, our study expands the knowledge of claudin family protein FigA in calcium homeostasis in response to extracellular stimuli.

Strains, Culture Condition, and Transformation
All fungal strains used in this study are listed in Supplementary  Table S1. TN02A7, a strain with a deletion in the gene required for non-homologous end joining in double-strand break repair was used in transformation experiments as a parental wild-type strain of A. nidulans . A1160, a parental wild-type strain of A. fumigatus was purchased from FGSC (Fungal Genetics Stock Center). All fungal strains were routinely cultured on MM with supplements to support the growth of relevant auxotrophic strains, as described previously (Zhang et al., 2014. Standard transformation procedures were performed according to a previously described method for A. nidulans and A. fumigatus (Osmani et al., 1988;May, 1989;Jiang et al., 2014).

Plate Assays
To assess the influence of extracellular calcium on fungal growth, MM was supplemented with 20 mM CaCl 2 , 100 mM CaCl 2 , or 1.5 mM EGTA. 2 µl of 1 × 10 7 /ml A. nidulans conidia were spotted onto the relevant media. To assess the influence of extracellular calcium on A. fumigatus, MM was supplemented with 100 mM CaCl 2 or 5 mM EGTA. 2 µl aliquots of 1 × 10 8 /ml A. fumigatus conidia from the indicated strains were spotted onto the relevant media. For the Congo Red sensitivity test, 2 µL aliquots (1 × 10 5 conidia/mL, 1 × 10 6 conidia/mL, 1 × 10 7 conidia/mL, respectively) of indicated strains were spotted onto MM and MM plus 0.75 µg/mL Congo Red. All strains were cultured at 37 • C for 2.5 days and then the colonies were observed and imaged.

Construction of A. nidulans FigA Point Mutation Strains
Using genomic DNA (gDNA) of A. nidulans wild-type as the template, a 2.7 kb figA DNA fragment including a 1 kb upstream promoter region, a 0.9 kb coding sequence, and a 3 flanking sequence was amplified with the primer pair FigAF/FigAR. The resulting fragment was digested with SmaI and PstI and was cloned into the SmaI and PstI digested plasmid pQa-pyroA to generate plasmid pFigA-pyroA. The Mut Express II Fast Mutagenesis kit (Vazyme TM ) was used to construct plasmids carrying site-directed mutations. In brief, using the resulting plasmid pAnFigA-pyroA as a template, a DNA fragment with the complete ORF including the site-directed mutation (glycine 97 -alanine mutation), promoter sequence and 3 UTR was amplified with the primers G97AF/G97AR. A similar strategy was used to construct plasmids carrying other mutations, including glycine 100 -alanine, cysteine 102alanine, cysteine 112 -alanine and the combined four mutated sites. All recombinant plasmids were individually transformed into the figA mutant strain to generate relevant FigA point mutants.

Constructions of A. fumigatus figA Deletion and Aequorin-Expressing Strains
To construct the AffigA deletion cassette in A. fumigatus, a fusion PCR based method was used as described previously . In brief, 5 and 3 flanking DNA fragments of AffigA were amplified using the primers AffigA-P1/AffigA-P3, AffigA-P4 /AffigA-P6 from A. fumigatus wild-type A1160 gDNA. As a selectable marker, a 2.1 kb DNA fragment of N. crassa pyr4 was amplified from the plasmid pAL5 using the primers Diag-pyr4-5 /Diag-pyr4-3 . The three PCR products were fused using primers AffigA-P2/AffigA-P5. The final PCR product was transformed into wild-type A1160 cells to construct the figA knockout strain. A diagnostic PCR was performed to ensure figA had been replaced by pyr-4 at the original figA locus, using primers AffigA-P1/Diag-pyr4-3 .
For construction of the aequorin-expressing strains, the plasmid pAEQS1-15 containing a codon-optimized aequorin (Nelson et al., 2004) and the selective markers riboB or hygB were co-transformed into the indicated mutants. Transformants were screened for aequorin expression using methods described previously (Osmani et al., 1988;Denis and Cyert, 2002) and a high aequorin expressing strain was selected after homokaryon purification involving repeated plating of single conidia. All primers used to design constructs are listed in Supplementary  Table S2.

Southern Blot
To perform Southern blotting, the genomic DNA from the wildtype and the AffigA strain were digested with BamHI/EcoRV and BamHI/SalI respectively, separated by electrophoresis and transferred to a nylon membrane (Zeta-probe+; Bio-Rad). The fragment amplified with primers AffigA -southern -F and AffigA -southern -R was used as a probe to detect the AffigA and wild-type strains, respectively. Labeling and visualization were performed using a digoxigenin (DIG) DNA labeling and detection kit according to the manufacturer's instructions (Roche Applied Science, Indianapolis, IN, United States).

Cross Complementation Assays for FigA Homologs
The figA genes from A. fumigatus and A. nidulans were cloned into pAN7-1 vector, which contained a hygromycin selection marker. Using gDNA of A. fumigatus wild-type A1160 as a template, a 2796 bp DNA fragment was amplified with the primer pair Af FigA-recon-P1/Af FigA-recon-P3, and then the fragment was cloned into plasmid pAN7-1 using the ClonExpress II OneStep Cloning Kit (Vazyme TM ) to generate the plasmid pAfFigA-hygB. A similar strategy was used to clone A. nidulans figA into plasmid pAN7-1 to generate pFigA-hygB plasmid using the primer pair FigA-recon-P1/FigA-recon-P3 with gDNA of A. nidulans TN02A7 as a template.
To clone A. fumigatus figA into the pQa-pyroA vector, a 2.7 kb DNA fragment was amplified with the primer pair Af FigAF/Af FigAR using A. fumigatus A1160 gDNA as a template. The fragment was digested with SmaI and PstI and ligated into SmaI and PstI digested pQa-pyroA to generate the recombinant plasmid pAf FigA-pyroA. The plasmids pAf FigA-hygB and pFigA-hygB were transformed into the AffigA mutant, and the plasmids pAf FigA-pyroA and pFigA-pyroA were transformed into figA mutant to generate the relevant complemented strains.

Intracellular [Ca 2+ ] c Measurement
Strains expressing the codon-optimized aequorin gene were grown on MM for 2.5 days to achieve maximal conidiation. 1 × 10 6 spores in liquid media were dispensed in each well of a 96-well microtiter plate (Thermo Fischer, United Kingdom). Each experiment was performed as six replicates in the same multi well plate. Aequorin was reconstituted by incubating mycelia in 100 µl PGM containing 2.5 µM coelenterazine native (Biosynth AG, Rietlistrasse, Switzerland) for 4 h at 4 • C in the dark. After reconstitution, mycelia were washed with two 1 ml washes in PGM and allowed to recover to room temperature for 1 h (Greene et al., 2002;Liu F.F. et al., 2015). To chelate extracellular Ca 2+ , 1 mM EGTA was added to each well 10 min prior to stimulus injection. At the end of each experiment, active aequorin was completely discharged by permeabilizing the cells with 20% (vol/vol) ethanol in the presence of excess Ca 2+ (3 M CaCl 2 ) to determine the total aequorin luminescence of each culture. Luminescence was measured with an LB 96P Microlumat Luminometer (Berthold Technologies, Germany), which was controlled by a dedicated computer running the Microsoft Windows-based Berthold WinGlow software. Relative light unit (RLU) values were converted into [Ca 2+ ] c concentrations using the following empirically derived calibration formula: pCa = 0.332588 (−logk) + 5.5593, where k is luminescence (in RLU) s −1 /total luminescence (in RLU) (Nelson et al., 2004). Error bars represent the standard error of the mean from six independent experiments and percentages in the figures represent peak [Ca 2+ ] c compared to that of wild-type (100%).

Statistical Analysis
Statistical differences were analyzed using GraphPad Prism 6 software (GraphPad Software). p-Values were calculated with one-way ANOVA for multiple comparisons and adjusted with Tukey correction and non-paired Student's t-test where two groups were compared.
FIGURE S2 | Quantitative data of the colony diameter of the indicated strains. The control strain (TN02A7) and the figA, midA, figA midA, cchA, figA cchA mutants were grown on MM at 37 • C for 2.5 days and then the hyphal radial growth was quantified. Error bars represent standard deviations from three replicates.
FIGURE S3 | Constructions of AffigA deletion strain. (A) Diagrams showing the strategy for generating AffigA deletion strain. The AffigA gene was replaced with the pyr4 expression cassette to create the AffigA mutant. (B) Diagnostic PCR was employed to verify the mutant. For lanes 1 and 2, the PCR primers Diag-del-AffigA-5 /Diag-del-AffigA-3 were used to detect whether AffigA existed in the genome. For lanes 3 and 5, the PCR primers AffigA-P1/Diag-pyr4-3 (lane 3) and Diag-pyr4-5 /AffigA-P6 (lane 5) were used, respectively, to verify homologs replacement of AffigA by pyr4 marker. For lanes 2, 4 and 6, genomic DNA of parental strain was used as PCR template; for lanes 1, 3 and 5, the template was genomic DNA of AffigA mutant. (C) Southern blot. The DIG-labeled probe bound to a 1072 and 2214 bp fragment in the wild-type and AffigA strains respectively, indicating the replacement of AffigA by pyr4.
FIGURE S4 | The morphologic observation of hyphae. The micrographs of Different Interference Constrast (DIC) of indicated strains. There was no significant difference in hyphal polarity between the AffigA strain and wild-type (A1160). Bars: 10 µm.