Identification and Characterization of Aspergillus nidulans Mutants Impaired in Asexual Development under Phosphate Stress

Abstract

The transcription factor BrlA plays a central role in the production of asexual spores (conidia) in the fungus Aspergillus nidulans. BrlA levels are controlled by signal transducers known collectively as UDAs. Furthermore, it governs the expression of CDP regulators, which control most of the morphological transitions leading to the production of conidia. In response to the emergence of fungal cells in the air, the main stimulus triggering conidiation, UDA mutants such as the flbB deletant fail to induce brlA expression. Nevertheless, ΔflbB colonies conidiate profusely when they are cultured on a medium containing high H₂PO₄^- concentrations, suggesting that the need for FlbB activity is bypassed. We used this phenotypic trait and an UV-mutagenesis procedure to isolate ΔflbB mutants unable to conidiate under these stress conditions. Transformation of mutant FLIP166 with a wild-type genomic library led to the identification of the putative transcription factor SocA as a multicopy suppressor of the FLIP (Fluffy, aconidial, In Phosphate) phenotype. Deregulation of socA altered both growth and developmental patterns. Sequencing of the FLIP166 genome enabled the identification and characterization of PmtC^P282L as the recessive mutant form responsible for the FLIP phenotype. Overall, results validate this strategy for identifying genes/mutations related to the control of conidiation.

Keywords: asexual development; conidiation; filamentous fungi; mutagenesis; protein O-mannosylation; signal transduction; transcriptional regulation.

Figure 1

Mutagenesis procedure followed to isolate FLIP (Fluffy in Phosphate) mutants: (A) Phenotype of the parental ΔflbB strain on Aspergillus minimal medium (AMM) supplemented with increasing concentrations of NaH₂PO₄. Petri dishes (diameter: 5.5 cm) were cultured at 37 °C for 72 h. A concentration of 0.65 M was established. (B) Mutagenesis procedure followed for the isolation of FLIP mutants: ΔflbB conidia were collected and inoculated (1250 conidia/plate; diameter: 14 cm) on AMM dishes supplemented with 0.65 M NaH₂PO₄. Conidia (all plates but one, which was used as negative control) were mutagenized by using UV radiation of 254 nm and exposure times of 80–100 s. After 72 h of culture at 37 °C, mutants unable to conidiate were transferred to 5.5-cm dishes filled with AMM plus 0.65 M NaH₂PO₄ before phenotypic characterization. (C) Growth and conidiation phenotypes of a group of FLIP mutants in AMM and AMM plus 0.65 M H₂PO₄⁻ compared to the parental null flbB strain and after 72 h of incubation at 37 °C (diameter of Petri dishes: 9 cm). The arrows designate FLIP mutants with different phenotypes (see also Figure 2).

Figure 2

Phenotypic classification of FLIP mutants: Phenotype of specific FLIP mutants after 72 h of culture at 37 °C on AMM, on AMM supplemented with NaH₂PO₄ (0.65 M), sucrose (1.0 M), KCl (0.6 M) plus MES (0.05 M), MgCl₂ (0.18 M), or H₂O₂ (6 mM), as well as on AMM in which carbon or nitrogen sources were diluted to one-fifth of the original concentration. The different FLIP phenotypes were compared to reference strains with a wild-type or aconidial (parental ΔflbB; ΔflbD, ΔflbC, and ΔbrlA) phenotypes. Five (G1–5) out of the six phenotypic groups are shown: G1—FLIP mutants with a ΔflbD-like phenotype on AMM with 0.65 M NaH₂PO₄ (FLIP33, FLIP99, and FLIP121 are shown; see also Figure S2); G2—FLIP mutants with a clearly aconidial phenotype on AMM with 0.65 M NaH₂PO₄ (i.e., FLIP51, FLIP144, FLIP166, FLIP176, and FLIP196); G3—FLIP mutants showing a reduced colony diameter (i.e., FLIP16 and FLIP94); G4—FLIP mutants with a reduced growth phenotype that can be reverted under specific culture conditions (FLIP204 and FLIP149); and G5—FLIP mutants with a wet, purportedly autolytic, phenotype in the center of the colony under specific culture conditions (FLIP199). Group G6 corresponds to those FLIP mutants not included in any of the previous five categories (not shown). Scale bar = 2 cm.

Figure 3

Analysis of socA sequence and protein evolution: (A) Sequence analysis of An8501/socA. The predictions of the intronic and exonic regions done by the AspGD Aspergillus genome (http://www.aspgd.org/) and FungiDB databases (https://fungidb.org/fungidb/) (top) are compared to DNA-seq (FLIP166) and RNA-seq (ΔflbB and a wild-type reference) experiments (middle). On the one hand, the right panel shows that the intronic mutation identified in FLIP166 in the fourth intron of socA was also present in the RNA-seq reads mapping to this intron in the reference strains (arrow *1), suggesting that it is not responsible for the FLIP166 phenotype. On the other hand, the left panel shows that the initiation codon (arrow *3) and the first intron were incorrectly annotated. Arrow *2 indicates the end of the first exon, and arrow *4 indicates the correct initiation codon. The bottom panel shows the prediction of the functional domains done by the InterPro website for the corrected amino acidic sequence of SocA, which includes two transcriptional regulatory domains. (B) Phylogenetic tree of SocA orthologs: The tree was built using MegaX (neighbor-joining method and 2500 replicates) and edited using iTOL. Red color designates SocA orthologs of species of the order Onygenales, while green and purple differentiate SocA orthologs of Aspergillaceae or Trichocomaceae species within Eurotiales, respectively. The dotted orange square marks the position of An8501/SocA in the tree. Note that the evolution of SocA includes a duplication event in specific Aspergilli and Penicilli.

Figure 4

Functional characterization of SocA: (A) Phenotypes of ΔsocA, SocA::GFP, and gpdA^p::SocA::GFP strains, all in both ΔflbB (left) and wild-type (right) backgrounds, after 72 h of culture at 37 °C on AMM and AMM plus 0.65 M H₂PO₄⁻ (diameter of Petri dishes: 5.5 cm): The color code is the same as that used in Figure 4B. (B) Quantification of conidia production by the ΔflbB strains shown in panel A (bar colors keyed to photos): Values are given as the mean of at least three replicates plus SD. p values are given, and statistically significant (p < 0.05) relations are indicated with a variable number of asterisks (* p < 0.05; **p < 0.01; ***p < 0.001; **** p < 0.0001). Double deletion of flbB and socA significantly decreases conidia production in AMM supplemented with H₂PO₄⁻ (C) Immunodetection of SocA::HA_3x, driven either by native or gpdA^p promoters, both in wild-type and ΔflbB backgrounds: Two clones per strains are shown. The Coomassie-stained gel is shown as a loading control. Driving expression through gpdA^p clearly increases SocA levels. (D) Immunodetection of SocA::HA_3x (driven by the native promoter and in the wild-type flbB background) in mycelial samples collected after 15 h of culture in AFM and 0, 15, 30, 45, 60, 120, 180, and 240 min after the transference of mycelia to AMM supplemented with H₂PO₄⁻. The Coomassie-stained gel is shown as a loading control. Addition of 0.65 M H₂PO₄⁻ to the culture medium does not increase SocA levels. (E) Subcellular localization of gpdA^p-driven SocA::GFP in germlings: The micrograph shows the altered germination pattern of the conidia (arrowheads), while fluorescence microscopy shows the nuclear localization of the putative transcriptional regulator (arrows). Scale bar = 5 µm. (F) Variation of the percentage of germinated conidia with the time of culture in liquid medium: Values of the gpdA^p::SocA::HA_3x strain (orange line) are compared to a wild-type reference (purple line) and are given as the mean of two replicates plus SD. The dotted line shows that, in order to achieve the level of germination of the reference, the strain constitutively expressing SocA::HA_3x is delayed approximately three hours.

Figure 5

Identification of pmtC as the mutated gene responsible for the FLIP166 phenotype: (A) Phenotype of strains ΔflbB, FLIP166, and FLIP166 transformed with a pRG3::flbB plasmid after 48 h of culture on AMM (top row) and AMM supplemented with 0.65 M H₂PO₄⁻ (bottom row). Scale bar = 2 cm. The aconidial phenotype of FLIP166 under phosphate stress conditions is the result of the addition of the effect of flbB deletion and the unknown mutation. (B) Phenotypes of FLIP166 and FLIP166 transformed with An12172::gfp, acdA::gfp or pmtC::gfp constructs (row 1) or ΔflbB and ΔflbB transformed with the mutant versions of the abovementioned constructs (rows 2 and 3) after 72 h of culture on AMM supplemented with 0.65 M H₂PO₄⁻. Diameter of Petri dishes: 5.5 cm. Expression of a wild-type pmtC form suppresses the FLIP166 phenotype, while the insertion of a mutant pmtC^Pro282Leu form in the null flbB background causes a FLIP phenotype with reduced growth. The two additional mutants of pmtC isolated (ΔflbB background), Arg762Gly and Tyr767Stop, show an aconidial phenotype on AMM supplemented with 0.65 M H₂PO₄⁻.

Figure 6

Evolutionary analysis of protein O-Mannosyltransferases of A. nidulans: Phylogenetic tree corresponding to 2169 fungal orthologs of A. nidulans PmtA/An5105, PmtB/AN4761, and PmtC/An1459. The tree was built using MegaX (neighbor-joining method and 1000 replicates) and edited using iTOL. The color key indicates the fungal class each ortholog belongs to. The clades corresponding to PmtA/B/C orthologs are indicated. Results suggest that the three protein O-mannosyltransferases are conserved in all fungal classes.

Figure 7

Domain organization and predicted structure of PmtC: (A) Domain analysis of PmtC based on InterPro and Phyre2. The extension of domains related to protein O-mannosyltransferase activity, as well as those of a signal peptide and the twelve transmembrane helices predicted are also indicated. Red and blue lines indicate orientation towards the ER lumen or the cytoplasm, respectively. (B) Prediction of the three-dimensional structure of PmtC carried out by Phyre2 and based on Protein Data Bank (PDB) entry 6P25. The rainbow-colored form on the left indicates the N- (blue) and C-terminal (red) ends of the protein, while the form on the right represents domains with secondary structure. The position of residues Pro282 (transmembrane, TM, helix 8) and Arg762 (C-terminus) are also indicated. (C) Assessment of the effect on interatomic interactions and protein stability of the Pro282 (left) to Leu (middle) substitution identified in the mutant FLIP166 (row 1) or the Arg762 (left) to Gly (middle) substitution identified in the transformation of the parental ΔflbB strain (row 2): Blue and red colors in the pictures on the right represent a gain of rigidity (Pro282Leu) or flexibility (Arg762Gly), respectively. The analysis was carried out using the DynaMut website.

Figure 8

Subcellular localization of PmtC: (A) Phenotypes of strains expressing a wild-type or a mutant Pro282Leu form of PmtC::GFP (wild-type background) after 60 h of culture on AMM (diameter of Petri dishes: 5.5 cm). Substitution of Pro282 by a Leu causes an inhibition of radial growth and an aconidial phenotype. (B) Subcellular localization of PmtC::GFP in wild-type or ΔflbB hyphae: PmtC localizes to structures resembling the ER [52,53]. Scale bars = 5 µm. (C) Localization of PmtC::GFP in wild-type hyphae before and 20, 40, and 60 min after the addition of 8 mM DTT [52]. Recovery of growth and native PmtC localization after the removal of DTT is also shown (bottom panel). Scale bar = 5 µm. (D) Micrographs of hyphae of strains expressing PmtC::GFP or PmtC^Pro282Leu::GFP chimeras (wild-type background). Arrowheads indicate the multiple adjacent branching sites observed in the mutant strain. (E) Subcellular localization of PmtC^Pro282Leu::GFP (wild-type or ΔflbB backgrounds) and PmtC^Arg762Gly::GFP (ΔflbB background) in hyphae. Both PmtC mutant forms maintain the localization of the wild-type form, suggesting that Pro282Leu or Arg762Gly substitutions affect PmtC activity but not its localization. Scale bar = 5 µm.