A Transient Receptor Potential-like Calcium Ion Channel in the Filamentous Fungus Aspergillus nidulans

Transient Receptor Potential (TRP) proteins constitute a superfamily that encodes transmembrane ion channels with highly diverse permeation and gating properties. Filamentous fungi possess putative TRP channel-encoded genes, but their functions remain elusive. Here, we report that a putative TRP-like calcium channel, trpR, in the filamentous fungus Aspergillus nidulans, performs important roles in conidiation and in adapting to cell wall disruption reagents in a high temperature-induced defect-dependent manner, especially under a calcium-limited culture condition. The genetic and functional relationship between TrpR and the previously identified high-affinity calcium channels CchA/MidA indicates that TrpR has an opposite response to CchA/MidA when reacting to cell wall disruption reagents and in regulating calcium transients. However, a considerable addition of calcium can rescue all the defects that occur in TrpR and CchA/MidA, meaning that calcium is able to bypass the necessary requirement. Nevertheless, the colocalization at the membrane of the Golgi for TrpR and the P-type Golgi Ca2+ ATPase PmrA suggests two channels that may work as ion transporters, transferring Ca2+ from the cytosol into the Golgi apparatus and maintaining cellular calcium homeostasis. Therefore, combined with data for the trpR deletion mutant revealing abnormal cell wall structures, TrpR works as a Golgi membrane calcium ion channel that involves cell wall integration.


Introduction
Transient receptor potential (TRP) proteins were first described in Drosophila [1], and the presence of their superfamilies, that possess highly diverse permeation and gating properties, has been reported in most eukaryotes [2][3][4]. To date, around 30 proteins have been reported to belong to the TRP superfamily and possess some similar structural characteristics. Based on sequence homology, these proteins have been subdivided into eight major branches and are divided into three broad groups: group one includes TRPC, TRPA, TRPM, TRPN, TRPV, group two includes TRPP and TRPML, and group three is the TRPY subfamily which currently only has one member, TRPY1 (or Yvc1). Furthermore, members of group one and two are all found in the metazoans but TRPY1(TRPY subfamily) was identified in fungi which putatively evolved from metazoans to fungi and resulted in a divergence [5]. However, TRP proteins seem to be absent in archaea, bacteria or higher plants [2,6,7].
In mammals, most TRP members are permeable for both Ca 2+ or Mg 2+ , and some are even highly permeable relative to the monovalent cations [2]. TRP channels are best recognized as sensors for environmental irritants, causing somatosensory modalities, such as pain, cold, itching, and other protective responses [3,6]. It has been found that TRP mutations are linked to human diseases, indicating the physiological importance of TRP proteins [4,7]. The mammalian TRP protein forms a tetramer, where each polypeptide

Strains, Media, and Growth Conditions
The A. nidulans strains used in this study have been summarized and listed (see Table 1). A. nidulans strains were grown on rich media YAG or YUU (YAG: 2% glucose, 1 mL·L −1 trace elements, 0.5% yeast extract, 2% agar (for solid). YUU: YAG + 5 mM uridine + 10 mM uracil). Experiments were performed in a minimal amount of medium PDR or PDRUU (PDR: 2% glucose, 50 mL·L −1 salts, 1 mL·L −1 1000 × trace elements solution, 2.5 µM pyridoxine, 6.6 µM riboflavin. PDRUU: PDR + 5 mM uridine + 10 mM uracil, 1000 × trace elements solution and 20 × Salts solution were prepared according to the formulation provided in the literature [26]. The recipe of the 1000 × trace elements solution is as follows: 77 mM ZnSO O 42 mM.), maintained at 37 • C. The Conidia were harvested on YAG plates using sterile H 2 O and placed, for long-term storage, in 50% glycerol at −80 • C. Additional instructions will be provided if special cultivation conditions are required.

Genetic Mutant Strain Construction
To construct the deletion mutant strains in this study, homologous recombination was used [27]. All primers used to design constructs are listed in Table 2. Fungal transformation was also performed as previously described [28]. For construction of ∆trpR, we used pyrG as a selectable nutritional marker and amplified the 5 and 3 flanking regions of the trpR open reading frame (ORF) from A. nidulans TN02A7 genomic DNA with primer pairs trpR P1/P3 and trpR P4/P6, respectively. Finally, combining these three fragments was achieved through fusion PCR using the primer pair trpR P2/P5. The PCR product was transformed into the recipient wild-type strain.
To construct the trpR complemented strain, we amplified the trpR gene including the promoter, ORF, and terminator, with the primer pairs trpR up/down from A. nidulans gDNA. We cloned the trpR gene into the plasmid pQa-pyroA, which contains a 1.7 kb pyroA fragment as a selectable marker. The new plasmid was transformed into the recipient ∆trpR strain to generate the trpR c strain.
To generate the TrpR-GFP strain, we followed the same principle used for building deletion mutant strains. In summary, the 5 and 3 flanking regions of the trpR stop codon were amplified from strain TN02A7 using primer pairs trpR-gfp P1/P3 and trpR-gfp P4/P6, respectively. The gfp-pyrG fragment was amplified from the plasmid pFNO3 using primers gfp up and pyrG down. The trpR-gfp-pyrG fusion PCR product (using primer pairs trpR-gfp P2/P5) was transformed into the recipient strain TN02A7. The TrpR-GFP mRFP-PH OSBP and TrpR-GFP RFP-PmrA strains were constructed by transforming the relative gene fragments into the recipient TrpR-GFP strain. The transformants were verified using diagnostic PCR and microscopic testing. The ∆pmrA gpd-trpR and ∆trpR gpd-pmrA strains were constructed using a similar strategy. To summarize, the gpd promoter was amplified with primer pairs gpd up/down, and the trpR gene fragment was amplified with primers gpd-trpR up/down. These two fragments were combined using primers gpd up and gpd-trpR down. Finally, the above fusion product was cloned into the plasmid pQa-pyroA to create a new plasmid. We transformed it into the recipient ∆pmrA strain to obtain the ∆pmrA gpd-trpR strain. Similar approaches were applied for the construction of the ∆trpR gpd-pmrA strain. And the transformers were verified using qRT-PCR assays.
The strains that expressed codon-optimized aequorin were constructed by co-transforming the plasmid pAEQ containing aequorin and the selective markers pyroA or riboB genes into the indicated mutants [29]. We screened the transformers for aequorin expression using diagnostic PCR assays and selected the suitable aequorin expressing strains for further purification.
The double deletion strain was generated by crossing two relative single deletion strains as previously described [30]. In brief, we inoculated two parent strains on a plate for 2 days at 37 • C. Then, the mixed-grown mycelia were transferred to a screening medium supplemented with 66 µM riboflavin and incubated for 15 days at 30 • C until cleistothecia became visible. A grain of cleistothecium was lifted, and the mycelia around it were cleaned, after which a cleaned cleistothecium was transferred to a 1.5-mL centrifuge tube. Then, 1 mL of sterilized H 2 O was added, and the solution was mixed well. Finally, a 10-µL suspension of ascospores was transferred to the rich YUU medium for culturing. The progenies were screened based on their phenotypes.

Phylogenetic Analysis
All of the amino acid sequences were obtained from the fungal databases FungiDB (http://fungidb.org/fungidb, accessed on 26 October 2021) and NCBI (https://www.ncbi. nlm.nih.gov, accessed on 26 October 2021). The Hidden Markov Model (HMM) profile for the TRP Pfam (PF06011) was downloaded from the protein family database (http: //pfam.xfam.org, accessed on 26 October 2021) and was used to identify the putative TRP channel genes from the A. nidulans genome with HMMER 3.0 (http://hmmer.janelia.org/, accessed on 31 July 2020) [31]. The phylogenetic tree was constructed via MEGA 7 software, using the neighbor-joining method and a bootstrap test with 1000 iterations.

Plate
To analyze the impact of thermal treatment on conidiation, wild-type and relevant strains were cultured at 30, 37 and 42 • C. To obtain the impact of cell wall stress, we tested the condition of fungal growth on a medium supplemented with the following agents: calcofluor white (CFW), congo red (CR), and caspofungin (CAS) (Sigma-Aldrich, St. Louis, MO, USA). In order to test the restorative effect of calcium on fungal growth, 50 mM of CaCl 2 was added to the medium. The operation process was as follows: 2.5 µL of the conidia (1 × 10 6 conidia·mL −1 ) of the indicated strains were spotted onto relevant media and cultured for 2.5 days. Finally, the colony diameter was measured, and the total spore quantity of all strains was counted. At least three replicates were performed for each experiment.

Microscopic Observation and Image Processing
In order to observe the hyphal growth, approximately 1 × 10 4 conidia of the relevant strains were incubated in a 1 mL liquid PDR or PDRUU medium and cultured in a petri dish containing a coverslip, at 37 • C for approximately 10 h and observed under a microscope. For the localization of the TrpR-GFP protein, we incubated the TrpR-GFP strain in PDR medium. After 10 h, we removed the medium and washed the mycelia three times with phosphate-buffered saline. Then, the mycelia were fixed with 4% paraformaldehyde (Polysciences, Warrington, PA, USA) for 40 min at room temperature in the dark. The paraformaldehyde was cleaned and visualized under a fluorescence microscope using a 63× objective oil lens. FM4-64 (Sigma-Aldrich, St. Louis, MO, USA) staining was conducted on the ice in accordance with the protocol manual. All the images were captured with a Zeiss Axio imager A1 microscope (Carl Zeiss, Jena, Germany).

RNA Isolation and Quantitative RT-PCR Assays
For RNA isolation, 1 × 10 8 fresh conidia from related strains were inoculated in 100 mL of a liquid PDR or PDRUU medium at 37 • C for 16 h, and then the mycelia were harvested and frozen in liquid nitrogen. RNA was extracted using liquid nitrogen and an RNAzol RT column kit (Sangon Biotech no. B511631-0100). Both the reverse transcription-PCR and qRT-PCR analyses were performed using HiScript II Reverse Transcriptase (Vazyme catalog no. R201-01) and SYBR Premix Ex Taq (TaKaRa catalog no. DRR041A), respectively. The specific operations complied with the instructions of the protocol manual, and the transcription levels were calculated according to the comparative threshold cycle (∆ CT ) method [27].

Cytoplasmic Ca 2+ Measurement
Cytoplasmic Ca 2+ determination was performed as previously described [19,32]. We briefly transformed the pAEQ vector harboring the codon-optimized aequorin gene into the indicated strains. The strains expressing the aequorin gene were cultured in a PDRUU medium, adjusted to 1 × 10 7 spores·ml −1 , and then distributed into wells in a 96-well microdroplet plate at 100 µL per well. Each strain was inoculated with 8 parallel replicates. After their incubation for 18 h at 37 • C, the medium was removed, and the mycelia were rinsed twice with PGM (50 mM glucose, 1 mM MgCl 2 and 20 mM PIPES (pH 6.7)). The aequorin was reconstituted by incubating mycelia in 100 µL 25 µM·ml −1 coelenterazine (Sigma-Aldrich, St. Louis, MO, USA) diluted with PGM for 4 h at 4 • C in the dark. After reconstitution, the mycelia were washed twice with PGM, and the plate was placed at room temperature for 1 h. The luminescence, excited by 10 mM CaCl 2 ,was measured with an LB 96P Microlumat luminometer (Berthold Technologies, Bad Wildbad, Germany) and the active aequorin was completely discharged using a discharge buffer (containing 20% (vol/vol) ethanol and 3 M CaCl 2 ). Finally, the relative light unit (RLU) values were converted into [Ca 2+ ]c concentrations through the use of the following calibration formula: pCa = 0.332588 (−log k) + 5.5593, where k is luminescence (in RLU) s −1 /total luminescence (in RLU) [29].

Statistical Analysis
Data were provided by means of ± SD. The statistical significance was estimated using either t-tests or multiple t-tests. The p-values of less than 0.05 were considered statistically significant.

Phylogenetic Evolution and Diversification of TrpR Homologs in Selected Eukaryotic Species
To study the phylogenetic relationship of all proteins harboring the TRP Pfam in A. nidulans database, a Hidden Markov Model profiling (hmmscan) was performed against the A. nidulans protein database using the TRP Pfam (PF06011) as a searching criterion [31]. As shown in Figure 1A, there are 4 putative TRP-like channels in A. nidulans which contain the TRP Pfam domain in their protein sequence. Among them, the gene numbered AN9146 is matched with a putative homolog of S. pombe trp1322 which had been reported for its functions of mediating the cytoplasmic Ca 2+ in yeasts. We then focused on the functions of AN9146 which we referred to as a trpR.
To further explore the conservative property of TrpR homologs, we performed an evolutionary analysis using the TrpR's total protein sequence to perform a BLAST search in selected eukaryotes, as presented in Figure 1B. As a result, all the selected ascomycete homologs present a relatively close relationship between them ( Figure 1B). Homologs in Aspergillus spp are very closely related, such that A. nidulans demonstrated, approximately, a 65-90% amino acid sequence, identifying with other species of Aspergillus. In comparison, the TrpR in A. nidulans (AN9146) has a 43% identification with Neurospora crassa whereas TrpR only presented 25% amino acid sequence identity with S. pombe, 24% with Cryptococcus neoformans, 22% with Candida albicans. Further phylogenetic analyses indicate that all selected ascomycete homologs possess signatures that are typical for the TRP_N domain and TRP Pfam, suggesting that the TrpR homologs are relatively conserved in fungi ( Figure 1B). [31]. As shown in Figure 1A, there are 4 putative TRP-like channels in A. nidulans which contain the TRP Pfam domain in their protein sequence. Among them, the gene numbered AN9146 is matched with a putative homolog of S. pombe trp1322 which had been reported for its functions of mediating the cytoplasmic Ca 2+ in yeasts. We then focused on the functions of AN9146 which we referred to as a trpR. To further explore the conservative property of TrpR homologs, we performed an evolutionary analysis using the TrpR's total protein sequence to perform a BLAST search in selected eukaryotes, as presented in Figure 1B. As a result, all the selected ascomycete homologs present a relatively close relationship between them ( Figure 1B). Homologs in Aspergillus spp are very closely related, such that A. nidulans demonstrated, approximately, a 65-90% amino acid sequence, identifying with other species of Aspergillus. In comparison, the TrpR in A. nidulans (AN9146) has a 43% identification with Neurospora crassa whereas TrpR only presented 25% amino acid sequence identity with S. pombe, 24% with Cryptococcus neoformans, 22% with Candida albicans. Further phylogenetic analyses indicate that all selected ascomycete homologs possess signatures that are typical for the TRP_N domain and TRP Pfam, suggesting that the TrpR homologs are relatively conserved in fungi ( Figure 1B).

Dynamic Localization of TrpR-GFP during Different Developmental Stages of A. nidulans
To further study the localization of TrpR, a TrpR-GFP strain with a green fluorescent protein (GFP) tag fused at the C-terminus of TrpR was constructed. We confirmed it to be successful by a diagnostic PCR (see Figure S1A). As shown in Figure 2A, the green fluorescence of the TrpR-GFP fusion protein possesses a multiple-type distribution under the different developmental stages. In swollen conidia, TrpR displayed a weak plasma membrane pattern in the form of a circle along the conidial cell wall, accompanied by deep-

Dynamic Localization of TrpR-GFP during Different Developmental Stages of A. nidulans
To further study the localization of TrpR, a TrpR-GFP strain with a green fluorescent protein (GFP) tag fused at the C-terminus of TrpR was constructed. We confirmed it to be successful by a diagnostic PCR (see Figure S1A). As shown in Figure 2A, the green fluorescence of the TrpR-GFP fusion protein possesses a multiple-type distribution under the different developmental stages. In swollen conidia, TrpR displayed a weak plasma membrane pattern in the form of a circle along the conidial cell wall, accompanied by deepgreen particles of the endomembrane system. In the germlings, TrpR-GFP predominantly accumulated in the apical region of hyphae. In mature hyphae, TrpR-GFP presented septum-type and cytoplasmic-punctuated locations.
To further explore the location of TrpR, we used the lipophilic marker FM4-64, a lipid membrane marker, to stain the TrpR-GFP strain. The results suggest that the majority of the TrpR-GFP foci was stained by FM4-64, especially in the subapical region (see Figure 2B). Additionally, we used the human oxysterol-binding protein PH domain (PH OSBP ), which is a well-established marker to label late/trans-Golgi compartments, to examine the possible colocalization association of TrpR-GFP with Golgi compartments. As shown in Figure 2C, TrpR exhibited partial puncta colocalization with the late Golgi marker mRFP-PH OSBP . Overall, TrpR is an endomembrane protein that is usually located in the membrane of Golgi and vesicle-like membrane structures.

Lack of TrpR Causes Hypersensitivity to Thermal Stresses and Cell Wall Destruction Reagents
To investigate the biological function of TrpR in A. nidulans, we constructed a null mutant carrying a deletion of the gene encoding TrpR (AN9146) by replacing the coding sequence with the pyrG selectable marker in the parental TN02A7 strain. The generation of the ∆trpR mutant was confirmed to be successful by a diagnostic PCR (see Figure S1B). As shown in Figure 3A-C, compared to the parental wild-type strain, removing the trpR did not affect the colony diameter on the PDRUU medium but the number of conidia in the ∆trpR mutant markedly reduced. Despite the fact that the parental wild type contained 100% of conidia, the ∆trpR mutant only reached 45% of WT at 30 • C and 10% of WT at 37 • C. Nevertheless, when cultured at 42 • C, the conidiation was almost abolished in ∆trpR. In comparison, the parental wild-type strain still displayed a robust conidia production at 42 • C, although WT also displayed relatively decreased conidia compared to its levels at 37 • C. To confirm that the defective phenotype was specifically caused by the trpR deletion, we constructed a trpR complemented strain by reintroducing trpR into the ∆trpR mutant. The results revealed that trpR c could resolve the defects of the ∆trpR mutant, suggesting that a lack of TrpR causes a marked reduction in conidial production, especially under thermal culture stresses (Figure 3).
Since the TRP channel superfamily in mammals and in yeasts performs critical functions in the response to external stimuli, and in the knowledge that its members are referred to as the "vanguards of the sensory system", we postulated as to whether TrpR in A. nidulans possesses the ability to "sense" external stimuli. Thus, we compared the growth of ∆trpR and the parental wild-type strain in the presence of different stress agents ( Figure S2). We found that ∆trpR was hypersensitive to cell wall stressors, including the chitin-binding agents calcofluor white (CFW) and congo red (CR), and the β-1,3-glucan synthase inhibitor caspofungin (CAS) compared to the parental wild-type strain, as shown in Figure 2D-F. Altogether, these results suggest that the ∆trpR mutant may have cell wall defects, and that a lack of TrpR results in a drastic reduction in the number of conidia produced in a high temperature-induced defect-dependent manner.  To further explore the location of TrpR, we used the lipophilic marker FM4-64, a lipid membrane marker, to stain the TrpR-GFP strain. The results suggest that the majority of the TrpR-GFP foci was stained by FM4-64, especially in the subapical region (see Figure  2B). Additionally, we used the human oxysterol-binding protein PH domain (PH OSBP ), which is a well-established marker to label late/trans-Golgi compartments, to examine the possible colocalization association of TrpR-GFP with Golgi compartments. As shown in Figure 2C, TrpR exhibited partial puncta colocalization with the late Golgi marker mRFP-PH OSBP . Overall, TrpR is an endomembrane protein that is usually located in the mem- In comparison, the parental wild-type strain still displayed a robust conidia production at 42 °C, although WT also displayed relatively decreased conidia compared to its levels at 37 °C. To confirm that the defective phenotype was specifically caused by the trpR deletion, we constructed a trpR complemented strain by reintroducing trpR into the ΔtrpR mutant. The results revealed that trpR c could resolve the defects of the ΔtrpR mutant, suggesting that a lack of TrpR causes a marked reduction in conidial production, especially under thermal culture stresses (Figure 3). Since the TRP channel superfamily in mammals and in yeasts performs critical functions in the response to external stimuli, and in the knowledge that its members are referred to as the "vanguards of the sensory system", we postulated as to whether TrpR in A. nidulans possesses the ability to "sense" external stimuli. Thus, we compared the growth of ΔtrpR and the parental wild-type strain in the presence of different stress agents ( Figure S2). We found that ΔtrpR was hypersensitive to cell wall stressors, including the chitin-binding agents calcofluor white (CFW) and congo red (CR), and the β-1,3-glucan synthase inhibitor caspofungin (CAS) compared to the parental wild-type strain, as shown in Figure 2D-F. Altogether, these results suggest that the ΔtrpR mutant may have cell wall defects, and that a lack of TrpR results in a drastic reduction in the number of conidia produced in a high temperature-induced defect-dependent manner.

Defects of the ∆trpR Mutant Can Be Rescued by Adding Extracellular Ca 2+
Previous studies reported that TRP channels perform crucial roles in the regulation of cytoplasmic Ca 2+ in fission yeast. To determine whether the defects mentioned above in trpR are related to intracellular calcium homeostasis, we added CaCl 2 to MM and then compared the growth of ∆trpR and the parental wild-type strain. As shown in Figure 4A-D, we found that an addition of calcium was able to not only dramatically restore the conidiation in the ∆trpR mutant but was also able to alter the hypersensitivity of ∆trpR for the insensitive phenotype under treatment with cell wall stress agents, which displayed phenotypes similar to that of the parental wild type. In addition, the phenotypic restoration had a dose-dependent manner. After the addition of 50 mM Ca 2+ , the defective phenotypes of ∆trpR was almost restored to the level the of parental wild-type strain (see Figure 4C,D). In contrast, the addition of calcium chelator-EGTA exacerbated the conidiation defects in the ∆trpR mutant ( Figure 4E,F).
To further test the specification of Ca 2+ , we added other divalent cations, including Mg 2+ Cu 2+ , Co 2+ , Mn 2+ at the indicated concentrations (see Figure S3E) into media and found that the addition of Mg 2+ could also partly restore the defective phenotypes (see Figure S3A-D), but other ions were unable to rescue the defects of ∆trpR.
Taken together, these results suggest that TrpR is involved in the Ca 2+ uptake when subject to low calcium conditions and that partially increasing the amount of extracellular calcium and Mg 2+ can bypass the requirement of TrpR in A. nidulans. tion had a dose-dependent manner. After the addition of 50 mM Ca 2+ , the defective phenotypes of ΔtrpR was almost restored to the level the of parental wild-type strain (see Figure 4C,D). In contrast, the addition of calcium chelator-EGTA exacerbated the conidiation defects in the ΔtrpR mutant ( Figure 4E,F).

Genetic and Functional Relationship between TrpR and the Previously Identified High-Affinity Calcium Channels CchA/MidA
Previous studies have identified that under calcium-limiting conditions, the high affinity calcium system (HACS)-CchA/MidA is required for colony growth. TrpR displayed a function similar to the function of MidA/CchA. We then wanted to investigate the relationship between the MidA/CchA complex and TrpR in regulating calcium homeostasis and adapting to cell wall stress. For the purpose of analyzing the genetic phenotype, we generated ∆trpR∆midA and ∆trpR∆cchA mutants via genetic crossing, as described in the Materials and Methods section. In our study, and consistent with previous reports, the ∆midA and ∆cchA mutants displayed a smaller colony size with a decreased level of conidium production compared to that of the parental wild-type strain when grown on a solid PDRUU medium. In comparison, the ∆trpR∆midA and ∆trpR∆cchA double mutants presented overlapping aggravated defects when compared with both ∆trpR and ∆cchA or ∆midA single mutants. (Figure 5A,B).
Furthermore, when the medium was supplemented with the cell wall destruction reagent-congo red (CR), the ∆trpR mutant became hypersensitive to CR and colony growth was not detectable. In contrast, the ∆midA and ∆cchA mutants were resistance to CR, displaying a colony diameter that is similar to the colony diameter of the parental wildtype strain. Interestingly, exogenous Ca 2+ substantially overcame all the defects seen in the single or double mutants under either thermal or CR stress ( Figure 5A,B). These data implied that TrpR may perform a function opposite to CchA/MidA under calcium-limiting conditions, while abundant calcium addition is able to bypass the requirement for three of them, which means that TrpR may also belong to a member of the high-affinity uptake system. Under high calcium concentrations, there are other uptake systems to regulate Ca 2+ homeostasis in the absence of TrpR and CchA or MidA.
To test the functional relationship of TrpR with CchA/MidA, we monitored the extracellular calcium-induced cellular calcium transients [Ca 2+ ]c in the living cells of A. nidulans using the expressed codon-optimized aequorin. When treated with 0.1 M CaCl 2 , the [Ca 2+ ]c concentration in wild-type cells transiently increased from a resting level of approximately 0.1 µM to a peak concentration of 0.6 µM. In comparison, the ∆trpR mutant displayed a greater increase of 115% compared to the parental wild type (100%) in the [Ca 2+ ]c amplitudes. In contrast, the ∆cchA mutant showed a reduction in the [Ca 2+ ]c amplitude by approximately 80% as compared to the parental wild type under the same stimulating conditions. Notably, unlike the single mutant TrpR or CchA, the ∆trpR∆cchA double mutant displayed a comparable [Ca 2+ ]c amplitude to that of the parental wild-type strain ( Figure 5C,D). These data suggest that TrpR and CchA have a reversed function in regulating cellular calcium transients [Ca 2+ ]c, implying that the HACS components of MidA and CchA are required for the Ca 2+ influx from the extracellular environment while TrpR is primarily responsible for the transport of calcium from the cytoplasm into the Golgi apparatus.

Lack of Golgi-Localized ATPase PmrA Severely Aggravates Defects of ∆trpR
The P-type Golgi Ca 2+ ATPase PmrA is an A. nidulans homolog of yeast Pmr1, which localizes at the Golgi and is responsible for the Ca 2+ transport from the cytoplasm into the Golgi. In order to investigate the relationship between TrpR and PmrA, we generated ∆pmrA and ∆trpR∆pmrA mutants. As shown in Figure 6A, the ∆pmrA mutant and the ∆trpR∆pmrA double deletion mutant showed a slight reduction in the hyphal radial growth and conidiation compared to the wild-type strain on a minimal PDRUU media. However, for the treatment of the cell wall perturbation with congo red (CR) and calcofluor white (CFW), the ∆trpR∆pmrA double deletion mutant did not show a colony sign (if any). Similarly, under the medium supplemented with the cell wall targeted antifungal CAS, ∆trpR∆pmrA displayed very severe colony defects with tiny fluffy colonies when cultured at 37 • C. Supplemented Ca 2+ in the medium was unable to rescue all defects in the ∆trpR∆pmrA mutant under cell wall stress conditions. These data suggest that a lack of both TrpR and PmrA is cannot be accounted for by other Ca 2+ uptake systems for cell wall stress tolerance in A. nidulans ( Figure 6A).   To further assess whether TrpR is colocalized with Golgi-localized PmrA, we labeled PmrA with an RFP tag in the TrpR-GFP background. A microscopic examination showed that most of the TrpR colocalized with PmrA ( Figure 6B). To further test the transient [Ca 2+ ]c change in these mutants, the ΔpmrA, ΔtrpR and ΔtrpRΔpmrA mutants were exposed to a 0.1 M CaCl2 stimulus, respectively, and the [Ca 2+ ]c amplitude of ΔtrpRΔpmrA showed a remarkably unusual increase to approximately 178% compared to 100% of the parental wild-type strain. The single deletion mutant ΔtrpR or ΔpmrA also resulted in a significantly higher [Ca 2+ ]c amplitude compared to the parental wild type but less than that of the double deletions of ΔtrpRΔpmrA, indicating that the perturbation of calcium homeostasis induced by the trpR deletion could be further aggravated by a loss of pmrA ( Figure 6C,D), and that TrpR may have a parallel function with PmrA in transporting the cellular [Ca 2+ ]c into the Golgi in response to extracellular calcium.
Given that PmrA and TrpR perform similar functions, we next wondered whether the defective phenotype caused by trpR deletion could be compensated for by an overexpressed pmrA and vice versa. Strains were overexpressed via the introduction of trpR and pmrA under the control of a constitutive promoter gpd into the ΔpmrA and ΔtrpR backgrounds, respectively. The RT-PCR verified that the expression of trpR in the ΔpmrA gpd-trpR strain was approximately 8 times higher than that of the parental wild-type strain, and pmrA in the ΔtrpR gpd-pmrA strain was nearly 20 times higher than that of the wildtype strain. (See Figure S4). These data confirmed that the gpd promoter induced a high To further assess whether TrpR is colocalized with Golgi-localized PmrA, we labeled PmrA with an RFP tag in the TrpR-GFP background. A microscopic examination showed that most of the TrpR colocalized with PmrA ( Figure 6B). To further test the transient [Ca 2+ ]c change in these mutants, the ∆pmrA, ∆trpR and ∆trpR∆pmrA mutants were exposed to a 0.1 M CaCl 2 stimulus, respectively, and the [Ca 2+ ]c amplitude of ∆trpR∆pmrA showed a remarkably unusual increase to approximately 178% compared to 100% of the parental wild-type strain. The single deletion mutant ∆trpR or ∆pmrA also resulted in a significantly higher [Ca 2+ ]c amplitude compared to the parental wild type but less than that of the double deletions of ∆trpR∆pmrA, indicating that the perturbation of calcium homeostasis induced by the trpR deletion could be further aggravated by a loss of pmrA ( Figure 6C,D), and that TrpR may have a parallel function with PmrA in transporting the cellular [Ca 2+ ]c into the Golgi in response to extracellular calcium.
Given that PmrA and TrpR perform similar functions, we next wondered whether the defective phenotype caused by trpR deletion could be compensated for by an overexpressed pmrA and vice versa. Strains were overexpressed via the introduction of trpR and pmrA under the control of a constitutive promoter gpd into the ∆pmrA and ∆trpR backgrounds, respectively. The RT-PCR verified that the expression of trpR in the ∆pmrA gpd-trpR strain was approximately 8 times higher than that of the parental wild-type strain, and pmrA in the ∆trpR gpd-pmrA strain was nearly 20 times higher than that of the wild-type strain. (See Figure S4). These data confirmed that the gpd promoter induced a high expression for its controlled genes at the mRNA level. However, as shown in Figure 6E, there was almost no difference between the mutant strains and the corresponding overexpressing strains. These results may imply that, although TrpR and PmrA had similar effects in regulating [Ca 2+ ]c, they still had independent characteristic functions, and a compensatory relationship probably did not exist between TrpR and PmrA.

TrpR Is Involved in the Normal Cell Wall Architecture and Composition
Given that the ∆trpR mutant was hypersensitive to cell wall perturbation stress reagents, a lack of TrpR may affect cell wall formation. To explore the effects of TrpR on cell wall architecture, the hyphal cell wall was inspected using transmission electron microscopy (TEM). The results revealed a significantly thinner cell wall in the ∆trpR mutant than in the wild-type strain, indicating that TrpR plays a pivotal role in cell wall architecture ( Figure 7A,B). In addition, we further analyzed the cell wall monosaccharide compositions in mutants and wild-type strains using high-performance ion chromatography. The results revealed abnormalities in the ∆trpR mutant compared to the parental strain ( Figure 7C). The loss of TrpR made an impact on cell wall composition, resulting in an increased proportion of glucosamine but a decreased level of glucose in the cell wall compared with the wild-type strain. To further test whether colony defects of ∆trpR result from cell wall dysfunction, we added sorbitol, an osmotic stabilizer to recover the phenotype of a fragile cell wall. As shown in Figure 7D,E, although sorbitol was able to facilitate a remarkably reduced conidiation for the wild-type strain, the ∆trpR mutant evidenced clear recovery of conidial production through the treatment of sorbitol compared to that which was observed using the minimal medium with the absence of sorbitol. Consequently, there was no significant difference for conidial number between ∆trpR and its parental wild type or between ∆trpR and the trpR complementary strain in the presence of sorbitol which suggest that the colony defects of ∆trpR may be a result of the cell wall dysfunction ( Figure 7D,E). Thus, these data suggest that TrpR plays a crucial role in cell wall composition and architecture and that the osmotic stabilizer is capable of rescuing the conidial defect for deletion of trpR.

Discussion
TRP proteins constitute a superfamily that encodes transmembrane ion channels with very diverse permeation and gating properties [2][3][4]. In mammals, TRP channels are best known as sensors for environmental irritants inducing somatosensory responses [33,34]. Members of the TRP family are intended to be conserved from fungi to mammals. However, the biological functions of TRP channels are not defined in filamentous fungi. In this study, we aimed to find some putative TRP proteins in filamentous fungus Aspergillus nidulans by using TRP Pfam (PF06011) as one of the search criteria in the hidden Markov Model analysis (hmmscan). According to a bioinformatics analysis, we found 4 TRP domaincontaining proteins that are putative members of TRP channels in filamentous fungus A. nidulans. Among them, deleting AN9146, which is referred to as TrpR, displayed colony defects especially under thermal, calcium-limited and cell-wall-stress cultural conditions. Through a phenotypic comparison using gene deletion, overexpression and mutant crossing techniques, we demonstrated that the putative transient receptor potential protein TrpR, a major calcium transporter, is involved in asexual conidiation and the response to cell wall stress adaption by affecting cellular calcium regulation in the filamentous fungus A. nidulans.
In mammals, much evidence has demonstrated that TRP channels perform important roles in physiological and pathological processes, and the protein structure and the specific inhibitor of some TRP members have been recognized to some extent [2]. TRP homologs were also identified [33,34] in yeast. The first TRP protein found in S. cerevisiae is TRPY1, is located in vacuoles and is responsible for the modulation of cytosolic calcium signaling by releasing Ca 2+ from the vacuole in response to hyperosmotic stress and its activation and inhibition are modulated by calcium and lipid, respectively [12,14]. However, the current analysis shows that TRPY1 is a member of V-type of the TRP cation channel subfamily (Interpro entry IPR024862) and that there is no TRP Pfam domain inside of TRPY1 s protein sequence, based on the protein information resource database InterPro (http://www.ebi.ac.uk/interpro/, accessed on 23 October 2021). We therefore could not find a TRPY1 homolog in A. nidulans by searching the TRP Pfam (PF06011). Besides, the three TRP-like ion channels were identified in S. pombe: pkd2, trp1322, and trp663. The deletion of either Trp1322 or Pkd2 lowers the ability to maintain intracellular Ca 2+ homeostasis [13]. The Pkd2 channel was also found to be involved in cell wall synthesis, membrane proteins transport and cytokinesis [15,16]. In this study, we found that most fungal homologs of TrpR display signatures typical of the TRP_N (PF02221) domain and TRP Pfam (PF06011), which are relatively conserved in various fungi. Previous studies identified that the TRP_N (or called ML) domain might be involved in mediating diverse biological functions through an interaction with specific lipids [35][36][37], which means that TRP homologs may need to recognize lipids in order to function properly.
Our findings in this study indicate that TrpR possesses dynamic cellular localizations at the internal membrane system and septa and a weak localization at the plasma membrane of germlings, while the majority of TrpR is highly localized at the membranes of the Golgi and vesicles. These data indicate that TrpR in A. nidulans may perform important functions during the different developmental stages. Notably, a lack of TrpR caused a marked conidiation reduction, and the conidiation in ∆trpR was almost nonexistent at 42 • C. In comparison, the deletion of trpR did not affect the colony size, suggesting that TrpR is more often required for asexual reproduction than for hyphal growth. In addition, we noticed that the trpR mutant was hypersensitive to cell wall destruction reagents (CR, CFW and an antifungal CAS), implying that cell wall integration requires the function of TrpR. Interestingly, these defective phenotypes could be completely resolved via the addition of extracellular calcium. In contrast, the defects in the ∆trpR mutant were exacerbated by adding the calcium chelator-EGTA. These data suggest that TrpR is calcium permeable and may affect cell wall integrity by regulating calcium homeostasis.
A crosstalk between the calcium signal pathway and cell wall integrity pathway has been reported in yeast and some filamentous fungi. Many studies have demonstrated that losses of function in the mutants of calcium regulators could lead to defects of the cell wall integrity [38]. Findings in this study suggest that cell wall defects that were induced by deletion of TrpR may be due to two possibilities. The first is the abnormal expression of cell wall synthetase as regulated by cellular calcium homeostasis in TrpR mutants since previous studies have reported that in A. fumigatus, a major calcium-related transcription factor CrzA was able to regulate the expression of chitin synthases by binding to the calcineurin-dependent response elements (CDRE) in their promoter [39,40]. The second possibility may be a result of the imbalance of calcium signaling in Golgi apparatus in ∆trpR results in abnormal cell-wall integrity since cell wall material transport requires the normal functioning of the Golgi apparatus [25].
In addition, the defects in the ∆trpR mutant could also be rescued by the addition of Mg 2+ , which suggests that TrpR may be a nonspecific transmembrane ion channel involving calcium and magnesium ion transportation, similar to that in mammalian TRP proteins. In human cells, the TRPM subfamily contains similar characteristic that can be permeable to calcium and magnesium ions [41]. Among the members of the TRPM subfamily, TRPM2 was found to be permeable to Ca 2+ , Mg 2+ , and monovalent cations [2]. TRPM6 and TRPM7 are most often studied with regard to their roles in mammals and cellular Mg 2+ homeostasis [42]. These data suggested that the TrpR may be a relative of the TRPM subfamily.
Moreover, the functional and genetic relationship between TrpR and the known calcium channels CchA/MidA was determined by monitoring the cellular calcium transients and colony phenotypes under calcium-limited culture conditions [22,23], indicating that TrpR may also belong to a member of the high-affinity Ca 2+ influx system, but TrpR seems to perform a function opposite to the function of the CchA/MidA complex in regulating cellular calcium transients since the double mutant ∆trpR ∆cchA displayed a recovery of the normal calcium transient compared to the calcium transient of the single mutant, resulting in a calcium response peak similar to that in the parental wild-type strain. In contrast, the double deletion of both TrpR and PmrA displayed an overlaid abnormal increase in calcium transients and hypersensitivity to cell wall destruction reagents. Nevertheless, the colocalization at the membrane of the Golgi for TrpR and PmrA suggested that these two channels may work as ion transporters, transferring Ca 2+ from the cytosol into the Golgi apparatus and maintaining cellular calcium homeostasis [24]. However, an overexpressed trpR in ∆pmrA or vice versa was unable to recover defects for the single mutation of trpR or pmrA, suggesting that TrpR might contain a parallel but irreplaceable function with that of P-type Golgi Ca 2+ ATPase-PmrA. However, we could not overlook the possibility that overexpressed strains may not induce normal functions at the protein level, since we only verified the overexpressed strains at the mRNA level. Furthermore, future work may provide insight in the relationship between TrpR with PmrA at the protein level.
Taken together, the data in this study demonstrated a bioinformatics predicted TRP ion channel, TrpR, which performs an important role in conidiation and cell wall integration by transferring Ca 2+ from the cytosol into the Golgi apparatus. Calcium homeostasis, which is maintained by TrpR, is required for cell wall integration and for the response to thermal and cell wall stresses.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/jof7110920/s1, Figure S1: Diagnostic PCR analyses of the indicated strains, Figure S2: ∆trpR mutant showed no hypersensitivity to various stress agents, Figure S3: The defects in the ∆trpR mutant can be restored by excess magnesium, Figure S4: Verification of the overexpression strains.