Acptp2,3 participates in the regulation of spore production, stress response, and pigments synthesis in Aspergillus cirstatus

Experimental materials

The pDHt-sk-hyg and pDht-sknt plasmids used in this study were stored in our laboratory. The wild type (WT) strain of A. cristatus (CGMCC 7.193) was isolated from Fuzhuan brick tea made by the Yiyang Tea Factory in Yiyang, China. To induce sexual and asexual development, the strains grew on MYA solid medium with low osmotic stress at 28 °C, and on on MYA solid medium with high osmotic stress at 37 °C (20 g of malt extract, 5 g of yeast extract, 30 g of sucrose, 170 g of sodium chloride, and 1000 mL of water, Shao et al., 2022), respectively. Using IM (K₂HPO₄ 2.05 g; KH₂PO₄ 1.45 g; NaCl 0.15 g; MgSO₄•7H₂O 0.5 g; CaCl2•6H₂O 0.1 g; FeSO₄•7H₂O 0.0025 g; (NH₄)₂SO₄ 0.5 g; glucose 2.0 g, 0.5%(W/V)glycerol) as the induction medium for transformation. The morphological observation medium consisted of MYA medium supplemented with various quantities of sucrose, sorbitol, hydrogen peroxide, Congo red, and SDS chemicals. The strains were photographed with a camera (Canon EOS 7D Mark II; Canon, Tokyo, Japan).

Experimental methods

Screening and identification of the ΔAcptp2,3 and ΔAcptp2,3-C strain

Acptp2,3’s whole CDS was deleted using homologous recombination techniques. The Acptp2,3 deletion cassette featuring hph (a resistance gene of hygromycin) as the selective marker was created by fusing the 5′-untranslated region (5′-UTR) and 3′-untranslated region (3′-UTR) of the Acptp2,3 gene. To construct the final knockout vector, up-Acptp2,3-pDHt/sk-hyg-Acptp2,3-down, the 5′-BamH I-Xho I UTR and 3′-Spe I-Xba I UTR were amplified using appropriate primer pairs from genomic DNA of the WT strain and cloned into the cloning sites of pDHt/sk-hyg plasmid (a schematic diagram of vector construction as shown in Fig. S1). To complement the Acptp2,3 mutant, the Acptp2,3 gene with its own promoter was amplified from genomic DNA using primers (Table S1) and inserted between Hind III and Kpn I of the pDHt/sknt plasmids. The plasmid sequences were confirmed by polymerase chain reaction (PCR), restriction enzyme digestion, and sequencing. The transformation was carried out exactly as described earlier (Tan, 2008). Agrobacterium tumefaciens strain LBA4404, comprising a previously produced vector, was cultivated in a liquid minimum medium at 28 °C for 48 h, with 50 ug/mL kanamycin. A 100 uL aliquot of the A. tumefaciens fluid solution was combined with an equal volume of a conidial suspension from the WT strain or ΔAcptp2,3 strain and cultivated at 28 °C for approximately 9 h. The mixture was plated on an IM plate and cultured at 28 °C for 48 h. MYA medium with 300 ug/mLAmpicillin, 50 ug/mL Hygromycin B, or 200 ug/mL Geneticin (G418) was plated on a coculture plate at 28 °C until transformants developed. The transformed strains were validated using both PCR and real-time quantitative PCR (RT-qPCR), the primers design principles required for the validation of ΔAcptp2,3 and ΔAcptp2,3-C strains were shown in Figs. S2 and S3.

Morphological observation

Various strains were inoculated on the matching medium, colony morphology was observed, colony diameter was measured, sexual and asexual spore production statistics, and various sensitivity tests in ΔAcptp2,3 and ΔAcptp2,3-C were performed.

Statistical analysis

Statistical analysis of gene expression, colony diameter, ascospore and conidia number: Set up three biological replicates, three measurements were recorded and the data were analyzed statistically. * p < 0.05: the lowest significance; ** p < 0.01: a moderate degree of significance; *** p < 0.001: the highest significance.

Real time-PCR detection

Real time-PCR (RT-PCR) was carried out as previously described in Shao et al. (2022). Total RNA was extracted at the tested time point. Then, 2 mg of RNA was utilized to synthesize cDNA using a RevertAid First Strand cDNA Synthesis Kit (catalogue#K1622; Thermo Fisher Scientific, Waltham, MA, USA). RT-qPCR was performed using a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) in a total volume of 10 uL, which consisted of 5 uL of SsoFast EvaGreen SuperMix (catalogue #172-5,201; Bio-Rad Laboratories), 1 uL of each primer (10 pmol/mL and 1 uL of template). GAPDH was selected as a candidate reference gene. The primers employed in the RT-qPCR experiments were designed using the Primer 3 online program and the resulting RT-qPCR products were tested via agarose gel electrophoresis. Each primer pair was tested with serial dilutions of cDNA to determine the linear range of the RT-qPCR assays. Three biological replicates were analysed (Shao et al., 2022). All the RT-qPCR primers used were listed in Table S1.

Screening and identification of the ΔAcptp2,3 and ΔAcptp2,3-C strain

Analysis of the gene sequence of Actp2,3 showed that ptp2,3 was highly conserved among Aspergillus specifications and ptp2,3 homologs as a well supported group with A. Melleus (Fig. S4). And it has a PTP-fungal domain spanning amino acids 512-777, which was identical to the conserved domain of the ptp2,3 gene in S.cerevisiae. Subsequently, the ΔAcptp2,3 knockout strain was constructed, and the enzyme digestion results showed that the size of the product was expected, indicating that the plasmid up-Acptp2,3-pDHt/sk-hyg-Actp2,3-down (Fig. S5A) and the complementation vector were successfully constructed (Fig. S52B). ΔAcptp2,3 knockout and ΔAcptp2,3-C complementation strains were obtained through ATMT-mediated transformation (Tan, 2008). Specific primers (the primers used in this study are shown in Table S1) were used to validate ΔAcptp2,3 and ΔAcptp2,3-C strains. The segments amplified from the WT and ΔAcptp2,3 genomic DNA templates were 3,160 bp and 4,206 bp respectively (Fig. S6A). RT-PCR was used to further validate the ΔAcptp2,3, and the results showed that the expression of the Acptp2,3 gene was not detected in the knockout strains (Fig. S6B). A total of 13 ΔAcptp2,3-C strains were confirmed by PCR (Fig. S6C). Further detection by RT-PCR revealed that the expression level of Acptp2,3 was nearly identical to that of the WT strain, indicating that Acptp2,3 was actually complemented in ΔAcptp2,3 strains (Fig. S6D). The above results revealed that knockout strains might be utilized to investigate phenotypic differences.

Acptp2,3 promoted the production of ascospores and conidia in A. cristatus

Studies showed that the absence of ptp2,3 leaded to the defection of sporulation in mutants, even the mutants almost lost the ability of sporulation (Yang, 2013). To investigate the impact of Acptp2,3 on sexual and asexual sporulation, the conidial liquid (1.0 × 10⁶/mL) of wild strains WT, ΔAcptp2,3, and ΔAcptp2,3-C were inoculated on 1 M MYA solid medium and cultivated at 28 °C for 7 days. Phenotype and microstructure were examined. The results revealed that the wild-type strains and ΔAcptp2,3 strains could continue on sexual development. There was no significant change in the shape of the cleistothecium on MYA medium with 1 M NaCl (Fig. 1A, columns 1–2). The cleistothecium was crushed and examined under a microscope. On the 14th day of culture, both ΔAcptp2,3 and wild-type strains generated typical ascospores (Fig. 1B). However, the wild-type strain produced 4.4 times more ascospores than the ΔAcptp2,3 strains (Fig. 1C). Similar findings were obtained in MYA medium with 1 M sorbitol and 1 M sucrose (Fig. 1A, columns 4–6).

The conidial liquid (1.0 × 10⁶/mL) of wild strains WT, ΔAcptp2,3, and ΔAcptp2,3-C were inoculated on 3 M MYA solid medium and cultivated at 37 °C for 7 days to investigate the effect of ΔAcptp2,3 strains on asexual sporulation. The diameter of ΔAcptp2,3 was smaller than that of the wild type (Fig. 2B), and the colony was yellow-green (Fig. 2A, first row). The conidial output was roughly 4.6 times lower than that of the wild type on the seventh day (Fig. 2C). Microscopic analysis revealed no significant variation in asexual sporulation structure between WT, ΔAcptp2,3, and ΔAcptp2,3-C (Fig. 2A, row 2).

Deleting Acptp2,3 gene effects the response of A. cristatus to high osmotic stress

As a key gene in the HOG pathway, one of the main functions of ptp2,3 is to regulate the osmotic stress of S. cerevisiae (Mattison & Ota, 2000). WT, ΔAcptp2,3, and ΔAcptp2,3-C strains were grown in MYA medium with 2 M/3 M sorbitol/sucrose at 37 °C. ΔAcptp2,3 grew similarly to the wild type, but at a slower rate on MYA medium with 2 and 3 M sorbitol (Figs. 3A–3B). The conidial number of WT, ΔAcptp2,3, and ΔAcptp2,3-C strains were statistically examined on a medium with 3 M sorbitol and 3 M sucrose. In the presence of sorbitol and sucrose, ΔAcptp2,3 produced considerably less conidia. Wild-type strain produced 3.14 and 2.2 times more conidia than ΔAcptp2,3 strains on MYA medium with 3 M sorbitol and sucrose (Fig. 3C). The results showed that ΔAcptp2,3 was more susceptible to high sucrose concentrations than sorbitol. Furthermore, Acptp2,3 might increase the conidial production of A. cristatus in sorbitol and sucrose-rich mediums.

Role of Acptp2,3 under oxidative stress

When conducting functional studies on the Fgptp gene in Fusariurri graminearurr, it was found that ΔFgptp strains were not sensitive to oxidative stress (Jiang, 2012). We would like to know the effect of Actp2,3 on the response of oxidative stress in A. cristatus. WT, ΔAcptp2,3, and ΔAcptp2,3-C conidial suspensions were inoculated on MYA medium with hydrogen peroxide (H₂O₂), whereas MYA medium without any agent was utilized as the control medium. Oxidative stress affects ΔAcptp2,3 strains. The colony diameter of ΔAcptp2,3 was substantially smaller than that of the wild type on medium with varied doses of H₂O₂ (10 mM, 30 mM, and 50 mM) (Figs. 4A–4B). The results showed that ΔAcptp2,3 was much more susceptible to H₂O₂, indicating that this gene might modulate the response of oxidative stress in A. cristatus.

Acptp2,3 was involved in the response of Congo red and sodium dodecyl sulfate in A. cristatus

In F. graminearurr, the absence of Fgptp2 slowed down the growth of ΔFgptp2 strain (Jiang, 2012). We wanted to know if there would be similar results in the absence of Actp2,3. WT, ΔAcptp2,3, and ΔAcptp2,3-C were inoculated on MYA medium (0.5 M NaCl) including agentia to detect the sensitivity of ΔAcptp2,3 to Congo red and SDS. MYA (0.5M NaCl) with no additions was employed as the control medium. The results demonstrated that both ΔAcptp2,3 and WT could grow on the medium containing Congo red. After 5 days of cultivation, the ΔAcptp2,3 strains had wider colony diameters than the WT strains, indicating reduced sensitivity to Congo red (Figs. 5A and 5B). WT and ΔAcptp2,3-C strains struggled to grow in a medium with 0.01% SDS, whereas ΔAcptp2,3 strains performed well (Figs. 5C and 5D). These findings revealed that Acptp2,3 was involved in the response to cell wall-damaging chemicals such as Congo red and SDS.

Deleting Acptp2,3 caused colonies to deepen in color

WT, ΔAcptp2,3, and ΔAcptp2,3-C were inoculated on MYA solid medium to compare pigments. As illustrated in Fig. 6A, the WT colony’s center was brown, with bright yellow margins. The ΔAcptp2,3 colony had a brown center and yellow margins. The colony color of ΔAcptp2,3-C resembled that of the wild type (Fig. 6A). RT-qPCR was used to detect the expression levels of four genes (SI65_08209, SI65_08202, SI65-08742 and SI65_08211) involved in pigments synthesis. The ΔAcptp2,3 strain had considerably greater expression levels of four pigments synthesis genes compared to the wild-type and ΔAcptp2,3-C strains (Fig. 6B), demonstrating that Acptp2,3 could decrease the pigments of A. cristatus.

RT-qPCR analysis of related gene expression

The wild type strains grew faster than ΔAcptp2,3 strains in high sucrose and H₂O₂ medium, but slower in Congo red and SDS media. We used real-time fluorescence PCR to detect the expression of associated genes. The expression levels of genes associated with osmotic stress and oxidative stress were found to be lower than those of the wild type, while genes associated with the Congo red response were notably higher (Fig. 7). These findings indicated that Acptp2,3 adversely regulated the expression of genes associated with osmotic stress and oxidative stress while positively regulating the expression of genes associated with the Congo red response.