Functional Characterization of the Sterol-Synthesis-Related Gene CgCYP51 in the Poplar Anthracnose-Causing Fungus Colletotrichum gloeosporioides

Abstract

Poplar is an economically and ecologically valuable tree species. Anthracnose, which severely affects poplar tree growth, is mainly caused by Colletotrichum gloeosporioides. In the infestation cycle of poplar anthracnose, the entry of C. gloeosporioides into the host tissue depends on the formation of an appressorium. The subsequent development of the appressorium determines the pathogenesis of poplar anthracnose and the degree of damage. Previous studies have found that the transcription factor CgSte12 affects appressorium formation and development by regulating the expression of a series of genes, including the sterol-synthesis-related gene CgCYP51, which influences appressorium formation and development. In this study, knockout and functional analyses of CgCYP51 revealed decreases in differentiation, darkening rate, and turgor pressure of appressoria in mutants. Additionally, compared with the wild-type appressorium, mutant appressoria secreted less mucus and exhibited abnormal penetration pore formation, ultimately leading to decreased pathogenicity. Moreover, CgCyp51 affected the sensitivity of C. gloeosporioides to sterol biosynthesis inhibitors. Considered together, the study findings indicate CgCYP51 is a key CgSte12-regulated gene that affects C. gloeosporioides appressorium formation and development. Furthermore, the study data provide new insights into the molecular basis of C. gloeosporioides appressorium formation and development.

1. Introduction

Poplar anthracnose is a highly destructive disease that mainly affects tree branches and leaves, resulting in rapid and substantial defoliation [1]. The poplar species most seriously affected by this disease in China is Beijing poplar (Populus × beijingensis), which is a fast-growing, adaptable, and widely planted tree [2]. The main pathogen responsible for poplar anthracnose is Colletotrichum gloeosporioides, a hemibiotrophic filamentous pathogenic fungus [3]. After C. gloeosporioides encounters a suitable host plant, its conidia secrete a mucilaginous substance that adheres to the plant surface. The conidia germinate to form a germ tube, after which the upper part of the germ tube expands to produce an important invasive structure (i.e., appressorium) that develops into a collar structure and penetration peg [4] at an appropriate infection site [5]. It can enter the host plant directly or through stomata and wounds [6]. In addition, appressoria secrete mucus that enables the fungus to attach firmly to the host surface; this mucus is usually distributed around the penetration pore, which contributes to the formation of appressorial turgor pressure [7].

To infect the host, plant pathogenic fungi must first detect host surface signals and then form infection structures that facilitate the invasion of the host plant. During the detection of these host signals, many signaling pathways are activated, including the mitogen-activated protein kinase (MAPK) signaling pathway, which is widespread and highly conserved in fungi. MAPKs are mainly involved in transducing a series of extracellular signals and regulating gene expression during different developmental processes [8]. The sequential induction of the associated signaling cascade activates transcription factors and the expression of specific genes in response to environmental stimuli. For example, Ste12, which was first characterized in Saccharomyces cerevisiae, is a key transcription factor that functions downstream of the MAPK signaling pathway and helps regulate the morphological changes and pathogenicity of most pathogenic fungi [9]. The conserved structure of Ste12 in filamentous fungi is similar to that of its homolog in S. cerevisiae; it generally consists of two C-terminal C2H2 zinc-finger structural domains, a Ste structural domain, and a novel structural domain. This transcription factor influences the pathogenicity of pathogenic fungi by regulating the formation of the appressorium and the subsequent penetration of susceptible host tissue. In Magnaporthe oryzae, knocking out Mst12 does not alter appressorium formation or germination, but it inhibits the production of penetration pegs, resulting in a lack of pathogenicity [10].

A previous study sequenced the transcriptome of the CgSTE12 knockout mutant, which identified EVM0014725 (CYP51) as a differentially expressed gene associated with sterol synthesis according to a KEGG pathway enrichment analysis [11]. The expression level of EVM0014725 (CYP51), which encodes a sterol 14α-demethylase important for ergosterol biosynthesis in fungi, is downregulated at 24 h. Earlier research revealed the impaired penetration pore formation of the CgSTE12 knockout mutant appressorium at 24 h [12]. This finding is consistent with the downregulated expression of EVM0014725 (CYP51) at 24 h, reflecting the likely importance of CYP51 for appressorium formation.

The cytochrome P450 (CYP) family consists of many members that typically catalyze monooxidation reactions, with key roles in primary and secondary metabolic pathways. Sterol 14α-demethylase (Cyp51) is the most functionally conserved CYP and catalyzes the 14α-methyl hydroxylation of sterol precursors during sterol synthesis [13,14,15]. In fungi, the inhibition of sterol 14α-demethylase activity disrupts sterol production, leading to cell membrane damage and cell death. Additionally, 14α-demethylase is an important target for applied research aimed at designing antifungal agents known as 14α-demethylase inhibitors (DMIs) [16]. This enzyme was first purified from S. cerevisiae in 1984 and classified in the Cyp51 family [17]. In M. oryzae, the deletion of MoCYP51A affects conidia, virulence, and susceptibility to DMI fungicides, whereas the deletion of MoCYP51B does not result in significant phenotypic changes [18]. In Fusarium graminearum, FgCYP51A is associated with the susceptibility to azoles, FgCYP51B encodes a sterol 14α-demethylase important for ascospore formation, and FgCYP51C affects virulence [19]. In Aspergillus fumigatus, a lack of CYP51A expression leads to increased susceptibility to certain azoles [20]. Cyp51 has long been widely studied as a target of antifungal agents. To date, Cyp51 has been analyzed in many fungi regarding its genetic characteristics, in vivo and ex vivo physiological properties, and resistance to DMIs. Although DMI resistance has been widely reported, only a few Cyp51 proteins have been examined in terms of their effects on appressoria. Moreover, the C. gloeosporioides gene encoding sterol 14α-demethylase has not been functionally annotated.

In this study, we analyzed the effects of CgCyp51, a sterol-synthesis-related protein, on C. gloeosporioides appressorium formation and development as well as C. gloeosporioides pathogenicity through knockout and phenotypic analyses. The results showed that a lack of CgCyp51 affects the appressorium formation rate, blackening rate, turgor pressure, mucus accumulation at the base of the appressorium, and penetration pore, ultimately leading to decreased virulence. The study findings have clarified the biological functions and underlying mechanisms of the P450 superfamily, while also providing new insights into the defense and control mechanisms of C. gloeosporioides.

2. Materials and Methods

2.1. Experimental Strains and Culture Methods

The wild-type (WT) C. gloeosporioides strain CFCC80308 [21] was isolated from Populus × beijingensis in Beijing, China, for this study. Potato dextrose agar (PDA) medium was inoculated with WT or mutant strains and incubated at 25 °C to induce spore production.

Transformed Escherichia coli DH5α cells were cultured on solid Luria Bertani (LB) medium (5 g yeast extract, 10 g tryptone, 10 g NaCl, and 15 g agar) or in liquid LB medium (5 g yeast extract, 10 g tryptone, and 10 g NaCl) at 37 °C. Solid TB3 medium (3 g yeast extract, 3 g casamino acids, 20% sucrose, and 0.7% agar) was used to screen for mutants.

2.2. Molecular Cloning of CgCYP51

The CgCyp51 amino acid sequence (Protein ID: 1748231) was determined by comparing the gene sequences obtained from the transcriptome data with sequences in the C. gloeosporioides genome database (https://mycocosm.jgi.doe.gov/Gloci1/Gloci1.home.html, accessed on 14 July 2023). The similarities in the sequences of CgCyp51 and its homologous proteins were assessed using ClustalX 2.1. Additionally, evolutionary relationships were analyzed by constructing phylogenetic trees using MEGA 7.0 [22]. Moreover, protein structural domains were predicted using the InterProScan tool (https://www.ebi.ac.uk/interpro, accessed on 20 July 2023) [23].

2.3. Knockout Mutants and Complementary Strains

CgCYP51 was replaced with a hygromycin B phosphotransferase gene (hph) via homologous recombination, while a CgCYP51 knockout vector was constructed using a split-marker method [24,25]. To obtain CgCYP51 deletion mutants, sequences flanking CgCYP51 (about 1.5 kb upstream and downstream) were amplified by PCR using the following primer pairs: CgCyp51-5f for/CgCyp51-5f rev and CgCyp51-3f for/CgCyp51-3f rev. The amplified upstream and downstream fragments were purified and ligated to the pMD-19 vector using a solution containing T4 DNA ligase. The resulting recombinant vectors were inserted into DH5α cells. The transformed cells were added to solid LB medium containing 50 μg/mL ampicillin in plates and then incubated overnight at 37 °C. Primers M13F and M13R were used to screen for positive clones. Next, CgCyp51-5f for/hyh-r and CgCyp51-3f rev/hyh-f primers, upstream and downstream flanking fragments, and the hph fragment with M13F and M13R were used for an overlapping PCR amplification. The upstream and downstream flanking fragments were fused to the first two-thirds of the hph fragment and to the last two-thirds of the hph fragment, respectively. The fused fragments were used to generate a CgCYP51 knockout vector. The two overlapping fragments were inserted into WT C. gloeosporioides protoplasts via a PEG-mediated protoplast transformation method. Transformants were initially screened using 300 μg/mL thaumatin. After a 2-day incubation, putative transformants were re-screened using PDA medium containing 350 μg/mL thaumatin. The ΔCgcyp51-1 and ΔCgcyp51-3 mutants were confirmed on the basis of a PCR analysis using the internal-CgCyp51for/internal-CgCyp51rev and external-CgCyp51for/external-CgCyp51rev primer pairs, respectively.

The complementation vector was constructed using the CgCYP51 knockout mutants. Specifically, the complete CgCYP51 coding sequence was used for the transformation of CgCYP51 knockout mutant protoplasts according to the above-mentioned PEG-mediated transformation method. Genomic DNA was extracted from the transformants using CTAB for a PCR analysis involving the Internal-CgCyp51 for/Internal-CgCyp51 rev primer pair. The verified complementation strain was named ΔCgcyp51/CYP51. The primers used in this experiment are shown in Table S1.

2.4. Growth Test

The WT and mutant strains were incubated on PDA plate medium for 5 days, after which an agar fungus chunk (0.5 cm diameter) containing mycelia from the outer edge of each colony was produced using a hole punch. The agar plugs were transferred to the center of fresh solid PDA medium in plates using an inoculating needle. These fungus chunks were then incubated at 25 °C for 4 days. The growth of each strain was examined by measuring the diameter of each colony. In addition, solid media with colonies were cut longitudinally and photographed to determine the thickness of the aerial mycelium. The experiment was repeated three times.

2.5. Conidial Germination and Development of Appressorium

WT, ΔCgcyp51-1, ΔCgcyp51-3, and ΔCgcyp51/CYP51 were inoculated in the center of solid PDA medium plates and incubated for 5 days. The surface of the colonies was then rinsed with sterile water to obtain a conidial suspension of each strain. A blood cell counting plate was used to adjust the conidial suspension concentration to 2 × 10⁴ conidia/mL. An equal volume (30 μL) of conidial suspension was selected for each strain and added dropwise to the hydrophobic surface of a GelBond membrane and then incubated in the dark for 12 h in a 25 °C incubator. The formation and blackening of adherent cells of each strain were analyzed using a light microscope and photographed. Two mutants, ΔCgcyp51-1 and ΔCgcyp51-3, were used in this experiment. The experiment consisted of 3 biological replicates and 3 technical replicates.

2.6. Appressorium Turgor Pressure Test

A 0.6 g/mL PEG 4000 solution was selected as a high-permeability agent to determine the turgor pressure inside appressoria. An equal volume (30 μL) of conidial suspension was added dropwise to the hydrophobic surface of the GelBond membrane and incubated for 10 h at 25 °C in the dark. The sporulation solution was replaced with 0.6 g/mL PEG 4000, which was followed by a 10 min incubation in darkness. Appressorium collapse was analyzed using a light microscope and photographed. There were four strains in this experiment, WT, ΔCgcyp51-1, ΔCgcyp51-3, and ΔCgcyp51/CYP51. At least 200 appressoria were examined for each strain, and the experiment was repeated three times.

2.7. Scanning Electron Microscopy

The hydrophobic surface of a GelBond membrane was inoculated with a 30 μL conidial suspension (2 × 10⁴ conidia/mL). The membrane was placed in a large dish containing moistened filter paper and incubated at 25 °C for 24 h. All samples were sheared into 0.4 × 0.4 cm pieces and then affixed to a metal stage with conductive adhesive. To examine the internal collar structure of the adherent cells and penetration pores, a method for rupturing the cell wall of well-developed adherent cells was applied (i.e., a disposable cell spatula was used to repeatedly scrape and rupture the adherent cell wall). After labeling, the samples were dried in an oven at 68 °C for 3 h and plated with gold. They were then examined and photographed using a field emission scanning electron microscope (SEM; Hitachi S-4700, HITACHI, Tokyo, Japan). Three biological and five technical replicates were included in this analysis. All images were analyzed using ImageJ v.2.0 to determine the size of the mucus area.

2.8. Cellophane Membrane Penetration Test

An experiment was performed in order to assess the ability of WT and mutant strains to penetrate a cellophane membrane. Specifically, the cellophane membrane was cut into 30 × 30 mm squares, sterilized, and placed on the surface of solid PDA medium in plates. The center of each cellophane membrane was inoculated with an agar plug (0.5 cm diameter) containing the mycelium of healthy strains. All plates were incubated at 25 °C for 2 days and then photographed. The cellophane membrane with mycelium was removed, and then new colonies growing on the PDA medium were examined and photographed after 2 days. The experiment was repeated three times.

2.9. Determination of Stress Sensitivity

The WT, ΔCgcyp51, and ΔCgcyp51/CYP51 strains were grown on solid PDA medium for 5 days, and then 5 mm mycelial blocks (collected at the colony edges) were used to inoculate fresh solid PDA medium containing 1.2 M NaCl, 1 M sorbitol, and 5 and 10 mM H₂O₂ (final concentration) in plates. A 5 mm mycelial block was added to solid PDA medium supplemented with 0.5 μg/mL terbinafine, 5 μg/mL lovastatin, 10 μg/mL spiroxamine, and 0.25 μg/mL prochloraz (final concentrations) in plates. After 4 days of dark incubation at 25 °C, the growth of each strain was observed, and the diameter of each colony was measured and photographed. All the experiments related to stress sensitivity were repeated three times.

2.10. Pathogenicity Test

Current-year Beijing poplar branches were hydroponically cultivated for 4–5 weeks. New leaves were collected and treated once with 75% ethanol and washed three times with sterile water. They were subsequently placed on filter paper to remove excess liquid before being added to a large Petri dish containing six layers of moistened filter paper. The leaf surface was inoculated with a 30 μL conidial suspension (20 × 10⁴ conidia/mL). Moistened cotton balls were placed on the petioles, and then the inoculated leaves were incubated at 25 °C for 1 week. During the incubation period, the leaves were photographed and examined for disease symptoms (e.g., number and size of spots). The experiment was repeated three times.

3. Results

3.1. Characterization and Deletion of CgCYP51 in C. gloeosporioides

CgCyp51 (Protein ID: 1748231) was identified on the basis of a BLASTP search of the C. gloeosporioides genome database (http://genome.jgi.doe.gov/Gloci1/Gloci1.home.html, accessed on 14 July 2023). A sequence analysis revealed the similarity between CgCyp51 and Cyp51 homologs in C. aenigma, C. viniferum, M. oryzae, Phialemonium atrogriseum, and Diaporthe ampelina (Figure 1A). A structural domain analysis showed that CgCyp51 has a CYP structural domain (Figure 1B). In addition, a phylogenetic analysis indicated C. gloeosporioides CgCyp51 is more closely related to Cyp51 from C. viniferum than to Cyp51 from other fungal species (Figure 1C). To functionally characterize CgCYP51, it was replaced with hph via PEG-mediated transformation (Figure 1D–F). The full-length CgCYP51 sequence was reintroduced into the ΔCgcyp51 mutant to obtain the complementation strain (Figure 1G).

3.2. CgCyp51 Does Not Regulate C. gloeosporioides Mycelial Growth

To investigate whether CgCyp51 affects the trophic growth of C. gloeosporioides, solid PDA media were inoculated with WT, ΔCgcyp51-1, ΔCgcyp51-3, and ΔCgcyp51/CYP51 (complementation strain) in plates, which were then incubated at 25 °C for 4 days. The WT, knockout mutant, and complementation strains did not differ in terms of colony morphology and colony diameter. We also cut the solid PDA medium in each plate to measure the thickness of the aerial mycelium, which revealed a lack of significant differences among the strains (Figure 2). These results suggest that CgCyp51 does not regulate C. gloeosporioides mycelial growth.

3.3. CgCyp51 Regulates Appressorium Formation and Melanization

The hydrophobic surface of a GelBond membrane was inoculated with a WT, ΔCgcyp51-1, ΔCgcyp51-3, or ΔCgcyp51/CYP51 conidial suspension and incubated for 12 h. Both WT and ΔCgcyp51/CYP51 produced many blackened appressoria (appressorium formation rate of 84.06% and 84.93% and appressorium blackening rate of 95.57% and 94.12%, respectively). Compared with WT, ΔCgcyp51-1 and ΔCgcyp51-3 had significantly lower appressorium formation rates (30.58% and 29.64%, respectively) and appressorium blackening rates (77.78% and 74.35%, respectively) (Figure 3). Hence, CgCyp51 appears to modulate appressorium formation and blackening in C. gloeosporioides.

3.4. CgCyp51 Regulates C. gloeosporioides Appressorium Turgor Pressure

GelBond membranes were inoculated with WT, ΔCgcyp51-1, ΔCgcyp51-3, and ΔCgcyp51/CYP51 conidial suspensions and incubated for 10 h, after which the sporulation solution was replaced with 0.6 g/mL PEG 4000. Following a 10 min incubation, the appressorium collapse rate was calculated for each strain. The appressorium collapse rate of the WT control (54.68%) was lower than that of ΔCgcyp51-1 and ΔCgcyp51-3 (91.74% and 90.38%, respectively), but was similar to that of ΔCgcyp51/CYP51 (55.18%) (Figure 4). The relatively high appressorium collapse rates of the knockout mutants reflected the substantial decrease in the turgor pressure of the C. gloeosporioides appressorium due to a lack of CgCYP51 expression.

3.5. CgCyp51 Regulates Appressorium Mucus Production and Penetration Pore Formation

The appressorium attaches to the host surface by secreting a glycoprotein-rich mucus, which is usually distributed around the penetration pore and contributes to an increase in the appressorium turgor pressure. The hydrophobic surface of a GelBond membrane was inoculated with a WT, ΔCgcyp51-1, ΔCgcyp51-3, or ΔCgcyp51/CYP51 conidial suspension and then incubated for 24 h. The samples were subsequently prepared and plated for the SEM examination, which detected a significant decrease in the amount of mucus around the base of the ΔCgcyp51-1 and ΔCgcyp51-3 appressoria. More specifically, the mucus content of the knockout mutants was nearly half of that of the WT and ΔCgcyp51/CYP51 strains (Figure 5A,B), implying that CgCyp51 regulates the production of mucus at the appressorium base. Additionally, in the C. gloeosporioides appressorium, plasma membrane invagination results in the production of a collar structure that is closely associated with penetration pore formation. The hydrophobic surface of a GelBond membrane was inoculated with a conidial suspension. Following a 24 h incubation, a cell-wall-breaking treatment was administered, and samples were prepared. According to the SEM examination, a mature open penetration pore formed at the base of WT and ΔCgcyp51/CYP51 appressoria. In addition, a collar structure that formed in the interior of the appressoria was connected to the penetration pore. In contrast, ΔCgcyp51-1 and ΔCgcyp51-3 appressoria lacked an open penetration pore but contained a collar structure (Figure 5C). These observations indicate that CgCyp51 helps regulate the formation of the C. gloeosporioides appressorium penetration pore.

3.6. CgCyp51 Does Not Affect the Ability of Colletotrichum gloeosporioides Mycelia to Penetrate a Cellophane Membrane

The surface of a cellophane membrane was inoculated with WT, ΔCgcyp51-1, ΔCgcyp51-3, or ΔCgcyp51/CYP51 agar plugs and incubated. The colonies of all strains on the cellophane membranes were similar in size. The cellophane membrane and mycelia were removed. After a 2-day incubation, new colonies on the solid PDA medium were detectable for all strains. Notably, there were no obvious differences in colony size among the analyzed strains (Figure 6). Accordingly, the ability of C. gloeosporioides mycelia to penetrate the cellophane membrane was unaffected by knocking out CgCYP51.

3.7. CgCYP51 Knockout Mutant Responses to High Osmotic and Oxidative Stresses

To investigate the potential effect of CgCyp51 on the C. gloeosporioides response to high osmotic stress, WT, ΔCgcyp51-1, ΔCgcyp51-3, and ΔCgcyp51/CYP51 agar plugs were used to inoculate solid PDA medium containing 1.2 M NaCl and 1 M sorbitol in plates. An examination of colony growth revealed the ΔCgcyp51-1 and ΔCgcyp51-3 colony growth rates and morphological characteristics did not differ significantly from those of the WT and complementation strains (Figure 7A,B), suggesting that CgCyp51 does not regulate the C. gloeosporioides response to high osmotic stress.

Because oxidative stress affects the invasion of plant hosts by pathogenic fungi, we investigated whether CgCyp51 regulates the oxidative stress response of C. gloeosporioides. Specifically, we analyzed WT, ΔCgcyp51-1, ΔCgcyp51-3, and ΔCgcyp51/CYP51 growth on solid PDA medium containing 5 or 10 mM H₂O₂ in plates. There were no significant differences in colony growth rates or morphology among the tested strains (Figure 7A,C), suggesting that CgCyp51 is not involved in the regulation of the C. gloeosporioides response to oxidative stress.

3.8. Determination of the Sensitivity of CgCYP51 Knockout Mutants to Sterol Biosynthesis Inhibitors

CgCyp51 is an important enzyme in the sterol synthesis pathway. To assess whether CgCyp51 influences the sensitivity of C. gloeosporioides to sterol biosynthesis inhibitors (SBIs), WT, ΔCgcyp51-1, ΔCgcyp51-3, and ΔCgcyp51/CYP51 agar plugs were used to inoculate solid PDA medium containing 0.5 μg/mL terbinafine, 5 μg/mL lovastatin, 10 μg/mL spiroxamine, and 0.25 μg/mL prochloraz in plates. Growth rates were lower for the knockout mutants than for the WT and complementation strains (Figure 8). Hence, CgCyp51 appears to affect the sensitivity of C. gloeosporioides to SBIs.

3.9. CgCyp51 Regulates Colletotrichum gloeosporioides Pathogenicity

In this study, appressorium formation and development were altered in the CgCYP51 knockout mutants. Because the appressorium is closely associated with C. gloeosporioides pathogenicity, we examined the possible effect of CgCyp51 on pathogenicity. Briefly, intact detached poplar leaves were inoculated with 30 μL conidial suspensions (20 × 10⁴ conidia/mL) of WT, ΔCgcyp51-1, ΔCgcyp51-3, and ΔCgcyp51/CYP51 and then examined for disease spots. At 4 days post-inoculation (dpi), leaves inoculated with WT or ΔCgcyp51/CYP51 conidial suspensions began to show black lesions. In contrast, leaves inoculated with ΔCgcyp51-1 or ΔCgcyp51-3 conidial suspensions did not develop black lesions. However, at 5 dpi, black lesions started to appear at the inoculation sites of the leaves inoculated with ΔCgcyp51-1 or ΔCgcyp51-3; these lesions gradually expanded over time. On the basis of the disease spot area at 6 dpi, the pathogenicity of ΔCgcyp51-1 and ΔCgcyp51-3 was significantly lower than that of WT and ΔCgcyp51/CYP51 (Figure 9). These results suggest that CgCyp51 regulates C. gloeosporioides pathogenicity.

4. Discussion

The CYP51 gene is present in almost all organisms, including lower eukaryotes, higher plants, bacteria, fungi, and mammals [26]. Multiple CYP51 genes have been identified in filamentous fungi, including M. oryzae, Aspergillus oryzae, F. graminearum, nidulans, and A. fumigatus [13,18,20,27,28]. In this study, we identified CgCyp51 in C. gloeosporioides as a gene encoding an essential transcription factor regulating appressorium development, turgor pressure changes, mucus production, penetration pore formation, sterol biosynthesis, and pathogenicity during spore germination.

In M. oryzae, MoCYP51A is required for effective conidiation, full virulence, and DMI susceptibility. Mutants in which MoCYP51A is deleted are morphologically indistinguishable from the allelopathic WT M. oryzae strain Guy11 in nutrient cultures but exhibit impaired conidiation and decreased virulence. An analysis of intracellular localization showed that MoCyp51A is mainly localized in the cytoplasm of mycelia and conidia. Moreover, the ΔMoCyp51A mutant is highly sensitive to DMIs [18]. In the current study, the ΔCgcyp51 mutants differed significantly from the WT control in terms of appressorium formation following conidial germination, with a significant decrease in the appressorium formation rate of the mutants, but the WT and knockout mutants were morphologically indistinguishable (Figure 2; Figure 3). Additionally, the ΔCgcyp51 mutants exhibited increased sensitivity to SBIs (Figure 8).

In an earlier study involving grapevines infected with C. gloeosporioides, CgCyp51 was revealed to contribute to mycelial growth, virulence, and spore production. Interestingly, all single-gene (i.e., CgCYP51A or CgCYP51B) deletion mutants and complementation strains failed to produce spores, but the single-gene deletion mutants were less pathogenic than the other strains [29,30]. However, in the current study, the CgCYP51 knockout mutant and complementation strains produced spores normally. More importantly, knocking out CgCYP51 disrupted C. gloeosporioides appressorium development. We also investigated the effect of CgCyp51 on the appressorium. Our experimental results showed that the ΔCgcyp51 mutant appressorium was unable to form an open penetration pore. Moreover, the amount of mucus at the base of the appressorium as well as the turgor pressure decreased significantly (Figure 4; Figure 5). Considering the observed decreased virulence of the ΔCgcyp51 mutant strains in this study (Figure 9), we hypothesized that knocking out CgCYP51 adversely affects pathogenicity mainly by altering appressorium formation and development.

The accumulation of mucus around the appressorium has long been recognized as a typical process during appressorium formation. The glycoprotein-rich mucus enables the pathogenic fungus to firmly attach to the host surface, while also affecting the secretion and function of various cuticle-degrading enzymes (e.g., serine proteases secreted by fungi in the genus Streptomyces) [31,32,33]. In the entomopathogenic fungus Metarhizium robertsii, the production of the extracellular polysaccharide acetylgalactosamine galactan (GAG) mediates mucus production, cuticle penetration, and the localized infestation of the insect host by the appressorium. Deletion of the GAG biosynthesis gene cluster results in the loss of mucus around the appressorium and adversely affects the ability of the appressorium to adhere to and penetrate the insect body wall. In addition, this gene cluster is involved in regulating the production of some appressorium proteins, including enzymes that degrade the insect cuticle [34]. Similarly, the deletion of the P450 sterol 14α-demethylase-encoding gene CgCYP51 in the present study resulted in a substantial decrease in mucus levels (Figure 5). Furthermore, the mucus around the appressorium contributes to turgor pressure. In M. oryzae, endogenous spermine limits the oxidative damage in the endoplasmic reticulum, which promotes the folding and secretion of glycoproteins as well as the production and adherence of mucus required to increase the turgor pressure, ultimately facilitating the penetration of host rice cells; however, deleting the gene encoding a spermine synthase (SPS1) decreases mucus production in the appressorium and results in solute leakage, with detrimental effects on cuticle penetration [7]. In the present study, deleting CgCYP51 resulted in a significant decrease in the appressorium turgor pressure, which we hypothesized was associated with a substantial decrease in the amount of mucus at the appressorium base. These findings suggest that CgCYP51 is a key gene affecting appressorium mucus production, but more research is required to comprehensively characterize mucus accumulation at the base of the C. gloeosporioides appressorium. More specifically, additional mucus-synthesis-related genes will need to be identified, and gene knockout and functional analyses remain to be conducted.

During the infection of poplar by C. gloeosporioides leading to anthracnose, several mechanisms mediating the penetration of host tissue and maximizing C. gloeosporioides growth and reproduction are activated [35]. The appressorium that forms is capable of producing a collar structure as well as a penetration pore, which subsequently forms penetration pegs that penetrate the cuticle and epidermal cell walls of poplar. In this process, the mechanisms underlying the formation of the collar structure and penetration pore as well as the connection between them are unclear. In C. gloeosporioides, the deletion of CgFim1 affects the penetration pore and results in the absence of a collar structure and penetration peg, leading to a loss of pathogenicity [36]. In the appressorium of the CgCYP51 knockout mutant, the collar structure was unaffected, but an open penetration pore did not form at 24 h, which resulted in decreased pathogenicity. These results indicate that although CgCyp51 is a key enzyme that regulates the membrane structure, it does not affect the collar structure formed via a plasma membrane invagination in the C. gloeosporioides appressorium, but it helps regulate the formation of a penetration pore. The CgCYP51 knockout mutants were still pathogenic, although they were less virulent than the WT strain. We hypothesized that this difference was due to the delayed development of the penetration pore in the knockout mutants (i.e., open penetration pore and penetration peg formed only after 24 h). To elucidate the associated mechanism, we will analyze appressorium penetration pore and penetration peg formation in the knockout mutants at 48 h using an SEM, while also continuing to functionally characterize genes related to sterol synthesis. The structural mechanism of the C. gloeosporioides appressorium will also be investigated in greater detail.

5. Conclusions

In summary, we revealed the important effects of the transcription factor CgCyp51 on C. gloeosporioides growth, appressorium formation, appressorium melanization, turgor pressure changes, mucus production, penetration pore formation, and pathogenicity. The results of this study reveal that CgCyp51 has multiple regulatory effects on C. gloeosporioides. Specifically, CgCyp51 is a key transcription factor affecting mucus production, penetration pore formation, and the ability of C. gloeosporioides to infect susceptible poplar tissues. We hope that these findings will contribute to further prevention and control of poplar anthracnose and can be used to comprehensively analyze the pathogenesis of Colletotrichum gloeosporioides and develop new target drugs.