The Pheromone-Regulated Membrane Protein CsPRM10 Plays an Essential Role in the Asexual Reproduction of the Pepper Anthracnose Fungus Colletotrichum scovillei

Abstract

The phytopathogenic fungus Colletotrichum scovillei causes a destructive anthracnose on pepper fruit worldwide. Conidiation plays an essential role in the dissemination of pathogenic fungi, yet the regulatory mechanisms underlying this process remain largely unknown. In this study, a pheromone-regulated membrane protein 10 (PRM10) was identified in C. scovillei, whose function has not been characterized in fungal plant pathogens previously. The targeted gene deletion mutant (ΔCsprm10) was normal in plant infection but showed a decrease in surface hydrophobicity compared to the wild-type strain. Notably, ΔCsprm10 was completely defective in conidiation. A microscopic observation further confirmed that ΔCsprm10 failed to form conidiophores, suggesting that CsPRM10 plays an essential role in the conidiation of C. scovillei by regulating conidiophore development. The transcriptomic analysis indicated that the loss of CsPRM10 caused differential expressions of genes related to membrane-associated processes and nuclear functions. Taken together, these findings suggest that CsPRM10 acts as a novel regulator of conidiation in C. scovillei and provide new insights into the molecular basis of fungal asexual development.

1. Introduction

Anthracnose caused by Colletotrichum species is a destructive disease occurring on many agriculturally important crops, vegetables, and fruits, leading to a considerable economic loss around the world annually [1,2,3]. Colletotrichum scovillei in the Colletotrichum acutatum species complex causes anthracnose on pepper fruits during preharvest and postharvest storage in many countries, especially those in East and Southeast Asia [3,4]. The infection of C. scovillei starts with conidia landing on and adhering to the surface of pepper fruits [5]. With the perception of host physical and chemical signals, conidia germinate and differentiate appressoria at the tips of germ tubes. Subsequently, the appressoria develop tiny pegs that penetrate and form highly branched hyphae visualized as dendroid structures inside the cuticle layer of pepper fruits [6]. Following the successful infection of fruit tissue, typical anthracnose lesions are formed, and abundant pinkish conidia are produced, which serve as the main inoculum for disease epidemics [5,7].

Conidiation is a critical process for the dissemination of plant pathogenic fungi [8,9]. Inhibiting conidiation has been considered a promising strategy to control fungal disease epidemics [10,11]. Although a number of commercial fungicides are effective in suppressing fungal growth and host infection, those specifically targeting conidiation remain undeveloped [12,13,14]. Conidiation is a highly regulated developmental process involving a series of complex cellular and morphological events. It is initiated by the formation of aerial hyphae, which subsequently differentiate into conidiophores [6,9,15]. The apical cells of conidiophores further develop into phialides, which ultimately produce conidia [16,17]. Unlike the saprotrophic filamentous fungus Aspergillus nidulans, the molecular mechanisms underlying the conidiation of plant pathogenic ascomycete fungi remain largely unexplored, and only a few genes have been identified as being associated with this process. For example, a T-DNA insertion mutant of COS1 was completely defective in conidiophore development in Magnaporthe oryzae [18]. The MoHOX2 deletion mutant could develop conidiophores but failed to produce conidia [15]. MoMsn2 promotes the differentiation of aerial hyphae into conidiophores of M. oryzae, and its deletion mutant showed a severe reduction in conidiation [19]. In Fusarium graminearum, the deletion of StuA, encoding an APSES transcription factor, resulted in severe defects in conidiophore development and conidiation [20]. The deletion of FgYCK1 blocked phialide development, thereby abolishing conidiation [21]. In the pepper anthracnose fungus C. scovillei, the transcription factors CsHOX2 and CsCZF9 were found to regulate conidia production but not conidiophore development [5,6].

The pheromone-regulated membrane protein 10 (PRM10) was first described as a stress response protein in Saccharomyces cerevisiae [22]. Subsequent studies have mainly focused on its transcriptional responses to stress conditions. For example, the PRM10 of S. cerevisiae was upregulated by exogenous treatments of mating pheromones (α-factors), the β-1,3-glucan synthase inhibitor (caspofungin), or sugar-induced osmotic stress and the overexpression of protein kinase C, small GTPase Rho2, or transcription factor Ste12 [23,24]. Furthermore, a point mutation in PRM10 enhanced the uptake of mevalonate in S. cerevisiae [25]. Recently, the PRM10 ortholog was found to be highly expressed in the basidiospores of Puccinia striiformis f. sp. tritici, the causal agent of wheat stripe rust [26]. However, the functional roles of PRM10 remain uncharacterized in plant pathogenic fungi.

In this study, we aimed at investigating the functions of CsPRM10, orthologous to S. cerevisiae PRM10, in the fungal differentiation and pathogenicity of C. scovillei by using the deletion mutant (ΔCsprm10). ΔCsprm10 was found to be completely defective in conidiation on both artificial medium and the host plant. The defect found in ΔCsprm10 was rescued in a complemented strain (Csprm10c). Pathogenicity assay showed that ΔCsprm10 was normal in plant infection, compared to the wild-type and Csprm10c strains. Collectively, our results suggest that CsPRM10 plays a fundamental role in the conidiation of C. scovillei.

2. Materials and Methods

2.1. Fungal Strains and Culture Conditions

The C. scovillei strain KC05 was used as the wild-type strain in this study [27]. All strains were cultured on V8 agar (V8A), minimal medium agar (MMA), potato dextrose agar (PDA), complete medium agar (CMA), and oatmeal medium agar (OMA) in a growth chamber, as previously described [28].

2.2. Bioinformatic Analysis

The sequences of CsPRM10 homologs were retrieved from the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/, accessed on 10 April 2025). Phylogenetic analysis was conducted using MEGA12 (version 12.0.11). The domains of CsPRM10 homologs were predicted using InterProScan.

2.3. Generation of Knock-Out and Complemented Strains

Genomic DNA was extracted following previously reported methods [27,29]. The CsPRM10 deletion construct was generated using the double-joint PCR [30]. The upstream and downstream regions of CsPRM10 were amplified using paired primers PRM10_5F/R and PRM10_3F/R, respectively, while the hygromycin resistance cassette (HPH) was amplified using paired primers HPH_F/R (Table S1). These three amplified fragments were fused by PCR with primers PRM10_5F/3R, and the fused fragments were amplified with primers PRM10_NF/NR (Table S1). The resulting constructs were transformed into protoplasts of the wild-type strain following a previous method [28]. The knock-out mutants growing on TB3 agar were first selected by a screening PCR using paired primers PRM10_SF/SR (Table S1) and then confirmed through Southern blotting. To construct the complemented strain, protoplasts of ΔCsPRM10 were transformed with the full length of CsPRM10 together with the geneticin resistance cassette. Complemented strains were verified through a reverse transcription (RT) PCR.

2.4. Analyses of Fungal Growth and Developments

To evaluate mycelial growth, mycelial agar plugs obtained from three-day-old MMA were inoculated to V8A, MMA, PDA, and CMA and cultured in a growth chamber at 25 °C and in darkness [31]. After six days, photographs of cultured mycelia were taken, and colony diameters were measured. To evaluate fungal stress adaptation, strains were grown on CMA containing 300 ppm Congo red (CR) and 0.005% sodium dodecyl sulfate (SDS) at 25 °C and in darkness, and the colony diameters were measured, and photographs were taken after five days. The surface hydrophobicity of mycelia was assessed by dropping sterile distilled water (SDW) and 0.1% SDS solution onto mycelia grown on PDA, CMA, and MMA. After 1 min, photographs were taken. To evaluate conidiation, conidia were harvested with 5 mL SDW from V8A and PDA and 3 mL SDW from seven-day-old lesions of pepper fruit and counted using a hemocytometer (Marienfeld-Superior, Berlin, Germany). To visualize the conidiophores, mycelial agar plugs obtained from three-day-old MMA were inoculated to OMA and cultured at 25 °C and in darkness for four days. Then, the obtained mycelial agar plugs from OMA were transferred to a humid box and cultured at 25 °C and in lightness for five hours. Aerial hyphae and conidiophores were stained with a modified lactophenol–aniline blue solution, washed with SDW, and observed under a light microscope. To visualize appressorium-like structures (ALSs), mycelial agar plugs from four-day-old OMA were inoculated onto the hydrophobic surface of coverslips placed inside a humid box and cultured at 25 °C and in darkness for five days. All experiments were independently conducted three times with at least three replicates each time.

2.5. Pathogenicity Assays

Mycelial agar plugs (5 mm in diameter), which did not contain any conidium, were obtained from four-day-old OMA. Artificial wounds were created on intact pepper fruits by piercing the pearl of intact fruits with a syringe needle (0.6 mm in diameter). Both artificially wounded and intact pepper fruits were inoculated with the mycelial agar plugs described above and incubated in a humid chamber at 25 °C. Photographs of lesions on wounded and intact pepper fruits were taken after six and ten days, respectively. The relative lesion size was estimated using ImageJ (version 1.53t). The pathogenicity assay was performed in three independent experiments. In each experiment, three pepper fruits were used for the treatment of the wild-type, ΔCsprm10, and Csprm10c strains, and each fruit was inoculated with two mycelial agar plugs.

2.6. RNA Sequencing

Fresh fungal mycelia grown in CM broth were transferred to V8 broth to induce conidiophore differentiation. Fungal tissues were separated using 3 layers of Miracloth and immediately frozen in liquid nitrogen for RNA isolation. Three biological replicates of extracted RNA were sequenced on an Illumina platform. A total of 45.8 Gb of clean data was obtained and mapped to the published genome (https://doi.org/10.6084/m9.figshare.26112565) using HISAT2 (version 2.2.1) [5]. Differentially expressed genes (DEGs) between the ΔCsprm10 and wild-type strains were analyzed using DESeq2 (version 1.36.0) [32]. Genes with an adjusted p value (false discovery rate, FDR) < 0.05 and |log₂ fold change| > 1 were considered differentially expressed.

3. Results

3.1. Phylogenetic Analysis of CsPRM10

CsPRM10 is predicted to encode a protein containing 976 amino acids. To determine the relationship between CsPRM10 and its homologs, we performed a phylogenetic analysis and domain prediction. The phylogenetic analysis showed that CsPRM10 is closely related to its orthologs from Colletotrichum spp. and distantly related to its ortholog in a human pathogen Cryptococcus neoformans (Figure 1). Domain prediction showed that all CsPRM10 orthologs contain a ThrE-like_N (threonine/serine exporter-like, N-terminal domain) domain, while only the CsPRM10 orthologs from A. nidulans, Schizosaccharomyces pombe, Saccharomyces cerevisiae, and C. neoformans have an additional domain, threonine/serine exporter, ThrE (threonine/serine exporter, ThrE) (Figure 1). These results suggest that CsPRM10 orthologs are conserved in plant pathogenic fungi.

3.2. Targeted Deletion of CsPRM10

To investigate the functions of CsPRM10, we generated a deletion mutant by using homologous recombination (Figure 2A). The resulting deletion mutant was verified through Southern blotting (Figure 2B). Genomic copies of CsPRM10 were re-transformed into ΔCsprm10 to generate a complemented strain, Csprm10c. As expected, the sequence within CsPRM10 was detected in the wild-type strain and Csprm10c but not in ΔCsprm10 by RT-PCR (Figure 2C).

3.3. CsPRM10 Is Involved in Surface Hydrophobicity

Mycelial growth was evaluated by measuring the colony diameters. The colony diameters of ΔCsprm10 were similar on V8A and MMA and slightly reduced on PDA and CMA, compared to those of the wild-type and Csprm10c strains (Figure 3A,B). Interestingly, the mycelia of ΔCsprm10 were found to be more wettable than those of the wild-type and Csprm10c strains on V8A and CMA. To test whether CsPRM10 is involved in the surface hydrophobicity of C. scovillei, drops of SDW and SDS were placed on mycelia. The result showed that SDW or SDS remained beaded on the mycelia of the wild-type and Csprm10c strains. However, the mycelia of ΔCsprm10 were easily soaked by SDW or SDS. This result suggests that CsPRM10 is involved in the surface hydrophobicity of C. scovillei.

3.4. CsPRM10 Is Essential for Conidiation

Microscopic observation showed that the wild-type and Csprm10c strains formed many conidia, whereas ΔCsprm10 failed to produce any conidia (Figure 4A). To determine whether ΔCsprm10 could form conidiophores, lactophenol–aniline blue was used to identify conidiophores by staining aerial hyphae. The result showed that ΔCsprm10 was unable to form conidiophores, compared to many conidiophores observed in the wild-type and Csprm10c strains (Figure 4B). We next performed a quantitative analysis of conidiation on V8A and PDA. Expectedly, ΔCsprm10 was completely defective in conidiation, whereas the wild-type and Csprm10c strains produced many conidia (Figure 4C). To further investigate whether CsPRM10 is related to conidiation on the host plant, we counted conidia on anthracnose lesions of pepper fruit. The result showed that anthracnose lesions caused by the wild-type and Csprm10c strains formed orange conidium masses, whereas only white mycelia were observed on anthracnose lesions caused by ΔCsprm10 (Figure 4D,E). Quantitative analysis showed that ΔCsprm10 did not produce any conidia, whereas the wild-type and Csprm10c strains formed (367.2 ± 53.4) × 10⁴ conidia/mL and (336.1 ± 52.1) × 10⁴ conidia/mL, respectively (Figure 4F). These results suggest that CsPRM10 is essential for the conidiation and conidiophore development of C. scovillei.

3.5. CsPRM10 Is Associated with Stress Adaptation

To test whether CsPRM10 is involved in adaptation to cell wall and membrane stresses, we evaluated the colony growth of ΔCsprm10 on CMA amended with CR and SDS. The result showed that the mycelial growth of ΔCsprm10 was significantly inhibited by SDS but not CR compared to the wild-type (Figure 5A,B). The defect of ΔCsprm10 in response to SDS was restored in Csprm10c (Figure 5A,B). This result suggests that CsPRM10 is involved in tolerance to cell membrane stress.

3.6. CsPRM10 Is Dispensable for Disease Development

As ΔCsprm10 is completely defective in conidiation, we performed pathogenicity assay by inoculating mycelial agar plugs onto healthy pepper fruits. The result showed that all intact and wounded pepper fruits inoculated with ΔCsprm10 developed typical anthracnose (Figure 6A), and the lesion size was comparable to those observed in fruits inoculated with the wild-type and Csprm10c strains (Figure 6B). We further evaluated ALS formation by inoculating mycelial agar plugs onto the hydrophobic surface of cover glass. ΔCsprm10 was found to form ALSs similar to the wild-type and Csprm10c strains (Figure 6C). These results suggest that CsPRM10 is dispensable for the pathogenicity of C. scovillei.

3.7. Transcriptomic Profiling of ΔCsprm10

To investigate the transcriptomic response following CsPRM10 deletion, an RNA-seq analysis was conducted to profile the gene expressions of ΔCsprm10, compared to the wild-type strain. This analysis identified 1968 DEGs (p < 0.05), with 1199 downregulated DEGs (log2 fold change < −1) and 769 upregulated DEGs (log2 fold change > 1) (Figure S1, Table S2). Gene Ontology (GO) enrichment analysis showed that the downregulated DEGs were enriched in membrane-associated GO categories, including plasma membrane, transmembrane transport, and transmembrane transporter activity (Figure 7A). Moreover, multiple transport-related biological processes were overrepresented, such as monoatomic ion transport, cation transmembrane transport, and import across the plasma membrane (Figure 7A). In contrast, upregulated DEGs were predominantly enriched in GO terms related to nuclear and nucleocytoplasmic processes, including nucleolus, nuclear transport, nucleocytoplasmic transport, and protein export from nucleus (Figure 7B). Several molecular function categories linked to energy-dependent and nucleic acid-associated activities, such as ATP hydrolysis activity, helicase activity, and ribonucleoside triphosphate phosphatase activity, were also overrepresented (Figure 7B). Together, these results indicate distinct GO enrichments between downregulated and upregulated DEGs in ΔCsprm10, with membrane-related transport processes represented among downregulated DEGs and nuclear-associated functions enriched among upregulated DEGs.

4. Discussion

In this study, we functionally characterized CsPRM10 in the pepper anthracnose fungus C. scovillei. Phylogenetic analysis indicated that CsPRM10 is closely related to its homologs from filamentous plant pathogenic fungi. Consistent with this evolutionary divergence, domain prediction showed that CsPRM10 homologs from plant pathogenic fungi harbor a ThrE-like_N domain but lack the ThrE domain (Figure 1). This domain architecture probably reflects an evolutionary adaptation of CsPRM10 to fungal differentiation and virulence in plant pathogenic fungi.

A major finding of this study is that the deletion of CsPRM10 resulted in a complete loss of conidiation in C. scovillei (Figure 4C,F). Microscopic examination further confirmed that ΔCsprm10 failed to form conidiophores, indicating that CsPRM10 plays a critical role at an early stage of conidiation. These data clearly distinguish CsPRM10 from previously characterized conidiation-related regulators in C. scovillei, such as CsHOX2 and CsCZF1, that are essential for conidium production but dispensable for conidiophore formation [5,6]. Therefore, CsPRM10 represents another class of developmental regulator required for the establishment of conidiophore architecture, rather than conidium generation. CsPRM10 was also found to be involved in the regulation of surface hydrophobicity, as evidenced by the mycelia of ΔCsprm10 displaying increased wettability compared to the wild-type strain (Figure 3C). Surface hydrophobicity has been widely recognized as a key determinant of fungal development and is required for the emergence of aerial hyphae, conidiophore formation, and conidium dispersal [33,34,35]. The decreased surface hydrophobicity of ΔCsprm10 suggests that CsPRM10 may be involved in membrane-associated functions that regulate cell wall assembly and hydrophobin deployment, thereby enabling the formation of aerial hyphae and conidiophores.

The RNA-seq analysis conducted at an early stage of conidiophore development revealed the altered expression of genes associated with membrane-related processes and nuclear functions, providing a mechanistic insight for the failure of conidiophore formation in the absence of CsPRM10 (Figure 7). Genes downregulated in ΔCsprm10 were predominantly enriched in membrane-associated transport processes, including transmembrane transport and ion transport. Proper membrane organization and solute transport are known to be involved in polarized growth and cellular differentiation during conidiophore development [36,37]. Notably, the ortholog of flbC was identified among the downregulated genes. flbC has been documented as an important regulator of aerial hyphal development and conidiation in A. nidulans and M. oryzae [38,39]. In addition, several genes encoding cell wall- and surface-associated proteins were downregulated, including those predicted to encode hydrophobins and chitin synthases. In filamentous fungi, hydrophobin and chitin synthase have been implicated in aerial hyphal formation and conidiation [35,40,41,42].

Despite its roles in regulating conidiation and surface hydrophobicity, the deletion of CsPRM10 did not affect the pathogenicity of C. scovillei (Figure 6A,B). This observation contrasts with previous studies which have reported a close relationship between surface hydrophobicity and fungal pathogenicity. Defects in surface hydrophobicity often lead to impaired host recognition and compromised host infection [43,44]. Considering that C. scovillei forms dendroid structures within the cuticle layer of pepper fruit [6], C. scovillei may employ alternative mechanisms, such as chemical signaling and enzymatic degradation at an early stage of host plant infection. In addition, ΔCsprm10 retained the ability to form ALSs at levels comparable to those of the wild-type strain (Figure 6C), suggesting that the differentiation of infection-related structures in C. scovillei is largely independent of surface hydrophobicity.