The Cell Wall Integrity MAP Kinase Signaling Pathway Is Required for Development, Pathogenicity, and Stress Adaption of the Pepper Anthracnose Fungus Colletotrichum scovillei

Abstract

The cell wall integrity (CWI) signaling pathway plays important roles in the dissemination and infection of several plant pathogenic fungi. However, its roles in the pepper fruit anthracnose fungus Colletotrichum scovillei remain uninvestigated. In this study, the major components of the CWI signaling pathway—CsMCK1 (MAPKKK), CsMKK1 (MAPKK), and CsMPS1 (MAPK)—were functionally characterized in C. scovillei via homology-dependent gene replacement. The ΔCsmck1, ΔCsmkk1, and ΔCsmps1 mutants showed impairments in fungal growth, conidiation, and tolerance to CWI and salt stresses. Moreover, ΔCsmck1, ΔCsmkk1, and ΔCsmps1 failed to develop anthracnose disease on pepper fruits due to defects in appressorium formation and invasive hyphae growth. These results suggest that CsMCK1, CsMKK1, and CsMPS1 play important roles in mycelial growth, conidiation, appressorium formation, plant infection, and stress adaption of C. scovillei. These findings will contribute to a better understanding of the roles of the CWI signaling pathway in the development of pepper fruit anthracnose disease.

1. Introduction

The plant pathogenic fungi are exposed to diverse environmental stresses, including osmotic stress, oxidative stress, nutrient limitations, chemical and mechanical stresses, and stresses from host immune system [1]. The fungal cell wall is a polymeric structure—mainly containing chitin, glucan, galactomannan, and glycosylated proteins—that serves as the primary barrier against environmental stresses to maintain cellular integrity [2,3]. Despite some fundamental diversity in cell wall structure among different species and even different developmental stages of the same fungal species, the maintenance and biosynthesis of the cell wall is mainly dependent on the conserved cell wall integrity (CWI) signaling pathway [1,4]. The CWI signaling pathway plays important roles in cell growth, differentiation, and stress adaption [4,5]. In Saccharomyces cerevisiae, the CWI signaling pathway includes several cell surface receptors coupled with a Rho1 GTPase, protein kinase C, and a sequentially activated mitogen-activated protein kinase (MAPK) cascade (Bck1/Mck1-Mkk1/2-Slt2/Mps1) [5].

In plant pathogenic fungi, components of the CWI signaling pathway have been shown to regulate growth, dissemination, infection-related morphogenesis, and suppression of host defense in several fungal pathogens that causes severe foliar and root diseases [6–8]. For example, the Magnaporthe oryzae genes Mck1, Mkk2, and Mps1 are required for conidiation and appressorium-mediated penetration [7,9,10], while Botrytis cinerea Bmp3, orthologous to Mps1, is involved in conidiation, surface sensing, and pathogenicity [6]. However, the roles of the CWI-related MAPKs have not been characterized in the pepper fruit anthranose pathogen Colletotrichum scovillei, a species belonging to the Colletotrichum acutatum species complex.

Anthracnose disease on pepper fruit caused by C. scovillei occurs during pre- and post-harvest stages, leading to huge economic losses in subtropical and temperate countries [11,12]. The C. scovillei produces a huge number of conidia under favor conditions, which is a determinant of anthracnose disease dissemination. In the early infection stage, C. scovillei develops a non-melanized appressorium, which serves as the major infection structure [13]. The appressorium then forms a tiny penetration peg, which subsequently differentiates into highly branched invasive hyphae in the cuticle layer of pepper fruits, called the dendroid structure [14,15]. Due to the agricultural impact and unique infection processes, anthracnose disease by C. scovillei on pepper fruit caused is becoming an interesting model to study molecular mechanisms governing the developments of fruit fungal disease. So far, several regulatory genes have been characterized to play essential roles in dissemination, appressorium formation, penetration, and invasive hyphae of C. scovillei [11,16]. However, the CWI-related MAPKs, known to associate with fungal differentiation, virulence, and stress adaptions in foliar fungal pathogens, have not been investigated in the pepper fruit anthracnose fungus, C. scovillei.

This study aims at determining the roles of CWI-related MAPKs in the pepper fruit anthracnose fungus, C. scovillei, using deletion mutants (ΔCsmck1, ΔCsmkk2, and ΔCsmps1). The results showed that ΔCsmck1, ΔCsmkk2, and ΔCsmps1 were defective in mycelial growth, conidiation, appressorium formation, pathogenicity, and tolerance to CWI and salt stresses, compared to the wild-type. However, the defects of deletion mutants were recovered in each corresponding complemented strain. Together, our results suggest that CWI signaling pathway plays pivotal roles in fungal developments, virulence, and stress adaptions of the pepper fruit anthracnose fungus, 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. The wild-type and its transformants were routinely cultured on the minimal medium agar (MMA), complete medium agar (CMA), V8 juice agar (V8A), and potato dextrose agar (PDA) were performed as previously described [11].

2.2. Bioinformatic tools

The amino acid sequences of CWI-related MAPKs and their orthologs used in this study were downloaded from the Comparative Fungal Genomics Platform (CFGP; http://cfgp.riceblast.snu.ac.kr/) and UniProt (https://www.uniprot.org/). Domains of all sequences were predicted using InterProScan (https://www.ebi.ac.uk/interpro/). All amino acid sequences were aligned using the ClustalW. The phylogenetic tree was constructed using the MEGA X software.

2.3. Targeted gene deletion and complementation

The deletion mutants and complemented strains were generated as previously described [11]. Briefly, the upstream and downstream sequences (about 1.5 kb) of each targeted gene were amplified with primers 5 F/5R and 3 F/3R, respectively (Table S1) [17]. The two amplified segments were fused to an hph cassette to generate a deletion construct, which was transformed into protoplasts of C. scovillei KC05, as previously described [18]. For the generation of complemented strains, genomic copies of the targeted gene were transformed with the geneticin-resistant cassette G418 to the protoplasts of deletion mutant. The deletion mutants and complemented strains were selected through rounds of screening polymerase chain reaction (PCR) [13,17], then further confirmed through Southern blotting and reverse-transcription (RT)-PCR, respectively.

2.4. Southern blotting and RT-PCR

The Southern blotting and RT-PCR were performed according to previously reported methods. Briefly, the probe DNA (about 500 bp in length) of Southern blotting was labeled with Biotin-High Prime (Roche, Indianapolis, IN, United States) to detect the restriction enzymes-digested genomic DNA in a nylon hybridization membrane [11]. The SuperScript® III First-strand Synthesis System (Invitrogen, Grand Island, NY, United States) was applied to synthesize the first-strand complementary DNA (cDNA) from total RNA (5 μg), extracted using the Easy-Spin™ Total RNA Extraction Kit (iNtRON Biotechnology, Seongnam, South Korea).

2.5. Assays of fungal developments and pathogenicity

Mycelial growth was tested by measuring colony diameters. mycelial agar plugs (5 mm in diameter) were inoculated onto PDA and incubated for five days in the dark followed by two days in the light. Conidiation was measured by counting conidia collected with 5 ml of distilled water from seven-day-old V8 agar. The concentrations of conidial suspensions were evaluated using a hemocytometer under a light microscope [11]. To test the conidial germination and appressorium formation, the conidial suspensions (5 × 10⁴ mL⁻¹) were dopped onto the hydrophobic surface of coverslips and incubated in a humid plastic box at 25 °C for 16 h. For pathogenicity assays, conidial suspensions (5 × 10⁵ mL⁻¹) were inoculated onto wounded and intact pepper fruits and incubated in a humid box at 25 °C for six and nine days, respectively [13]. All data were collected from at least three independent experiments with three replicates per experiment. A Duncan’s test (p < .05) was used to estimate the significant difference.

3. Results and discussion

3.1. Phylogenetic analysis, domain structure, and targeted gene deletion

To isolate CWI-related MAPKs in C. scovillei, amino acid sequences of MCK1 (MGG_00883), MKK2 (MGG_06482), and MPS1 (MGG_04943) were used to blast query the whole genome of C. scovillei using the CFGP. Three loci—CAP_011231, CAP_005029, and CAP_001359—were identified and named CsMCK1, CsMKK1, and CsMPS1, respectively. A phylogenetic analysis of CsMCK1, CsMKK1, and CsMPS1 and their orthologs was performed by a neighbor-joining method with 1000 bootstrap replicates [13,19]. The results showed that CsMCK1, CsMKK1, and CsMPS1 from C. scovillei are closely related to their homologs in other fungi (Figure 1). Domain prediction showed that all selected CWI-related MAPKs contain a protein kinase domain (IPR000719) (Figure 1). These results suggest that CsMCK1, CsMKK1, and CsMPS1 are conserved among fungi.

Figure 1.

Phylogenetic analysis and domain prediction. Phylogenetic tree of CsCMK1, CsMKK2, CsMPS1, and their orthologs constructed using a neighbor-joining method with 1000 bootstraps in MEGA X. InterProScan was used to predict domains in all protein sequences. All sequences were downloaded from UniProt (https://www.uniprot.org/), NCBI (https://www.ncbi.nlm.nih.gov/), or the Comparative Fungal Genomics Platform (http://cfgp.riceblast.snu.ac.kr/). The protein kinase domain (IPR000719) and amino acid sequence are indicated by a grey box and black line, respectively.

To functionally characterize the roles of CsMCK1, CsMKK1, and CsMPS1 in C. scovillei, each of the targeted genes was deleted via homology-dependent replacement (Figure 2(A)). The deletion mutants and complemented strains were confirmed through Southern blotting and RT-PCR, respectively (Figure 2(B,C)).

Figure 2.

Generation of transformants. (A) Target gene deletion strategy. The open reading frame of the targeted gene was replaced using an hph cassette via fusion PCR; (B) Verification of deletion mutants. The deletion mutants were selected by rounds of PCR screening with primers SF/SR. The genomic DNA of the wild-type and selected deletion mutants was individually digested with the indicated restriction enzymes and transferred to a nylon membrane. The specific probe (∼500 bp), amplified with PF/PR (Table S1), was labeled with Biotin High Prime and hybridized to the nylon membrane; (C) Confirmation of complemented strains. Genomic copies of each target gene were reintroduced to protoplasts of the corresponding deletion mutants. The complemented strains were confirmed via reverse transcription PCR with primers RTF/RTR (Table S1).

3.2. Roles of CWI-related MAPKs in mycelial growth and stress adaption

Because the CWI-related MAPK signaling cascade is involved in mycelial growth and stress adaption [8,10,20,21], the colony diameters of mycelial growth in ΔCscmk1, ΔCsmkk1, and ΔCsmps1 were evaluated on potato dextrose agar (PDA). After five days, colony diameters of ΔCsmck1 (43.6 ± 1.8 µm), ΔCsmck1 (43.1 ± 1.6 µm), and ΔCsmck1 (43.2 ± 1.1 µm) were significantly reduced compared to the wild-type (54.1 ± 1.3 µm) (Figure 3(A,B)). The defect in mycelial growth rate was restored in each corresponding complemented strain (Figure 3(B)). This result suggests that CsCMK1, CsMKK1, and CsMPS1 are involved in mycelial growth. To test whether CsCMK1, CsMKK1, and CsMPS1 are involved in stress adaption of C. scovillei, inhibition of mycelial growth was tested on PDA containing stress chemicals, including Congo red, sorbitol, and NaCl. Compared to the wild-type and each complemented strain, the mycelial growth of ΔCscmk1, ΔCsmkk1, and ΔCsmps1 was significantly inhibited by Congo red and sorbitol but not NaCl (Figure 3(A,B)), suggesting that CsCMK1, CsMKK1, and CsMPS1 are involved in tolerance to CWI and osmotic stresses. These results are in accordance with several research previous reported [8,9,20], which highlights the conserved roles of CWI-related MAPKs in fungal growth and stress adaptions.

Figure 3.

Stress adaptions of mycelia. Mycelial agar plugs were inoculated onto potato dextrose agar (PDA) with or without addition of chemical stressors [Congo red (300 ppm), NaCl (0.4 M), or sorbitol (1 M)] and incubated at 25 °C in the dark. (A) Visualization of colony growth. Photographs were taken after six days; (B) Quantitative measurement of colony growth. Colony diameters were measured after six days. An asterisk (*) indicates a significant difference (Duncan’s test, p < .05).

3.3. Role of CWI-related MAPKs in asexual production

Conidiation play important roles in the dissemination and propagation of plant fungal pathogens [22]. A microscopic visualization indicated that ΔCscmk1, ΔCsmkk1, and ΔCsmps1 produced fewer conidia compared to the wild-type (Figure 4(A)). A further quantitative measurement showed that the concentrations of conidial suspensions of ΔCscmk1 [(7.0 ± 1.5) × 10⁴ mL⁻¹], ΔCsmkk1 [(9.0 ± 1.3) × 10⁴ mL⁻¹], and ΔCsmps1 [(9.0 ± 1.4) × 10⁴ mL⁻¹] were significantly lower than the wild-type [(84.0 ± 7.9) × 10⁴ mL⁻¹] (Figure 4(B)). The conidiation defect was recovered in each complemented strain (Figure 4(B)). These results suggest that CsCMK1, CsMKK2, and CsMPS1 are involved in conidiation of C. scovillei. Considering that generation of conidia from phialide of conidiophores requires cell wall remodeling [23,24], the significantly reduced conidiation in the ΔCscmk1, ΔCsmkk1, and ΔCsmps1 would result from their defects in cell wall integrity.

Figure 4.

Evaluation of conidiation. (A) Visualization of conidiation. Mycelial agar plugs (5 mm diameter) were inoculated onto oatmeal agar (OMA) and incubated at 25 °C in the dark for three days. The obtained OMA mycelial agar plugs were placed on the hydrophilic surface of a glass slide and incubated in a humid plastic box at 25 °C in the light for 5 h. Scale bar = 50 µm; (B) Quantitative evaluation of conidiation. Three-day-old OMA mycelial agar plugs (5 mm diameter) were inoculated to potato dextrose agar (PDA) and incubated at 25 °C for five days in darkness followed by two days under light. Conidia were harvested with 5 mL of sterilized distilled water.

3.4. Role of CWI-related MAPKs in appressorium formation

The cell wall mediates signal transduction from the external environment to intracellular events and plays important roles in pre-infection developments [10,25,26]. Thus, the conidial germination and appressorium formation of ΔCscmk1, ΔCsmkk1, and ΔCsmps1 were examined. The results showed that conidial germination rates of ΔCscmk1, ΔCsmkk1, and ΔCsmps1 were normal compared to the wild-type. However, ΔCscmk1, ΔCsmkk1, and ΔCsmps1 were completely defective in appressorium formation (Figure 5), whereas the wild-type and complemented strains developed appressorium with > 90% formation rates. This result suggests that CsCMK1, CsMKK1, and CsMPS1 are essential for appressorium formation of C. scovillei. Intriguingly, loss functions of each CWI-related MAPKs in M. oryzae did not abolish appressorium formation [9,10]. However, deletion of CgMCK1, CgMKK1, or CgMPS1 in Colletotrichum gloeosporioides resulted in a complete defect in appressorium development [20]. These data indicated that CWI-related MAPKs share different mechanisms in the regulation of appressorium formation among different fungal pathogens.

Figure 5.

Appressorium formation. Conidia were harvested from seven-day old OMA with sterilized distilled water. The conidial suspensions (5 × 10⁴ conidia mL⁻¹) were dropped onto the hydrophobic surface of coverslips and incubated in a humid plastic box at 25 °C. (A) Visualization of appressorium formation. Photographs were taken after 16 h. Scale bar = 10 µm; (B) Quantitative measurement of appressorium formation. An asterisk (*) indicates a significant difference (Duncan’s test, p < .05).

3.5. Role of CWI-related MAPKs in pathogenicity

To determine whether CsCMK1, CsMKK1, and CsMPS1 are involved in pathogenicity, conidial suspensions were inoculated onto intact pepper fruits. After nine days, the wild-type and complemented strains caused typical anthracnose disease. However, ΔCscmk1, ΔCsmkk1 and ΔCsmps1 failed to cause disease (Figure 6), suggesting that CsCMK1, CsMKK1, and CsMPS1 are essential for pathogenicity of C. scovillei. To investigate whether CsCMK1, CsMKK1, and CsMPS1 are associated with invasive hyphae growth, the conidial suspensions were further inoculated into wounded pepper fruits. After six days, ΔCscmk1 and ΔCsmkk1 failed to cause anthracnose disease, while ΔCsmps1 led to disease with reduced severity compared to the wild-type and complemented strains (Figure 6). This result suggests CsMCK1, CsMKK1, and CsMPS1 are involved in invasive hyphae growth of C. scovillei.

Figure 6.

Pathogenicity assay. Drops (20 µL) of conidia suspensions (50 × 10⁴ conidia mL⁻¹) were inoculated onto wounded and intact pepper fruits and incubated in a humid plastic box at 25 °C. Photographs of wounded and intact pepper fruits were taken after six and nine days, respectively.

Important roles of the CWI-related MAPKs on pathogenicity have been determined in several plant pathogenic fungi [8–10,20]. For example, in the M. oryzae, MCK1, MKK2, and MPS1 are required for appressorium-mediated penetration [9,10]. In C. gloeosporioides, CgMCK1, CgMKK1, and CgMPS1 are essential for appressorium formation and invasive hyphae growth [20]. Our data showed that the ΔCscmk1 and ΔCsmkk1 were completely defective on both appressorium formation and invasive hyphae growth (Figures 5 and 6). Although the ΔCsmps1 failed to form appressorium, it stilled form invasive hyphae, because the ΔCsmps1 caused anthracnose disease with reduced severity on wounded pepper fruit (Figure 6). Collectively, our results demonstrated that the CWI-related MAPKs, CsMCK1, CsMKK1, and CsMPS1, are important for growth, development, virulence, and stress adaption of the pepper fruit anthracnose fungus C. scovillei.

Funding Statement

This study was supported by the Basic Science Research Program through the National Research Foundation of Korea grant (NRF-2020R1A2C100550700) funded by the Ministry of Education, Science and Technology, Republic of Korea. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).