A CRISPR/Cas9-Based Study of CgloRPCYG, a Gene That Regulates Pathogenicity, Conidial Yield, and Germination in Colletotrichum gloeosporioides

Abstract

The filamentous fungus Colletotrichum gloeosporioides is the causative agent of one of the most serious diseases that damage plant fruit. In this study, we discovered and experimentally characterized a new gene in Colletotrichum gloeosporioides named CgloRPCYG. The CRISPR/Cas9 knockout mutant and complementary strain of CgloRPCYG were then obtained by polyethylene glycol (PEG)-mediated protoplast transformation to determine the related phenotypes. Compared with the wild-type strain and complementary mutant, the pathogenicity of the CRISPR/Cas9 knockout mutant was significantly decreased, the conidial yield was significantly reduced, and conidial germination was significantly delayed. These data indicate that CgloRPCYG contributes to pathogenicity, conidial yield, and germination in C. gloeosporioides. The successful application of the CRISPR/Cas9 system in C. gloeosporioides also confirms its utility in filamentous fungi for fundamental research and practical application. Furthermore, CgloRPCYG is a potential target gene for use in the development of plant protection technologies, such as spray-induced gene silencing, with the aim of controlling plant anthracnose disease caused by C. gloeosporioides.

1. Introduction

Filamentous fungi have long been known to play a significant role in agricultural production, which is severely disrupted due to disease occurrence and mycotoxin production. With the advancement of sequencing technology and fungal genome databases, a more extensive range of opportunities for genetic manipulation of filamentous fungi is available [1]. Colletotrichum spp. is a model system for fundamental, biochemical, physiological, and genetic studies [1]. Most Colletotrichum species are hemibiotrophic, with features including conidial yield, conidial germination, germ tubes development, melanized appressorium formation, infection pegs that penetrate host plant cell walls [1], biotrophic intracellular hyphae, and eventual progression into a necrotrophic phase in dead host plant cells [1]. Currently, the Colletotrichum genus includes more than 600 species. C. gloeosporioides complex species is ubiquitous and can infect over 470 plant species; it can infect leaves, flowers, and fruit, with symptoms including irregularly shaped lesions, mainly black in color, and typical reddish-pink conidia piles. The resulting disease is commonly referred to as anthracnose. C. gloeosporioides complex species is a globally distributed pathogenic fungus that causes fruit anthracnose in a vast range of hosts [2], such as avocado (Persea americana) [3], strawberry (Fragaria × ananassa) [4], mango (Mangifera indica) [5], citrus (Citrus sinensis) [6], apple (Malus pumila) [7], pepper (Capsicum annuum) [8,9], pear (Pyrus bretschneideri), and tomato (Solanum lycopersicum) [10,11]. C. gloeosporioides complex species breaches the fruit cuticle of tropical and subtropical fruits but remains quiescent until fruit ripening signals a switch to necrotrophy, culminating in devastating fruit anthracnose disease [10,12]. Previous research has confirmed that C. gloeosporioides species has evolved a sophisticated mechanism to infect hosts via various types of pathogenicity factors such as secondary metabolites, effectors, plant cell wall hydrolase and other enzymes, and transcription factors, among others. Discovering new pathogenicity genes is still the key to explaining the pathogenic mechanisms.

The clustered regularly interspaced short palindromic repeat/CRISPR-related nuclease 9 (CRISPR/Cas9) system evolved in bacterial and archaeal systems as an innate component of their adaptive immune response, and represents a new, efficient genome-editing tool for gene insertion, knockout, and replacement [13]. The system contains two crucial modules, endonuclease Cas9 and a chimeric single-guide RNA (sgRNA) [14]. The Cas9–sgRNA ribonucleoprotein complex can bind to a specific DNA sequence upstream of the protospacer adjacent motif (PAM) (5′-NGG-3′) via complementary base pairing, inducing DNA double-strand breaks and simultaneously editing multiple genes [15,16,17,18]. The CRISPR/Cas9 system is now used for specific targeted genome modification in a wide range of eukaryotes. With its high efficiency and easy operation, this technology has been successfully applied in a variety of organisms, including animals (zebrafish (Danio rerio) [19], mice [20], pigs [21], plants (Arabidopsis [22,23,24,25], tobacco [22], sorghum [22], rice [22,26], and papaya [27]), and fungi and oomycetes (Aspergillus oryzae [28], Trichoderma reese [29], Magnaporthe oryzae [30], Phytophthora capsici [31], and P. sojae [32]). The CRISPR/Cas9 system used Phytophthora to demonstrate that PcAvh1 [33] and PlAvh142 [34] are essential for pathogenicity. However, while the CRISPR/Cas9 system has been used for different plant pathogens, there has been limited application in the hemibiotrophic fungus C. gloeosporioides. The construction of a CRISPR/Cas9 genome-editing toolkit for C. gloeosporioides could be an important supplement to the molecular technology of functional genomics awaiting construction.

In this study, the CRISPR/Cas9 system was used to functionally characterize a gene potentially important for pathogenicity in C. gloeosporioides. For this purpose, we obtained knockout mutants through the CRISPR/Cas9 system, which can delete long fragments of DNA. Then, knockout mutants with long fragment deletion were complemented, and complementary mutants were finally obtained. The new gene was named CgloRPCYG based on its function in regulating pathogenicity, conidial yield, and germination in C. gloeosporioides. This preliminary exploration is expected to provide a foundation for research on functional genes in the filamentous fungus C. gloeosporioides.

2. Materials and Methods

2.1. Fungal Strain, Growth Conditions, and Plant Fruits

C. gloeosporioides wild-type (WT) strain 171-1 and backbone vectors pHS-Cas9-Backbone and pOE-Backbone used in this study were provided by the Institute of Environment and Plant Protection, Chinese Academy of Tropical Agricultural Sciences. The C. gloeosporioides strain was routinely grown on potato dextrose agar (PDA) and maintained at 28 °C [11]. Escherichia coli DH5α competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd. (Cat# DL1001) (Shanghai, China). Conidiation was assayed in 3-day-old PD broth cultures. Fruits used in the pathogenicity assay included apple (M. pumila, cultivar Hongfushi), pear (P. bretschneideri, cultivar Xuehua), and mango (M. indica, cultivar Guifei).

2.2. Sequence Alignment and Phylogenetic Analysis

The genomic sequence was obtained from the sequence database of C. gloeosporioides (https://www.ncbi.nlm.nih.gov/; GenBank assembly accession no. GCA_011800055.1, 30 September 2018). Physicochemical properties, including the amino acid sequence length, isoelectric point, and stability, were predicted using the ProtParam tool in the ExPASY server [35]. The amino acid sequences of CgloRPCYG were analyzed using the Protein Basic Local Alignment Search Tool (BlastP) of the National Biotechnology Information Center (NCBI) for multiple sequence alignment. According to the similarity with the sequence of CgloPRCYG, 54 amino acid sequences were screened, including 34 of the same genus and 20 of different genera. The 54 amino acid sequences were derived from Colletotrichum spp. (XP 031887974.1, KAF4899211.1, KAF0317795.1, KAF4820371.1, XP 037173807.1, Cg-14 EQB55550.1, XP 045256771.1, KAF4868737.1, KAH0431835.1, KAH9227001.1, XP 038738854.1, XP 036575154.1, TDZ39120.1, TEA19528.1, TDZ44972.1, MAFF 240422 TDZ16536.1, KAF6790751.1, XP 022480039.1, KXH66003.1, PJ7 EXF76443.1, XP 049151650.1, KXH32929.1, XP 035326615.1, M1.001 XP 008097816.1, KDN71047.1, XP 049133866.1, TKW59130.1, CCF38965.1, TQN70612.1, IMI 349063 XP 018161448.1, KZL86094.1, GJC78716.1, KZL77832.1, OLN87683.1), Lasiodiplodia theobromae (KAF9631773.1, KAB2573554.1), Diplodia corticola (XP 020134537.1), Neofusicoccum parvum (UCRNP2 EOD47268.1), Macrophomina phaseolina (MS6 EKG15908.1), Saccharata proteae (CBS 121410 KAF2087595.1), Xylariales sp. (PMI 506 KAH8661846.1), Dactylonectria estremocensis (KAH7139677.1), Clonostachys solani (CAH0056040.1), Jackrogersella minutella (KAI1098867.1), Hypoxylon rubiginosum (KAI6085517.1), Daldinia sp. (XP 047787477.1, XP 049095194.1, EC12 OTB20813.1, KAI1477780.1), Ophiobolus disseminans (KAF2833591.1), Pyrenochaeta sp. (MPI-SDFR-AT-0127 KAH7357626.1), Plenodomus lindquistii (KAI8934303.1), and Leptosphaeria maculans (KAH9881555.1, JN3 XP 003835460.1). CgloRPCYG was also compared with other amino acid sequences, and the phylogenetic tree was constructed based on 1000 bootstrap replicates and using the neighbor-joining method in DNAMAN v5.2.2 software (Lynnon Biosoft, San Ramon, CA, USA) and MEGA v6.06 software [36]. The Sanger sequencing results were analyzed with DNAMAN v5.2.2 and Chromas v2.22 software (Technelysium Pty Ltd., South Brisbane, QLD, Australia).

2.3. Plasmid Construction

In this work, we used a Cas9 nuclease and the usual PAM sequence (NGG) [35]. Using the online Eukaryotic Pathogen gRNA Design Tool (EuPaGDT, available at http://grna.ctegd.uga.edu, 30 September 2018) [37], a 20 nt sgRNA that can guide the Cas9 nuclease to the target for genomic DNA cleavage was designed according to the sequence of CgloRPCYG. The sequence of the sgRNA CgloRPCYG sgRNA02 is 5′-AACCACCTGACGATGCTTGT-3′. The fusion vector pHS-Cas9-CgloRPCYG was obtained by integrating the sgRNA into the backbone vector pHS-Cas9-Backbone (with hygromycin resistance cassette (Hyg^r), ampicillin resistance cassette (Amp^r), Cas9 nuclease cassette (hCas9), and origin of replication (Ori)). The fusion plasmid was verified via Sanger sequencing analysis, and the pHS-Cas9-Backbone was used as the control plasmid (EV). The pHS-Cas9-Backbone plasmid was purchased from Beijing Syngentech Company Limited. For the complementary construct, a fragment consisting of pgpdA promoter, CgloRPCYG open reading frame, and TtrpC terminator sequences were cloned into complementary vector pEO (containing the bialaphos resistance gene, Bar). EcoRI and XbaI restriction endonuclease sequences and protective bases were added to the 5′ and 3′ ends, respectively, of the CgloRPCYG gene. Then, the pEO vector was constructed by restriction endonuclease and T4 ligase. The successful complementary vector was named pEO-CgloRPCYG. All successfully fused plasmids were propagated in E. coli DH5α competent cells. PCR was used for the identification and sequencing of E. coli-positive clones.

2.4. Polyethylene Glycol-Mediated Protoplast Transformation

Protoplasts were subjected to polyethylene glycol (PEG)-mediated transformation according to a previously published protocol [38]. For transformation, at least 1.0 × 10⁷ protoplasts and 20 μg of pHS-Cas9-RPCYG plasmid were mixed with up to 2 mL PEG buffer [38]. The protoplast mixture (3 mL) was added to potato dextrose broth (PDB) containing 0.8 M sucrose and cultured at 28 °C for 24 h with light for regeneration [38]. The culture was plated onto PDA containing 0.8 M sucrose and 150 μg/mL hygromycin B (VWR (Shanghai), Co. Ltd., Shanghai, China) and cultured at 28 °C for 24 h. A second cladding layer of PDA containing 100 μg/mL hygromycin B was added to the plates and cultured at 28 °C until mycelia grew out of the medium [39]. Then, the complementary vector was introduced into the CgloRPCYG knockout mutant protoplasts, and transformants were screened by PDS medium with glufosinate and verified by PCR, which resulted in the complementary mutant strain CΔCgloRPCYG-1.

2.5. Extraction of DNA and Verification of Transformants

Monoclonal colonies were selected for further verification. The genomic DNA of the transformants was isolated using cetyltrimethylammonium bromide (CTAB) and phenol–chloroform extraction, as previously described [40], and the DNA templates were used for the verification of transformants [40]. Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA) was used for PCR amplification. The primers used are listed in Text S1 and Table S1. Agarose gel electrophoresis and Sanger sequencing were performed for verification of the transformants.

2.6. Characterization of C. gloeosporioides Mutants

To study the fungal growth, the wild-type (WT) strain, CgloRPCYG knockout mutants (ΔCgloRPCYG-1, KO), WT strain transformed with the empty vector (EV), CgloRPCYG complementary mutant (CΔCgloRPCYG-1, CKO), and WT strain transformed with the complementary empty vector (CEV) were cultured on PDA plates at 28 °C, and the diameter of colonies was measured daily. For the stress tolerance assay, strains were grown on minimal medium (MM; 50 g/L dextrose, 1.0 g/L ammonium nitrate, 0.5 g/L potassium dihydrogen phosphate, 1.5 g/L disodium hydrogen phosphate, 1.0 g/L sodium chloride, and 0.2 g/L magnesium sulfate heptahydrate, pH 6.5, control) containing 1 M NaCl, 1 M KCl, 5 mM H₂O₂, 0.01% sodium dodecyl sulfate (SDS), and 300 μg/mL Congo red (CR) [41]. To determine the conidial yield, mature conidia of the strains were harvested from 3-day-old PDB cultures. A hemocytometer was used to count the conidia. For the conidial germination assay, a 25 μL conidial suspension (1 × 10⁵ conidium/mL in ddH₂O) of each strain was dripped onto hydrophobic coverslips and cultured in a moistened incubator at 28 °C [39]. Conidial germination was observed and recorded at 10 h [39].

2.7. Pathogenicity Assay

The pathogenicity assay was performed as described in [42], including assessment of pathogenicity, conidial yield, and germination. Physiologically ripe apples, pears, and mangoes without diseased spots were used for pathogen inoculation. The fruits were inoculated with 5 mm diameter plugs of 7-day-old cultures of WT strain and mutants grown on PDA at 28 °C. Before inoculation, fruits were wounded with a sterilized microneedle holder to facilitate penetration of the fungus into fruit tissue. Inoculated fruits were kept at 25 °C and >90% relative humidity for 4 days. The diameters of the fruit anthracnose were measured by cross intersection method and the symptoms were photographed. Each treatment contained 3 replicates of 6 fruits, and the entire experiment was repeated 3 times.

2.8. Statistical Analysis

In this study, all quantitative data were derived from at least 3 replicates. The significance of the data was analyzed using SAS 9.2 software (SAS Institute Inc., Cary, NC, USA) and WPS 2022 (Kingsoft, Beijing, China). We used the One-Way ANOVA test for analysis and Duncan’s multiple range test. In this study, different lowercase letters above bars indicate significant differences between means (p < 0.05).

3. Results

3.1. Bioinformatics and Phylogenetic Analyses of the CgloRPCYG Gene

The CgloRPCYG gene was identified in the C. gloeosporioides genomic sequence (GenBank assembly accession no. GCA_011800055.1). CgloRPCYG contains a 684 bp open reading frame containing two introns (intron 1, 57 bp; intron 2, 61 bp). The CgloRPCYG gene encodes 246 amino acid residues. The prediction results show that the molecular formula of the CgloRPCYG protein is C₁₁₃₆H₁₇₆₃N₂₉₁O₃₆₆S₁₄, with a molecular weight of 25,802 Dalton. The isoelectric point and instability index are 4.67 and 42.96, respectively, indicating that it is an unstable protein. A phylogenetic tree of 55 amino acid sequences from 16 genera shows that CgloRPCYG is most similar to sequences in Colletotrichum spp. followed by Lasiodiplodia (Figure 1). In Colletotrichum spp., CgloRPCYG and homologous proteins have little difference and cluster into a large group. Other genera of filamentous fungi cluster in different groups. Similar results were found for the Lasiodiplodia, Daldinia, and Leptosphaeria genera. The BLAST searches suggest that the CgloRPCYG protein has a similar sequence in C. tropicale strain CgS9275 (KAF4820371.1). The phylogenetic analysis shows that homologous proteins of CgloRPCYG are widely present in filamentous fungi and that the sequence is conserved (Figure 1).

3.2. Successful Creation of Fusion Plasmid, Knockout, and Complementary Mutants

The fusion vector pHS-Cas9-CgloRPCYG was obtained by successfully integrating the sgRNA into the backbone vector pHS-Cas9-Backbone, which was confirmed via sequencing analysis (Figure 2A). Transformants were obtained by PEG-mediated protoplast transformation and selection on PDA plates containing 100 μg/mL hygromycin B. PCR amplification with primers CgloRPCYG-D-F/CgloRPCYG-D-R and Hyg-F/Hyg-R and agarose gel electrophoresis showed that two deletion mutants, ΔCgloRPCYG-1 and ΔCgloRPCYG-2, were obtained (Figure 2B,C), and Sanger sequencing also confirmed that the two mutants are deletion mutants (Figure 2D,E). As shown in Figure 2E, compared with the sequence of WT, a 150 bp deletion occurred in ΔCgloRPCYG-1, while a 149 bp deletion was detected in ΔCgloRPCYG-2. The mutated sequences in the ΔCgloRPCYG-1 and ΔCgloRPCYG-2 mutants were found to be respectively located 3 and 4 bp upstream of the PAM sequence (NGG). The efficiency of >100 bp deletion in the CRISPR/Cas9 knockout is 8.33%. In this study, CgloRPCYG knockout mutants were successfully obtained, but the deletion efficiency was low.

The complementary vector pOE-CgloRPCYG was obtained by successfully integrating the CgloRPCYG ORF into the backbone vector pOE-Backbone, which was confirmed via sequencing analysis (Figure 3A). PCR amplification of transformant DNA was performed using primers Bar-F/Bar-R with CgloRPCYG-F/CgloRPCYG-R and agarose gel electrophoresis. The PCR products were sequenced using the Sanger method. The DNA of the wild-type strain was used as a control. The Bar-F/Bar-R primer pair amplified specific 552 bp bands for complementary mutant and complementary backbone vector pOE-Backbone (Figure 3B). At the same time, a specific bright band of 684 bp for complementary mutant was found in the amplified transformants with the CgloRPCYG-F/CgloRPCYG-R primer pair, indicating that CgloRPCYG ORF was successfully transferred into the transformants (Figure 3C). The efficiency of positive transformation was 34.28%. To sum up, the knockout mutants and complementary strains of CgloRPCYG were successfully obtained and named ∆CgloRPCYG-1, ∆CgloRPCYG-2, and C-∆CgloRPCYG, respectively.

3.3. Role of CgloRPCYG in Mycelial Growth and Stress Tolerance

To address the role of CgloRPCYG in environmental adaptation, the WT, EV, CEV, KO, and CKO strains were inoculated on MM agar plates containing 5 mM H₂O₂, 0.01% SDS, 1 M NaCl, 1 M KCl, or 300 mg/L Congo red. Compared with the WT strain (Figure 4A,B), the ΔCgloRPCYG-1 mutant (KO) showed similar sensitivity to the various abiotic stresses above (Figure 4B). The data show that there are no significant differences in the colony diameter of the WT, EV, CEV, KO, and CKO strains after 3, 5, and 7 days of culture on MM plates. Additionally, the colony colors and mycelial sparsity of ΔCgloRPCYG-1 show differences. For a pathogen to successfully infect a host, it must overcome host-derived reactive oxygen species (ROS). Thus, we used H₂O₂ to mimic host-derived ROS. We found that deletion of the CgloRPCYG gene did not alter the sensitivity of the pathogen to ROS. This result indicates that CgloRPCYG is not involved in mediating responses to oxidative stress. Salt stress is an important basis for assessing fungal adaptation to the environment. We used KCl and NaCl to mimic environmental salt stress and found that CgloRPCYG gene deletion has different effects on the salt stress of the pathogen. The results showed that in the MM media containing NaCl for 3 days, the colony diameters of KO strain were reduced by 30.88%, compared with WT strain. In addition, after 5 and 7 days of mycelial growth in MM medium containing NaCl, the colony diameter of KO strains increased by 29.95% and 40.43% compared to WT strains, respectively.

3.4. CgloRPCYG Plays a Crucial Role in Pathogenicity, Conidial Yield, and Germination

To investigate whether deletion of CgloRPCYG would affect the pathogenicity of C. gloeosporioides, mycelial plugs were inoculated by microneedle wounded ripe apple, pear, and mango fruits. After 4 d of infection, the size of lesions on fruits caused by the indicated strains was significantly reduced compared with the lesions induced by WT and CKO (Figure 4B and Figure 5A). Compared with the WT strain, the diameter of lesions caused by knockout mutant on wounded apple, pear, and mango decreased by 86.06, 44.70, and 39.47%, respectively (Figure 4B and Figure 5A). On wounded apple fruit, the diameter of lesions caused by the ΔCgloRPCYG-1 mutant was 0.23 cm in contrast to 1.65 cm for the WT strain and 1.57 cm for the complementary mutant CΔCgloRPCYG-1. On wounded pear fruit, the diameter of lesions caused by the ΔCgloRPCYG-1 mutant was 0.73 cm in contrast to 1.32 cm for the WT strain and 1.17 cm for the complementary mutant CΔCgloRPCYG-1. On wounded mango fruit, the diameter of lesions caused the ΔCgloRPCYG-1 mutant was 0.46 cm in contrast to 0.76 cm for the WT strain and 0.66 cm for the complementary mutant CΔCgloRPCYG-1. These data suggest that CgloRPCYG plays a vital role in determining the pathogenicity of C. gloeosporioides.

Next, to investigate whether CgloRPCYG plays a role in C. gloeosporioides conidial yield, mature conidia harvested from 3-day-old PDB cultures were counted with a hemocytometer. The conidial yield and germination of strains were observed under an optical microscope. The relative conidiation of the KO mutant was significantly lower than that of the other four mutants (Figure 5C). In addition, to identify whether the deletion of CgloRPCYG had an effect on conidial germination, we recorded and compared conidial germination among the different strains within 8, 12, and 24 h. The results show that the conidia germination rate of ΔCgloRPCYG-1 was delayed in comparison to WT, EV, CV, and CΔCgloRPCYG-1 on the glass slides (Figure 5D). These results indicate that CgloRPCYG is essential for conidial yield and germination in C. gloeosporioides.

4. Discussion

The CRISPR/Cas systems can be divided into six types and more than 20 subtypes based on the differences in Cas9, Cas12, and Cas13 effectors [43,44,45,46]. The most common of these is CRISPR/Cas9 system, which is efficient, easy to operate, and has many successful cases [46]. There is also an online software designed with sgRNA. In this study, we analyzed the genomic sequence of C. gloeosporioides and designed an sgRNA target using EuPaGDT online software [37]. EuPaGDT allows users to tune the design of gRNA and characterization of on/off targets, including different CRISPR/Cas9 orthologs and PAM sequences, such as NGG, NAG, and NGA [37]. We referred to the tables of on-target/off-target scores to visually check them and corresponding PAMs, as well as the alignment of gRNA with the CgloRPCYG gene sequence, selecting the one with the highest score and finally obtaining sgRNA02: 5′-AACCACCTGACGATGCTTGT-3′. Next, we successfully constructed a CgloRPCYG gene editing vector using the CRISPR/Cas9 system, resulting in the deletion of the CgloRPCYG fragment, which provides a reference for the application of CRISPR/Cas9 in the filamentous fungus C. gloeosporioides. The results of this study confirm the applicability of EuPaGDT in C. gloeosporioides. This system can be used in the long term to study the function of other genes by only changing the sequence of sgRNA.

In Claviceps purpurea, CRISPR/Cas9 pyr4 mutations are mostly located 3 bp upstream of the PAM sequence, with insertions and deletions of larger DNA fragments in addition [47]. This shows that the effect of applying CRISPR/Cas9 in C. purpurea is similar to that in C. gloeosporioides. Meanwhile, the gene editing efficiency of the CgloRPCYG gene is higher than that of pyr4 gene, TrpE gene in C. purpurea [47]. Recently, several studies have reported the successful development of this technology in filamentous fungi, including the industrial filamentous fungi A. fumigatus [48], T. reesei [29], and Myceliophthora thermophila [49], as well as plant-pathogenic fungi Ustilago maydis [50], M. oryzae [30], and Ustilaginoidea virens [41]. For instance, Liu et al. [49] successfully developed a CRISPR/Cas9 system for efficient multiplexed genome engineering in Myceliophthora species. However, few studies on C. gloeosporioides have used this system. Hence, the novel CRISPR/Cas9 technique has been shown to have great potential in the study of functional genes in filamentous fungi. On this basis, a CRISPR/Cas9-mediated in situ complementation method for C. gloeosporioides could also be developed, with reference to P. sojae [51].

The research reports suggest that CgloRPCYG is responsible for the pathogenicity of filamentous fungi. Certainly, the deletion of CgloRPCYG led to lower pathogenicity in apple, pear, and mango fruits in this study, indicating that CgloRPCYG functions as a pathogenicity factor. In C. gloeosporioides, knockout of the CgHOS2 [52], CgHSF1 [53], CgGa1 [54], CgCPS1 [55], CgOPT2 [56], and CgEnd3 [57] genes showed the same result. C. gloeosporioides produce conidia, exhibit conidial germination and appressorium, form penetration pegs, and invade hosts, thus causing plant disease. We analyzed the effects of gene deletion on conidial yield and germination rate. Deletion of CgloRPCYG caused an obvious decrease in conidial yield and delayed conidial germination. Compared with the wild-type strain, the conidial yield was decreased by 60.60% in the knockout mutant. Similarly, conidial germination rates at 8, 12, and 24 h decreased by 36.11, 31.71, and 60.24%, respectively. A study reported that the deletion of BcCFEM1 in B. cinerea is responsible for a decrease in conidia production [58]. In C. gloeosporioides, CgHOS2 gene regulates the length of the germ tube [52]. CgloRPCYG is a potential target gene for use in the development of both plant protection technologies, such as spray-induced gene silencing, as well as new fungicides targeted to control plant anthracnose disease caused by C. gloeosporioides.

5. Conclusions

In conclusion, we experimentally characterized a new gene in C. gloeosporioides named CgloRPCYG. We successfully developed a CRISPR/Cas9 system for gene editing in C. gloeosporioides, a filamentous fungus with a wide range of plant hosts. The length of the knockout fragment is about 150 bp. Through this deletion system and complementary mutants, we demonstrated that the CgloRPCYG protein is involved in conidial yield and germination of C. gloeosporioides, as well as pathogenicity in three hosts (apple, pear, and mango). We provided a new pathogenic gene from C. gloeosporioides. It can be used to develop plant protection technologies and develop new fungicide targets. At the same time, our study extends upon the knowledge regarding the pathogenic molecular mechanisms of C. gloeosporioides.