The Histone Acetyltransferase CgHat1 Regulates Growth, Development, and Pathogenicity of Colletotrichum gloeosporioides

Abstract

Colletotrichum gloeosporioides causes anthracnose on a wide range of plants, resulting in serious economic losses worldwide. However, the molecular mechanisms underlying its pathogenicity still remain largely unknown. In the past 20 years, the importance of acetylation/deacetylation modification in the pathogenicity of phytopathogens has already been highlighted, but how it functions in C. gloeosporioides is obscure. Here, we identified and functionally characterized a histone acetyltransferase CgHat1 in C. gloeosporioides. As suspected, CgHat1 is localized to the nucleus and regulates the acetylation levels of histone H4K5 and H4K12. Targeted gene deletion revealed that CgHat1 plays crucial roles in growth, colony pigmentation, and conidiation. Furthermore, we provided evidence showing that ΔCghat1 mutant is defective in conidial germination, appressorial formation, and response to endoplasmic reticulum (ER) stress. These combined effects lead to the decreased pathogenicity of ΔCghat1 mutant. Our studies not only firstly shed light on the pleiotropic roles of histone acetyltransferase in C. gloeosporioides, but also offer a potential fungicide target for anthracnose control.

1. Introduction

Colletotrichum spp. is one of the top 10 pathogens in plant-pathogenic fungi and almost every crop grown in the world is susceptible to one or more species of Colletotrichum [1,2]. Among them, C. gloeosporioides is one of the most common and widely distributed species in the world, which causes anthracnose on a wide range of plants, including Gossypium hirsutum, Morus alba, Malus pumila, and Capsicum annuum, resulting in economic losses of 30–60% or even more [3]. Apart from its economic importance, C. gloeosporioides has also emerged as a model for scientists to study fungal pathogenicity. For example, the actin-bundling protein CgFim1-organized F-actin dynamics during appressorium development regulates the pathogenicity of C. gloeosporioides in Hevea brasiliensis [4]. The conserved MAPK kinase CgMkk1 phosphorylates the transcriptional factor CgCrzA to respond to the CFW-induced stress and underpin the virulence of C. gloeosporioides in Cunninghamia lanceolate [5]. The secreted protein CgCsa-mediated Fe³⁺ homeostasis regulates the pathogenicity of C. gloeosporioides in Capsicum spp. [6]. Although much progress has been made in the study of its pathogenesis, the molecular mechanisms underlying pathogenicity still remain largely unknown.

The transcriptome analyses revealed that Colletotrichum spp. reprograms fungal gene expression to infect host plants [2]. The acetylation and deacetylation of histones are pivotal epigenetic mechanisms for reprograming gene transcription [7]. Histone acetylation promotes the opening of chromatin and the further binding of transcription factors, thus promoting gene transcription. Conversely, histone deacetylation reduces the accessibility of transcription factors by forming a closed chromatin conformation, thus inhibiting gene transcription [7,8]. The histone acetyltransferase Hat1 is one of the first identified histone acetyltransferases. It mainly promotes gene transcription by promoting the acetylation of lysine K5 and K12 in histone H4 [9,10].

Although the mechanism of Hat1 regulating histone acetylation is highly conserved, its biological function is species-specific. The ScHat1 in Saccharomyces cerevisiae is dispensable for growth, while the absence of HAT1 gene in Candida albicans could cause elongated cells [11,12]. There are a few reports about the important roles of Hat1 on virulence in pathogenic fungi, but its pathogenic mechanism is different in different fungi. In the plant pathogenic fungus Magnaporthe oryzae, MoHat1 governs the pathogenicity mainly by regulating the turgor pressure of appressorium, while in the animal pathogenic fungus Candida albicans, CaHat1 affects the pathogenicity mainly by regulating the formation of infection structure [12,13]. These raise the question of how CgHat1 functions in C. gloeosporioides.

In the presented study, we identified the histone acetyltransferase CgHat1 in C. gloeosporioides and characterized its function. We revealed that nucleus-locating protein CgHat1 plays crucial roles in the acetylation of H4K5 and H4K12. Furthermore, we found that CgHat1 is involved in growth, colony pigmentation, and conidiation. Importantly, we provided evidence demonstrating the importance of this protein in responses to ER stress, appressorium development, and pathogenicity of C. gloeosporioides.

2. Materials and Methods

2.1. Strains and Culture Conditions

The C. gloeosporioides strain, isolated from M. alba leaves and identified by molecular and morphological analysis, was used as WT in this study and was preserved in Yuelushan Laboratory at −80 °C. All strains were cultured on CM, MM or PDA agar plates in darkness at 28 °C. Liquid CM was used to culture strains for extracting total proteins and harvesting conidia.

2.2. Targeted Gene Deletion, Complementation and Subcellular Localization

Targeted gene deletion was carried out by one-step gene replacement strategy [5]. First, two sequences of around 1.0 kb flanking the CgHAT1 were amplified by PCR with primer pairs (Table S1). Then, the upstream and downstream flanking sequences were overlapped with the flanks of hygromycin resistance cassette (HPH), respectively. Finally, the resulting 3.4 kb fragments were introduced into the protoplasts of WT strain for gene deletion. For complementation assays, the CgHAT1 gene and its native promoter regions were amplified by PCR with primer pairs (Table S1) using high-fidelity DNA polymerase and inserted into pYF11 vector. After sequencing, the construct was introduced into the protoplasts of ΔCghat1 mutant as described previously [14]. For the subcellular localization of CgHat1, the mycelia and conidia of the strains co-expressing CgHat1-GFP and H1-RFP were observed and visualized under a fluorescent microscope. The merged GFP fluorescence and RFP fluorescence indicated their co-localization in nucleus.

2.3. Growth and Stress Response Assays

To test hyphal growth, small agar blocks of the strains were cut from the periphery of the colonies and cultured on fresh CM, MM, and PDA agar plates at 28 °C in darkness. After 3 days incubation, the colony diameters were measured. To test stress response, the strains were cultured on fresh CM or CM supplemented with ER stress (2.5 mM DTT), osmotic stresses (1 M NaCl or 1 M KCl), cell wall integrity stresses (0.01% SDS or 400 μg/mL Congo Red), oxidative stress (5 mM H₂O₂), and rapamycin stress (25 nM rapamycin).

2.4. Conidiation, Appressorial Formation and Pathogenicity Assays

To test conidiation, the strains were first cultured in 200 mL liquid CM for 3 days and then the mycelia were filtered by three layers of lens paper, harvesting the conidia in filtrates for statistical analysis. The harvested conidia were also further washed by ddH₂O twice and were resuspended to a concentration of 2 × 10⁵ spores per milliliter. Finally, resuspended conidia were inoculated on cover slips (hydrophobic) for conidial germination and appressorial formation. To test pathogenicity, the strains were inoculated onto the M. alba leaves in the high-humidity plates. After incubation in darkness at 28 °C for 3 days, the lesions were observed, photographed and assessed by ImageJ software [15].

2.5. Protein Extraction and Acetylation Analysis

The strains were cultured in liquid CM for 36 h and then the mycelia of each strain were harvested. Next, the mycelia were ground into fine powder in a mortar containing liquid nitrogen and resuspended with 1 mL RIPA (radio immunoprecipitation assay) buffer with protease inhibitors. The lysates were placed on the ice for 30 min and vortex-mixed once 10 min for protein lysing, followed by centrifugation at 13,400× g for 20 min at 4 °C to remove the cell debris. The supernatant lysates were collected as extracted proteins and were analyzed by 10% SDS-PAGE followed by Western blotting with anti-H4K5Ac (PTM BIO, Hangzhou, Zhejiang, China, PTM-119, 1:2000), anti-H4K12Ac (PTM BIO, Hangzhou, Zhejiang, China, PTM-121, 1:2000), anti-H4 (EpiZyme, Shanghai, Shanghai, China, P010074, 1:1000), and anti-Tubulin (Engibody, Dover, DE, USA, AT0003, 1:10,000) for detection.

2.6. Bioinformatics and Statistical Analysis

The phylogenetic tree of Hat1 proteins was constructed by MEGA5.05 using a neighbor-joining method with 1000 bootstrap replicates. The sequences of Hat1 proteins were collected from the NCBI database (https://www.ncbi.nlm.nih.gov/, accessed on 12 August 2025). The domain of CgHat1 were predicted using the SMART website (http://smart.embl-heidelberg.de/, accessed on 12 August 2025). For statistical analysis, all experiments were performed with at least three technical replicates and repeated three times, which showed consistent results. Data were assessed for normality and then analyzed by Student’s t-test with SPSS 20.0 software. p < 0.05 was considered as a significant difference.

3. Results

3.1. Identification and Bioinformatic Analysis of CgHat1

Using S. cerevisiae Hat1 sequence as a query, we acquired its homolog by a BLAST_P search in the C. gloeosporioides genome database and named it CgHat1. Phylogenetic analysis of CgHat1 and its homologs from other fungi revealed that Hat1 proteins are highly conserved, with MoHat1 being most homologous to Cnhat1 in C. noveboracense (98% identify and 99% similarity) and most distant to yeast Hat1p (still 30% identify and 47% similarity) (Figure 1A). CgHat1 encodes a 481-amino acid (aa) polypeptide and is predicted with a conserved N-terminal HAT domain (7–163 aa) by the SMART website (Figure 1B).

3.2. CgHat1 Is Localized to the Nucleus

To observe the subcellular localization of CgHat1, a green fluorescent protein (GFP) tag was fused to the C-terminus of CgHat1. Spot green fluorescent signals were observed under fluorescent microscope. To test whether the spot signals indicate nucleus, a nucleus marker H1-RFP was introduced into the CgHat1-GFP expressed strains. We found that all the green fluorescence was co-localized with green fluorescence in mid and tip regions of the vegetative hyphae (Figure 2A). Furthermore, we also found that CgHat1-GFP was co-localized well with H1-RFP in conidia (Figure 2B). These results indicate that CgHat1 is localized to the nucleus in both vegetative hyphae and conidia.

3.3. CgHat1 Regulates the Acetylation of H4K5 and H4K12

To test the roles of CgHat1 in nucleus, we obtained the CgHAT1 gene deletion mutant ΔCghat1 according to the homologous recombination principle (Figure S1A,B). Since two mutant strains were indistinguishable in general phenotypes, we randomly selected ΔCghat1#15 for further study. The yeast ScHat1 acetylates the histone H4K5 and H4K12 [9,10]. Thus, we examined the acetylation levels of H4K5 and H4K12. Compared to wide-type (WT) and complemented strain ΔCghat1/CgHAT1, ΔCghat1 showed significantly decreased acetylation levels of both H4K5 and H4K12 (Figure 3). These results suggest that CgHat1 regulates the acetylation of H4K5 and H4K12.

3.4. CgHat1 Is Important for the Growth on PDA Media and for the Colony Pigmentation

To address the role of CgHat1 in growth, the WT, ΔCghat1, and ΔCghat1/CgHAT1 strains were cultured on CM, MM, and PDA agar plates. The results showed that all the strains exhibit similar growth levels on CM and MM agar plates, whereas the ΔCghat1 mutant showed significantly decreased growth rates compared to WT and ΔCghat1/CgHAT1 on PDA agar plates (Figure 4). Additionally, compared to the colony pigmentation in WT and ΔCghat1/CgHAT1, the ΔCghat1 mutant showed absolutely no pigmentation (Figure 5). These results suggest that CgHat1 plays important in growth on PDA media and in colony pigmentation.

3.5. CgHat1 Is Critical for Asexual Development

As asexual conidia play an important role in the disease cycle and infection of Colletotrichum [16,17], the conidiation production of the WT, ΔCghat1, and ΔCghat1/CgHAT1 strains were collected, quantified, and analyzed. After 3 days of incubation, the WT and ΔCghat1/CgHAT1 strains produced about 300 × 10⁴ conidia per milliliter, compared to no more than 200 × 10⁴ conidia/mL produced by ΔCghat1 mutant (Figure 6). This result indicates that CgHat1 is critical for asexual development.

3.6. CgHat1 Is Required for Full Virulence

As a phytopathogen, we focus more of our interest on examining the roles of CgHat1 in virulence. The WT, ΔCghat1, and ΔCghat1/CgHAT1 strains were inoculated onto the M. alba leaves. After 4 days incubation, the lesions caused by WT and ΔCghat1/CgHAT1 strains showed obviously larger than that caused by ΔCghat1 mutant (Figure 7A). Furthermore, the statistical analysis revealed that the WT and ΔCghat1/CgHAT1 caused more than 0.3 cm² lesion areas on M. alba leaves, compared to about 0.1 cm² lesion areas caused by ΔCghat1 mutant (Figure 7B). This result indicates that CgHat1 is required for full virulence of C. gloeosporioides.

3.7. CgHat1 Plays Important Roles in Conidial Germination and Appressorial Formation

To investigate the possible reasons for the decreased virulence in ΔCghat1 mutant, we tested the appressorial development. The conidia of WT, ΔCghat1, and ΔCghat1/CgHAT1 strains were incubated on artificial hydrophobic surfaces. The percentages of conidial germination were more than 85% in WT and ΔCghat1/CgHAT1, compared to about 60% of that in the ΔCghat1 mutant (Figure 8). Similar decreased rates of ΔCghat1 mutant were also observed in appressorial formation, compared to WT and ΔCghat1/CgHAT1 strains (Figure 8). This result indicates that CgHat1 plays important roles in conidial germination and appressorial formation.

3.8. CgHat1 Regulates the Response to ER Stress

In order to grow normally and infect plants, pathogenic fungi must overcome many stresses [18,19]. Therefore, we tested whether CgHat1 may also associate with the response to environmental stresses. We found that the ΔCghat1 mutant showed significantly higher inhibition rates to endoplasmic reticulum (ER) stress (DTT) than that in WT and ΔCghat1/CgHAT1 strains (Figure 9). Meanwhile, we also found that the ΔCghat1 mutant showed similar sensitivity with WT and ΔCghat1/CgHAT1 strains to osmotic stresses (NaCl and KCl), cell wall integrity stresses (SDS and CR), oxidative stress (H₂O₂) and rapamycin stress (Figure S2). These results suggest that CgHat1 regulates the response to ER stress.

4. Discussion

In the long-term and complicated arms race with host plants, pathogens, including Colletotrichum spp., successfully infect host plants through various strategies such as gene transcription rearrangement [2]. HATs are necessary co-activators for transcriptional activation by modifying histones [20,21]. In the past 20 years, the importance of HATs in the pathogenicity of fungi have already been highlighted [22,23,24]. The Hat1 protein is one of the first identified histone acetyltransferases and mainly promotes gene transcription by promoting the acetylation of lysine K5 and K12 in histone H4 [9,10], which is consistent with our study showing that CgHat1 is localized in nucleus to regulate the H4K5Ac and H4K12Ac in C. gloeosporioides. Furthermore, the phylogenetic analysis revealed that the sequence of Hat1 proteins is highly similar in fungi, especially in Colletotrichum species. The conserved role of Hat1 in acetylation and sequence similarity of such proteins might forecast its conservative biological functions in different organisms.

Actually, CgHat1 plays important roles in conidiation, which is consistent with its homologues in Pestalotiopsis microspora [25]. Meanwhile, its biological function might also be species-specific. Our results revealed that CgHat1 is dispensable for growth in synthetic medium and positively regulates growth in semisynthetic medium. However, the yeast ScHat1 is not involved in growth, while the absence of the HAT1 gene in C. albicans could cause elongated cells [11,12]. Furthermore, although both of the Hat1 proteins in M. oryzae and C. albicans participate in the pathogenic process, their pathogenic mechanisms vary across different species. To achieve successful infection, M. oryzae employs Hat1 to regulate the turgor pressure of appressorium, whereas C. albicans primarily utilizes Hat1 to influence pathogenicity by controlling the formation of its infection structures [12,13]. These raise the question of how CgHat1 functions in the pathogenicity of C. gloeosporioides.

The significantly decreased virulence of ΔCghat1 revealed that CgHat1 is required for pathogenicity. We reasoned that the defect in virulence of ΔCghat1 mutant was directly due to the decreased conidial germination and appressorial formation, which is essential for the penetration and host-colonization of Colletotrichum spp. [16,17]. Moreover, the studies in M. oryzae showed that the host-derived ER stress is a common barrier for the successful infection of pathogens [26,27]. Therefore, the susceptibility of ΔCghat1 mutant to ER stress might also be a reason for its pathogenicity defect. Additionally, the melanin layer is essential for the penetration and pathogenicity of M. oryzae and Alternaria alternata [28,29,30]. The reduced pigmentation in ΔCghat1 mutant might be another reason for its reduced pathogenicity. However, it is unclear whether there is a direct correlation between pigmentation and pathogenicity in Colletotrichum and further studies are highly warranted.

5. Conclusions

Taken together, we identified and functional characterized the histone acetyltransferase CgHat1 in C. gloeosporioides. Our findings showed that CgHat1 is localized in the nucleus and regulates the H4K5Ac and H4K12Ac. The ΔCghat1 mutant is pleiotropic in defects, ranging from growth to conidiation and responses to ER stress. Our studies provide new insight for exploring such a protein as a potential fungicide target for anthracnose control. However, the genes regulated by CgHat1 are not yet clear in Colletotrichum. Further studies will focus on the interactive analysis between transcriptome and acetylomics.