The C. parasitica wildtype strain EP155 (ATCC 38755), its isogenic hypovirus (CHV1-EP713)-infected strain EP155/CHV1-EP713 (designated EP155/+virus; ATCC 52571) (Chen et al., 1994), highly efficient homologous recombinant strain KU80 (ΔCpku80 of EP155) (Lan et al., 2008), and derived mutant strains were incubated on potato glucose agar (PDA) medium at 26°C with a 12/12-h light/dark cycle for phenotypic analyses. PDA medium was also used for DNA, RNA, and protein extraction, as previously described (Li et al., 2022).

The ΔCpSmt3 deletion mutant was constructed using a homologous recombination method based on the KU80 strain. Using EP155 genome DNA as the template, the upstream (1,051 bp) and downstream (1,150 bp) regions flanking the CpSmt3 gene were amplified using the primers smt3-left-F/smt3-left-R and smt3-right-F/smt3-right-R, respectively. The hygromycin B resistance gene hph was amplified using the primers hph-F/hph-R, and then the three amplification products were ligated by fusion polymerase chain reaction (PCR) to obtain a 4.1-kb cassette. The purified products were transformed into KU80 protoplasts mediated by polyethylene glycol (PEG), which were regenerated and screened on medium containing 30 μg/mL hygromycin B (Thermo Fisher Scientific, USA). The transformed strains were consecutively cultured on PDA containing hygromycin B for three generations, and were subsequently identified. According to the standard protocol described by Sambrook and Russell (2001), Southern blot and PCR methods were used to identify the ΔCpSmt3 deletion mutant. The construction of ΔCpUbc2 and ΔCpUbc9 deletion mutants were performed using the same protocol with corresponding primer sets.

The ΔCpSmt3-Com complementation strain was generated by amplifying the entire CpSmt3 gene (open reading frame and promoter sequence) by PCR. Subsequently, the amplified fragment was cloned into the transformation vector pCPXG418, with a geneticin resistance (G418) cassette. The resulting construct pCPXG418-CpSmt3 was transformed into ΔCpSmt3. ΔCpSmt3-Com was then confirmed by PCR using primer smt3-qPCR-F/smt3-qPCR-R.

To construct the CpSmt3-OE overexpression strain, the CpSmt3 open reading frame was amplified and then inserted into the vector pCPXG418, with the gpd promoter for the transgene. The resulting construct pCPXG418-CpSmt3 was then introduced into wildtype (EP155) protoplasts. Positive clones were confirmed by PCR and quantitative reverse transcription PCR (qRT-PCR). ΔCpSmt3 overexpressing CpRho1 (designated ΔCpSmt3/CpRho1-OE) was constructed using the same protocol with a corresponding primer set. Primers used in this study are compiled in Supplementary Table 1.

CpRho1 fragment (including 182 bp intron and 412 bp exon) was amplified by PCR and cloned into the short hairpin RNA (shRNA) expression vector WRNAIPG (constructed by our group), between the Pgpd promoter and TrapC terminator, with hygromycin B resistance as the selection marker. The RNAi construct was then transformed into wildtype (EP155) protoplasts, and confirmed by qRT-PCR. Primers used in this study are compiled in Supplementary Table 1.

Phenotypic traits (growth rate, pigmentation, sporulation, and stress tolerance) were assessed using previously established methods (Kim et al., 1995). Briefly, for assessing sporulation, fungal strains were cultured on PDA medium at 26°C for 14 days. Conidia were collected and quantified using a hemacytometer. For assessing stress tolerance, strains were cultured on PDA media supplemented with stress chemicals. Three independent replicates were performed for each experiment. For mycelium dry weight assays, the same size blocks (0.5 mm²× 0.5 mm²) cut from 5-day-old PDA cultures were inoculated into EP liquid medium (Puhalla, 1971). After incubation at 26°C for 7 days, the mycelia in EP liquid medium were filtered and washed. Then, the mycelium was dried at 60°C for 48 h. The dry weight of each mycelium was determined by an electronic balance (Sartorius, Germany).

Light microscopy was performed using an Olympus BX51 fluorescent microscope (Olympus, Japan). Transmission electron microscopy (TEM) was performed using a JEM-1400-FLASH Transmission Electron Microscope (JEOL, Japan). For in situ mitochondria analysis, fungal hyphae were cultured in PDA medium for 10 days, scraped, and fixed with 2.5% glutaraldehyde in 100 mM phosphate buffer (pH 7.2) at 4°C overnight. After rinsing with phosphate buffer (50 mM, pH 6.8), the samples were dehydrated, embedded with Epon 812, then sliced into ultrathin sections, stained with uranium acetate (2%), and poststained with lead citrate (Shi et al., 2019). Three biological replicates were performed for each sample.

Mitochondrial membrane potential was assessed using a JC-1 fluorescent probe (Beyotime, China), and the ratio of JC-1 red/green fluorescence intensity was utilized as a representation of MMP. Briefly, the strains were cultured on PDA medium with a piece of cellophane for 10 days. The harvested mycelia were placed in JC-1 staining buffer containing 10 μM JC-1, incubated at 37°C in the dark for 20 min, and then washed with JC-1 washing buffer twice. The red fluorescence of JC-1 dimers represents normal MMP, while the green fluorescence of JC-1 monomers signifies a depolarized MMP. A transition from red to green fluorescence indicates a reduction in MMP.

The virulence of the strains was analyzed using dormant stems of Chinese chestnut (Castanea mollissima), as described previously (Shi et al., 2014). Canker sizes were measured and photographed 28 days post inoculation. The virulence assay was repeated three times for each fungal strain.

Total fungal ROS production was detected by 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. The fungal mycelia were incubated with 10 μM DCFH-DA (Beyotime, China) in the dark at 37°C for 30 min, and then washed with PBS (10 mM, pH 7.5) three times. At a maximum excitation wavelength of 480 nm and a maximum emission wavelength of 525 nm, fluorescence microscopy was used to detect fluorescence signals. For mitochondrial ROS (mtROS) level detection, MitoSOX red mitochondrial superoxide dismutase indicator (MEC, USA) was used as described previously (Luo et al., 2021). Briefly, mycelia collected from PDA medium were washed three times with PBS, incubated with 10 μM MitoSOX red for 30 min at room temperature, washed three times with PBS again, and observed using fluorescence microscopy.

To obtain SUMO conjugates, a plasmid expressing 3 × FLAG–CpSmt3 fusion protein was constructed and transformed into the wildtype strain, and transformants were identified based on G418 resistance. A transformant was confirmed by western blot analysis using an anti-FLAG antibody (1: 2000, ABclonal) as the primary antibody. The verified strain was incubated in liquid EP medium for 3 days then harvested. Total protein was extracted from the cells using NP-40 Lysis Buffer (Beyotime, China) with 1 mM phenylmethylsulfonyl fluoride (PMSF), incubated with anti-FLAG antibody (ABclonal) overnight at 4°C and then mixed with protein A/G agarose (Sangon, China) for 2 h, which was then washed using 1× immunoprecipitation (IP) buffer and 1 × 0.1 IP buffer three and six times, respectively. Next, the lysates were incubated with loading buffer at 95°C for 5 min and then centrifuged at 25°C and 1,200 rpm for 1 min. The proteins were collected, digested using trypsin, and analyzed by LC-MS/MS (Q Exactive HF, Thermo Scientific). The resulting MS/MS data were used to search against the nonredundant C. parasitica protein database of the Joint Genome Institute (JGI).¹ Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed using eggnog-mapper² and Tbtools software (Chen et al., 2020).

Fungal strains were cultured on PDA medium with a piece of cellophane for 10 days, and then the mycelium was collected for proteome sequencing. Protein isolation was conducted using a fungal protein extraction kit (ProExcell™, China). Next, 20 μg protein was digested overnight using 1 μg sequencing-grade trypsin (Promega Corp., USA). Thereafter, the peptides were prepared for nano-LC-MS/MS by C18 Zip-Tip purification according to the manufacturer’s protocol (Millipore Inc., USA). Three biological replicate samples were then suspended in water with 0.1% formic acid (v/v) and subjected to nano-LC-MS/MS (Q Exactive HF, Thermo Scientific, USA). Briefly, 1 μg peptide sample was injected into a reverse-phase BEH C18 column (100 μm × 100 mm; particle size: 1.7 μm; pore size: 300 Å) (Waters Corp., Massachusetts, USA) for LC using a Waters nanoACQUITY LC system. Peptides eluting from the column were analyzed by data-dependent MS/MS on a Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific Inc., Massachusetts, USA). The data were searched against the C. parasitica genome database and a decoy database using the Sequest HT search engine in Proteome Discoverer 1.4 software (Thermo Fisher Scientific Inc., Massachusetts, USA).

Differentially expressed proteins (DEPs) between the wildtype strain and ΔCpSmt3 were determined using the t-test function in R language, with fold change > 1.2 and p < 0.05 indicating statistical significance. The number of DEPs was then calculated. GO enrichment analysis of the DEPs was conducted using DAVID software,³ with p < 0.05 indicating statistical significance. DEPs were also annotated using KEGG Mapper.⁴ The KEGG enrichment analysis of the DEPs was conducted using DAVID software.⁵ Fisher’s exact test was employed as the statistical test, with p < 0.05 indicating statistical significance.

The SUMO ortholog of C. parasitica was identified by searching the C. parasitica genome database⁶ using the S. cerevisiae Smt3 protein sequence as a query (GenBank: QHB07964). A protein comprising 106 amino acids, designated CpSmt3 (JGI ID: 356715), was found to share 68.3% similarity with the S. cerevisiae Smt3. The CpSmt3 gene contains three exons and two introns (Supplementary Figure 1A). Domain analysis revealed that it has a conserved Ubl_Smt3_like domain (coordinates 25–97). Phylogenetic analysis showed that CpSmt3 is most closely related to the SUMO proteins of Valsa mali and Neurospora crassa (Supplementary Figure 1B).

To investigate the function of the CpSmt3 gene in C. parasitica, we constructed a CpSmt3 deletion mutant (ΔCpSmt3) and a CpSmt3 overexpression strain (CpSmt3-OE) (Supplementary Figure 2). Southern blot and RT-PCR analyses of ΔCpSmt3 showed that CpSmt3 was successfully deleted from the genome of C. parasitica (Supplementary Figures 2B–D). In addition, western blot analysis using anti-SUMO1 antibody showed that the wildtype and complementation strain (ΔCpSmt3-Com) had a ∼16 kDa band, while ΔCpSmt3 did not (Supplementary Figure 2E). This indicated that the CpSmt3 was involved in SUMOylation of C. parasitica. Compared to the wildtype strain EP155 and the parental strain KU80, ΔCpSmt3 exhibited reduced aerial hyphae, intense pigmentation, slow colony growth, and no spore formation (Figures 1A–C). Microscopy showed that the hyphae of ΔCpSmt3 were swollen and accompanied by multinucleated cells, and the hyphal diameter was also significantly larger than that of the wildtype strain (Figure 1D and Supplementary Figure 2F). However, no significant changes were observed in CpSmt3-OE (Supplementary Figures 2G, H).

To determine the role of SUMO in the stress response, wildtype and ΔCpSmt3 were inoculated on PDA media supplemented with cell stress agents Congo red, sodium dodecyl sulfate (SDS), NaCl, and H₂O₂. ΔCpSmt3 was more sensitive to the stress than the wildtype strain, especially to H₂O₂ and SDS, with inhibition rates of 78.02 and 97.69%, respectively (Figures 1E, F).

To explore the role of CpSmt3 in C. parasitica virulence, chestnut stems were inoculated with the wildtype strain, KU80, EP155/+virus, ΔCpSmt3, and complementation strain to assess their virulence. The canker formed by ΔCpSmt3 was significantly smaller than that of wildtype strain or KU80, but comparable to the canker formed by the hypovirus-infected wildtype strain. The reduced virulence of ΔCpSmt3 was fully restored by re-induction of the wildtype copy of CpSmt3 (Figures 1G, H).

As SUMOylation has been reported to be involved in mitochondrial and cell wall morphology (Yamada et al., 2021; Azizullah et al., 2023), we wondered whether deletion of CpSmt3 would have similar effects in C. parasitica. TEM revealed that ΔCpSmt3 exhibited swollen mitochondria and a thickened cell wall compared to the wildtype strain (Figures 2A, B).

To further explore the mitochondrial dysfunction caused by CpSmt3 deletion, JC-1 staining was used to measure the MMP. There was a significant decrease in MMP upon CpSmt3 deletion (Figure 2C). In addition, TEM analysis showed that autophagic bodies were evident in the mycelia of ΔCpSmt3 (Figure 2A). Monodansylcadaverine (MDC) staining further proved that autophagic bodies accumulated in ΔCpSmt3 cells, but not in the wildtype strain (Figure 2D). Together, these results suggested that deletion of CpSmt3 caused organelle defects and autophagy.

Previous studies have shown that SUMOylation play essential roles in the regulation of ROS homeostasis by controlling ROS production and clearance, and reduced SUMOylation may lead to higher ROS production (Stankovic-Valentin and Melchior, 2018). As deletion of CpSmt3 caused decreased MMP, we measured the intracellular ROS in ΔCpSmt3 using the DCFH-DA fluorescence assay. Compared to the wildtype strain, ΔCpSmt3 had a significantly higher ROS level (Figures 3A, B). To further identify the source of ROS in ΔCpSmt3, we assessed the mtROS by using MitoSOX Red staining. A large amount of mtROS was observed in ΔCpSmt3 (Figures 3C, D), suggesting that the ROS burst was derived from mitochondria. To confirm this speculation, we supplemented the PDA medium with the ROS inhibitor N-Acetyl-L-cysteine (NAC) or the mtROS-specific inhibitor Mito-TEMPO. After the treatment, the ΔCpSmt3 colonies became larger and the ROS level was significantly reduced. In contrast, treatment with the ERO1-α inhibitor EN460 failed to increase the ΔCpSmt3 colony size or reduce the ROS level (Figures 3E, F).

In animals, SUMOylation has been reported to activate the immune system to counteract viral infections (Imbert and Langford, 2021). To examine the role of SUMOylation in the interaction between C. parasitica and the hypovirus CHV1-EP713, the hypovirus was introduced into ΔCpSmt3 (ΔCpSmt3/CHV1-EP713, designated ΔCpSmt3/+virus) via hyphal anastomosis with the isogenic hypovirus-infected wildtype strain (EP155/+virus). The resultant colony size was significantly increased compared to ΔCpSmt3, suggesting that hypovirus infection partially reversed the growth impairment resulting from CpSmt3 deletion. In contrast, the hypovirus-infected CpSmt3-OE did not exhibit a change in colony size compared to the hypovirus-infected wildtype strain (Figures 4A and Supplementary Figure 3D). However, when ΔCpSmt3 and ΔCpSmt3/+virus inoculated on chestnut branches, the canker sizes of them were the same (Supplementary Figure 3A). Interestingly, hypovirus-infected ΔCpSmt3 accumulated about five times more viral dsRNA than the hypovirus-infected wildtype strain, while hypovirus-infected ΔCpSmt3-Com regained the ability to inhibit viral dsRNA accumulation. Hypovirus-infected CpSmt3-OE did not exhibit an altered viral dsRNA accumulation compared to the hypovirus-infected wildtype strain (Figures 4B and Supplementary Figure 3E).

To verify whether SUMOylation influences hypovirus replication, we constructed SUMO E1 subunit Uba2 and E2 enzyme Ubc9 mutant strains of C. parasitica, designated ΔCpUba2 (JGI ID: 255521) and ΔCpUbc9 (JGI ID: 324396), respectively (Supplementary Figures 3B, C). They both exhibited a phenotype very similar to that of ΔCpSmt3, and hypovirus infection of both strains partially rescued the colony size and growth (Figure 5A and Supplementary Figure 3D) but viral dsRNA accumulation was elevated five- and ten-fold, respectively (Figures 5B and Supplementary Figure 3E).

To investigate the molecular mechanism underlying the increased viral dsRNA, we measured the transcript level of CpDcl2, a key gene in the antiviral RNA silencing pathway. There was no significant difference in CpDcl2 expression between the wildtype strain and ΔCpSmt3. However, CpDcl2 was highly upregulated (13-fold) in the hypovirus-infected wildtype strain, but only upregulated ∼4-fold in the hypovirus-infected ΔCpSmt3 strain (Figure 5C). These results suggest that SUMOylation is required for strong inhibition of viral dsRNA accumulation, likely by upregulating CpDcl2 to suppress viral replication.

It is generally believed that SUMOylation increases protein stability through competitive binding to the ubiquitination modification sites in the substrate proteins (Bae et al., 2004; Prudent et al., 2015; Nakagawa et al., 2016). To explore the molecular mechanism of SUMOylation in phenotype, we performed a label-free proteomic analysis of wildtype vs. ΔCpSmt3 mycelia. A total of 3,970 and 3,254 proteins were identified in these strains, respectively, representing 35.82% (4,158 proteins) of the 11,609 annotated proteins from the whole genome (Supplementary Table 2). Compared to the wildtype strain, 1,869 differentially expressed proteins (DEPs), 1,702 downregulated and 167 upregulated, were observed in ΔCpSmt3 (Supplementary Table 2).

To define the SUMOylation target spectrum of CpSmt3 in C. parasitica, we enriched the SUMOylated proteins in a wildtype (EP155) transformant expressing 3 × FLAG–CpSmt3 fusion protein, using affinity pull-down assays involving anti-FLAG beads. Western blot analysis with anti-FLAG antibody showed that the fusion protein was successfully expressed (Supplementary Figure 4A). The anti-FLAG bead-captured proteins were then trypsin-digested and subjected to LC-MS/MS analysis. A total of 500 SUMOylated proteins were identified in three independent biological replicates. A total of 1,229 SUMOylation sites and 644 SUMO interaction motifs (SIM) were identified within these proteins using the SUMO site prediction software GPS-SUMO 2.0 online tool⁷ (Supplementary Figures 4B, C and Supplementary Table 3). Interestingly, the majority (408 out of 500) of these potential SUMOylated proteins were the DEPs identified in ΔCpSmt3 (Figure 6A). Intriguingly, most of these SUMOylated DEPs (397 out of 408) were downregulated in ΔCpSmt3, including the five pathogenesis-related proteins listed in Supplementary Table 3.

Gene Ontology enrichment analysis indicated that the downregulated SUMOylated proteins in ΔCpSmt3 were significantly enriched in structural constituent of ribosome, ribonucleoprotein complex, cytoplasm, cytosol, protein folding, and response to stress (Figure 6B and Supplementary Table 4). KEGG enrichment analysis showed that these proteins were significantly enriched in translation, ribosome, exosome, citrate cycle, translation factors, and oxidative phosphorylation pathway (Figure 6C and Supplementary Table 5), implying that SUMOylation is essential for maintaining ribosomal and mitochondrial function and survival in response to environmental stress.

To further explore the regulated mechanism of CpSmt3, we attempted to find out the proteins related to phenotypic defects of ΔCpSmt3 mutant from SUMO substrates with known functions (Table 1). We found Rho1 was related to cell cycle, which has also been reported in Aspergillus fumigatus that regulates cell wall integrity and stress response to H₂O₂ stress (Zhang et al., 2018). In addition, CpRho1 (JGI ID: 97360) was downregulated SUMOylated DEPs in ΔCpSmt3 (Supplementary Tables 2, 3). Therefore, we selected CpRho1 as the target for further study. To examine the contribution of this protein in C. parasitica, we attempted to generate a CpRho1 deletion mutant. However, we failed to obtain a CpRho1-knockout strain after screening hundreds of candidate strains. Thus, we opted to generate a CpRho1-silenced strain. Three independent strains, CpRho1-RNAi-1, CpRho1-RNAi-2, and CpRho1-RNAi-3, with 64, 73, and 51% CpRho1 mRNA levels, respectively (Supplementary Figures 5A, B), were selected for further analysis. They exhibited 84 to 93% reduction in colony size, had no spore formation, and their hyphae were swollen, similar to that of ΔCpSmt3. However, CpRho1 overexpression in ΔCpSmt3 (ΔCpSmt3/CpRho1-OE) partially restored the reduced colony size of ΔCpSmt3 (Figures 7A, B and Supplementary Figure 5C), suggesting a link between CpSmt3 and CpRho1 in modulating fungal traits. TEM showed autophagic vacuoles and significantly thickened hyphal cell walls in the CpRho1-RNAi mutants, but the mitochondrial morphology remained normal (Figures 7C, D). Cellular analysis showed that the CpRho1-RNAi mutants had increased accumulation of autophagic vacuoles, elevated ROS production, and multinucleation. Importantly, these aberrations were greatly ameliorated by CpRho1 overexpression in ΔCpSmt3 (ΔCpSmt3/CpRho1-OE) (Figures 7E–H), implying that CpSmt3 may exert its effects by maintaining a proper CpRho1 level to ensure the normal cellular functions of the fungus.