The SUMO gene MrSmt3 is involved in SUMOylation, conidiation and stress response in Metarhizium robertsii

Abstract

Smt3, as a small ubiquitin-like modifier (SUMO), play an essential role in the regulation of protein SUMOylation, and thus this process can affect various important biological functions. Here, we investigated the roles of MrSmt3 (yeast SUMO/Smt3 homologs) in the entomopathogenic fungus Metarhizium robertsii. Our results of subcellular localization assays demonstrated that MrSmt3 was present in the cytoplasm and nucleus, whereas MrSmt3 was largely localized in the nucleus during oxidative stress. Importantly, disruption of MrSmt3 significantly decreased the level of protein SUMOylation under heat stress. Deletion of MrSmt3 led to a significant decrease in conidial production, and increased sensitivity to various stresses, including heat, oxidative, and cell wall-disturbing agents. However, bioassays of direct injection and topical inoculation demonstrated that deletion of MrSmt3 did not affect fungal virulence. Furthermore, RNA-seq analysis identified 1,484 differentially expressed genes (DEGs) of the WT and ΔMrSmt3 during conidiation, including 971 down-regulated DEGs and 513 up-regulated DEGs, and further analysis showed that the expression level of several classical conidiation-associated genes, such as transcription factor AbaA (MAA_00694), transcription factor bZIP (MAA_00888) and transcription factor Ste12 (MAA_10450), was down-regulated in the ΔMrSmt3 mutant. Specifically, the major downregulated DEGs were mainly associated with a variety of metabolic regulatory processes including metabolic process, organic substance metabolic process and primary metabolic process. Collectively, our findings highlight the important roles of the SUMO gene MrSmt3 in modulating SUMOylation, conidiation and stress response in M. robertsii.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-73039-x.

Introduction

Entomopathogenic fungi play an important role in modulating the insect populations by infecting insects via direct cuticle penetration, and thus rarely cause the resistance of insects^1–3. The representative entomopathogenic fungi including Metarhizium have been widely used for exploring the interactions of fungal pathogen-host and as biocontrol agents^4,5. Metarhizium robertsii, a well-known entomopathogenic fungus, exhibits virulence against a broad-spectrum of hosts and has been applied widely as mycopesticides⁶. As a biocontrol agent, the control potential of M. robertsii relies on conidial production and fitness (including virulence factors and tolerances to various stresses)⁷. Therefore, it is important to address the molecular mechanism for augmenting the conidial yield and fitness in M. robertsii^8,9. Recently, post-translation modifications, such as ubiquitination^10,11, and SUMOylation^12–14, have been extensively studied as bringing an essential link between phenotype (including development and environmental stress response) and genotype in most pathogenic fungi. However, there are few reports on elucidating the regulatory roles of ubiquitination and SUMOylation in M. robertsii.

SUMOylation with small ubiquitin-like modifier (SUMO), similar with ubiquitination, is a crucial post-translational modification in eukaryotic cells and involves in a series of process mediated by specific enzymes, such as E1 SUMO-activating enzyme, E2 SUMO-conjugating enzyme, E3 SUMO ligase, and SUMO proteases^15–18. Usually, SUMOylation get started with SUMO mature form by exposing a C-terminal diglycine (GG) motif¹⁶. Subsequently, SUMO is activated by E1 and transferred to E2, and then E3 aids in conjugating a glycine residue of SUMO with a lysine residue on the substrate protein by using an iso-peptide bond. Finally, the SUMO protease releases SUMO for recycling through cleaving iso-peptide bond¹⁹, and thus SUMOylation has been shown to regulate a variety of biological roles in various species^20,21.

As the key regulators in SUMOylation, SUMOs contain approximately 100 amino acids, and bind to substrate proteins for regulating different cellular functions²². Typically, there are only one SUMO (encoded by a single gene Smt3) in most fungi, and previous studies have shown Smt3 plays distinct roles in various fungi^15,18. For example, in the yeast Saccharomyces cerevisiae, a single SUMO gene was determined as a high copy suppressor of a mutation in the centromere protein Mif2 and thus designated as Suppressor of Mif Two (Smt3)^22,23. The Smt3 deletion strains are not viable²⁴, and the different core amino acid (lysine) residues of Smt3 is essential for various stress conditions, including high temperature and heavy metal stress²². In Candida albicans, Smt3 plays a crucial role in phenotypic changes and stress resistance²⁵. In Aspergillus nidulans, Smt3 contributes to pleiotropic effects on growth and development²⁶. Specifically, Smt3 was also recently found to be essential for vegetative growth, asexual development, response to stress, DNA damage and virulence in several pathogenic fungi, including A. flavus^27,28, Magnaporthe oryzae^14,29, Fusarium graminearum¹³, F. oxysporum^30,31, Beauveria bassiana³², and Botrytis cinerea¹⁷. Nevertheless, the biological roles of MrSmt3 in the entomopathogenic fungus M. robertsii is still unknown. Here, the functions of the SUMO-encoding gene MrSmt3 were investigated in M. robertsii. It was found that MrSmt3 is involved in SUMOylation, and required for conidiation and stress response in M. robertsii. Specifically, RNA-seq analysis showed that MrSmt3 deletion resulted in several classical conidiation-associated transcription factors genes with downregulated expression in M. robertsii during conidiation.

Results

Identification and subcellular localization of the SUMO gene in M. robertsii

A single SUMO gene MrSmt3 (MAA_00207) was acquired from the search of M. robertsii genome. Sequence analysis showed that the predicted protein sequence of MrSmt3 contains 98 amino acids with a predicted molecular weight of approximately 11.44 kDa, which include multiple conserved domains (a UBQ domain (PF11976). The multiple sequence alignment analysis revealed that MrSmt3 was homologous to previously reported fungal SUMO orthologs, including Smt3 in S. cerevisiae and MoSmt3 in M. oryzae. Additionally, the diglycine residues motif conserved among MrSmt3 and its homologs was also identified (Fig. 1A). Phylogenetic analysis of Smt3 orthologs in other fungi found that MrSmt3 shared a closer evolutionary relationship from insect pathogenic fungi including M. acridum and M. rileyi (Fig. 1B).

Fig. 1.

Sequence characteristics of MrSmt3 in M. robertsii. (A) Alignment of Smt3 from M. robertsii, Purpureocillium lilacinum, M. oryzae, A. nidulans and S. cerevisiae. Sequences of amino acid were acquired by BLAST (Accession numbers) and aligned with ClustalW. Similar and identical residues were denoted by gray regions and gray characters, respectively. (B) A phylogenetic analysis of Smt3 in M. robertsii with other fungal SUMO homologs from the database of NCBI (Accession numbers) was conducted by using the neighbor-joining method. The scale bar denotes branch length proportional to genetic distance.

To investigate the subcellular localization of MrSmt3, the plasmids containing eGFP-tagged MrSmt3 fusion protein under regulation of the strong promoter was transformed into WT, and the localization of MrSmt3 was analyzed by a fluorescence microscope. The results showed that the MrSmt3 was found to be present in cytoplasm and nucleus, including in hyphae and conidia (Fig. 2A). Specifically, the MrSmt3-eGFP fusion protein was mainly detected in the nucleus under the treatment of oxidative conditions (30% H₂O₂) (Fig. 2B).

Fig. 2.

Subcellular localization of MrSmt3 in M. robertsii. (A) The MrSmt3 was present in the nucleus and the cytoplasm under normal cultivation. Hyphae and conidia were stained with DAPI. Scale: 5 μm. (B) The MrSmt3 was mainly localized to the nucleus under oxidative stress (30% H₂O₂ treated conditions). Scale bar, 5 μm. DIC differential interference contrast.

MrSmt3 is involved in SUMOylation

To determine whether the SUMOylation-related gene, MrSmt3 is involved in the SUMOylation in M.robertsii, deletion of MrSmt3 was obtained by a selectable marker-bar (glufosinate ammonium) cassette (Fig. S1A). The occurrence of expected homologous recombination for MrSmt3 targeting disruption and complementation were validated by PCR and RT-PCR analyses (Fig. S1B,C,D and Table S1). Accordingly, the expression of MrSmt3 in the cDNA samples acquired from 3-day-old PDA cultures was absent in ΔMrSmt3 but present in the WT and complemented strains (C-ΔMrSmt3 strains) (Fig. S1B,C).

Here, thermal stress was used to investigate the differences of SUMOylation between the wild-type and ΔMrSmt3 mutant. Western blotting analysis showed that the level of protein SUMOylation in the wild-type was markedly increased under high temperature conditions (45 °C for 90 min) (Fig. 3A–C lane 1,2). However, the levels of SUMOylated proteins in the ΔMrSmt3 mutant remained undetectable changes after heat treatment (Fig. 3A–C lane 3,4). Our findings indicate that MrSmt3 is involved in SUMOylation in M.robertsii.

Fig. 3.

Effects of MrSmt3 disruption on SUMOylation levels of total proteins during heat stress. (A) The SUMOylation levels of total proteins were detected by using an anti-SUMO antibody. (B) Total proteins of different strains treated by high temperature conditions were separated by SDS-PAGE gel, and stained with Coomassie brilliant blue (as CK for total proteins). (C) Grayscale analysis on the SUMOylation levels of total protein were performed for quantification by using ImageJ software (https://imagej.net/ij/index.html). The original blots are presented in Supplementary Fig. S4.

Disruption of MrSmt3 results in reduced conidial yield and germination

To assess the role of MrSmt3 in M. robertsii, three different strains as illustrated above, including WT, ∆MrSmt3, and C-ΔMrSmt3 strains, were applied. For the conidiation-associated assay, conidial production and germination was investigated, and our results demonstrated that the ∆MrSmt3 strain exhibited markedly reduced conidial production, compared to WT strain. For instance, the conidial yield incubated on PDA for 7 days was significantly decreased from (2.57 ± 0.35) × 10⁷ conidia cm^− 2 in the WT strain to (0.96 ± 0.17) × 10⁷ conidia cm^− 2 in the ∆MrSmt3 strain (Fig. 4A). Specifically, the conidial production in the ∆MrSmt3 mutant strain was significantly reduced compared with WT, as shown in the microscopic image analysis (Fig. 4B). For the conidial germination, our results found that the half-time of germination (GT₅₀) was significantly advanced in the ∆MrSmt3 strain (Fig. 4C).

Fig. 4.

Effects of MrSmt3 deletion on conidial production and germination. (A) Conidial production of different strains incubated on PDA for 7 and 14 days. (B) Micrograph analysis for conidiation of different strains during early stage of conidial production (cultured on PDA for 3–5 days at 25 °C). Scale: 10 μm. (C) The germination half-time (GT₅₀) for the different strains (after cultivation for 24 h at 25 °C). The error bars represent the standard deviation (SD) of three biological replicates. * p < 0.05.

Additionally, to explore whether MrSmt3 is involved in the hyphal growth, we investigated the growth of different strains cultured on PDA, SDAY, and 1/4 SDAY, respectively. Our observations showed that there are no significant alterations for colony phenotype among the WT, ∆MrSmt3, and C-ΔMrSmt3 strains, while ΔMrSmt3 mutant incubated on SDAY media exhibited slightly reduced colony sizes at 25 °C in darkness for 10 days (Fig. S2). Our findings suggest that MrSmt3 is not essential for hyphal growth in M. robertsii.

Deletion of MrSmt3 affects stress tolerance

To investigate the effect of MrSmt3 deletion under environmental stresses, the conidial germination under high temperature stress was measured. Our results showed that conidial thermotolerance of ΔMrSmt3 was significantly reduced, for example, the conidial germination and mean 50% inhibition time (IT₅₀) of ΔMrSmt3 was lower than the control strains (Fig. 5A,B). Moreover, microscopic analysis for the 3-h heat treatment group showed that the conidial germination of ΔMrSmt3 was indeed lower than of control strains (Fig. 5C). These observations suggest that MrSmt3 is required for the conidial tolerance to high temperature in M. robertsii.

Fig. 5.

Effects of MrSmt3 disruption on conidial tolerance to high temperature. (A) The conidial heat tolerance (conidial germination percentages) of respective strains were investigated during high temperature conditions. Conidia were subjected to 45 °C for varying durations, followed by incubation for 20 h at 25 °C to determine conidial germination. (B) The average 50% inhibition time (IT₅₀) of respective strains during high temperature conditions. (C) Micrograph analysis for conidial germination of respective strains after cultured at 45 °C for 3 h, followed by a 20-hour incubation at 25 °C. Scale: 5 μm. The error bars represent the standard deviation (SD) of three biological replicates. * p < 0.05.

To analyze the function of MrSmt3 under different chemical stresses, hyphal growth of three different strains was investigated on PDA plates with various chemical agents, including SDS, CFW, Menadione, H₂O₂, and NaCl (Fig. 6A), and the growth inhibition rate was obtained by assessing fungal colony diameters (Fig. 6B). Deletion of MrSmt3 resulted in reduced tolerance to cell wall integrity stressors (SDS and CFW) and oxidative stresses (menadione and H₂O₂), but no apparent difference to hyperosmotic stresses (NaCl) (Fig. 6A,B), compared to the control strains. These observations suggest that MrSmt3 is also involved in the chemical stress tolerance.

Fig. 6.

Effects of MrSmt3 disruption on various chemical stresses tolerance. (A) Colony morphology of respective strains incubated on PDA with different chemical agents (including SDS, CFW, Menadione, H₂O₂, and NaCl). The images were acquired after cultivation at 25 °C for 10 days. Scale: 1 cm. (B) Statistical analysis for the growth inhibition rates under various chemical stresses. The error bars represent the standard deviation (SD) of three biological replicates. * p < 0.05.

MrSmt3 deletion did not affect fungal virulence

To investigate whether MrSmt3 participate in fungal pathogenicity of M. robertsii, bioassay was performed based on the topical immersion or injection with conidial suspensions of three different strains. The results demonstrated that there are no evident variations for LT₅₀ among WT, ∆MrSmt3, and C-ΔMrSmt3 strains, including the assay of topical immersion (Fig. S3A) and injection (Fig. S3B). These observations suggested that disruption of MrSmt3 has no impact on fungal virulence in M. robertsii.

Identification of DEGs modulated by the MrSmt3 during conidiation

To further explore the regulatory role of the Smt3 during conidiation, RNA-seq analysis was conducted by comparing differentially expressed genes (DEGs) of the WT and ΔMrSmt3, and our results showed that there were 1484 DEGs in ΔSmt3 versus WT, including 971 down-regulated DEGs and 513 up-regulated DEGs (Fig. 7A and Table S2). Among the identified DEGs, several downregulated DEGs involved in conidiation are illustrated in Fig. 7B, including classical conidiation-regulating transcription factor coding gene AbaA and Ste12. Furthermore, Gene Ontology (GO) functional analysis revealed that these DEGs were classified into 41 GO categories (Table S3), with the top 20 GO terms displayed in Fig. 7C. Specifically, the classification results demonstrated that the major downregulated DEGs were primarily involved in a variety of metabolic regulatory processes, such as metabolic processes (380 DEGs), primary metabolic process (308 DEGs), organic substance metabolic process (340 DEGs) (Fig. 7C and Table S3). Moreover, the KEGG (Kyoto encyclopedia of genes and genomes) pathway analysis found that the significant enrichment pathways of the major downregulated DEGs were valine, leucine and isoleucine degradation, tryptophan metabolism and purine metabolism, which was also associated with fungal metabolism (Table S4). These findings suggest that MrSmt3 was deeply involved in various metabolic processes in M. robertsii.

Fig. 7.

RNA-seq analysis for the DEGs modulated by MrSmt3 during conidiation. (A) Volcano plot for the distribution of the differential expression genes (DEGs) between the WT and ΔMrSmt3 strains during conidiation. (B) The downregulated expression genes associated with the regulation of conidiation process were acquired by RNA-seq analysis. (C) GO enrichment analysis of the DEGs.

Discussion

Previous researches have demonstrated that the SUMO orthologues (Smt3) and SUMOylation pathway play essential roles in various pathogenic fungi^{13–15,17,18}. Here, we investigated the biological functions of MrSmt3, a homolog of yeast Smt3, in the insect pathogenic fungus M. robertsii and found that MrSmt3 plays an important role in SUMOylation, fungal development and stress response. In particularly, our results demonstrated that the MrSmt3 deletion strains exhibited reduced protein SUMOylation levels under high temperature stress, which is consistent with recent studies suggesting that Smt3 (acts as a SUMO) is involved in the modulation of SUMOylation processes^14,28,29. Furthermore, our results demonstrated that the disruption of MrSmt3 markedly decreased the fungal tolerance to heat shock and chemical stresses. Interestingly, the deletion of MrSmt3 did not affect the fungal virulence. More importantly, MrSmt3 disruption largely reduced the conidial yield, and RNA-seq analysis was employed to investigate the possible DEGs regulated by MrSmt3 during conidiation.

It has been reported that conidia are key components in mycopesticide based on entomopathogenic fungi including M. robertsii³³, and thus it is crucial for exploring the molecular improvement of conidial production and conidiation-regulating processes^7,9. In this study, disruption of MrSmt3 led to decreased conidial yield and delayed conidial germination, further RNA-seq analysis identified 1484 DEGs in ΔMrSmt3 versus WT during conidiation. Among these DEGs, several genes associated with the conidiation processes were identified to be significantly downregulated. For instance, the expression level of some classical conidiation-regulating genes, including key regulatory transcription factors coding gene AbaA, Ste12 and protein FluG gene (Fig. 7B, Table S2 and S5)^34–36, were reduced in the MrSmt3 deletion strains, which may be partially accounted for reduced conidial production. A similar phenomenon was also reported in Smt3 deletion from M. oryzae¹⁴, A. flavus²⁸, and A. nidulans²⁶. Previously, it has also been reported that Smt3 act as a modulator of transcription factors, and thus regulating several conidiation-associated genes^27,37. More importantly, functional annotation of these DEGs in ΔMrSmt3 versus WT during conidiation determined a series of metabolic processes for the major downregulated DEGs, which indicated the underlying linkage of SUMOylation pathway with various metabolic processes involved in the conidiation. These finding is also consistent with previous researches that Smt3, as the key components of SUMOylation, take part in transcriptional regulation systems which involved in different biological roles^27,38,39. Taken together, our data suggest that MrSmt3 and SUMOylation is essential for conidiation in M. robertsii.

Stress response is vital for fungal survival and infection in entomopathogenic fungi, and Metarhizium may experience and respond to diverse stresses during storage and field application^40,41. In the present study, we found that the SUMOylation-related gene MrSmt3 play key roles in heat stress and chemical stresses resistance. For heat stress, our findings are consistent with recent report in yeast^22,42, C. albicans^25,43, and B. cinerea¹⁷, that is, Smt3 deletion strains exhibited reduced tolerance to high or low temperatures. Recent studies have shown that deletion of BbSmt3 enhances the tolerance of B. bassiana to high temperature stress³². However, deletion of MrSmt3 led to a significant decrease in the tolerance to heat stress in M. robertsii. Therefore, we speculate that Smt3 may play different roles in different species, which is also consistent with report that homologous proteins with similar domains may exert different biological roles in the respective fungi⁶. Moreover, MrSmt3 inactivation significantly reduced the SUMOylation level under high temperature stress. Although there were no significance differences for the classical genes of heat shock protein in this study, however, the expression levels of several genes related to ubiquitin were largely down-regulated in the MrSmt3 gene deletion strains based on our RNA-seq analysis (Table S6). Therefore, we speculate that MrSmt3 is involved in the heat tolerance by regulating the ubiquitin-associated genes/pathways. In fact, the ubiquitin and ubiquitin-like associated pathway was markedly induced by high temperature in M. robertsii from our previous RNA-seq analysis⁴⁴. However, the SUMOylated protein substrates involved in heat stress response remain to be identified in further analysis. Additionally, for chemical stress response, our results showed that disruption of Smt3 led to increased sensitivity to oxidative, and cell wall stresses, which is also similar with previous researches in yeast⁴⁵, C. albicans²⁵, M. oryzae²⁹, and B. cinerea¹⁷. Moreover, in B. bassiana, the relative growth inhibition rate of ∆BbSmt3 strains increased by 32.2% after 6 days of growth on SDS supplemented medium³². In F. oxysporum, the mycelial growth of ∆FonSmt3 strains was significantly inhibited compared to WT, when grown on PDA medium supplemented with CR and CFW³¹. In this study, the expression levels of some genes associated with cell wall integrity were also markedly decreased in ΔMrSmt3 mutant based on the RNA-seq analysis (Table S7), which indicate that the expression of cell wall integrity-related genes may be regulated by MrSmt3 in M. robertsii.

For the pathogenicity assays, our results showed that the ΔMrSmt3 strains did not display decreased fungal virulence when compared with the control strains. However, the SUMO gene was found to be essential for pathogenicity in most pathogenic fungi, including M. oryzae^14,29, (A) flavus²⁸, (B) cinerea¹⁷, and B. bassiana³². Specifically, similar functions of MrSmt3 were shared with Smt3 homologs in yeast²² and M. oryzae^14,29 during conidiation and stress response, whereas distinct functions of MrSmt3 and Smt3 orthologs exhibited in fungal virulence^14,29,32. These observations are consistent with the recent reports that homologous proteins with similar domains may play distinct biological roles in different fungi^6,46. Furthermore, it has widely been reported the ability of these fungal pathogens such as Beauveria and Metarhizium to exist as endophytes and safeguard their colonized host plants against pests^47,48. Therefore, we speculated that MrSmt3 might play an essential role in the interaction of Metarhizium-plant during root colonization. Regarding the different pathogenicity of Smt3 inactivation in Beauveria and Metarhizium, similar findings for the polyubiquitin gene UBI4 between Beauveria and Metarhizium were also reported previously^49,50. However, further investigation for the detailed possible mechanisms is required.

In conclusion, we identified the roles of SUMO gene MrSmt3 in M. robertsii, and we also found that MrSmt3 is involved in SUMOylation, and required for conidiation and stress response in M. robertsii. Specifically, RNA-seq analysis showed that MrSmt3 inactivation resulted in some classical conidiation-associated gene with downregulated expression in M. robertsii during conidiation. Our results provide a basis for exploring the SUMOylation in M. robertsii. However, the other components of the SUMOylation machinery and target substrate candidate remain to be further investigated in M. robertsii.

Materials and methods

Fungal strains

The wild-type (WT) strain we used was M. robertsii ARSEF 2575. The wild-type strains, deletion mutants, and complementary strains were incubated on potato dextrose agar (PDA) medium for 10 days, and then conidia were collected and mixed with a 0.05% Tween 80 solution for sufficient shaking to obtain a conidial suspension.

Sequence analysis

Based on the Smt3 protein in S. cerevisiae (KZV12750), sequence alignment was performed by using National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/) to acquire the corresponding Smt3 protein in M. robertsii (XP_007816396.1). The Smt3 protein sequences of other pathogenic fungi were acquired from NCBI, and the homology of Smt3 protein sequences was compared using multiple sequence alignment by CLUSTALW (https://www.genome.jp/tools-bin/clustalw). The phylogenetic analysis was conducted by using MEGA7 (https://www.megasoftware.net/)⁵¹.

Gene deletion and complementation

Using the DNA of WT as a template, the 5’-flanking sequences (1417 bp, using primers MrSmt3-Up-F/MrSmt3-Up-R, with the restriction enzyme cutting site being Pst I) and 3’-flanking sequences (1738 bp, using primer MrSmt3-Dn-F/MrSmt3-Dn-R, with the restriction enzyme cutting site being Xba I) (Table S1) were amplified by using 2 × Phanta Max Master Mix (Vazyme, Nanjing, China). The amplified PCR product was ligated to the vector plasmid pDHt-SK-bar (conferring resistance to glufosinate-ammonium) through recombinant ligase (2 × MultiF Seamless Assembly Mix, ABclonal, Wuhan, China), which produced a replacement vector P-bar-MrSmt3 (Fig. S1A) for fungal transformation to obtain the two MrSmt3 deletion mutant (ΔMrSmt3, Fig. S1B, C) according to our previous researches^52,53. To obtain the complementary strains (C-ΔMrSmt3), the MrSmt3 and its own promoter fragment was amplified by the primers C-MrSmt3-5 F/C-MrSmt3-3R (with the restriction enzyme cutting site being Pst I) (Table S1), and the obtained fragment was connected to the vector plasmid pDHt-ben (conferring resistance against Benomyl) using recombinant ligase. Subsequently, the recombinant plasmid was applied to fungal transformation to obtain the two complementary strains by using ΔMrSmt3 as background (Fig. S1B, C). PCR validation was performed using primers MrSmt3-ID-F (P1)/MrSmt3-ID-R (P2), and RT-PCR verification was used by primers MrSmt3-L-F (P3)/MrSmt3-L-R (P4) and GAPDH-F/GAPDH-R (Table S1).

Subcellular localization of Smt3 in M. robertsii

According to our previous report⁵², the coding sequence of MrSmt3 fragment was amplified by the MrSmt3-cDNA-F/MrSmt3-cDNA-R primer (Table S1), which was then fused to the C-terminus of green fluorescent protein to construct a fusion plasmid pDHt-gpdA-MrSmt3-GFP-Ttrpc-bar containing a glyceraldehyde-3-phosphate dehydrogenase promoter. The fusion plasmid was integrated into the wild-type using Agrobacterium tumefaciens transformation method⁵³. DAPI was used to stain the nuclei of conidia and hyphae, while another group was treated with 30% hydrogen peroxide solution for oxidative stress. Subsequently, the subcellular localization of MrSmt3 was observed under a laser confocal microscope⁵².

Western blot analysis

Aliquots (100 µL) of conidial suspensions (10-day-old conidia with 1 × 10⁷ conidia/mL) were inoculated onto PDA media and incubated for 2.5 days, and then the samples were collected to extract total protein according to our previous report⁶. 20 µg of total protein were separated on 10% SDS-PAGE, and then transferred to nitrocellulose filter membrane (Pall, 66485). The SUMOylation level of total protein was identified by anti-SUMO antibody (1:100 dilution, PTM-1109) and anti-mouse secondary antibody (1:5000 dilution, Thermo, 31430).

Phenotypic assays

For phenotypic assays, the experimentations were performed for different strains (WT, ΔMrSmt3, and C-ΔMrSmt3 strains) based on our researches described previously^6,52,53.

For conidial germination, 10 µL of conidial suspensions (1 × 10⁶ conidia/mL) were inoculated onto PDA media, and placed in a 25℃ incubator for cultivation. Starting from 2 h after inoculation, the conidial germination was observed under a microscope, and the number of conidial germinated and those that did not germinate were recorded. The observation was conducted every 2 h until all conidia germinated. SPSS software (Version 16.0, SPSS, Inc., Chicago, IL, USA; https://www.ibm.com/spss) was used to calculate the average 50% conidial germination time (GT₅₀). For conidial production, 40 µL of conidial suspensions (1 × 10⁶ conidia/mL) from different strains were uniformly coated on PDA plates (35 mm diameter) and incubated at 25℃. Subsequently, all samples after cultivation of 7 and 14 days were scraped into 40 mL of 0.05% Tween 80 solution and shaken thoroughly with a shaker for 15 min. The conidial concentration was calculated by hemocytometers. The images of conidia production were taken under a microscope (Olympus BX51) after inoculation of 3, 4, and 5 days.

For hyphal growth assay, 1 µL of spore suspension (1 × 10⁷ conidia/mL) was inoculated onto PDA, SDAY, and 1/4 SDAY media, respectively, and placed in a 25 ℃ incubator for 14 days. The colony morphology was photographed and measured for diameter.

For conidial heat tolerance assay, 1 mL of spore suspensions (1 × 10⁶ conidia/mL) were added to 1.5 mL of micro-centrifuge tubes and placed in a 45℃ constant temperature water bath for different heat treatment time. Then, 10 µL of spore suspension were taken and inoculated onto PDA plates. After cultivation of 20 h in a 25℃ incubator, and then the number of germinated and non-germinated conidia were counted. The time for inhibited spore germination by 50% after heat treatment (IT₅₀) was analyzed by SPSS software.

For chemical stress tolerance assay, 1 µL of spore suspension (1 × 10⁷ conidia/mL) was inoculated onto PDA media (control) or PDA media containing corresponding chemical agents, such as Sodium dodecyl sulfate (SDS), Calcofluor white (CFW), Menadione, H₂O₂, and NaCl. The colony diameter was measured after cultivation of 10 days in a 25℃ incubator, and the growth inhibition rate was determined as described in our previous research, that is, growth inhibition rate = ((D_control – D_treated)/D_control) × 100% (D represents the mean colony diameter of different strains).

Fungal virulence was analyzed by using Galleria mellonella larvae. For immersing infection, the larvae were immersed in conidial suspensions (1 × 10⁷ conidia/mL) for 60 s. For direct injection, 10 µL of spore suspension (1 × 10⁵ conidia/mL) were injected into the hemocoel of the larvae. The survival rate of larvae was recorded every 12 h over a 15-day period, and the mean lethal time for 50% mortality (LT₅₀) was analyzed by SPSS software.

RNA-seq analysis

To investigate the DEGs affected by MrSmt3 deletion during conidiation, RNA-seq analysis was conducted based on our researches described previously⁴⁴. Aliquots (100 µL) of conidial suspensions (10-day-old conidia with 1 × 10⁷ conidia/mL) were inoculated onto PDA media and cultured for 2.5 days, and then the samples of WT and ΔMrSmt3 strains with three biological replicates were collected. Total RNAs were extracted for sequencing at BGI (Shenzhen, China). Subsequently, genes with a Q-value ≤ 0.05 and an absolute value of the log₂ (ΔMrSmt3/WT ratio) ≥ 1 were considered as DEGs. To further explore the biological roles and metabolic pathways, the DEGs were analyzed by using GO functional annotation analysis and KEGG pathway enrichment analysis (KEGG database, https://www.genome.jp/kegg/)⁵⁴.

Statistical analysis

Data are presented as the mean ± standard deviations (SD) of three biological replicates. Statistical analysis was conducted by using one-way analysis of variance (ANOVA). Tukey’s multiple comparison test was used, and p-value ≤ 0.05 was considered significantly different.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

B.H. and Z.W. conceived and designed the study. J.S., H.C., and Z.W. wrote and revised the manuscript. J.S., H.C., D.X., J.L., and Z.W. performed the experiments and data analysis. The published version of the manuscript was reviewed and approved by all authors.

Data availability

The raw sequence data that support the findings of this study have been deposited in the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov) with the Sequence Reading Archive (SRA) Study accession number SRP490621.

Declarations

Competing interests

The authors declare no competing interests.