Through NCBI annotation, the predicted protein encoded by the gene SsCI33130 is 100% similar to S. scitamineum OTU1-deubiquitylation enzyme and consists of 109 amino acid residues (NCBI: protein accession no. CDU24320.1). The isoelectric point (PI) of the protein was 6.06, the molecular weight (MW) was 12.16 kDa, the full length of the gene was 330 bp (Figure 1A). Phylogenetic analysis of the protein encoded by the SsCI33130 gene showed that the protein encoded by the gene was highly conserved in smut fungi (Figure 1B). The subcellular localization prediction indicated that the protein encoded by the SsCI33130 gene was localized in the cytoplasm of S. scitamineum (Figure 1C).

To further investigate the functions of SsCI33130, gene deletion and complementary strains were constructed as described in the section “Materials and Methods.” Each flanking fragment was amplified by PCR with the primer pairs SsCI33130-LB-F/R and SsCI33130-RB-F/R. The band sizes were 917 and 1032 bp, respectively (Figures 2A,B). Then two fused fragments were amplified by PCR with the primer pairs SsCI33130-LB-F/Hpt-LB-226 and SsCI33130-RB-F/Hpt-RB-R. The band sizes were about 3 and 2.5 Kb, respectively (Figures 2C,D). They were used for S. scitamineum wild type protoplasts transformation. As expected, two positive deletion mutants ΔSs33130⁺ and ΔSs33130^– (ΔSs33130⁺ is from the wild type Ss16⁺, ΔSs33130^– is from the wild type Ss16^–) were obtained. The SsCI33130-positive deletion mutants were confirmed by PCR using the primer pairs SsCI33130-OF/SsCI33130-OR and SsCI33130-IF/SsCI33130-IR. Use of the primer pair SsCI33130-IF/SsCI33130-IR resulted in a 295-bp band from the SsCI33130 gene in the wild type (Ss16⁺ and Ss16^–), but not in the deletion mutants, while the primer pair SsCI33130-OF/SsCI33130-OR produced a 5089-bp band in the deletion mutants and a 2408-bp band in the wild types, indicating that the gene SsCI33130 was deleted in the wild type strains (Ss16⁺ and Ss16^–) (Figure 2E). The SsCI33130-positive complementary mutants were confirmed with PCR using the primer pairs Zeocin-JC-F/Zeocin-JC-R (583 bp) and SsCI33130-IF/SsCI33130-IR (295 bp) (Figure 2F). Fortunately, two positive complementary mutants (COM33130⁺ and COM33130^–) were obtained.

The response of the SsCI33130 gene knockout mutants, complementary mutants, and wild type haploids to cell wall stress, osmotic stress, and reactive oxygen stress on solid plate medium was observed. The results showed that the phenotypes of the SsCI33130 gene deletion mutants, complementary mutants, and their wild types were not significantly different on YePSA medium and MM medium (Figure 3). It is speculated that the SsCI33130 gene is not involved in physiological functions such as oxidation, osmosis, and cell wall integrity.

There was almost no difference in the growth phenotype of the wild type, knockout mutant, and complementary mutant of S. scitamineum in YePSA solid medium (Figure 4A). In YePS liquid culture, the growth curves of the SsCI33130 gene knockout mutant, complementary mutant, and wild type were basically the same (Figures 4B,C). The results showed that the SsCI33130 gene did not affect the growth of S. scitamineum.

Under the inverted fluorescence microscope (100 times oil microscope), the SsCI33130 gene knockout mutant, complementary mutant, and wild type haploid were in the shape of one long rod or two long rods connected at the division stage (Figure 5), which indicated that the deletion of the gene did not affect the spore morphology of S. scitamineum.

The sexual mating between the wild types (Ss16⁺ + Ss16^–) produced a large number of white villous hyphae, while the white villous hyphae produced between the knockout mutants (ΔSs33130⁺ + ΔSs33130^–) and between the knockout mutant and wild type (Ss16⁺ + ΔSs33130^–, Ss16^– + ΔSs33130⁺) were less than those produced between the wild types (Ss16⁺ + Ss16^–), which indicated that the knockout mutants had weaker sexual mating ability. The sexual mating ability between the complementary mutants (COM33130⁺ + COM33130^–) and between the complementary mutant and wild type (Ss16⁺ + COM33130^–, Ss16^– + COM33130⁺) returned to the level of the wild type (Ss16⁺ + Ss16^–) (Figure 6A). However, knockout of the SsCI33130 gene did not affect the hyphal structure (Supplementary Figure 1). On the YePSA medium supplemented with 0.02 mM tryptophol or 5 mM cAMP, the sexual mating between the knockout mutants (ΔSs33130⁺ + ΔSs33130^–) and between the wild types and the knockout mutants (Ss16⁺ + ΔSs33130^–, Ss16^– + ΔSs33130⁺) was restored. It was thus speculated that this gene is involved in the sexual mating of S. scitamineum (Figure 6A). During 0–48 h of sexual mating, the expression level of the gene ARO8 related to tryptophol biosynthesis in the knockout mutant combination (ΔSs33130⁺ + ΔSs33130^–) was significantly lower than that in the wild type combination (Ss16⁺ + Ss16^–) or complementary mutant combination (COM33130⁺ + COM33130^–) (Figure 6B), while the expression level of the gene ARO8 in the complementary mutant combination and in the wild type combination was almost the same (Figure 6B). Similarly, the expression level of the gene UAC1 related to cAMP biosynthesis in the SsCI33130 knockout mutant combination (ΔSs33130⁺ + ΔSs33130^–) was significantly lower than that in the wild type combination (Ss16⁺ + Ss16^–) and complementary mutant combination (COM33130⁺ + COM33130^–), while the expression level of the gene UAC1 was almost equal in the complementary mutant combination and wild type combination (Figure 6C). We hypothesized that the SsCI33130 gene regulates the expression of genes related to the synthesis of the small molecule signaling substances (cAMP or tryptophol) required for the sexual mating of S. scitamineum, thereby affecting the synthesis of signaling substances.

Compared with the wild types (Ss16⁺ or Ss16^–), the OTU1-deubiquitinase activity of the SsCI33130 gene knockout mutants (ΔSs33130⁺ or ΔSs33130^–) decreased significantly, and the OTU1-deubiquitinase activity of the SsCI33130 gene complementary mutants (COM33130⁺ or COM33130^–) was restored to the level of the wild type. In addition, the OTU1-deubiquitinase activities of the knockout mutants, complementary mutants, and wild types peaked at 36 h (Figure 7), indicating that the SsCI33130 gene affected the OTU1-deubiquitinase activity of S. scitamineum.

During the haploid spore culture process from 0 to 72 h, the expression level of the SsCI33130 gene in the wild types or complementary mutants showed a trend of increasing first and then slightly decreasing, and the maximum expression level was observed at 60 h, but almost no expression was detected in the knockout mutants (ΔSs33130⁺ or ΔSs33130^–) (Figure 8A). Similarly, in the process of sexual mating, the expression level of the SsCI33130 gene showed a trend of increasing at first and then slightly decreasing, and the expression was the highest at 60 h, but almost no expression was detected between the knockout mutants (ΔSs33130⁺ + ΔSs33130^–) (Figure 8B). In the process of sugarcane bud infection, the expression levels of this gene in the combinations containing wild types or complementary mutants showed gradually increasing trend, with the highest expression level detected at 72 h, but almost no expression was detected in the knockout mutant combination (ΔSs33130⁺ + ΔSs33130^–) (Figure 8C). In the process of haploid spore culture, sexual mating, and sugarcane bud infection, the expression level of the SsCI33130 gene in the complementary mutants returned to the wild type expression level and was even overexpressed (Figure 8).

The typical symptom of sugarcane smut is the formation of black whip from the growing point of the sugarcane plant. In this study, the symptoms of sugarcane smut were observed (Figure 9A), and the incidence rate of sugarcane smut was investigated (Figure 9B). The incidence rate of sugarcane smut between the knockout mutants (ΔSs33130⁺ + ΔSs33130^–) and between the wild types and knockout mutants (Ss16⁺ + ΔSs33130^–, Ss16^– + ΔSs33130⁺) was 30.77, 47.3, and 41.8%, respectively, which was significantly lower than the incidence rate (71.13%) between wild types (Ss16⁺ + Ss16^–). The incidence rate of sugarcane smut between the complementary mutants (COM33130⁺ + COM33130^–) and between the complementary mutant and wild type (Ss16⁺ + COM33130^–, Ss16^– + COM33130⁺) was 68.33, 72.33, and 71.33%, respectively, which was not significantly different from the incidence rate (71.13%) between the wild types (Ss16⁺ + Ss16^–). This suggested that the pathogenicity of the complementary mutants basically recovered to the level of the wild type.

Based on transcriptome sequencing data, a gene SsCI33130 encoding OTU1-deubiquitinase was identified as a significantly (P ≤ 0.05) differentially expressed gene in haploids of the same mating type in isolates Ss16 (strong pathogenic strain with 75% incidence rate of sugarcane smut in the susceptible variety “ROC22”) and Ss47 (weak pathogenic strain with 25% incidence rate of sugarcane smut in the susceptible variety “ROC22”) of S. scitamineum with different pathogenicities (Wu et al., 2020). Blast comparison based on the amino acid sequences (DNA sequences into amino acid sequences)¹ was performed on the NCBI database² to obtain the conserved domain of the protein encoded by the SsCI33130 gene. The SsCI33130 encoding the protein sequence was analyzed using the Compute pI/M_w tool³ to determine the theoretical pI and MW. Phylogenetic analysis of the protein encoded by the SsCI33130 gene was conducted in MEGA 7, and the phylogenetic tree was constructed using the neighbor-joining method (Saitou and Nei, 1987; Kumar et al., 2016). The ProComp 9.0 online tool⁴ was used to predict the subcellular localization of the SsCI33130-encoding protein.

The wild type haploid isolates Ss16⁺ and Ss16^– of S. scitamineum were isolated and identified in our laboratory and stored at −80°C (Deng Q. Q. et al., 2018). The culture media used in this study included YePSA medium (yeast extract 1%, peptone 2%, sucrose 2%, and agar 2%), YePS liquid medium (yeast extraction 1%, peptone 2%, sucrose 2%, pH 7.0), YePS soft medium (yeast extract 1%, peptone 2%, sugar 2%, agar 0.65%), YePSS medium (yeast extract 1%, peptone 2%, sugar 2%, D-sorbitol 18.17%, agar 2%), and MM medium (K₂HPO₄ 0.205%, KH₂PO₄ 0.145%, NH₄NO₃ 0.05%, (NH4)₂SO₄ 0.03%, FeSO₄ 0.001%, CaCl₂ 0.001%, Glucose 0.2%, 500 μL Z-Buffer, pH 6.7–7.0). For the mating/filamentation assay, equal volumes of wild type, deletion mutant, or complementary mutant haploid sporidia of opposite mating-types were mixed and plated on YePSA solid medium in the absence or presence of 5 mM cAMP and 0.02 mM tryptophol and then kept in the dark in a 28°C incubator for 42 h before photographing (Sun et al., 2019). For stress tolerance assessment, the sporidial culture at OD₆₀₀ = 1.0 and its 10-fold serial dilutions were inoculated on YePSA medium or MM medium in the absence or presence of stress inducers, including 50 μg/mL Congo red (CR), 50 μg/mL SDS, 2 mM H₂O₂, and 500 mM NaCl, and then incubated in the dark at 28°C for 48 h before assessment and photographing. For the growth assay, the sporidia of wild type S. scitamineum (Ss16⁺ and Ss16^–), deletion mutant (ΔSs72380⁺ and ΔSs72380^–), and complementary mutant (COM72380⁺ and COM72380^–) were cultured in 50 mL of YePS liquid medium at 28°C with shaking at 200 rpm for 24 h. Aliquots of cultured sporidia were then diluted with fresh YePS liquid medium, the cell density was adjusted to 10⁵ cells per mL, and samples were then cultured for another 48 h under the same conditions. The OD₆₀₀ was measured with a spectrophotometer (NanoDrop 2000C) every 6 h to monitor the yeast-like (budding) growth of the wild type, deletion mutant, or complementary mutant strains.

Fungal genomic DNA was extracted using a modified CTAB method (Shen et al., 2006). The PCR amplification was performed using Phanta High-Fidelity DNA Polymerase (Vazyme, P505). Purification of DNA fragments was conducted using a FastPure Gel DNA Extraction Mini Kit (Vazyme, DC301). Total RNA was extracted with TRIzol (Vazyme, R401), and HISCRIPT III RT SuperMix (Vazyme, R323) was used for cDNA synthesis. A NanoDrop ND-1000 (Thermo Fischer Scientific, Wilmington, DE, United States) was used for measuring the concentration and purity.

The construction of two fragments for the replacement of the SsCI33130 gene by the Hpt (encodes a phosphotransferase conferring hygromycin resistance) gene was based on previous methods (Yang and Chung, 2012; Li et al., 2014; Chang et al., 2018). The flanking DNA of the SsCI33130 gene was PCR-amplified using wild type S. scitamineum genomic DNA (Ss16⁺ and Ss16^–). The Hpt gene in the plasmid pDAN (a circled vector) was the template. Gene knockout mutants were obtained by PEG-mediated protoplast transformation (Deng Y. Z. et al., 2018; Cai et al., 2020). All primers involved in the construction and validation of the knockout mutants are indicated in Table 1.

The complementation of the SsCI33130 gene followed a previous strategy (Chang et al., 2018). The complemented gene not only carries the hygromycin homologous fragment to replace the hygromycin fragment in the knocked-out mutants but also carries the zeocin resistance marker gene to screen the complements. Complementary mutants were obtained by PEG-mediated protoplast methods (Chang et al., 2018; Cai et al., 2020). All primers involved in the construction and validation of the complementary mutants are listed in Table 1.

We assessed the effect of the SsCI33130 gene on OTU1-deubiquitinase activity every 12 h over a period of 48 h under haploid sporidia culture conditions (YePS liquid medium, 28°C, 200 rpm) based on the determination of the OTU1-deubiquitinase activity of the knockout mutants, complementary mutants, and wild types of the SsCI33130 gene. The OTU1-deubiquitinase activity determination was conducted in accordance with the instructions of the fungal OTU1-deubiquitinase enzyme linked immunosorbent assay (ELISA) kit (Shanghai Enzyme-linked Biotechnology). This kit uses double antibody sandwich method to determine the level of fungal OTU1-deubiquitinase in samples.

To evaluate the effect of SsCI33130 on the expression of genes related to the synthesis of sexual mating signal substances in S. scitamineum, we used quantitative real-time (qRT)-PCR to detect the expression levels of the genes Aro8 and Uac1, which are related to the synthesis of signaling substances (tryptophol and cAMP) (Chang et al., 2018; Wang et al., 2019). Three sexual mating combinations, i.e., a wild type combination (Ss16⁺ + Ss16^–), knockout mutant combination (ΔSs33130⁺ + ΔSs33130^–), and complementary mutant combination (COM33130⁺ + COM33130^–), were designed. During the 0–48 h sexual mating process, samples were taken every 12 h, the total RNA of the samples was extracted, and gene expression was detected by qRT-PCR. For the qRT-PCR, we used a ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711), and the reaction was run on a real-time PCR system (CFX96, Bio-Rad). Relative expression values were calculated with the 2^–ΔΔCt method using ACTIN as an internal control (Livak and Schmittgen, 2001). Three biological repeats each containing three technical replicates for each sample were performed. The nucleotide sequences of primers Aro8-qF/qR, Uac1-qF/qR, and ACTIN-qF/qR are listed in Table 1.

We assessed the transcriptional profile of the SsCI33130 gene every 12 h over a period of 72 h under haploid and mating conditions as well as during the infection process (after inoculation of the sugarcane buds of the smut susceptible variety “ROC22.” For detailed inoculation methods, refer to section “Assay of the Pathogenicity of the Knockout and Complement Mutants of the SsCI33130 Gene” (section “Materials and Methods” below) using qRT-PCR. The method of qRT-PCR and the calculation of gene relative expression values were the same as those stated above. The nucleotide sequences of the primers SsCI33130-qF/qR and ACTIN-qF/qR are listed in Table 1.

Sporidial colonies of the wild types (Ss16⁺ and Ss16^–), knockout mutants (ΔSs33130⁺ and ΔSs33130^–), or complementary mutants (COM33130⁺ and COM33130^–) were inoculated into 50 mL of YePS liquid medium and cultured at 28°C with shaking at 200 rpm for 2 days. Sporidia were collected by centrifugation and washed twice with ddH₂O, after which they were re-suspended in YePS liquid medium at a final concentration of 2 × 10⁹ spores/mL. Sporidia of opposite mating types were then mixed in equal volumes, after which 200 μL of this mixture was syringe-injected into growth point of seedlings with 4–5 leaves from the highly susceptible sugarcane variety “ROC22” (20 plants were inoculated in each treatment) (Shen et al., 2014; Chang et al., 2018). A wild type mixture (Ss16⁺ and Ss16^–) served as a positive control, and sterile water was used as a negative control. Inoculated plants were kept in a greenhouse (30°C, relative humidity 70%) for 4 months. Investigation of the occurrence of sugarcane smut began after 1 month. At the end of each investigation, the diseased plants were labeled to avoid repeated investigations, black whip symptoms were covered with plastic bags to prevent the spread of teliospores, and the number of diseased plants (including anatomically confirmed plants with early symptoms of smut) and the morbidity were calculated.

Images were taken using an Axio Observer Z1 microscope (Zeiss, Jena, Germany) equipped with a sCMOS camera (PCO Edge, Kelheim, Germany).

Data were expressed as the means ± standard error (SE). Differences among different treatments were analyzed using GraphPad Prism 8 software (GraphPad, United States).