The wild-type strain Neostagonosporella sichuanensis SICAUCC 16–0001 was acquired from the Culture Collection at Sichuan Agricultural University (SICAUCC) and activated on PDA medium at 25°C for 7 days and then subcultured on fresh PDA medium at 25°C for 30 days. Nicotiana benthamiana was cultivated at 25°C under a 14-h light and 10-h dark cycle in an artificial climate incubator. Healthy one-year fishscale bamboo seedlings were cultivated at 25°C under a 12-h light and 12-h dark cycle in an artificial climate incubator after transplanted from Zhougongshan Town (103°2′59.87″E, 29°50′8.56″N) in Ya’an City, Sichuan Province, China, to the Chengdu Academy of Agriculture and Forestry Sciences (103°85′73.17″E, 30°70′32.61″N) in Chengdu City, Sichuan Province, China.

Xylanase family proteins were identified by querying the HMM profile of the Glyco_hydro_11 (GH11) domain (Pfam ID: PF00457) against the Neostagonosporella sichuanensis SICAUCC 16–0001 genome and nine other phaeosphaeriacous species (Ampelomyces quisqualis, Leptosphaeria microscopica, Ophiobolus disseminans, Paraphoma chrysanthemicola, Parastagonospora nodorum, Phaeosphaeria poagena, Stagonospora sp., Setomelanomma holmii, and Setophoma terrestris) using HMMER v3.4 (Potter et al., 2018) with an E-value threshold of ≤1 × 10⁻⁵. The returned hits with scores > 30 were selected. The genome sequences used for the xylanase proteins screening of four strains, Leptosphaeria microscopica, Paraphoma chrysanthemicola, Phaeosphaeria poagena, and Setophoma terrestris were downloaded from the JGI Genome Portal (Nordberg et al., 2014), and the genome sequences of the remaining five species were downloaded from the NCBI Genome (Sayers et al., 2022). Subsequently, these candidate proteins were searched for the Glutamic acid enzymatic activity sites and conserved glycoside hydrolase families 11 motifs using the ProSITE webserver (Sigrist et al., 2012). These sequences without enzyme active sites were discarded. The remaining sequences were submitted to SignalP v5.0 (Almagro Armenteros et al., 2019), TMHMM v2.0 (Krogh et al., 2001), BIG-PI Fungal Predictor (Eisenhaber et al., 2004), and WoLF PSORT (Horton et al., 2007) for secretory protein prediction. Protein sequences possessing a signal peptide, devoid of the transmembrane region and anchor location, and extracellular subcellular localization were identified as xylanase proteins and analyzed further. The phylogenetic tree was constructed with these selected xylanase proteins using MEGA v11 (Tamura et al., 2021) with the neighbor-joining method and visualized through Evolview v3.0 webserver (Subramanian et al., 2019). ClustalX v2.0 (Larkin et al., 2007) and ESpriPT3.0 webserver (Robert and Gouet, 2014) performed multiple sequence alignment. SOMPA (Geourjon and Deléage, 1995) and SWISS-MODEL web tools (Waterhouse et al., 2024) were used to predict the secondary and tertiary structures with the AlphaFold v2 method, respectively. The quality of the predicted models was assessed by SAVES v6.1 (Bowie et al., 1991; Lüthy et al., 1992; Colovos and Yeates, 1993; Laskowski et al., 1996) and Qmean (Benkert et al., 2011).

Fresh Neostagonosporella sichuanensis mycelia were collected for total RNA extraction utilizing the Quick RNA Isolation Kit (Huayueyang, Beijing, China). The concentration and purity of RNA were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The isolated RNA was preserved at-80°C for subsequent study. High-quality total RNA was reverse transcribed into cDNA with the PrimeScript™ RT Reagent Kit (Perfect Real Time; Takara, Beijing, China). Only two candidate xylanase genes (Nsxyn1 and Nsxyn2) were successfully cloned from the cDNA of Neostagonosporella sichuanensis SICAUCC 16–0001. Primers (Supplementary Table S1) were designed by Primer Premier 5.0 (Premier Biosoft International, Palo Alto, CA, USA). The target fragments were amplified using 2 × TransTaq^® High Fidelity (HiFi) PCR SuperMix II (−dye) (TRANS, Beijing, China) and purified using a Universal DNA Purification Kit (TIANGEN, Beijing, China). Then, they were connected to pMD™-19 T vectors (Takara, Beijing, China), respectively, by the manufacturer’s guidelines. The ligation mixture was transformed into Escherichia coli DH5α cells, which were then spread onto antibiotic-containing Luria-Bertani (LB) agar. Positive clones were screened by colony PCR and verified by sequencing.

Nsxyn1 and Nsxyn2 fragments (Amplified primers were in Supplementary Table S1), devoid of the predicted signal peptides and stop codons, were introduced into the pET-32a plasmid at the EcoR I and Xho I sites, respectively, and subsequently transformed into Escherichia coli BL21(DE3) for expression. To get adequate soluble proteins for subsequent analysis, expression conditions were optimized by inducing with IPTG at various concentrations and temperatures, followed by an analysis of expression levels by SDS-PAGE (Zhao et al., 2022). Large-scale protein production was carried out under optimal conditions, and the His6-tagged protein was purified utilizing a His-tag protein purification kit (Reductant&Chelator-resistant; Beyotime, Shanghai, China). Two Glu residues of the Nsxyn1rec/Nsxyn2rec mutant were replaced with Lys residues utilizing the Hieff Mut™ Site-Directed Mutagenesis Kit (Yeasen, Shanghai, China) to eliminate xylanase activity of Nsxyn1 and Nsxyn2. Following verification through sequence alignment, the Nsxyn1rec and Nsxyn2rec mutants were expressed under optimal conditions and purified as described above. Protein concentrations were then determined using a BCA protein assay kit (Beyotime, Shanghai, China).

For qualitative analysis, recombinant proteins were introduced to wells containing a medium with hardwood xylan (Beyotime, Shanghai, China) as the exclusive carbon source (Meddeb-Mouelhi et al., 2014). After 2 h of incubation, the medium was treated with 1% Congo red solution for 1 h and then eluted with 1 mol/L NaCl solution for 3 h. Hydrolytic zones were then examined using sterile eluents as negative controls. Xylanase activity was quantified by a modified DNS (dinitrosalicylic acid) assay (Fu et al., 2019) with some modifications. A mixture of 90 μL of 1% hardwood xylan solution (pH 6.5) and 10 μL of recombinant xylanase solution was incubated in PCR tubes at 45°C for 30 min. Subsequent to the reaction, 100 μL of DNS solution was immediately added, and the mixture was heated in a boiling water bath for 10 min before cooling to ambient temperature. The negative controls comprised 90 μL of 1% xylan solution (pH 6.5) and 10 μL of heat-inactivated recombinant xylanase. Absorbance was measured at 540 nm. All experiments were performed in triplicate.

To assess the potential necrotic activity of the purified proteins, leaves of Nicotiana benthamiana were infiltrated with 10 μmol/L of each purified xylanase protein using a needleless syringe. The infiltration sites were marked, and the plants were incubated under controlled conditions at 25°C under a 14-h light and 10-h dark cycle. Leaf tissue was observed for the development of necrosis throughout 5 days post infiltration, with leaves infiltrated with buffer only used as negative controls.

The transcript levels of Nsxyn1 and Nsxyn2 at different infection times in fishscale bamboo were quantified by qRT-PCR on a CFX96™ real-time system (Bio-Rad, Hercules, CA, USA). Specific primers for amplification were designed according to the coding regions of Nsxyn1 and Nsxyn2, with the Neostagonosporella sichuanensis elongation factor 1 alpha gene as an internal control (the coding sequence is detailed in Supplementary Table S2). The qRT-PCR reaction setup and conditions followed those described by Liu et al. (2019). A no-template control (ddH₂O) was included. The infection timepoint exhibiting the lowest expression served as the baseline (assigned a value of 1.0). Relative expression levels were calculated utilizing the 2^-∆∆Ct method (Ritter and Schulz, 2004; Zhu et al., 2015). Each qRT-PCR experiment was performed in triplicate. Data were analyzed and plotted using GraphPad version 8.4.2, with statistical significance assessed by unpaired Student’s t-test.

To detect whether the signaling peptides of Nsxyn1 and Nsxyn2 proteins possess secretory functions, the relevant signaling peptide sequences were inserted into the pSUC2 vector and transformed into Saccharomyces cerevisiae YTK12 competent cells, which are lacking invertase secretion. Transformants were selected on CMD-W medium and cultured at 30°C for 48 h. Positive colonies were transferred to the YPRAA medium to evaluate the secretory activity and incubated at 30°C for 48 h. The CMD-W and YPRAA mediums were prepared according to Huang’s method (Huang C. P. et al., 2023). The secretory function was further confirmed by staining with 2,3,5-triphenyl tetrazolium chloride (TTC), as described by Yin et al. (2018). The appearance of a red precipitate indicated successful secretion of the expressed proteins.

To investigate the subcellular localization of Nsxyn1 and Nsxyn2 proteins in planta and assess their necrosis-inducing activity, the coding sequences of Nsxyn1 and Nsxyn2 (without stop codons) were ligated to the 5′ end of the enhanced green fluorescent protein (EGFP) gene, driven by the MAS promoter (Amplified primers were in Supplementary Table S1). These constructs were then inserted into the pCAMBIAsuper1300-EGFP vector at the KpnI and XbaI restriction sites to generate recombinant expression vectors, pCAMBIAsuper1300:Nsxyn1-EGFP and pCAMBIAsuper1300:Nsxyn2-EGFP.

Onion epidermal cells and Arabidopsis thaliana protoplasts were used for subcellular localization analysis. Disinfected onion inner epidermal slices (~1 cm²) were incubated in Agrobacterium suspensions (GV3101) containing either Nsxyn1-EGFP, Nsxyn2-EGFP, or the empty pCAMBIAsuper1300-EGFP vector as a control, for 30 min. The epidermal slices were then placed on MS medium and incubated at 25°C under a 14-h light and 10-h dark cycle. Fluorescence was observed 24 h later using an Ultra-High Resolution Confocal Microscope (Leica, Wetzlar, Germany), with excitation at 488 nm. Moreover, protoplasts were prepared from the leaves of 3–4 week-old A. thaliana seedlings for protoplast localization. The Nsxyn1 and Nsxyn2 plasmids were co-transformed with an SV40 NLS plasmid into the protoplasts via PEG4000-mediated transformation. After overnight incubation at 23°C under low light conditions, the protoplasts were centrifuged at 400 rpm for 5 min, and the supernatant was discarded. Fluorescent signals were visualized using a confocal laser microscope (FV1000). Fluorescence detection parameters were GFP excitation at 488 nm, mCherry excitation at 561 nm, and chloroplast autofluorescence at 640 nm.

For necrosis induction assays, Agrobacterium tumefaciens GV3101 (pSoup-19) harboring the Nsxyn1 or Nsxyn2 constructs was infiltrated into the leaves of Nicotiana benthamiana. Empty vector pCAMBIAsuper1300-EGFP-MGS served as a negative control. After 3 days of incubation at 25°C with a 14-h light and 10-h dark cycle, the infiltrated leaves were collected, and the development of necrosis was assessed.

Nsxyn1 and Nsxyn2 mutants were created via targeted gene disruption using homologous recombination. Gene-specific left, and right flanking regions (1,000–1,500 bp) along with the hygromycin resistance flanking region were amplified and inserted into the pCE-Zero vector (detailed primers are in Supplementary Table S1). Using the method described by Liang et al. (2024), the knockout construct was introduced into Neostagonosporella sichuanensis SICAUCC 16–0001 via protoplast transformation. Successful mutant generation was verified by PCR analysis.

For the complementation of mutants, the full-length Nsxyn1 and Nsxyn2 genes, including their native promoter regions, were inserted into the pEASY-NeoR vector, which contains a neomycin resistance (NeoR) marker (detailed primers are in Supplementary Table S1). These constructs were reintroduced into the respective mutant strains. Complemented strains were selected on G418-containing mediums, and successful integration was verified by PCR.

To assess the impact of Nsxyn1 and Nsxyn2 deletions on fungal development and stress adaptation, a comprehensive phenotypic analysis was conducted on the gene deletion mutants, complemented strains, and wild-type controls. Growth rates were determined by measuring colony diameters on a PDA medium incubated at 25°C over a 30-day incubation period. Sporulation was quantified by counting conidia produced on the PDA plates after 30 days using a hemocytometer. Spore morphology was examined under a light microscope. At the same time, conidial germination rates were assessed by incubating conidia in sterile distilled water for 24 h at 25°C, followed by counting germinated spores under the microscope.

Oxidative and osmotic stress assays were performed to evaluate stress tolerance. Strains were grown on a PDA medium supplemented with 10 μmol/L Congo red for oxidative stress and either 1 mol/L NaCl or 0.02% SDS for osmotic stress. Colony growth was monitored by measuring the diameters after a 30-day incubation period. To evaluate the capacity of these strains to degrade primary cell wall components, they were cultured on the PDA mediums containing 1% (w/v) cellulose, xylan, or pectin as the sole carbon source. Enzyme activity was quantified by measuring the diameter of clear zones after Congo red staining, providing insight into the ability to degrade cell wall polysaccharides of these strains.

For virulence assessment, Nsxyn1 and Nsxyn2 gene deletion mutants, complemented strains, and the wild-type strains were cultured on PDA at 25°C for 30 days. Single conidia were isolated and transferred to fresh PDA medium for 15-day incubation. Conidia were harvested and diluted to a concentration of 1 × 10⁶ conidia/mL in sterile distilled water. Surface-sterilized host plant leaves were wounded with a sterile needle, and 10 μL of the conidial suspension was applied to each wound. Inoculated plants grow at 25°C with 80% relative humidity. Disease symptoms were monitored daily. Lesion sizes were recorded at 30 days post-inoculation to determine virulence. The assays were repeated in triplicate in each treatment.

Data analysis was performed using One-way ANOVA followed by Duncan’s multiple range test in SPSS version 27.0 (SPSS Inc., Shanghai, China). Graphical data visualization was conducted using GraphPad Prism 8.4.2 to illustrate significant differences in disease indices among strains.

Three xylanases containing GH11 domain proteins (Nsxyn1-Nsxyn3) were identified in the genome of Neostagonosporella sichuanensis SICAUCC 16–0001 through bioinformatics analysis. These xylanases were identified as secreted proteins with signaling peptides, lacking transmembrane regions and GPI-anchor signals (Supplementary Table S3). The same screening method predicted 51 xylanase proteins from nine other phaeosphaeriacous species (Supplementary Table S3). These xylanase proteins and Nsxyn1-Nsxyn3 were subjected to phylogenetic analysis with the neighbor-joining method. The generated phylogenetic tree clustered the proteins into six distinct clades, designated Branch1 to Branch6, widely represented among the analyzed phaeosphaeriacous species. Most xylanases exhibited relatively higher sequence similarity within the same branches, indicating a degree of conservation within the Phaeosphaeriaceae family (Figure 1). However, sequence alignment analysis revealed low homology between the GH11 domains of the three xylanases from N. sichuanensis, with pairwise identities of 44.04% between Nsxyn1 and Nsxyn2, 29.44% between Nsxyn1 and Nsxyn3, and 47.67% between Nsxyn2 and Nsxyn3 (Figure 2). Despite the low sequence homology, all three xylanases shared two conserved catalytic sites associated with glutamic acid residues (Figure 2).

The number and locations of alpha helices and extended strands were similar between Nsxyn1, Nsxyn2, and Nsxyn3 proteins according to the analysis of their secondary structures (Figures 3A–C) and tertiary structures (Figures 3D–F), leading us to hypothesize that all three proteins have similar functions. The tertiary structure quality of the three xylanase proteins was assessed using the SAVES pipeline and Qmean. The overall quality factor of the tertiary structure of Nsxyn1, Nsxyn2, and Nsxyn3 was 90.45, 89.11, and 88.89, respectively, as determined by the ERRAT program and Nsxyn1 and Nsxyn3 passed the quality test of the Verify3D program, in contrast to Nsxyn2 where less than 80% (77.97%) of the residues had an averaged 3D-1D score > = 0.1. Nevertheless, the Ramachandran plots generated by PROCHECK showed that over 90% of the amino acid residues of all three proteins were in acceptable regions (Figures 3G–I). On the other hand, the Nsxyn1, Nsxyn2, and Nsxyn3 models evaluated by Qmean had Z-scores of-1.27, −1.49, and-1.40, respectively (Figures 3J–L). The SAVES pipeline and the Qmean evaluation results revealed that the tertiary structures of all three protein models predicted by the SWISS-MODEL using the Alphafold v2 method were consistently of good quality and suitable for further experimental study.

Two candidate xylanase genes (Nsxyn1 and Nsxyn2) were cloned from the cDNA of Neostagonosporella sichuanensis SICAUCC 16–0001 (Supplementary Figure S1); however, Nsxyn3 was not obtained despite numerous attempts. The sequences of two genes obtained by cloning, Nsxyn1 and Nsxyn2, were identical to the sequences from the genome annotation of Neostagonosporella sichuanensis SICAUCC 16–0001, reflecting the accuracy of the genome assembly and annotation results. The functions of Nsxyn1 and Nsxyn2 genes were then further investigated.

Recombinant plasmids pET-32a-Nsxyn1 and pET-32a-Nsxyn2 were successfully constructed and transformed into Escherichia coli BL21 (DE3) competent cells (Supplementary Figure S2). Expression parameters, including IPTG concentration, induction duration, and temperature, were carefully optimized to improve Nsxyn1 and Nsxyn2 protein yield and enhance protein solubility. The findings indicated that IPTG concentration did not impact Nsxyn1 and Nsxyn2 protein expression, so the lowest IPTG concentration of this experiment, 0.2 mM, was chosen to continue optimizing other expression conditions (Supplementary Figures S3A,B). Similarly, the expression of Nsxyn1 was not affected by the duration of induction, so 2 h was optimal (Supplementary Figure S3C). Conversely, 5 h was chosen for pET-Nsxyn2 induction due to the significantly increased amount of induced protein compared to the previous hours (Supplementary Figure S3D). The expression of Nsxyn1 and Nsxyn2 proteins was highest at 30°C (Supplementary Figures S3E,F). The optimal screening conditions induced the expression of Nsxyn1 and Nsxyn2 proteins before purification.

To determine whether the two Glu sites in Nsxyn1 and Nsxyn2 proteins are essential for the enzyme activity, we conducted site-directed mutagenesis genes, Nsxyn1^SM113–205 and Nsxyn2^SM121–212. Subsequently, they were transferred to E. coli BL21 (DE3) for overexpression using the previously optimized conditions.

Solubility detection experiments showed that Nsxyn1, Nsxyn2, Nsxyn1^SM113–205, and Nsxyn2^SM121–212 were present as soluble proteins (Supplementary Figures S3G,H). The Nsxyn1, Nsxyn2, Nsxyn1^SM113–205, and Nsxyn2^SM121–212 proteins were purified before detection of enzyme activity.

The Nsxyn1 and Nsxyn2 recombinant proteins have xylanase activity and can induce the necrosis phenotype of Nicotiana benthamiana independently of xylanase activity.

The purified Nsxyn1 and Nsxyn2 proteins exhibited the ability to hydrolyze xylan to form hydrolytic rings when they were added to the small holes of the medium with xylan as the sole carbon source (Figures 4A,B). On the other hand, Nsxyn1^SM113–205 and Nsxyn2^SM121–212 lost the ability to degrade xylan (Figure 4C). The above findings align with the quantitative enzyme activity analysis of the purified protein using xylan as substrate (Figures 4D,E).

Injection of purified Nsxyn1 and Nsxyn2 proteins into Nicotiana benthamiana leaves resulted in necrosis approximately 5 days after treatment (Figures 4F,G). To determine whether this necrosis was related to the enzymatic activity of these proteins, site-directed mutagenesis was used to generate catalytic site mutants (Nsxyn1^SM113–205 and Nsxyn2^SM121–212) in both proteins. Despite the loss of enzymatic activity, the mutant proteins still induced necrosis in N. benthamiana leaves, comparable to the unmutated proteins (Figures 4H,I). These findings suggested that the necrosis-inducing activity of these xylanase proteins is independent of their enzymatic function.

To assess the involvement of Nsxyn1 and Nsxyn2 in the infection process of Neostagonosporella sichuanensis, their expression levels at different periods of infection with fishscale bamboo were determined using qRT-PCR (Figure 5). Under non-inductive conditions, the expression levels of both genes were at their lowest, normalized to 1. Expression analysis revealed significant up-regulation of Nsxyn1 and Nsxyn2 during the infection process, with Nsxyn1 showing a particularly strong increase, reaching an 80-fold up-regulation at 15 days post-inoculation. These results suggest that Nsxyn1 and Nsxyn2 are strongly induced during infection.

The Nsxyn1 and Nsxyn2 genes were inserted into the pSUC2 vector and subsequently transformed into the YTK12 yeast strain. Both transformants grew on CMD-W and YPRAA selective media, indicating successful gene expression (Figure 6). Similarly, the positive control strain harboring the pSUC2-Avr1b^SP construct also exhibited normal growth, whereas the negative control showed no growth, confirming the validity of the experimental system. Further TTC staining analysis revealed that YTK12 strains expressing Nsxyn1 and Nsxyn2 produced a red precipitate. These results strongly suggest that the proteins encoded by these genes contain functional signal peptides that facilitate secretion.

The plasmid maps and electrophoretic analysis during vector construction are presented in Supplementary Figure S4. All fluorescence signals were effectively recorded using confocal laser scanning microscopy in the onion epidermal cells (Figure 7A) and Arabidopsis thaliana protoplasts (Figure 7B) expressing the Nsxyn1 and Nsxyn2. Results from the transient expression system showed that onion epidermal cells with empty PCAMBIAsuper1300-GFP carriers emitted green fluorescence throughout the cell structure. However, EGFP green fluorescence was mainly distributed in the nuclear onion cells with pCAMBIAsuper1300-Nsxyn1-EGFP or pCAMBIAsuper1300-Nsxyn2-EGFP overexpression carriers. Furthermore, when examined in A. thaliana protoplasts, the Nsxyn1 and Nsxyn2 xylanases displayed a dual localization pattern in both the nucleus and the cytoplasm. Together, Nsxyn1 and Nsxyn2 proteins were localized in the nucleus and cytoplasm.

Transient expression of Nsxyn1-EGFP and Nsxyn2-EGFP in Nicotiana benthamiana leaves resulted in localized cell necrosis, as evidenced by visible necrotic lesions at the infiltration sites (Figures 7C,D). In contrast, the EGFP control did not induce any signs of necrosis, and the leaves remained healthy. This suggested that Nsxyn1 and Nsxyn2 possessed necrosis-inducing activity in plant cells, independent of the fluorescent tag.

Deletion and complementation strains for both genes were generated to investigate the role of Nsxyn1 and Nsxyn2 in cell wall degradation (Supplementary Figures S5, S6). It was observed that Neostagonosporella sichuanensis exhibited minimal growth on plates containing either 90 μg/mL hygromycin B or 200 μg/mL G418 (Supplementary Figure S7), leading to the selection of these concentrations for transformant screening. The deletion strains of Nsxyn1 and Nsxyn2 were verified by PCR analysis (Supplementary Table S1), and positive transformants were chosen for further study (Supplementary Figures S8, S9).

The capacity of the deletion mutants (ΔNsxyn1 and ΔNsxyn2) and the complementation strains (ctNsxyn1 and ctNsxyn2) to degrade cell wall components was evaluated on the C’zapek media that was devoid of carbon sources but supplied with xylan, cellulose, or pectin. On the C’zapek medium with xylan as the sole carbon source, colony diameters of ΔNsxyn1 and ΔNsxyn2 mutants were significantly smaller than those of the wild type (Figure 8). Additionally, the colony diameter of the ΔNsxyn1 mutants was unexpectedly decreased on C’zapek media with cellulose or pectin, while the ΔNsxyn2 mutants showed a similar reduction only when grown on C’zapek medium with pectin. The complementation strains, ctNsxyn1, and ctNsxyn2, completely restored cellulose and pectin degradation capacity and partially restored the potential for xylan degradation.

To examine the impact of Nsxyn1 and Nsxyn2 gene deletion on the growth of Neostagonosporella sichuanesis, mycelial plugs of wild type, ΔNsxyn1 and ΔNsxyn2 mutants, and the ctNsxyn1 and ctNsxyn2 complement strains were inoculated on PDA medium under light/dark (12 h/12 h) conditions at 25°C for 30 days. A significant decrease in colony diameter and spore production was observed in the ΔNsxyn1 mutant relative to the wild type (Figures 9A–C). In contrast, no strong negative effect on the growth rate was observed in the ΔNsxyn2 mutant (Figures 9A,B). However, it was found that the colony morphology of the ΔNsxyn2 mutant changed, shifting from a smooth surface to a cotton-like appearance, with a corresponding reduction in microconidia production compared to the wild type (Figures 9A,C). Spore production only statistically partially recovered, and the growth rate was restored after complementation. Additionally, the conidial morphology of all transformant strains did not differ significantly from that of the wild type, and all strains were able to germinate normally (Figure 9D).

The integrity of the fungal cell wall is crucial for successful host cell infection, as it maintains the shape of the fungal cell and regulates interactions with the external environment (Cabib et al., 2001). Continuous cell wall remodeling is required for proper fungal growth and development (Bowman and Free, 2006). We assessed the effects of cell wall-disrupting agents on the growth of the ΔNsxyn1 and ΔNsxyn2 mutant strains (Figure 10). Colony size measurements were employed to quantify the growth inhibition rate, revealing that the ΔNsxyn1 and ΔNsxyn2 strains exhibited significant sensitivity to cell wall stressors, including Congo red, SDS, and NaCl. Notably, the ΔNsxyn2 strain displayed greater sensitivity than the ΔNsxyn1 strain.

Pathogenicity assays were conducted to determine the roles of Nsxyn1 and Nsxyn2 in rhombic-spot disease on living fishscale bamboo culms. The wild-type strain caused typical necrotic symptoms. In contrast, while the ΔNsxyn1 and ΔNsxyn2 mutants displayed similar symptoms, their virulence was significantly reduced (Figure 11). Complementation strains ctNsxyn1 and ctNsxyn2 restored most of the virulence phenotype. These findings underscore the critical roles of Nsxyn1 and Nsxyn2 in the pathogenicity of N. sichuanensis.