The ApCtf1β2-ApCUT3 module mediates host cuticle penetration and modulates immune responses thereby influencing blight resistance in Bambusa pervariabilis × Dendrocalamopsis grandis

Arthrinium phaeospermum is a globally distributed pathogen with a broad host range and is one of the principal causal agents of blight on B. pervariabilis × D. grandis. Ctf1 facilitates degradation of the plant cuticle, as well as early infection and colonization; however, the transcriptional regulation and pathogenic mechanisms of the Ctf1β subfamily remain unclear. We found that the ApCtf1β family contains conserved core domains and variable terminal regions. Through synteny analysis combined with interaction transcriptomics, we identified ApCtf1β2 as a key virulence gene that is significantly upregulated during infection. Functional analyses revealed that ApCtf1β2 is involved in fatty acid metabolism, stress responses, and sporulation, and that Δ ApCtf1β2 significantly reduces virulence. Integrated CUT&Tag and RNA-seq analyses identified ApCUT3 as a primary target; Y1H, EMSA, and LCA confirmed that ApCtf1β2 directly activates ApCUT3 expression by binding to the GCC(n5)CGG motif in the ApCUT3 promoter. The effector ApCUT3 possesses a functional signal peptide, is secreted, localizes to extranuclear compartments, and plays a pivotal role in degrading the host cuticle. Single-gene knockouts (Δ ApCtf1β2 or Δ ApCUT3 ) each attenuate pathogenicity, whereas the double knockout (Δ ApCtf1β2 / ApCUT3 ) exhibits a pronounced hypovirulent phenotype with synergistic interaction. Hosts infected with the double mutant maintain relatively intact cuticles, exhibit substantially weakened JA and SA defense signaling, and display markedly reduced defense enzyme activities. In summary, the ApCtf1β2-ApCUT3 module promotes fungal infection by transcriptionally activating the effector ApCUT3, thereby compromising the host physical barrier and suppressing defense activation. These findings provide a theoretical basis for targeted control of fungal diseases. The ApCtf1β2-ApCUT3 module mediates host cuticle penetration and modulates immune responses thereby influencing blight resistance in Bambusa pervariabilis × Dendrocalamopsis grandis Peng Yan 1† ; Yi Liu 1† ; Sijia Liu 1 ; Han Zhao 1, 2, 3 ; Zhanbo Liu 1, 2, 3 ; Shujiang Li 1, 2, 3, 4, * 1 College of Forestry, Sichuan Agricultural University, Chengdu 611130, China. 2 Forest Ecology and Conservation in the Upper Reaches of the Yangtze River Key Laboratory of Sichuan Province, Chengdu 611130, China. 3 Sichuan Mt. Emei Forest Ecosystem National Observation and Research Station, Chengdu 611130, China. 4 Bamboo Diseases and Pests Control and Resources Development Key Laboratory of Sichuan Province, Leshan, 614000, China. * Corresponding author: Shujiang Li, [email protected], Tel.: +86-186-0835-7494. † These authors contributed equally to this study. Summary Arthrinium phaeospermum is a globally distributed pathogen with a broad host range and is one of the principal causal agents of blight on B. pervariabilis × D. grandis. Ctf1 facilitates degradation of the plant cuticle, as well as early infection and colonization; however, the transcriptional regulation and pathogenic mechanisms of the Ctf1β subfamily remain unclear. We found that the ApCtf1β family contains conserved core domains and variable terminal regions. Through synteny analysis combined with interaction transcriptomics, we identified ApCtf1β2 as a key virulence gene that is significantly upregulated during infection. Functional analyses revealed that ApCtf1β2 is involved in fatty acid metabolism, stress responses, and sporulation, and that Δ ApCtf1β2 significantly reduces virulence. Integrated CUT&Tag and RNA-seq analyses identified ApCUT3 as a primary target; Y1H, EMSA, and LCA confirmed that ApCtf1β2 directly activates ApCUT3 expression by binding to the GCC(n5)CGG motif in the ApCUT3 promoter. The effector ApCUT3 possesses a functional signal peptide, is secreted, localizes to extranuclear compartments, and plays a pivotal role in degrading the host cuticle. Single-gene knockouts (Δ ApCtf1β2 or Δ ApCUT3 ) each attenuate pathogenicity, whereas the double knockout (Δ ApCtf1β2 / ApCUT3 ) exhibits a pronounced hypovirulent phenotype with synergistic interaction. Hosts infected with the double mutant maintain relatively intact cuticles, exhibit substantially weakened JA and SA defense signaling, and display markedly reduced defense enzyme activities. In summary, the ApCtf1β2-ApCUT3 module promotes fungal infection by transcriptionally activating the effector ApCUT3, thereby compromising the host physical barrier and suppressing defense activation. These findings provide a theoretical basis for targeted control of fungal diseases.

Arthrinium phaeospermum ; B. pervariabilis × D. grandis ; ApCtf1β2-ApCUT3 module; synergistic interaction; cuticle penetration; immune responses 1 Introduction B. pervariabilis × D. grandis combines rapid growth with ease of vegetative propagation, conferring significant economic and ecological value, and has consequently been widely deployed along the Yangtze River for urban greening, papermaking, bamboo shoot production, handicrafts, and ornamental purposes (Liu et al., 2024; Yan et al., 2024). In recent years, widespread dieback outbreaks have caused substantial economic losses, and studies have identified A. phaeospermum as the primary causal agent of shoot blight in this hybrid (Luo et al., 2025). A. phaeospermum is a globally distributed pathogenic fungus with a broad host range; its hosts include 56 plant species, such as legumes, bamboo, sugarcane, and olive, as well as animals and humans (Li et al., 2020). The fungus demonstrates strong ecological adaptability and is found in diverse environments, including air, soil, and plant debris. A. phaeospermum secretes a specific proteinaceous toxin (AP-Toxin) that directly disrupts host mitochondrial membrane structure and function (Li et al., 2013). Additionally, it produces a range of cell wall-degrading enzymes that work synergistically to dismantle the host’s physical barriers, thereby facilitating hyphal penetration (Fang et al., 2021, 2022). However, the mechanisms by which the pathogen precisely regulates the spatiotemporal expression of virulence factors during infection—specifically, how it senses host-specific signals and initiates early infection programs—remain unresolved questions central to research in pathogen biology and plant–microbe interactions. The cuticle covering the epidermis of higher plants is a biopolyester primarily composed of C16 and C18 hydroxy fatty acids, as well as epoxy fatty acids, which are polymerized to form cutin (Nawrath, 2006). Its hydrophobic nature restricts the loss of water vapor and solutes, while also providing protection against ultraviolet radiation, mechanical damage, and pathogen invasion (Chassot et al., 2007; Chen et al., 2013; Domínguez et al., 2011). Thus, the cuticle serves a critical role in plant defense. Moreover, the integrity of the cuticle is closely associated with the maintenance of microbial community homeostasis and the regulation of plant immune responses (Golzan et al., 2023; Hossain et al., 2023; Martínez and Maicas, 2021; Vasselli and Shaw, 2022). For instance, in Sclerotinia sclerotiorum and Macrophomina phaseolina, the expression of cutinases increases during appressoria maturation and penetration; inhibition or impairment of cutinase activity leads to a marked decrease in infection capacity (Gong et al., 2022; Liu et al., 2023). Conversely, in certain pathogens such as Nectria haematococca and Fusarium graminearum, cutinases are not strictly necessary for pathogenicity, indicating functional redundancy or the existence of alternative pathways (Davies et al., 2000; Dickman, 1986; Sweigard et al., 1992). The contribution of cutinases to pathogenesis extends beyond breaching the physical barrier: some cutinases function as pathogen-associated molecular patterns (PAMPs), directly eliciting host defense responses and facilitating the activation of host immune signaling (Gong et al., 2022; De Lorenzo et al., 2011). Additionally, cutinases are implicated in inducing host-derived signals, facilitating fungal spore adhesion, and supporting carbon acquisition during the saprophytic phase (Cheung et al., 2009; Järvinen et al., 2009). As a result, the spatial and temporal precision in cutinase expression required for these diverse biological functions is regulated by specific transcription factors. Elucidating how transcription factors orchestrate cutinase gene expression, and thereby precisely govern cuticle penetration and immune activation, remains a central question in the study of pathogenic mechanisms. In plant pathogenic fungi, the cutinase transcription factor Ctf1 and its α/β subtypes mediate the transcription of cutinase genes through complex regulatory networks, and their roles in pathogenicity display pronounced species specificity. In F. solani, F. oxysporum, and Aspergillus nidulans, Ctf1 induces cutinase activity and regulates the expression of related genes; however, deletion of Ctf1 does not significantly impact virulence during root infection by F. oxysporum (Li et al., 2002; Li and Kolattukudy, 1997; Rogers et al., 1994; Voigt et al., 2005)。 Ctf1α has been identified as a key positive regulator, and its deletion leads to marked reductions in cutinase activity and virulence in F. solani f. sp. pisi and F. verticillioides (Li et al., 2002; Peng et al., 2024). By contrast, the molecular mechanisms and biological functions of Ctf1β remain largely uncharacterized. Early studies, which noted its weak transcriptional activation capacity, proposed that Ctf1β primarily maintains basal cutinase expression under noninducing conditions and provides an initial signal for Ctf1α induction (Li et al., 2002). However, in Fusarium verticillioides, the Ctf1β homolog FvFARB is strongly upregulated during infection and directly regulates the cutinase gene FvCUT3, thereby influencing both virulence and carbon utilization efficiency (Peng et al., 2024). In B. bassiana, BbCtf1β and BbCtf1α jointly regulate development and pathogenicity (Wang et al., 2020). These findings suggest that Ctf1β may play an independent transcriptional regulatory role; however, the molecular basis for this function remains incompletely elucidated. The key to elucidating the molecular mechanism by which Ctf1β acts as an independent transcriptional regulator is the identification of its specific DNA binding sequence—the core binding motif. These conserved cis-acting elements serve as the basis for transcription factor target recognition, directly determining regulatory specificity and efficiency (Peng et al., 2024). In fungi, G-rich response elements, commonly referred to as G boxes with a characteristic motif of AGGGG, have been reported in the promoters of multiple cell wall-degrading enzyme genes and are associated with the maintenance of basal transcription (Chen et al., 2021; Peng et al., 2024). For the Ctf1 family, Ctf1α from F. solani recognizes a palindromic sequence, CCGAGG, and mediates cutin monomer-induced gene expression; similarly, the FarA and FarB homologs in A. nidulans can bind the same or closely related DNA elements in vitro (Bravo-Ruiz et al., 2013; Lin et al., 2022; Luo et al., 2016; Rocha et al., 2008). In contrast, the DNA binding properties and core motif of Ctf1β remain to be systematically characterized. Based on current functional insights, we propose two potential mechanisms: first, Ctf1β may bind to the Ctf1α recognition motif, such as the GCC box, though with lower affinity, thereby enabling transcriptional fine-tuning through competitive binding; alternatively, Ctf1β may recognize a novel and unique DNA motif, thus conferring independent DNA binding specificity and regulatory capacity. Comprehensive investigation of the specific regulatory functions of Ctf1β, as well as the potential regulatory modules it forms with downstream target genes, is crucial for refining the transcriptional regulatory model of cutinase expression in plant pathogenic fungi and for advancing our understanding of their pathogenic mechanisms. First, this study will undertake a comprehensive phylogenetic and structural analysis of ApCtf1β2, followed by genetic approaches to elucidate its functional characteristics. Utilizing CUT&Tag, we will acquire genome-wide binding profiles of ApCtf1β2, identify its core binding motif, and construct an atlas of downstream target genes. Focusing on the representative target gene ApCUT3, we will conduct functional validation to systematically dissect how the ApCtf1β2–ApCUT3 regulatory module mediates cuticle penetration and pathogenesis. These investigations will clarify whether ApCtf1β2 functions as an independent transcriptional regulator, provide direct experimental evidence for the molecular mechanisms underpinning the Ctf1β2 subtype in pathogenic fungi, and establish a conceptual framework for designing novel disease intervention strategies targeting this regulatory pathway. 2 Result 2.1 Evolutionary features of the ApCtf1β transcription factor family and screening for key virulence genes The ApCtf1β family primarily comprises three domains: Fungal TF MHR, GAL4, and ZIP. Members of Group 1 retain only the MHR domain, whereas Groups 2 and 3 commonly possess the GAL4 domain, with some members also containing a ZIP domain. These domain architectures are conserved at the sequence level and are consistent with the phylogenetic topology; members within the same clade share similar domain configurations. Notably, Group 1 lacks the Zn 2 Cys 6 module. The number and arrangement of motifs vary substantially among the genes, with most members containing 5–7 motifs. Motif 1 is present in most proteins and occupies a conserved relative position across clades. ORF comparisons revealed gene lengths ranging from approximately 1.2 to 3.6 kb, and while exon counts varied widely among family members, they remained relatively consistent within each group (Fig. 1A). Analysis of physicochemical properties indicated significant variability within the ApCtf1β family (Table S1). We identified six segmental duplications and one tandem duplication ( ApCtf1β11 / 12 ) within the ApCtf1β family, suggesting that segmental duplication is the principal driver of family expansion (Fig. 1B). Interspecies synteny analysis demonstrated a negative correlation between ApCtf1β conservation and taxonomic distance: only two homologous gene pairs were detected between A. phaeospermum and F. oxysporum (different orders), whereas nine and fifteen homologous pairs were identified between A. phaeospermum and N. moseri (same family) and A. hydei (same genus), respectively. Notably, ApCtf1β2 and ApCtf1β13 each exhibit syntenic relationships with at least two homologous gene pairs, suggesting that they may represent key nodes driving the phylogenetic diversification of the ApCtf1β family (Fig. 1C). Based on Dual RNA-seq analysis, we examined ApCtf1β family expression at four time points (S1: 0 d, S2: 7 d, S3: 14 d, S4: 21 d). The results indicated that Cluster A includes six ApCtf1β genes whose expression changed significantly over time, with marked upregulation at S3 and S4; Cluster B comprises ten genes with consistently low and non-significant expression (Fig. 1D). By integrating synteny and transcriptome datasets, we selected ApCtf1β2 for in-depth functional characterization. Secondary and tertiary structure analyses showed that ApCtf1β2 contains 18 α-helices, two β-sheets, and a highly conserved zinc-finger motif characteristic of Zn 2 Cys 6 -type zinc-finger transcription factors (Fig. 1E; Fig. S1). Figure 1. Bioinformatics and expression profiling of the ApCtf1β family. (A) Phylogenetic tree of the ApCtf1β family in conjunction with conserved motif, domain architecture, and gene structure analyses. (B) Chromosomal distribution and intraspecific synteny analysis of ApCtf1β family members. (C) Interspecies synteny analysis between A. phaeospermum, A. hydei, N. moseri, and F. oxysporum . (D) Circular heatmap of ApCtf1β expression profiles with cluster analysis. (E) Tertiary structure model of ApCtf1β2 annotated with functional motifs. 2.2 ApCtf1β2 mediates pathogen virulence through regulation of metabolic and stress‑response genes Yeast transcriptional activation assays confirmed that ApCtf1β2 possesses transcriptional activation activity (Fig. 2A). Transformants expressing ApCtf1β2mCherry were generated via Agrobacterium-mediated transformation. Confocal microscopy revealed that the red fluorescence of the ApCtf1β2-mCherry fusion protein was predominantly localized in the nucleus and colocalized with DAPI staining, further confirming the nuclear localization of ApCtf1β2 (Fig. 2B). Carbon source utilization assays demonstrated that the Δ ApCtf1β2 mutant exhibited normal growth on linoleic acid, sodium oleate, and olive oil, but showed a significantly reduced growth rate on sodium propionate. Growth inhibition reached approximately 80% on sodium acetate and sodium butyrate (Fig. 2C, D). These findings indicate that ApCtf1β2 plays a critical role in fatty acid metabolism and utilization. Stress response assays revealed that the Δ ApCtf1β2 mutant was significantly more sensitive to SDS, calcofluor white (CFW), and H 2 O 2, with markedly higher inhibition rates compared to the control (Fig. 2C, D), suggesting that ApCtf1β2 is involved in cellular responses to cell wall integrity and oxidative stresses. qRT-PCR analysis indicated that five fatty acid/carbon metabolism genes ( FAS2, ACC, FBP1, ICL1, PDH1 ) and five cell wall biosynthesis genes ( CHS1, CHS6, CHS7, FKS1, RHO1 ) were significantly downregulated in the mutant (Fig. 2E), suggesting that Δ ApCtf1β2 may affect fatty acid metabolism, carbon utilization, and cell wall integrity by modulating the expression of these genes. CWDE activity assays showed a significant reduction in cutinase activity in the Δ ApCtf1β2 mutant, whereas pectinase and cellulase activities remained unchanged (Fig. 2F). Sporulation assays on potato dextrose agar (PDA) plates demonstrated that the mutant produced significantly fewer spores than both the wild-type (WT) and complemented strains (Fig. 2G). Pathogenicity assays indicated that, at 7 days post-inoculation (dpi), the Δ ApCtf1β2 mutant exhibited markedly attenuated virulence on young shoots of B. pervariabilis × D. grandis, while virulence was restored to WT levels in the complemented strain (Fig. 2H, I), confirming a crucial role for ApCtf1β2 in the pathogenicity of A. phaeospermum . Figure 2. ApCtf1β2 mediates virulence by regulating metabolic and stress response pathways. (A) Yeast transcriptional activation assay demonstrating that ApCtf1β2 possesses transcriptional activation activity. Yeast transformants carrying pGBKT7- ApCtf1β2 were plated on SD/−Trp and SD/−Trp/−His to assess growth; pGBKT753 and empty pGBKT7 were used as positive and negative controls, respectively. (B) Nuclear localization of ApCtf1β2 observed through DAPI staining. (C) Comparative analysis of nutrient utilization on media supplemented with long-chain and short-chain carbon sources, along with assessment of tolerance to cell wall and oxidative stress using media containing H₂O₂, Congo red (CR), SDS, or calcofluor white (CFW). (D) Quantification and statistical evaluation of hyphal growth inhibition rates induced by short-chain and long-chain carbon sources, as well as by cell wall and oxidative stresses. (E) qRT-PCR quantification of the expression levels of five carbon metabolism genes and five cell wall biosynthesis genes in WT and mutant strains. (F) Enzymatic activity assays of three cell wall-degrading enzymes—cutinase, pectinase, and cellulase—were conducted in WT and mutant strains. (G) Sporulation was measured on potato dextrose agar (PDA) at 25°C after seven days of incubation, with results statistically compared between WT and mutant strains. (H, I) Spores of A. phaeospermum were inoculated onto young shoots of B. pervariabilis × D. grandis, and lesion formation was evaluated at 7 dpi. Lesion areas were quantified using ImageJ and subjected to statistical analysis. 2.3 Mapping ApCtf1β2 binding across the genome and identification of the core metabolic targets (ApCUT) CUT&Tag analysis indicated that ApCtf1β2 binding sites are predominantly located at the transcription start site (TSS) and transcription end site (TES), corresponding to typical promoter and intergenic region binding patterns. This distribution suggests that ApCtf1β2 may regulate target gene expression at the transcriptional level by binding to cis-regulatory elements in these regions. A heatmap of signal intensity centered on peak regions demonstrated strong enrichment at peak centers, further supporting the reliability of the peak calling results (Fig. 3A). ApCtf1β2 binding sites were distributed across all 19 chromosomes, with 64.22% located in promoter regions, 19.95% in exons, 11.22% in intergenic regions, and 4.61% in introns (Fig. 3B). De novo motif analysis identified three core motifs that are significantly associated with ApCtf1β2 binding (Fig. 3C). Intersection analysis of 803 differentially expressed genes from RNA-seq with 1,229 CUT&Tag target genes identified 289 overlapping genes (Fig. 3D). Gene Ontology (GO) enrichment analysis demonstrated that these genes are enriched in biological processes such as small molecule metabolism, organic substance metabolism, and biosynthesis. In terms of molecular function, they are significantly enriched in catalytic activity, particularly hydrolase activity and oxidoreductase activity. For cellular component, these genes are primarily localized to the extracellular region (Fig. 3E). KEGG pathway analysis revealed significant enrichment in core metabolic pathways, including carbohydrate metabolism (Fig. 3F). Notably, the gene CUT was annotated in multiple enriched molecular function terms, such as hydrolase activity and oxidoreductase activity, as well as in KEGG pathways like tryptophan metabolism, suggesting that it represents a functionally relevant and high-confidence core regulatory target. Based on this evidence, CUT was selected as the candidate gene for subsequent functional validation. Figure 3. Integrated CUT&Tag and RNA-seq analysis of the ApCtf1β2 target binding profile. (A) CUT&Tag signal density and heatmap across the transcription start site region. (B) Chromosomal distribution of ApCtf1β2 binding sites and corresponding genomic feature annotations. (C) De novo motifs identified from ApCtf1β2 CUT&Tag data. (D) Candidate downstream target genes identified by integrating CUT&Tag and RNA-seq datasets. (E, F) Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis for ApCtf1β2 downstream target genes. 2.4 ApCtf1β2 directly binds to and activates ApCUT3 expression qRT-PCR results revealed significant differential responses of CUT family genes to ApCtf1β2 regulation, as identified by integrated analysis. In WT strains following host induction, both ApCUT3 and ApCUT5 were significantly upregulated; in OE- ApCtf1β2 strains, ApCUT3 was specifically activated, exhibiting expression levels markedly higher than those observed in WT and other CUT family members. These findings preliminarily indicate that ApCUT3 is a key downstream target of ApCtf1β2 and may play an essential role in the infection process of A. phaeospermum . IGV visualization of CUT&Tag peaks further confirmed ApCtf1β2 binding at the ApCUT3 promoter region (Fig. 4B). Motif analysis using MEME Suite revealed significant enrichment of “CCG” and “CGG” core motifs within ApCtf1β2 binding sequences. These motifs form a 5′ CCG(n)CGG spaced palindromic recognition unit, consistent with the binding characteristics of the Zn 2 Cys 6 transcription factor family. To validate the CUT&Tag results, multiple independent assays were performed, including Y1H, EMSA, and dual-luciferase reporter assays. Y1H assays indicated that yeast co-transformed with pGADT7- ApCtf1β2 and pAbAi- ApCUT3 -Pro was able to grow under selective conditions (Fig. 4D). EMSA results demonstrated that the ApCtf1β2 protein binds to the ApCUT3 promoter probe, whereas mutation of the 5′ CCG(n5)CGG motif to 5′ AAA(n5)AAA abolished binding, indicating sequence specificity. Dual-luciferase reporter assays further demonstrated that co-transfection of ApCtf1β2 with an ApCUT3 promoter-driven LUC reporter in 62SK cells significantly increased luciferase activity (Fig. 4F), confirming direct activation of the ApCUT3 promoter by ApCtf1β2. In summary, ApCtf1β2 positively regulates ApCUT3 expression by specifically binding to the palindromic motif within the ApCUT3 promoter, thereby contributing to the pathogenic process of A. phaeospermum . Figure 4. ApCtf1β2 specifically binds the ApCUT3 promoter. (A) qRT-PCR analysis of the relative expression levels of ApCUT family genes in WT and OE- ApCtf1β 2 strains over a 0–96 hpi time course on host-derived bamboo tissue medium. (B) Representative CUT&Tag peaks of ApCtf1β2 observed at ApCUT promoters. (C) MEME Suite motif analysis identifies ApCtf1β2 binding sites within the ApCUT3 promoter, specifically at GCC (P = 1.6e-4) and GGC (P = 1.71e-4) motifs. (D) Yeast one-hybrid (Y1H) assay: pGADT7- ApCtf1β2 and pAbAi- ApCUT3 -Pro were co-transformed into the Y1H strain and cultured on SD/-Leu medium; co-transformation of the empty pGADT7 vector with pAbAi- ApCUT3 -Pro was used as the negative control (see Fig. 4D). (E) Electrophoretic mobility shift assay (EMSA) confirms in vitro binding of ApCtf1β2 to the 5′ CCG(n5)CGG motif within the ApCUT3 promoter. The labeled probe (Pr) was synthesized from the ApCUT3 promoter sequence, whereas the mutant probe (mPr) contains the motif substitution 5′ AAA(n5)AAA. MBP alone served as a negative control. (F) Dual-luciferase assay demonstrates the interaction between ApCtf1β2 and the ApCUT3 promoter: 62SK- ApCtf1β2 and LUC- ApCUT3 -Pro were co-infiltrated into Nicotiana benthamian a leaves; 62SK and LUC were used as negative controls. 2.5 Signal Peptide Secretion and Extranuclear Localization of ApCUT3 and Its Key Role in Pathogenicity Based on secondary and tertiary structure predictions (Fig. 5A and Fig. S5), the 3D structure of ApCUT3 features a core composed of five parallel β-strands, surrounded and connected by several α-helices; a Ser-His-Asp catalytic triad at the apex of this core is characteristic of cutinases. The signal peptide function of ApCUT3 was assessed using a yeast secretion assay, in which pSUC2- Avr1b SP served as a positive control and YTK12 as well as the empty vector (EV) were used as negative controls. YTK12 cells transformed with pSUC2- ApCUT3 SP exhibited growth on selective plates comparable to the positive control and produced the red, insoluble TPF following TTC treatment (Fig. 5B), indicating that the ApCUT3 signal peptide mediates secretion. To evaluate the effect of signal peptide deletion, a truncated construct (ApCUT3 ΔSP ) was transiently expressed in N. benthamiana . ApCUT3 ΔSP retained the ability to suppress BAX-induced cell death at levels similar to the full-length ApCUT3 (Fig. 5C), indicating that interference with plant pattern-triggered immunity (PTI) is independent of the signal peptide. Confocal microscopy revealed that ApCUT3-mCherry exhibited a predominantly extranuclear, punctate/granular signal in the cytoplasm and nucleus with minimal DAPI overlap, whereas ApCUT3 ΔSP -mCherry colocalized strongly with DAPI and was primarily localized to the nucleus, suggesting that the signal peptide is essential for secretion-dependent extranuclear localization (Fig. 5D). Colony morphology of the WT, Δ ApCUT3, and the complemented strain (Δ ApCUT3 +) showed no evident differences (Fig. 5E). Cutinase activity was significantly reduced in Δ ApCUT3 compared with WT (Fig. 5F), while the mycelial growth rate and spore production on potato dextrose agar (PDA) were not significantly different among the strains (Fig. 5G, H). In inoculation assays on young stems of the host bamboo, Δ ApCUT3 exhibited significantly reduced virulence at 7 days post-inoculation (dpi) compared with WT, and virulence was restored in the complemented strain (Fig. 5I), indicating that ApCUT3 is critical for the pathogenicity of A. phaeospermum . Figure 5. Secretion, Subcellular Localization, and Pathogenic Functions of ApCUT3 Signal Peptide. (A) Three-dimensional structure of ApCUT3 and its catalytic site. (B) Validation of ApCUT3 signal peptide secretion activity: YTK12 cells transformed with pSUC2- ApCUT3 SP were plated on CMDM and YPRAA media and assessed for growth and TTC staining; empty pSUC2 was used as the negative control, while pSUC2- Avr1b SP served as the positive control. (C) ApCUT3 interferes with plant immune responses: full-length ApCUT3 and signal peptide–truncated ApCUT3 (ApCUT3 ΔSP ) were transiently expressed in N. benthamiana leaves, which were subsequently stained with trypan blue; BAX was utilized as a positive control. (D) DAPI staining revealed that ApCUT3 is present both inside and outside the nucleus, whereas ApCUT3 ΔSP localizes predominantly to the nucleus. (E, G, H) Analysis of colony morphology, radial growth rate, and sporulation for wild-type and mutant strains cultivated on PDA for 7 days at 25°C. (F) Cutinase activity assays were conducted to compare wild-type and mutant strains. (I) Pathogenicity assay: spores of A. phaeospermum were inoculated onto B. pervariabilis × D. grandis stems, and lesion formation was examined at 7 dpi. Lesion areas were quantified using ImageJ and analyzed statistically. 2.6 ApCtf1β2-ApCUT3 mediated cuticle degradation leads to host barrier disruption and dysregulation of defense responses Quantitative and qualitative analyses consistently showed that deletion of either gene in the ApCtf1β2-ApCUT3 module (Δ ApCtf1β2 or Δ ApCUT3 ) significantly reduced virulence relative to the WT, resulting in markedly smaller stem lesions; the double knockout (Δ ApCtf1β2/ApCUT3 ) produced the smallest lesions, indicating the greatest impairment in lesion initiation and expansion (Fig. 6A). DAB staining revealed continuous, deep-brown H 2 O 2 precipitate bands in the longitudinal vascular regions surrounding inoculation sites in the WT and the complemented strain; single knockouts exhibited weaker and more dispersed signals, while the double knockout displayed the least H 2 O 2 accumulation, suggesting that the module triggers an H 2 O 2 burst (Fig. 6B). Aniline blue fluorescence imaging demonstrated large, continuous punctate callose deposits at cell walls and vascular bundle margins in the WT and complemented strains; single knockouts exhibited primarily scattered puncta, whereas the double knockout displayed only sparse and weak fluorescence, indicating that the module induces PTI-associated callose deposition in the host (Fig. 6C). WGA/PI confocal imaging further showed that the WT and complemented strains formed continuous, highly branched hyphal networks at the vascular bundles; single knockouts presented with reduced and fragmented hyphal signals, and the double knockout exhibited only sporadic weak signals, indicating the lowest colonization capacity and underscoring the importance of this module for fungal colonization (Fig. 6D). SEM analysis revealed pronounced cuticle damage (thinning, rupture, and exposure of underlying tissues) in the WT and complemented strains; single knockouts led to only mild wrinkling with largely preserved overall structure; the double knockout retained an essentially intact surface with tightly closed cells, highlighting the module’s crucial role in compromising the host physical barrier (Fig. 6E). GC–MS quantification demonstrated significant differences in the total amounts of cuticular components among strains: the double knockout (Δ ApCtf1β2/ApCUT3 ) had the highest total, followed by Δ ApCtf1β2 and Δ ApCUT3, whereas the WT and the complemented strain (Δ ApCtf1β2/ApCUT3 +) had the lowest totals. The compositional profile was consistent across strains, with fatty acids as the predominant class, followed by alkanes; alcohols and other compounds were present at considerably lower levels (Fig. 6F and S7). A heatmap of the top five compounds within each of the four compound classes confirmed this trend: the double knockout showed the highest abundances, the single knockouts were intermediate, and WT and complemented strains were lowest, mirroring the pattern of total cuticular content. In the double knockout, fatty acids and long-chain alkanes were significantly enriched, whereas alcohols and other compounds were less abundant and exhibited smaller variation, further supporting a critical role for the ApCtf1β2-ApCUT3 module in disrupting host cuticle structure (Fig. 6G). Transcriptional analyses indicated that expression of jasmonic acid (JA)-pathway genes ( JAZ1, PR3, MYC2 ) and salicylic acid (SA)-pathway genes ( PR1, NDR1 ) was significantly reduced in the single and double knockouts compared to WT, with the double knockout displaying the lowest expression; the complemented strain was restored to WT levels (Fig. 6H). Assessment of defense-related enzyme activities and oxidative stress markers (PPO, POD, PAL, CAT, and MDA) revealed the highest levels in WT and complemented strains, moderate reductions in most indicators in the single knockouts, and further significant decreases across indicators in the double knockout (Fig. 6I and S8). In summary, deletion of either ApCtf1β2 or ApCUT3 considerably impairs the pathogen’s ability to elicit host defense responses, and the double knockout produces an synergistic interaction. Independent transcriptional and biochemical evidence consistently identify the ApCtf1β2-ApCUT3 module as a key factor in the pathogenic process. Figure 6. ApCtf1β2-ApCUT3 module-mediated cuticle degradation and disruption of host defenses. Pathological, physiological, and biochemical assays were performed on B. pervariabilis × D. grandis 7 days after inoculation with spores from both wild-type and mutant strains. (A) Lesion areas were quantified using ImageJ and statistically analyzed. (B) Reactive oxygen species (ROS) accumulation in young stems was evaluated by DAB staining, and hydrogen peroxide (H 2 O 2 ) content was determined using the titanium sulfate method. (C) Callose deposition in young stems was visualized via aniline blue staining and quantitatively measured using an ELISA kit. (D) Hyphal structures were labeled with WGA, and plant cell walls/nuclei were counterstained with PI; red/green fluorescence pixel ratios were calculated in Fiji. (E) Scanning electron microscopy (SEM) images show stem surfaces and cross-sections. (F, G) GC-MS analyses were conducted to assess cuticular components across treatments, followed by heatmap visualization of the major constituents. (H) Quantitative reverse transcription PCR (qRT-PCR) was employed to determine gene expression levels related to the jasmonic acid (JA) and salicylic acid (SA) signaling pathways. (I) Physiological and biochemical indices were measured and normalized for each treatment. Figure 7. Model of ApCtf1β2-ApCUT3 module mediated A. phaeospermum infection of B. pervariabilis × D. grandis . ApCtf1β2 specifically binds to the ApCUT3 promoter and activates its transcription, thereby promoting the secretion of cuticle-degrading enzymes. Individual deletion of ApCtf1β2 or ApCUT3 significantly attenuates A. phaeospermum virulence, while simultaneous deletion of both further diminishes virulence, demonstrating a strong synergistic interaction. Accordingly, the ApCtf1β2-ApCUT3 module represents a nonredundant, interdependent core pathogenicity unit that drives early cuticle degradation, initiates localized immune responses, and facilitates hyphal colonization within vascular bundles, thus playing a pivotal role in pathogenesis. 3.Discussion Collectively, this study reveals that expansion of the ApCtf1β family occurred primarily through segmental duplications and local tandem repeats (e.g., ApCtf1β11/12 ), providing a molecular substrate for the recombination of cis-regulatory elements and subsequent divergence of expression profiles (Chang and Ehrlich, 2013). Within this family, members exhibit a conserved core domain with variable termini: the N-terminal fungal transcription factor domain is highly conserved across paralogs, maintaining both DNA-binding and dimerization functions, whereas the modular evolution of the C-terminus has fostered functional diversification. This diversification has enabled certain paralogs—such as ApCtf1β2 —to specialize as infection-stage regulators, while other copies have undergone functional decay (Long et al., 2003; Sandhya et al., 2017). This evolutionary trajectory accounts for the pronounced upregulation of ApCtf1β2 during host infection and its retention of the canonical Zn 2 Cys 6 zinc-cluster motif. These features are consistent with selection for a functionally essential paralog mediating infection-specific transcriptional regulation. In contrast, the loss of such domains, as observed for FarC in Aspergillus, can result in functional attenuation without overt phenotypic effects (Luo et al., 2016). The unique DNA-recognition mechanism of Zn 2 Cys 6 zinc-cluster transcription factors provides a mechanistic basis for the retained DNA-binding capacity of ApCtf1β2 and its function as an upstream regulator (Garg et al., 2018; Macpherson et al., 2006). The Ctf1α and Ctf1β subtypes have distinct roles in lipid metabolism: Ctf1α mainly regulates long-chain fatty acid metabolism, whereas Ctf1β predominantly facilitates short-chain fatty acid utilization (Hynes et al., 2006; Wang et al., 2020). Consistent with this, the Δ ApCtf1β2 mutant displayed markedly impaired growth on short-chain fatty acids, confirming a central function for ApCtf1β2 in this metabolic pathway. Furthermore, ApCtf1β2 influences sporulation, oxidative stress tolerance, and cell wall integrity. The Δ ApCtf1β2 strain produced significantly fewer spores, exhibited heightened sensitivity to extracellular oxidative stress and cell wall disruption, and showed reduced expression of genes involved in carbon metabolism and cell wall biosynthesis, supporting a key regulatory role in nutrient-driven growth and multi-stress resilience. The effect of Ctf1 loss on pathogen virulence is species-specific, likely reflecting differences in infection strategies (Peng et al., 2024). During A. phaeospermum infection, Δ ApCtf1β2 mutants demonstrated severely impaired secretion of cuticle-degrading enzymes (e.g., cutinases), significantly reduced early colonization, and attenuated virulence, indicating that ApCtf1β2 directly regulates the expression of crucial effectors required for cuticle penetration and early infection establishment. Taken together, these findings indicate that ApCtf1β2 is a multifunctional transcription factor in A. phaeospermum, occupying a central regulatory node for lipid substrate utilization, nutrient-dependent growth, environmental stress tolerance, and host infection. Ctf1/Far proteins, which contain a highly conserved Zn 2 Cys 6 zinc cluster domain, mediate transcriptional regulation of target genes by binding to a conserved palindromic promoter motif, GCC(n)GGC (Rocha et al., 2008). CUT&Tag profiling indicated that Ctf1β demonstrates multitiered DNA-binding specificity, preferentially recognizing complete binding sites with a GC-rich core and near-palindromic symmetry (Fig. S4), a recognition pattern consistent with the dimeric binding mechanism of Zn 2 Cys 6 transcription factors (Recio et al., 2023). Additionally, Ctf1β can bind single-side half sites and various noncanonical motifs; this flexible binding repertoire provides a molecular basis for its nuanced regulation of diverse target genes (Corton et al., 1998; Marmorstein et al., 1992). CUT genes commonly occur as multicopy families in fungal genomes (3–17 copies), underscoring the central role of cuticle-degrading enzymes in pathogen ecological adaptation (Skamnioti et al., 2008; Villafana and Rampersad, 2020). Integrated RNA-seq and CUT&Tag analyses identified ApCUT1–5 as direct transcriptional targets of ApCtf1β2, suggesting that the ApCUT gene family has undergone duplication followed by functional diversification. Based on promoter elements and spatiotemporal expression patterns, ApCUT genes can be categorized as constitutive ( ApCUT1, ApCUT2, ApCUT4 ) or inducible ( ApCUT3, ApCUT5 ). Constitutive genes sustain basal expression and release cuticle degradation products that serve as induction signals, thereby promoting high-level expression of inducible genes and establishing a positive feedback loop. This diversification in gene architecture and expression strategies is closely associated with the pathogen’s life-history traits and niche adaptation (Gui et al., 2018; Liu et al., 2016). validated that ApCtf1β2 binds the 5′GCC(n5)GGC motif within the ApCUT3 promoter and thereby induces its expression. Cuticle-degrading enzymes function as core effectors during early infection, and their loss markedly compromises attachment, penetration, and early colonization in pathogens such as F. solani and S. sclerotiorum, resulting in substantially reduced virulence (Arya and Cohen, 2022; Gong et al., 2022); By contrast, in species harboring multiple CUT copies or possessing complementary cell-wall–degrading enzymes, such as U. virens and B. cinerea, single-gene CUT knockouts are often compensated for and do not substantially affect virulence (Lu et al., 2018, 2025). ApCUT3 encodes a protein with a canonical effector signal peptide, and the Δ ApCUT3 mutant exhibits significantly reduced virulence, indicating insufficient secretion of cuticle-degrading enzyme activity during infection to effectively degrade the host cuticle. These findings support a dual role for ApCUT3 as both an effector and a degradative enzyme, which may promote fungal proliferation in part by inducing host cell death. In summary, ApCtf1β2 specifically binds the 5′GCC(n5)GGC element in the ApCUT3 promoter to precisely activate this critical effector gene, thereby forming a core transcriptional regulatory module that drives early infection. The ApCtf1β2 transcription factor and the cuticle-degrading enzyme ApCUT3 constitute a regulatory module that operates in a nonredundant and synergistic manner during A. phaeospermum infection. The double knockout strain (Δ ApCtf1β2/ApCUT3 ) exhibited a significantly attenuated phenotype, substantially more severe than either single knockout, as evidenced by minimal lesion formation, the lowest H 2 O 2 accumulation and callose deposition, and an almost complete absence of hyphal networks within vascular bundles. These observations suggest that ApCtf1β2 and ApCUT3 are neither fully redundant nor entirely independent; rather, they display interdependence at both the transcriptional and biochemical levels. Specifically, ApCtf1β2 directly activates ApCUT3 transcription, but ApCUT3 maintains low residual transcriptional activity in the absence of ApCtf1β2, suggesting the presence of alternative compensatory pathways. The double knockout disrupts both the direct activation and compensatory mechanisms, resulting in an synergistic phenotype. This finding aligns with patterns observed in B. cinerea and S. sclerotiorum, where multiple interdependent genes collectively regulate pathogenicity (Bi et al., 2021; Derbyshire and Raffaele, 2023; Newman et al., 2023). Following infection, the cuticle remained largely intact in the double knockout strain, whereas wild type and complemented strains displayed pronounced cuticle damage, including thinning, cracking, and exposure of underlying tissues. These structural alterations were directly associated with GC–MS analysis of fatty acid enrichment: the Δ ApCtf1β2/ApCUT3 treatment displayed the highest levels of host fatty acids and alkanes, indicative of insufficient cuticle degradation. The integrity of this physical barrier was significantly correlated with host defense gene expression. Cuticle degradation products serve as damage-associated molecular patterns (DAMPs), recognized by the host to activate jasmonic acid (JA) and salicylic acid (SA) signaling pathways and downstream defense genes. The absence of cuticle-degrading enzymes prevents DAMP release, thereby diminishing host defense activation. Deletion of the ApCtf1β2-ApCUT3 module caused significant reductions in expression of host JA/SA pathway genes and multiple defense enzyme activities, thereby revealing a positive correlation between pathogen infection severity and host defense responses (Gui et al., 2018; Hou et al., 2019). Together, these findings establish the ApCtf1β2-ApCUT3 module as a key pathogenic unit necessary for early cuticle degradation, initiation of localized immune responses, and facilitation of hyphal colonization in vascular bundles. The synergistic effect observed in the double knockout underscores a unique interdependent yet functionally distinct relationship between ApCtf1β2 and ApCUT3 during A. phaeospermum infection. Collectively, through systematic molecular, biochemical, and genetic analyses, this study demonstrates that the multifunctional transcription factor ApCtf1β2 and the core effector ApCUT3 together constitute a transcriptional regulatory module that cooperatively facilitates early cuticle degradation, activates host defense responses, and promotes hyphal colonization within vascular bundles. These findings clarify the direct regulatory relationship and the synergistic mechanism between a fungal transcription factor and an effector, providing a key molecular basis for elucidating pathogen transcriptional networks and multilayered pathogenic processes. Notably, cuticle-degrading enzyme genes frequently exist as multicopy families in fungi and exhibit temporally and spatially distinct expression patterns, enabling functional compensation following single-gene knockout. In certain pathogens, disruption of a principal cuticle-degrading gene still permits residual infection, suggesting that the relative significance of the Ctf1β-CUT module differs across pathogen–host systems and is species specific. 4Materials and Methods Experimental materials A. phaeospermum (GenBank accession OK626768) was employed in this study. The strain is deposited at the China Forestry Culture Collection Center (CFCC86860). One-year-old plants of B. pervariabilis × D. grandis (height: 1.0 m, culm length: 0.5 m) were obtained from Sichuan Agricultural University, where the local annual average temperature and relative humidity are approximately 25.2 °C and 81%, respectively. N. benthamiana were cultivated in a greenhouse at Sichuan Agricultural University under controlled conditions: light intensity of 90 μmol·m⁻²·s⁻¹, temperature of 25 °C, relative humidity of 60%, and a photoperiod of 16 h light/8 h dark. Gene‑family bioinformatic analyses The A. phaeospermum genome (GenBank GCA_006503535.1) and the Dual RNA-seq dataset (GenBank SAMN19312317) were used as references to retrieve candidate sequences containing ApCtf1β2 conserved domains through BLASTP and HMMER searches. Maximum likelihood phylogenetic trees were constructed with IQ-TREE using automatic model selection and bootstrap support (n = 1000). Conserved motifs were identified using MEME Suite, and domain annotations were performed with SMART. MCScanX was employed to detect segmental and tandem duplication events, and to quantify intra- and interspecies homolog pairs for conservation assessment. Visualization and additional analyses were carried out in TBtools. Protein sequence alignments and secondary structure annotations were generated with ESPript and PredictProtein, while tertiary structure models were built in SWISSMODEL, model quality was evaluated, and structural visualization was performed in PyMOL. Strains and culture conditions Overexpression and subcellular localization constructs were assembled using the pCAMBIA1303 backbone. The recombinant plasmids ApCtf1β2-mCherry and ApCUT3-mCherry were introduced into Agrobacterium tumefaciens AGL1 and subsequently transferred into A. phaeospermum by Agrobacterium-mediated transformation (ATMT) (Thai et al., 2021). Positive transformants were identified via confocal microscopy (Zeiss LSM 880) following nuclear counterstaining. Gene deletions were generated by replacing ApCtf1β2 and ApCUT3 with Hyg and G418 selection cassettes, respectively. Complementation constructs containing ApCtf1β2 or ApCUT3 were selected using Ble or NRS resistance markers; the selection concentrations are detailed in Fig. S2, and validation of transformants is shown in Fig. S6. Both gene deletion and complementation procedures were conducted using ATMT. To evaluate carbon source utilization, WT and Δ ApCtf1β2 strains were inoculated on minimal medium (MM) containing single carbon sources. Growth assays utilized MM supplemented with one of the following as the sole carbon source: short-chain organic acids (e.g., sodium acetate or sodium propionate at 40 mM) or lipid substrates (3 mM linoleic acid, 3 mM sodium oleate, or 0.2% olive oil). Plates were incubated at 25 °C for 7 d, after which colony diameters were measured. Cell wall integrity assays were performed on PDA supplemented with 100 μg/mL calcofluor white (CFW), 100 μg/mL Congo red (CR), or 0.01% sodium dodecyl sulfate (SDS); growth comparisons between strains were subsequently conducted. RNA‑Seq and quantitative real‑time PCR (qRT‑PCR) Total RNA was extracted from WT and OE- ApCtf1β2 strains and subsequently reverse transcribed into cDNA. Sequencing libraries were constructed and sequenced on an Illumina HiSeq 2000 platform to produce 150 bp paired-end reads. High-quality reads were mapped to the A. phaeospermum genome using HISAT2. Differentially expressed genes (DEGs) were determined with DESeq2, with the criteria of |log₂ fold change| > 1 and P < 0.05. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed utilizing clusterProfiler (Yu et al., 2012). qRT-PCR assays were conducted on an Applied Biosystems™ 7500 instrument with TransScript® Green One Step qRT-PCR SuperMix (TransGen). Tubulin (TUB) and Actin served as internal reference genes for A. phaeospermum and bamboo samples, respectively. Primer sequences are detailed in Table S2, and transcriptome validation results are presented in Fig. S3. CUT&Tag and data analysis CUT&Tag assays were conducted using the NovoNGS CUT&Tag 3.0 high-sensitivity kit (N259 YH01 01A, Novo Protein, Jiangsu, China) with the OE- ApCtf1β2 strain used as input. Sequencing libraries were generated and processed on the Illumina NovaSeq 6000 platform in paired-end 150 bp (PE150) mode. Raw sequence reads underwent adapter trimming and removal of low-quality sequences to obtain clean reads. Peak calling was performed using MACS2, and peak annotation was accomplished with ChIPseeker (Kaya-Okur et al., 2019). Yeast one‑ and two‑hybrid assays Yeast one-hybrid (Y1H) and yeast two-hybrid (Y2H) assays were performed in accordance with the manufacturer’s instructions (Ouyi Biotech). For Y1H analysis, ApCtf1β2 was inserted into the pGADT7 vector while the ApCUT3 promoter was integrated into pAbAi, and both constructs were subsequently transformed into the Y1H yeast strain. Transformants were plated onto selective medium (SD/-Trp-His) and additionally onto medium containing 200 ng/mL Aureobasidin A (AbA). The empty pGADT7 vector served as the negative control, and protein-DNA interaction was evaluated based on colony growth. For Y2H assays, ApCtf1β2 was cloned into the pGBKT7 vector and transformed into the Y2H strain. Transformants were cultured on SD/-Trp and SD/-Trp-His plates supplemented with X-α-Gal and incubated at 30°C for three days. Transcriptional activation and protein-protein interactions were determined by assessing colony growth and blue color development. Electrophoretic mobility shift assay (EMSA) ApCtf1β2 coding sequence was cloned into pMALc5x and subsequently expressed in E. coli BL21. The recombinant MBP-tagged protein was purified. Biotin-labeled double-stranded probes corresponding to the ApCUT3 promoter cis-element were synthesized by Shanghai Sangon Biotech. An unlabeled probe was used as a competitor, while MBP alone served as a negative control. The EMSA procedures were performed according to established published protocols (Xiao et al., 2018). Dual‑luciferase transient expression assay The ApCUT3 promoter was cloned into pGreenII 0800‑LUC and ApCtf1β2 into pGreenII 62‑SK (Yang et al., 2023). Empty pGreenII 62‑SK was used as a negative control. Recombinant plasmids were introduced into A. tumefaciens GV3101 and co‑infiltrated into N. benthamiana leaves in the indicated combinations. After 48 h, firefly (LUC) and Renilla (REN) luciferase activities were measured using a dual‑luciferase assay kit (Novizan). The LUC/REN ratio was used to evaluate promoter activation. Prior to measurement, leaves were sprayed with 1 mM D‑luciferin potassium salt; imaging was performed with a Promega GloMax 96 in vivo imager after a 5‑min incubation. Pathology and physiological‑biochemical assays B. pervariabilis × D. grandis stems were inoculated by applying 10 μL drops of A. phaeospermum spore suspension (1 × 10 6 cfu/mL) onto intact stem surfaces. The inoculated plants were maintained in a greenhouse at 25 °C with 70–90% relative humidity and a 16 h light/8 h dark photoperiod. Lesion areas were documented by photography at 7 dpi and quantified using ImageJ software. Accumulation of H 2 O 2 was assessed qualitatively via DAB staining, followed by destaining with 70% ethanol prior to imaging. H 2 O 2 content was subsequently quantified using the titanium sulfate method. Callose deposition was visualized through aniline blue staining (protected from light for 30–60 min), and images were acquired under UV excitation. The amount of callose was quantified employing a plant callose ELISA kit (Shanghai EnzymeLinked Biotechnology). WGA/PI co-staining combined with confocal Z-stack imaging was utilized to assess the distribution of hyphae and vascular colonization: hyphae were stained with WGA-Alexa Fluor, while plant cell walls and nuclei were labeled with propidium iodide. Red/green fluorescence pixel ratios were analyzed using Fiji software. For scanning electron microscopy (Hitachi S4800), samples were processed by fixation, dehydration, mounting, and coating with gold/platinum according to standard protocols, followed by imaging at various magnifications. Extracellular enzyme activities were determined as follows: cutinase activity via the p-nitrophenyl butyrate (pNPB) assay, and pectinase and cellulase activities by the DNS method. Additional physiological and biochemical analyses were performed using established methods: catalase (CAT) by UV absorption, peroxidase (POD) by the guaiacol assay, polyphenol oxidase (PPO) by the o-diphenol method, superoxide dismutase (SOD) by the NBT reduction assay, malondialdehyde (MDA) by the TBA method, and phenylalanine ammonia-lyase (PAL) by UV spectrophotometry (Qin & Tian, 2005; Shah & Nahakpam, 2012). Statistical analysis Data processing and statistical analyses were conducted using Microsoft Excel, GraphPad Prism 9.5, TBtools, and IBM SPSS Statistics 25. For comparisons among multiple groups, one-way ANOVA was performed, followed by Duncan’s multiple range test (P < 0.05). For pairwise comparisons, Student’s t-test was utilized, with significance thresholds set at P < 0.05 and P < 0.01. Different lowercase letters denote statistically significant differences (p < 0.05). 5 Author contributions P.Y. and Shu.J.L. conceived and designed the experiments. 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Authors Metrics & Citations Metrics Article Usage 140views 81downloads Citations Download citation Peng Yan, Yi Liu, Sijia Liu, et al. The ApCtf1β2-ApCUT3 module mediates host cuticle penetration and modulates immune responses thereby influencing blight resistance in Bambusa pervariabilis × Dendrocalamopsis grandis. Authorea. 03 February 2026. DOI: https://doi.org/10.22541/au.177009341.12428182/v1 DOI: https://doi.org/10.22541/au.177009341.12428182/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu.