The ApHSF–ApCtf1β2–ApCUT3 Cascade Couples Host ROS Detoxification to Cuticle Penetration During Fungal Pathogenesis

ABSTRACT

Successful infection by plant‐pathogenic fungi requires both penetration of the host cuticle and tolerance of the reactive oxygen species (ROS) burst associated with host immunity. However, how these early infection events are temporally coordinated remains unclear. Here, we identified a three‐tier transcriptional cascade, ApHSF–ApCtf1β2–ApCUT3, in Arthrinium phaeospermum, the causal agent of shoot blight in hybrid bamboo ( Bambusa pervariabilis × grandis). Yeast one‐hybrid, electrophoretic mobility shift and dual‐luciferase assays showed that ApHSF directly binds the CTAGAA core motif in the ApCtf1β2 promoter and activates its transcription. Functional analyses further showed that ApHSF promotes detoxification of host‐derived ROS by activating the fungal antioxidant system, whereas pharmacological suppression of host ROS accumulation substantially restored the pathogenicity defect of the ΔApHSF mutant. ApHSF neither bound nor independently activated the ApCUT3 promoter. Instead, yeast three‐hybrid and combinatorial dual‐luciferase assays showed that, under early oxidative stress, ApHSF acts as a cofactor to enhance ApCtf1β2‐dependent activation of ApCUT3. In vivo fluorescence imaging further demonstrated that ROS detoxification alone was insufficient for full pathogenicity, which additionally required an intact ApCtf1β2–ApCUT3 module for cuticle penetration and colonization. Phos‐tag immunoblotting, λ‐protein phosphatase treatment and kinase inhibition assays showed that heat and oxidative stress induced phosphorylation of ApHSF and ApCtf1β2 and that these phosphorylation responses depended on p38 MAPK activity. By contrast, host cuticle‐derived cues preferentially induced the ApCtf1β2–ApCUT3 module. Together, these findings reveal how a plant‐pathogenic fungus integrates oxidative and cuticle‐associated signals to coordinate early infection.

The ApHSF–ApCtf1β2–ApCUT3 transcriptional cascade links fungal adaptation to host‐derived ROS with cuticle penetration during infection. ApHSF promotes ROS detoxification and activates ApCtf1β2, which induces ApCUT3 expression and cuticle degradation, thereby coordinating oxidative stress adaptation with host barrier penetration.

1. Introduction

Hybrid bamboo ( Bambusa pervariabilis × grandis) combines the resilience of B. pervariabilis with the rapid growth and high yield of B. grandis . In the Yangtze River basin of China, this hybrid bamboo is a multifunctional species of considerable value for both the forestry economy, particularly papermaking and shoot production, and ecological restoration (Liu et al. 2024). In recent years, shoot blight caused by Arthrinium phaeospermum has resulted in extensive bamboo mortality, with the proportion of severely affected stands often exceeding 50%. This destructive disease substantially impairs the carbon sequestration capacity and ecological regulatory functions of bamboo forests (Luo et al. 2025). During plant–pathogen coevolution, the cuticle secreted by epidermal cells has evolved as the outermost barrier against both biotic and abiotic stresses (Heredia et al. 2024). This hydrophobic barrier is composed primarily of a cutin polymer matrix and cuticular waxes. It serves three major roles in plant immune defence. First, its surface microstructure limits water retention and thereby directly inhibits pathogen spore attachment and germination. Second, enzymatic degradation products generated during infection can act as damage‐associated molecular patterns (DAMPs). These DAMPs are recognized by the host and trigger immune responses. In addition, cuticle‐associated compounds, such as triterpenes and fatty aldehydes, exhibit strong antimicrobial activity. Together, these function as a physical barrier, immune activator, and chemical defence system that constitute the first line of plant defence. Therefore, successful colonization by pathogenic fungi depends largely on breaching the host cuticle (González‐Valenzuela et al. 2023; Moreira et al. 2024).

Cutinases play dual roles in cuticle degradation and immune modulation during fungal pathogenesis. During the early stages of infection, trace amounts of constitutively secreted cutinases directly mediate the strong adhesion of spores, such as those of Uromyces viciae‐fabae, to the host surface (Deising et al. 1992). Moreover, the released cutin monomers act as cross‐kingdom signalling molecules that induce appressorium formation and downstream pathogenicity‐related gene expression in Magnaporthe oryzae and Colletotrichum trifolii (Dickman et al. 2003; Skamnioti and Gurr 2007). During the intermediate stages of infection, pathogens secrete large amounts of inducible cutinases to extensively degrade the cuticle, thereby facilitating hyphal invasion. Deletion of core cutinase genes, such as SsCut1 and Pbc1, results in a marked reduction in pathogenicity or even complete loss of penetration ability in Sclerotinia sclerotiorum and Pyrenopeziza brassicae (Gong et al. 2022; Li et al. 2003). During the intermediate and late stages of infection, cutinases and their degradation products are also intimately involved in plant immune interactions. Free cutin fragments can function as DAMPs that trigger host defence responses, including reactive oxygen species (ROS) bursts (L'Haridon et al. 2011). In addition, some cutinases function as non‐enzymatic effectors. For example, RcCUT1 from Rhizoctonia cerealis and VdCUT11 from Verticillium dahliae directly induce host cell necrosis, thereby accelerating disease progression during the necrotrophic stage (Gui et al. 2018; Lu et al. 2018). This functional pleiotropy, spanning enzymatic degradation and immune manipulation, highlights the highly evolved infection strategies of pathogenic fungi (Arya and Cohen 2022). However, the large‐scale production of extracellular degradative enzymes imposes a substantial metabolic burden. Accordingly, transcription of cutinase genes is typically regulated by fungus‐specific Zn(II)₂Cys₆ transcription factors, termed cutinase transcription factors (CTFs). In Fusarium solani f. sp. pisi, CTF1α is activated by trace amounts of plant‐derived cutin monomers and specifically binds the cut1 promoter to initiate transcription (Li et al. 2002). This regulatory mechanism appears to be highly conserved among filamentous fungi. In Aspergillus nidulans, the homologous transcription factors FarA and FarB respond to cutin degradation products of different chain lengths and regulate distinct sets of hydrolases (Hynes et al. 2006). In M. oryzae and Aspergillus flavus, the homologues FAR1/2 and FarA/B directly regulate the secretion of epidermis‐degrading enzymes, including cutinases, and serve as core virulence determinants for tissue penetration and colonization (Luo et al. 2016; bin Yusof et al. 2014). Notably, CTF1 is dispensable for root infection in the soil‐borne fungus Fusarium oxysporum (Rocha et al. 2008), suggesting that this regulatory module is adapted to specific infection microenvironments. Current studies have primarily focused on the direct regulation of downstream cutinase genes by CTFs. However, pathogenic fungi also encounter a host‐derived ROS burst during cuticle penetration. Whether upstream stress‐responsive regulators, such as heat shock transcription factors (HSFs), can sense ROS signals and coordinate antioxidant defence with the cuticle degradation module (CTF–CUT) remains an unresolved molecular question.

The ROS burst triggered by plant basal immunity constitutes the primary biochemical barrier encountered by pathogenic fungi during host cuticle penetration. To colonize this highly oxidative microenvironment, fungi depend on global stress‐response networks. HSFs are highly conserved transcriptional regulators in eukaryotes. They not only mediate heat stress responses but also serve as central hubs for sensing and scavenging excess ROS. In Saccharomyces cerevisiae , HSF1 directly coordinates with oxidative stress pathways to promote ROS scavenging (Raitt et al. 2000). This antioxidant mechanism appears to be highly conserved during the evolution of plant‐pathogenic fungi (Park and Son 2024; Sheng et al. 2015). Current studies indicate that the virulence of multiple plant pathogens depends heavily on HSF‐mediated ROS detoxification systems. In Phytophthora sojae, the transcription factor PsHSF1 is essential for detoxifying the host‐derived oxidative burst and maintaining root colonization capacity (Sheng et al. 2015). In S. sclerotiorum, the SsHsf1–Snf5 module promotes pathogenesis by precisely regulating ROS homeostasis (Xiao et al. 2024). Furthermore, the regulatory scope of HSFs extends beyond antioxidant networks to encompass pathogenic development and metabolic pathways. For instance, CgHSF1 in Colletotrichum gloeosporioides directly controls the formation of specialized infection structures, such as appressoria (Gao et al. 2022). In addition, HsfA in Aspergillus fumigatus regulates the cell wall integrity (CWI) pathway and complex lipid metabolism (Fabri et al. 2023).

Our previous study established the ApCtf1β2–ApCUT3 module as a key pathogenic regulatory module that enables A. phaeospermum to breach the host cuticular barrier (Yan et al. 2026). However, how this pathogen senses the host‐derived ROS burst and activates the degradation pathway during early infection remains unclear. Here, we identified the core heat shock transcription factor ApHSF through yeast one‐hybrid (Y1H) screening. Electrophoretic mobility shift (EMSA) and dual‐luciferase reporter (DLR) assays demonstrated that ApHSF directly binds the ApCtf1β2 promoter and activates its transcription. In contrast, yeast two‐hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays detected no direct protein–protein interaction between ApHSF and ApCUT3, whereas Y1H and DLR assays detected neither binding of ApHSF to the ApCUT3 promoter nor independent promoter activation by ApHSF. Yeast three‐hybrid (Y3H) and combinatorial DLR assays further revealed that ApHSF enhances ApCtf1β2‐mediated activation of ApCUT3. In vivo phosphorylation analyses additionally uncovered the post‐translational regulation and dynamic activation of the ApHSF–ApCtf1β2–ApCUT3 module in response to host ROS‐mediated immunity. Collectively, these findings define the ApHSF–ApCtf1β2–ApCUT3 transcriptional cascade and reveal a molecular strategy by which pathogenic fungi coordinate ROS scavenging with cuticle degradation to ensure successful infection under host immune pressure.

2. Results

2.1. ApHSF Specifically Binds to the ApCtf1β2 Promoter and Activates Its Expression

We performed 5′ promoter deletion analysis in combination with β‐glucuronidase (GUS) activity assays. Deletion of the ApCtf1β2 promoter region from −1500 to −1000 bp significantly reduced GUS activity to near‐basal levels (Figure 1A), indicating that this 500‐bp region contains the critical cis‐acting elements required for ApCtf1β2 transcription. Using this fragment as bait in a Y1H library screen, we identified 40 candidate interacting proteins, including the heat shock transcription factor ApHSF (Figure S1 and Table S2). Direct Y1H assays confirmed this interaction. Yeast co‐transformed with pGADT7‐ApHSF and pAbAi‐ApCtf1β2‐Pro grew normally on SD/−Leu medium supplemented with 200 ng/mL aureobasidin A (AbA) (Figure 1B). Motif prediction analysis revealed that this region contains typical HSF and bZIP binding motifs (Figure 1C). We then performed site‐directed mutagenesis followed by Y1H assays to validate the predicted binding sites. When the core HSF motif in the target sequence was mutated to GGGGGG (D4), yeast growth was abolished (Figure 1D). In contrast, ApHSF did not interact with the predicted bZIP motifs within this region (Figure 1E). These results indicate that CTAGAA is the core cis‐element specifically recognized by ApHSF. In vitro electrophoretic mobility shift assay (EMSA) further confirmed that recombinant MBP‐ApHSF specifically bound to the probe containing the HSF motif. In addition, the unlabelled competitor probe effectively abolished the mobility shift (Figure 1G). Molecular docking analysis provided a predictive structural model of the ApHSF–CTAGAA complex and suggested several potential hydrogen‐bonding contacts at the protein–DNA interface (Figure S2). DLR assays showed that co‐transfection of 62SK‐ApHSF and LUC‐ApCtf1β2‐Pro significantly increased relative luciferase activity (Figure 1F). Taken together, these results demonstrate that ApHSF positively regulates ApCtf1β2 expression by specifically binding to the CTAGAA core motif in its promoter. This regulatory interaction likely plays an important role during infection by A. phaeospermum.

FIGURE 1.

ApHSF specifically binds to and activates the ApCtf1β2 promoter. (A) GUS activity analysis of 5′ deletion fragments of the ApCtf1β2 promoter. The left panel shows the promoter deletion constructs and the distribution of predicted transcription factor‐binding motifs. The right panel shows the corresponding GUS staining and relative enzymatic activity (n = 3). (B) Yeast one‐hybrid (Y1H) assay showing the interaction between ApHSF and the ApCtf1β2 promoter. pGADT7‐ApHSF and pAbAi‐ApCtf1β2‐Pro were co‐transformed into the Y1H strain. Transformants were plated on SD/−Leu medium supplemented with 200 ng/mL aureobasidin A (AbA) for selection. Co‐transformation of the empty pGADT7 vector with pAbAi‐ApCtf1β2‐Pro served as the negative control. (C) MEME Suite motif analysis showing the sequence features of the predicted HSF‐ and bZIP‐binding sites in the ApCtf1β2 promoter region. (D) Y1H assay of site‐directed mutations in the core HSF‐binding motif. D1 represents the wild‐type motif (CTAGAA), whereas D2–D4 represent site‐directed mutant sequences. (E) Y1H assay of site‐directed mutations in the predicted bZIP‐binding motif. D1 represents the wild‐type sequence (CCACGGTGA), whereas D2–D4 represent site‐directed mutant sequences. (F) Dual‐luciferase reporter (DLR) assay showing transcriptional activation of the ApCtf1β2 promoter by ApHSF. 62SK‐ApHSF and LUC‐ApCtf1β2‐Pro were co‐expressed in Nicotiana benthamiana leaves. Co‐expression of the empty 62SK and LUC vectors served as the negative control. The bar graph shows the quantification of relative luciferase activity (n = 3). (G) In vitro electrophoretic mobility shift assay (EMSA) verifying the binding of ApHSF to the CTAGAA motif in the ApCtf1β2 promoter. The wild‐type probe contained the CTAGAA core motif, whereas the mutant probe (mPr) contained a GGGGGG substitution. Each binding reaction contained 2 μg purified MBP‐ApHSF protein and 20 fmol 5′‐biotin‐labelled double‐stranded probe. MBP alone served as the protein negative control. Unlabelled wild‐type probes were added at 10‐, 20‐, and 50‐fold molar excesses for competition assays, and the labelled mutant probe was used to evaluate the sequence dependence of binding. Data in the bar graphs (A, F) are presented as means ± SD (n = 3). Different lowercase letters indicate significant differences among treatment groups (one‐way ANOVA followed by Tukey's HSD test, p < 0.05).

2.2. ApHSF Positively Regulates Multiple Stress Responses and Intracellular ROS Homeostasis in A. phaeospermum

Yeast transcriptional activation assays confirmed that ApHSF functions as a transcriptional activator. Yeast transformants harbouring pGBKT7‐ApHSF grew normally and turned blue on amino acid‐deficient medium (SD/−Trp‐His) (Figure 2A). Confocal microscopy revealed that the red fluorescence signal of the ApHSF‐mCherry fusion protein was predominantly localized in the nucleus and overlapped with the DAPI signal (Figure 2B). These results confirm the nuclear localization of ApHSF. Phenotypic analyses showed that, compared with the wild‐type (WT) and complemented (ΔApHSF+) strains, the ΔApHSF mutant exhibited significantly increased sensitivity to high temperature (30°C), oxidative stress (H₂O₂ and menadione), cell wall‐perturbing agents (Congo Red [CR], SDS, and Calcofluor White [CFW]), and osmotic stress (KCl, NaCl, and sorbitol) (Figure 2C,D). Furthermore, its growth rate was significantly reduced under carbon‐deficient (MM − C) and nitrogen‐deficient (MM − N) conditions. Quantitative ROS analysis indicated that high‐temperature and oxidative stress treatments resulted in significantly greater intracellular ROS accumulation in the ΔApHSF mutant than in the wild‐type (WT) strain (Figure 2E). These findings indicate that ApHSF is essential for maintaining cell wall integrity, antioxidant defence, and osmotic stress tolerance in A. phaeospermum. Reverse transcription‐quantitative PCR (RT‐qPCR) analysis showed that high‐temperature (HT) and oxidative (menadione) stresses significantly induced the expression of several heat shock protein genes (HSP70, HSP90, and HSP104) and antioxidant enzyme genes (SOD1, CAT1, and GPX1) in the WT and complemented strains. However, induction of these target genes was significantly attenuated in the ΔApHSF mutant (Figure 2F). Collectively, these results indicate that the transcription factor ApHSF mediates multiple stress‐response and nutrient‐adaptation processes in A. phaeospermum by regulating downstream target genes, including heat shock proteins and antioxidant enzymes.

FIGURE 2.

Subcellular localisation of ApHSF and its roles in multiple environmental stress responses. (A) Yeast transcriptional activation assay. Growth and colour development of pGBKT7‐ApHSF transformants were assessed on dropout medium (SD/−Trp−His) with or without X‐α‐gal. pGBKT7‐53 and the empty pGBKT7 vector served as the positive and negative controls, respectively. (B) Subcellular localisation of the ApHSF‐mCherry fusion protein in hyphae. DAPI was used for nuclear staining (scale bar = 10 μm). (C, D) Mycelial growth phenotypes of the wild‐type (WT), knockout mutant (ΔApHSF), and complemented strain (ΔApHSF+) under various stress conditions (C). These conditions included different temperatures, oxidative stress (H₂O₂ and menadione), cell wall stress (Congo Red [CR], SDS, and Calcofluor White [CFW]), osmotic stress (KCl, NaCl, and sorbitol), and nutrient‐deficient media (MM, MM − N, and MM − C). The corresponding quantitative analysis of mycelial growth inhibition is shown in (D). (E) Reactive oxygen species (ROS) staining and heatmap analysis of the indicated strains under high‐temperature (30°C) and oxidative stress (40 mM H₂O₂ or 30 μM menadione) treatments. (F) Reverse transcription‐quantitative PCR analysis of the relative expression levels of heat shock protein genes (HSP70, HSP90, and HSP104) and antioxidant enzyme genes (SOD1, CAT1, and GPX1) in each strain (n = 3). Expression was analysed under normal culture conditions (CK) and after induction by high temperature (HT) or oxidative stress (menadione). Data in the bar graphs (D, F) are presented as means ± SD. Different lowercase letters indicate significant differences among treatment groups (one‐way ANOVA followed by Tukey's HSD test, p < 0.05).

2.3. ApHSF‐Mediated ROS Detoxification Is Crucial for Successful Infection by A. phaeospermum

Pathogenicity assays showed that, under normal inoculation conditions, the lesion area caused by the ΔApHSF mutant on young hybrid bamboo stems was significantly smaller than that caused by the WT and complemented (ΔApHSF+) strains. However, when host ROS production was blocked using the NADPH oxidase inhibitor diphenyleneiodonium (DPI), the pathogenicity defect of the ΔApHSF mutant was significantly rescued (Figure 3A,B). To provide an independent genetic test complementary to the DPI‐based pharmacological inhibition, we compared the pathogenicity of these strains on WT Arabidopsis thaliana Col‐0 and the ROS burst‐defective rbohD mutant. Lesion expansion caused by the ΔApHSF mutant was severely restricted on Col‐0 leaves but was significantly enhanced on rbohD leaves (Figure 3C,D). This genetic evidence supports the conclusion that attenuation of the host ROS burst alleviates the pathogenicity defect associated with loss of ApHSF. Histochemical ROS detection by 3,3′‐diaminobenzidine (DAB) staining during infection showed that, from 12 to 72 h post‐inoculation (hpi), the WT and complemented strains effectively scavenged infection‐induced ROS. In contrast, tissues inoculated with the ΔApHSF mutant exhibited extensive dark‐brown staining of ROS deposition (Figure 3E). Quantitative H₂O₂ measurements confirmed that H₂O₂ levels in tissues inoculated with the ΔApHSF mutant increased sharply at 12 h post‐inoculation (hpi) and remained significantly higher than those in WT‐inoculated tissues throughout the subsequent stages (12–72 hpi) (Figure 3F). These findings indicate that the mutant is severely impaired in its ability to scavenge host‐derived ROS during the host immune response. Expression profiling during infection showed that, during the critical infection window (12–24 hpi), heat shock genes (HSP70, HSP90, and HSP104) were upregulated in the WT and complemented strains. In addition, antioxidant enzyme genes (SOD1, CTA1, and GPX1) showed even stronger induction. However, induction of these target genes was markedly attenuated in the ΔApHSF mutant throughout infection (Figure 3G). Together, the pharmacological, genetic, and biochemical evidence indicates that ApHSF promotes fungal pathogenicity by activating downstream antioxidant systems and counteracting the host‐derived ROS burst during infection.

FIGURE 3.

ApHSF enables the pathogen to overcome the host reactive oxygen species (ROS) burst and maintain full pathogenicity. (A, B) Pathogenicity phenotypes (A) and quantitative analysis of lesion areas (B) of the wild‐type (WT), knockout mutant (ΔApHSF), and complemented strain (ΔApHSF+) on young hybrid bamboo stems under normal inoculation (CK, control) and NADPH oxidase inhibitor (diphenyleneiodonium, DPI) treatment (n = 6, scale bar = 2 mm). (C, D) Pathogenicity phenotypes (C) and quantitative analysis of lesion areas (D) of each strain on leaves of wild‐type Arabidopsis thaliana (Col‐0, control) and the ROS burst‐defective mutant rbohD (n = 6, scale bar = 2 mm). (E) 3,3′‐diaminobenzidine (DAB) histochemical staining of plant leaves at different infection time points (0–72 h post‐inoculation [hpi]). Dark‐brown regions indicate ROS accumulation within the tissues. (F) Quantification of H₂O₂ levels in plant tissues at the corresponding infection time points (n = 3). (G) Heatmap showing temporal changes in the relative expression levels of heat shock protein genes (HSP70, HSP90, and HSP104) and core antioxidant enzyme genes (SOD1, CAT1, and GPX1) in the WT and ΔApHSF mutant during infection (0–72 hpi). Quantitative data (B, D, F) are presented as means ± SD. Different lowercase letters indicate significant differences among treatment groups (one‐way ANOVA followed by Tukey's HSD test, p < 0.05).

2.4. ApHSF Acts as a Cofactor to Enhance ApCtf1β2‐Mediated Activation of the Downstream Target Gene ApCUT3

Y2H analysis showed that transformants co‐expressing ApHSF and ApCUT3 grew on double‐dropout medium (SD/−Leu/−Trp). However, these transformants failed to grow on quadruple‐dropout medium (SD/−Leu/−Trp/−His/−Ade) and showed no X‐α‐gal colour development (Figure 4A). Bimolecular fluorescence complementation (BiFC) assays further showed that co‐expression of ApHSF and ApCUT3 in Nicotiana benthamiana epidermal cells did not produce specific YFP interaction signals (Figure 4B). To determine whether ApHSF directly regulates the ApCUT3 promoter, direct Y1H and DLR assays were performed. Yeast co‐transformed with pGADT7‐ApHSF and pAbAi‐ApCUT3‐Pro grew normally on SD/−Leu medium but failed to grow on SD/−Leu medium supplemented with 200 ng/mL AbA, similar to the negative control (Figure 4C). These results indicate that no direct interaction between ApHSF and the ApCUT3 promoter was detected under the tested conditions. DLR assays further showed that co‐expression of 62SK‐ApHSF and LUC‐ApCUT3‐Pro did not significantly alter relative luciferase activity compared with the empty‐effector control (Figure 4D). Taken together, these results indicate that ApHSF neither detectably binds to nor independently activates the ApCUT3 promoter under the tested conditions. Together, these in vitro and in vivo assays indicate that ApHSF does not directly interact with the cutinase ApCUT3. Y3H assays were performed in the presence of relatively high concentrations of AbA (200 and 400 ng/mL) to suppress background autoactivation. Under these conditions, the reporter gene was moderately activated by co‐expression of ApCtf1β2 with the ApCUT3 promoter reporter. Co‐expression of ApHSF significantly enhanced this activation. Moreover, addition of 0.15 mM H₂O₂ to simulate an oxidative microenvironment further enhanced this activation (Figure 4E,F). DLR assays showed that expression of ApCtf1β2 alone moderately activated the ApCUT3 promoter under normal conditions at 25°C. Co‐expression of ApHSF with ApCtf1β2 significantly enhanced this activation. Addition of 0.2 mM H₂O₂ to mimic the ROS burst during early infection further enhanced this activation, resulting in peak relative luciferase activity (Figure 4G,H). Taken together, these in vitro and in vivo results demonstrate that ApHSF does not directly activate the downstream target gene. Instead, under early oxidative stress (H₂O₂), ApHSF acts as a transcriptional cofactor to potentiate ApCtf1β2‐mediated activation of the pathogenicity gene ApCUT3.

FIGURE 4.

ApHSF cooperates with ApCtf1β2 under oxidative stress to activate transcription of the pathogenicity gene ApCUT3. (A) Yeast two‐hybrid (Y2H) assay testing the direct physical interaction between ApHSF and ApCUT3. Constructed bait (pGBKT7‐ApHSF or pGBKT7‐ApCUT3) and prey (pGADT7‐ApCUT3 or pGADT7‐ApHSF) vectors were co‐transformed into the yeast strain. Growth and colour development were assessed on double‐dropout (SD/−Leu/−Trp) and quadruple‐dropout (SD/−Leu/−Trp/−His/−Ade) media supplemented with 20 ng/mL X‐α‐gal. pGBKT7‐53 + pGADT7‐T and pGBKT7‐Lam + pGADT7‐T served as the positive and negative controls, respectively. (B) Bimolecular fluorescence complementation (BiFC) assay testing the interaction between ApHSF and ApCUT3 in Nicotiana benthamiana epidermal cells. Agrobacterium carrying different recombinant plasmids [pSPYNE(R)173 and pSPYCE(M)] was co‐infiltrated into leaves according to the indicated combinations. pSPYNE(R)173‐bZIP63 + pSPYCE(M)‐bZIP1 served as the positive control. Specific YFP interaction signals were observed under UV illumination. DIC indicates the corresponding bright‐field view. (C) Yeast one‐hybrid (Y1H) assay testing the interaction between ApHSF and the ApCUT3 promoter. pGADT7‐ApHSF and pAbAi‐ApCUT3‐Pro were co‐transformed into the Y1H strain. Transformants were plated on SD/−Leu medium with or without 200 ng/mL aureobasidin A (AbA). Co‐transformation of the empty pGADT7 vector with pAbAi‐ApCUT3‐Pro served as the negative control. (D) Dual‐luciferase reporter (DLR) assay testing the transcriptional activation of the ApCUT3 promoter by ApHSF. 62SK‐ApHSF and LUC‐ApCUT3‐Pro were co‐expressed in N. benthamiana leaves. Co‐expression of the empty 62SK vector and LUC‐ApCUT3‐Pro served as the negative control. The bar graph shows the quantification of relative luciferase activity (LUC/REN ratio; n = 3). (E, F) Qualitative growth phenotypes (E) and quantitative analysis of yeast culture OD values (F) showing the transcriptional synergistic effect in the yeast three‐hybrid (Y3H) system (n = 3). The pAbAi plasmid carrying the ApCUT3 promoter was used as the reporter vector. This construct was co‐transformed with AD‐Bridge vectors carrying different effector proteins into yeast. AbA (200 or 400 ng/mL) was added to SD/−Leu selection medium to suppress autoactivation, and 0.15 mM H₂O₂ was included to mimic oxidative stress. (G, H) DLR assay showing the efficiency of transcriptional activation in vivo in N. benthamiana. Effector vectors (empty 62SK or recombinant 62SK plasmids carrying target genes) and the reporter vector containing the ApCUT3 promoter (LUC‐ApCUT3‐Pro) were co‐infiltrated into leaves. (G) shows in vivo fluorescence imaging of infiltration sites under normal conditions (25°C) and oxidative stress conditions supplemented with 0.2 mM H₂O₂. (H) shows the corresponding quantitative analysis of relative luciferase activity (LUC/REN ratio) (n = 3). Quantitative data in the bar graphs (D, F) are presented as means ± SD. Different lowercase letters indicate significant differences among treatment groups (one‐way ANOVA followed by Tukey's HSD test, p < 0.05).

2.5. The ApHSF–ApCtf1β2–ApCUT3 Module Synergistically Coordinates Fungal ROS Scavenging and Host Cuticle Penetration

In vitro growth assays performed on potato dextrose agar (PDA) supplemented with 40 mM H₂O₂ or 0.2 mM menadione showed that the ΔApHSF mutant exhibited markedly impaired growth. Quantitative ROS measurements following short‐term treatment with 40 mM H₂O₂ or 30 μM menadione revealed substantial intracellular ROS accumulation in this mutant. Overexpression of ApCtf1β2 in the ΔApHSF background (ΔApHSF + ApCtf1β2) significantly restored growth and reduced ROS accumulation. These results indicate that ApCtf1β2 provides partial compensation for antioxidant capacity. By contrast, overexpression of the downstream cutinase gene ApCUT3 in the ΔApHSF background (ΔApHSF + ApCUT3) failed to rescue the growth defect. Conversely, overexpression of ApHSF in the double‐knockout background (ApHSF+ΔApCtf1β2/ΔApCUT3) restored antioxidant capacity to near‐WT levels (Figure 5A–C). This finding confirms that ApHSF has a ROS‐scavenging function independent of the downstream ApCtf1β2–ApCUT3 module. To further examine whether ApHSF affects additional infection‐associated genes, 25 candidates were selected by integrating the A. phaeospermum genome annotation with our previously generated A. phaeospermum–hybrid bamboo interaction transcriptome. The gene IDs and NR annotations of these 25 candidate genes are provided in Table S3. These genes represented five functional categories and displayed distinct temporal expression patterns during the pathogen–host interaction (Figure S6A). RT‐qPCR analysis showed that deletion of ApHSF reduced or delayed the infection‐associated induction of multiple candidate genes, particularly those associated with ROS detoxification and protein homeostasis, whereas most altered expression patterns were fully or partially restored in the ΔApHSF+ strain (Figure S6B). These results indicate that ApHSF‐dependent transcriptional responses extend beyond the characterized ApCtf1β2–ApCUT3 module. Arabidopsis inoculation assays showed that virulence of the ΔApHSF mutant and downstream pathway knockout strains was significantly reduced on Col‐0 leaves. On the rbohD mutant, virulence of the ΔApHSF strain was significantly restored, whereas the double‐knockout strain (ΔApCtf1β2/ΔApCUT3) failed to recover (Figure 5D,E). These results suggest that ApHSF‐mediated ROS scavenging is essential for establishment of early infection. However, after suppression of host ROS defences, the pathogen still depends specifically on the ApCtf1β2–ApCUT3 module to complete cuticle penetration. On young stems of B. pervariabilis × grandis, the WT strain caused extensive necrosis and abundant aerial hyphae. By contrast, combinatorial strains lacking the downstream ApCtf1β2–ApCUT3 module showed the most pronounced reduction in virulence. In these strains, lesion expansion was markedly restricted, and only localized browning was observed at the inoculation sites (Figure 5F,G). In vivo wheat germ agglutinin (WGA)/propidium iodide (PI) fluorescence staining showed that the WT strain established an extensive fungal network within host tissues, whereas the ΔApHSF, ΔApHSF + ApCUT3, and strains lacking the downstream ApCtf1β2–ApCUT3 module exhibited markedly reduced green fluorescence and lower green‐to‐red fluorescence intensity ratios. Expression of ApCtf1β2 in the ΔApHSF background partially restored the fluorescence signal (Figure 5H,I). Consistent with these observations, quantitative PCR (qPCR) analysis showed reduced fungal biomass in the ΔApHSF and ΔApHSF + ApCUT3 strains, partial recovery in ΔApHSF + ApCtf1β2, and the lowest biomass in ΔApCtf1β2/ΔApCUT3. Overexpression of ApHSF in the double‐knockout background resulted in only partial recovery (Figure 5J). These results indicate that the downstream ApCtf1β2–ApCUT3 module is required for efficient fungal colonization of host tissues. Infection‐stage expression profiling further showed that ApHSF, ApCtf1β2, and ApCUT3 were sequentially induced during the early, middle, and late stages of infection, respectively (Figure 5K). Together, these results indicate that ApHSF‐mediated ROS detoxification and the downstream ApCtf1β2–ApCUT3 module coordinately contribute to fungal infection and host tissue colonization.

FIGURE 5.

Functions of the ApHSF–ApCtf1β2–ApCUT3 cascade in oxidative stress tolerance and infection‐associated colonization. (A, C) Qualitative growth phenotypes under in vitro oxidative stress (A) and the corresponding quantitative analysis of mycelial growth inhibition (C) (n = 3). The wild‐type (WT) and the indicated combinatorial mutant strains were inoculated onto potato dextrose agar supplemented with 40 mM H₂O₂ or 0.2 mM menadione, and growth was recorded after 7 days of incubation at 25°C in the dark. (B) Microplate‐based colourimetric detection of intracellular reactive oxygen species (ROS) accumulation in hyphae and heatmap analysis of the corresponding absorbance values (n = 3). (D, E) Pathogenicity phenotypes from in vivo Arabidopsis thaliana inoculation assays (D) and the corresponding quantitative analysis of lesion areas (E) (n = 6). Each combinatorial strain was inoculated onto leaves of wild‐type A. thaliana (Col‐0) and the ROS burst‐defective mutant rbohD. (F, G) Pathogenicity phenotypes on young hybrid bamboo ( Bambusa pervariabilis × grandis) stems (F) and the corresponding quantitative analysis of lesion areas (G) (n = 6). Necrotic lesion expansion and aerial hyphae formation caused by each strain were documented. (H, I) In situ WGA fluorescence staining under confocal laser scanning microscopy (CLSM) showing fungal infection networks (H) and the corresponding quantitative analysis of green‐to‐red fluorescence intensity ratios (I) (n = 3). Fungal hyphae were labelled with a chitin‐specific WGA–Alexa Fluor probe and are shown in green, whereas host cell walls and nuclei were counterstained with propidium iodide (PI) and are shown in red. The WGA‐to‐PI fluorescence intensity ratio was used as a relative fungal colonization index. (J) Relative fungal biomass in infected hybrid bamboo tissues determined by quantitative PCR (qPCR) using a fungal‐specific gene normalized to a host reference gene (n = 6). Values were normalized to the WT‐inoculated group. (K) Reverse transcription‐qPCR analysis of transcription of core target genes during infection (n = 3). Infected host tissues were collected from 0 to 72 h post‐inoculation (hpi). Relative expression levels (fold change [FC]) of ApHSF, ApCtf1β2, and ApCUT3 were calculated using the 2^−ΔΔCt method and normalized to the 0 hpi control. Quantitative data in the bar graphs and box plots (C, E, G, I, J) are presented as means ± SD. Different lowercase letters indicate significant differences among treatment groups (one‐way ANOVA followed by Tukey's HSD test, p < 0.05).

2.6. Differential Expression and Phosphorylation Responses of the ApHSF–ApCtf1β2–ApCUT3 Module

RT‐qPCR analysis under cutin induction showed that treatment with 1 or 5 mg/mL cutin did not alter transcription of ApHSF. However, cutin treatment induced expression of the downstream genes ApCtf1β2 and ApCUT3 (Figure 6A). Immunoblot analysis showed that ApHSF protein levels remained constitutively stable under cutin induction. By contrast, ApCtf1β2 and ApCUT3 proteins showed strong inducible accumulation (Figure 6B). These results indicate that host‐derived cutin preferentially induces the downstream transcriptional and enzymatic branch of this module. Under heat stress (30°C) and oxidative stress induced by menadione, the ERK1/2, p38, and JNK mitogen‐activated protein kinase (MAPK) pathways all showed marked phosphorylation activation (Figure 6C–E). Moreover, this activation was effectively blocked by the corresponding pathway inhibitors U0126, SB203580, and SP600125. Immunoblotting showed that ApHSF displayed two basal bands under normal conditions. Under heat and oxidative stress, however, both ApHSF and ApCtf1β2 displayed clear mobility shifts accompanied by the appearance of slow‐migrating forms. Pathway inhibition analysis revealed that, under both heat and oxidative stress, only the p38 inhibitor SB203580 abolished the stress‐induced slow‐migrating forms of ApHSF and ApCtf1β2, restoring their basal banding patterns. By contrast, neither the ERK inhibitor U0126 nor the JNK inhibitor SP600125 had any detectable effect (Figure 6F). ApCUT3 showed no detectable phosphorylation‐dependent mobility shift, and its protein abundance remained largely unchanged during the short‐term stress treatments (Figure 6F). To verify whether these mobility shifts were phosphorylation‐dependent, protein extracts from untreated, heat‐treated, and menadione‐treated mycelia were subjected to λ‐protein phosphatase treatment followed by Phos‐tag SDS–PAGE analysis. λ‐Protein phosphatase treatment abolished the stress‐induced slow‐migrating forms of ApHSF and ApCtf1β2, and the signals shifted to their corresponding fast‐migrating forms. The upper basal band of ApHSF detected under untreated conditions also disappeared after phosphatase treatment (Figure 6G). The original uncropped immunoblot images corresponding to Figure 6B–G are provided in Figure S7. These results confirm that the mobility shifts of ApHSF and ApCtf1β2 were phosphorylation‐dependent and demonstrate that their stress‐induced phosphorylation depends on p38 MAPK activity, although direct phosphorylation by p38 MAPK remains to be established. To examine potential membrane‐associated components acting upstream of the p38 MAPK pathway, nine candidate proteins were identified by integrating genome annotation with signal‐peptide and membrane‐topology predictions. These candidates comprised five Wsc/Mid2‐like proteins, one Msb2/Hkr1‐like signalling mucin, one Sho1‐like protein, one Opy2‐like membrane component and one Sln1/TcsB‐like histidine kinase (Figure S8B and Table S4). Eight candidate genes showed detectable expression in the interaction transcriptome and exhibited distinct temporal expression patterns during infection, whereas ApWSC2‐like2 remained undetected under the conditions examined (Figure S8A). Four candidates representing different predicted signalling classes were further analysed by RT–qPCR following H₂O₂ treatment. ApWSC1 and ApSHO1 reached their highest transcript levels at 1 h, ApMUC‐like1 showed a progressive increase and peaked at 2 h, and ApSLN1‐like exhibited an earlier transient response that peaked at 0.5 h (Figure S8C). These results identify several oxidative‐stress‐responsive membrane‐associated candidates, although their direct roles in ROS perception and p38 MAPK activation remain unresolved. Together, these molecular data reveal functional specialization and differential regulatory mechanisms within the ApHSF–ApCtf1β2–ApCUT3 module in response to complex environmental signals. The upstream regulator ApHSF is constitutively expressed and is primarily controlled by stress‐induced, p38 MAPK activity‐dependent phosphorylation. The central hub ApCtf1β2 is subject to dual regulation through cutin‐induced expression and stress‐induced, p38 MAPK activity‐dependent phosphorylation. In contrast, ApCUT3 is not directly regulated through stress‐responsive phosphorylation. Host cutin serves as a major inductive signal for ApCUT3 expression, whereas oxidative stress indirectly enhances ApCUT3 promoter activation through the p38 MAPK‐dependent ApHSF–ApCtf1β2 regulatory module. The predicted membrane‐associated candidates extend this regulatory framework upstream of p38 MAPK but remain provisional components requiring functional validation. Based on these findings, we propose a regulatory model in which the p38 MAPK activity‐dependent ApHSF–ApCtf1β2–ApCUT3 cascade temporally coordinates detoxification of host‐derived ROS with penetration of the host cuticle during A. phaeospermum infection (Figure 7).

FIGURE 6.

Differential expression and phosphorylation of the ApHSF–ApCtf1β2–ApCUT3 module induced by host cutin and specifically mediated by p38 MAPK. (A) Reverse transcription‐quantitative PCR analysis of transcription of core target genes under cutin induction (n = 3). Hyphae were treated with 1 or 5 mg/mL cutin. Relative expression levels of ApHSF, ApCtf1β2, and ApCUT3 were calculated using the 2^−ΔΔCt method. (B) Immunoblot analysis of protein accumulation patterns of the module under cutin induction. After treatment with the indicated cutin concentrations, protein accumulation of ApHSF, ApCtf1β2, and ApCUT3 was analysed by immunoblotting. Actin served as the loading control. (C–E) Validation of MAPK pathway activation and inhibitor‐mediated suppression under environmental stress. Samples were exposed to heat stress (30°C) or menadione‐induced oxidative stress for 1 h. Specific kinase inhibitors (U0126, SB203580, and SP600125) or the corresponding solvent control (dimethyl sulphoxide [DMSO]) were applied as indicated. Phosphorylation levels of p‐ERK1/2 (C), p‐p38 (D), and p‐JNK (E) were detected using specific antibodies. (F) Analysis of post‐translational modification patterns of the module under environmental stress and MAPK pathway inhibition. Total proteins were extracted from the indicated stress and inhibitor treatment groups. Specific antibodies (α‐HSF1, α‐His, and α‐HA) were used to detect phosphorylation‐dependent mobility shifts of ApHSF and ApCtf1β2, as well as steady‐state protein levels of ApCUT3, respectively. (G) Phosphatase validation of the stress‐induced mobility shifts of ApHSF and ApCtf1β2. Protein extracts from untreated mycelia and mycelia exposed to heat stress (30°C) or menadione‐induced oxidative stress were treated with or without λ‐protein phosphatase, as indicated, followed by Phos‐tag SDS–PAGE and immunoblot analysis. Quantitative data in the bar graph (A) are presented as means ± SD. Different lowercase letters indicate significant differences among treatment groups for the same gene (one‐way ANOVA followed by Tukey's HSD test, p < 0.05).

FIGURE 7.

Regulatory model of the ApHSF–ApCtf1β2–ApCUT3 transcriptional cascade during Arthrinium phaeospermum infection. Early infection and antioxidant response: At the initial stage of infection, host immunity triggers a reactive oxygen species (ROS) burst. The pathogen senses this oxidative stress through the p38 MAPK pathway, which promotes phosphorylation of the transcription factors ApHSF and ApCtf1β2. Phosphorylated ApHSF activates the downstream antioxidant system to scavenge host‐derived ROS, thereby overcoming the host biochemical defence barrier. Intranuclear transcriptional cascade: ApHSF directly binds the CTAGAA motif in the ApCtf1β2 promoter and activates its transcription. Under early oxidative stress, although ApHSF does not directly bind ApCUT3, it acts as a cofactor to enhance ApCtf1β2‐mediated transcriptional activation of ApCUT3. Late infection and physical barrier breach: During the later stage of infection, the host cuticle and cutin‐derived signals specifically stimulate ApCUT3 expression. The secreted ApCUT3 protein (cutinase) degrades the host cuticle, thereby facilitating epidermal penetration and invasive colonization. Solid arrows indicate direct activation, signalling, or process progression. Dashed arrows indicate cooperative enhancement. The symbol labelled “No direct binding” indicates the absence of direct interaction between ApHSF and ApCUT3. Flat‐headed lines indicate inhibition or ROS‐scavenging effects.

3. Discussion

Breaching the host cuticle and overcoming the ROS burst triggered by pattern‐triggered immunity (PTI) are prerequisites for successful infection by plant‐pathogenic fungi. In this study, we elucidated the pathogenic regulatory mechanism of the ApHSF–ApCtf1β2–ApCUT3 transcriptional cascade in A. phaeospermum. Our results demonstrate that the heat shock transcription factor ApHSF maintains intracellular redox homeostasis and mediates detoxification of host‐derived ROS. In parallel, ApHSF specifically recognizes the CTAGAA motif in the ApCtf1β2 promoter and directly activates its transcription. Direct Y1H and DLR assays detected neither binding of ApHSF to the ApCUT3 promoter nor independent promoter activation by ApHSF under the tested conditions. However, under early oxidative stress and p38 MAPK‐mediated phosphorylation, ApHSF functions as a transcriptional cofactor to enhance ApCtf1β2‐mediated activation of ApCUT3. During infection, this module exhibits pronounced spatiotemporal specificity in gene expression and distinct functional specialization at the post‐translational level. Specifically, the upstream regulator ApHSF is constitutively expressed and regulated by phosphorylation to coordinate the early antioxidant response. By contrast, the downstream ApCtf1β2–ApCUT3 module specifically responds to host cutin signals to drive cuticle degradation and tissue penetration during the intermediate and late stages of infection. Consistent with the WGA/PI fluorescence patterns, qPCR‐based fungal biomass measurements further showed that disruption of the downstream ApCtf1β2–ApCUT3 module restricts fungal colonization within host tissues. Collectively, these findings define the biological functions of the ApHSF–ApCtf1β2–ApCUT3 cascade. They reveal a molecular strategy by which the fungus senses environmental cues and executes temporally coordinated regulation through this module. By coordinating early PTI suppression with cuticle degradation during the intermediate and late stages of infection, the pathogen ultimately achieves successful colonization.

HSF‐mediated stress‐response and antioxidant networks are highly conserved during fungal pathogen evolution (Andrási et al. 2021; Li et al. 2025; Xiao et al. 2022). Consistent with the role of HSF1 in maintaining intracellular ROS homeostasis and oxidative stress adaptation in fungi such as S. cerevisiae and S. sclerotiorum (Raitt et al. 2000; Sheng et al. 2015; Xiao et al. 2024; Zhou et al. 2018), deletion of ApHSF in A. phaeospermum significantly increased sensitivity to oxidative stress, including H₂O₂. This defect compromises the pathogen's ability to scavenge excess ROS generated by host pattern‐triggered immunity (PTI) during early infection. These findings support a central role for HSFs in counteracting early host defences in pathogenic fungi. Previous studies have primarily emphasized the individual roles of HSFs in stress adaptation and developmental regulation. These roles include regulating infection structure development in C. gloeosporioides (Gao et al. 2022) and maintaining cell wall integrity in A. fumigatus (Fabri et al. 2023). Consistent with this broader regulatory scope, the targeted expression analysis showed that deletion of ApHSF altered the infection‐stage expression of genes involved not only in ROS detoxification and protein homeostasis, but also in redox metabolism, stress signalling, and infection‐associated cellular functions (Figure S6). The recovery of most of these expression patterns in the complemented strain further supports a broader ApHSF‐dependent transcriptional response beyond the characterized ApCtf1β2–ApCUT3 module. Although ROS can function as environmental cues that promote pathogenic development in plant‐pathogenic fungi such as Magnaporthe grisea, Botrytis cinerea, and V. dahliae (Egan et al. 2007; Segmüller et al. 2008; Zhao et al. 2016), molecular evidence that HSFs directly regulate physical barrier degradation remains limited. Here, we show that ApHSF specifically binds the CTAGAA element in the promoter of the cutinase transcription factor ApCtf1β2 and activates its expression. This finding suggests that host‐derived ROS are not merely defence molecules but can also be exploited by the pathogen as environmental cues. Upon sensing this signal, ApHSF coordinates antioxidant responses to maintain intracellular homeostasis while simultaneously activating the ApCtf1β2–ApCUT3 cuticle degradation module. This transcriptional mechanism, which integrates antagonism of biochemical defences through ROS scavenging with dismantling of physical barriers through cuticle degradation, provides new molecular insight into how pathogenic fungi overcome multiple host defence layers (Arya et al. 2021; Jian et al. 2023; Wan et al. 2021).

Furthermore, this study systematically dissects the cascade regulatory mechanism governing cutinase expression. Building on the established CTF‐mediated transcriptional regulation of cutinases, we reveal a synergistic regulatory relationship between oxidative stress responses and cuticle degradation. Fungal penetration of the plant cuticle depends heavily on the secretion of cutinases (Arya and Cohen 2022). Their transcription is typically regulated by fungus‐specific Zn(II)₂Cys₆ zinc‐finger transcription factors, including CTF1α in F. solani f. sp. pisi (Li et al. 2002), FarA/FarB in A. nidulans (Bermúdez‐García et al. 2019), FAR1/2 in M. oryzae (bin Yusof et al. 2014), and members of the Far family in A. flavus (Luo et al. 2016). As an intermediate node in the cutinase regulatory network of A. phaeospermum, ApCtf1β2 activates the downstream pathogenicity gene ApCUT3, consistent with the conserved functions of CTF/Far family members in other fungi. However, the ApCtf1β2–ApCUT3 pathogenic module alone cannot explain how the pathogen simultaneously copes with the intense oxidative stress imposed by the host oxidative burst during early infection while coordinating initiation of the cuticle degradation programme. Building on the CTF‐centered regulatory framework, this study reveals a synergistic mechanism linking oxidative stress responses and cuticle degradation. Y2H and BiFC analyses detected no direct protein–protein interaction between ApHSF and ApCUT3. Y1H and DLR assays further detected neither binding of ApHSF to the ApCUT3 promoter nor independent promoter activation by ApHSF under the tested conditions. Nevertheless, Y3H and combinatorial DLR assays confirmed that, under H₂O₂ stress, ApHSF acts as a transcriptional cofactor to significantly enhance ApCtf1β2‐mediated activation of ApCUT3. Immunoblotting and inhibitor assays using the p38 MAPK inhibitor SB203580 further demonstrated that this synergistic transcriptional effect is regulated by p38 MAPK‐dependent phosphorylation. The Hog1/p38 MAPK pathway is a core signalling module that coordinates osmotic and oxidative stress responses in diverse fungi. For example, MoOsm1 in M. oryzae participates in hyperosmotic responses and adaptation to host‐derived ROS, whereas FgHog1 in Fusarium graminearum plays vital roles in mycelial growth, stress responses, and plant infection. These observations indicate that this pathway serves as a key hub through which pathogens integrate external stress signals and maintain infection‐associated adaptability (Dixon et al. 1999; Jiang et al. 2018; Liu et al. 2020; Zheng et al. 2012). To examine the possible upstream inputs of this pathway, we identified nine membrane‐associated signalling candidates belonging to the Wsc/Mid2, Msb2/Hkr1, Sho1, Opy2 and Sln1/TcsB classes. Most of the corresponding genes were expressed during infection, and ApWSC1, ApMUC‐like1, ApSHO1 and ApSLN1‐like displayed distinct transcriptional responses to H₂O₂ treatment. These results associate several predicted membrane components with the oxidative‐stress response of A. phaeospermum, but do not establish that they directly perceive ROS or activate p38 MAPK. The pathogen likely relies on membrane‐associated signalling components and the p38 MAPK pathway to convert early extracellular ROS cues into intracellular transcriptional outputs that initiate the ApHSF–ApCtf1β2 regulatory network. Subsequent cuticle degradation may release chemical signals such as cutin monomers, which can induce appressorium formation (Francis et al. 1996; Murray et al. 2013; Ryder and Talbot 2015). In addition, these degradation products may function as DAMPs that activate plant immunity or directly contribute to pathogenesis (Gui et al. 2018; Hou et al. 2019; de Lorenzo and Cervone 2022; Moreira et al. 2024). Accordingly, we propose a stage‐dependent regulatory model that integrates oxidative and cuticle‐derived signals. During early infection, the pathogen senses host‐derived ROS through the p38 MAPK cascade, which mediates phosphorylation of ApHSF and ApCtf1β2. During the intermediate and late stages of infection, ApCtf1β2 responds to cutin signals and activates ApCUT3 transcription, with activated ApHSF enhancing this transcriptional response. Compared with regulation by a single transcription factor, this p38 MAPK‐mediated cascade, which integrates dual environmental cues (ROS and cutin), coordinates the stage‐dependent expression of genes involved in oxidative‐stress adaptation and cuticle penetration.

In this study, we identified and systematically characterized the ApHSF–ApCtf1β2–ApCUT3 transcriptional regulatory network in A. phaeospermum, which is jointly driven by two host‐derived signals, ROS and cutin. Through stress‐induced phosphorylation requiring p38 MAPK activity and temporally coordinated transcriptional regulation, this module precisely integrates early ROS adaptation with subsequent cuticle penetration and tissue colonization. This coordination provides a mechanistic basis for infection establishment and pathogenicity development. These findings extend current understanding of how HSF‐associated regulation and cutinase activation are coupled during fungal infection. They also identify potential molecular entry points for future disease‐control strategies against bamboo shoot blight and related forest diseases. Although the core pathogenic functions of this transcriptional module have been clarified, several underlying molecular mechanisms remain to be resolved. First, targeted expression profiling of 25 representative genes indicated that ApHSF‐dependent transcriptional responses extend beyond the characterized ApCtf1β2–ApCUT3 module (Figure S6). However, this targeted analysis does not define the complete ApHSF regulon or distinguish direct ApHSF targets from indirect expression changes. Comparative RNA‐seq of the WT, ΔApHSF, and complemented strains during early infection, combined with genome‐wide ApHSF‐binding analysis and functional validation of key candidates, will therefore be required to resolve the broader ApHSF regulatory network. Second, bioinformatic screening and expression analyses identified nine putative membrane‐associated signalling components, several of which showed infection‐associated expression and distinct transcriptional responses to H₂O₂ treatment (Figure S8 and Table S4). However, these results do not establish which candidate, if any, directly perceives host‐derived ROS or acts upstream of p38 MAPK. Targeted deletion of representative candidates, followed by analysis of p38 MAPK phosphorylation and downstream ApHSF–ApCtf1β2 responses, will be required to define the membrane‐proximal components that initiate this signalling pathway. Third, given the trans‐acting synergistic properties of ApHSF, whether it dynamically remodels chromatin states to regulate transcription of downstream target genes remains to be determined.

4. Experimental Procedures

4.1. Experimental Materials

The A. phaeospermum strain used in this study is deposited in GenBank under accession number OK626768. The wild‐type (WT) strain is deposited in the China Forestry Culture Collection Center under accession number CFCC86860. All mutant strains were cultured on potato dextrose agar (PDA) at 25°C in the dark. One‐year‐old plants of hybrid bamboo ( B. pervariabilis × grandis) were obtained from Sichuan Agricultural University. These plants were approximately 1.0 m in height and had rhizomes approximately 0.5 m in length. The plants were maintained under ambient conditions with an annual mean temperature of 25.2°C and a relative humidity of 81%. Seeds of Arabidopsis thaliana (Col‐0), the ROS burst‐defective mutant rbohD, and N. benthamiana were purchased from AraShare Technology Co. Ltd. (China). These plants were grown in a greenhouse at 25°C and 60% relative humidity under a 16‐h light/8‐h dark photoperiod with a light intensity of 90 μmol m⁻² s⁻¹.

4.2. ApCtf1β2 Promoter Truncation and GUS Activity Assay

To define the core transcriptional regulatory region of the ApCtf1β2 promoter, the 2000‐bp sequence upstream of the start codon was designated as the full‐length promoter and serially truncated in 500‐bp increments from the 5′ end. The full‐length and truncated promoter fragments were individually cloned into the pCAMBIA1303 vector containing the gusA reporter gene. The recombinant plasmids were introduced into Agrobacterium tumefaciens AGL‐1 competent cells by the freeze–thaw method. Using the A. tumefaciens ‐mediated transformation (ATMT) method (Sun et al. 2023), recombinant Agrobacterium cells and WT mycelia were co‐cultured on medium supplemented with 200 μM acetosyringone (AS) for 48 h. The co‐cultures were subsequently transferred to PDA supplemented with 350 μg/mL hygromycin and 200 μg/mL cefotaxime for selection of positive transformants. Stable transformants were obtained after five rounds of single‐spore purification. For qualitative GUS activity analysis, fresh mycelia from each transformant were collected, incubated in X‐Gluc staining buffer at 37°C in the dark, and monitored for colour development. For quantitative GUS activity analysis, total mycelial protein was extracted from each strain, and fluorescence was measured in a microplate assay using 4‐methylumbelliferyl‐β‐D‐glucuronide (4‐MUG) as the substrate. Relative GUS activity was normalized to total protein concentration.

4.3. Construction of the Subcellular Localisation Vector and Microscopic Observation

Using pCAMBIA1303 as the backbone vector, the full‐length coding sequence of ApHSF was amplified. The stop codon was removed, and the amplified fragment was fused in frame with the mCherry coding sequence to generate the ApHSF‐mCherry subcellular localization vector. The recombinant vector was introduced into the A. phaeospermum WT strain using the ATMT method. Stable transformants were inoculated into potato dextrose broth (PDB) and cultured at 25°C in the dark for 3 days. Mycelia were then harvested and stained with DAPI for nuclear visualization. Fluorescence signals were observed using an LSM 880 confocal laser scanning microscope (Carl Zeiss), and images were acquired and merged using ZEN software.

4.4. Construction of Gene Knockout, Complementation, and Combinatorial Mutant Strains

All single‐ and double‐gene knockout, complemented, and combinatorial mutant strains were generated using the ATMT system. Based on homologous recombination, approximately 1.0‐kb flanking sequences upstream and downstream of each target gene were fused with hygromycin (Hyg) or G418 resistance cassettes and cloned into pCAMBIA1300 to generate knockout constructs. For complementation, a fragment containing the full‐length open reading frame (ORF) of ApHSF together with a bleomycin (Ble) resistance marker was cloned into pBHt2. To examine the temporal dependency within the ternary module, sequential transformations were performed using plasmids carrying different selectable markers to construct the downstream double‐knockout strain (ΔApCtf1β2/ΔApCUT3). Subsequently, overexpression vectors carrying the target genes were introduced into the corresponding mutant backgrounds to generate ΔApHSF + ApCtf1β2, ΔApHSF + ApCUT3, and the combinatorial mutant overexpressing the upstream gene in the double‐knockout background (ApHSF+ΔApCtf1β2/ΔApCUT3). A schematic overview of the construction strategy is provided in Figure S5. The resulting plasmids were introduced into A. tumefaciens AGL‐1 and co‐cultured with fungal mycelia for 48 h. Transformants were subsequently selected using antibiotics corresponding to the resistance markers carried by each vector. All primers used for vector construction and mutant identification are listed in Table S1. Molecular verification of the mutant strains is shown in Figure S5.

4.5. Stress Tolerance Analysis

To evaluate stress tolerance, uniform mycelial plugs (5 mm in diameter) were placed at the centre of PDA or minimal medium (MM) plates supplemented with different chemical agents. Stress conditions were as follows: oxidative stress (20 or 40 mM H₂O₂ or 0.2 mM menadione); cell wall stress (100 μg/mL Congo Red [CR], 0.01% SDS, or 100 μg/mL Calcofluor White [CFW]); osmotic stress (0.5 M KCl, 0.5 M NaCl, or 1 M sorbitol); and nutrient limitation using carbon‐deficient (MM − C) or nitrogen‐deficient (MM − N) medium. After 7 days of incubation at 25°C in the dark, colony diameters were measured using the cross‐line method, and growth inhibition rates were calculated. To detect intracellular H₂O₂ accumulation, fresh mycelia were collected after the following treatments: culture at 25°C for 3 days; heat stress at 30°C for 3 days; heat stress at 30°C for 1 day followed by recovery at 25°C for 2 days; or induction in PDB containing 40 mM H₂O₂ or 30 μM menadione for 1 h. For qualitative DAB staining, approximately 25 mg of mycelia was placed in a 24‐well plate containing 1 mL of DAB staining solution, incubated in the dark for 2 h, and photographed. In parallel, independently treated mycelial samples were used to prepare crude extracts, and intracellular H₂O₂ levels were determined using the titanium sulphate method.

4.6. Plant Pathogenicity Assay, ROS Histochemistry, and Microscopic Observation

Healthy young stems of hybrid bamboo or leaves of A. thaliana were selected for inoculation. A 10‐μL spore suspension (1 × 10⁶ spores/mL) was drop‐inoculated onto intact surfaces, and the inoculated tissues were maintained under humid conditions at 25°C and 80% relative humidity. For ROS‐blocking experiments, plant tissues were pretreated with the NADPH oxidase inhibitor diphenyleneiodonium (DPI) before inoculation. As an independent genetic validation of the contribution of the host ROS burst, the fungal strains were also inoculated onto leaves of A. thaliana Col‐0 and the ROS burst‐defective rbohD mutant under the same conditions. Between 0 and 72 h post‐inoculation (hpi), infected tissues were collected at the indicated time points for analysis of the ROS burst. ROS accumulation in leaves was qualitatively visualized by DAB histochemical staining, and images were captured after decolourization with 70% ethanol. Dynamic H₂O₂ levels in infected plant tissues were quantified using the titanium sulphate method. To visualize pathogen colonization and infection networks within host tissues, WGA–Alexa Fluor probes were used to specifically label fungal chitin, whereas propidium iodide (PI) was used to label plant cell walls and nuclei. Multichannel z‐stack images were acquired using a Zeiss LSM 880 confocal microscope, and the WGA‐to‐PI fluorescence intensity ratio was quantified using Fiji software as a relative fungal colonization index. For independent quantification of fungal biomass, total genomic DNA was extracted from infected hybrid bamboo tissues at 7 days post‐inoculation (dpi). Relative fungal biomass was determined by qPCR using the fungal Tubulin gene (AP‐Z1304754) and the bamboo Actin gene (evm.model.scaffold_7.1359) as the fungal and host targets, respectively. Relative fungal biomass was calculated using the 2^−ΔΔCt method and normalized to that of the WT‐inoculated group. Six independent biological replicates were analysed for each strain. The primers used for fungal biomass quantification are listed in Table S1.

4.7. Y1H, Y2H and Y3H Assays

Yeast hybrid assays (Y1H, Y2H, and Y3H) were performed strictly according to the manufacturer's instructions for the corresponding kits (OE Biotech). For Y1H assays, different truncated fragments or site‐directed mutant sequences of the ApCtf1β2 promoter, as well as the ApCUT3 promoter fragment, were cloned into the pAbAi vector, and ApHSF was inserted into pGADT7. Following transformation, cells were plated on SD/−Leu medium supplemented with 200 ng/mL AbA for selection. Strains co‐transformed with the corresponding pAbAi reporter and the empty pGADT7 vector served as negative controls. Following transformation, cells were plated on SD/−Leu medium supplemented with 200 ng/mL AbA for selection. Strains co‐transformed with the empty pGADT7 vector served as the negative control. For Y2H assays, ApHSF and ApCUT3 were cloned into pGBKT7 and pGADT7, respectively. The co‐transformed Y2H strains were plated on SD/−Leu/−Trp medium and SD/−Leu/−Trp/−His/−Ade medium supplemented with 20 ng/mL X‐α‐Gal to assess protein interactions and autoactivation. For Y3H assays, the pAbAi plasmid containing the ApCUT3 promoter served as the reporter vector, and the ORFs of ApHSF and ApCtf1β2 were combinatorially inserted into the AD‐Bridge vector (vector construction is shown in Figure S3). To simulate the oxidative microenvironment of early infection, 0.15 mM H₂O₂ was added to the selection system containing 200 or 400 ng/mL AbA. The efficiency of ternary synergistic transcriptional activation was quantitatively assessed by measuring the OD values of the yeast cultures.

4.8. Electrophoretic Mobility Shift Assay (EMSA)

The coding sequence of ApHSF was cloned into the pMAL‐c5x vector and introduced into Escherichia coli BL21 for recombinant protein expression. Following induction, the MBP‐ApHSF fusion protein was purified. A double‐stranded probe containing the wild‐type CTAGAA motif and a corresponding mutant probe in which CTAGAA was replaced by GGGGGG were synthesized by Sangon Biotech (Shanghai, China). The probes used for detection were labelled with biotin at the 5′ end, and their complete sequences are listed in Table S1. Each 20‐μL binding reaction contained 2 μg purified MBP‐ApHSF protein, 20 fmol biotin‐labelled double‐stranded probe, 10 mM Tris–HCl (pH 7.5), 50 mM KCl, 1 mM dithiothreitol (DTT), 5 mM MgCl₂, 2.5% glycerol, 0.05% NP‐40, and 50 ng/μL poly(dI–dC). The reaction mixtures were incubated at 25°C for 20 min. For competition assays, the unlabelled wild‐type probe was added at 10‐, 20‐, or 50‐fold molar excess before addition of the labelled probe. The labelled mutant probe was used to assess the sequence dependence of binding, and an equivalent amount of maltose‐binding protein (MBP) alone served as the protein negative control. Poly (dI–dC) was included as nonspecific competitor DNA, whereas no additional unrelated oligonucleotide competitor was used. Protein–DNA complexes were separated on a 6% native polyacrylamide gel in 0.5× TBE buffer and detected by chemiluminescence according to Wang et al. (2022).

4.9. BiFC and DLR Assays in N. benthamiana

For the BiFC assay, ApHSF and ApCUT3 were cloned into pSPYNE(R)173 and pSPYCE(M), respectively. The recombinant plasmids were introduced into A. tumefaciens GV3101 and co‐infiltrated into N. benthamiana leaves according to the indicated combinations. At 48 h post‐infiltration, specific YFP interaction signals in epidermal cells were observed under the YFP and differential interference contrast (DIC) channels using a confocal laser scanning microscope (Zeiss, LSM 880). For the DLR assay, target gene promoters (ApCtf1β2‐Pro or ApCUT3‐Pro) were cloned into pGreenII‐0800‐LUC, and effector coding sequences were cloned into pGreenII‐62‐SK. To assess the independent transcriptional activation of the ApCUT3 promoter by ApHSF, 62SK‐ApHSF or the empty 62SK vector was co‐infiltrated with LUC‐ApCUT3‐Pro into N. benthamiana leaves. At 48 h after infiltration with the mixed Agrobacterium suspension, some treatments were additionally exposed to 0.2 mM H₂O₂ to simulate oxidative stress, and D‐luciferin potassium salt was sprayed onto the leaves. After a 5‐min reaction, luminescence images were acquired using a Biolight PlantView100 in vivo imaging system. For quantitative analysis, tissues from infiltrated areas were collected, and firefly luciferase (LUC) and Renilla luciferase (REN) activities were measured using a Dual‐Luciferase Reporter Assay Kit (Vazyme) on a microplate reader (Promega GloMax 96). The relative LUC/REN ratio was calculated. Optimization of leaf infiltration and yeast concentration is shown in Figure S4.

4.10. Identification and Expression Analysis of Putative Membrane‐Associated Signalling Candidates

To identify putative membrane‐associated signalling components potentially acting upstream of the p38 MAPK pathway, the predicted proteome of A. phaeospermum was screened by integrating genome annotation with predicted membrane features. Proteins annotated as Wsc/Mid2‐, Msb2/Hkr1‐, Sho1‐, Opy2‐ or Sln1/TcsB‐like signalling components were retained for further analysis. Protein length, molecular mass and theoretical isoelectric point were calculated from the predicted amino acid sequences using the ProtParam module of Biopython. Classical signal peptides were predicted using SignalP 6.0 with the Eukarya model, whereas transmembrane helices and membrane topology were predicted using DeepTMHMM. Candidate names were provisionally assigned according to their genome annotations and predicted structural features. Expression values for the nine candidate genes were extracted from the previously generated A. phaeospermum–hybrid bamboo interaction transcriptome at four infection stages, corresponding to 0, 7, 14 and 21 days post‐inoculation (dpi). Three biological replicates were retained for each stage. Values recorded as not detected or NA were assigned a value of zero. For heatmap visualization, expression values from the 12 biological samples were subjected to row‐wise min–max normalization for each gene according to (x − min)/(max − min). The resulting values ranged from 0 to 1 and were used to visualize temporal expression patterns within each gene rather than to compare absolute transcript abundance among different genes.

4.11. Temporal and Inducible Gene Expression Analysis by RT‐qPCR

Total mycelial RNA was extracted using the TRIzol method under different treatment conditions (high temperature [30°C], 30 μM menadione, 40 mM H₂O₂, menadione stress, or induction with 1 or 5 mg/mL host cutin) and at different infection time points (0, 6, 12, 24, 48, and 72 hpi). For analysis of the oxidative‐stress responses of putative membrane‐associated signalling candidates, WT mycelia were cultured in PDB at 25°C for 3 days and subsequently treated with 40 mM H₂O₂. Mycelial samples were collected at 0, 0.5, 1, and 2 h after treatment, immediately frozen in liquid nitrogen and stored at −80°C until RNA extraction. Four representative genes, ApWSC1, ApMUC‐like1, ApSHO1, and ApSLN1‐like, were selected because they represented distinct predicted membrane‐signalling classes and showed detectable expression in the interaction transcriptome. Three independent biological replicates were analysed for each treatment time. After reverse transcription, cDNA was amplified on an Applied Biosystems 7500 instrument using TransScript Green One‐Step qRT‐PCR SuperMix (TransGen). The target genes analysed included stress‐related genes (HSP70, HSP90, HSP104, SOD1, CAT1, and GPX1) and cascade module genes (ApHSF, ApCtf1β2, and ApCUT3), and the four representative membrane‐associated candidate genes. For the H₂O₂‐responsive expression analysis, the untreated 0‐h sample was used as the calibrator for each candidate gene. For the targeted expression analysis, the genome annotation of A. phaeospermum was cross‐referenced with our previously generated transcriptomic dataset of the A. phaeospermum–hybrid bamboo interaction. Twenty‐five genes were selected according to their infection‐responsive expression patterns and functional annotations related to ROS detoxification, redox metabolism, stress signalling and transcriptional regulation, molecular chaperones and protein homeostasis, and cell wall‐, membrane‐, and infection‐associated functions. For heatmap visualization, the transcript abundance of each gene across the interaction‐transcriptome samples was independently normalized to a range of 0–1. The expression of these genes was further analysed in the WT, ΔApHSF, and ΔApHSF+ strains at 0, 6, 12, 24, 48, and 72 hpi. Three independent biological replicates were analysed for each strain and time point. The tubulin gene (TUB) was used as the internal reference for normalization in A. phaeospermum. Relative expression levels were calculated using the 2^−ΔΔCt method, and primer sequences are listed in Table S1.

4.12. Western Blot and Protein Phosphorylation Analysis

Total proteins were extracted from untreated strains, strains exposed to 30°C heat stress, or strains treated with menadione for western blotting. In kinase pathway inhibition assays, 20 μM U0126, 20 μM SB203580, or 20 μM SP600125 (MedChemExpress) was added to the culture system before stress treatment to selectively inhibit the corresponding MAPK pathway. Protein extracts were separated by SDS‐PAGE and transferred onto PVDF membranes. Phosphorylation levels of mitogen‐activated protein kinases were detected using anti‐p‐ERK1/2 (Phospho‐p44/42 MAPK, 4370, 1:2000; CST), anti‐p‐p38 (Phospho‐p38 MAPK, 4511, 1:1000; CST), and anti‐p‐JNK (Phospho‐SAPK/JNK, 4668, 1:1000; CST) antibodies. To detect stress‐induced mobility shifts of ApHSF and ApCtf1β2, including phosphorylation‐dependent mobility shifts, protein samples were separated on SuperSep Phos‐tag gels (Wako). For phosphatase validation, equal aliquots of protein extracts from untreated, heat‐treated, and menadione‐treated mycelia were adjusted to a final volume of 50 μL in 1× NEBuffer for Protein MetalloPhosphatases supplemented with 1 mM MnCl₂. Samples were incubated with 400 U of Lambda Protein Phosphatase (New England Biolabs) at 30°C for 30 min. Paired control samples without Lambda Protein Phosphatase were incubated under identical conditions. Following phosphatase treatment, the samples were separated by Phos‐tag SDS–PAGE. Before electrotransfer, the Phos‐tag gels were gently washed twice for 10 min in transfer buffer containing 10 mM EDTA, followed by one 10‐min wash in EDTA‐free transfer buffer, and were subsequently transferred onto PVDF membranes. Immunodetection was performed using anti‐Hsf1 (ABE1044, 1:2000; Sigma‐Aldrich), anti‐His (D291‐3, 1:5000; MBL), and anti‐HA (M180‐3, 1:5000; MBL) antibodies. Actin was detected in parallel by conventional SDS–PAGE using anti‐Actin (A2066, 1:5000; Sigma‐Aldrich) and served as the loading control. After incubation with goat anti‐rabbit IgG‐HRP (A9169, 1:5000; Sigma‐Aldrich) or the corresponding secondary antibodies, signals were visualized using a chemiluminescence imaging system (Tanon).

4.13. Statistical Analysis

All qualitative and quantitative experiments were performed with at least three independent biological replicates. Data analysis and visualization were performed using Excel, GraphPad Prism 9.5, TBtools, and IBM SPSS Statistics 25. Quantitative data are presented as means ± standard deviation (SD). For comparisons among multiple groups, one‐way ANOVA followed by Tukey's HSD test was used (p < 0.05). Comparisons between two groups were performed using Student's t‐test (p < 0.05 or p < 0.01). For RT‐qPCR analyses, statistical comparisons were performed using ΔC _t values rather than the 2^−ΔΔCt values used for graphical presentation. For the H₂O₂‐responsive expression analysis, treatment times were compared separately for each candidate gene using one‐way ANOVA followed by Tukey's HSD test. Different lowercase letters indicate significant differences among treatment times within the same gene at p < 0.05.

Author Contributions

Lin Li: visualization. Yutong Liu: methodology. Han Zhao: project administration. Shujiang Li: writing – review and editing, resources, funding acquisition. Peng Yan: writing – original draft, methodology, software. Sijia Liu: data curation. Zhanbo Liu: supervision.

Funding

This research was funded by the National Natural Science Foundation of China, grant number 32171795; the Young Talents of Sichuan Tianfu Qingcheng Program, grant number 2023SCQC1680.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: Yeast one‐hybrid (Y1H) library screening in Arthrinium phaeospermum and validation of the interaction between ApHSF and the ApCtf1β2 promoter. (A) Autoactivation test of the bait strain. The bait plasmid pAbAi‐ApCtf1β2‐Pro was transformed into the Y1HGold yeast strain and plated on SD/−Ura medium supplemented with different concentrations of aureobasidin A (AbA; 0, 100, 150, and 200 ng/mL). Colony growth was monitored to determine the minimum AbA concentration required to completely suppress background autoactivation; 200 ng/mL AbA was established as the optimal screening concentration. (B) Primary screening of the cDNA library. The A. phaeospermum cDNA prey library constructed in the pGADT7 vector was transformed into the bait strain described above and plated on SD/−Leu selective medium supplemented with 200 ng/mL AbA to screen for potential positive transformants. (C) Spot assay validation of primary positive clones. Candidate positive clones obtained from the primary screen were respotted onto SD/−Leu selective medium containing 200 ng/mL AbA for secondary screening. A total of 50 candidate positive clones grew normally, of which 40 were successfully identified by sequencing (see Table S2 for details). In the figure, “+” indicates the positive control (co‐transformation of pGADT7‐Rec53 and p53‐AbAi), whereas “−” indicates the negative control (co‐transformation of the empty pGADT7 vector and pAbAi‐ApCtf1β2‐Pro). (D) One‐to‐one validation of ApHSF. The sequenced pGADT7‐ApHSF plasmid was re‐co‐transformed with pAbAi‐ApCtf1β2‐Pro into the Y1HGold strain and plated on SD/−Leu medium with or without AbA (150 or 200 ng/mL) to exclude library background interference and confirm binding of ApHSF to the ApCtf1β2 promoter.

Figure S2: Predicted molecular docking model of the interaction between ApHSF and the CTAGAA motif in the ApCtf1β2 promoter. (A) Overall view of the predicted ApHSF–ApCtf1β2 promoter DNA complex. ApHSF is shown in green, whereas the promoter target sequence is shown as an orange‐yellow double helix. (B) Predicted three‐dimensional structure of the DNA‐binding domain (DBD) of ApHSF. (C) Detailed view of the putative protein–DNA interface in the docking model. Amino acid residues and nucleotide bases predicted to participate in the interaction are shown as red and orange sticks, respectively. Yellow dashed lines indicate predicted hydrogen‐bonding contacts, and the numbers indicate the corresponding calculated distances (Å). The model suggests potential contacts between PHE‐155 and DG‐85 (3.2 Å), TYR‐213 and DG‐85 (2.6 Å), LYS‐199 and DC‐86 (3.5 Å), SER‐205 and DT‐87 (2.8 Å), ARG‐208 and DG‐13 (2.4 and 2.6 Å), LYS‐217 and DA‐11 (3.3 Å), and LYS‐217 and DT‐10 (2.5 and 3.2 Å). These predicted contacts represent a possible binding configuration and require further experimental validation.

Figure S3: Schematic diagram of construction of the yeast three‐hybrid vector pGADT7‐Bridge. (A) Schematic diagram of construction of the single‐factor vectors. The transcription factor ApHSF or ApCtf1β2 was inserted into the MCS I site of the pGADT7 backbone vector to generate the corresponding basic vectors for subsequent modification of the bridge construct. (B) Insertion of the bridge expression cassette. A second expression cassette consisting of the MET17 promoter, MCS II, and the PGK1 terminator was directionally inserted into the pGADT7 backbone through the BamHI and XhoI sites, thereby introducing a second independent cloning site in addition to MCS I and generating the dual‐expression bridge vector framework. (C) Final structure of the pGADT7‐Bridge vector. The transcription factor ApHSF was inserted into MCS I, whereas ApCtf1β2 was inserted into MCS II, enabling their co‐expression in the same yeast cell for use in the yeast three‐hybrid system to evaluate the cooperative regulatory effect of ApHSF on ApCtf1β2‐mediated transcriptional activation of the target promoter.

Figure S4: Optimisation of stress conditions and selection of appropriate treatment concentrations for the Arthrinium phaeospermum, yeast, and Nicotiana benthamiana experimental systems. (A) Evaluation of heat stress tolerance of Arthrinium phaeospermum under different temperature gradients (25°C–37°C). The left panel shows colony morphology on potato dextrose agar (PDA) plates, whereas the line graph and bar graph on the right represent colony diameter (mm) and relative ion leakage (%), respectively, at the corresponding temperatures. The results established 30°C as an appropriate sublethal heat‐stress condition. (B) Qualitative and quantitative analyses of yeast responses to oxidative stress induced by different concentrations of H₂O₂ (0–0.25 mM). The upper bar graph shows optical density (OD) values measured in liquid culture, and the lower panel shows qualitative spot‐growth phenotypes of yeast suspensions under the corresponding concentrations. A concentration of 0.15 mM H₂O₂ was selected as the optimal condition for simulating the early oxidative microenvironment in the yeast three‐hybrid (Y3H) assay. (C) Assessment of membrane damage in Nicotiana benthamiana leaf discs treated with different concentrations of H₂O₂ (0–0.3 mM). The bar graph shows relative ion leakage of the leaf discs, and the lower panel shows browning and necrotic phenotypes of leaves after treatment with the indicated concentrations. The results established 0.2 mM H₂O₂ as an appropriate stress concentration for the in vivo dual‐luciferase reporter (DLR) assay. Quantitative data are presented as means ± standard deviation (SD). Different lowercase letters in the bar graphs or line graphs indicate significant differences among treatment groups (one‐way ANOVA followed by Tukey's HSD test, p < 0.05).

Figure S5: Construction strategy and molecular verification of target gene knockout, complementation, and combinatorial mutant strains. (A) Schematic diagrams of homologous recombination‐based strategies for gene knockout (upper) and complementation/overexpression (lower). LHA and RHA denote the approximately 1.0‐kb left and right homologous arms, respectively. Hyg and G418 represent the hygromycin and geneticin resistance cassettes, respectively, used to replace the target genes. Ble denotes the bleomycin resistance cassette. (B) PCR‐based molecular verification of the mutant strains. Transformants were verified by PCR using specific primer pairs targeting different selectable markers (Hyg, G418, and Ble) and the corresponding target fragments. The verified strains included the single‐gene knockout mutant (ΔApHSF, Hyg‐positive), the complemented strain (ΔApHSF+, Hyg‐ and Ble‐positive), overexpression strains in the knockout background (ΔApHSF + ApCUT3 and ΔApHSF + ApCtf1β2, Hyg‐ and Ble‐positive), the downstream double‐knockout mutant (ΔApCtf1β2/ΔApCUT3, Hyg‐ and G418‐positive), and the combinatorial mutant overexpressing the upstream gene in the double‐knockout background (ApHSF + ΔApCtf1β2/ΔApCUT3, Hyg‐, G418‐, and Ble‐positive). DL 5K DNA Marker is shown on the left.

Figure S6: Expression profiles of representative candidate genes associated with ApHSF‐mediated responses during infection. (A) Heatmap of 25 candidate genes selected by integrating the genome annotation of Arthrinium phaeospermum with the previously generated A. phaeospermum–hybrid bamboo interaction transcriptome. S1–S4 represent 0, 7, 14, and 21 days post‐inoculation (dpi), respectively, corresponding to 0, 1, 2, and 3 weeks after inoculation, with three biological replicates for each group. Transcript abundance was normalized relative to S1 and scaled from 0 to 1 for each gene. (B) Reverse transcription‐quantitative PCR analysis of the selected genes in the wild type (WT), ΔApHSF, and ΔApHSF+ strains at 0, 6, 12, 24, 48, and 72 h post‐inoculation (hpi). Relative transcript levels were calculated using the 2^−ΔΔCt method with TUB as the internal reference, and the heatmap shows the mean values of three independent biological replicates. In the functional annotation bar, A–E indicate reactive oxygen species (ROS) detoxification, redox metabolism, stress signalling and transcriptional regulation, molecular chaperones and protein homeostasis, and cell wall‐, membrane‐, and infection‐associated functions, respectively. ΔApHSF, ApHSF deletion mutant; ΔApHSF+, complemented strain.

Figure S7: Original, uncropped immunoblot images corresponding to Figure 6. Figure 6B shows the original, full‐length immunoblot images of ApHSF, ApCtf1β2, and ApCUT3 protein accumulation under cutin induction, together with the corresponding Actin loading control. Figure 6C–E show the original, full‐length immunoblot images of phosphorylation levels of p‐ERK1/2 (Figure 6C), p‐p38 (Figure 6D), and p‐JNK (Figure 6E) in the canonical MAPK pathways under 30°C heat stress or menadione‐induced oxidative stress, with or without specific kinase inhibitors. Figure 6F shows the original, full‐length Phos‐tag SDS‐PAGE immunoblot images of stress‐induced mobility shifts of ApHSF, ApCtf1β2, and ApCUT3 under 30°C heat stress or menadione‐induced oxidative stress in the presence or absence of MAPK pathway inhibitors. Figure 6G shows the original, full‐length Phos‐tag SDS–PAGE immunoblot images of ApHSF and ApCtf1β2 from untreated, heat‐treated, and menadione‐treated samples with or without λ‐protein phosphatase treatment.

Figure S8: Expression profiles, predicted membrane architectures and oxidative‐stress responses of putative membrane‐associated signalling candidates in Arthrinium phaeospermum. (A) Expression profiles of nine candidate genes in the previously generated A. phaeospermum–hybrid bamboo interaction transcriptome. Three biological replicates are shown for each infection stage (S1–S4). Transcript abundance was independently subjected to min–max normalization within each gene and is displayed on a scale from 0 to 1. The colour bar above the heatmap indicates the four infection stages. (B) Predicted membrane architectures of the nine candidate proteins. Protein length, classical signal peptides and membrane topology were determined from the predicted amino acid sequences using SignalP 6.0 and DeepTMHMM. Yellow, blue, green and pink regions indicate DeepTMHMM‐predicted signal regions, extracellular regions, transmembrane helices and cytoplasmic regions, respectively. Each protein was scaled independently according to its full length. SP, signal peptide; CS, cleavage site; TMH, transmembrane helix; N‐out/C‐in, extracellular N‐terminus and cytoplasmic C‐terminus; N‐in/C‐in, cytoplasmic N‐ and C‐termini. C, Relative transcript levels of four representative candidate genes in WT mycelia following treatment with 40 mM H₂O₂ for 0, 0.5, 1 and 2 h. Relative expression levels were calculated using the $2^{-\mathrm{\Delta \Delta }{C}_{\mathrm{t}}}$ method, with the untreated 0‐h sample used as the calibrator for each gene. Data are presented as means ± SD from three independent biological replicates, and individual points represent biological replicates. Different lowercase letters indicate significant differences among treatment times within the same gene according to one‐way ANOVA followed by Tukey's HSD test (p < 0.05).

Table S1: Primers used in this study.

Table S2: Sequence identification and functional annotation of ApCtf1β2 promoter‐interacting proteins identified via yeast one‐hybrid screening.

Table S3: Gene IDs and NR annotations of the 25 candidate genes used to analyse ApHSF‐dependent transcriptional responses during infection.

Table S4: Physicochemical properties and predicted membrane features of putative stress sensor candidates in Arthrinium phaeospermum.

Data Availability Statement

The data that supports the findings of this study are available in the Supporting Information—S1 of this article.