AP-2α 相关研究

Figure 1 | Identification and subcellular localization of VdAP-2α in Verticillium dahliae.

(A) Morphological Characterization of the WT V. dahliae and the T-DNA mutant 166 cultivated on PDA plates.

(B) and (C) Pathogenicity Evaluation of the WT and T-DNA mutant 166 strain on G. hirsutum and Shandong maple, with water treatment as a negative control.

(D) Phylogenetic analysis of the VdAP-2α protein in V. dahliae and its homologs in other fungi. Protein sequences were downloaded from the NCBI database (https://www.ncbi.nlm.nih.gov/). The phylogenetic tree was constructed using the neighbor-joining method in MEGA 6.0, with bootstrap values derived from 1,000 iterations.

(E) Domain Architecture Prediction of the VdAP-2α protein sequence using bioinformatics tools such as SMART, InterPro, and Pfam. The Adaptin N and Alpha adaptin C2 domains were annotated using Illustrator for Biological Sequences (IBS). Scale bar = 50 amino acids.

(F) Subcellular localization of VdAP-2α in V. dahliae. The VdAP-2α-GFP fusion protein was expressed in the WT strain AT13. Conidia suspensions of positive transformants were incubated on hydrophobic glass slides at 25 °C under dark conditions for 14 h. GFP fluorescence was visualized using a Zeiss Axio Imager.M2. Scale bar = 10 μm.

Figure 2 | VdAP-2α regulates vegetative growth and sporulation in Verticillium dahliae. (A) Morphological characterization of the WT, T-DNA mutant 166, ΔVdAP-2α mutant, and ECΔVdAP-2α strain on PDA, MM, and V8 media. (B) and (D) Radial growth measurements of strains depicted in (A) and (C). Error bars denote standard errors derived from triplicate plates; experiments were replicated three times. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-test). (C) Colony phenotypes of the WT, T-DNA mutant 166, ΔVdAP-2α mutants, and ECΔVdAP-2α strains on Czapek agar supplemented with distinct carbon substrates (sucrose, starch, pectin, or sodium carboxymethyl cellulose (CMC-Na)). Strains were cultured at 25 °C in the dark for 7 days. Each strain was inoculated on at least three plates, and the experiment was repeated three times. (E) Conidia morphology of the WT, T-DNA mutant 166, ΔVdAP-2α mutant, and ECΔVdAP-2α strain. Scale bar = 10 μm. (F) and (G) Sporulation capacity and conidia size of the WT, T-DNA mutant 166, ΔVdAP-2α mutant, and ECΔVdAP-2α strain. Conidial suspensions were cultivated in 50 mL CM liquid medium at 25 °C in darkness with orbital shaking at 150 rpm for 48 h. Conidial production and dimensions were quantified. Error bars represent standard errors from triplicate assays; experiments were repeated three times. *P < 0.05, **P < 0.01 (Student’s t-test). (H) Detection of VdAP-2α expression pattern in WT strain. The conidia suspension of AT13 strain (1×106 conidia/mL) was spread on PDA plates overlaid with sterile cellophane, and incubated at 25 °C under darkness. Fungal bodies were harvested at 0, 2, 3, 4, 5, 7, 10, and 14 days, with day 0 serving as the baseline control. The expression profile of VdAP-2α was analyzed by RT-qPCR. The experiment was repeated three times. *P < 0.05, **P < 0.01, ***P < 0.001 (Student's t-test).

Figure 3 | VdAP-2α Regulates Microsclerotia Development and Melanin Deposition in Verticillium dahliae. (A) Stereoscopic visualization of microsclerotia morphology in the WT, ΔVdAP-2α mutants, and ECΔVdAP-2α strains. Conidial suspensions (50 μL, 1×106 conidia/mL) were evenly distributed on cellophane-overlaid BMM agar and incubated at 25 °C under complete darkness for 7 and 14 days. Microsclerotial structures were examined using a Zeiss SteREO Discovery.V20. Each experimental condition was replicated three times, analyzing three independent plates per iteration. Scale bar = 0.2 mm. (B) and (C) Computational assessment of melanized area percentage and microsclerotial enumeration in the studied strains. Microsclerotial quantification was performed across three distinct fields (each 1 mm²) following 7 and 14 days of incubation on BMM medium at 25 °C in complete darkness. The experiment was repeated three times. *P < 0.05, **P < 0.01, ***P < 0.001 (Student's t-test). (D) Expression levels of melanin biosynthesis-related genes during microsclerotia development. Samples were cultured in BMM medium at 25 °C in the dark for 7 days, and gene expression was quantified using RT-qPCR. The experiment was repeated three times. **P < 0.01 (Student’s t-test).

Figure 4 | Comparative transcriptomic analysis of the functional regulation of VdAP-2α in Verticillium dahliae.

(A) Comparative transcriptomic analysis of the ΔVdAP-2α mutant, and WT strain. Differentially expressed genes (DEGs) in the V. dahliae DK185 genome were annotated by Gene Ontology (GO), covering transcriptional regulation, cellular metabolic synthesis, membrane architecture, carbon utilization, nitrogen assimilation and transport mechanisms. DEG identification between the ΔVdAP-2α mutant and WT strain was conducted with a stringent threshold of log2 fold change ≥2.

(B) Regulatory scope of VdAP-2α on DEG expression profiles. A GO-based functional enrichment analysis was performed to elucidate the biological roles of DEGs identified between the WT strain and the ΔVdAP-2α mutant. The genes of cellular biosynthetic process exhibit a high degree of overlap with those associated with membrane constituents and carbon and nitrogen metabolism. The DEGs were filtered based on a log2 fold change threshold of ≥2.

(C) Pathway-based annotation of DEGs via KEGG enrichment analysis. A comprehensive KEGG pathway enrichment analysis was executed to map the DEGs identified in the WT V. dahliae and the ΔVdAP-2α mutant.

(D) Hierarchical clustering of DEGs associated with key functional modules. A transcriptional heatmap was constructed to illustrate the expression patterns of DEGs linked to critical biological functions, including hyphal morphogenesis, vesicular trafficking, oxidoreductase cascades, nitrogenous compound metabolism, and carbohydrate catabolism. The log2-transformed fold change values were visualized alongside their respective gene loci (VdLs.17 strain).

Figure 5 | VdAP-2α affects polar growth and development in Verticillium dahliae (A) Hyphal tip morphology and CFW staining in the WT, VdAP-2α deletion mutant, and complemented strain. Scale bar = 10 μm. (B) Percentage of hyphal tips exhibiting normal growth in the WT, ΔVdAP-2α mutant, and ECΔVdAP-2α strain. Conidia suspensions (50 μL, 2×104/mL) were incubated on hydrophobic slides in PDA liquid medium at 25 °C under dark conditions for 20 h. Hyphal tip morphology was examined, and the percentage of normally growing tips was determined from a sample size of 100 hyphae. (C) and (D) Assessment of bipolar growth of hyphae in the WT, ΔVdAP-2α mutant, and ECΔVdAP-2α strain. Conidia suspensions (50 μL, 5×104 conidia/mL) were incubated on hydrophobic slides in PDA liquid medium at 25 °C under dark conditions for 10 h. Bipolar germination rates were calculated. Error bars represent standard errors from three replicates, and the experiment was repeated three times. **P < 0.01 (Student’s t-test). Scale bar = 20 μm (E) Comparative analysis of hydrophobicity in the WT, ΔVdAP-2α mutant, and ECΔVdAP-2α strain. Penetration of water-soluble aniline blue into hyphae was measured to assess hydrophobicity. The experiment was repeated three times. **P < 0.01 (Student's t-test). (F) Quantification of the penetration area relative to the total plate area. ImageJ software was employed for precise measurement. The experiment was repeated three times. ***P < 0.001 (Student's t-test).

Figure 6 | VdAP-2α influences ergosterol biosynthesis in Verticillium dahliae (A) HPLC chromatograms of squalene in the WT, ΔVdAP-2α mutant, and ECΔVdAP-2α strain. All strains were cultivated in Czapek medium supplemented with or without antifungal agents (2 μg/100 mL voriconazole or 0.6 μg/100 mL terbinafine) for 5 days, followed by squalene extraction. (B) Quantitative assessment of squalene levels in the WT, ΔVdAP-2α mutant, and ECΔVdAP-2α strain. Error bars denote standard errors from triplicate experiments, with the experiment conducted in triplicate. Statistical significance was determined using Student’s t-test (**P < 0.01, ***P < 0.001). Standard errors for (D) and (F) were calculated following the same methodology as in (B). (C) HPLC chromatograms of lanosterol in the WT, ΔVdAP-2α mutant, and ECΔVdAP-2α strain. (D) Lanosterol concentration in the WT, ΔVdAP-2α mutant, and ECΔVdAP-2α strain. (E) HPLC chromatograms of ergosterol in the WT, ΔVdAP-2α mutant, and ECΔVdAP-2α strain. (F) Ergosterol accumulation in the WT, ΔVdAP-2α mutant, and ECΔVdAP-2α strain. (G) The transmission electron micrographs of V. dahliae. The intracellular structures of the WT, ΔVdAP-2α mutant, and ECΔVdAP-2α strain were observed using a JEM-1400 transmission electron microscope. Scale bar = 1 μm. The orange arrow indicates the plasma membrane.

图 7 |VdAP-2α 影响元宝筠大丽轮枝菌的致病性。（A） 在用非生物胁迫物（0.015% SDS、1 M 山梨糖醇、0.7 M NaCl、150 μg/mL CR 或 20 μg/mL CFW）修饰的 Czapek 琼脂上培养的 WT、ΔVdAP-2α 突变体和 ECΔVdAP-2α 菌株的集落形态。将培养物在 25 °C 下黑暗中维持 7 天。制备每个菌株一式三份的板，并将实验独立重复 3 次。（B） 非生物胁迫条件下的菌株敏感性曲线 （SDS、山梨糖醇、NaCl、CR 和 CFW）。抑制率是根据 （A） 中的菌落直径计算的。误差线表示来自三个重复的标准误差，并且实验重复了 3 次。**P < 0.01（学生 t 检验）。（C） 山东枫树苗中 WT、ΔVdAP-2α 突变体和 ECΔVdAP-2α 菌株的致病性测定。用各自的真菌菌株接种测试植物，用水接种的树苗作为阴性对照。黄萎病症状进展记录为 35 dpi。进行了 3 个独立实验，每个实验包括 10 棵接种的树苗。从感染的植物中重新分离病原真菌，并在 25 °C 的 V8 培养基上黑暗中繁殖 4 天。（D） 接种的山东枫树中真菌生物量的比较定量。从基干片段 25-30 dpi 中提取基因组 DNA，以 VdEF-1α 为靶基因，山东枫 18S 核糖体 DNA 为参考序列进行定量 PCR。使用 2-ΔΔCT 方法计算相对真菌生物量，基于一式三份的生物学重复。P < 0.001 （学生 t 检验）。

补充图 S1.突变体 166 中 T-DNA 插入位点的分析。

（A） 突变体 166 中 T-DNA 侧翼序列与野生型菌株基因组序列的比较分析。使用 hiTAIL-PCR 技术对突变体 166 中的靶基因进行鉴定和测序，然后在野生型菌株中定位相应的序列。

（B） T-DNA 插入突变体 166 的示意图。IBS 软件（一种生物序列映射工具）用于注释外显子、内含子和 T-DNA 序列。

补充图 S2.分析VdAP-2α 基因丽轮枝菌）。

（a） 和 （b） vdAP-2α。VdAP-2α 基因序列和潮霉素抗性基因 （Hyg的两个转化体中对VdAP-2α。以 AT13 菌株为对照。

（c） 和 （d） VdAP-2α 互补的验证。VdAP-2α 基因序列和遗传素抗性基因 （G418的两个转化体中对VdAP-2α。以 AT13 菌株为对照。

补充图 S3.差异表达基因 （DEG） 的转录组学分析。

（A） 火山图说明了 ΔVdAP-2α 和野生型样品之间的差异基因表达。x 轴表示基因表达（治疗/对照）的倍数变化，而 y 轴表示 -log10（p 值），值越高表示差异越显著。在图中，红点表示基因显著上调，蓝点表示基因显著下调，灰点表示基因无显著差异。图左侧的点对应于下调的基因，右侧的点对应于上调的基因。点越接近边界，差异就越显著。

（B） EggNOG 分类直方图。由欧洲分子生物学实验室 （EMBL） 开发的 EggNOG 数据库为选定的基因集（Up 和 Down）提供详细的直系同源群 （OG） 分析。x 轴表示 EggNOG 功能类别（标记为 A-Z），y 轴表示分配给每个类别的基因/转录本的数量。

（C） GO 分类直方图。基因本体论 （GO） 数据库根据生物过程、细胞成分和分子功能对基因进行分类。y 轴列出 GO 次要术语，x 轴显示分配给每个术语的基因/转录本的数量，颜色代表不同的基因集。

（D） 通路分类直方图。KEGG 数据库用于根据基因参与代谢途径或功能类别对基因进行分类。x 轴列出 KEGG 通路名称，y 轴表示注释到每个通路的基因/转录本的数量。

补充图 S4.WT、ΔVdAP-2α、ECΔVdAP-2α 菌株对伏立康唑和特比萘芬的应激反应，以及角鲨烯、羊毛甾醇和麦角甾醇定量的标准曲线。（A） WT、VdAP-2α 缺失突变体和互补菌株对伏立康唑和特比萘芬的耐受性评价。将分生孢子悬浮液 （3 μL， 1×106 孢子/mL） 接种到 Czapek 培养基上，并在 25°C 下避光孵育 7 天。实验一式三份进行，每次分析三个板。（B） 伏立康唑和特比萘芬对 WT、VdAP-2α 缺失突变体和互补菌株的抑制率的测定。相对抑制率 = （ （阴性对照菌落直径 - 处理过的菌落直径） / 阴性对照菌落直径 ） × 100%。实验重复 3 次。**P < 0.01，***P < 0.001（学生 t 检验）。（C） 沃特世联盟 e2695 高效液相色谱 （HPLC） 检测的角鲨烯、羊毛甾醇和麦角甾醇标准品的保留时间。（D）、（E） 和 （F） 角鲨烯、羊毛甾醇和麦角甾醇的标准曲线，分别通过绘制峰面积与浓度的关系生成。y 表示浓度，x 表示峰面积，0.0036、0.18818 和 0.00545 表示 y 截距，R² 表示相关系数。使用 Origin 软件进行计算。

补充图 S5.野生型 VdAP-2α 突变体和互补菌株对棉花幼苗的致病性。（A） 野生型 VdAP-2α 突变体和互补菌株感染的棉花茎的病害表型和纵切面。（B） 受感染棉花幼苗中大丽轮枝菌的生物量定量。使用植物基因组 DNA 提取试剂盒 （TianGen） 从棉花幼苗中提取总 DNA。以 VdEF-1α 基因为靶点，棉花 18S rRNA 为参考，通过 qPCR 对真菌生物量进行定量。使用 2–ΔΔCt 方法进行相对定量。（C） 野生型 VdAP-2α 突变体和互补菌株感染的棉花幼苗的疾病指数和严重程度分级。根据枯萎叶的数量对疾病严重程度进行分级：0（无枯萎的叶子）、1（一片枯萎的叶子）、2（两片枯萎的叶子）和 3（超过三片枯萎的叶子或植物死亡）。每个处理至少评估 20 株植物，并重复实验 3 次。误差线表示来自三个仿行的标准误差。*P < 0.05，***P < 0.001（学生 t 检验）。（D） VdAP-2α 在玻璃纸上的渗透试验。对玻璃纸上的 WT 、 ΔVdAP-2α 和 ECΔVdAP-2α 菌株进行浸润分析。将每个菌株接种在玻璃纸膜上，48 小时后，去除玻璃纸。然后将 PDA 板在 25°C 避光中孵育 5 天，以观察和收集渗透表型。