To explore the molecular mechanism through which the novel fungicide N-(naphthalen-1-yl) phenazine-1-carboxamide (NNPCN) inhibits Rhizoctonia solani, we clarified the target and mode of action, explored lead compounds, and developed novel fungicides. Methods: Growth observation, scanning electron microscopy, transmission electron microscopy, transcriptome sequencing technology, quantitative real-time PCR (qRT-PCR), physiological and biochemical determination, and reverse molecular docking technology were used to study the effects of this compound on the microscopic morphology of R. solani. The differentially expressed genes (DEGs), functions, and metabolic pathways were analyzed. The genes displaying significant differences were randomly selected for qRT-PCR verification and confirmed by physiological and biochemical determination to construct their binding mode with key targets. The results showed that the mycelium treated with NNPCN produced a red secretion and exhibited progressive creeping growth. Under a scanning electron microscope, hyphal swelling, uneven thickness, fractures, deformities, and hyphal surface warts increased. Under a transmission electron microscope, the cell wall was separated, the subcellular organelles were disintegrated, and the septum disappeared. Furthermore, there were 6838 DEGs under NNPCN treatment, including 291 significant DEGs, of which 143 were upregulated and 148 downregulated. Ten DEGs were randomly selected for qRT-PCR verification, and the gene expression trend was consistent with the transcriptome sequencing results. Gene Ontology enrichment analysis showed that the DEGs were significantly enriched in cell wall glucan decomposition and metabolism, cell membrane synthesis, metabolism, composition, organic hydroxyl compounds, oxidoreductase activity, and transition metal ion binding. Metabolic pathway enrichment analysis showed that there were 16 significant metabolic pathways, such as steroid biosynthesis and ABC transporters. Further study found that genes, such as the glycosyl hydrolase family 10 domain-containing protein, which is related to glucan catabolic process function as tied to the cell wall, were downregulated. Lipid oxidation, modification, and other genes related to the cell membrane were also downregulated. Secondly, genes related to lipid modification, lipid metabolism processes, integral components of the membrane, and other ABC transporters were downregulated. Fatty-acid oxidation and carbohydrate metabolic processes, which are related to antioxidant and metabolic functions, displayed significant differences in their target genes. Nitrite reductase [NADH] activity and mitochondrial organization gene expression were downregulated. These results revealed that target genes may involved in the cell wall, cell membrane, antioxidant and metabolism, nitrogen metabolism, and mitochondria. The results of the physiological and biochemical tests showed that NNPCN decreased the β-1,3-glucanase, malondialdehyde, and ATPase activities and nucleic acid leakage but increased the activity of nitrate reductase. The results of the reverse molecular docking showed that NNPCN could freely bind to target proteins such as β-1,3-glucanase, ABC transporter, and NADPH nitrate reductase, whereby NNPCN could bind to glucanase via van der Waals and electrostatic forces and to ABC transporter and NADPH nitrate reductase via hydrogen bonding. Conclusion: The mechanism via which NNPCN inhibits R. solani may be related to the cell wall structure, cell membrane damage, antioxidant activity, and metabolism.

中文翻译：

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新型杀菌剂N-(naphthalen-1-yl)吩嗪-1-甲酰胺对立枯丝核菌的抑制分子机制

为了探索新型杀菌剂N- (naphthalen-1-yl) 吩嗪-1-甲酰胺 (NNPCN) 抑制立枯丝核菌的分子机制，我们明确了靶点和作用方式，探索了先导化合物，并开发了新型杀菌剂。方法：采用生长观察、扫描电镜、透射电镜、转录组测序技术、实时定量PCR（qRT-PCR）、生理生化测定、反向分子对接技术研究该化合物对显微组织的影响。R. solani 的形态. 分析了差异表达基因 (DEG)、功能和代谢途径。随机选择表现出显着差异的基因进行qRT-PCR验证，并通过生理生化测定确认，构建其与关键靶点的结合模式。结果表明，用 NNPCN 处理的菌丝体产生红色分泌物并表现出渐进的爬行生长。扫描电镜下菌丝肿胀、粗细不均、断裂、畸形，菌丝表面疣体增多。透射电镜下，细胞壁分离，亚细胞器解体，隔膜消失。此外，NNPCN处理下有6838个DEGs，其中显着291个DEGs，其中上调143个，下调148个。随机选取10个DEG进行qRT-PCR验证，基因表达趋势与转录组测序结果一致。基因本体富集分析表明，DEGs在细胞壁葡聚糖分解代谢、细胞膜合成、代谢、组成、有机羟基化合物、氧化还原酶活性、过渡金属离子结合等方面均显着富集。代谢途径富集分析表明，有16条重要的代谢途径，如类固醇生物合成和ABC转运蛋白。进一步的研究发现，与与细胞壁相关的葡聚糖分解代谢过程功能相关的基因，例如含糖基水解酶家族 10 结构域的蛋白质，被下调。脂质氧化、改性、和其他与细胞膜相关的基因也被下调。其次，与脂质修饰、脂质代谢过程、膜的组成部分和其他 ABC 转运蛋白相关的基因被下调。与抗氧化和代谢功能相关的脂肪酸氧化和碳水化合物代谢过程在其靶基因上表现出显着差异。亚硝酸还原酶 [NADH] 活性和线粒体组织基因表达下调。这些结果表明靶基因可能参与细胞壁、细胞膜、抗氧化和代谢、氮代谢和线粒体。生理生化试验结果表明，NNPCN降低了β-1,3-葡聚糖酶、丙二醛、和 ATPase 活性和核酸泄漏，但增加了硝酸还原酶的活性。反向分子对接结果表明，NNPCN可以与β-1,3-葡聚糖酶、ABC转运蛋白和NADPH硝酸还原酶等靶蛋白自由结合，从而NNPCN可以通过范德华力和静电力与葡聚糖酶和ABC结合转运蛋白和 NADPH 硝酸盐还原酶通过氢键。结论：NNPCN 抑制的机制R. solani可能与细胞壁结构、细胞膜损伤、抗氧化活性和代谢有关。