PEG-Mediated Protoplast Transformation of Penicillium sclerotiorum (scaumcx01): Metabolomic Shifts and Root Colonization Dynamics.

IF 4.2 2区生物学 Q2 MICROBIOLOGY

Journal of Fungi Pub Date : 2025-05-17 DOI:10.3390/jof11050386

Israt Jahan, Qilin Yang, Zijun Guan, Yihan Wang, Ping Li, Yan Jian

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引用次数: 0

Abstract

Protoplast-based transformation is a vital tool for genetic studies in fungi, yet no protoplast method existed for P. sclerotiorum-scaumcx01 before this study. Here, we optimized protoplast isolation, regeneration, and transformation efficiency. The highest protoplast yield (6.72 × 10⁶ cells/mL) was obtained from liquid mycelium after 12 h of enzymatic digestion at 28 °C using Lysing Enzymes, Yatalase, cellulase, and pectinase. Among osmotic stabilizers, 1 M MgSO₄ yielded the most viable protoplasts. Regeneration occurred via direct mycelial outgrowth and new protoplast formation, with a 1.02% regeneration rate. PEG-mediated transformation with a hygromycin resistance gene and GFP tagging resulted in stable GFP expression in fungal spores and mycelium over five generations. LC/MS-based metabolomic analysis revealed significant changes in glycerophospholipid metabolism, indicating lipid-related dynamics influenced by GFP tagging. Microscopy confirmed successful colonization of tomato roots by GFP-tagged scaumcx01, with GFP fluorescence observed in cortical tissues. Enzymatic (cellulase) seed pretreatment enhanced fungal colonization by modifying root surface properties, promoting plant-fungal interaction. This study establishes an efficient protoplast transformation system, reveals the metabolic impacts of genetic modifications, and demonstrates the potential of enzymatic seed treatment for enhancing plant-fungal interactions.

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聚乙二醇介导的菌核青霉原生质体转化：代谢组学变化和根定植动力学。

原生质体转化是真菌遗传研究的重要工具，但在此之前，尚没有原生质体方法对菌核菌种进行转化。在此，我们优化了原生质体的分离、再生和转化效率。在28℃条件下，利用Lysing酶、Yatalase、纤维素酶和果胶酶对液体菌丝体进行酶切12 h，原生质体产量最高，为6.72 × 106个细胞/mL。在渗透稳定剂中，1 M MgSO4产生的原生质体存活率最高。通过菌丝直接外生和原生质体形成再生，再生率为1.02%。peg介导的湿霉素抗性基因转化和GFP标记在真菌孢子和菌丝中稳定表达了五代。基于LC/ ms的代谢组学分析显示甘油磷脂代谢发生了显著变化，表明GFP标记影响了脂质相关动力学。显微镜证实GFP标记的scumcx01成功定植番茄根，在皮层组织中观察到GFP荧光。酶（纤维素酶）种子预处理通过改变根表面特性增强真菌定植，促进植物与真菌的相互作用。本研究建立了一个高效的原生质体转化系统，揭示了遗传修饰的代谢影响，并证明了酶促种子处理在增强植物与真菌相互作用方面的潜力。

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来源期刊

Journal of Fungi Medicine-Microbiology (medical)

CiteScore

6.70

自引率

14.90%

发文量

1151

审稿时长

11 weeks

期刊介绍： Journal of Fungi (ISSN 2309-608X) is an international, peer-reviewed scientific open access journal that provides an advanced forum for studies related to pathogenic fungi, fungal biology, and all other aspects of fungal research. The journal publishes reviews, regular research papers, and communications in quarterly issues. Our aim is to encourage scientists to publish their experimental and theoretical results in as much detail as possible. Therefore, there is no restriction on paper length. Full experimental details must be provided so that the results can be reproduced.