PEG-Mediated Protoplast Transformation of <i>Penicillium sclerotiorum</i> (scaumcx01): Metabolomic Shifts and Root Colonization Dynamics
Protoplast-based transformation is a vital tool for genetic studies in fungi, yet no protoplast method existed for <i>P. sclerotiorum</i>-scaumcx01 before this study. Here, we optimized protoplast isolation, regeneration, and transformation efficiency. The highest protoplast yield (6.72...
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| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2025-05-01
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| Series: | Journal of Fungi |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2309-608X/11/5/386 |
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| Summary: | Protoplast-based transformation is a vital tool for genetic studies in fungi, yet no protoplast method existed for <i>P. sclerotiorum</i>-scaumcx01 before this study. Here, we optimized protoplast isolation, regeneration, and transformation efficiency. The highest protoplast yield (6.72 × 10<sup>6</sup> 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 MgSO<sub>4</sub> 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 <i>GFP</i> tagging resulted in stable <i>GFP</i> 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 <i>GFP</i> tagging. Microscopy confirmed successful colonization of tomato roots by <i>GFP</i>-tagged scaumcx01, with <i>GFP</i> 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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| ISSN: | 2309-608X |