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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2025-05-01
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| author | Israt Jahan Qilin Yang Zijun Guan Yihan Wang Ping Li Yan Jian |
| author_facet | Israt Jahan Qilin Yang Zijun Guan Yihan Wang Ping Li Yan Jian |
| author_sort | Israt Jahan |
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| description | 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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| institution | Kabale University |
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| language | English |
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| series | Journal of Fungi |
| spelling | doaj-art-11c9a6fe0bdb4c44a0803d72b711b5c72025-08-20T03:47:54ZengMDPI AGJournal of Fungi2309-608X2025-05-0111538610.3390/jof11050386PEG-Mediated Protoplast Transformation of <i>Penicillium sclerotiorum</i> (scaumcx01): Metabolomic Shifts and Root Colonization DynamicsIsrat Jahan0Qilin Yang1Zijun Guan2Yihan Wang3Ping Li4Yan Jian5Key Laboratory of Agro-Environment in the Tropics, Ministry of Agriculture and Rural Affairs, Guangdong Provincial Key Laboratory of Eco-Circular Agriculture, Guangdong Engineering Research Centre for Modern EcoAgriculture, College of Natural Resources and Environment, South China Agricultural University, Guangzhou 510642, ChinaKey Laboratory of Agro-Environment in the Tropics, Ministry of Agriculture and Rural Affairs, Guangdong Provincial Key Laboratory of Eco-Circular Agriculture, Guangdong Engineering Research Centre for Modern EcoAgriculture, College of Natural Resources and Environment, South China Agricultural University, Guangzhou 510642, ChinaKey Laboratory of Agro-Environment in the Tropics, Ministry of Agriculture and Rural Affairs, Guangdong Provincial Key Laboratory of Eco-Circular Agriculture, Guangdong Engineering Research Centre for Modern EcoAgriculture, College of Natural Resources and Environment, South China Agricultural University, Guangzhou 510642, ChinaKey Laboratory of Agro-Environment in the Tropics, Ministry of Agriculture and Rural Affairs, Guangdong Provincial Key Laboratory of Eco-Circular Agriculture, Guangdong Engineering Research Centre for Modern EcoAgriculture, College of Natural Resources and Environment, South China Agricultural University, Guangzhou 510642, ChinaKey Laboratory of Agro-Environment in the Tropics, Ministry of Agriculture and Rural Affairs, Guangdong Provincial Key Laboratory of Eco-Circular Agriculture, Guangdong Engineering Research Centre for Modern EcoAgriculture, College of Natural Resources and Environment, South China Agricultural University, Guangzhou 510642, ChinaKey Laboratory of Agro-Environment in the Tropics, Ministry of Agriculture and Rural Affairs, Guangdong Provincial Key Laboratory of Eco-Circular Agriculture, Guangdong Engineering Research Centre for Modern EcoAgriculture, College of Natural Resources and Environment, South China Agricultural University, Guangzhou 510642, ChinaProtoplast-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.https://www.mdpi.com/2309-608X/11/5/386<i>Penicillium sclerotiorum</i>protoplast transformation<i>GFP</i> taggingmetabolomic analysisplant–fungal interaction |
| spellingShingle | Israt Jahan Qilin Yang Zijun Guan Yihan Wang Ping Li Yan Jian PEG-Mediated Protoplast Transformation of <i>Penicillium sclerotiorum</i> (scaumcx01): Metabolomic Shifts and Root Colonization Dynamics Journal of Fungi <i>Penicillium sclerotiorum</i> protoplast transformation <i>GFP</i> tagging metabolomic analysis plant–fungal interaction |
| title | PEG-Mediated Protoplast Transformation of <i>Penicillium sclerotiorum</i> (scaumcx01): Metabolomic Shifts and Root Colonization Dynamics |
| title_full | PEG-Mediated Protoplast Transformation of <i>Penicillium sclerotiorum</i> (scaumcx01): Metabolomic Shifts and Root Colonization Dynamics |
| title_fullStr | PEG-Mediated Protoplast Transformation of <i>Penicillium sclerotiorum</i> (scaumcx01): Metabolomic Shifts and Root Colonization Dynamics |
| title_full_unstemmed | PEG-Mediated Protoplast Transformation of <i>Penicillium sclerotiorum</i> (scaumcx01): Metabolomic Shifts and Root Colonization Dynamics |
| title_short | PEG-Mediated Protoplast Transformation of <i>Penicillium sclerotiorum</i> (scaumcx01): Metabolomic Shifts and Root Colonization Dynamics |
| title_sort | peg mediated protoplast transformation of i penicillium sclerotiorum i scaumcx01 metabolomic shifts and root colonization dynamics |
| topic | <i>Penicillium sclerotiorum</i> protoplast transformation <i>GFP</i> tagging metabolomic analysis plant–fungal interaction |
| url | https://www.mdpi.com/2309-608X/11/5/386 |
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