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Genetic transformation of lignin degrading fungi facilitated by Agrobacterium tumefaciens.

Sharma KK, Kuhad RC - BMC Biotechnol. (2010)

Bottom Line: The fungal transformants were confirmed by PCR and Southern hybridization.The transformation efficiency was maximum at 20°C whereas no transfer was observed at temperature above 29°C.These findings provide a rapid and reproducible transformation method without external addition of acetosyringone, which could be useful for improving white-rot fungi for their various biotechnological applications.

View Article: PubMed Central - HTML - PubMed

Affiliation: Lignocellulose Biotechnology Laboratory, Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi, India.

ABSTRACT

Background: White-rot fungi are primarily the major degraders of lignin, a major obstacle for commercial exploitation of plant byproducts to produce bioethanol and other industrially important products. However, to improve their efficacy for lignin degradation, it has become necessary to genetically modify these organisms using appropriate vectors. Agrobacterium tumefaciens, a soil phytopathogenic bacterium, generally transforms plants by delivering a portion of the resident Ti- plasmid, the T-DNA (transfer DNA). The trans-Kingdom gene transfer is initiated by the activity of Ti-plasmid encoded vir (virulence) genes in response to low-molecular-mass phenolic compounds such as acetosyringone. A. tumefaciens played a major role in plant genetic engineering and basic research in molecular biology, accounting for nearly 80% of the transgenic plants produced so far. Initially, it was believed that only dicotyledons, gymnosperms and a few monocotyledonous species could be transformed by this bacterium; but recent reports have totally changed this scenario by demonstrating that many 'recalcitrant' species not included in its natural host range can also be transformed, especially filamentous fungi.

Results: This paper describes an efficient and convenient Agrobacterium-mediated gene transformation system for successful delivery of T-DNA, carrying the genes coding for β-glucuronidase (uidA), green fluorescent protein (gfp) and hygromycin phosphotransferase (hpt) to the nuclear genome of lignin degrading white-rot fungi such as Phanerochaete chrysosporium, Ganoderma sp. RCKK-02, Pycnoporous cinnabarinus, Crinipellis sp. RCK-1, Pleurotus sajor-caju and fungal isolate BHR-UDSC without supplementation of acetosyringone. The fungal transformants were confirmed by PCR and Southern hybridization. The expression vector pCAMBIA 1304-RCKK was constructed by the addition of GPD promoter from plasmid p416 to the binary vector backbone pCAMBIA1304, which controls uidA and gfp gene. Transmission Electron Microscope (TEM) analysis revealed the attachment of bacterial cells to the fungal hyphae. Transformation frequency varied from 50 to 75% depending on the fungal species used in this study. The transformation efficiency was maximum at 20°C whereas no transfer was observed at temperature above 29°C.

Conclusion: These findings provide a rapid and reproducible transformation method without external addition of acetosyringone, which could be useful for improving white-rot fungi for their various biotechnological applications.

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Agrobacterium mediated in-vitro gene transfer as measured through UV-florescence using different excitation filters (BP460-490) and their hygromycin selection. (A). Ganoderma sp. RCKK-02; (E). Crinipellis sp. RCK-1; (I). P. cinnabarinus (M). P. sojur-caju; (N). P. chrysosporium; (O). Fungal isolate BHR-UDSC. (B,F,J) Images under Bright field; (C, D, G, H, K, L) GFP-tagged mycelia using different excitation filters (BP460-490) and barrier filter BA520IF.
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Figure 3: Agrobacterium mediated in-vitro gene transfer as measured through UV-florescence using different excitation filters (BP460-490) and their hygromycin selection. (A). Ganoderma sp. RCKK-02; (E). Crinipellis sp. RCK-1; (I). P. cinnabarinus (M). P. sojur-caju; (N). P. chrysosporium; (O). Fungal isolate BHR-UDSC. (B,F,J) Images under Bright field; (C, D, G, H, K, L) GFP-tagged mycelia using different excitation filters (BP460-490) and barrier filter BA520IF.

Mentions: The fungal cultures when co-cultured with A. tumefaciens harboring hpt gene resulted in development of hygromycin-resistant (Hygr) mycelia (Figure 2B). The DNA transfer events were studied by monitoring the transgene associated with T-DNA (gfp, uidA and hpt) in Hygr colonies. GUS histochemical assays after co-cultivation showed high frequency of transformation. A high percentage GUS positive cells were detected in the transformed mycelia (about 75% in Crinipellis sp. RCK-1 &P. chrysosporium), indicating that the cells received T-DNA (Table. 1). A standard transformation system of P. chrysosporium was used as a positive control which has been reported earlier [11]. Stable transformants showed the expression of GFP as detected by confocal microscopy (Figure 3). Interestingly Hygr phenotype was retained even after sub-culturing the transformants in hygromycin- free medium for a period of 2 months.


Genetic transformation of lignin degrading fungi facilitated by Agrobacterium tumefaciens.

Sharma KK, Kuhad RC - BMC Biotechnol. (2010)

Agrobacterium mediated in-vitro gene transfer as measured through UV-florescence using different excitation filters (BP460-490) and their hygromycin selection. (A). Ganoderma sp. RCKK-02; (E). Crinipellis sp. RCK-1; (I). P. cinnabarinus (M). P. sojur-caju; (N). P. chrysosporium; (O). Fungal isolate BHR-UDSC. (B,F,J) Images under Bright field; (C, D, G, H, K, L) GFP-tagged mycelia using different excitation filters (BP460-490) and barrier filter BA520IF.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2944332&req=5

Figure 3: Agrobacterium mediated in-vitro gene transfer as measured through UV-florescence using different excitation filters (BP460-490) and their hygromycin selection. (A). Ganoderma sp. RCKK-02; (E). Crinipellis sp. RCK-1; (I). P. cinnabarinus (M). P. sojur-caju; (N). P. chrysosporium; (O). Fungal isolate BHR-UDSC. (B,F,J) Images under Bright field; (C, D, G, H, K, L) GFP-tagged mycelia using different excitation filters (BP460-490) and barrier filter BA520IF.
Mentions: The fungal cultures when co-cultured with A. tumefaciens harboring hpt gene resulted in development of hygromycin-resistant (Hygr) mycelia (Figure 2B). The DNA transfer events were studied by monitoring the transgene associated with T-DNA (gfp, uidA and hpt) in Hygr colonies. GUS histochemical assays after co-cultivation showed high frequency of transformation. A high percentage GUS positive cells were detected in the transformed mycelia (about 75% in Crinipellis sp. RCK-1 &P. chrysosporium), indicating that the cells received T-DNA (Table. 1). A standard transformation system of P. chrysosporium was used as a positive control which has been reported earlier [11]. Stable transformants showed the expression of GFP as detected by confocal microscopy (Figure 3). Interestingly Hygr phenotype was retained even after sub-culturing the transformants in hygromycin- free medium for a period of 2 months.

Bottom Line: The fungal transformants were confirmed by PCR and Southern hybridization.The transformation efficiency was maximum at 20°C whereas no transfer was observed at temperature above 29°C.These findings provide a rapid and reproducible transformation method without external addition of acetosyringone, which could be useful for improving white-rot fungi for their various biotechnological applications.

View Article: PubMed Central - HTML - PubMed

Affiliation: Lignocellulose Biotechnology Laboratory, Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi, India.

ABSTRACT

Background: White-rot fungi are primarily the major degraders of lignin, a major obstacle for commercial exploitation of plant byproducts to produce bioethanol and other industrially important products. However, to improve their efficacy for lignin degradation, it has become necessary to genetically modify these organisms using appropriate vectors. Agrobacterium tumefaciens, a soil phytopathogenic bacterium, generally transforms plants by delivering a portion of the resident Ti- plasmid, the T-DNA (transfer DNA). The trans-Kingdom gene transfer is initiated by the activity of Ti-plasmid encoded vir (virulence) genes in response to low-molecular-mass phenolic compounds such as acetosyringone. A. tumefaciens played a major role in plant genetic engineering and basic research in molecular biology, accounting for nearly 80% of the transgenic plants produced so far. Initially, it was believed that only dicotyledons, gymnosperms and a few monocotyledonous species could be transformed by this bacterium; but recent reports have totally changed this scenario by demonstrating that many 'recalcitrant' species not included in its natural host range can also be transformed, especially filamentous fungi.

Results: This paper describes an efficient and convenient Agrobacterium-mediated gene transformation system for successful delivery of T-DNA, carrying the genes coding for β-glucuronidase (uidA), green fluorescent protein (gfp) and hygromycin phosphotransferase (hpt) to the nuclear genome of lignin degrading white-rot fungi such as Phanerochaete chrysosporium, Ganoderma sp. RCKK-02, Pycnoporous cinnabarinus, Crinipellis sp. RCK-1, Pleurotus sajor-caju and fungal isolate BHR-UDSC without supplementation of acetosyringone. The fungal transformants were confirmed by PCR and Southern hybridization. The expression vector pCAMBIA 1304-RCKK was constructed by the addition of GPD promoter from plasmid p416 to the binary vector backbone pCAMBIA1304, which controls uidA and gfp gene. Transmission Electron Microscope (TEM) analysis revealed the attachment of bacterial cells to the fungal hyphae. Transformation frequency varied from 50 to 75% depending on the fungal species used in this study. The transformation efficiency was maximum at 20°C whereas no transfer was observed at temperature above 29°C.

Conclusion: These findings provide a rapid and reproducible transformation method without external addition of acetosyringone, which could be useful for improving white-rot fungi for their various biotechnological applications.

Show MeSH
Related in: MedlinePlus