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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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Related in: MedlinePlus

(A). Stages of co-cultivation of fungus and Agrobacterium plates. Inner plate contains fungal culture and outer contains Agrobacterium culture. (i) Innocula on inner plate with lignin (0.1%) (ii) Full-grown fungal mycelia and (iii) Fungal cultures with Agrobacterium under co-cultivation conditions. (B). Diagrammatic illustration of Agrobacterium mediated transformation of different white-rot fungi.
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Figure 2: (A). Stages of co-cultivation of fungus and Agrobacterium plates. Inner plate contains fungal culture and outer contains Agrobacterium culture. (i) Innocula on inner plate with lignin (0.1%) (ii) Full-grown fungal mycelia and (iii) Fungal cultures with Agrobacterium under co-cultivation conditions. (B). Diagrammatic illustration of Agrobacterium mediated transformation of different white-rot fungi.

Mentions: Two Petri dishes of different diameter were kept in a concentric position, placing smaller one in the centre of bigger Petri dish (Figure 2A). First, the inner Petri dish was poured with 2% MEA supplemented with 0.1% lignin and augmentin (100 μg/ml) and then bigger plate was poured with yeast extract agar (YEA) containing kanamycin at a concentration of 50 μg/ml. Both the plates were poured very carefully so that media do not mix with each other. The MEA plate (inner one) was inoculated at the centre with a fungal disc, incubated at 28°C and it was allowed to grow till the hyphae cross the periphery of the inner plate and move on to the outer plate. Thereafter, Agrobacterium culture was plated carefully on the medium in outer plate, which already contains growing mycelia and co-cultivated at temperatures ranging from 20-29°C for 48 h (Table 1 and Figure 2A, B).


Genetic transformation of lignin degrading fungi facilitated by Agrobacterium tumefaciens.

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

(A). Stages of co-cultivation of fungus and Agrobacterium plates. Inner plate contains fungal culture and outer contains Agrobacterium culture. (i) Innocula on inner plate with lignin (0.1%) (ii) Full-grown fungal mycelia and (iii) Fungal cultures with Agrobacterium under co-cultivation conditions. (B). Diagrammatic illustration of Agrobacterium mediated transformation of different white-rot fungi.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: (A). Stages of co-cultivation of fungus and Agrobacterium plates. Inner plate contains fungal culture and outer contains Agrobacterium culture. (i) Innocula on inner plate with lignin (0.1%) (ii) Full-grown fungal mycelia and (iii) Fungal cultures with Agrobacterium under co-cultivation conditions. (B). Diagrammatic illustration of Agrobacterium mediated transformation of different white-rot fungi.
Mentions: Two Petri dishes of different diameter were kept in a concentric position, placing smaller one in the centre of bigger Petri dish (Figure 2A). First, the inner Petri dish was poured with 2% MEA supplemented with 0.1% lignin and augmentin (100 μg/ml) and then bigger plate was poured with yeast extract agar (YEA) containing kanamycin at a concentration of 50 μg/ml. Both the plates were poured very carefully so that media do not mix with each other. The MEA plate (inner one) was inoculated at the centre with a fungal disc, incubated at 28°C and it was allowed to grow till the hyphae cross the periphery of the inner plate and move on to the outer plate. Thereafter, Agrobacterium culture was plated carefully on the medium in outer plate, which already contains growing mycelia and co-cultivated at temperatures ranging from 20-29°C for 48 h (Table 1 and Figure 2A, B).

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