Development of a simple and rapid Agrobacterium tumefaciens-mediated transformation system for the entomopathogenic fungus Metarhizium anisopliae var. acridum

Duarte, Rubens Tadeu Delgado; Staats, Charley Christian; Fungaro, Maria Helena Pelegrinelli; Schrank, Augusto; Vainsten, M.H.; Furlaneto-Maia, Luciana; Nakamura, C. V.; Souza, Wanderley de; Furlaneto, Márcia Cristina

doi:10.1111/j.1472-765x.2006.02092.x

Cited by 27 publications

(10 citation statements)

References 25 publications

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“…The TEM results revealed bacterial attachment to fungal cells. This method of co-culturing might have resulted in a substantial increase in concentration of bacteria which eventually facilitated transfer of T-DNA to fungal cells without wound formation [31,32]. …”

Section: Resultsmentioning

confidence: 99%

Genetic transformation of lignin degrading fungi facilitated by Agrobacterium tumefaciens

Sharma

Kuhad

2010

BMC Biotechnology

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BackgroundWhite-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.ResultsThis 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.ConclusionThese 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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Section: Resultsmentioning

confidence: 99%

Genetic transformation of lignin degrading fungi facilitated by Agrobacterium tumefaciens

Sharma

Kuhad

2010

BMC Biotechnology

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“…All disruption and complement plasmids were separately mobilized into A. tumefaciens AGL-1 and subsequently transformed into WT or the knockout mutant of each gene using the previous protocol [28], [36]. The disruption and complement mutants of BbSod2 and BbSod3 were screened based on the bar and sur resistance to PPT (200 µg/ml) and chorimuron ethyl (10 µg/ml) included in M-100 plates [29], respectively, and detected for the presence or absence of each target gene by PCR with the primers I1/I2 and I3/I4.…”

Section: Methodsmentioning

confidence: 99%

Additive Contributions of Two Manganese-Cored Superoxide Dismutases (MnSODs) to Antioxidation, UV Tolerance and Virulence of Beauveria bassiana

et al. 2012

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The biocontrol potential of entomopathogenic fungi against arthropod pests depends on not only their virulence to target pests but tolerance to outdoor high temperature and solar UV irradiation. Two Beauveria bassiana superoxide dismutases (SODs), BbSod2 and BbSod3, were characterized as cytosolic and mitochondrial manganese-cored isoenzymes (MnSODs) dominating the total SOD activity of the fungal entomopathogen under normal growth conditions. To probe their effects on the biocontrol potential of B. bassiana, ΔBbSod2, ΔBbSod3, and three hairpin RNA-interfered (RNAi) mutants with the transcripts of both BbSod2 and BbSod3 being suppressed by 91–97% were constructed and assayed for various phenotypic parameters in conjunction with ΔBbSod2/BbSod2, ΔBbSod3/BbSod3 and wild-type (control strains). In normal cultures, the knockout and RNAi mutants showed significant phenotypic alterations, including delayed sporulation, reduced conidial yields, and impaired conidial quality, but little change in colony morphology. Their mycelia or conidia became much more sensitive to menadione or H2O2-induced oxidative stress but had little change in sensitivity to the hyperosmolarity of NaCl and the high temperature of 45°C. Accompanied with the decreased antioxidative capability, conidial tolerances to UV-A and UV-B irradiations were reduced by 16.8% and 45.4% for ΔBbSod2, 18.7% and 44.7% for ΔBbSod3, and ∼33.7% and ∼63.8% for the RNAi mutants, respectively. Their median lethal times (LT50s) against Myzus persicae apterae, which were topically inoculated under a standardized spray, were delayed by 18.8%, 14.5% and 37.1%, respectively. Remarkably, the effects of cytosolic BbSod2 and mitochondrial BbSod3 on the phenotypic parameters important for the fungal bioncontrol potential were additive, well in accordance with the decreased SOD activities and the increased superoxide levels in the knockout and RNAi mutants. Our findings highlight for the first time that the two MnSODs co-contribute to the biocontrol potential of B. bassiana by mediating cellular antioxidative response.

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“…It has been reported that A. tumefaciens is also able to transfer its DNA to various filamentous fungi, including members of the Ascomycetes, Basidiomycetes, and Zygomycetes phyla, as efficiently as to plants (Michielse et al 2005;Sun et al 2009;Chen et al 2009). The ATMT approach generates a high number of transformants and does not require special equipment (Duarte et al 2007). These advantages of ATMT make this method a valuable tool for molecular genetic manipulation of fungi.…”

Section: Introductionmentioning

confidence: 99%

Development of a simple and efficient transformation system for the basidiomycetous medicinal fungus Ganoderma lucidum

Shi

Fang

et al. 2011

World J Microbiol Biotechnol

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In this study, we report the development of a simple and efficient system for genetic transformation of the medicinal fungus Ganoderma lucidum. Various parameters were optimized to obtain successful Agrobacterium tumefaciens-mediated transformation. Co-cultivation of bacteria and protoplast at a ratio of 1,000:1 at 25°C in medium containing 0.2 mM acetosyringone was found to be the optimum condition for high efficiency transformation. Four plasmids, each carrying a different promoter driving the expression of an antibiotic resistance marker, were tested. The construct carrying the Ganoderma lucidum glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter showed good transformation efficiency, whereas constructs with the GPD promoter from ascomycetes were ineffective. Our analysis showed that over 70% of the transformants tested remained mitotically stable even after five successive rounds of subculturing. We were able to detect the expression of EGFP and GUS reporter genes in the Ganoderma lucidum transformants by fluorescence imaging and histochemical staining assays respectively. Our results demonstrate a new transgenic approach that will facilitate Ganoderma lucidum research.

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Development of a simple and rapid Agrobacterium tumefaciens-mediated transformation system for the entomopathogenic fungus Metarhizium anisopliae var. acridum

Cited by 27 publications

References 25 publications

Genetic transformation of lignin degrading fungi facilitated by Agrobacterium tumefaciens

Genetic transformation of lignin degrading fungi facilitated by Agrobacterium tumefaciens

Additive Contributions of Two Manganese-Cored Superoxide Dismutases (MnSODs) to Antioxidation, UV Tolerance and Virulence of Beauveria bassiana

Development of a simple and efficient transformation system for the basidiomycetous medicinal fungus Ganoderma lucidum

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