Efficient GFP expression in the mushrooms Agaricus bisporus and Coprinus cinereus requires introns

Burns, Claire; Gregory, KE; Kirby, Melissa; Cheung, Man Kit; Riquelme, Meritxell; Elliott, Timothy J.; Challen, Michael P.; Bailey, Andy M.; Foster, Gary D.

doi:10.1016/j.fgb.2004.11.005

Cited by 90 publications

(81 citation statements)

References 43 publications

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“…including Agaricus bisporus and Coprinus cinereus, in which inclusion of introns is hypothesized to stabilize protein expression (6). Recently, the GFP gene has been optimized for C. neoformans codon usage to enhance fluorescence (41).…”

Section: Idnurm Et Al Eukaryot Cellmentioning

confidence: 99%

Peroxisome Function Regulates Growth on Glucose in the Basidiomycete Fungus Cryptococcus neoformans

Idnurm¹,

Giles²,

Perfect

et al. 2007

Eukaryot Cell

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The function of the peroxisomes was examined in the pathogenic basidiomycete Cryptococcus neoformans. Recent studies reveal the glyoxylate pathway is required for virulence of diverse microbial pathogens of plants and animals. One exception is C. neoformans, in which isocitrate lyase (encoded by ICL1) was previously shown not to be required for virulence, and here this was extended to exclude also a role for malate synthase (encoded by MLS1). The role of peroxisomes, in which the glyoxylate pathway enzymes are localized in many organisms, was examined by mutation of two genes (PEX1 and PEX6) encoding AAA (ATPases associated with various cellular activities)-type proteins required for peroxisome formation. The pex1 and pex6 deletion mutants were unable to localize the fluorescent DsRED-SKL protein to peroxisomal punctate structures, in contrast to wild-type cells. pex1 and pex6 single mutants and a pex1 pex6 double mutant exhibit identical phenotypes, including abolished growth on fatty acids but no growth difference on acetate. Because both icl1 and mls1 mutants are unable to grow on acetate as the sole carbon source, these findings demonstrate that the glyoxylate pathway can function efficiently outside the peroxisome in C. neoformans. The pex1 mutant exhibits wild-type virulence in a murine inhalation model and in an insect host, demonstrating that peroxisomes are not required for virulence under these conditions. An unusual phenotype of the pex1 and pex6 mutants was that they grew poorly with glucose as the carbon source, but nearly wild type with galactose, which suggested impaired hexokinase function and that C. neoformans peroxisomes might function analogously to the glycosomes of the trypanosomid parasites. Deletion of the hexokinase HXK2 gene reduced growth in the presence of glucose and suppressed the growth defect of the pex1 mutant on glucose. The hexokinase 2 protein of C. neoformans contains a predicted peroxisome targeting signal (type 2) motif; however, Hxk2 fused to fluorescent proteins was not localized to peroxisomes. Thus, we hypothesize that glucose or glycolytic metabolites are utilized in the peroxisome by an as yet unidentified enzyme or regulate a pathway required by the fungus in the absence of peroxisomes.

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Section: Idnurm Et Al Eukaryot Cellmentioning

confidence: 99%

Peroxisome Function Regulates Growth on Glucose in the Basidiomycete Fungus Cryptococcus neoformans

Idnurm¹,

Giles²,

Perfect

et al. 2007

Eukaryot Cell

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show abstract

“…To perform the silencing constructs of atr1 and chk1 genes, we constructed a recipient plasmid carrying the gpdII promoter, an efficient constitutive promoter from Agaricus bisporus previously used in C. cinerea (Burns et al 2005); as a fungal transcriptional terminator we used the 39-UTR region from the hygromycin-resistant gene from the pNEB-Hyg plasmid, which is used in U. maydis for transformation (Castillo-Lluva et al 2004); and as a selectable marker we used the pab1 gene from C. cinerea, which encodes PABA synthase, necessary for para-aminobenzoic acid production (Bottoli et al 1999). pBS (+)KS plasmid was used as backbone.…”

Section: Rnai Proceduresmentioning

confidence: 99%

A DNA Damage Checkpoint Pathway Coordinates the Division of Dikaryotic Cells in the Ink Cap MushroomCoprinopsis cinerea

et al. 2013

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The fungal fruiting body or mushroom is a multicellular structure essential for sexual reproduction. It is composed of dikaryotic cells that contain one haploid nucleus from each mating partner sharing the same cytoplasm without undergoing nuclear fusion. In the mushroom, the pileus bears the hymenium, a layer of cells that includes the specialized basidia in which nuclear fusion, meiosis, and sporulation occur. Coprinopsis cinerea is a well-known model fungus used to study developmental processes associated with the formation of the fruiting body. Here we describe that knocking down the expression of Atr1 and Chk1, two kinases shown to be involved in the response to DNA damage in a number of eukaryotic organisms, dramatically impairs the ability to develop fruiting bodies in C. cinerea, as well as other developmental decisions such as sclerotia formation. These developmental defects correlated with the impairment in silenced strains to sustain an appropriated dikaryotic cell cycle. Dikaryotic cells in which chk1 or atr1 genes were silenced displayed a higher level of asynchronous mitosis and as a consequence aberrant cells carrying an unbalanced dose of nuclei. Since fruiting body initiation is dependent on the balanced mating-type regulator doses present in the dikaryon, we believe that the observed developmental defects were a consequence of the impaired cell cycle in the dikaryon. Our results suggest a connection between the DNA damage response cascade, cell cycle regulation, and developmental processes in this fungus. IN fungal cells, mating-the process equivalent to fertilizationbrings together two haploid nuclei in the same cytoplasm. It is generally thought that this process is essentially followed by nuclear fusion, resulting in a diploid nucleus that either enters meiosis immediately (as occurs in the fission yeast Schizosaccharomyces pombe) or is maintained and proliferates in the diploid state (as happens in the budding yeast Saccharomyces cerevisiae). However, in a large number of fungi, mating does not result in diploid nuclei. Instead, they form dikaryons, cells that contain one haploid nucleus from each mating partner sharing the same cytoplasm for a period of time without undergoing nuclear fusion or meiosis. These dikaryons continue to propagate, and eventually nuclear fusion will take place, which will be followed by meiosis, closing the sexual cycle (Brown and Casselton 2001).Dikaryon cell division, also called conjugate division, represents a big challenge to the cell since it has to ensure that each daughter dikaryon inherits a balance of each parental genome. For that, a complex cell cycle is required that involves distinct mechanisms to maintain heterokaryosis after cell division. For example, for a large number of Basidiomycota (e.g., the mushroom Coprinopsis cinerea), conjugate division includes the formation of a structure known as the clamp connection or clamp cell, as well as the sorting of one of the nuclei to this structure (Casselton 1978;Brown and Casselton 2001). In the...

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“…Moreover, using the same vector, Combier et al (2003) were able to obtain the integration of the EGFP construct as part of the T-DNA into the H. cylindrosporum genome but did not observe GFP fluorescence. Recently, Burns et al (2005) successfully transformed the mushroom Agaricus bisporus with a GFP expressing vector and concluded that efficient GFP expression in A. bisporus requires introns. Introns were also used for GFP expression in the two previously transformed homobasidiomycetes Schizophyllum commune (Lugones et al 1999) and Phanerochaete chrysosporium (Ma et al 2001).…”

Section: Microscopic Fluorescence Analysismentioning

confidence: 99%

Functional expression of the green fluorescent protein in the ectomycorrhizal model fungus Hebeloma cylindrosporum

et al. 2006

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Hebeloma cylindrosporum is a model fungus for mycorrhizal studies because of its fast growth rate, simple nutritional requirements, and completion of its life cycle in vitro, and because it is amenable to transformation. To advance cell biological research during establishment of symbiosis, a tool that would enable the direct visualisation of fusion proteins in the different symbiotic tissues [namely, the expression of reporter genes such as Green Fluorescent Protein (GFP)] was still a missing tool. In the present study, H. cylindrosporum was transformed using Agrobacterium carrying the binary plasmid pBGgHg containing the Escherichia coli hygromycin B phosphotransferase (hph) and the EGFP genes, both under the control of the Agaricus bisporus glyceraldehyde-3-phosphate dehydrogenase promoter. EGFP expression was successfully detected in transformants. The fluorescence was uniformly distributed in the hyphae, while no significant background signal was detected in control hyphae. The suitability of EGFP for reporter gene studies in Hebeloma cylindrosporum was demonstrated opening up new perspectives in the Hebeloma genetics.

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Efficient GFP expression in the mushrooms Agaricus bisporus and Coprinus cinereus requires introns

Cited by 90 publications

References 43 publications

Peroxisome Function Regulates Growth on Glucose in the Basidiomycete Fungus Cryptococcus neoformans

Peroxisome Function Regulates Growth on Glucose in the Basidiomycete Fungus Cryptococcus neoformans

A DNA Damage Checkpoint Pathway Coordinates the Division of Dikaryotic Cells in the Ink Cap MushroomCoprinopsis cinerea

Functional expression of the green fluorescent protein in the ectomycorrhizal model fungus Hebeloma cylindrosporum

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