Green fluorescent protein is lighting up fungal biology

Abstract

Prasher ( 42 ) cloned a cDNA for the green fluorescent protein (GFP) gene from the jellyfish Aequorea victoria in 1992. Shortly thereafter, to the amazement of many investigators, this gene or derivatives thereof were successfully expressed and conferred fluorescence to bacteria and Caenorhabditis elegans cells in culture ( 10 , 31 ), followed by yeast ( 24 , 39 ), mammals ( 40 ), Drosophila ( 66 ), Dictyostelium ( 23 , 30 ), plants ( 28 , 49 ), and filamentous fungi ( 54 ). The tremendous success of GFP as a reporter can be attributed to unique qualities of this 238-amino-acid, 27-kDa protein which absorbs light at maxima of 395 and 475 nm and emits light at a maximum of 508 nm. The fluorescence of GFP requires only UV or blue light and oxygen, and therefore, unlike the case with other reporters (β-glucuronidase, β-galacturonidase, chloramphenicol acetyltransferase, and firefly luciferase) that rely on cofactors or substrates for activity, in vivo observation of gfp expression is possible with individual cells, with cell populations, or in whole organisms interacting with symbionts or environments in real time. Complications caused by destructive sampling, cell permeablization for substrates, or leakage of products do not occur. Furthermore, the GFP protein is extremely stable in vivo and has been fused to the C or N terminus of many cellular and extracellular proteins without a loss of activity, thereby permitting the tagging of proteins for gene regulation analysis, protein localization, or specific organelle labeling. The mature protein resists many proteases and is stable up to 65°C and at pH 5 to 11, in 1% sodium dodecyl sulfate or 6 M guanidinium chloride (reviewed in references 17 and 67 ), and in tissue fixed with formaldehyde, methanol, or glutaraldehyde. However, GFP loses fluorescence in methanol-acetic acid (3:1) and can be masked by autofluorescent aldehyde groups in tissue fixed with glutaraldehyde. Fluorescence is optimal at pH 7.2 to 8.0 ( 67 ). Limitations on GFP as a reporter for some applications are its low turnover rate, 2-h lag time for autoactivation of its chromophore, improper folding at high temperatures (37°C), which results in nonfluorescent and insoluble forms of the protein, and requirement for oxygen, which is not present in equal concentrations in all subcellular locations or cell types (reviewed in references 17 and 67 ). These characteristics of GFP, however, have not posed a problem for many applications, and mutant forms of GFP that have an ability to fold properly at high temperatures, increased solubility and fluorescence, reduced photobleaching ( 16 , 17 , 51 ), and reduced half-lives ( 1 ) have been developed. Coupled with fluorescence-activated cell sorting, confocal microscopy or quantitative image analysis techniques, GFP technology can be used to isolate transformed cells or specific cell types from populations of cells ( 14 ), to quantify gene expression of individual cells within whole organisms ( 8 ), or to assess the dispersal and biomass of organisms in complex environments, such as in animal or plant hosts ( 38 , 59 ), in biofilms ( 55 ), in fermentors ( 41 ), on leaf surfaces ( 53 , 61 ), or in soils ( 2 ). The vast majority of studies utilizing GFP expression in fungi have been with yeast (reviewed in reference 13 ). Ustilago maydis was the first filamentous fungus for which successful expression of gfp was reported ( 54 ), followed closely by Aspergillus nidulans ( 22 , 57 ) and Aureobasidium pullulans ( 61 ). Presently, gfp expression has been reported for 16 species comprising 12 genera of filamentous fungi, including Colletotrichum ( 21 , 44 ), Mycosphaerella ( 52 ), Magnaporthe ( 32 , 35 ), Cochliobolus ( 38 ), Trichoderma ( 2 , 70 ), Podospora ( 5 ), Sclerotinia ( 63 ), Schizophyllum ( 37 ), Aspergillus ( 20 , 47 , 50 ) and Phytophthora ( 7 , 62 ). In this review we draw on published reports, with the goal of providing an overview of GFP technology as it applies to the biology of filamentous fungi. These reports are not exhaustive of potential applications of GFP technology, as examples of genomic approaches to utilizing GFP in bacterial and yeast systems attest ( 4 , 46 , 60 , 65 ). Expression of gfp in filamentous fungi requires a gfp variant that is efficiently translated in fungi, a transformation system, and a fungal promoter that satisfies the requirements of a given experimental objective. Transformation of fungi has recently been reviewed by Gold et al. ( 26 ). Robinson and Sharon ( 44 ) suggest that GFP can actually be used to optimize transformation protocols. In addition to reporting the construction of a new fungal transformation vector that expresses SGFP under the control of the ToxA gene promoter from Pyrenophora tritici-repentis ( 12 ) and demonstrating its use in plant pathogens belonging to eight different genera of filamentous fungi ( Fusarium, Botrytis, Pyrenophora, Alternaria, Cochliobolus, Sclerotinia, Colletotrichum , and Verticillium ), in this review we also enumerate and describe a comprehensive list of vectors for expressing GFP in fungi.