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Review
. 2001 May;67(5):1987-94.
doi: 10.1128/AEM.67.5.1987-1994.2001.

Green fluorescent protein is lighting up fungal biology

J M Lorang  1 , R P Tuori, J P Martinez, T L Sawyer, R S Redman, J A Rollins, T J Wolpert, K B Johnson, R J Rodriguez, M B Dickman, L M Ciuffetti
Affiliations
Review

Green fluorescent protein is lighting up fungal biology

J M Lorang et al. Appl Environ Microbiol. 2001 May.
No abstract available

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Figures

FIG. 1
FIG. 1
Micrographs of fungi transformed with pCT74. Epifluorescence (A through I), Nomarski (A and C, second panel), or confocal (E) microscopy of P. tritici-repentis conidia (magnification, ×400) (A), a mint root infected with V. dahliae (magnification, ×200) (B), C. sativus conidia (magnification, ×1,000) (C), microsclerotia of V. dahliae (magnification, ×400) (D), two serial sections of a C. magna germinated conidia with germ tube and appressoria (E), B. cinerea conidiophores (magnification, ×100) (F), A. alternata hyphae and conidia (magnification, ×400) (G), cross-section of an S. sclerotiorum sclerotium (magnification, ×200) (H), and F. sambucinum hyphae (magnification, ×400) (I).
FIG. 2
FIG. 2
A GFP expression vector for filamentous fungi, pCT74. pCT74 is based on pBlue-SGFP-TYG-nos-KS (from Jen Sheen, Department of Molecular Biology, Massachusetts General Hospital, Harvard University), which contains SGFP-TYG on an NcoI/NotI DNA fragment inserted into the EcoRV site and the nos terminator on a PstI/EcoRI DNA fragment inserted into the EcoRI site of pBluescript KS (Stratagene, La Jolla, Calif.). An NcoI site containing the start codon of SGFP and a NotI site replaced EcoRV in this process. A 417-bp PCR product containing ∼325 bp of the promoter region and ∼90 bp of the 5′ untranslated region of the ToxA gene (12) was generated by PCR amplification from a genomic template with primers 28 (5′-TAGTGGACTGATTGGAATGCATGGAGGAGT-3′) and 29 (5′-GATAGAACCCATGGCCTATATTCATTCAAT-3′). Engineered ClaI and NcoI sites, respectively, are in bold, and naturally occurring BamHI and NcoI sites within the ToxA promoter region were destroyed in this process. This 417-bp fragment was ligated into the NcoI/ClaI sites of pBlue-SGFP-TYG-nos-KS, resulting in pCT73. A 1.4-kb SalI fragment containing the modified hygromycin resistance gene hph under the control of the trpC promoter (9) was ligated into the SalI site of pCT73, resulting in pCT74. Note that NotI and NcoI are not unique restriction sites in pCT74, and not all unique sites in the pBluescript KS polylinker are shown.
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