Green fluorescent protein as a reporter for gene expression in the mucoralean fungus Absidia glauca

Christina Schilde · Johannes W?stemeyer ·Anke Burmester

Received: 16 June 2000 / Revised: 15 September 2000 / Accepted: 28 September 2000 / Published online: 18 November 2000Arch Microbiol (2001) 175:1–7DOI 10.1007/s002030000228

?Springer-Verlag 2000

toplasts were analyzed for transient expression under the fluorescence microscope. Only a few protoplasts showed green fluorescence (data not shown). In subsequent exper-iments, several transformants were obtained that grew on selective, neomycin-containing solid medium. Transfor-mants with paGFP, paGFP-RAG and peGFP-SEG were analyzed in detail. Their mycelia exhibited green fluores-cent areas. Typical microscopic views are presented in Fig.2. As A. glauca has some intrinsic autofluorescence, the choice of filter combinations is crucial. Autofluores-cence of the mycelium is highest with filter combination 1 with an excitation wavelength of 365 nm. The lowest background was obtained with filter combination 3. All fluorescent micrographs in Fig.2 were obtained with this filter combination. However, combination 3 requires strong gfp expression. Therefore, for routine screening purposes, combination 2 is preferred. It leads to increased fluorescence signals and an acceptable degree of autoflu-orescence.

Transformant colonies form sectors with respect to GFP fluorescence (Fig.2 C, G). Sporangia with intensely green fluorescent spores could be found after introducing peGFP-SEG or paGFP (Fig.2 A, E). In control experi-ments with untransformed mycelia, fluorescent sporangia were not observed. Sectoring of the fluorescent phenotype is expected. Protoplasts contain between 10 and 30 nuclei and thus are almost certainly heterokaryotic with respect to the gfp gene after transformation and subsequent growth. Plasmid loss during mitosis is inevitable due to the autonomous character of replicons and represents the main reason for the formation of sectors.

Southern blot analysis of A. glauca transformants Sectors from fungal colonies transformed with paGFP and peGFP-SEG were subcultured on Petri dishes with neomycin-containing selective medium. These mycelia were used for inoculating neomycin-containing liquid cul-tures, a procedure that selects efficiently for plasmid-con-taining material. DNAs from such cultures were restricted with Hin dIII, transferred to nylon membranes and hy-bridized with a labeled probe containing exclusively bac-terial parts of the vector and structural information of the gfp gene (Fig.3). DNA from sector 1 of the peGFP-SEG transformant shows the predicted pattern, with two bands of 5 kb and 3.5 kb (Fig.3A, lane 2). Similar patterns could be obtained with DNA from sectors 2–4 of the same pri-mary transformant. Weak signals were obtained for the 5-kb band, whereas the 3.5-kb band was much stronger. This indicates that plasmid spectra are heterogeneous be-tween sectors (Fig.3B, lanes 1–3), an observation that was confirmed by retransformation experiments in E. coli. Several plasmids show the original pattern, whereas oth-ers belong to a new class of deletion derivatives with a to-tal length that corresponds to the 3.5-kb fragment visible in Southern blots. In five sectors from a colony trans-formed with paGFP, the expected 7-kb band was not de-tected. Instead, bands with lengths of 5 and 3.5 kb with varying relative signal strengths were obtained (Fig.3B, lanes 5–9). The sectors of this transformant propagate dif-ferentially rearranged plasmids. Stable integration of vec-

Fig.2A–H Microscopy of A. glauca transformed with GFP vec-tors.A,B Sporangia and C,D mycelium of peGFP-SEG transfor-mants.E,F Sporangia and G,H mycelium of paGFP transfor-mants. The bar (B) corresponds to 50 μm (C,D,G,H) or 25 μm (A,B,E,F)

Fig.3A–C Southern-blot analysis of DNA from A. glauca trans-formants. A fragment containing bacterial vector parts and the gfp gene was used as hybridization probe. Identical amounts of DNA were loaded in all lanes. The 1 kb ladder (Gibco, BRL) was used as size marker (M).A Lane 1 Hin dIII-restricted DNA of wild type

A. glauca,lane 2 Hin dIII-restricted DNA of sector 1 of the peGFP-

SEG transformant.B Lanes 1–3 Hind dIII-restricted DNA of sec-tors 2–4 of the peGFP-SEG transformant,lane 4 Hin dIII-restricted DNA of wild type A. glauca,lanes 5–9 Hin dIII-restricted DNA of sectors 1–5 of the paGFP transformant.C Lane 1 Hin dIII-re-stricted DNA of wild type A. glauca,lane 2uncut DNA of sector

4 of the peGFP-SEG transformant,lane 3 Hin dIII-restricted DNA

of sector 4 of the peGFP-SEG transformant

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