gfp genes, optimized for expression in plants, function in Aspergillus
Initial experiments with the native gfp gene, cloned in the pAL5 expression vector, failed to produce detectable fluorescence when transformed into Aspergillus. We therefore explored the possibility that some of the modified gfp genes produced for use in other species could be used directly to overcome this problem. We cloned four different gfp genes into the alcohol-inducible expression vector, pAL5 (Fig. 1A; Doonan et al., 1991). We used this inducible expression system as expression of high levels of GFP could be toxic but neither the GFP nor the GFP fusions reported here had significant effects on fungal growth.
Figure 1. . Construction of Aspergillus GFP expression plasmids. Schematic representation of the different gfp clones in the pAL5 expression vector. In all constructs the gfp gene is flanked by the alcA promoter (alcA P) and the histone terminator (His2B T). Cloning sites and specific gfp mutations are also shown. A. gfp expression constructs. pAL5GFP2 contains a gfp2 gene with two mutations S65T and V163A. pMCB16 contains a region (diagonally shaded box) derived from mgfp4 that has been codon optimized for higher plants (Siemering et al., 1996). pMCB17 contains a region (diagonally shaded box) from mgfp5, codon optimized for higher plants and containing three additional mutations (Siemering et al., 1996). pMCB32 contains the sgfp gene that has been completely codon adapted for expression in animal and plant cells (Haas et al., 1996, Chiu et al., 1996). B. ER-directed GFP constructs with a secretion signal peptide derived from the chitinase gene (ChSP) and an ER retention signal (striped triangle). pMCB40 contains mgfp5 -ER (gift from J. Haseloff); pMCB41 contains the sgfp gene fused to ChSP and ER retention signals in addition to a HA epitope tag; pMCB45 contains the gfp2-5 gene from pMCB17 fused to ChSP and ER retention signals. C. Nuclear directed GFP construct, pMCB42, containing the GAL4 DNA-binding domain (GAL4 BD), a HA epitope tag and the sgfp gene.
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To examine the expression of GFP in hyphae, spores were patched on to ethanol induction medium and grown at 25°C. Plates containing five gfp transformants for each construct and a vector-only control were examined using an epifluorescence microscope. Transformants containing the gfp2 construct did not display any detectable green fluorescence when grown on ethanol media (Fig. 2A, GFP2) and were essentially indistinguishable from GR5 transformants containing an empty pAL5 vector (data not shown). The GFP2 protein contains the S65T mutation, which has a single excitation peak centred at 490 (Heim et al., 1995) and the V163A substitution, which enhances solubility, perhaps via improving protein folding (Siemering et al., 1996). However, transformants containing the gfp2-4, gfp2-5 and sgfp constructs were readily detectable, although fluorescence intensity varied depending on the construct. Germlings carrying each construct were photographed and printed under identical conditions, shown in Fig. 2A. gfp2-4 contains the S65T substitution in addition to a region with optimal plant codon usage and produces a readily detectable level of fluorescence. GFP2-5 cells were brighter than GFP2-4, but GFP2-5 has three additional substitutions (V163A, I167T, and S175G) that may contribute to enhanced fluorescence (Heim et al., 1995; Siemering et al., 1996). sgfp, containing only the S65T mutation, produced the brightest fluorescence, despite the absence of the V163A, I167T, and S175G mutations.
Figure 2. . Expression of GFP in Aspergillus. A. Confocal microscopy of GFP expression in Aspergillus hyphae. The GFP channel is shown on the left and the same field of view as seen by transmitted light is shown on the right. GFP2, pAL5GFP2; GFP2-4, pMCB16; GFP2-5, pMCB17; sGFP, pMCB32. Magnification ×1500 B. SDS–PAGE gel (15%) of protein extracts from Aspergillus transformed with pAL5 vector (V); pAL5GFP2 (2); pMCB16 (16); pMCB17 (17) or pMCB32 (32) and grown under non-inducing or inducing conditions. C. western blot of SDS–PAGE gel with anti-GFP antibodies. The lanes are as described in B. D. Fluorimetry of GFP transformants in repressing and inducing media. Fluorescence units are arbitrary. E. Time course of GFP production after switching hyphae into ethanol media.
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The absence of fluorescence from transformants carrying the gfp2 construct could be due to a number of reasons, including failure to produce mRNA or protein. Suboptimal codon usage has been reported for a number of other fungal species (Spellig et al., 1996; Cormack et al., 1997) and in plants the native gfp transcript contains inappropriate intron splice sites resulting in excision of part of the ORF (Haseloff et al., 1997). As a first step towards defining the reason why gfp2 does not produce any useful fluorescence in Aspergillus and why transfromants containing the other constructs vary in brightness we examined the amount of protein produced (Fig. 2B and C), as compared to the intensity of GFP fluorescence (D). This experiment clearly shows that the intensity of fluorescence in the protein extract from a given gfp transfromant is influenced primarily by the amount of GFP protein produced. First, gfp2 constructs did not produce any detectable GFP protein under either induced or non-induced conditions: gfp2 transformants (lane 2) were indistinguishable from pAL5 transformants (lane V) under both induced and non-induced conditions, as judged by both western blotting (Fig. 2C) and fluorimetry (Fig. 2D). gfp2-4, gfp2-5 and sgfp transfromants grown under inducing conditions produced protein (lanes 16, 17 and 32 in Fig. 2) detectable by western blotting (C) and observable as an extra band on SDS–PAGE gels (arrowed in B). gfp2-4 and gfp2-5 transformants produced approximately equal amounts of protein, as judged by western blotting, but the brightness of gfp2-5 transformants was consistently 1.5- to 2-fold greater than gfp2-4 transformants. The substitutions, V163A, I167T, and S175G, found in GFP2-5 therefore seem to have a significant enhancing effect. In fluorimetry experiments, sgfp transformants tended to produce higher intensities than gfp2-4 or gfp2-5. However, western blot experiments show that sgfp transformants produce higher levels of protein, thereby contributing to the increased fluorescent signal. These differences are also reflected at the transcript level: the gfp 2 transcript cannot be detected on Northern blots, whereas sgfp produces stronger signals than gfp2-4 or gfp2-5. This suggests that the sgfp gene, having been modified throughout its entire length, may produce a more stable transcript than the partially modified gfp2-4 and gfp2-5, which, in turn, are more stable than the unmodified gfp gene. The sequence changes made in the modified genes tend to increase the overall GC content of the transcript and may lead to more stable transcripts. According to Haseloff et al. (1997) the changes made in the gfp sequence affect the central region of the gene (from NdeI to AccI sites, incorporated in our gfp2-4 and gfp2-5 versions) and affect not only the site of processing of the cryptic intron but also the usage of many codons in this region. The changes effectively prevent mis-splicing but no direct effect on translatability can be drawn from their data. Interestingly, sgfp was designed with modifications of the codon usage to adapt it to animal cells (Haas et al., 1996), but these modifications allowed improved expression in plant cells and removed the cryptic splicing points (Chiu et al., 1996). We (J. M. Fernández-Ábalos, unpublished) have found that only the sgfp gene functions in Streptomyces, a prokaryotic organism with highly biased codon usage (Wright and Bibb, 1992). Taken together, these observations suggest that the increase in GC content of the modified gfp genes increases the stability of the transcripts in these microorganisms.
To determine the extent to which gfp could be used as a dynamic reporter of gene expression, we investigated the time course of alcohol induction and repression of GFP expression from the alcA promoter. Cells transformed with pMCB32 (sgfp) were grown overnight (about 12 h) in shaken rich glucose medium (YG) at 37°C and then switched to minimal ethanol medium at 30°C. Protein extracts were made from samples taken every 30 min for the appearance of green fluorescence. No fluorescence was detected until 1.5 h and thereafter accumulated over a period of several hours (Fig. 2E). When pMCB32 cells grown on ethanol were switched to minimal glucose medium, fluorescence (per μg of protein) was maintained at a high level for at least 4 h and then declined by about 40% every hour (data not shown), As the nuclear doubling time is about 90–120 min under these conditions, this decline represents the combined effect of dilution by cell growth and protein degradation. Considering that the alcA promoter responds quickly to changes in carbon source (Pateman et al., 1983), this suggests that GFP protein is turned over rather slowly in Aspergillus.
Location of GFP within the hyphae
One of the major attractions of GFP is the ability to study dynamic events associated with protein targeting and its regulation in vivo. The constructs pMCB16, 17 and 32 were all designed to produce soluble GFP. As judged by microscopic observation, soluble GFP was present throughout the cytoplasm and was able to enter the nucleus. The nuclei often appeared brighter than surrounding cytoplasm, suggesting that GFP accumulated within them. However, GFP was excluded from vacuoles and mitochondria, which were present along the length of the hyphae. Immunogold staining of hyphae using antibodies against GFP confirmed that GFP was absent from the mitochondria and vacuoles but abundant in the cytoplasm and nuclei (data not shown).
Subcellular localization of GFP using targeting sequences
The ability of gfp transcriptional fusions to monitor protein trafficking has been demonstrated in a number of systems (reviewed by Cubitt et al., 1995). To test if GFP could be targeted to a specific location, in this case the ER, we cloned mgfp5 (Siemering et al., 1996, J. Haseloff, personal communication) containing ER retention signals into pAL5. Two other versions of GFP, GFP2-5 and sGFP, were also engineered to place the plant chitinase export signal at the N-terminus of GFP along with the ER retention peptide signal on the C-terminus. These constructs directed GFP to a tubular network within the cell. GFP2-5 produced the brightest fluorescence. A z-series of optical sections through hyphae (Fig. 3) shows GFP2-5 directed to a branching tubular network containing variously shaped brighter nodes. This network extends throughout the cell to within 1–2 μm from the tip and surrounds the nucleus. Vacuoles, the cytoplasm and the nucleus all contained low levels of GFP. Equivalent constructs in plants have been demonstrated to target to the ER (Boevink et al., 1996), illuminating a network similar to that which we describe here.
Figure 3. . Plant ER-targeting and retention signals direct GFP to a tubular network within the hyphae. A z-series of ER-retained GFP produced by a transformant containing pMCB45 (ER-GFP2-5). The images are 3 μm apart. The final micrograph show a phase image of the cell. Magnification × 3000.
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GFP can be used to follow nuclear division in Aspergillus
GFP is widely used to follow organelle behaviour (for example Rizzuto et al., 1995). To follow nuclear division we used a GAL4 DNA-binding domain–GFP fusion, which we know locates to the nucleus of yeast cells (J. M. Fernández-Ábalos, unpublished). In Aspergillus the GAL4 BD–sGFP fusion is targeted to the nucleoplasm and the spindle plaque, producing an image very similar to the DNA-specific dye DAPI. The nucleolus contains very low levels of GFP. A useful aspect of the GAL4 BD–sGFP protein is its constant association with the nucleus throughout the cell cycle. This permits us to follow the dynamics of nuclear division. Time-lapse photography (Fig. 4) was used to follow a nucleus from the early stages of mitosis (panels 0 and 1,5), through mid-phase (panels 5 and 9), anaphase (panels 14.5 and 16.5) telophase (panels 18.5 and 21) and decondensation as the daughter nuclei re-enter interphase (panels 21, 24 and 29). Under these conditions (effective observation temperature, 19°C), mitosis takes between 15 and 22 min.
Figure 4. . A Yeast GAL4 DNA-binding domain–GFP fusion is targeted to the nucleus and provides the means to directly follow mitotic events in living cells. A time lapse series of nuclear division as visualized by nuclear targeted GFP produced by a transformant containing pMCB42. The time in minutes is given in each frame. Magnification ×1500.
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By careful examination of optically sectioned nuclei in the early stages of mitosis, we can discriminate up to seven discrete strands in individual nuclei. The GFP image of early mitosis differs markedly form that observed using DNA-specific dyes on fixed material in that these strands are not normally observed. However, as the later stages of mitosis as observed by GAL4 BD–sGFP closely resemble that of fixed material and the organism has eight genetic linkage groups, we suggest that these strands may represent individual chromosomes. During the early stages of mitosis, the GFP-stained chromosomes appear to be attached at a single point, initially at their extremes but as mitosis proceeds the region of attachment becomes more diffuse and tends to move towards the middle of the strands. As the nuclei approach mid-phase, the chromosomes condense further so that discrete strands are impossible to resolve (panel 5). Anaphase A movement occurs very quickly, taking less than 1 min to resolve the chromatids into two groups, about 1–1.5 μm apart. After the initial separation, the daughter nuclei remain connected by a thin bridge of GFP-stained material and may hold this configuration for several minutes. Checks on DNA segregation may occur during this apparent pause as we have observed occasional bidirectional exchanges of material between the two chromatid masses: small amounts of GFP-stained material move from one nucleus into the other and then back (data not shown). When such exchanges do occur, this phase of mitosis seems to be prolonged, taking up to 15 min in one extreme case (as against the norm of 3–4 min). These observations would be compatible with the existence of a mid-anaphase checkpoint as observed in budding yeast (Yang et al., 1997) implicated as a final check on the correct segregation of the genetic material before the two daughters become separate physical entities. After anaphase, the separation between daughter nuclei increases to a maximum of 3–5 μm over a period of about two minutes. As the nuclei re-enter interphase (as judged by the reappearance of a nucleolus) they may recoil together again slightly before taking up their final positions. Nuclear movement during interphase is relatively slow (compared with mitotic movements). (See note added in proof.)
Unmodified gfp genes fail to produce useful green fluorescence when expressed in Aspergillus. We have taken advantage of the modified forms of gfp that have recently been produced for use in plants to demonstrate their use for following gene expression, protein localization and nuclear behaviour in Aspergillus. Expression of GFP in Arabidopsis is low because of the presence of a cryptic intron, 84 bp long (Haseloff and Amos, 1995; Haseloff et al., 1997). Haseloff's group modified gfp to specifically prevent intron splicing by changing the codons in this region (modifications that are present in our gfp2-4 version) and introduced enhancing mutations (present in our gfp2-5). Haas et al. (1996) have changed codon usage throughout the entire gene producing the sgfp gene, which should also eliminate the splice recognition sites and, because of improved codon usage, may enhance protein production (Chiu et al., 1996). Recently, the sgfp gene has been used as a vital marker in the phytopathogenic fungus Ustilago maydis (Spellig et al., 1996) and Cormack et al. (1997) have shown that codon adaptation is also necessary for expression of GFP in Candida. Here, we demonstrate that both the intron-modified gfp and the completely synthetic sgfp genes are both effective in Aspergillus. These data, combined with the observation that the sgfp gene is the only one expressed in other species with extreme codon bias such as Streptomyces (incidentally, the codon usage in sgfp closely approaches the codon preference in Streptomyces, J. M. Fernández-Ábalos, unpublished), suggest that it may be necessary to produce a specifically adapted gfp for each group of organisms.
Our results also indicate that subcellular localisation signals from other species, such as the ER retention signals from plants and the nuclear localization signals present in GAL4 DNA-binding domain from yeast, are functionally conserved. Thus, GFP fusion proteins containing such signals are effective for marking organelles and subcellular compartments in filamentous fungi without major alterations and should be useful for following protein trafficking. However, we did not find that it was necessary to restrict GFP to the ER to obtain acceptable levels of fluorescence as is the case in Arabidopsis (Haseloff et al., 1997). Finally, the GAL4 BD-GFP fusion marks nuclei and chromatin, allowing us to describe the dynamics of nuclear division in Aspergillus for the first time.