Intron requirement for AFP gene expression in Trichoderma viride

Jun Xu and Zhen Zhen Gong

State Key Laboratory of Molecular Biology, Box 16, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, PR China

Correspondence
Zhen Zhen Gong
zzgong{at}sibs.ac.cn


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The 430 bp ORF of the Aspergillus giganteus antifungal protein (AFP) gene, containing two small introns, was fused between the promoter and the terminator of the Aspergillus nidulans trpC gene. The AFP gene in this vector produced detectable levels of spliced mRNA in Trichoderma viride. In contrast, in the same vector configuration, its 285 bp intronless derivative showed no accumulation of mRNA when transformed into T. viride. Such expression results were confirmed at the protein level. This fact demonstrated that the introns were required for AFP gene expression in T. viride. This is thought to be a novel phenomenon found in filamentous fungi. Although the mechanism of splicing in filamentous fungi might be similar to that in other eukaryotes, little is known of how it affects expression. This study suggests that the small introns in filamentous fungal genes may not only act as intervening elements, but may also play crucial roles in gene expression by affecting mRNA accumulation. Furthermore, it may provide new evidence for intron-dependent evolution.


Abbreviations: AFP, antifungal protein


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The coding sequences of most eukaryotic genes are interrupted by introns (intervening sequences) that are eventually removed by a precise process termed splicing. Much progress has been made in the understanding of the splicing mechanism. However, knowledge about the biological significance of introns has emerged more slowly.

It has been suggested that introns might play a role in chromatin structure and gene function (Svaren & Chalkley, 1990), as well as in the evolutionary process through ‘exon shuffling’ (Gilbert, 1985; Kolkman & Stemmer, 2001). Introns have been shown to regulate gene expression at the level of transcription (Salgueiro et al., 2000; Boeda et al., 2001; Clancy & Hannah, 2002; Morello et al., 2002). It has been suggested that introns could regulate gene expression post-transcriptionally (McKeown, 1992; Rose & Last, 1997; Sivak et al., 1999). Experiments with SV40 virus indicated that splicing was a prerequisite for the production of stable cytoplasmic mRNAs (Gruss et al., 1979; Khoury et al., 1979; Villarreal & White, 1983). The requirement of introns for efficient mRNA cytoplasmic accumulation has been demonstrated for many eukaryotic genes (Callis et al., 1987; Neuberger & Williams, 1988; Chung & Perry, 1989; Nesic et al., 1993). Moreover, it has also been shown that certain genes cannot be expressed with their introns removed (Hamer & Leder, 1979; Jonsson et al., 1992). It has been proposed that the presence of introns might protect pre-mRNA from undergoing degradation in the nucleus, facilitate polyadenylation, or target mRNA to the cytoplasm (Liu & Mertz, 1995).

In contrast to plant and mammalian genes, introns of filamentous fungi are usually short, on average less than 100 bp (Gurr et al., 1987). Little is known about their roles in the regulation of gene expression in fungal cells. An antifungal protein (AFP) (51 aa, pI~10·65) and its gene have been characterized in Aspergillus giganteus MDH18894 (Wnendt et al., 1994). The 430 bp ORF that encodes the AFP precursor is interrupted by two introns (89 and 56 bp) which have conserved splice sites and the expected features of filamentous fungal gene architecture. It should be noted that no sequence homologous to AFP genes has been observed in other organisms. To test whether the introns play a role in AFP gene expression, we constructed two expression vectors containing either the 430 bp AFP gene or its 285 bp intronless derivative. Subsequently, we transformed the constructs into Trichoderma viride (another species of the filamentous fungi from the Moniliaceae family, like A. giganteus) and then characterized the AFP expression levels. Here we present our data on the role of small fungal introns in gene expression.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strains, media and plasmids.
Filamentous fungi (T. viride, A. giganteus MDH18894) were routinely cultured in beef complete medium (1·5 % beef extract, 2 % peptone, 2 % corn starch and 0·5 % NaCl). Plasmids pDH25 (Cullen et al., 1987), pAN7-1 (Punt & van den Hondel, 1992) and pUC-19 were used for construction of recombinant vectors. All plasmids were propagated in Escherichia coli strain TGI according to Sambrook et al. (1989).

Construction of expression vectors pANAFP and pANAFP*.
The 3 kb EcoRI (partial)/XbaI-digested fragment of pDH25 that contained the promoter/terminator sequences of the Aspergillus nidulans trpC and hygromycin B phosphotransferase (hph) genes was subcloned into the corresponding restriction sites of pUC-19, generating the plasmid pUChph.

The AFP gene (430 bp) was amplified by PCR from genomic DNA of A. giganteus. Its intronless derivative (AFP cDNA) was amplified by RT-PCR from total RNA of A. giganteus. Two primers, P1 (5'-CCATCGATATGAAGTTCGTTTCTCTCGC-3') and P2 (5'-CCGGATCCCTAGCAGTAGCACTTCC-3') were designed and synthesized according to the published sequences of the AFP gene of A. giganteus. The underlined sequences indicate the positions of the ClaI and BamHI restriction sites. The amplified fragments were subcloned into the ClaI and BamHI restriction sites of pUChph, respectively, which generated two plasmids designated pUCAFP (AFP gene) and pUCAFP* (AFP cDNA). PCR was performed using P1 and P2 under the following conditions: 94 °C for 5 min; then 94 °C for 30 s, 58 °C for 30 s and 72 °C for 30 s, for 30 cycles; and 72 °C for 7 min. The RT reaction was conducted using an oligo-dT18 primer under the conditions described below. Complete sequence analyses of both versions of the AFP gene fragments inserted into pUCAFP and pUCAFP* were performed using P1 and P2 on an ABI 3700 automated DNA sequencer and the sequence data of the inserted fragments were consistent with the published sequences (430 bp AFP gene and 285 bp AFP cDNA; Genbank accession no. X60771).

The 2·4 kb EcoRI/XbaI-digested fragment of pUCAFP containing the AFP gene was filled in with the Klenow fragment of E. coli DNA polymerase and was ligated to pAN7-1 which was digested with HindIII and was also filled in with Klenow fragment to give the expression plasmid pANAFP. In the same way, the 2·27 kb XbaI/EcoRI-digested fragment of pUCAFP* containing AFP cDNA was subcloned into pAN7-1 to generate the expression plasmid pANAFP*.

Transformation of T. viride.
Conidia (107) were cultivated in 60 ml PYGS basic medium (infusion from potatoes, 0·5 % yeast extract, 2 % glucose, 2 % sucrose) for 24 h at 28 °C. About 1 g mycelium (wet wt) was filtered through a sterilized nylon gauze, washed twice with sterile water and incubated in 10 ml 0·5 M MgSO4 containing 200 mg Lywallzyme (produced by The Microbiology Institute of Guangdong Province, China) for 2·5 h at 37 °C on an orbital shaker (120 r.p.m.). Protoplasts were separated from the mycelia by filtration through another sterilized nylon gauze, incubated on ice for 10 min, collected by centrifugation (4000 g, 10 min, 4 °C) and washed twice with STC1700 (1·2 M sorbitol, 10 mM Tris/HCl, pH 7·5, 50 mM CaCl2, 35 mM NaCl). Finally, protoplasts were resuspended (107–108 ml-1) in STC1700.

Transformation was carried out according to Punt & van den Hondel (1992). The final suspensions of protoplasts and plasmids in STC1700 were plated onto PYGS regeneration plates containing 0·6 M sucrose and 1·5 % agar. After incubation at 28 °C for 12 h, 8–10 ml PYGS selective medium containing 0·6 M sucrose, 0·8 % agar and 60 µg hygromycin B ml-1 were spread over the first layer. Plates were incubated at 28 °C for 72 h. After incubation, hygromycin B-resistant colonies were then transferred to PYGS basic agar plates.

DNA and RNA isolation.
Conidia (106) of fungi were cultured in 60 ml beef complete medium at 28 °C for 72 h on an orbital shaker (180 r.p.m.). Mycelia were harvested by filtration through sterile nylon gauze, frozen and powdered in liquid nitrogen. Genomic DNA was isolated as described by Yelton et al. (1984). Total RNA was isolated according to Chomczynski & Sacchi (1987) and treated with RNase-free DNase before use.

Southern blots.
Southern blot analysis was performed according to Sambrook et al. (1989). The probes were radiolabelled with [{alpha}-32P]dATP using a nick translation kit (Promega).

RT-PCR analysis.
Approximately 1·0 µg total RNA was used as template. The RT reaction was conducted using primer P2 according to the manufacturer's instructions for using AMV Reverse Transcriptase (Promega). Subsequent PCR was conducted with P1 and P2 under the conditions described above. The products of RT-PCR were analysed by 5 % PAGE.

Western blotting.
Conidia (107) were cultured at 28 °C for 72 h in 250 ml beef complete medium. Culture supernatant was loaded onto a CM-Cellulose 52 column (2x6 cm) pre-equilibrated with PB buffer (10 mM sodium phosphate, pH 7·0). After washing with PB buffer, the bound basic proteins were eluted from the column with 1·5 M NaCl in PB buffer. The eluted fraction was dialysed against water, lyophilized and redissolved in water. The protein samples were separated by 15 % SDS-PAGE and then transferred onto nitrocellulose membranes. Membranes were blocked with 5 % non-fat milk dissolved in TBST buffer (10 mM Tris/HCl, pH 7·2–7·4, 100 mM NaCl, 0·1 % Tween 20) for 1 h and incubated with anti-AFP sera diluted (1 : 1000) in TBST for 1 h. Membranes were washed in TBST, incubated with horseradish-peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology), diluted (1 : 10 000) in TBST for 1·5 h and washed as above, followed by the addition of Supersignal (Pierce) to detect bound immunocomplexes. Anti-AFP sera were raised in New Zealand white rabbits. After the first subcutaneous injection of 0·3 mg AFP in Freund's complete adjuvant, a second injection of 0·15 mg AFP in Freund's incomplete adjuvant was performed 4 weeks later, boosted with an injection of 0·15 mg AFP in Freund's incomplete adjuvant 2 weeks later, then anti-AFP sera were harvested 2 weeks after that.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Transformation of T. viride
The expression vectors pANAFP and pANAFP* were transformed into T. viride according to the method described above. Transformants were identified by PCR analysis and were further confirmed by Southern blot analysis.

Analysis of transformants by PCR
Approximately 100 ng genomic DNA isolated from hygromycin B-resistant colonies (transformants) or wild-type T. viride was used as the template. Genomic DNA of A. giganteus and plasmid DNA (pUCAFP*) were used as positive controls. PCR analysis was conducted using P1 and P2 under the conditions described above. The amplified products were analysed on 5 % PAGE. Eight potential T transformants (AFP gene) and seven potential T* transformants (intronless AFP gene) were identified by PCR analysis (Fig. 1). No corresponding amplified fragment could be detected in the wild-type T. viride.



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Fig. 1. PCR analysis of genomic DNA isolated from transformants and wild-type T. viride. The amplified products were analysed by 5 % PAGE. Lanes M, DNA marker. (a) Lanes: 1, genomic DNA of A. giganteus; 2–9, genomic DNA of T1, T2, T6, T7, T9, T12, T14 and T16, respectively. The arrow on the right indicates the amplified fragments of the AFP gene (430 bp). (b). Lanes: 1, plasmid pUCAFP*; 2, genomic DNA of the wild-type T. viride; 3–9, genomic DNA of T1*, T2*, T3*, T6*, T8*, T9* and T12*, respectively. The arrow on the right indicates the amplified fragment of the 285 bp intronless AFP gene (i.e. AFP cDNA).

 
Southern blot analysis of transformants
Southern blot analysis was further performed according to the method described above with genomic DNA isolated from wild-type T. viride and from the potential transformants identified by PCR analysis. Ten micrograms of genomic DNA was thoroughly digested with EcoRI or BamHI. The EcoRI-digested plasmid DNA (pUCAFP) was used as a positive control. The 430 bp [{alpha}-32P]dATP-labelled AFP gene was used as a probe. The results showed that expression plasmids integrated (multiple copies) into the genome of the recipient in T1, T7, T9, T16, T1*, T2*, T6*, T8*, T9* and T12* (Fig. 2). However, no hybridization signals were observed for T2, T6, T12, T14, T3* or wild-type T. viride in this study.



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Fig. 2. Southern blot analysis. Lanes: M, DNA marker; 1, plasmid pUCAFP digested with EcoRI. (a) Lanes: 2–6, genomic DNA of the wild-type T. viride, T1*, T2*, T1 and T2 digested with EcoRI, respectively; 7–11, genomic DNA of the wild-type T. viride, T1*, T2*, T1 and T2 digested with BamHI, respectively. (b). Lanes 2–7, genomic DNA of T6, T7, T9, T12, T14 and T16 digested with EcoRI, respectively; 8–13, genomic DNA of T6, T7, T9, T12, T14 and T16 digested with BamHI, respectively. (c). Lanes: 2–6, genomic DNA of T3*, T6*, T8*, T9* and T12* digested with EcoRI, respectively; 7–11, genomic DNA of T3*, T6*, T8*, T9* and T12* digested with BamHI, respectively.

 
Requirement of introns for the accumulation of AFP transcripts
RT-PCR analysis was performed on total RNA isolated from the positive T and T* transformants that were confirmed by Southern blot analysis. The total RNAs of A. giganteus and wild-type T. viride were both analysed as parallel controls. The data showed that accumulation of mRNA of the AFP gene could only be detected in T transformants (AFP gene) (Fig. 3). No detectable level of transcript, however, was observed in T* transformants (intronless AFP gene) and the recipient. This suggested that introns are required for the accumulation of AFP transcripts in T. viride.



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Fig. 3. Analysis of transcripts by RT-PCR on 5 % PAGE. Lanes: M, DNA marker; 1, PCR amplified products from plasmid pUCAFP*; 2–11, total RNA of A. giganteus, T1, T7, T9, T16, wild-type T. viride, T1*, T2*, T6* and T12*, respectively.

 
Analysis of AFP synthesis
Proteins isolated from culture supernatants of the recipient and transformants by CM-Cellulose 52 were analysed by Western blotting (Fig. 4). The results were consistent with those of RT-PCR analysis. A protein identical to the mature AFP (51 aa) was observed in transformants T1, T7, T9 and T16, but not in the recipient or transformants T1*, T2*, T6* and T12*.



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Fig. 4. Western blot analysis. Lanes: 1, proteins isolated from A. giganteus; 2–5, proteins isolated from T1, T7, T9 and T16, respectively; 6, proteins isolated from wild-type T. viride; 7–10, proteins isolated from T1*, T2*, T6* and T12*, respectively.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this study, we showed that introns are required for AFP gene expression in T. viride cells, as demonstrated by the level of mRNA and confirmed by analysis of AFP synthesis. Our study suggests that the small introns in filamentous fungal genes may not only act as intervening elements, but may also play crucial roles in gene expression.

Introns have been demonstrated to be pivotal for the expression of many mammalian and plant genes (Hamer & Leder, 1979; Callis et al., 1987; Buchman & Berg, 1988; Neuberger & Williams, 1988; Chung & Perry, 1989; Jonsson et al., 1992; Nesic et al., 1993). Recent studies with yeast, Xenopus, Drosophila and mammalian cells have shown that splicing facilitates mRNA export and that mutations that affect splicing could inhibit mRNA export (Linder & Stutz, 2001). Although it has been suggested that the mechanism of intron splicing in filamentous fungi is similar to that in other eukaryotes (Tollervey & Mattaj, 1987; Seraphin, 1995), little is known of how it affects expression. Our observations support the post-transcriptional model, in which the small introns of the AFP gene may regulate expression by enhancing the stability of the mRNA or by facilitating the export of mRNA. Owing to the absence of splicing, the intronless transcripts could be degraded more easily or may not be transported efficiently into the cytoplasm, and thus no detectable level of transcript from an intronless AFP gene could be observed.

The chromosomal position of random integration may have some effects on the level of AFP gene transcription. It might be argued that all of the plasmids containing an intronless AFP gene have integrated into a region of genome that is poorly transcribed, which could also lead to the effect observed in this study. But the fact that there is an all-or-none effect between two classes of transformants makes this interpretation highly unlikely. A recent study showed that complex response elements existing in the promoter region of the A. giganteus AFP gene are responsible for its efficient transcription (Meyer et al., 2002). However, whether the effect of the introns on expression of the AFP gene in T. viride is related to specific regulating elements in A. giganteus remains unknown.

Soon after the discovery of split genes, a theory of intron-dependent evolution was proposed; that introns may participate in the evolutionary process of primordial genes through the shuffling of exonic sequences and that new genes were produced in this way (Gilbert, 1985; Kolkman & Stemmer, 2001). This theory has been supported by many studies (Stone et al., 1985; Courseaux & Nahon, 2001; Kolkman & Stemmer, 2001; Long, 2001). It has been suggested that the intron/exon structure of a gene corresponds to its evolutionary history (Duester et al., 1986; Hartung et al., 2002). By comparison of the intron/exon structures of various alcohol dehydrogenase genes and related enzyme genes, Duester et al. (1986) showed that the evolution of the oligonucleotide-binding domain was intron-dependent. A recent study indicated that AFP has the characteristic features of an oligonucleotide-binding domain (Martinez Del Pozo et al., 2002). In view of our current study and the evidence summarized above, it is tempting to speculate that the evolution of the AFP gene might be intron-dependent.


   ACKNOWLEDGEMENTS
 
We thank Dr Vera Meyer and Mr Torsten Theis for the gift of purified AFP proteins for preparation of anti-AFP sera, and Drs D. Cullen and C. A. van den Hondel for the gifts of fungal expression plasmids. We also thank several scientists for critical reading and providing comments on this manuscript.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 27 May 2003; revised 11 July 2003; accepted 17 July 2003.



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