*Centro Studio Micologia del Terreno-CNR
and
Dipartimento di Biologia Vegetale dellUniversità, Turin, Italy;
and
Istituto di Patologia Generale Veterinaria, Milan, Italy;
and
§Department of Biology, University of York, York, England
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Abstract |
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Introduction |
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Sterile mycelia with different colony morphologies have also been described as common symbionts of ericoid roots in North America (Stoyke, Egger, and Currah 1992
; Hambleton and Currah 1997
), Europe (Duclos and Fortin 1983
; Perotto et al. 1990, 1996
), South Africa (Straker and Mitchell 1985
), and Australia (Hutton, Dixon, and Sivasithamparam 1994
; Liu, Chambers, and Cairney 1998
), where they often form the majority of mycorrhizal isolates. Their taxonomic position, however, is unknown because they lack the morphological structures that could be used for identification.
The nuclear ribosomal genes have been extensively used for taxonomic purposes in fungi (e.g., Berbee and Taylor 1993
; Gargas et al. 1995
). Therefore, we have begun to sequence these genes to determine the genetic diversity of ericoid mycelia collected worldwide, focusing on the nuclear small subunit (SSU) rDNA genes and the internal transcribed sequences (ITSs). While sequencing the SSU rDNA, we have discovered introns in most of the ericoid isolates. The presence and sequence of an intron in the SSU rDNA of one H. ericae isolate has already been reported by Egger, Osmond, and Goodier (1995)
. In this paper, we analyzed the entire SSU rDNA of several isolates of H. ericae and of Oidiodendron spp., as well as representatives of 16 groups of sterile mycelia. The sites of intron insertion have been mapped in all isolates and shown to occur at five different positions, including sites rarely described in fungi. Sequence analysis demonstrates that many of these introns belong to group I.
Group I introns are a structural and functional group with a widespread but irregular distribution (Dujon 1989
) and are frequently found in lower eukaryotes, especially algae and fungi (Dujon 1989
; Johansen, Muscarella, and Vogt 1996
). They occur at several locations along the chloroplast and mitochondrial genome, including protein coding genes, but in the nuclear genome they seem to be restricted to the rDNA genes. Several have been shown to splice both in vitro and in vivo due to the autocatalytic properties of the intron RNA. Insertion in intronless copies of the same gene, a process called homing (Cech 1990
), is usually catalyzed by an intron-encoded DNA endonuclease (Belfort and Roberts 1997
), although alternative mechanisms have been suggested (Roman and Woodson 1998
).
In fungi, nuclear group I introns have been found both in the Ascomycetes and the Basidiomycetes. In most cases described in the literature, one or two introns have been found in the SSU rDNA of the same organism (see Gargas, DePriest, and Taylor [1995
] and Johansen, Muscarella, and Vogt [1996
] for references). An exception is the group of lichen-forming fungi, one of the most complex systems, where relatively small insertions have been described in as many as eight different sites in Lecanora dispersa (Gargas, DePriest, and Taylor 1995
).
As the databases on ribosomal fungal sequences are becoming larger, reports on ribosomal insertions are also increasing rapidly. We used a large set of intron sequences to investigate the relationships existing among ericoid introns occurring at the different insertion sites and those of other fungi.
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Materials and Methods |
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PCR Amplification
The reaction components for the PCR reaction and the thermal cycling conditions were those described by Gardes and Bruns (1993)
. Taq polymerase from Dynazyme (Celbio) was used, and the amplification products were analyzed by electrophoresis on agarose gel after staining with ethidium bromide. Primers to conserved regions of the rDNA corresponded to those designed by White et al. (1990)
. Additional primers were designed on conserved sequences found preferentially in the Ascomycetes, and the position of their 3' nucleotide on the SSU rDNA is given relative to Saccharomyces cerevisiae. Forward primers were 18SA (5'-CCTGGTTGATCCTGCCAGT, nucleotide 21), 18SB (5'-ATTAAAGTTGTTGCAGTTAAA, nucleotide 621), and 18SB1 (5'-GTGGTGGTGCATGGCCGTT, nucleotide 1279). Reverse primers were 18SC (5'-GCACTCTAATTTGTTCAAAGTA, nucleotide 745), 18SD (5'-ATTGCGATAACGAACGAGACC, nucleotide 1310), and 18SE (5'-ATGATCCTTCCGCAGGTTCAC, nucleotide 1776). The position and orientation of the primers used in this study is indicated in the diagram of the rDNA gene in figure 1
.
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ResultsVariable Sizes of the Nuclear SSU rDNA Genes |
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An RT-PCR experiment carried out on representatives of the different ericoid isolates showed that these insertions are spliced during rRNA maturation independent of their sizes and positions along the ribosomal gene (fig. 3 ).
Characterization of DNA Insertions
PCR-amplified fragments containing DNA insertions were sequenced in representative strains of the different ericoid groups (table 1 ). The exact positions and sequences of all rDNA insertions were established (fig. 4
), with the exception of the optional insertion of H. ericae in position 1506, whose sequence was already published by Egger, Osmond, and Goodier (1995)
. Insertion sites were numbered in accordance with the Escherichia coli SSU rDNA (Gargas, DePriest, and Taylor 1995
). No insertions were found in the 5' half of the SSU rDNA, whereas five possible insertion sites were identified in the 3' half. Comparison of isolates with DNA insertions in the same positions revealed fragments of variable size (fig. 4
). Only a partial sequence of about 1,300 bp was obtained for the larger insertion of sterile mycelium C5, and this was not considered in the subsequent alignments. Insertion sizes ranged from almost 1,800 bp for sterile isolate PSIV down to 185 bp for sterile mycelia I2 and Duclos VI (fig. 4 ).
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After PCR amplification with primers 18SB/NS4, only O. maius 89 showed a DNA fragment of the expected size, whereas all other isolates gave DNA fragments of the same size but larger than expected. When a region of the SSU rDNA farther downstream was amplified, about half of these isolates showed an additional insertion (fig. 5 ). Sequencing of this optional insertion revealed that it did not contain any of the short sequences characteristic of group I introns. Its position (nucleotide 989, relative to E. coli) corresponds to an insertion site not found in other ericoid fungi (fig. 4 ) and rarely described for the nuclear SSU rDNA (see table 2 ).
Each of the seven H. ericae isolates had one intron either in position 943 (revealed by primers NS5/NS6 in fig. 6 A) or in position 1506 (revealed by primers NS7/NS8 in fig. 6 B), but none had introns at both sites.
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An apparently contradictory result, later explained by sequencing, demonstrated the same phenomenon for a number of sterile mycelia that showed single bands after PCR amplification. Amplifications with NS5/NS8 primers revealed insertions in this DNA segment, but when genomic DNA was amplified with internal primers to map these insertions more precisely (NS5/NS6 and NS7/NS8; see fig. 1 ), these isolates gave amplified fragments corresponding in size to intronless DNA copies (data not shown). DNA sequencing, however, revealed that all introns that failed to appear during PCR mapping were inserted at position 1199, which interrupts the annealing site of NS6 and NS7 primers.
Intron Sequence Analysis
The ericoid intron sequences were excised from the flanking DNA, and a first alignment of the complete sequence was attempted with CLUSTAL X. Insertions at positions 989 and 1199, which did not contain the conserved sequences of group I introns, were too divergent to be aligned with the others and were considered separately. The remaining group I introns clustered together according to their insertion sites, independent of intron length (data not shown).
To test the hypothesis that fungal introns in the same position along the SSU rDNA are related, about 70 additional intron sequences were retrieved from gene banks and analyzed (table 2 ). Most of them contained the typical conserved sequences of group I introns and aligned with the group I introns from ericoid fungi. Some of the retrieved intron sequences that did not show the P, Q, R, and S regions could be aligned with the ericoid introns also lacking these features. Some of the retrieved sequences (table 2 ) could not be aligned with confidence with any of the ericoid introns and were not considered further. This was also the case for the two short ericoid introns in the isolates Duclos VI and I2, both of 185 bp and of very similar sequences. Many, although not all, of the sequences that were too divergent to be aligned were found at sites different from those identified in ericoid fungi (table 2 ).
Because the sequences were different in size and contained very variable regions, two different alignments were carried out. A first alignment considered both variable and conserved regions, and manual modifications of the CLUSTAL X alignment involved only the exclusion of the most peripheral sequence regions and the realignment of the S region in some isolates (intron sequences H.eri100 943; H.eriCV5 943; DVIII 943; PSIV 943, and P.lign 943) due to the presence of long intervening sequences between the R and S regions (alignment accession number DS39187). Even though the alignment of the variable regions is uncertain for the most distant comparisons, we used this alignment to generate the unrooted tree shown in figure 7 because this gives a clear picture of the overall similarity of the sequences. When the analysis was confined to the most conserved regions (alignment accession number DS39188), the tree had a similar topology except that the introns at site 789 were nested within the clade of introns at site 1506 (see fig. 8 ). This indicates that introns at site 789 are related to those at site 1506 in the more conserved regions, but their more variable regions identify them as a separate, well-supported group. Introns at site 789 seem to be quite rare in fungi. In ericoid fungi, they were found exclusively in O. maius isolates and in the sterile mycelium Duclos IX. The lichen-forming species Lecanora dispersa and Myriosclerotinia scirpicola (table 2 ) were the only other fungi (both Ascomycetes) in which introns at this position were reported. However, the intron from L. dispersa could not be aligned with the ericoid introns.
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Some insertions identified in ericoid isolates are not typical group I introns because they lack the short conserved sequences that characterize this group. With the exception of the smallest introns of strains Duclos VI and mycelium I2 (both 185 bp), all sequences of ericoid introns at positions 989 and 1199 could be aligned with a number of unclassified introns retrieved from the gene bank (table 2 ). These introns were found in the same two positions as the ericoid introns (989 and 1199), as well as in positions 287 and 516, two positions not found in ericoid fungi. Four regions strongly conserved among the different introns could be identified (fig. 9 ), and this alignment was used to generate the unrooted tree shown in figure 10 . Introns at position 516 formed a well-supported group, those in position 989 formed a poorly supported cluster (53%), and those at positions 287 and 1199 formed a single mixed group. When the alignment considered only the most conserved regions, indicated in boxes in figure 9 , the corresponding unrooted tree showed that the cluster of introns at position 516 was still highly supported, whereas all the others formed a mixed group (data not shown).
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DiscussionIntrons Are a Common Feature of the Nuclear SSU rDNA of Most Ericoid Isolates |
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Sizes of ericoid fungal introns are very variable and correspondd to those most commonly observed for other fungal introns (about 200500 bp). In some groups, however, they tend to be quite large. Five fungal groups contained insertions longer than 800 bp, with a maximum length of about 1,800 bp in sterile mycelium PSIV, which is to our knowledge the largest group I intron identified so far in the nuclear SSU rDNA of eukaryotes. Concerning the sites of insertion identified in the SSU rDNA of ericoid fungi, some, such as sites 943 and 1506, are conserved and very commonly found in lower eukaryotes whereas others, like sites 789 and 989, are rare and have been identified only sporadically.
Intragenomic Variability in the Presence of Group I Introns
In filamentous fungi, nuclear rDNA genes are present in tandem repeats ranging from about 60 copies in Coprinus (Cassidy et al. 1984
) to 220 in Neurospora crassa (Russell et al. 1984
). The homing ability of group I introns allows them to insert at specific sites of intronless rDNA genes and spread over gene repeats (Cech 1990;
Lambowitz and Belfort 1993
; Belfort and Roberts 1997
). However, data on Ascomycetes (DePriest 1993
) and Basidiomycetes (Hibbett 1996
) indicate that some constraints on intron transposition may exist, since introns are not necessarily found on all rDNA repeats within the genome. The same observation was made in this study for several ericoid fungal isolates, for which the relative number of intronless and intron-containing rDNA repeats seems to be quite variable. For many sterile isolates, intronless rDNA repeats probably made up a minor percentage in the genome, as they could be amplified and generate a DNA fragment only with primers that excluded intron-containing copies by annealing directly to the intron insertion site. In other cases, two bands of equal intensity were amplified, suggesting a more balanced situation. In this study, we may have underestimated the presence of introns occurring at low frequencies in the rDNA repeats because we did not screen the genomes of ericoid isolates with intron-specific primers, as described, for example, by Hibbett (1996)
.
It remains an open question whether the heterogeneity observed in the rDNA repeats occurs within a single haploid nucleus,or whether rDNA copies respectively lacking and containing introns are separated into different nuclei of a heterokaryotic mycelium. Heterogeneity in ribosomal gene sequences has been shown within a single coenocytic spore of arbuscular mycorrhizal fungi (Lloyd-MacGilp et al. 1996
; Lanfranco, Delpero, and Bonfante 1996;
Trouvelot et al. 1999
). In the case of Scutellospora castanea, in situ hybridization has revealed that these different sequences are found within the same nucleus (Trouvelot et al. 1999
).
Mobility of Group I Introns
Horizontal transmission of group I introns among taxa has been discussed since their discovery on account of their scattered distribution and presence/absence in related taxa. In fungi, this hypothesis has been supported by phylogenetic analysis of the Homobasidiomycetes (Hibbett 1996
) and the archiascomycetous Protomyces (Nishida, Tajiri, and Sugiyama 1998
). Its investigation in ericoid fungi, however, will first require determination of the phylogenetic relationship of the sterile ericoid mycelia with each other and with H. ericae and the genus Oidiodendron.
Sequence analysis of the fungal introns indicates that insertions sharing the same site on the SSU rDNA gene form distinct lineages and are likely related. This hypothesis was already suggested by Hibbett (1996)
and Bhattacharya, Friedl, and Damberger (1996)
and is now supported in fungi by the analysis of a much larger number of intron sequences. In particular, introns in position 943 (relative to E. coli) are clearly distinct from those at site 1506. These two sites are certainly very ancient, as they are found in several protists (see Gargas, DePriest, and Taylor 1995
). Site 789 is an uncommon and probably more recent insertion site, and introns in this position also form a well-supported group.
It remains an open question whether the clusters gathering together insertions found at the same position in different taxa result from their occurrence in a common progenitor, with a loss in all the intermediate lineages, or from horizontal transfer across different phyla. Although the cost of intron gain and intron loss is not known, the wide phylogenetic distance between some of the organisms in the tree (both Basidiomycetes and Ascomycetes) would suggest the second hypothesis as the most parsimonious. If this is the case, the mechanisms that allow introns to home in a predefined position must be very efficient, since no exceptions were found for the introns at position 943, for example. Lateral movement along the rDNA gene may nevertheless be possible in some cases and has been suggested by several authors (e.g., Bhattacharya, Friedl, and Damberger 1996
; Hibbett 1996
). If introns in position 789 are a more recent acquisition, their closer similarities with introns at position 1506 suggest that they may have originated from the transposition of introns originally in position 1506. The close relatedness between the intron at position 1199 of the ericoid isolate I1 and that found at site 287 in the yeastlike endosymbiont of an anobiid beetle, Stegobium paniceum, also suggests intron transposition. Close similarity between introns at positions 287 and 1199 was also found by Bhattacharya, Friedl, and Damberger (1996)
in the lichen-forming fungus Lecanora dispersa, although these sequences could not be aligned with those of ericoid introns.
Phylogenetic analysis has also indicated that horizontal transmission of group I introns may have occurred between organelles (Turmel et al. 1995
) and also between fungi and plants during the intimate contacts established during pathogenic or symbiotic interactions (Nishida and Sugiyama 1995
; Adams, Clements, and Vaughn 1998
). In ericoid symbiosis, the fungal and plant cytoplasms are separated only by a thin interface (Perotto et al. 1995
), so a possibility exists that some introns may thus have been transmitted from the fungus to the host, although this aspect has not been investigated.
Isolates of the Same Species Contain Optional Introns
Screening of ericoid isolates also raised the question of how these introns are inherited/transmitted at a lower taxonomic rank. This aspect could be investigated in O. maius and H. ericae, for which the taxonomic identification of isolates is certain. Heterogeneity among isolates of the same species, expressed by the presence/absence of specific introns, was found in both species. For H. ericae, our results are supported by recent findings on genetically related isolates from ectomycorrhizal roots, showing the variable presence of the intron at position 1506 (T. Vrålstad, personal communication). Heterogeneity has often been reported in eukaryotes (see Johansen, Muscarella, and Vogt 1996
). In fungi, it has been described in natural populations of the lichen complex Cladonia merochlorophaea (DePriest and Been 1992
; DePriest 1993
), in the ectomycorrhizal Ascomycetes Cenococcum geophilum (Shinohara, Lobuglio, and Rogers 1996
), and in Sclerotinia sclerotiorum isolates (Carbone, Anderson, and Kohn 1995
), in which an optional intron was found in the mitochondrial SSU rDNA.
Mechanisms of Intron Mobility
Mobile group I introns generally encode a site-specific endonuclease that cleaves near the site of intron insertion (see Lambowitz and Belfort 1993
). Genes coding for endonucleases have been reported for several introns occurring in the cell organelles, where they share either the LAGLI-DAGLI or the GIY-YIG motifs (Johansen, Embley, and Willassen 1993
; Lambowitz and Belfort 1993
; Belfort and Roberts 1997
). In contrast, very few intron-encoded endonucleases are known for nuclear rDNA. They were first described in extrachromosomal rDNA of the slime molds Physarum polycephalum and Didymium iridis and the amoeboflagellate Naegleria (Einvik, Elde, and Johansen 1998
). In fungi, intron-encoded endonucleases have recently been reported in the nuclear SSU rDNA of Nectria galligena (Johansen and Haugen 1999
). In this species, a polymorphic insertion element either containing or lacking an endonuclease-encoding open reading frame (ORF) was observed in different isolates (Crockard et al. 1998
). All endonucleases encoded by nuclear rDNA introns seem to be members of a distinct family defined by a conserved 30-amino-acid segment including a His-Cys motif (Johansen, Embley, and Willassen 1993
).
Two of the longest introns identified in the nuclear SSU rDNA of ericoid fungi may also code for endonucleases, as one of the possible reading frames revealed strong homologies with the conserved 30-amino-acid region of P. polycephalum and Naegleria. Similar to N. galligena, the region coding for the His-Cys box in the 1199 intron of ericoid isolate I1 was found on the antisense rDNA strand, thus indicating that a separate transcription event would be needed to generate an mRNA for the endonuclease.
Alternative mechanisms based on the reversal of the splicing reaction at the RNA level have been proposed to explain mobility of introns in the absence of endonuclease-coding sequences (Lambowitz and Belfort 1993
; Roman and Woodson 1998
). Since analysis of most ericoidand, in general, fungalintrons excludes the presence of possible coding sequences, mechanisms based on reverse splicing and loss may explain the scattered distribution of nuclear rDNA introns. However, it cannot be excluded that endonuclease-coding sequences were once present in a larger number of fungal species but were lost over time. It is well documented that ORFs coding for endonucleases can be mobile themselves (see Lambowitz and Belfort 1993
).
Some Insertion Elements Lack the Consensus Sequences of Typical Group I Introns
Several fungal intron sequences deposited in gene banks do not contain the consensus sequences of group I introns. Sequence studies of lichen-forming fungi led to the suggestion that some of the smaller DNA insertions lacking the P, Q, R, and S regions may be remnants of larger group I introns that went through incorrect splicing (Grube, Gargas, and DePriest 1996
; Stenroos and DePriest 1998
). Duclos VI and Sterile mycelium I2, which feature the smallest insertions (185 bp) in position 1199, are closely related by ITS sequencing to sterile mycelium I1 (unpublished data), which displays a large intron (1,330 bp) in the same position. However, alignment to this larger insertion did not support the hypothesis of incorrect splicing of a previously larger intron related to the I1 intron.
The large sizes of some of these introns (up to 1,330 bp), their occurrence at specific sites on the SSU rDNA distinct from those colonized by the subgroup IC1 group I introns, and the presence of four well-conserved sequence regions indicate that these less abundant nuclear ribosomal introns are not relics but represent a distinct population of insertional elements in fungi. These conserved regions had not been previously identified, although they seem to be involved in the formation of a secondary structure similar to other group I introns, as recently suggested by Johansen and Haugen (1999)
for the N. galligena intron at position 1199. Analysis of the mature rRNA of ericoid isolates shows that all introns are correctly spliced during rRNA processing, indicating that proper folding is obtained for members of this intron subgroup.
In conclusion, we have identified a high level of polymorphism in the nuclear SSU rDNA genes due to insertion, at distinct positions, of a heterogeneous population of introns that are different in size and sequence. Two distinct subgroups of introns colonize the rDNA of ericoid fungi, one showing the conserved regions of subgroup IC1 group I introns and the other featuring conserved (but distinct) nucleotide sequences. Comparisons with other fungal sequences has allowed us to strengthen the hypothesis that, if transferred horizontally across taxa as suggested by phylogenetic evidence (Hibbett 1996
), group I introns usually home very specifically into the same insertion site. Homing may be less specific, or transposition events may occur more frequently, for the second group of introns, as we found high similarity for insertions in at least three different sites. The finding of sequences related to endonuclease genes in some of the ericoid introns may provide an additional clue to lead to an understanding of the mechanisms of intron mobility in nuclear introns.
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Acknowledgements |
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Footnotes |
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1 Keywords: ericoid fungi,
group I introns,
Hymenoscyphus ericae,
Oidiodendron maius,
rDNA genes,
endonucleases.
2 Address for correspondence and reprints: Silvia Perotto, Centro Studio Micologia del Terreno, V.le Mattioli 25, 10125 Torino, Italy. E-mail: perotto{at}bioveg.unito.it
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