A Diverse Population of Introns in the Nuclear Ribosomal Genes of Ericoid Mycorrhizal Fungi Includes Elements with Sequence Similarity to Endonuclease-Coding Genes
Silvia Perotto
2
Paola Nepote-Fus
0
Laura Saletta
2
Claudio Bandi
1
J. Peter W. Young
3
0
Dipartimento di Biologia Vegetale dell'Universita`
,
Turin
,
Italy
1
Istituto di Patologia Generale Veterinaria
,
Milan
,
Italy
2
Centro Studio Micologia del Terreno-CNR
3
Department of Biology, University of York
,
York, England
Ericoid mycorrhizal fungi form symbioses with the roots of members of the Ericales. Although only two genera have been identified in culture, the taxonomic diversity of ericoid symbionts is certainly wider. Genetic variation among 40 ericoid fungal isolates was investigated in this study. PCR amplification of the nuclear small-subunit ribosomal DNA (SSU rDNA) and of the internal transcribed spacer (ITS), followed by sequencing, led to the discovery of DNA insertions of various sizes in the SSU rDNA of most isolates. They reached sizes of almost 1,800 bp and occurred in up to five different insertion sites. Their positions and sizes were generally correlated with morphological and ITS-RFLP grouping of the isolates, although some insertions were found to be optional among isolates of the same species, and insertions were not always present in all SSU rDNA repeats within an isolate. Most insertions were identified as typical group I introns, possessing the conserved motifs characteristic of this group. However, other insertions lack these motifs and form a distinct group that includes other fungal ribosomal introns. Alignments with almost 70 additional sequences from fungal nuclear SSU rDNA introns indicate that introns inserted at the same site along the rDNA gene are generally homologous, but they also suggest the possibility of some horizontal transfers. Two of the ericoid fungal introns showed strong homology with a conserved motif found in endonuclease genes from nuclear rDNA introns.
Introduction
Ericoid mycorrhizal fungi are a diverse group of
fungi that form symbiotic associations with plants in the
Ericales (Perotto et al. 1995; Read 1996; Straker 1996).
The fine roots of these plants are colonized in nature by
both Ascomycetes and Basidiomycetes (Bonfante 1980;
Peterson, Mueller, and Englander 1980), but taxonomic
identification is confined to the Ascomycetes, as
Basidiomycetes symbionts have not yet been isolated in pure
culture. Species from two genera are reported as
mycorrhizal partners of ericaceous plants: Hymenoscyphus
ericae (Read) Korf and Kernan (Read 1974), with its
anamorph Scytalidium vaccinii Dalpe, Litten, and
Siegler (Egger and Siegler 1993), and Oidiodendron spp.
(Couture, Fortin, and Dalpe 1983; Dalpe 1986), with
their teleomorphs in the Gymnoascaceae and
Mixothricaceae (Dalpe 1989; Hambleton et al. 1998).
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 (...truncated)