Evolution of mitochondrial SSU-rDNA variable domain sequences and rRNA secondary structures, and phylogeny of the Agrocybe aegerita multispecies complex.

Mitochondrial small subunit (mtSSU) rDNA variable (V1, V2, V4, V6, V8 and V9) domain sequences and rRNA secondary structures evidenced eight molecular groups within 32 strains of the Agrocybe aegerita multispecies complex from different continents. mtSSU-rRNA secondary structure evolution occurred mainly by insertion/deletion of sequences from 8 to 57nt long. Preferential insertion/deletion sites correlated with loops of the mtSSU-rRNA secondary structures, and suggested that these events occurred in regions without interactions in the ribosomal-protein assembly. Indels modified the stem length (V1 and V4 domains) or the size and loop number (V6 and V9 domains). Three indels inserted in the V1 and V4 domains had 76.5% to 94.7% identity with short sequences of the mitochondrial cytochrome c oxidase gene; this fact and the presence of inverted repeated motifs within indel sequences suggested a mechanism of evolution based on insertion/deletion of sequences from another region of the mitochondrial genome. Phylogenetic relationships inferred using both ribosomal DNA sequences and rRNA secondary structures were congruent and evidenced three clades within the A. aegerita complex: European, Argentinean, and a more distant Asian-American clade including A. aegerita and A. chaxingu strains. These results suggested that numerous genetic exchanges occurred between Asian-American strains after isolation of the European clade. V4-V6-V9 concatenated sequences of European and Argentinean clades had 86.1% identity, similar to the value calculated between two Agrocybe closely related species, suggesting that these clades could represent different species. A cleaved amplified polymorphic sequence test for rapid characterization of strains was developed.

A CAPS test allowing a rapid distinction of Penicillium expansum among fungal species collected on grape berries, inferred from the sequence and secondary structure of the mitochondrial SSU-rRNA.

Penicillium expansum is a fungal species highly damageable for the postharvest conservation of numerous fruits. In vineyards, this fungus is sometimes isolated from grape berries where its presence may lead to the production of geosmin, a powerful earthy odorant, which can impair grapes and wines aromas. However, the discrimination of P. expansum from related fungi is difficult because it is based on ambiguous phenotypic characters and/or expensive and time-consuming molecular tests. In this context, the complete sequences and secondary structures of Penicillium expansum and Penicillium thomii mitochondrial SSU-rRNAs were achieved and compared with those of two other phylogenetically related Ascomycota: Penicillium chrysogenum and Emericella nidulans. The comparison has shown a high conservation in size and sequence of the core and of the variable domains (more than 80% of nt identity) of the four SSU-rRNAs, arguing for a close phylogenetic relationship between these four species of the Trichocomaceae family. Large (from 10 to 18 nt) inserted/deleted (indel) sequences were evidenced in the V1, V5 and V6 variable domains. The size variations (10 to 18 nt) of the V1 indel sequence allowed the distinction of the four species; the V5 indel (15 nt) was specifically recovered in E. nidulans; the V6 indel (16 nt), shared by the three Penicillium species, was lacking in E. nidulans. A couple of conserved primers (UI/R2) were defined to generate a PCR product containing the V1 to V5 variable domains. This product contained the two regions of the four SSU-rRNAs showing the highest rates of nt substitutions, namely the V2 variable domain and, surprisingly, a helix (H17) of the core. The H17 sequence was shown to specifically possess in P. expansum a recognition site for the ClaI restriction endonuclease. Hence, this enzyme generates a digestion pattern of the PCR product with two bands (350 bp+500 bp), specific to P. expansum and easily separable by agarose gel electrophoresis. This leads to a CAPS test usable for P. expansum discrimination among grape berries isolated filamentous fungi. The CAPS test was validated by a comparative analysis involving 29 strains belonging to 17 species currently isolated from grape berries in the Bordeaux vineyards.

The mitochondrial apocytochrome b genes of two Agrocybe species suggest lateral transfers of group I homing introns among phylogenetically distant fungi.

The Agrocybe chaxingu and Agrocybe aegerita mitochondrial apocytochrome b coding sequences are highly similar (97% of nt identity), but have highly different sizes (2312 and 4867nt, respectively), due to the presence of three large group IB introns: two (iAae1 and iAae2) in A. aegerita, one (iAch1) in A. chaxingu. All these introns encode a homing endonuclease (HE) similar to those described in introns of mitochondrial genes (cob, cox1, and nad5) from various organisms. Phylogenetic trees were built with these HE sequences. From these trees, the Agrocybe coding introns argue for recent lateral transfers, i.e., occurring after the separation of the two Agrocybe species, involving phylogenetically distant fungi such as members of the Ascomycota phylum (for iAch1 and iAae2) and, for the first time to our knowledge, a member of the Chytridiomycota phylum (for iAae1). The grouping of the HE gene (HEG) sequences according to the mitochondrial gene (cob, cox1, and nad5) where they are inserted, suggests modifications of the interactions between the HE and the recognized sequences, leading to new target genes. The largest distribution of the iAch1 HE, shared by several cob and cox1 mitochondrial genes from Ascomycota, Basidiomycota, and Chytridiomycota phyla, suggests a higher target flexibility of this HE, perhaps related to the presence of two different LAGLIDADG motifs in the catalytic site of the enzyme.

Characterization of Penicillium species isolated from grape berries by their internal transcribed spacer (ITS1) sequences and by gas chromatography-mass spectrometry analysis of geosmin production.

Geosmin (trans-1,10-dimethyl-trans-9-decalol), an earthy-musty compound, has been identified in wines and in grape juice, in which its presence is highly detrimental to the aromatic quality. Geosmin has a biological origin, and the analysis of rotten grape microflora has been done on two grape varieties (Semillon, Cabernet Sauvignon) from six parcels of the Bordeaux region over 3 years (1999, 2000, 2001). Forty-three Penicillium-related species have been analyzed by gas chromatography-mass spectrometry (GC-MS) for their geosmin production. GC-MS analysis has demonstrated that the earthy odor was always correlated with the presence of geosmin. Phenotypic characterization of Penicillium spp. being ambiguous, a molecular characterization by rDNA internal transcribed spacer (ITS1) sequencing was performed for all strains. The results evidenced that all strains producing geosmin belonged to only one species, P. expansum, and that the other strains, not producing geosmin, belonged to three species: P. purpurogenum, P. thomii, and Talaromyces wortmanii.

The Aa-Pri4 gene, specifically expressed during fruiting initiation in the Agrocybe aegerita complex, contains an unusual CT-rich leader intron within the 5' uncoding region.

The Aa1-Pri4 gene was cloned from the edible mushroom Agrocybe aegerita. The gene, specifically expressed during fruiting initiation, encodes a glycine-rich protein of 116 amino acids, with no homology to already known proteins. Homologous genes were amplified from two other strains belonging to the Agr. aegerita complex and originating from South-East Asia; and a comparison of the three genes revealed a high conservation of the coding sequences (72.8-97.8%). The PRI4 putative protein sequences were highly similar (87.5-100.0%); and all of them contained two protein kinase C sites, suggesting a potential supplementary regulation by phosphorylation at the protein level. The 5' uncoding regions all presented a leader intron, very variable in sequence (45.7% identity), but with a high C+T content (74.5-79.0%). The presence of such CT-rich sequences previously described in the promoter of highly expressed fungal genes suggests that the leader intron of the Aa1-Pri4 gene could be involved in the high-level, stage-specific expression.

Secondary structure and molecular evolution of the mitochondrial small subunit ribosomal RNA in Agaricales (Euagarics clade, Homobasidiomycota).

The complete sequences and secondary structures of the mitochondrial small subunit (SSU) ribosomal RNAs of both mostly cultivated mushrooms Agaricus bisporus (1930 nt) and Lentinula edodes (2164 nt) were achieved. These secondary structures and that of Schizophyllum commune (1872 nt) were compared to that previously established for Agrocybe aegerita. The four structures are near the model established for Archae, Bacteria, plastids, and mitochondria; particularly the helices 23 and 37, described as specific to bacteria, are present. Within the four Agaricales (Homobasidiomycota), the SSU-rRNA "core" is conserved in size (966 to 1009 nt) with the exception of an unusual extension of 40 nt in the H17 helix of S. commune. The four core sequences possess 76% of conserved positions and a cluster of C in their 3' end, which could constitute a signal involved in the RNA maturation process. Among the nine putative variable domains, three (V3, V5, V7) do not show significant length variations and possess similar percentages of conserved positions (69%) than the core. The other six variable domains show important length variations, due to independent large size inserted/deleted sequences, and higher rates of nucleotide substitutions than the core (only 31% of conserved positions between the four species). Interestingly, the inserted/deleted sequences are located in few preferential sites (hot spots for insertion/deletion) where they seem to arise or disappear haphazardly during evolution. These sites are located on the surface of the tertiary structure of the 30S ribosomal subunit, at the beginning of hairpin loops; the insertions lead to a lengthening of existing hairpins or to branching loops bearing up to five additional helices.

Molecular characterization of the Pri3 gene encoding a cysteine-rich protein, specifically expressed during fruiting initiation within the Agrocybe aegerita complex.

Coding and regulating sequences of Pri3 (a novel gene specifically expressed in both nucleus types of dikaryons during fruiting initiation) were compared within nine strains of the Agrocybe aegerita complex originating from Europe, Asia and Latin America. Highly homologous coding sequences were found; and the 95-amino-acid-long PRI3 deduced proteins all shared eight cysteines and eight glycines, a putative signal peptide suggesting an extra-cellular localization, a protein kinase C site (T(70)) and a cAMP-dependent kinase site (S(74)). The PRI3 proteins presented no significant homologies with already known proteins and constitute a new class of small cysteine-rich proteins. Within the 5' uncoding regions, two leader introns and a putative upstream regulating sequence resembling the QA1-F activator site were highly conserved. Comparison of the gene sequences within the A. aegerita complex suggested a divergent evolution of the European and Asian/Latin American groups of genes.

Phylogenetic relationships of Pleurotus species according to the sequence and secondary structure of the mitochondrial small-subunit rRNA V4, V6 and V9 domains.

A comparative study of the V4, V6 and V9 domains of the mitochondrial small-subunit (SSU) rRNA was conducted to evaluate the use of these sequences to investigate phylogenetic relatedness within the genus Pleurotus. The PCR products encompassing these regions from 48 isolates belonging to 16 Pleurotus species were sequenced and compared. From this comparison, the length and sequence of the three domains were found to be constant within a species. Significant inter-species variations due to insertion/deletion events were found, in most cases occurring in regions not directly involved in the maintainance of the standard SSU rRNA secondary structure. Phylogenetic analysis based upon these mitochondrial sequences was in agreement with relationships previously established by morphological descriptions and with previous studies based upon the nuclear genome or isozymes; moreover such analysis resolved some ambiguities in earlier analyses. It was confirmed that P. ostreatus and P. florida represent a single species, as well as P. pulmonarius and P. sajor-caju. The phylogenetic analysis also made it possible to assess the relative positions of P. rattenburyi, P. lampas, P. sapidus, P. colombinus and P. eryngii. The results clearly showed that sequences of the V4, V6 and V9 domains of the mitochondrial SSU rRNA could provide good markers for use in the taxonomy and phylogeny of species of Basidiomycota. Because of their nucleotide conservation, the major advantage of these species-specific markers was the possibility to study only one isolate from each species to determine phylogenetic relatedness.