Fungal Genetics and Biology 46 (2009) 837–848
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The Cryphonectria parasitica mitochondrial rns gene: Plasmid-like elements, introns and homing endonucleases Claudia B. Monteiro-Vitorello a,*, Georg Hausner a,1, Denise B. Searles a, Ewan A. Gibb b, Dennis W. Fulbright c, Helmut Bertrand a a b c
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824-4320, USA Department of Microbiology, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada Department of Plant Pathology, Michigan State University, East Lansing, MI 48824, USA
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Article history: Received 14 February 2009 Accepted 8 July 2009 Available online 14 July 2009 Keywords: Cryphonectria parasitica Mitochondria Plasmid-like element Hypovirulence rns gene Group I intron Group II intron
a b s t r a c t The mt-rns gene of Cryphonectria parasitica is 9872 bp long and includes two group I and two group II introns. An analysis of intronic protein-encoding sequences revealed that LAGLIDADG ORFs, which usually are associated with group I introns, were transferred at least twice into group II introns. A plasmidlike mitochondrial element (plME) that appears in high amounts in previously mutagen-induced mit1 and mit2 hypovirulent mutants of the Ep155 standard virulent strain of C. parasitica was found to be derived from a short region of the mt-rns gene, including the exon 1 and most of the first intron. The plME is a 4.2-kb circular, multimeric DNA and an autonomously-replicating mtDNA fragment. Although sexual transmission experiments indicate that the plME does not directly cause hypovirulence, its emergence is one manifestation of the many complex molecular and genetic events that appear to underlie this phenotype. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction In a previous study on the chestnut blight fungus Cryphonectria parasitica (Monteiro-Vitorello et al., 1995), mitochondrial defects were induced in the virulent Ep155 standard laboratory strain by a mutagenic treatment that was intended to cause preferentially mtDNA breaks. Among the respiratory mutants that were recovered from this treatment, two, mit1 and mit2, had hypovirulence phenotypes that could be transmitted to virulent strains by hyphal contact and were maternally inherited in sexual crosses. Since cytoplasmically-transmissible hypovirulence phenotypes that are not affiliated with respiratory defects or transmitted sexually are associated with infections of C. parasitica by certain types of double-stranded RNA (dsRNA) viruses (reviewed by Nuss, 1992, 2005; Bertrand, 2000), it is important to note that the mutants were selected in Ep155, a strain which commonly is used as the virus-free, virulent standard in studies on the pathogenicity of this fungus. In order to distinguish the hypovirulence trait that is associated with the mit mutants from that which is elicited by viruses,
* Corresponding author. Present address: Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Rua Santa Adélia, 166, 09210-170, Santo André, SP, Brazil. Fax: +55 11 4437 8530. E-mail address:
[email protected] (C.B. Monteiro-Vitorello). 1 Present address: Department of Microbiology, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada. 1087-1845/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.fgb.2009.07.005
it has been named ‘‘mitochondrial hypovirulence” (Monteiro-Vitorello et al., 1995). During the characterization of the mit1 and mit2 mutants, it was observed that both had acquired high amounts of mtDNA-derived small plasmid-like elements. Plasmid-like elements that are derived from regions of the mitochondrial DNA (mtDNA) have been found in a variety of fungi (Griffiths, 1992; Hausner, 2003) and have been named plasmidlike mitochondrial elements (plME) to distinguish them from true mitochondrial plasmids. Circular mtDNA-derived plMEs that exist in multimeric forms also have been found to be associated with a degenerative disease in the plant-pathogenic fungus Ophiostoma novo-ulmi (Charter et al., 1993; Abu-Amero et al., 1995). In the filamentous fungi, the mechanisms involved in the initial formation of mitochondrial plMEs, their amplification, mode of inheritance and physiological effects are still poorly understood (reviewed in Griffiths, 1992, 1995, 1996; Griffiths et al., 1995; Kempken, 1995; Hausner et al., 2006a). However, there are instances where plMEs can have important functions in mtDNA maintenance; for example, in some yeasts (e.g. Candida parapsilosis) that have linear mitochondrial chromosomes, specialized plMEs, referred to as telomeric circles (t-circles), are involved in telomere maintenance (Tomaska et al., 2002). To gain further insights into the molecular basis of mitochondrial hypovirulence, mit plMEs were characterized. Briefly, the findings reported in this paper indicate that plMEs were found to have originated from a region of the mtDNA that partially overlaps
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the mt-rns gene, which is 9872 bp long and interrupted by two group I introns and two group II introns. Although group I and II introns are self-splicing, sometimes the process is expedited by intron-encoded maturases (Saldanha et al., 1993; Ho et al., 1997; Bassi et al., 2002; Bolduc et al., 2003; Belfort, 2003; Caprara and Waring, 2005). In some instances, the intronencoded maturase proteins also act as intron mobility-facilitating homing endonucleases (HE; Dujon, 1989; Dujon and Belcour, 1989; Belfort and Roberts, 1997; Belfort et al., 2002; Haugen et al., 2004, 2005; Stoddard, 2005). Some intronic homing endonuclease genes (HEGs) themselves are mobile elements that can move from an ORF-containing intron to an ‘‘ORF-less” intron (Mota and Collins, 1988; Sellem et al., 1996; Sellem and Belcour, 1997). It appears that self-splicing introns provide the HEGs with a neutral location, thus reducing the chance of the HEG damaging the host genome (Goddard and Burt, 1999; Lambowitz et al., 1999; Belfort et al., 2002; Schäfer, 2003; Stoddard, 2005). HEGs and introns are quite invasive and contribute towards the size of fungal mtDNA genomes, mtDNA polymorphisms, and mtDNA rearrangements (Dujon, 1989; Charter et al., 1996; Belcour et al., 1997; Salvo et al., 1998; Hamari et al., 1999; Gobbi et al., 2003; Gibb and Hausner, 2005; Gogarten and Hilario, 2006). These elements are of interest since introns-associated mtDNA instabilities have been observed in yeast as well as an assortment of filamentous fungi (Osiewacz and Esser, 1984; Michel and Cummings, 1985; Cummings et al., 1986, 1990; Dujon and Belcour, 1989; Gillham, 1994; Abu-Amero et al., 1995), thus a detailed description of the mt-rns gene and its introns is presented.
2. Materials and methods 2.1. Fungal strains and growth conditions The C. parasitica mitochondrial mutants mit1 and mit2 (Monteiro-Vitorello et al., 1995) along with the wild type strain Ep155 were used in this study. The maintenance of stock cultures was as described by Puhalla and Anagnostakis (1971) and Anagnostakis (1979). The media and culture conditions for obtaining large amounts of mycelium for the preparation of mitochondria were described previously (Monteiro-Vitorello et al., 1995; Bell et al., 1996). 2.2. DNA extraction and restriction endonuclease digestion The procedure used for the purification of mitochondria was based on the flotation-gradient method described by Lambowitz (1979) as modified by Monteiro-Vitorello et al. (1995) and Bell et al. (1996). DNA was extracted from mitochondria (Bell et al., 1996) and digested with restriction endonucleases under conditions described by Sambrook et al. (1989) with modifications recommended by the manufacturer of the enzymes (Roche Diagnostics, Indianapolis, IN, USA). Restriction fragments or PCR products were separated by gel electrophoresis through 0.7% to 1.0% agarose (Invitrogen, Carlsbad, CA, USA) gels in TBE buffer and visualized under UV-light after staining of the gels with ethidium bromide. DNA fragments were sized using the 1-kb molecular weight ladder (Invitrogen) as a standard. 2.3. Molecular cloning To obtain clones of this plasmid-like element, the mitochondrial DNA from the mit2 strain was digested with HindIII (Invitrogen) and the resulting DNA fragments were ligated into a HindIII-digested Bluescript KS vector (Stratagene, La Jolla, CA, USA) and cloned in Escherichia coli DH5a. Bacterial transformations, and subsequent plating and screening were done by standard protocols (Sambrook
et al., 1989). DNA restriction fragments or DNA segments obtained by the polymerase chain reaction (PCR) were extracted from 1% low-melting point agarose gels (FMC SeaPlaque; Mandel, Guelph, ON, Canada) and purified using Wizard Miniprep or PCR Prep columns, as described by the supplier (Promega, Madison, WI, USA). The 4.2-kb monomer of a plME, which appears as an amplified HindIII fragment on electropherograms of the mtDNA from the mit2 mutant, was extracted from an agarose gel, labeled with digoxigenin (DIG) by the randomly-primed DNA replication procedure and used as a probe on Southern blots according to the protocols provided by the manufacturer (Roche Applied Science, Indianapolis, IN 46250, USA) to identify cloned plasmids that contained the entire plME HindIII monomer as an insert. To identify regions of the mtDNA from which the plMEs of mit1 and mit2 were derived, Southern blots of XbaI- or HindIII-cleaved Ep155 mtDNA were hybridized to a probe prepared from the HindIII clone of the plasmid-like element from mit2. Southern blots were prepared on Hybond-N + nylon membrane (Amersham Biosciences, Piscataway, NJ, USA) using standard techniques (Sambrook et al., 1989). For sequencing, the HindIII monomer of the mitochondrial plME from the mit2 mutant was inserted into the pBluescript KS vector (Stratagene) in both orientations with respect to the location of the T3 and T7 primer sites. This facilitated the generation of deletion subclones with the exonuclease III double-stranded Nested Deletion kit (Pharmacia Biotech, Kalamazoo, MI, USA) according to the approach described by Henikoff (1984). The entire mt-rns gene was cloned using a shotgun strategy. Libraries containing HindIII or XbaI fragments of the mtDNA of the EP155 wild type as inserts in the pBluescript II KS (+/) vector (Stratagene) and mtDNA digested were established as described previously (Bell et al., 1996). Conditions for ligation, transformation and screening for potential recombinants were done according Sambrook et al. (1989). The E. coli colonies in these libraries were screened for rns-containing plasmids by Southern blot hybridization with heterologous gene probes from Neurospora crassa (Bell et al., 1996) and by PCR; utilizing primers designed from known mt-rns sequences. 2.4. DNA sequencing Sequencing of DNA was carried out using plasmid DNA purified with the WizardTM Minipreps DNA Purification System (Promega, Madison, WI) according to the manufacturer’s recommended protocols. Dideoxy DNA sequencing was performed initially according to the protocols described by Sanger et al. (1977) with the modifications recommended by Zhang et al. (1991) and [a33P] dATP (Amersham Biosciences) in the labeling reaction, and later by automated sequencing with fluorescent dyes using Applied Biosystems 3100 Genetic Analyzers (Applied Biosystems, Forest City, CA, USA). DNA sequences were determined for both strands of cloned DNAs by the progressive extension of sequences obtained with the vector-based T7 and T3 primers (Stratagene) as well as appropriately designed, sequence-extending primers synthesised by the Research Technology Support Facility at Michigan State University. The continuity of sequences across the restriction sites that were involved in the cloning of DNA was confirmed by the sequencing of PCR products obtained from a template consisting of purified Ep155 mtDNA with pairs of primers that annealed to sequences located approximately 200 base pairs upstream and downstream, respectively, of each of the pertinent locations. 2.5. Two-dimensional gel electrophoresis Mitochondrial DNA for analysis by two-dimensional (2D) agarose gel electrophoresis was prepared from 24 h non-synchronized cultures of the mit2 strain. Neutral/neutral 2D agarose gel
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electrophoresis were done on the uncut mtDNA of the mit2 mutant as described by Brewer and Fangman (1987) with the following modifications: the first dimension was performed in a 0.7% agarose gel in 1 TBE (85 mM TRIS-base, 89 mM borate and 2.5 mM EDTA) at 40 V over 20 h at room temperature, the second dimension was performed in a 1.2% agarose (Invitrogen) gel at 4 °C in 1 TBE buffer containing 3 mg/ml ethidium bromide at 100 V for 60 h. First and second dimension gels were Southern-blotted and hybridized as described above. 2.6. Phylogenetic analyses and RNA folding All sequences obtained from the C. parasitica plME HindIII clone and cloned mtDNA XbaI fragments were initially compared with mtDNA sequences available for N. crassa. Nucleotide sequences for the plME segments were assembled into contigs (GenBank accession no. AF029891). Sequences for the mt-rns regions were analyzed with PCGENE, annotated and submitted to GenBank (accession no. AF029891). Fungal mt-rns sequences and sequences related to intron-encoded LAGLIDADG ORFs were extracted from GenBank using the C. parasitica sequences as queries in BLASTp (Altschul et al., 1990) searches. Nucleotide sequence alignments were done with the ClustalX program (Thompson et al., 1997), and the alignments were manually refined with GeneDoc (v2.5.010; Nicholas et al., 1997). For phylogenetic analysis, the amino acid sequence alignments were generated with the online PRALINE multiple sequence alignment program (Simossis and Heringa, 2003, 2005) and then refined further with the GeneDoc program. We generated phylogenetic estimates for the nucleotide or amino acid sequence alignments using the PHYLIP package (Felsenstein, 2006; Version 3.66; http://evolution.genetics.washington.edu/phylip/getme.html). Neighbor-joining (NJ) analysis employing either DNADIST or PROTDIST was used to obtain distance matrices (settings: F84 for DNA or Dayhoff’s PAM 250 for amino acid sequences), which were then analyzed with the NEIGHBOR program. Parsimony analysis was done with DNAPARS or PROTPARS for DNA or amino acids sequences, respectively. Bootstrap analysis (SEQBOOT, n = 1000) was performed in combination with NJ and Parsimony analysis. Majority rule consensus trees based on either parsimony or NJ analysis were obtained with the CONSENSE program. The MrBayes (version 3.1) program (Ronquist and Huelsenbeck, 2003; Ronquist, 2004) was used for Bayesian analysis. The NEXUS file format necessary for the alignment (input) file was generated with the file converting option available within DAMBE (Xia, 2001). The amino acid substitution model setting for Bayesian analysis was as follows: Blosum62 + I + G + F, gamma distribution with four gamma rate parameters. The model was chosen based on evaluating the amino acid alignment with the ProtTest program (Abascal et al., 2005). The Bayesian inference of phylogenies was initiated from a random starting tree and four chains were run simultaneously for 2 106 generations; trees were sampled every 100 generations. The first 25% of trees generated were discarded (‘‘burn-in”) and the remaining trees were used to construct a 50% majority rule consensus tree and to compute the posterior probability values. For nucleotide sequences, the GTR model with gamma distribution was applied to the data set and four chains were run simultaneously for 106 generations with a sampling frequency of 100 and a ‘‘burn-in” corresponding to the first 25% of sampled trees. The final phylogenetic trees were drawn with the TreeView program (Page, 1996) using PHYLIP tree outfiles or MrBayes tree files; these were annotated with Corel DrawTM (Corel Corporation, Ottawa, Ontario, Canada).
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The secondary structure models of the putative rns group I introns (rns introns 2 and 4) were generated with the online program mfold (http://www.bioinfo.rpi.edu/~zukerm/rna/; Zuker, 2003). However, constraints were applied to the folds based on RNA secondary models previously published by Michel et al. (1982), Waring and Davies (1984), Waring et al. (1984), and Michel and Westhof (1990). The intron stem–loop structural elements were individually identified following the convention of Burke et al. (1987), Burke (1988), Cech (1988, 1990), Cech et al. (1994) and Michel and Westhof (1990). The flanking 50 exon sequences were included in the folds to identify the P1 helix, and thereby the putative internal guide sequence. The final structures were drawn with Corel DrawTM.
3. Results 3.1. A plasmid-like element in the mit1 and mit2 mutants of C. parasitica As reported in a previous study (Monteiro-Vitorello et al., 1995), HindIII digests of mtDNAs from the mit1 and mit2 hypovirulent mitochondrial mutants of C. parasitica contained a novel 4.2-kb fragment which was absent in digests of the mtDNA from the virulent EP155 wild-type progenitor strain. When the 4.2 kb DNA fragment was labeled and used as probe on Southern blots of gels containing partially-digested mtDNA from the mit2 mutant, it not only hybridized with the characteristic 4.2-kb fragment, but also produced signals corresponding to 8.4 kb and, to a lesser degree, 12.6 kb linear molecules (Fig. 1A), most likely representing linear dimers and trimers of the same repeat. Moreover, when the same probe was hybridized to Southern blots of uncut mutant mtDNA, it produced signals at positions corresponding to molecules approximately 5, 9, 12 and >14 kb in size (Fig. 1A). These bands probably represent ‘‘relaxed” circular multimers of the 4.2 kb element, which, due to their topologies, are expected to migrate at reduced rates. In the fully-digested mit2 mtDNA sample shown in Fig. 1A, the putative multimers that appeared in the undigested and partially digested DNAs are reduced completely to 4.2 kb linear fragments. The results are consistent with the interpretation that the plMEs of the mit2 mutant are circular DNA molecules consisting of a variable number of repeats of the 4.2 kb sequence. The completely digested mtDNAs from the mutants as well as the Ep155 wild type included a 9.8 kb fragment that hybridized to the plME probe, suggesting that part or all of the 4.2-kb repeat sequence is derived from the mitochondrial chromosome. Hybridization of the cloned plME HindIII monomer probe to a Southern blot of XbaI-cleaved Ep155 wild-type mtDNA showed that it shares nucleotide sequences with three fragments cloned previously in E. coli as plasmids pX17 (=2.3 kb), pX24 (=1.9 kb), and pX56 (=3.7 kb), respectively, for the construction of a physical map of the C. parasitica mtDNA (Bell et al., 1996). To confirm that the plME is an autonomously replicating circular molecule, purified mtDNA from the mit2 mutant was analyzed by two-dimensional gel electrophoresis. In this system, electrophoresis in the first dimension separates DNA in proportion to mass, whereas second dimension electrophoresis in the presence of ethidium bromide separates molecules of different topologies (Bell and Byers, 1983; Friedman and Brewer, 1995), such as linear or circular molecules. Southern analysis of the first dimension gel (Fig. 1B) shows the putative circular monomer of the plasmid-like element and a discrete band of molecules that appear to be the circular version of dimers. The presence of versions of the plME smaller than the dominant monomer band are also indicated, but based on the intensity of the hybridization signals these appear to be in low amounts. These bands could represent supercoiled versions
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of the circular monomers, which are expected to migrate at a faster rate than the relaxed, nicked circular and linear equivalents. However, we cannot exclude the possibility that deletion variants of the plMEs do exist and account for the smaller bands. Southern analysis of the second dimension gel (Fig. 1B) shows a continuous arc of randomly-ended linear molecules that probably consists of sheared mtDNA as well as plasmid-like molecules. A discrete spot of high intensity hybridization on the two dimensional gel can be correlated with the position of the plME DNA on the first dimension gel. This spot is located above the arc of linear molecules, therefore represents the more slowly migrating, relaxed circles of the plME. Supercoiled molecules of the plME were not apparent in the second dimension gels. Such molecules are expected to be faster migrating than linear molecules, thus should have been located below the linear-DNA arc (Hausner et al., 2006a). Because of their size, multimers of the plME larger than dimers were not detected, most likely because large circular molecules are not efficiently resolved in either dimension by 2D agarose gel electrophoresis. These observations show that the plME is an autonomous molecule rather than a reiterated sequence within the mtDNA that can be cleaved into repeat units by HindIII.
3.2. Sequence analysis and inheritance pattern of the plME To ascertain the origin and molecular characteristics of the plME of the mit1and mit2 mutants, the complete nucleotide sequence of the 4.2 kb HindIII monomer was determined for plME of mit2 (Figs. 1 and 2C). The mit1 and mit2 plMEs based on their sizes, restriction maps, and the nucleotide sequences of PCR products across their 30 –50 junctions were assumed to be identical. Using the mit2 sequence as a BLASTn query within NCBI data bases revealed that only a stretch of 923 bp within the plME showed 81.9% sequence similarity to the 50 terminal region of the N. crassa mt-rns gene (Chiang et al., 1994). The apparent lack of relatedness of most of the DNA of the plME to any known genetic elements prompted the sequencing of not only the cloned XbaI and HindIII fragments of the mtDNA of the C. parasitica Ep155 mtDNA that hybridized with the cloned HindIII 4.2-kb monomer, but also adjacent cloned restriction fragments. An analysis of the resultant 11565-kb sequence (GenBank accession no. AF029891) (Fig. 2) showed that it contains the entire mt-rns gene, which includes four large introns, together with 1513 and 180 of upstream and downstream flanking base pairs, respectively. Alignment of the complete
Fig. 1. (A) Ethidium-bromide gel electrophoresis and southern blot analysis of the plME used as probe on Southern blots of gels containing uncut mtDNA (U); mtDNA partially cut with HindIII (P); and mtDNA completely cut with HindIII (C) from the mit2 mutant and the Ep155 strain of C. parasitica. The sizes (kb) of DNA standards are shown on the left side of the gel. (B) Autoradiograph showing the results of the first (1D) and second dimension (2D) gel electrophoresis of uncut mtDNA obtained from the mit2 mutant. The position of the slow-migrating circular monomers of the plasmid-like element is indicated by the letters CP. The arc of linear fragments of mtDNA and the plasmid-like element is labeled LA. The position of the monomer-sized linear molecules of the plME on the linear-DNA arc is indicated by the letters LP. (C) Schematic representation of a plME circular version and showing the 50 and 30 end sequences involved in the formation of this circular element. The location of a putative promoter region based on N. crassa (Kennell and Lambowitz, 1989; Tracy and Stern, 1995) is also shown, the latter is composed of a potential 11-nucleotide consensus sequence preceded by an AT-rich segment (Kubelik et al., 1990).
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Fig. 2. Schematic representation of a segment of the C. parasitica Ep155 mitochondrial DNA deposited in GenBank (accession no. AF029891). Numbers indicate the size of fragments in base pairs. (A) Sequence analysis showing the location of the gene encoding the mitochondrial small subunit ribosomal RNA (mt-rns), plME and its end terminal sequences, and the location of DNA-dependent RNA polymerase (rpo) remnant. The locations of XbaI, HindIII and SacI restriction sites are indicated. Positions of the group-I and -II introns and the respective intronic ORFs are indicated. Based on BLASTn analysis, the sequence upstream of the mt-rns gene in the mtDNA of C. parasitica shows similarity to remnants of a putative DNA-directed RNA polymerases (rpo) of the pfam00940 DNA-dependent single chain RNA polymerase family, usually found in bacteriophage and mitochondrial plasmids (Oeser and Tudzynski, 1989; Court and Bertrand, 1992; Hermanns and Osiewacz, 1994; Cermakian et al., 1997; Cahan and Kennell, 2005). (B) Locations of the cloned fragments used for sequencing are shown as arrows. The physical map of the Ep155 mt DNA has been published previously by Bell et al. (1996).
sequence of the 4.2-kb HindIII monomer with this nucleotide sequence showed that the plME is a circular form of a continuous segment of the Ep155 wild-type mtDNA that includes an 1130bp, AT-rich upstream region as well as the first exon and most of the first intron of the mt-rns locus (Fig. 2). The target site that results in cutting of the plME into 4.2 kb monomer fragments by HindIII is located near the 30 end of intron 1 in mt-rns (Fig. 1 and 2C). Based on the criteria provided by Toor and Zimmerly (2002), the first intron of the rns gene is a group II intron. The sequence of the 4210 base pairs of the plME is identical to the sequence of the nucleotides beginning at position 361 and ending at position 4570 of the Ep155 mtDNA sequence (GenBank accession no. AF029891). Although nucleotide stretches that could enhance base-pairing, therefore favor recombinational events, were found in the proximity of the plME end-points, we failed to detect sequences, particularly inverted repeats, which might have been involved directly in the excision of the plME sequence from the mtDNA by intramolecular homologous recombination. The 30 end of the plME sequence is GGGGGGAAA (Fig. 1C), a sequence which at the RNA level is part of domain VI (helix) of the group II intron, with the three As forming a bulge (see Toor and Zimmerly, 2002). This sequence is located within a region where group II intron encoded reverse transcriptases initiate cDNA synthesis (Morozova et al., 2002), suggesting the possibility that this plME was formed by a process that involved reverse transcription of the rns transcript. A putative promoter region for rns based on N. crassa and other filamentous fungi sequences is proposed (Fig. 1C) (Kennell and Lambowitz, 1989; Kubelik et al., 1990; Tracy and Stern, 1995; Pantou et al., 2006). The region is composed of a potential 11-nucleotide consensus sequence preceded by an AT-rich segment located inside of the plasmid, which could allow transcription of the plME. Although this is an attractive hypothesis, the
biogenesis and replication of plME needs to be explored experimentally. Reciprocal sexual crosses with virulent strains have demonstrated that the genetic determinants of the mitochondrial hypovirulence phenotypes of mit1 and mit2 are transmitted exclusively from the maternal parent to the progeny (Monteiro-Vitorello et al., 1995). In crosses where mitochondrial mutants were used as the female parent, all the progeny showed the characteristics associated with mitochondrial hypovirulence, such as slow growth, reduced conidiation and elevated levels of cyanide-insensitive respiration (Monteiro-Vitorello et al., 1995). However, the sexual transmission pattern of the plME was not examined during this initial characterization of the mitochondrial hypovirulence trait. Subsequent HindIII restriction analysis of the mtDNAs failed to detect the presence of the plME in any of the hypovirulent progeny from the same crosses, even though maternal transmission of mitochondrial DNA was detected by the transmission of RFLP markers. Hybridization of Southern blots of mtDNAs extracted from the hypovirulent progeny with the plME-specific probe also failed to detect the presence of the characteristic 4.2-kb restriction fragments. We have analyzed from eight to ten individuals of each reciprocal cross (data not shown). The failure of the transmission of plMEs through sexual crosses also has been observed in other systems, including N. crassa (Hausner et al., 2006b) and O. novo-ulmi (Charter et al., 1993). The initial report on the transmission of the hypovirulence traits from the mit1 and mit2 mutants through hyphal anastomoses to virulent C. parasitica recipients (MonteiroVitorello et al., 1995) did not include tests for the asexual transfer of plMEs or their possible effects on the virulence of C. parasitica. However, subsequent analyses of the mtDNAs of the converted recipients showed clearly that the plMEs are transmitted efficiently to the virulent recipients during their conversion to
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hypovirulence by hyphal contact with mit1 and mit2 hypovirulent donors. Thus, the plMEs of C. parasitica display the same sexual and asexual transmission characteristics as some of the small rnl derived plMEs of N. crassa (Hausner et al., 2006b) although the respective plMEs differ in size and with regards to the mtDNA regions from which they originated (see below). 3.3. Features of the C. parasitica mitochondrial rns gene The C. parasitica mt-rns gene is 9872 bp long, of which an estimated 1875 nucleotides comprise the combined exon sequences, at least as deduced from similarities with other mitochondrial rns sequences. The entire mt-rns sequence, including upstream and downstream flanking regions is schematically represented in Fig. 2. While there is significant similarity between the mt-rns genes of C. parasitica, Podospora anserina, and N. crassa, this similarity is not distributed evenly throughout the gene, but rather is concentrated in regions that contain the universally conserved domains (U1, U2, etc.). The C. parasitica mt-rns sequence shows 63.7% and 66.4% similarity to the corresponding sequences of P. anserina and N. crassa, respectively. The 50 and 30 flanking regions showed little similarity to sequences in the GenBank database and their informational content could not be accurately identified through simple sequence comparisons. The mt-rns gene contains four introns, which are located in three of the eight universally conserved domains comprising the rns core secondary structure, i.e. U4, U5 (two introns) and U6. The higher order structures of 16S-like rRNAs have been reviewed by Woese et al. (1983) and we follow the nomenclature for the variable and conserved stems and/or loops of the rns product as defined by Gutell et al. (1986) and Schnare et al. (1986). Introns were named as follows according to the convention proposed by Johansen and Haugen (2001) where the number following the abbreviated scientific name refers to the location of the intron with respect to the E. coli 16S RNA gene (GeneBank accession no. J01695): Cpa.mS785, Cpa.mS915, Cpa.mS953 and Cpa.mS1209 (Fig. 2). The 50 and 30 termini of the four introns were deduced from sequence identities to P. anserina and N. crassa mt-rns genes. The sizes of the four introns are as follows: Cpa.mS785 is 2081 bp, Cpa.mS915 is 2412 bp, Cpa.mS953 is 2067 bp and Cpa.mS1209 is 1334 bp. 3.4. The introns of the mt-rns gene of C. parasitica Two introns, Cpa.mS915 and Cpa.mS1209, were recognized as group I introns on the basis of structural features (Michel and Westhof, 1990), and the predicted structures (RNA folds) suggest that they belong to the 1C group (Supplementary Fig. 1). The remaining two introns, Cpa.mS785 and Cpa.mS953, have recently been folded and described by Toor and Zimmerly (2002) as group II introns. Sequence analyses revealed that all four introns contain open reading frames (Fig. 2), each potentially encoding a protein with two LAGLIDADG dodecapeptide motifs separated from each other by approximately 140 amino acids. These ORFs are characteristic of maturases and homing endonucleases frequently encoded by group I introns (Belfort et al., 2002). The ORFs encoded by the Cpa.mS953 and Cpa.mS1209 introns contain additional repetitive elements referred to as rA and rB (Cummings et al., 1989). The Cpa.mS915 and Cpa.mS953 introns are located within the rns U5 domain, separated by an exon of only 37 nucleotides. Sequence analyses also suggest that the ORFs of Cpa.mS915 and Cpa.mS1209 overlap the conserved core elements of their respective host introns. Similar arrangements have been described relatively recently (Carbone et al., 1995) even though the earliest
papers dealing with this topic ascertained that the majority of ORFs lay outside or between the core elements (Davies et al., 1982). 3.5. Phylogenetic analysis of the mtDNA rns gene and the intron ORFs The rns sequence of C. parasitica was aligned with 39 additional fungal mtDNA rns sequences (see Table 1). Although many of the rns sequences deposited in GenBank are incomplete, sequences of sufficient length from Ascomycetes with affinities to the Hypocreales, Dothediales, Sordariales, Euroticales, and Saccharomycetales could be recovered. The latter sequences served as the outgroup in the analysis. The data set comprised a total of 1389 unambiguously alignable nucleotide positions and was analyzed by programs contained within PHYLIP and with MrBayers (Bayesian analysis). Neighbour-joining, maximum parsimony and Bayesian analysis yielded similar phylogenetic estimates (Fig. 3). On the basis of morphological criteria, the genus Cryphonectria has been assigned to the order Diaporthales which is included in the class the Pyrenomycetes. The rns phylogenetic tree is consistent with the view that members of the Diaporthales share a common ancestor with the Sordariales (represented by N. crassa and P. anserina); the node supporting monophyly of these groups received a level of confidence of 85% in the NJ analysis and 84% in the parsimony analysis, whereas Bayesian analysis yielded a 100% posterior probability value for this node. The rns phylogenetic tree also confirms the monophyly of the Hypocreales and the monophyly of the Eurotiales with high levels of support. In general the node that supports the monophyly of the members of the Ascomycota, excluding members of the Saccharomycetales, received a level of bootstrap support of 100%. Overall the rns phylogeny is consistent with the current concept of the evolution of perithecial Ascomycetes (reviewed in Blackwell et al., 2006). A BLASTp database search, using the predicted amino acid sequences encoded by the ORFs within the C. parasitica mt-rns introns as the queries, identified other LAGLIDADG type ORFs. A phylogenetic analysis (Fig. 4) of the conserved core sequences of 35 intron-encoded proteins resulted in phylogenetic trees showing two major clades (nodes 1 and 2) that are strongly support by Bootstrap analysis and posterior probability values from Bayesian analysis. In one clade, the C.p.rnsi2 ORF groups with the ORF of P.a.ND5i1, and the analysis suggests that the ORFs P.a.cobi1, P.a.rnli1, O.i.rnli1, O.m.rnli1 and C.p.rnsi2 might be derived from a common ancestral gene. In the largest clade (node 2), the C.p.rnsi3 and C.p.rnsi4 ORFs group among LAGLIDADG ORFs that contain the rA and rB elements. Here the C.p.rnsi3 ORF appears to be related to rns group II intron-encoded ORFs of species in the genus Cordyceps (Fig. 4, node 3). The C.p.rnsi4 ORF appears to be closely related to an rns-encoded intron ORF found in strains of Agrocybe aegerita (Basidiomycota) (Fig. 4, node 4). Overall, ORFs within this clade can be located in group I or group II (i.e. C.p.rnsi3) introns and some of the members of this clade have been reported to be free standing (HyjefMp02 and HyjefMp12, NC_003388). The phylogenetic position of the C.p.rnsi1 ORF was not resolved in this analysis, however we were able to identify at least one potentially closely related sequence G.l.rnsi1 (Ganoderma lucidum, Basidiomycota, GenBank accession no. AF214475, see Fig. 4, node 5). The G. lucidum rns intron sequence was examined by the online RNAWeasel program (Lang et al., 2007; http://megasun.bch.umontreal.ca/RNAweasel/) and the program identified the conserved group II domain V sequence and fold. Thus, it appears that the C.p.rnsi1 and G.l.rnsi1 ORFs are located in group II introns, indicating that LAGLIDADG ORFs have invaded mtDNA rns group II introns on at least two, perhaps even more, occasions (Toor and Zimmerly, 2002). Overall, the analysis suggests not only that the ORFs in the C.p. rns introns were derived from different evolutionary lines, but also that ORFs found in rns or rnl genes are not necessarily phylo-
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Location
Intron name (ORF designation)
GenBank accession
Amoebidium parasiticum Agrocybe aegerita Cordyceps konnoana Cordyceps ramosopulvinata Cordyceps sp. 97009 Cyrphonectria parasitica Cyrphonectria parasitica Cyrphonectria parasitica Cyrphonectria parasitica Cyrphonectria parasitica Cyrphonectria parasitica Crinipellis perniciosa Ganoderma lucidum Hypocrea jecorina Hypocrea jecorina Neurospora crassa Neurospora crassa Neurospora crassa Penicillium marneffei Podospora anserina Podospora anserina Podospora anserina Podospora anserina Podospora anserina Podospora anserina Podospora anserina Podospora anserina Podospora anserina Podospora anserina Ophiostoma ips Ophiostoma minus Ophiostoma ulmi Ophiostoma novo-ulmi subsp. americana Ophiostoma novo-ulmi subsp. americana Sclerotinia sclerotiorum Schizosaccharomyces pombe
rnl rns rns rns rns rnl rns rns rns rns NDa5 ND4 rns free standingb free standingb ND5 ATPase subunit 6 ND4L COXc 1 COBd ND1 ND3 ND4 ND4L ND4L ND5 ND5 COX1 rnl rnl rnl COX1 rnl rnl rns COX1
A.p.rnli2 A.a.rnsi1 C.k.rnsi1 C.r.rnsi1 C.97009.rnsi1 C.p.rpmi C.p.rnsi1 C.p.rnsi2 C.p.rnsi3 C.p.rnsi4 C.p.ND5i2 Cri.p.ND4i1 G.l.rnsi1 HyjefMp02 HyjefMp12 N.c.ND5i1 N.c.ATP6i2 N.c.ND4Li1 P.m.cox1i4 P.a.cobi1 P.a.ND1i4 P.a.ND3i1 P.a.ND4i1 P.a.ND4Li1 P.a.ND4Li2 P.a.ND5i1 P.a.ND5i2 P.a.cox1i5 P.a.rnli1 O.i.rnli1 O.m.rnli1 O.u.cox1i3 O.n.u.rnli1 I-Onu1-P S.s.rnsi1 S.p.cox1i1
AF538042 AAB50391 AB031194 AB027348 AB027356 AAC24230 AF029891 AF029891 AF029891 AF029891 AAO14101 YP_025865 AAO13729 NP_570143 NP_570153 CAA28764 T50468 CAA28761 NP_943727 NP_074921 NP_074960 NP_074914 P15564 NP_074942 CAA38797 NP_074944 NP_074945 NP_074928 NP_074910 ABI15908 ABI15909 AAU07878 ABI15906 AAY59060 AAC48982 S78196
a b c d
ND genes: coding for subunits of the mitochondrial NADH dehydrogenase protein complex. Free standing based on GenBank accession no. AAL74167. Cytochrome oxidase I. COB = apocytochrome b.
genetically related with each other or confined to a specific set of genes. For example, the C.p.rnsi4 ORF groups within a clade that includes intron ORFs located in a variety of different host genes such as rns, rnl, nad3, nad4, atp6, and cox1. 4. Discussion 4.1. Plasmid-like elements Molecular events that are characteristically associated with fungal hypovirulence are of interest for the construction of comprehensive account of the contribution and interactions of the diverse genetic and physiological factors that are involved not only in the appearance of this phenotype, but also its persistence in natural populations of C. parasitica and other pathogenic fungi (Monteiro-Vitorello et al., 2000; Dawe and Nuss, 2001; Bertrand and Baidyaroy, 2002; Hoegger et al., 2003; Allen and Nuss, 2004; Nuss, 2005). Previously, Monteiro-Vitorello et al. (1995) were able to demonstrate by systematically targeting the mtDNA of the virus-free Ep155 laboratory strain of C. parasitica with a mutagen can produce mutations (mit1 and mit2) that conveyed a cytoplasmically-transmissible hypovirulence phenotype. One prominent phenomenon associated with the mit1 and mit2 mutations was the spontaneous appearance and proliferation of plMEs in the hyphae of the fungus. However the lack of transmission of these elements in sexual crosses indicates that they are not responsible for the maternally-inherited hypovirulence phenotype. In the EtBr-
induced mit1 and mit2 hypovirulent mutants of C. parasitica, the plMEs appear to have been generated by some kind of recombination event resulting in the apparent excision of a 4251-bp segment of the mtDNA that includes 1130 bp upstream sequence, exon 1 and most of the first intron of the mitochondrial rns gene. The mechanisms involved in the formation of plMEs in the filamentous fungi are either unknown, or have been speculated to involve an RNA intermediate, excision of mtDNA by processes involved in mtDNA repair, or illegitimate recombination events (reviewed in Hausner et al., 2006a). In several instances, group I or group II introns appear to be components of the plMEs, and these elements may have arisen by the reverse transcription of the spliced-out intronic RNA (Osiewacz and Esser, 1984; Michel and Lang, 1985) or generated by intramolecular recombination (Abu-Amero et al., 1995). The latter could be promoted by the presence of related introns in close proximity to one another thus acting like direct repeats promoting recombination events (Sethuraman et al., 2008). As an alternative to the above speculations, it can be argued that the ethidium-bromide treatment that was used to induce mitochondrial mutations (Monteiro-Vitorello et al., 1995) was directly responsible for the appearance of plMEs in the mit1 and mit2 mutants of C. parasitica. Since ethidium bromide can intercalate into mtDNA, the treatment could have caused DNA-repair related non-homologous crossover events that produced small circular molecules that eventually were amplified as plMEs. This proposal gathers support not only from a report indicating that
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Fig. 3. Phylogenetic tree based on Bayesian analysis of rns sequence data for species of the Ascomycota. The numbers at the nodes indicate the level of support based on Bayesian and bootstrap analyses. The number above the line represents the posterior probability values obtained from the 50% majority consensus tree generated using Bayesian analysis (Huelsenbeck and Ronquist, 2001; Huelsenbeck et al., 2001). The second and third numbers are bootstrap numbers based on NJ and parsimony analysis, respectively. Numbers are only provided for those nodes that received >50% support. In order for nodes to be considered statistically significant they should receive posterior probability values P99% and for bootstrap support the numbers should be P95% (Felsenstein, 1985). The topology and branch lengths are based on the Bayesian analysis, branch lengths are proportional to the number of substitutions per site (see scale bar). The sources of the sequences are provided after the species names (i.e. GenBank accession numbers). Ordinal relationships for the species are indicated on the right site, higher taxonomic rankings (Classes) are also denoted at the relevant nodes. Note: Ashbya gossypii = Eremothecium gossypii.
ethidium bromide induces mtDNA deletions in N. crassa (Niagro and Mishra, 1989), but also from the well-documented observation that the exposure of cells of the budding yeast Saccharomyces cereviseae to DNA intercalating agents, particularly ethidium bromide, results in massive mtDNA rearrangements and the accumulation of plasmid-like excision sequences, the so-called rho repetitive DNA elements (Faugeron-Fonty et al., 1983; De Zamaroczy et al., 1983). Mitochondrial plasmids and plasmid like elements are known to be associated with specific phenotypes in some filamentous fungi (reviewed by Kempken, 1995). We have concluded that the plME of C. parasitica does not cause a discrete phenotype because it is not transmitted in sexual crosses even though the hypovirulence phenotype is maternally inherited, as expected of a trait that is determined by a mutant form of mtDNA. The purging of certain types of plMEs during sexual reproduction also occurs in N. crassa (Hausner et al., 2006b), possibly because they lack sequence elements required for the assortment of mtDNA during some stage of meiotic sporogenesis. At this time, we do not know whether the hypovirulent progeny of sexual crosses eventually will acquire the same kind of plME as was observed in the parental strains after a
prolonged period of asexual propagation. Rearrangements in the rns region also have been observed recently in the mtDNAs of naturally-occurring dsRNA-free hypovirulent isolates of C. parasitica (Baidyaroy, Fulbright and Bertrand, unpublished data), suggesting that this segment of the mitochondrial chromosome might be important in the generation of the asexually-transmissible hypovirulence phenotypes that appear spontaneously in virus-free strains. 4.2. Phylogenetic relationships of the C. parasitica rns intron encoded ORFs Although the rns gene of C. parasitica appears to have been vertically transmitted during the course of the evolution of this fungus (Fig. 3), the phylogenetic relationships of the group I intron ORFs contained within the rns gene are more complex. Phylogenetic analyses revealed that the ORFs that are located within the introns of the C. parasitica rns gene are not necessarily closely related to each other (Fig. 4). Moreover, LAGLIDADG ORFs that appear to belong to the same clade, namely C.p.rnsi3 and C.p.rnsi4, can be
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Fig. 4. Phylogenetic tree based on Bayesian analysis of double motif LAGLIDADG HEG-like elements related to the C. parasitica rns intron-encoded ORFs. The two nodes that identify monophyletic groups are marked 1 and 2. The clade denoted by node 1 includes the C.p.rnsi2 ORF along with LAGLIDADG type ORFs related to mL1699 intronencoded ORFs (see Sethuraman et al., 2008) and node 2 identifies a clade that includes the group II intron encoded C.p.rnsi3 ORF, the group I intron encoded C.p.rnsi4 ORF together with other intron-encoded ORFs previously designated as Clade 1 in Dalgaard et al. (1997); also see Haugen and Bhattacharya (2004). For the designation of the numbers at the nodes see the legend for Fig. 3.
inserted into different types of introns. Specifically, the C.p.rnsi3 ORF is located in the Cpa.mS953 group II intron whereas the C.p.rnsi4 ORF is a component of the Cpa.mS1209 group I intron. Usually Group II introns either lack an ORF or encode reverse transcriptase-like ORFs; the latter appear to have tightly coevolved with their host group II intron unlike the more independent evolution of group I introns and their associated ORFs (Toor et al., 2001; Toor and Zimmerly, 2002). The nature of the LAGLIDADG type ORFs and their relationships with group II introns are not yet understood (Toor and Zimmerly, 2002). The Cpa.mS953 ORF is part of a family of LAGLIDADG ORFs that are associated with group II introns (Fig. 4, node 3) but appear to be related and probably derived from ORFs normally present within group I introns. This ‘‘intron host jump” is another example of the pervasive nature of HEGs and the pressure on HEGs to continuously invade new
niches or acquire new functions in order to avoid extinction (Goddard and Burt, 1999; Gogarten and Hilario, 2006). The C.p.rnsi3 ORF within the Cpa.mS785 intron is another example of a LAGLIDADG-type element that has inserted into a group II intron and yet it appears to be only distantly related to the Cpa.mS953 ORF family, indicating that different lineages of HEG-like elements have invaded rDNA group II introns on numerous occasions. The C.p.rns3 and C.p.rnsi4 intronic ORFs belong to LAGLIDADG protein clade 1 as designated by Dalgaard et al. (1997). A detailed analysis of the rDNA-inserted members of this clade by Haugen and Bhattacharya (2004) showed that double LAGLIDADG HEG motifs originated from a duplication event within a single motif HEG or fusion of two single motif HEGs. Haugen and Bhattacharya (2004) also showed that the double-motif HEGs appear to have been very successful in spreading with their host introns into
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new (divergent) rDNA sites. The phylogeny of the clade 1 LAGLIDADG ORFs presented in this paper reflects the observations of Haugen and Bhattacharya and further documents the successful spread of HEGs within a broad pool of rDNAs and protein-coding genes (see Fig. 4 and Table 1). The clade that contains the C.p.rnsi2 ORF of the Cpa.mS915 group I intron is a good example of the complex distribution of HEG-like elements. This clade contains a set of HEG-like elements that are restricted to the rnl U7 region (mL1699 intron, an A1 group I intron), suggesting vertical transmission, whereas early-branching members of this clade are located within the cob gene. Also, the C.p.rnsi2 ORF appears allied to an ORF inserted within a ND5 intron in P. anserina. This family of HEG-like elements recently has been phylogenetically analyzed in more detail by Sethuraman et al. (2008). Briefly, the latter and our study demonstrate the distribution of phylogenetically related HE/maturase-like sequences in unrelated genes (rns and rnl, nd1, nd3, nd4, nd5, cob, cox1, etc.) in different fungal species; i.e. the absence of a correlation between the branching pattern in the HE/maturase phylogeny with that of the host genes and organisms. These observations hint at the possibility of the horizontal transfer of same HEGs across species lines (reviewed in Dalgaard et al., 1997; Gimble, 2000; Stoddard, 2005). Acknowledgements G.H. research on HEGs is supported by a Discovery Grants from the Natural Sciences and Engineering Research Council of Canada. C.B.M.-V. research is supported by a Grant from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/Brazil). The research on hypovirulence was supported by National Research Initiative Competitive Grant 95-37303-1785 from the USDA. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.fgb.2009.07.005. References Abascal, F., Zardoya, R., Posada, D., 2005. ProtTest: selection of best-fit models of protein evolution. Bioinformatics 21, 2104–2105. Abu-Amero, S.N., Charter, N.W., Buck, K.W., Brasier, C.M., 1995. Nucleotidesequence analysis indicates that a DNA plasmid in a diseased isolate of Ophiostoma novo-ulmi is derived by recombination between two long repeat sequences in the mitochondrial large subunit ribosomal RNA gene. Curr. Genet. 28, 54–59. Allen, T.D., Nuss, D.L., 2004. Linkage between mitochondrial hypovirulence and viral hypovirulence in the chestnut blight fungus revealed by cDNA microarray analysis. Euk. Cell 3, 1227–1232. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic logical alignment search tool. J. Mol. Biol. 215, 403–410. Anagnostakis, S.L., 1979. Sexual reproduction of Endothia parasitica in the laboratory. Mycologia 71, 213–215. Bassi, G.S., de Oliveira, D.M., White, M.F., Weeks, K.M., 2002. Recruitment of intronencoded and co-opted proteins in splicing of the bI3 group I intron RNA. Proc. Natl. Acad. Sci. USA 99, 128–133. Belcour, L., Rossignol, M., Koll, F., Sellem, C.H., Oldani, C., 1997. Plasticity of the mitochondrial genome in Podospora. Polymorphism for 15 optional sequences: group-I, group-II introns, intronic ORFs and an intergenic region. Curr. Genet. 31, 308–317. Belfort, M., 2003. Two for the price of one: a bifunctional intron-encoded DNA endonuclease-RNA maturase. Genes Develop. 17, 2860–2863. Belfort, M., Roberts, R.J., 1997. Homing endonucleases: keeping the house in order. Nucleic Acids Res. 25, 3379–3388. Belfort, M., Derbyshire, V., Parker, M.M., Cousineau, B., Lambowitz, A.M., 2002. Mobile introns: pathways and proteins. In: Craig, N.L., Craigie, R., Gellert, M., Lambowitz, A.M. (Eds.), Mobile DNA II. ASM Press, Washington, DC, pp. 761– 783. Bell, L., Byers, B., 1983. Separation of branched from linear DNA by two-dimensional gel electrophoresis. Anal. Biochem. 130, 527–535. Bell, J.A., Monteiro-Vitorello, C.B., Hausner, G., Fulbright, D.W., Bertrand, H., 1996. Physical and genetic map of the mitochondrial genome of Cryphonectria parasitica Ep155. Curr. Genet. 30, 34–43.
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