Recurrent invasion of mitochondrial group II introns in specimens of <Emphasis Type="Italic">Pylaiella</Emphasis><Emphasis Type="Italic">littoralis</Emphasis> (brown alga), collected worldwide

The mitochondrial genome of a filamentous brown alga Pylaiella littoralis (strain CCMP 1907) has been reported to contain four group IIB introns in the LSU rRNA gene and three group IIA introns in the cox1 gene. We found extreme variability in the number of group II introns for these two genes by analyzing eight P. littoralis specimens collected at worldwide habitats. The first intron of the LSU rRNA gene from a specimen collected in France and the fourth intron from a specimen harvested in Japan exhibited an exceptionally long evolutionary distance when compared with the cognate introns found in P. littoralis specimens. Moreover, these introns harbored an intact or nearly intact tripartite ORF, suggesting they are the result of a recent invasion of cognate introns. Based on the fact that many of the target sites were intronless, we propose that opportunity of intron infection is the bottleneck step of the group II intron cycle which consists of invasion, degeneration, and complete loss from the target site. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Distribution of mitochondrial introns in the species Schizosaccharomyces pombe and the origin of the group II intron in the gene encoding apocytochrome b

Summary The mitochondrial genome size of 26 different Schizosaccharomyces pombe strains varies between 17.6 and 24.6 kilobase pairs due to the presence or absence of introns. One of these is the group II intron in the gene encoding apocytochrome b (cob: intron cobI1 ). Partial DNA sequences of continuous cob genes from six strains (including strain EF1: Trinkl et al. 1985) revealed identical nucleotide sequence in the region where the group II intron is inserted in the mosaic form of the gene. In contrast, analysis of the mosaic cob, gene in strain UCD-Fstl revealed several base pair changes in the exon regions flanking the splice point, compared with the continuous genes and with the mosaic cob gene in strain 50 (Lang et al. 1985). The base pair differences between the exons of the two mosaic cob genes and the identity of exons in all continuous cob genes argue in favour of the two cob introns in strains 50 and UCD-FstI as independent later acquisitions of the genes, rather than loss of the intron from a common mosaic ancestor of all strains.Other introns present in some but not all strain include two group I introns without open reading frame in the gene encoding subunit 1 of cytochrome c oxidase (cox1: introns cox1I2a and cox1I3), and two group I introns with open reading frames in the same gene (introns cox1I1 and cox1I2b).     

Structure of nuclear-localized <Emphasis Type="Italic">cox3</Emphasis> genes in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> and in its colorless close relative <Emphasis Type="Italic">Polytomella</Emphasis> sp.

Several chlorophyte algae do not have the cox3 gene, encoding subunit III of cytochrome c oxidase, in their mitochondrial genomes. The cox3 gene is nuclear-encoded in the photosynthetic alga Chlamydomonas reinhardtii and in the colorless alga Polytomella sp. In this work, the genomic sequences of the cox3 genes of these two closely related algae are reported. The cox3 genes of both C. reinhardtii and Polytomella sp. contain four introns in the region encoding the putative mitochondrial-targeting sequences. These four introns show low sequence identities, but their locations are conserved between these species. The cox3 gene of C. reinhardtii has five additional introns in the region encoding the mature subunit III of cytochrome c oxidase. Sequence analysis of intron 6 of the cox3 gene of C. reinhardtii revealed similarity with two sequence elements present in introns of several other nuclear genes from this green alga. In the majority of the genes, these conserved sequences are located either near the 3' end or near the 5' end of the introns. Based on these data, we propose that the colorless genus Polytomella separated from C. reinhardtii after the cox3 gene was transferred to the nucleus. The data also support the evolutionary hypothesis of a recent acquisition of introns in C. reinhardtii.     

Functional and structural characterisation of <Emphasis Type="Italic">Ag</Emphasis>MNPV <Emphasis Type="Italic">ie1</Emphasis>

We have located and cloned the Anticarsia gemmatalis multicapsid nucleopolyhedrovirus isolate 2D (AgMNPV-2D) genomic DNA fragment containing the immediate early 1 ORF and its flanking regions. Computer assisted analysis of the complete ie1 locus nucleotide sequence information was used to locate regulatory signals in the upstream region and conserved nucleotide and amino acid sequences. Comparative studies led to the identification of several characteristic protein motifs and to the conclusion that AgMNPV-2D is more closely related to Choristoneura fumiferana defective NPV than to other Group I nucleopolyhedrovirus. We have also shown that the AgMNPV IE1 protein was able to transactivate an early Autographa californica MNPV promoter and its own promoter in transient expression assays. In order to investigate the biological functionality of the ie1 promoter, the ie1 upstream activating region (UAR) was molecularly dissected and cloned upstream of the E. coli lacZ ORF. The results obtained, after transfection of UFL-AG-286 insect cells, leading us to find that the −492 and −357 versions contains sequence motifs important for the level of the lacZ reporter gene expression. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users. The GenBank accession number of the sequence reported in this paper is AF368905.     

Impact of a mutation in the mitochondrial <Emphasis Type="Italic">LSU rRNA</Emphasis> gene from <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> on the activity and the assembly of respiratory-chain complexes

Two substitutions A1090G and A1098C (together called the m mutation) located in the conserved GTPase domain of the mitochondrial LSU rRNA gene were recently shown to weakly compensate for the phenotypical effect of a –1T frameshift mutation in the mitochondrial cox1 gene of C. reinhardtii. In order to analyze the impact of the m mutation on the mitochondrial translational machinery, a strain carrying the m mutation but wild-type for the cox1 gene was isolated. We found that the growth and the respiratory rate of the m mutant were affected and that the activities of complexes I, III, and IV, all containing mitochondria-encoded subunits, were lowered. In contrast the activities of complex II and of the alternative oxidase, both encoded exclusively by the nuclear genome, were not modified. The steady-state levels of complex I enzyme and of several components of the respiratory complexes I, III, and IV were also reduced in the mutant. We moreover showed that m did not suppress other frameshift or UGA stop mutations which affect mitochondrial genes.     

Multiple group I introns detected in the nuclear small subunit rDNA of the autosporic green alga <Emphasis Type="Italic">Selenastrum capricornutum</Emphasis>

A phylogenetic investigation of the autosporic chlorophycean alga species Selenastrum capricornutum using the small subunit (SSU) rRNA gene revealed the unusual presence of six group IC1 introns. Previous studies showed that numerous green algal taxa contain group IC1 introns. However, whereas some algal species harbor multiple introns in a single ribosomal gene, none have contained as many as S. capricornutum. Three of the S. capricornutum introns are located at conserved algal intron sites and the remaining three are located at novel eukaryotic positions. The SSU rRNA genes and their introns have been sequenced and putative secondary structures are proposed for the introns. Also, their similarity to other group IC1 introns from algal, fungal, and viral sources is investigated. Results suggest the initial presence of introns at conserved locations, followed by duplication and insertion to novel positions within the SSU rRNA gene.     

Association of positional and functional candidate genes<Emphasis Type="Italic"> FGF1</Emphasis>,<Emphasis Type="Italic"> FBN2</Emphasis>, and<Emphasis Type="Italic"> LOX</Emphasis> on 5q31 with intracranial aneurysm

We previously performed a genome-wide linkage study of intracranial aneurysm (IA) and found positive evidence of linkage at chromosomes 5q22–31, 7q11, and 14q22. In the present study, we focus on 5q31, where three candidate genes, fibroblast growth factor 1 (FGF1), fibrillin 2 (FBN2), and lysyl oxidase gene (LOX) lie, and evaluate associations with IA. Genomic DNAs were obtained from 172 IA patients and 192 controls. Association analysis was performed with ten, five, and four single-nucleotide polymorphisms (SNPs) identified in FGF1, FBN2, and LOX, respectively. A difference in allelic frequency was observed for only the SNP at intron 4 in FGF1 (χ²=4.44, df=1, P=0.035). Although a haplotype association was observed with the combination of ten SNPs in FGF1 (χ²=16.04, df=1, P=0.00006), significant haplotype associations were not observed when haplotypes were constructed with the three, two, and four SNPs in FGF1 according to the linkage disequilibrium structure. No associations of FBN2 and LOX with IA were detected in the present study.     

The complete DNA sequence of the mitochondrial genome of the dermatophyte fungus Epidermophyton floccosum

We report here the complete nucleotide sequence of the 30.9-kb mitochondrial genome of the dermatophyte fungus Epidermophyton floccosum. All genes are encoded on the same DNA strand and include seven subunits of the reduced nicotinamide adenine dinucleotide ubiquinone oxireductase (nad1, nad2, nad3, nad4, nad4L, nad5, and nad6), three subunits of cytochrome oxidase (cox1, cox2, and cox3), apocytochrome b (cob), three subunits of ATP synthase (atp6, atp8, and atp9), the small and large ribosomal RNAs (rns and rnl), and 25 tRNAs. A ribosomal protein gene (rps5) is present as an intronic ORF in the large ribosomal subunit. The genes coding for cob and cox1 carry one intron and nad5 carries two introns with ORFs. The mtDNA of E. floccosum has the same gene order as Trichophyton rubrum mtDNA, with the exception of some tRNA genes. Maximum likelihood phylogenetic analysis confirms T. rubrum as a close relative of E. floccosum. This is the first complete mitochondrial sequence of a species of the order Onygenales. This sequence is available under GenBank accession number AY916130.     

Identification of the <Emphasis Type="Italic">het-r</Emphasis> vegetative incompatibility gene of <Emphasis Type="Italic">Podospora anserina</Emphasis> as a member of the fast evolving <Emphasis Type="Italic">HNWD</Emphasis> gene family

In fungi, vegetative incompatibility is a conspecific non-self recognition mechanism that restricts formation of viable heterokaryons when incompatible alleles of specific het loci interact. In Podospora anserina, three non-allelic incompatibility systems have been genetically defined involving interactions between het-c and het-d, het-c and het-e, het-r and het-v. het-d and het-e are paralogues belonging to the HNWD gene family that encode proteins of the STAND class. HET-D and HET-E proteins comprise an N-terminal HET effector domain, a central GTP binding site and a C-terminal WD repeat domain constituted of tandem repeats of highly conserved WD40 repeat units that define the specificity of alleles during incompatibility. The WD40 repeat units of the members of this HNWD family are undergoing concerted evolution. By combining genetic analysis and gain of function experiments, we demonstrate that an additional member of this family, HNWD2, corresponds to the het-r non-allelic incompatibility gene. As for het-d and het-e, allele specificity at the het-r locus is determined by the WD repeat domain. Natural isolates show allelic variation for het-r. Sequence data reported here are available in the Genbank database under accession numbers FJ269240 and FJ269239 for het-r and het-R, respectively.     

Behaviour of <Emphasis Type="Italic">Sinapis alba</Emphasis> chromosomes in a <Emphasis Type="Italic">Brassica napus</Emphasis> background revealed by genomic <Emphasis Type="Italic">in-situ</Emphasis> hybridization

Genomic in-situ hybridization (GISH) was applied to study the behaviour of addition chromosomes in first and second backcross (BC) progenies of hybrids between Brassica napus ssp. napus L. (AACC, 2n = 38) and Sinapis alba L. (SS, 2n = 24) produced by electrofusion. With GISH using genomic DNA of S. alba was used as probe it was possible to clearly distinguish both of the parental genomes and effectively monitor the fate of S. alba chromosomes in the BC₁ and BC₂ progenies. GISH analysis confirmed the sesquidiploid genome composition (AACCS) of the BC₁ progenies, which contained 38 chromosomes from B. napus and 12 chromosomes from S. alba. Genome painting in the pollen mother cells (PMCs) of the BC₁ plants revealed intergenomic association between B. napus and S. alba chromosomes, whereby a maximum of 4 trivalents between AC and S chromosomes were identified at metaphase I. In the BC₂ progenies, aneuploids with different numbers of additional chromosomes from S. alba, ranging from 1 to 7, were confirmed. Three putative monosomic alien addition lines were characterized, and the results are discussed with respect to the potential for intergenomic chromosome recombination.     

Spontaneous capture of oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis>) chloroplasts by wild <Emphasis Type="Italic">B</Emphasis>. <Emphasis Type="Italic">rapa</Emphasis>: implications for the use of chloroplast transformation for biocontainment

Environmental concerns over the cultivation of Genetically Modified (GM) crops largely centre on the ecological consequences following gene flow to wild relatives. One attractive solution is to deploy biocontainment measures that prevent hybridization. Chloroplast transformation is the most advanced biocontainment method but is compromised by chloroplast capture (hybridization through the maternal lineage). To date, however, there is a paucity of information on the frequency of chloroplast capture in the wild. Oilseed rape (Brassica napus, AACC) frequently hybridises with wild Brassica rapa (AA, as paternal parent) and yields B. rapa-like introgressed individuals after only two generations. In this study we used chloroplast CAPS markers that differentiate between the two species to survey wild and weedy populations of B. rapa for the capture of B. napus chloroplasts. A total of 464 B. rapa plants belonging to 14 populations growing either in close proximity to B. napus (i.e. sympatric <5 m) or else were allopatric from the crop (>1 km) were assessed for chloroplast capture using PCR (trnL-F) and CAPS (trnT-L-Xba I) markers. The screen revealed that two sympatric B. rapa populations included 53 plants that possessed the chloroplast of B. napus. In order to discount these B. rapa plants as F₁ crop-wild hybrids, we used a C-genome-specific marker and found that 45 out of 53 plants lacked the C-genome and so were at least second generation introgressants. The most plausible explanation is that these individuals represent multiple cases of chloroplast capture following introgressive hybridisation through the female germ line from the crop. The abundance of such plants in sympatric sites thereby questions whether the use of chloroplast transformation would provide a sufficient biocontainment for GM oilseed rape in the United Kingdom.     

Mitochondrial genome sequences and comparative genomics of <Emphasis Type="Italic">Phytophthora ramorum</Emphasis> and <Emphasis Type="Italic">P. sojae</Emphasis>

The sequences of the mitochondrial genomes of the oomycetes Phytophthora ramorum and P. sojae were determined during the course of complete nuclear genome sequencing (Tyler et al., Science, 313:1261,2006). Both mitochondrial genomes are circular mapping, with sizes of 39,314 bp for P. ramorum and 42,977 bp for P. sojae. Each contains a total of 37 recognizable protein-encoding genes, 26 or 25 tRNAs (P. ramorum and P. sojae, respectively) specifying 19 amino acids, six more open reading frames (ORFs) that are conserved, presumably due to functional constraint, across Phytophthora species (P. sojae, P. ramorum, and P. infestans), six ORFs that are unique for P. sojae and one that is unique for P. ramorum. Non-coding regions comprise about 11.5 and 18.4% of the genomes of P. ramorum and P. sojae, respectively. Relative to P. sojae, there is an inverted repeat of 1,150 bp in P. ramorum that includes an unassigned unique ORF, a tRNA gene, and adjacent non-coding sequences, but otherwise the gene order in both species is identical. Comparisons of these genomes with published sequences of the P. infestans mitochondrial genome reveals a number of similarities, but the gene order in P. infestans differed in two adjacent locations due to inversions and specific regions of the genomes exhibited greater divergence than others. For example, the breakpoints for the inversions observed in P. infestans corresponded to regions of high sequence divergence in comparisons between P. ramorum and P. sojae and the location of a hypervariable microsatellite sequence (eight repeats of 24 bp) in the P. sojae genome corresponds to a site of major length variation in P. infestans. Although the overwhelming majority of each genome is conserved (81–92%), there are a number of genes that evolve more rapidly than others. Some of these rapidly evolving genes appear specific to Phytophthora, arose recently, and future evaluation of their function and the effects of their loss could prove fruitful for understanding the phylogeny of these devastating plant pathogens. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The complete mitochondrial genome of the vascular wilt fungus <Emphasis Type="Italic">Verticillium dahliae</Emphasis>: a novel gene order for <Emphasis Type="Italic">Verticillium</Emphasis> and a diagnostic tool for species identification

The complete sequence (27,184 bp) of the mitochondrial (mt) genome of the phytopathogenic fungus Verticillium dahliae has been determined. It contains 14 protein-coding genes related to oxidative phosphorylation, two rRNA genes and a set of 25 tRNA genes. A single intron, that harbors an intronic ORF coding for a putative ribosomal protein (rps), is located within the large rRNA gene (rnl). Gene order comparisons of V. dahliae mtDNA and complete mt genomes of Pezizomycotina revealed four units of synteny for Sordariomycetes, namely rnl-trn _(11–12)-nad2-nad3, nad4L-nad5-cob-cox1, nad1-nad4-atp8-atp6 and rns-trn _(1–5)-cox3-trn _(1–5)-nad6-trn _(2–5). These four units, in different combinations, merged to single continuous unit in the orders of Hypocreales and Sordariales. V. dahliae (Phyllachorales) and all members of the genus showed a unique feature which is the translocation of the nad1-nad4-atp8-atp6-rns-cox3-nad6 region in between genes nad3 and atp9 of the Hypocreales mtDNA gene order. Analysis of mt intergenic sequences of Verticillium species permitted the design of a species-specific primer allowing the discrimination of V. longisporum against V. dahliae and V. albo-atrum. By considering the protein-coding gene sequences as one unit, a phylogenetic comparison with representatives of Ascomycota complete mtDNA was performed.Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized usersNucleotide sequence data reported are available in the GenBank database under the accession numbers DQ351941–DQ351957.     

Propagation of the [<Emphasis Type="Italic">PIN</Emphasis><Superscript><Emphasis Type="Italic">+</Emphasis></Superscript>] prion by fragments of Rnq1 fused to GFP

Prions are viewed as enigmatic infectious entities whose genetic properties are enciphered solely in an array of self-propagating protein aggregate conformations. Rnq1, a yeast protein with yet unknown function, forms a prion named [PIN ⁺ ] for its ability to facilitate the de novo induction of another prion, [PSI ⁺ ]. Here we investigate a set of RNQ1 truncations that were designed to cover major Rnq1 sequence elements similar to those important for the propagation of other yeast prions: a region rich in asparagines and glutamines and several types of oligopeptide repeats. Proteins encoded by these RNQ1 truncations were tested for their ability to (a) join (decorate) pre-existing [PIN ⁺ ] aggregates made of wild-type Rnq1 and (b) maintain the heritable aggregated state in the absence of wild-type RNQ1. While the possible involvement of particular sequence elements in the propagation of [PIN ⁺ ] is discussed, the major result is that the efficiency of transmission of [PIN ⁺ ] from wild-type Rnq1 to a fragment decreased with the fragment’s length. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

An autophagy gene, <Emphasis Type="Italic">MgATG5</Emphasis>, is required for cell differentiation and pathogenesis in <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Autophagy is a conserved degradation pathway that is involved in the maintenance of normal cell differentiation and development. The Saccharomyces cerevisiae ATG5 gene is an important component of the autophagy process. In this study, we identified MgATG5 as an autophagy-related gene in Magnaporthe oryzae that is homologous to ATG5. Using targeted gene replacement, an Mgatg5∆ mutant was generated and fungal autophagy was blocked. Cytological analysis revealed that the mutant had poor fungal morphogenic development, including a shortened aerial hyphae lifespan, decreased conidiation and perithecia formation, delayed conidial germination and appressorial formation, postponement of conidial cytoplasm transfer during appressorium formation, and reduction in formation of the penetration peg. Turnover of endogenous matter in the Mgatg5∆ mutant was also affected, as demonstrated by defects in the formation of conidial lipid droplets, and in the degradation of conidial glycogen deposits during appressorium formation. Lipid droplets and glycogen are necessary to generate adequate turgor in appressoria for invading the host surface. As a result of the decreased appressorium turgor and differentiation in the penetration peg, Mgatg5∆ pathogenicity was deficient in two host plants tested. The developmental and pathogenic phenotypes were restored by the introduction of an intact copy of MgATG5 into Mgatg5∆, demonstrating that the MgATG5 deletion was responsible for the cellular defects. Taken together, these findings suggest that autophagy promotes cell differentiation through turnover of endogenous matter during fungal development, and is thus essential for the pathogenicity of the rice blast fungus. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. J.-P. Lu and X.-H. Liu contributed equally to this work and are regarded joint first authors.     

Identification and characterization of a novel <Emphasis Type="Italic">Tritimovirus</Emphasis> species isolated from wild <Emphasis Type="Italic">Trisetum flavescens</Emphasis> L., family <Emphasis Type="Italic">Poaceae</Emphasis>

Yellow oat-grass plants (Trisetum flavescens L.) with mild mosaic and pronounced dwarfing symptoms were observed at different locations in the Czech Republic. Electron microscope observations of symptomatic plants revealed the presence of filamentous particles and inclusion bodies characteristic of the family Potyviridae. The virus was readily mechanically transmitted to its original host plus a narrow host range of monocot species. Serological assays of infected plant extracts using antiserum specific to the closest species in the family Potyviridae were negative. The 3′ end of the viral genome was cloned, sequenced and compared to sequences of species in the family Potyviridae. The virus is more closely related to viruses in the genus Tritimovirus than to other genera within the Potyviridae. Based on phylogenetic analyses of the coat protein cistron and flanking genomic regions, we propose this is a distinct viral species of the genus Tritimovirus, tentatively named Yellow oat-grass mosaic virus (YOgMV).     

Detection and Localization of <Emphasis Type="Italic">Rice stripe virus</Emphasis> Gene Products <Emphasis Type="Italic">In Vivo</Emphasis>

The genome of the Tenuivirus, Rice stripe virus (RSV) comprises four RNAs, the smallest three of which each contain two open reading frames (ORFs) arranged in an ambisense manner. The expression of the ORFs from RNAs 2–4 in plants and the insect vector, Laodelphax striatellus, was studied using antisera raised against the gene products. In Western blotting of the proteins from infected plants, the molecular masses of p2, p3, pc3 (nucleocapsid protein, N) and p4 (major non-structural protein, NCP) were as expected; that of pc4 appeared larger than expected. Antisera to the N- and C-terminal parts of the complementary ORF on RNA 2, analogous to that encoding glycoproteins on genomes of bunyaviruses and tospoviruses, revealed banding patterns suggestive of processing of the product; the possible processing is discussed. Four types of inclusion bodies were identified by immunofluorescent and immunogold microscopy of thin sections of infected leaves. Most electron-dense amorphous semi-electron-opaque inclusion bodies (dASO) contained only p4 while some contained at least p2, pc2-N, p3, pc3 as well as p4. A ring-like structure containing at least pc2-N, p4 and pc4 was also identified in infected plant cells. Fibrillar amorphous semi-electron-opaque inclusion bodies (fASO) contained only p4. Filamentous electron-opaque inclusion bodies (FEO), which consist of pc2-N^.and p4, were found both in infected plant cells and in the mid-gut lumen and mid-gut epithelial cells of L. striatellus. This suggests an interaction between p4 and pc2-N and a function of pc2-N distinct from that of its-homologue in Bunyaviridae. Our results confirm the in vivo ambisense coding strategy of Tenuivirus RNA 2 and provide further evidence that RSV does not produce enveloped virions in infected rice plants.