Chromosome-scale assemblies of Acanthamoeba castellanii genomes provide insights into Legionella pneumophila infection-related chromatin reorganization.

The unicellular amoeba Acanthamoeba castellanii is ubiquitous in aquatic environments, where it preys on bacteria. The organism also hosts bacterial endosymbionts, some of which are parasitic, including human pathogens such as Chlamydia and Legionella spp. Here we report complete, high-quality genome sequences for two extensively studied A. castellanii strains, Neff and C3. Combining long- and short-read data with Hi-C, we generated near chromosome-level assemblies for both strains with 90% of the genome contained in 29 scaffolds for the Neff strain and 31 for the C3 strain. Comparative genomics revealed strain-specific functional enrichment, most notably genes related to signal transduction in the C3 strain and to viral replication in Neff. Furthermore, we characterized the spatial organization of the A. castellanii genome and showed that it is reorganized during infection by Legionella pneumophila Infection-dependent chromatin loops were found to be enriched in genes for signal transduction and phosphorylation processes. In genomic regions where chromatin organization changed during Legionella infection, we found functional enrichment for genes associated with metabolism, organelle assembly, and cytoskeleton organization. Given Legionella infection is known to alter its host's cell cycle, to exploit the host's organelles, and to modulate the host's metabolism in its favor, these changes in chromatin organization may partly be related to mechanisms of host control during Legionella infection.

Diploid-dominant life cycles characterize the early evolution of Fungi.

Most of the described species in kingdom Fungi are contained in two phyla, the Ascomycota and the Basidiomycota (subkingdom Dikarya). As a result, our understanding of the biology of the kingdom is heavily influenced by traits observed in Dikarya, such as aerial spore dispersal and life cycles dominated by mitosis of haploid nuclei. We now appreciate that Fungi comprises numerous phylum-level lineages in addition to those of Dikarya, but the phylogeny and genetic characteristics of most of these lineages are poorly understood due to limited genome sampling. Here, we addressed major evolutionary trends in the non-Dikarya fungi by phylogenomic analysis of 69 newly generated draft genome sequences of the zoosporic (flagellated) lineages of true fungi. Our phylogeny indicated five lineages of zoosporic fungi and placed Blastocladiomycota, which has an alternation of haploid and diploid generations, as branching closer to the Dikarya than to the Chytridiomyceta. Our estimates of heterozygosity based on genome sequence data indicate that the zoosporic lineages plus the Zoopagomycota are frequently characterized by diploid-dominant life cycles. We mapped additional traits, such as ancestral cell-cycle regulators, cell-membrane- and cell-wall-associated genes, and the use of the amino acid selenocysteine on the phylogeny and found that these ancestral traits that are shared with Metazoa have been subject to extensive parallel loss across zoosporic lineages. Together, our results indicate a gradual transition in the genetics and cell biology of fungi from their ancestor and caution against assuming that traits measured in Dikarya are typical of other fungal lineages.

Recent expansion of metabolic versatility in Diplonema papillatum, the model species of a highly speciose group of marine eukaryotes.

BACKGROUND: Diplonemid flagellates are among the most abundant and species-rich of known marine microeukaryotes, colonizing all habitats, depths, and geographic regions of the world ocean. However, little is known about their genomes, biology, and ecological role. RESULTS: We present the first nuclear genome sequence from a diplonemid, the type species Diplonema papillatum. The ~ 280-Mb genome assembly contains about 32,000 protein-coding genes, likely co-transcribed in groups of up to 100. Gene clusters are separated by long repetitive regions that include numerous transposable elements, which also reside within introns. Analysis of gene-family evolution reveals that the last common diplonemid ancestor underwent considerable metabolic expansion. D. papillatum-specific gains of carbohydrate-degradation capability were apparently acquired via horizontal gene transfer. The predicted breakdown of polysaccharides including pectin and xylan is at odds with reports of peptides being the predominant carbon source of this organism. Secretome analysis together with feeding experiments suggest that D. papillatum is predatory, able to degrade cell walls of live microeukaryotes, macroalgae, and water plants, not only for protoplast feeding but also for metabolizing cell-wall carbohydrates as an energy source. The analysis of environmental barcode samples shows that D. papillatum is confined to temperate coastal waters, presumably acting in bioremediation of eutrophication. CONCLUSIONS: Nuclear genome information will allow systematic functional and cell-biology studies in D. papillatum. It will also serve as a reference for the highly diverse diplonemids and provide a point of comparison for studying gene complement evolution in the sister group of Kinetoplastida, including human-pathogenic taxa.

An Unexpectedly Complex Mitoribosome in Andalucia godoyi, a Protist with the Most Bacteria-like Mitochondrial Genome.

The mitoribosome, as known from studies in model organisms, deviates considerably from its ancestor, the bacterial ribosome. Deviations include substantial reduction of the mitochondrial ribosomal RNA (mt-rRNA) structure and acquisition of numerous mitochondrion-specific (M) mitoribosomal proteins (mtRPs). A broadly accepted view assumes that M-mtRPs compensate for structural destabilization of mt-rRNA resulting from its evolutionary remodeling. Since most experimental information on mitoribosome makeup comes from eukaryotes having derived mitochondrial genomes and mt-rRNAs, we tested this assumption by investigating the mitochondrial translation machinery of jakobids, a lineage of unicellular protists with the most bacteria-like mitochondrial genomes. We report here proteomics analyses of the Andalucia godoyi small mitoribosomal subunit and in silico transcriptomic and comparative genome analyses of four additional jakobids. Jakobids have mt-rRNA structures that minimally differ from their bacterial counterparts. Yet, with at least 31 small subunit and 44 large subunit mtRPs, the mitoriboproteome of Andalucia is essentially as complex as that in animals or fungi. Furthermore, the relatively high conservation of jakobid sequences has helped to clarify the identity of several mtRPs, previously considered to be lineage-specific, as divergent homologs of conserved M-mtRPs, notably mS22 and mL61. The coexistence of bacteria-like mt-rRNAs and a complex mitoriboproteome refutes the view that M-mtRPs were ancestrally recruited to stabilize deviations of mt-rRNA structural elements. We postulate instead that the numerous M-mtRPs acquired in the last eukaryotic common ancestor allowed mt-rRNAs to pursue a broad range of evolutionary trajectories across lineages: from dramatic reduction to acquisition of novel elements to structural conservatism.

Characterization of the mitogenome of Gongronella sp. w5 reveals substantial variation in Mucoromycota.

Gongronella is a genus of fungi in Mucorales (Mucoromycota). Some of its members have important biotechnological applications, but until now, not a single mitogenome has been characterized in Gongronella. Here, we present the complete mitogenome assembly of Gongronella sp. w5, a soil isolate known to interact with plants and several fungi. Its 36,593-bp circular mitogenome encodes the large and small subunit rRNAs, 14 standard mitochondrial proteins, 24 tRNAs, three free-standing ORF proteins, and the RNA subunit of RNase P (rnpB). These genes arrange in an order novel to known fungal mitogenomes. Three group I introns are present in the cob, cox1, and nad5 genes, respectively, and they are probably acquired by horizontal gene transfer. Phylogenetic analysis based on mitochondrion-encoded proteins supports the grouping of Gongronella sp. w5 with Absidia glauca, forming the Cunninghamellaceae clade within Mucoromycota. Gongronella and most other Mucoromycota species are predicted to use the standard genetic code in mitochondrial translation, rather than code 4 assigned by GenBank. A comparison among seven publicly available mitogenomes in Mucoromycota reveals the presence of the same 14 typical protein-coding genes plus rnpB, yet substantial variation in mitogenome size, intron number, gene order, and orientation. In this comparison, the uniqueness of Gongronella is evident from similarly large differences to its closest phylogenetic neighbor, A. glauca. This study promotes our understanding of fungal evolution in Mucoromycota. KEY POINTS: • This study reports the first mitogenome in Gongronella, which presents a novel gene order. • Different Mucoromycota mitogenomes show substantial variation of gene organizations. • Most Mucoromycota species use the standard genetic code to translate mitochondrial genes.

The draft nuclear genome sequence and predicted mitochondrial proteome of Andalucia godoyi, a protist with the most gene-rich and bacteria-like mitochondrial genome.

BACKGROUND: Comparative analyses have indicated that the mitochondrion of the last eukaryotic common ancestor likely possessed all the key core structures and functions that are widely conserved throughout the domain Eucarya. To date, such studies have largely focused on animals, fungi, and land plants (primarily multicellular eukaryotes); relatively few mitochondrial proteomes from protists (primarily unicellular eukaryotic microbes) have been examined. To gauge the full extent of mitochondrial structural and functional complexity and to identify potential evolutionary trends in mitochondrial proteomes, more comprehensive explorations of phylogenetically diverse mitochondrial proteomes are required. In this regard, a key group is the jakobids, a clade of protists belonging to the eukaryotic supergroup Discoba, distinguished by having the most gene-rich and most bacteria-like mitochondrial genomes discovered to date. RESULTS: In this study, we assembled the draft nuclear genome sequence for the jakobid Andalucia godoyi and used a comprehensive in silico approach to infer the nucleus-encoded portion of the mitochondrial proteome of this protist, identifying 864 candidate mitochondrial proteins. The A. godoyi mitochondrial proteome has a complexity that parallels that of other eukaryotes, while exhibiting an unusually large number of ancestral features that have been lost particularly in opisthokont (animal and fungal) mitochondria. Notably, we find no evidence that the A. godoyi nuclear genome has or had a gene encoding a single-subunit, T3/T7 bacteriophage-like RNA polymerase, which functions as the mitochondrial transcriptase in all eukaryotes except the jakobids. CONCLUSIONS: As genome and mitochondrial proteome data have become more widely available, a strikingly punctuate phylogenetic distribution of different mitochondrial components has been revealed, emphasizing that the pathways of mitochondrial proteome evolution are likely complex and lineage-specific. Unraveling this complexity will require comprehensive comparative analyses of mitochondrial proteomes from a phylogenetically broad range of eukaryotes, especially protists. The systematic in silico approach described here offers a valuable adjunct to direct proteomic analysis (e.g., via mass spectrometry), particularly in cases where the latter approach is constrained by sample limitation or other practical considerations.

Genome sequence of the opportunistic human pathogen Magnusiomyces capitatus.

The yeast Magnusiomyces capitatus is an opportunistic human pathogen causing rare yet severe infections, especially in patients with hematological malignancies. Here, we report the 20.2 megabase genome sequence of an environmental strain of this species as well as the genome sequences of eight additional isolates from human and animal sources providing an insight into intraspecies variation. The distribution of single-nucleotide variants is indicative of genetic recombination events, supporting evidence for sexual reproduction in this heterothallic yeast. Using RNAseq-aided annotation, we identified genes for 6518 proteins including several expanded families such as kexin proteases and Hsp70 molecular chaperones. Several of these families are potentially associated with the ability of M. capitatus to infect and colonize humans. For the purpose of comparative analysis, we also determined the genome sequence of a closely related yeast, Magnusiomyces ingens. The genome sequences of M. capitatus and M. ingens exhibit many distinct features and represent a basis for further comparative and functional studies.

Programmed translational bypassing elements in mitochondria: structure, mobility, and evolutionary origin.

Programmed translational bypassing enables ribosomes to 'ignore' a precise mRNA interval of several dozen nucleotides. Well-characterized bypassed sequences include hop and byp elements, present in bacteriophage T4 and mitochondria of the yeast Magnusiomyces capitatus, respectively. The bypassing mechanism of byps is probably similar to that of hop, yet the former appears more effective and less constrained as to sequence context. Furthermore, both elements are mobile but hop moves as part of a cassette including a homing endonuclease, whereas byps seem to spread like miniature DNA transposable elements known as GC clusters. Here, we argue that hop and byps arose independently by convergent evolution, and that byps evolved in magnusiomycete mitochondria due to (as yet unknown) alterations of the mitochondrial translation machinery.

Bacterial proteins pinpoint a single eukaryotic root.

The large phylogenetic distance separating eukaryotic genes and their archaeal orthologs has prevented identification of the position of the eukaryotic root in phylogenomic studies. Recently, an innovative approach has been proposed to circumvent this issue: the use as phylogenetic markers of proteins that have been transferred from bacterial donor sources to eukaryotes, after their emergence from Archaea. Using this approach, two recent independent studies have built phylogenomic datasets based on bacterial sequences, leading to different predictions of the eukaryotic root. Taking advantage of additional genome sequences from the jakobid Andalucia godoyi and the two known malawimonad species (Malawimonas jakobiformis and Malawimonas californiana), we reanalyzed these two phylogenomic datasets. We show that both datasets pinpoint the same phylogenetic position of the eukaryotic root that is between "Unikonta" and "Bikonta," with malawimonad and collodictyonid lineages on the Unikonta side of the root. Our results firmly indicate that (i) the supergroup Excavata is not monophyletic and (ii) the last common ancestor of eukaryotes was a biflagellate organism. Based on our results, we propose to rename the two major eukaryotic groups Unikonta and Bikonta as Opimoda and Diphoda, respectively.

An ancestral bacterial division system is widespread in eukaryotic mitochondria.

Bacterial division initiates at the site of a contractile Z-ring composed of polymerized FtsZ. The location of the Z-ring in the cell is controlled by a system of three mutually antagonistic proteins, MinC, MinD, and MinE. Plastid division is also known to be dependent on homologs of these proteins, derived from the ancestral cyanobacterial endosymbiont that gave rise to plastids. In contrast, the mitochondria of model systems such as Saccharomyces cerevisiae, mammals, and Arabidopsis thaliana seem to have replaced the ancestral α-proteobacterial Min-based division machinery with host-derived dynamin-related proteins that form outer contractile rings. Here, we show that the mitochondrial division system of these model organisms is the exception, rather than the rule, for eukaryotes. We describe endosymbiont-derived, bacterial-like division systems comprising FtsZ and Min proteins in diverse less-studied eukaryote protistan lineages, including jakobid and heterolobosean excavates, a malawimonad, stramenopiles, amoebozoans, a breviate, and an apusomonad. For two of these taxa, the amoebozoan Dictyostelium purpureum and the jakobid Andalucia incarcerata, we confirm a mitochondrial localization of these proteins by their heterologous expression in Saccharomyces cerevisiae. The discovery of a proteobacterial-like division system in mitochondria of diverse eukaryotic lineages suggests that it was the ancestral feature of all eukaryotic mitochondria and has been supplanted by a host-derived system multiple times in distinct eukaryote lineages.

Massive programmed translational jumping in mitochondria.

Programmed translational bypassing is a process whereby ribosomes "ignore" a substantial interval of mRNA sequence. Although discovered 25 y ago, the only experimentally confirmed example of this puzzling phenomenon is expression of the bacteriophage T4 gene 60. Bypassing requires translational blockage at a "takeoff codon" immediately upstream of a stop codon followed by a hairpin, which causes peptidyl-tRNA dissociation and reassociation with a matching "landing triplet" 50 nt downstream, where translation resumes. Here, we report 81 translational bypassing elements (byps) in mitochondria of the yeast Magnusiomyces capitatus and demonstrate in three cases, by transcript analysis and proteomics, that byps are retained in mitochondrial mRNAs but not translated. Although mitochondrial byps resemble the bypass sequence in the T4 gene 60, they utilize unused codons instead of stops for translational blockage and have relaxed matching rules for takeoff/landing sites. We detected byp-like sequences also in mtDNAs of several Saccharomycetales, indicating that byps are mobile genetic elements. These byp-like sequences lack bypassing activity and are tolerated when inserted in-frame in variable protein regions. We hypothesize that byp-like elements have the potential to contribute to evolutionary diversification of proteins by adding new domains that allow exploration of new structures and functions.

Evolution of tRNA Repertoires in Bacillus Inferred with OrthoAlign.

OrthoAlign, an algorithm for the gene order alignment problem (alignment of orthologs), accounting for most genome-wide evolutionary events such as duplications, losses, rearrangements, and substitutions, was presented. OrthoAlign was used in a phylogenetic framework to infer the evolution of transfer RNA repertoires of 50 fully sequenced bacteria in the Bacillus genus. A prevalence of gene duplications and losses over rearrangement events was observed. The average rate of duplications inferred in Bacillus was 24 times lower than the one reported in Escherichia coli, whereas the average rates of losses and inversions were both 12 times lower. These rates were extremely low, suggesting a strong selective pressure acting on tRNA gene repertoires in Bacillus. An exhaustive analysis of the type, location, distribution, and length of evolutionary events was provided, together with ancestral configurations. OrthoAlign can be downloaded at: http://www.iro.umontreal.ca/~mabrouk/.

Widespread occurrence of organelle genome-encoded 5S rRNAs including permuted molecules.

5S Ribosomal RNA (5S rRNA) is a universal component of ribosomes, and the corresponding gene is easily identified in archaeal, bacterial and nuclear genome sequences. However, organelle gene homologs (rrn5) appear to be absent from most mitochondrial and several chloroplast genomes. Here, we re-examine the distribution of organelle rrn5 by building mitochondrion- and plastid-specific covariance models (CMs) with which we screened organelle genome sequences. We not only recover all organelle rrn5 genes annotated in GenBank records, but also identify more than 50 previously unrecognized homologs in mitochondrial genomes of various stramenopiles, red algae, cryptomonads, malawimonads and apusozoans, and surprisingly, in the apicoplast (highly derived plastid) genomes of the coccidian pathogens Toxoplasma gondii and Eimeria tenella. Comparative modeling of RNA secondary structure reveals that mitochondrial 5S rRNAs from brown algae adopt a permuted triskelion shape that has not been seen elsewhere. Expression of the newly predicted rrn5 genes is confirmed experimentally in 10 instances, based on our own and published RNA-Seq data. This study establishes that particularly mitochondrial 5S rRNA has a much broader taxonomic distribution and a much larger structural variability than previously thought. The newly developed CMs will be made available via the Rfam database and the MFannot organelle genome annotator.

Natural reassignment of CUU and CUA sense codons to alanine in Ashbya mitochondria.

The discovery of diverse codon reassignment events has demonstrated that the canonical genetic code is not universal. Studying coding reassignment at the molecular level is critical for understanding genetic code evolution, and provides clues to genetic code manipulation in synthetic biology. Here we report a novel reassignment event in the mitochondria of Ashbya (Eremothecium) gossypii, a filamentous-growing plant pathogen related to yeast (Saccharomycetaceae). Bioinformatics studies of conserved positions in mitochondrial DNA-encoded proteins suggest that CUU and CUA codons correspond to alanine in A. gossypii, instead of leucine in the standard code or threonine in yeast mitochondria. Reassignment of CUA to Ala was confirmed at the protein level by mass spectrometry. We further demonstrate that a predicted tRNA(Ala)UAG is transcribed and accurately processed in vivo, and is responsible for Ala reassignment. Enzymatic studies reveal that tRNA(Ala)UAG is efficiently recognized by A. gossypii mitochondrial alanyl-tRNA synthetase (AgAlaRS). AlaRS typically recognizes the G3:U70 base pair of tRNA(Ala); a G3A change in Ashbya tRNA(Ala)UAG abolishes its recognition by AgAlaRS. Conversely, an A3G mutation in Saccharomyces cerevisiae tRNA(Thr)UAG confers tRNA recognition by AgAlaRS. Our work highlights the dynamic feature of natural genetic codes in mitochondria, and the relative simplicity by which tRNA identity may be switched.

Dinoflagellate tandem array gene transcripts are highly conserved and not polycistronic.

Dinoflagellates are an important component of the marine biota, but a large genome with high-copy number (up to 5,000) tandem gene arrays has made genomic sequencing problematic. More importantly, little is known about the expression and conservation of these unusual gene arrays. We assembled de novo a gene catalog of 74,655 contigs for the dinoflagellate Lingulodinium polyedrum from RNA-Seq (Illumina) reads. The catalog contains 93% of a Lingulodinium EST dataset deposited in GenBank and 94% of the enzymes in 16 primary metabolic KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways, indicating it is a good representation of the transcriptome. Analysis of the catalog shows a marked underrepresentation of DNA-binding proteins and DNA-binding domains compared with other algae. Despite this, we found no evidence to support the proposal of polycistronic transcription, including a marked underrepresentation of sequences corresponding to the intergenic spacers of two tandem array genes. We also have used RNA-Seq to assess the degree of sequence conservation in tandem array genes and found their transcripts to be highly conserved. Interestingly, some of the sequences in the catalog have only bacterial homologs and are potential candidates for horizontal gene transfer. These presumably were transferred as single-copy genes, and because they are now all GC-rich, any derived from AT-rich contexts must have experienced extensive mutation. Our study not only has provided the most complete dinoflagellate gene catalog known to date, it has also exploited RNA-Seq to address fundamental issues in basic transcription mechanisms and sequence conservation in these algae.

Mitochondrial DNA of Clathrina clathrus (Calcarea, Calcinea): six linear chromosomes, fragmented rRNAs, tRNA editing, and a novel genetic code.

Sponges (phylum Porifera) are a large and ancient group of morphologically simple but ecologically important aquatic animals. Although their body plan and lifestyle are relatively uniform, sponges show extensive molecular and genetic diversity. In particular, mitochondrial genomes from three of the four previously studied classes of Porifera (Demospongiae, Hexactinellida, and Homoscleromorpha) have distinct gene contents, genome organizations, and evolutionary rates. Here, we report the mitochondrial genome of Clathrina clathrus (Calcinea, Clathrinidae), a representative of the fourth poriferan class, the Calcarea, which proves to be the most unusual. Clathrina clathrus mitochondrial DNA (mtDNA) consists of six linear chromosomes 7.6-9.4 kb in size and encodes at least 37 genes: 13 protein codings, 2 ribosomal RNAs (rRNAs), and 24 transfer RNAs (tRNAs). Protein genes include atp9, which has now been found in all major sponge lineages, but no atp8. Our analyses further reveal the presence of a novel genetic code that involves unique reassignments of the UAG codons from termination to tyrosine and of the CGN codons from arginine to glycine. Clathrina clathrus mitochondrial rRNAs are encoded in three (srRNA) and ≥6 (lrRNA) fragments distributed out of order and on several chromosomes. The encoded tRNAs contain multiple mismatches in the aminoacyl acceptor stems that are repaired posttranscriptionally by 3'-end RNA editing. Although our analysis does not resolve the phylogenetic position of calcareous sponges, likely due to their high rates of mitochondrial sequence evolution, it confirms mtDNA as a promising marker for population studies in this group. The combination of unusual mitochondrial features in C. clathrus redefines the extremes of mtDNA evolution in animals and further argues against the idea of a "typical animal mtDNA."

Insights into the origin of metazoan filopodia and microvilli.

Filopodia are fine actin-based cellular projections used for both environmental sensing and cell motility, and they are essential organelles for metazoan cells. In this study, we reconstruct the origin of metazoan filopodia and microvilli. We first report on the evolutionary assembly of the filopodial molecular toolkit and show that homologs of many metazoan filopodial components, including fascin and myosin X, were already present in the unicellular or colonial progenitors of metazoans. Furthermore, we find that the actin crosslinking protein fascin localizes to filopodia-like structures and microvilli in the choanoflagellate Salpingoeca rosetta. In addition, homologs of filopodial genes in the holozoan Capsaspora owczarzaki are upregulated in filopodia-bearing cells relative to those that lack them. Therefore, our findings suggest that proteins essential for metazoan filopodia and microvilli are functionally conserved in unicellular and colonial holozoans and that the last common ancestor of metazoans bore a complex and specific filopodial machinery.

Yeast mitochondrial RNase P, RNase Z and the RNA degradosome are part of a stable supercomplex.

Initial steps in the synthesis of functional tRNAs require 5'- and 3'-processing of precursor tRNAs (pre-tRNAs), which in yeast mitochondria are achieved by two endonucleases, RNase P and RNase Z. In this study, using a combination of detergent-free Blue Native Gel Electrophoresis, proteomics and in vitro testing of pre-tRNA maturation, we reveal the physical association of these plus other mitochondrial activities in a large, stable complex of 136 proteins. It contains a total of seven proteins involved in RNA processing including RNase P and RNase Z, five out of six subunits of the mitochondrial RNA degradosome, components of the fatty acid synthesis pathway, translation, metabolism and protein folding. At the RNA level, there are the small and large rRNA subunits and RNase P RNA. Surprisingly, this complex is absent in an oar1Δ deletion mutant of the type II fatty acid synthesis pathway, supporting a recently published functional link between pre-tRNA processing and the FAS II pathway--apparently by integration into a large complex as we demonstrate here. Finally, the question of mt-RNase P localization within mitochondria was investigated, by GFP-tracing of a known protein subunit (Rpm2p). We find that about equal fractions of RNase P are soluble versus membrane-attached.

A biofertilizing fungal endophyte of cranberry plants suppresses the plant pathogen Diaporthe.

Fungi colonizing plants are gaining attention because of their ability to promote plant growth and suppress pathogens. While most studies focus on endosymbionts from grasses and legumes, the large and diverse group of ericaceous plants has been much neglected. We recently described one of the very few fungal endophytes promoting the growth of the Ericaceae Vaccinium macrocarpon (American cranberry), notably the Codinaeella isolate EC4. Here, we show that EC4 also suppresses fungal pathogens, which makes it a promising endophyte for sustainable cranberry cultivation. By dual-culture assays on agar plates, we tested the potential growth suppression (or biocontrol) of EC4 on other microbes, notably 12 pathogenic fungi and one oomycete reported to infect not only cranberry but also blueberry, strawberry, tomato plants, rose bushes and olive trees. Under greenhouse conditions, EC4 protects cranberry plantlets infected with one of the most notorious cranberry-plant pathogens, Diaporthe vaccinii, known to cause upright dieback and berry rot. The nuclear genome sequence of EC4 revealed a large arsenal of genes potentially involved in biocontrol. About ∼60 distinct clusters of genes are homologs of secondary metabolite gene clusters, some of which were shown in other fungi to synthesize nonribosomal peptides and polyketides, but in most cases, the exact compounds these clusters may produce are unknown. The EC4 genome also encodes numerous homologs of hydrolytic enzymes known to degrade fungal cell walls. About half of the nearly 250 distinct glucanases and chitinases are likely involved in biocontrol because they are predicted to be secreted outside the cell. Transcriptome analysis shows that the expression of about a quarter of the predicted secondary-metabolite gene clusters and glucan and chitin-degrading genes of EC4 is stimulated when it is co-cultured with D. vaccinii. Some of the differentially expressed EC4 genes are alternatively spliced exclusively in the presence of the pathogen, altering the proteins' domain content and subcellular localization signal, thus adding a second level of proteome adaptation in response to habitat competition. To our knowledge, this is the first report of Diaporthe-induced alternative splicing of biocontrol genes.

Rooting the eukaryotic tree with mitochondrial and bacterial proteins.

By exploiting the large body of genome data and the considerable progress in phylogenetic methodology, recent phylogenomic studies have provided new insights into the relationships among major eukaryotic groups. However, confident placement of the eukaryotic root remains a major challenge. This is due to the large evolutionary distance separating eukaryotes from their closest relatives, the Archaea, implying a weak phylogenetic signal and strong long-branch attraction artifacts. Here, we apply a new approach to the rooting of the eukaryotic tree by using a subset of genomic information with more recent evolutionary origin-mitochondrial sequences, whose closest relatives are α-Proteobacteria. For this, we identified and assembled a data set of 42 mitochondrial proteins (mainly encoded by the nuclear genome) and performed Bayesian and maximum likelihood analyses. Taxon sampling includes the recently sequenced Thecamonas trahens, a member of the phylogenetically elusive Apusozoa. This data set confirms the relationships of several eukaryotic supergroups seen before and places the eukaryotic root between the monophyletic "unikonts" and "bikonts." We further show that T. trahens branches sister to Opisthokonta with significant statistical support and question the bikont/excavate affiliation of Malawimonas species. The mitochondrial data set developed here (to be expanded in the future) constitutes a unique alternative means in resolving deep eukaryotic relationships.