Genomics of biotrophic, plant-infecting plasmodiophorids using in vitro dual cultures

The plasmodiophorids are a phylogenetically distinct group of parasitic protists that infect plants and stramenopiles, causing several important agricultural diseases. Because of the obligate intracellular part of their lifecycle, none of the plasmodiophorids has been axenically cultured. Further, the molecular biology of the plasmodiophorids is poorly understood because pure cultures are not available from any species. We report on an in-vitro dual culture system of the plasmodiophorids Plasmodiophora brassicae and Spongospora subterranea with their respective plant hosts, Brassica rapa and Solanum tuberosum. We show that these plasmodiophorids are capable of initiating and maintaining stable, long-term plant cell callus cultures in the absence of exogenous plant growth regulators. We show that callus cultures harbouring S. subterranea provide an excellent starting material for gene discovery from this organism by constructing a pilot-scale DNA library. Bioinformatic analysis of the sequences established that almost all of the DNA clones from this library were from S. subterranea rather than the plant host. The Spongospora genome was found to be rich in retrotransposable elements, and Spongospora protein-coding genes were shown to contain introns. The sequence of a near full-length non-LTR retrotransposon was obtained, the first transposable element reported from a cercozoan protist.     

A novel polyubiquitin structure in Cercozoa and Foraminifera: evidence for a new eukaryotic supergroup

Ubiquitin is a 76 amino acid protein with a remarkable degree of evolutionary conservation. Ubiquitin plays an essential role in a large number of eukaryotic cellular processes by targeting proteins for proteasome-mediated degradation. Most ubiquitin genes are found as head-to-tail polymers whose products are posttranslationally processed to ubiquitin monomers. We have characterized polyubuiquitin genes from the photosynthetic amoeboflagellate Chlorarachnion sp. CCMP 621 (also known as Bigelowiella natans) and found that they deviate from the canonical polyubiquitin structure in having an amino acid insertion at the junction between each monomer, suggesting that polyubiquitin processing in this organism is unique among eukaryotes. The gene structure indicates that processing likely cleaves monomers at the amino terminus of the insertion. We examined the phylogenetic distribution of the insertion by sequencing polyubiquitin genes from several other eukaryotic groups and found it to be confined to Cercozoa (including Chlorarachnion, Lotharella, Cercomonas, and Euglypha) and Foraminifera (including Reticulomyxa and Haynesina). This character strongly suggests that Cercozoa and Foraminifera are close relatives and form a new "supergroup" of eukaryotes.     

Polyubiquitin insertions and the phylogeny of Cercozoa and Rhizaria

A single or double amino acid insertion at the monomer-monomer junction of the universal eukaryotic protein polyubiquitin is unique to Cercozoa and Foraminifera, closely related 'core' phyla in the protozoan infrakingdom Rhizaria. We screened 11 other candidate rhizarians for this insertion: Radiozoa (polycystine and acantharean radiolaria), a 'microheliozoan', and Apusozoa; all lack it, supporting suggestions that Foraminifera are more closely related to Cercozoa than either is to other eukaryotes. The insertion's size was ascertained for 12 additional Cercozoa to help resolve their basal branching order. The earliest branching Cercozoa generally have a single amino acid insertion, like all Foraminifera, but a large derived clade consisting of all Monadofilosa except Metopion, Helk-esimastix, and Cercobodo agilis has two amino acids, suggesting one doubling event and no reversions to a single amino acid. Metromonas and Sainouron, cercozoans of uncertain position, have a double insertion, suggesting that they belong in Monadofilosa. An alternative interpretation, suggested by the higher positions for Metopion and Cercobodo on Bayesian trees compared with most distance trees, cannot be ruled out, i.e. that the second insertion took place earlier, in the ancestral filosan, and was followed by three independent reversions to a single amino acid in Chlorarachnea, Metopion and Cercobodo.     

Revised small subunit rRNA analysis provides further evidence that Foraminifera are related to Cercozoa

There is accumulating evidence that the general shape of the ribosomal DNA-based phylogeny of Eukaryotes is strongly biased by the long-branch attraction phenomenon, leading to an artifactual basal clustering of groups that are probably highly derived. Among these groups, Foraminifera are of particular interest, because their deep phylogenetic position in ribosomal trees contrasts with their Cambrian appearance in the fossil record. A recent actin-based phylogeny of Eukaryotes has proposed that Foraminifera might be closely related to Cercozoa and, thus, branch among the so-called crown of Eukaryotes. Here, we reanalyze the small-subunit ribosomal RNA gene (SSU rDNA) phylogeny by removing all long-branching lineages that could artifactually attract foraminiferan sequences to the base of the tree. Our analyses reveal that Foraminifera branch together with the marine testate filosean Gromia oviformis as a sister group to Cercozoa, in agreement with actin phylogeny. Our study confirms the utility of SSU rDNA as a phylogenetic marker of megaevolutionary history, provided that the artifacts due to the heterogeneity of substitution rates in ribosomal genes are circumvented.     

Phylogenetic position of Gromia oviformis Dujardin inferred from nuclear-encoded small subunit ribosomal DNA

Gromia oviformis Dujardin is a common marine protist characterised by a large, globular test and filose pseudopodia. First considered a foraminifer, Gromia was later placed within the Filosea and recently included among amoebae of uncertain affinities. In order to clarify the phylogenetic position of this genus, we sequenced the complete small-subunit ribosomal DNA gene of G. oviformis collected at five different geographic localities. The high divergence of obtained sequences suggests that G. oviformis is a species complex composed of several genetically distinct sibling species. Sequence analyses show Gromia to be a member of the Cercozoa, a heterogeneous assemblage which includes filose amoebae, the amoeboflagellate cercomonads, the chlorarachniophytes and the plasmodiophorid plant pathogens. Contrary to traditional classification, Gromia is not closely related to other testate filose amoebae (the Euglyphida), but seems to branch early among the Cercozoa. Our analyses also show a close relationship between the Cercozoa and the Acantharea. Because the Cercozoa are related to the Foraminifera based on other molecular data, we propose that most protists possessing filopodia, reticulopodia and axopodia have a common origin.     

Identification of genes from the obligate intracellular plant pathogen, Plasmodiophora brassicae

Plasmodiophora brassicae is an intracellular pathogen that infects plants in the Brassicaceae family. Although an important pathogen group, information on the genomic makeup of the plasmodiophorids is almost completely lacking. We performed suppression subtractive hybridization (SSH) between RNA from P. brassicae-infected and uninfected Arabidopsis tissue, then screened 232 clones from the resulting SSH library. In addition, we used an oligo-capping procedure to screen 305 full-length cDNA clones from the infected tissue. A total of 76 new P. brassicae gene sequences were identified, the majority of which were extended to full length at the 5' end by the use of RACE amplification. Many of the unisequences were predicted to contain signal peptides for ER translocation. Although we located few sequences in total, these markedly increase available data from the plasmodiophorids, and provide new opportunities to examine plasmodiophorid biology. Our study also points towards the best methods for future plasmodiophorid gene discovery.     

Foraminifera and Cercozoa are related in actin phylogeny: two orphans find a home?

In recent years, the increased sampling of protein-coding genes from diverse eukaryotes has revealed that many aspects of each gene tree are at odds with other phylogenies. This has led to the belief that each gene tree has unique strengths and weaknesses, suggesting that an accurate picture of eukaryotic relationships will be achieved only through comparative phylogeny using several different genes. To this end, actin genes were characterized from two genera of chlorarachniophytes, Chlorarachnion and Lotharella, and three species of the cercomonad flagellate Cercomonas: Phylogenetic trees including these new actin genes confirm the recently proposed relationship between chlorarachniophytes and cercomonads (Cercozoa) and, more importantly, also show a close relationship between Cercozoa and Foraminifera. Both of these are major eukaryotic groups encompassing extremely diverse organisms, yet there is no strong evidence for the evolutionary position of either from morphological or molecular data. The union of Cercozoa and Foraminifera suggested by actin phylogeny represents a novel step in the long process of determining the broad relationships between all major eukaryotic groups.     

Evolution of Rhizaria: new insights from phylogenomic analysis of uncultivated protists

Background  

Recent phylogenomic analyses have revolutionized our view of eukaryote evolution by revealing unexpected relationships between and within the eukaryotic supergroups. However, for several groups of uncultivable protists, only the ribosomal RNA genes and a handful of proteins are available, often leading to unresolved evolutionary relationships. A striking example concerns the supergroup Rhizaria, which comprises several groups of uncultivable free-living protists such as radiolarians, foraminiferans and gromiids, as well as the parasitic plasmodiophorids and haplosporids. Thus far, the relationships within this supergroup have been inferred almost exclusively from rRNA, actin, and polyubiquitin genes, and remain poorly resolved. To address this, we have generated large Expressed Sequence Tag (EST) datasets for 5 species of Rhizaria belonging to 3 important groups: Acantharea (Astrolonche sp., Phyllostaurus sp.), Phytomyxea (Spongospora subterranea, Plasmodiophora brassicae) and Gromiida (Gromia sphaerica).     

New barcoded primers for efficient retrieval of cercozoan sequences in high‐throughput environmental diversity surveys,with emphasis on worldwide biological soil crusts

We describe the performance of a new metabarcoding approach to investigate the environmental diversity of a prominent group of widespread unicellular organisms, the Cercozoa. Cercozoa is an immensely large group of protists, and although it may dominate in soil and aquatic ecosystems, its environmental diversity remains undersampled. We designed PCR primers targeting the hypervariable region V4 of the small subunit ribosomal RNA (SSU or 18S) gene, which is the recommended barcode marker for Cercozoa. The length of the amplified fragment (c. 350 bp) is suitable for Illumina MiSeq, the most cost‐effective platform for molecular environmental surveys. We provide barcoded primers, an economical alternative to multiple libraries for multiplex sequencing of over a hundred samples. In silico, our primers matched 68% of the cercozoan sequences of the reference database and performed better than previously proposed new‐generation sequencing primers. In mountain grassland soils and in biological soil crusts from a variety of climatic regions, we were able to detect cercozoan sequences encompassing nearly the whole range of the phylum. We obtained 901 operational taxonomic units (OTUs) at 97% similarity threshold from 26 samples, with c. 50,000 sequences per site, and only 8% of noncercozoan sequences. We could report a further increase in the diversity of Cercozoa, as only 43% of the OTUs were 97%–100% similar to any known sequence. Our study thus provides an advanced tool for cercozoan metabarcoding and to investigate their diversity and distribution in the environment.     

Cercozoa comprises both EF-1α-containing and EFL-containing members

Elongation factor 1α (EF-1α) and elongation factor-like protein (EFL) are considered to be functionally equivalent proteins involved in peptide synthesis. Eukaryotes can be fundamentally divided into ‘EF-1α-containing’ and ‘EFL-containing’ types. Recently, EF-1α and EFL genes have been surveyed across the diversity of eukaryotes to explore the origin and evolution of EFL genes. Although the phylum Cercozoa is a diverse group, gene data for either EFL or EF-1α are absent from all cercozoans except chlorarachniophytes which were previously defined as EFL-containing members. Our survey revealed that two members of the cercozoan subphylum Filosa (Thaumatomastix sp. and strain YPF610) are EFL-containing members. Importantly, we identified EF-1α genes from two members of Filosa (Paracercomonas marina and Paulinella chromatophora) and a member of the other subphylum Endomyxa (Filoreta japonica). All cercozoan EFL homologues could not be recovered as a monophyletic group in maximum-likelihood and Bayesian analyses, suggesting that lateral gene transfer was involved in the EFL evolution in this protist assemblage. In contrast, EF-1α analysis successfully recovered a monophyly of three homologues sampled from the two cercozoan subphyla. Based on the results, we postulate that cercozoan EF-1α genes have been vertically inherited, and the current EFL-containing species may have secondarily lost their EF-1α genes.     

Morphology and molecular phylogeny of a marine interstitial tetraflagellate with putative endosymbionts: <Emphasis Type="Italic">Auranticordis quadriverberis</Emphasis> n. gen. et sp. (Cercozoa)

Background  

Comparative morphological studies and environmental sequencing surveys indicate that marine benthic environments contain a diverse assortment of microorganisms that are just beginning to be explored and characterized. The most conspicuous predatory flagellates in these habitats range from about 20–150 μm in size and fall into three major groups of eukaryotes that are very distantly related to one another: dinoflagellates, euglenids and cercozoans. The Cercozoa is a diverse group of amoeboflagellates that cluster together in molecular phylogenies inferred mainly from ribosomal gene sequences. These molecular phylogenetic studies have demonstrated that several enigmatic taxa, previously treated as Eukaryota insertae sedis, fall within the Cercozoa, and suggest that the actual diversity of this group is largely unknown. Improved knowledge of cercozoan diversity is expected to help resolve major branches in the tree of eukaryotes and demonstrate important cellular innovations for understanding eukaryote evolution.     

Radiolaria divided into Polycystina and Spasmaria in combined 18S and 28S rDNA phylogeny

Radiolarians are marine planktonic protists that belong to the eukaryote supergroup Rhizaria together with Foraminifera and Cercozoa. Radiolaria has traditionally been divided into four main groups based on morphological characters; i.e. Polycystina, Acantharia, Nassellaria and Phaeodaria. But recent 18S rDNA phylogenies have shown that Phaeodaria belongs within Cerocozoa, and that the previously heliozoan group Taxopodida should be included in Radiolaria. 18S rDNA phylogenies have not yet resolved the sister relationship between the main Radiolaria groups, but nevertheless suggests that Spumellaria, and thereby also Polycystina, are polyphyletic. Very few sequences other than 18S rDNA have so far been generated from radiolarian cells, mostly due to the fact that Radiolaria has been impossible to cultivate and single cell PCR has been hampered by low success rate. Here we have therefore investigated the mutual evolutionary relationship of the main radiolarian groups by using the novel approach of combining single cell whole genome amplification with targeted PCR amplification of the 18S and 28S rDNA genes. Combined 18S and 28S phylogeny of sequences obtained from single cells shows that Radiolaria is divided into two main lineages: Polycystina (Spumellaria+Nassellaria) and Spasmaria (Acantharia+Taxopodida). Further we show with high support that Foraminifera groups within Radiolaria supporting the Retaria hypothesis.     

Molecular Biology of <Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis>

Initially, molecular techniques were used to detect and distinguish Plasmodiophora pathotypes in soil. Meanwhile, chromosomes from 2.2 Mb to 680 kb are characterized and the total genome size is estimated to be approximately 20 Mb. Furthermore, the genomic gene structure and the cDNA structure of several genes have been revealed, and the expression of those genes has been linked to development of clubroot to some extent. In addition, the sequence data have reinforced the inclusion of the plasmodiophorids within the Cercozoa. The recent successes in molecular biology have produced new approaches for clubroot research.     

甘蓝夜蛾和八字地老虎肌动蛋白基因的克隆与序列分析

肌动蛋白是细胞骨架微丝的主要组成成分,在肌肉收缩、细胞骨架形成、细胞移动等方面起重要作用。以鳞翅目夜蛾科昆虫甘蓝夜蛾Mamestra brassicae L.和八字地老虎Agrotis c-nigrum 3龄幼虫整个虫体为材料提取总RNA,利用RT-PCR和cDNA末端快速扩增技术(RACE),分别扩增得到2种昆虫的肌动蛋白的cDNA序列,甘蓝夜蛾肌动蛋白的cDNA序列含有1441个碱基,而八字地老虎肌动蛋白的cDNA序列含有1411个碱基。2种昆虫的该基因的cDNA序列均包括1个1131个碱基的开放阅读框,编码1个含376个氨基酸的蛋白。甘蓝夜蛾肌动蛋白分子量约为41.8kDa;八字地老虎肌动蛋白分子量约为41.9kDa。Prosite软件分析结果表明,甘蓝夜蛾和八字地老虎肌动蛋白氨基酸序列中存在3个肌动蛋白特征片段。GenBank数据库搜索及序列比对结果表明,甘蓝夜蛾肌动蛋白属于肌肉特异型肌动蛋白,八字地老虎肌动蛋白属于细胞质特异型肌动蛋白。2个基因的cDNA序列已经登录GenBank并获得登录号,甘蓝夜蛾肌动蛋白cDNA序列登录号为EU035314,八字地老虎肌动蛋白cDNA序列登录号为EU035315。利用RT-PCR技术在八字地老虎4龄、5龄、6龄幼虫、蛹期4个不同发育阶段和6龄期的肠道、体壁、脂肪体3种不同组织中都检测到了肌动蛋白基因在mRNA水平的表达。     

菜豆多聚泛肽基因在重金属胁迫下的表达

差别筛选HgCl2胁迫的菜豆(PhaseolusvulgarisL.)幼苗叶片cDNA库,分离出两个重金属胁迫相应基因PvSR5和PvSR51(Phaseolusvulgarisstress_relatedgene)片段。cDNA和氨基酸序列分析表明PvSR5和PvSR51分别编码一种多聚泛肽。Northernblot分析表明多聚泛肽是组成性表达蛋白,主要在根中表达,叶片和茎中表达较少;Hg、Cd、Cu和Zn等重金属、高温和水杨酸能强烈地刺激其在叶片中的表达,而受伤几乎没有影响。推测多聚泛肽在抵抗重金属胁迫和提高植物的抗逆性方面有重要作用。     

Evolution of human cytoplasmic actin gene sequences: chromosome mapping and structural characterizations of three cytoplasmic actin-like pseudogenes including one on the Y chromosome

Eight recombinant phage clones containing cytoplasmic actin-like gene sequences have been isolated from a human genomic library for structural characterization. Kpn I family repeat sequences flank six of these actin genes isolated, and Alu family repeats are scattered throughout the DNA inserts of all eight phage clones. Three of these genes are γ actin-like, and the other five are β actin-like. The complete nucleotide sequence analysis of one β and one γ actin-like genes and their flanking regions demonstrates that they both are processed pseudogenes. Using unique DNA sequences flanking these two pseudogenes as hybridization probes for human-mouse somatic cell hybrid DNAs, we have mapped the two actin pseudogenes on human chromosomes 8 and 3, respectively. We have also determined the DNA sequence of a human Y chromosome-linked, processed actin pseudogene. The different values of sequence divergence of these processed pseudogenes and their functional counterparts allow us to estimate the time of generation of the pseudogenes. The results suggest that the cDNA insertion events generating the human cytoplasmic actin-like pseudogenes have occurred at significantly different times during the evolution of primates, after their separation from other mammalian species.     

Global eukaryote phylogeny: Combined small- and large-subunit ribosomal DNA trees support monophyly of Rhizaria, Retaria and Excavata

Resolution of the phylogenetic relationships among the major eukaryotic groups is one of the most important problems in evolutionary biology that is still only partially solved. This task was initially addressed using a single marker, the small-subunit ribosomal DNA (SSU rDNA), although in recent years it has been shown that it does not contain enough phylogenetic information to robustly resolve global eukaryotic phylogeny. This has prompted the use of multi-gene analyses, especially in the form of long concatenations of numerous conserved protein sequences. However, this approach is severely limited by the small number of taxa for which such a large number of protein sequences is available today. We have explored the alternative approach of using only two markers but a large taxonomic sampling, by analysing a combination of SSU and large-subunit (LSU) rDNA sequences. This strategy allows also the incorporation of sequences from non-cultivated protists, e.g., Radiozoa (=radiolaria minus Phaeodarea). We provide the first LSU rRNA sequences for Heliozoa, Apusozoa (both Apusomonadida and Ancyromonadida), Cercozoa and Radiozoa. Our Bayesian and maximum likelihood analyses for 91 eukaryotic combined SSU+LSU sequences yielded much stronger support than hitherto for the supergroup Rhizaria (Cercozoa plus Radiozoa plus Foraminifera) and several well-recognised groups and also for other problematic clades, such as the Retaria (Radiozoa plus Foraminifera) and, with more moderate support, the Excavata. Within opisthokonts, the combined tree strongly confirms that the filose amoebae Nuclearia are sisters to Fungi whereas other Choanozoa are sisters to animals. The position of some bikont taxa, notably Heliozoa and Apusozoa, remains unresolved. However, our combined trees suggest a more deeply diverging position for Ancyromonas, and perhaps also Apusomonas, than for other bikonts, suggesting that apusozoan zooflagellates may be central for understanding the early evolution of this huge eukaryotic group. Multiple protein sequences will be needed fully to resolve basal bikont phylogeny. Nonetheless, our results suggest that combined SSU+LSU rDNA phylogenies can help to resolve several ambiguous regions of the eukaryotic tree and identify key taxa for subsequent multi-gene analyses.     

Higher-level phylogeny of Foraminifera inferred from the RNA polymerase II (RPB1) gene

Macroevolutionary relations among main lineages of Foraminifera have traditionally been inferred from the small subunit ribosomal genes (SSU rDNA). However, important discrepancies in the rates of SSU rDNA evolution between major lineages led to difficulties in accurate interpretation of SSU-based phylogenetic reconstructions. Recently, actin and beta-tubulin sequences have been used as alternative markers of foraminiferal phylogeny and their analyses globally confirm results obtained with SSU rDNA. In order to test new protein markers, we sequenced a fragment of the largest subunit of the RNA polymerase II (RPB1), a nuclear encoded single copy gene, for 8 foraminiferal species representing major orders of Foraminifera. Analyses of our data robustly confirm previous SSU rDNA and actin phylogenies and show (i) the paraphyly and ancestral position of monothalamid Foraminifera; (ii) the independent origin of miliolids; (iii) the monophyly of rotaliids, including buliminids and globigerinids; and (iv) the polyphyly of planktonic families Globigerinidae and Candeinidae. Additionally, the RPB1 phylogeny suggests Allogromiidae as the most ancestral foraminiferal lineage. In the light of our study, RPB1 appears as a valuable phylogenetic marker, particularly useful for groups of protists showing extreme variations of evolutionary rates in ribosomal genes.