The introduction of plastids into different heterotrophic protists created lineages of algae that diversified explosively, proliferated in marine and freshwater environments, and radically altered the biosphere. The origins of these secondary plastids are usually inferred from the presence of additional plastid membranes. However, two examples provide unique snapshots of secondaryendosymbiosis-in-action, because they retain a vestige of the endosymbiont nucleus known as the nucleomorph. These are chlorarachniophytes and cryptomonads, which acquired their plastids from a green and red alga respectively. To allow comparisons between them, we have sequenced the nucleomorph genome from the chlorarachniophyte Bigelowiella natans: at a mere 373,000 bp and with only 331 genes, the smallest nuclear genome known and a model for extreme reduction. The genome is eukaryotic in nature, with three linear chromosomes containing densely packed genes with numerous overlaps. The genome is replete with 852 introns, but these are the smallest introns known, being only 18, 19, 20, or 21 nt in length. These pygmy introns are shown to be miniaturized versions of normal-sized introns present in the endosymbiont at the time of capture. Seventeen nucleomorph genes encode proteins that function in the plastid. The other nucleomorph genes are housekeeping entities, presumably underpinning maintenance and expression of these plastid proteins. Chlorarachniophyte plastids are thus serviced by three different genomes (plastid, nucleomorph, and host nucleus) requiring remarkable coordination and targeting. Although originating by two independent endosymbioses, chlorarachniophyte and cryptomonad nucleomorph genomes have converged upon remarkably similar architectures but differ in many molecular details that reflect two distinct trajectories to hypercompaction and reduction.plastid ͉ secondary endosymbiosis ͉ intron ͉ endosymbiosis T he origin of plastids through endosymbiosis of a cyanobacterium-like prokaryote transferred photosynthesis into eukaryotes and launched a massive wave of diversification that subsequently generated a tremendous range of algae and plants (1). This initial event is referred to as primary endosymbiosis ( Fig. 1) and created a plastid with two membranes such as those of green algae, plants, red algae, and glaucophyte algae (1). Transfer of genes from the endosymbiont to the nuclear genome of the host initially led to dependence of the endosymbiont on the host that was necessary to stabilize the partnership (2). Ongoing transfer has resulted in reduction of the prokaryotic genome, so that plastid DNA now represents probably Ͻ10% of its original gene content, and increasingly sophisticated regulation of the endosymbiont by the host has resulted in endosymbiont replication, gene expression, metabolic activity, and even death being managed by the eukaryotic host (3). Indeed, primary plastids seem to retain some autonomy only in the synthesis and deployment of redox proteins involved in photosynthetic electron transfer (4).A...

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The plastid (apicoplast) of the malaria-causing parasite Plasmodium falciparum was derived via a secondary endosymbiotic process. As in other secondary endosymbionts, numerous genes for apicoplast proteins are located in the nucleus, and the encoded proteins are targeted to the organelle courtesy of a bipartite N-terminal extension. The first part of this leader sequence is a signal peptide that targets proteins to the secretory pathway. The second, so-called transit peptide region is required to direct proteins from the secretory pathway across the multiple membranes surrounding the apicoplast. In this paper we perform a pulse-chase experiment and N-terminal sequencing to show that the transit peptide of an apicoplast-targeted protein is cleaved, presumably upon import of the protein into the apicoplast. We identify a gene whose product likely performs this cleavage reaction, namely a stromal-processing peptidase (SPP) homologue. In plants SPP cleaves the transit peptides of plastid-targeted proteins. The P. falciparum SPP homologue contains a bipartite N-terminal apicoplast-targeting leader. Interestingly, it shares this leader sequence with a ⌬-aminolevulinic acid dehydratase homologue via an alternative splicing event.Plasmodium spp., the causative agents of malaria, belong to a family of intracellular parasites called the Apicomplexa. Plasmodium infects approximately 300 million people annually, causing over 1 million deaths, the great majority of which are caused by one species, Plasmodium falciparum (1). P. falciparum infects both humans and mosquitoes during its life cycle, with the pathogenic part of this cycle occurring predominantly in the erythrocytes of humans. The discovery of a non-photosynthetic plastid (the apicoplast) in the Apicomplexa has opened up a new area of anti-malarial drug targets (2, 3). However, the rational development of drugs targeting the apicoplast requires knowledge of apicoplast function. Preliminary studies indicate that the apicoplast is a site of fatty acid and isoprenoid biosynthesis (4 -6), and drugs targeting these pathways have been shown to kill P. falciparum (4, 6, 7).Like plant plastids, the apicoplast contains a reduced bacterial-like genome (8), from which a small number of proteins are expressed. The great majority of apicoplast proteins, as in plant plastids, are encoded in the nucleus and must be post-translationally targeted to the plastid. In plants, nuclear-encoded plastid proteins require a cleavable, N-terminal sequence called the transit peptide, which directs these proteins across the two membranes surrounding plant plastids (for reviews, see Refs. 9 and 10). Once in the plastid stroma, this transit peptide is cleaved by a stromal-processing peptidase (SPP 1 ; Refs. 11 and 12). Apicoplasts, however, are bound by four membranes (Ref. 2, but see Ref. 13), and proteins targeted to this organelle have been shown to require a bipartite N-terminal leader sequence (5,14,15). By fusing these N-terminal leader sequences to green fluorescent reporter protein (GFP)...

Chlorarachniophytes are amoeboflagellate cercozoans that acquired a plastid by secondary endosymbiosis. Chlorarachniophytes are the last major group of algae for which there is no completely sequenced plastid genome. Here we describe the 69.2-kbp chloroplast genome of the model chlorarachniophyte Bigelowiella natans. The genome is highly reduced in size compared with plastids of other photosynthetic algae and is closer in size to genomes of several nonphotosynthetic plastids. Unlike nonphotosynthetic plastids, however, the B. natans chloroplast genome has not sustained a massive loss of genes, and it retains nearly all of the functional photosynthesis-related genes represented in the genomes of other green algae. Instead, the genome is highly compacted and gene dense. The genes are organized with a strong strand bias, and several unusual rearrangements and inversions also characterize the genome; notably, an inversion in the small-subunit rRNA gene, a translocation of 3 genes in the major ribosomal protein operon, and the fragmentation of the cluster encoding the large photosystem proteins PsaA and PsaB. The chloroplast endosymbiont is known to be a green alga, but its evolutionary origin and relationship to other primary and secondary green plastids has been much debated. A recent hypothesis proposes that the endosymbionts of chlorarachniophytes and euglenids share a common origin (the Cabozoa hypothesis). We inferred phylogenies using individual and concatenated gene sequences for all genes in the genome. Concatenated gene phylogenies show a relationship between the B. natans plastid and the ulvophyte-trebouxiophyte-chlorophyte clade of green algae to the exclusion of Euglena. The B. natans plastid is thus not closely related to that of Euglena, which suggests that plastids originated independently in these 2 groups and the Cabozoa hypothesis is false.

Self-incompatibility (SI) is a genetic mechanism that restricts inbreeding in flowering plants. In the nightshade family (Solanaceae) SI is controlled by a single multiallelic S locus. Pollen rejection in this system requires the interaction of two S locus products: a stylar (S)-RNase and its pollen counterpart (pollen S). pollen S has not yet been cloned. Our understanding of how this gene functions comes from studies of plants with mutations that affect the pollen but not the stylar SI response (pollen-part mutations). These mutations are frequently associated with duplicated S alleles, but the absence of an obvious additional allele in some plants suggests pollen S can also be deleted. We studied Nicotiana alata plants with an additional S allele and show that duplication causes a pollen-part mutation in several different genetic backgrounds. Inheritance of the duplication was consistent with a competitive interaction model in which any two nonmatching S alleles cause a breakdown of SI when present in the same pollen grain. We also examined plants with presumed deletions of pollen S and found that they instead have duplications that included pollen S but not the S-RNase gene. This finding is consistent with a bipartite structure for the S locus. The absence of pollen S deletions in this study and perhaps other studies suggests that pollen S might be required for pollen viability, possibly because its product acts as an S-RNase inhibitor.S elf-incompatibility (SI) in many plant families is controlled by a multiallelic S locus that enables a style to reject any pollen expressing the same S allelic specificity as itself (1). In the Solanaceae, the family that includes tobacco, tomato, and petunia, SI is described as gametophytic because the allelic specificity of each pollen grain is determined by its own haploid genotype. The S locus in this family encodes a secreted extracellular RNase [stylar (S)-RNase] that accumulates in the style (2). Recognizing which S allele each pollen grain expresses is thought to require an interaction between the S-RNase and an unknown product(s) of a second S locus gene called pollen S (3, 4).As part of a strategy to identify pollen S, we isolated Nicotiana alata plants with gamma ray induced mutations that specifically affect the SI phenotype of pollen but not the SI phenotype of the style (5). Such plants are called pollen-part mutants (PPMs). Because ionizing radiation can cause either the deletion of part of a chromosome or chromosomal aberrations such as translocations, inversions, and fragments (6), the mutations in PPMs are likely to be complex because they can arise through one of a few different types of lesion.Among the PPMs described so far, the most frequent types of lesion are either translocations or small ''centric'' fragments (short extra chromosomes) that carry a duplicated copy of an S allele (5, 7-10). Breakdown of the pollen SI response in these plants occurs because of a ''competitive interaction'' that enables pollen with two different S alleles (but not two i...

It has long been held that the malaria parasite, Plasmodium sp., is incapable of de novo fatty acid synthesis. This view has recently been overturned with the emergence of data for the presence of a fatty acid biosynthetic pathway in the relict plastid of P. falciparum (known as the apicoplast). This pathway represents the type II pathway common to plant chloroplasts and bacteria but distinct from the type I pathway of animals including humans. Specific inhibitors of the type II pathway, thiolactomycin and triclosan, have been reported to target this Plasmodium pathway. Here we report further inhibitors of the plastid-based pathway that inhibit Plasmodium parasites. These include several analogues of thiolactomycin, two with sixfold-greater efficacy than thiolactomycin. We also report that parasites respond very rapidly to such inhibitors and that the greatest sensitivity is seen in ring-stage parasites. This study substantiates the importance of fatty acid synthesis for blood-stage parasite survival and shows that this pathway provides scope for the development of novel antimalarial drugs.

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Cerebral malaria is a major health problem in the developing world. Widespread resistance to existing drugs by the parasite Plasmodium falciparum has coincided with an increase in mortality, particularly in children. One potential source of new drugs comes from plant natural products. We found that commercially available, pharmaceutical grade eucalyptus oil and its principal component 1,8-cineole inhibited the growth and development of chloroquine-sensitive and chloroquine-resistant P. falciparum. This was true both when the oil was added directly to the parasite cultures and when cultures were exposed to the vapours. The development of the parasite was arrested at the early trophozoite stage, irrespective of when the oil was introduced. We used a new approach where the concentration of monoterpenes actually taken up by the cultures was measured directly using HS-GC. We found that the critical concentration required to inhibit and kill the parasite did not adversely affect the host erythrocytes, placing it in the range suitable for drug development. Given the ready availability and existing quality control of eucalyptus oils, this may represent an economically viable adjunct to current antimalarial therapies. Figure 1. Changes in P. falciparum after exposure to eucalyptus oil vapour (IC 90 ). Cultures were synchronized at the ring stage and observed over 96 h (two life cycles). (a) Control, without eucalyptus oil.(b) Culture exposed to eucalyptus oil for 96 h. (c) Culture exposed to eucalyptus oil for 48 h followed by 48 h recovery. Graphs represent the percentage of parasites at a certain life stage as a proportion of the total number of parasites, measured every 24 h Figure 2. Confocal microscopy images of live P. falciparum cells exposed to eucalyptus oil vapour (IC 90 ). Cells were synchronized at the ring stage and followed over 96 h (two life cycles). Cells were CS(l)YFP/ACP(l)DsRed double transfectants, co-labelled with the nuclear dye Hoechst 33 258. 16 Scale bar = 2 μm. (a) Control culture not exposed to eucalyptus oil, at 30 h: late trophozoite stage. (b) Culture exposed to eucalyptus oil at 30 h: stalled at the early trophozoite stage. (c) Control culture, now at 72 h: mid-schizont stage. (d) Exposed culture, now at 72 h: still stalled at the early trophozoite stage