The polyketide synthase StlA is involved in inducing aggregation in Polysphondylium violaceum.

In the social amoeba Dictyostelium discoideum, the polyketide MPBD (4-methyl-5-pentylbenzene-1,3-diol) regulates the gene expressions of cAMP signaling to make cells aggregation-competent and also induces spore maturation. The polyketide synthase StlA is responsible for MPBD biosynthesis in D. discoideum and appears to be conserved throughout the major groups of the social amoeba (Dictyostelia). In this study, we analyzed the function of StlA in Polysphondylium violaceum by identifying the gene sequence and creating the knockout mutants. We found that Pv-stlA- mutants had defects only in cell aggregation but not in spore maturation, indicating that the function of StlA in inducing spore maturation is species-specific. We also found that MPBD could rescue the aggregation defect in Pv-stlA- mutants whereas the mutants normally exhibited chemotaxis to their chemoattractant, glorin. Our data suggest that StlA is involved in inducing aggregation in P. violaceum by acting on signaling pathways other than chemotaxis in P. violaceum.

Adenylate cyclase A acting on PKA mediates induction of stalk formation by cyclic diguanylate at the Dictyostelium organizer.

Coordination of cell movement with cell differentiation is a major feat of embryonic development. The Dictyostelium stalk always forms at the organizing tip, by a mechanism that is not understood. We previously reported that cyclic diguanylate (c-di-GMP), synthesized by diguanylate cyclase A (DgcA), induces stalk formation. Here we used transcriptional profiling of dgca- structures to identify target genes for c-di-GMP, and used these genes to investigate the c-di-GMP signal transduction pathway. We found that knockdown of cAMP-dependent protein kinase (PKA) activity in prestalk cells reduced stalk gene induction by c-di-GMP, whereas PKA activation bypassed the c-di-GMP requirement for stalk gene expression. c-di-GMP caused a persistent increase in cAMP, which still occurred in mutants lacking the adenylate cyclases ACG or ACR, or the cAMP phosphodiesterase RegA. However, both inhibition of adenylate cyclase A (ACA) with SQ22536 and incubation of a temperature-sensitive ACA mutant at the restrictive temperature prevented c-di-GMP-induced cAMP synthesis as well as c-di-GMP-induced stalk gene transcription. ACA produces the cAMP pulses that coordinate Dictyostelium morphogenetic cell movement and is highly expressed at the organizing tip. The stalk-less dgca- mutant regained its stalk by expression of a light-activated adenylate cyclase from the ACA promoter and exposure to light, indicating that cAMP is also the intermediate for c-di-GMP in vivo. Our data show that the more widely expressed DgcA activates tip-expressed ACA, which then acts on PKA to induce stalk genes. These results explain why stalk formation in Dictyostelia always initiates at the site of the morphogenetic organizer.

A MADS-box transcription factor regulates a central step in sporulation of the oomycete Phytophthora infestans.

MADS-box transcription factors play significant roles in eukaryotes, but have not yet been characterized in oomycetes. Here, we describe a MADS-box protein from Phytophthora infestans, which causes late blight of potato. P. infestans and most other oomycetes express a single MADS-box gene. PiMADS is not transcribed during vegetative growth, but is induced early during asexual sporulation. Its mRNA levels oscillate in response to light, which suppresses sporulation. The protein was not detected in nonsporulating mycelia, but was found in sporulating mycelia and spores. Both mRNA and protein levels decline upon spore germination. A similar expression pattern as well as nuclear localization was observed when the protein was expressed with a fluorescent tag from the native promoter. Gene silencing triggered by a construct expressing 478 nt of MADS sequences indicated that PiMADS is required for sporulation but not hyphal growth or plant colonization. A comparison of wild type to a silenced strain by RNA-seq indicated that PiMADS regulates about 3000 sporulation-associated genes, and acts before other genes previously shown to regulate sporulation. Analysis of the silenced strain also indicated that the native gene was not transcribed while the transgene was still expressed, which contradicts current models for homology-dependent silencing in oomycetes.

4-Methyl-5-Pentylbenzene-1,3-Diol Regulates Chemotactic Cell Aggregation and Spore Maturation Via Different Mechanisms in Dictyostelium discoideum.

4-Methyl-5-pentylbenzene-1,3-diol (MPBD), a product of the polyketide synthase SteelyA, is a signaling molecule that regulates Dictyostelium discoideum development. During early development, MPBD controls chemotactic cell aggregation by regulating the expression of genes in the cAMP signaling pathway; however, during culmination at late development, it induces spore maturation. In the present study, we analyzed the effects of MPBD, its derivatives, and a putative MPBD-derived metabolite on developmental defects in the MPBD-less stlA null mutant. Using structure-activity relationship studies, it was observed that in MPBD, the functional groups that were essential for induction of spore maturation were different from those essential for induction of cell aggregation. Dictyoquinone, a putative MPBD metabolite rescued the aggregation defect in stlA null mutant in early development, but not the spore maturation defect at the later stage. Our data suggest that MPBD regulates chemotactic cell aggregation and spore maturation via different mechanisms.

Real-Time PCR and LAMP Assays for the Detection of Spores of Alternaria solani and Sporangia of Phytophthora infestans to Inform Disease Risk Forecasting.

Real-time loop-mediated isothermal amplification (LAMP) assays for the detection of sporangia of the causal pathogen of late blight, Phytophthora infestans, and spores of the main causal pathogen of early blight, Alternaria solani, were developed to facilitate the in-field detection of airborne inoculum to improve disease forecasting. These assays were compared with an existing real-time PCR assay for P. infestans and a newly developed real-time PCR assay for A. solani. Primers were designed for real-time LAMP of P. infestans and A. solani. The specificity of the P. infestans real-time LAMP assay was similar to that of an existing real-time PCR assay: DNA of P. infestans was consistently amplified as was DNA of the taxonomically closely related species Phytophthora mirabilis, Phytophthora phaseoli, and Phytophthora ipomoea; no amplification of DNA from the potato pathogens Phytophthora erythroseptica or Phytophthora nicotianae occurred. Real-time LAMP and PCR assays were developed for A. solani, and the specificity was compared with an existing conventional PCR assay. Importantly, the A. solani real-time LAMP and PCR assays did not amplify the species Alternaria alternata. However, cross-reactivity with Alternaria dauci was observed with the real-time PCR assay and Alternaria brassicae with the real-time LAMP assay. The sensitivity of all assays for the detection of DNA extracted from sporangia/spores of the target pathogens was evaluated. The P. infestans real-time LAMP assay reliably detected 5 pg of DNA, equivalent to ∼1 sporangia per reaction. By comparison, 20 fg of DNA was detectable with the existing real-time PCR assay. In the case of A. solani, real-time LAMP detected 4.4 pg of DNA, equivalent to ∼1 spore per reaction, and real-time PCR detected 200 fg of DNA. In-field air samplers were deployed in two trial plots planted with potato: one infected with P. infestans, and the other infected with A. solani. Four additional samplers were located in commercial potato fields. Air samples were taken through the season, and detection of airborne inoculum of P. infestans and A. solani with both real-time PCR and LAMP was assessed.

Proto-oncogenes in a eukaryotic unicellular organism play essential roles in plasmodial growth in host cells.

BACKGROUND: The eukaryotic unicellular protist Plasmodiophora brassicae is an endocellular parasite of cruciferous plants. In host cortical cells, this protist develops a unicellular structure that is termed the plasmodium. The plasmodium is actually a multinucleated cell, which subsequently splits and forms resting spores. The mechanism for the growth of this endocellular parasite in host cell is unclear. RESULTS: Here, combining de novo genome sequence and transcriptome analysis of strain ZJ-1, we identified top five significant enriched KEGG pathways of differentially expressed genes (DEGs), namely translation, cell growth and death, cell communication, cell motility and cancers. We detected 171 proto-oncogenes from the genome of P. brassicae that were implicated in cancer-related pathways, of which 46 were differential expression genes. Three predicted proto-oncogenes (Pb-Raf1, Pb-Raf2, and Pb-MYB), which showed homology to the human proto-oncogenes Raf and MYB, were specifically activated during the plasmodial growth in host cortical cells, demonstrating their involvement in the multinucleate development stage of the unicellular protist organism. Gene networks involved in the tumorigenic-related signaling transduction pathways and the activation of 12 core genes were identified. Inhibition of phosphoinositol-3-kinase relieved the clubroot symptom and significantly suppressed the development process of plasmodia. CONCLUSIONS: Proto-oncogene-related regulatory mechanisms play an important role in the plasmodial growth of P. brassicae.

Encystation: the most prevalent and underinvestigated differentiation pathway of eukaryotes.

Not long ago, protists were considered one of four eukaryote kingdoms, but recent gene-based phylogenies show that they contribute to all nine eukaryote subdomains. The former kingdoms of animals, plants and fungi are now relegated to lower ranks within subdomains. Most unicellular protists respond to adverse conditions by differentiating into dormant walled cysts. As cysts, they survive long periods of starvation, drought and other environmental threats, only to re-emerge when conditions improve. For protists pathogens, the resilience of their cysts can prevent successful treatment or eradication of the disease. In this context, effort has been directed towards understanding the molecular mechanisms that control encystation. We here firstly summarize the prevalence of encystation across protists and next focus on Amoebozoa, where most of the health-related issues occur. We review current data on processes and genes involved in encystation of the obligate parasite Entamoeba histolytica and the opportunistic pathogen Acanthamoeba. We show how the cAMP-mediated signalling pathway that controls spore and stalk cell encapsulation in Dictyostelium fruiting bodies could be retraced to a stress-induced pathway controlling encystation in solitary Amoebozoa. We highlight the conservation and prevalence of cAMP signalling genes in Amoebozoan genomes and the suprisingly large and varied repertoire of proteins for sensing and processing environmental signals in individual species.

The immunophilin repertoire of Plasmodiophora brassicae and functional analysis of PbCYP3 cyclophilin.

Plasmodiophora brassicae is a soil-borne pathogen that belongs to Rhizaria, an almost unexplored eukaryotic organism group. This pathogen requires a living host for growth and multiplication, which makes molecular analysis further complicated. To broaden our understanding of a plasmodiophorid such as P. brassicae, we here chose to study immunophilins, a group of proteins known to have various cellular functions, including involvement in plant defense and pathogen virulence. Searches in the P. brassicae genome resulted in 20 putative immunophilins comprising of 11 cyclophilins (CYPs), 7 FK506-binding proteins (FKBPs) and 2 parvulin-like proteins. RNAseq data showed that immunophilins were differentially regulated in enriched life stages such as germinating spores, maturing spores, and plasmodia, and infected Brassica hosts (B. rapa, B. napus and B. oleracea). PbCYP3 was highly induced in all studied life stages and during infection of all three Brassica hosts, and hence was selected for further analysis. PbCYP3 was heterologously expressed in Magnaporthe oryzae gene-inactivated ΔCyp1 strain. The new strain ΔCyp1+ overexpressing PbCYP3 showed increased virulence on rice compared to the ΔCyp1 strain. These results suggest that the predicted immunophilins and particularly PbCYP3 are activated during plant infection. M. oryzae is a well-studied fungal pathogen and could be a valuable tool for future functional studies of P. brassicae genes, particularly elucidating their role during various infection phases.

Characterization and expression analysis of a new small heat shock protein Hsp20.4 from Eimeria tenella.

Small heat shock proteins (sHsps) are ubiquitous and diverse molecular chaperones. Found in almost all organisms, they regulate protein refolding and protect cells from stress. Until now, no sHsp has been characterized in Eimeria tenella. In this study, the novel EtsHsp20.4 gene was cloned from E. tenella by rapid amplification of cDNA ends based on a previously identified expressed sequence tag. The full-length cDNA was 1019bp in length and contained an open reading frame of 558bp that encoded a 185-amino acid polypeptide with a calculated molecular weight of 20.4 kDa. The EtsHsp20.4 protein contained a distinct HSP20/alpha-crystallin domain that is the key determinant of their function as molecular chaperones and belongs to the HSP20 protein family. EtsHsp20.4 mRNA levels were higher in sporulated oocysts than in sporozoites or second-generation merozoites by real-time quantitative PCR, the transcription of EtsHsp20.4 was barely detectable in unsporulated oocysts. Immunolocalization with EtsHsp20.4 antibody showed that EtsHsp20.4 was mainly located on the surface of sporozoites, first-generation merozoites and second-generation merozoites. Following the development of parasites in DF-1 cells, EtsHsp20.4 protein was uniformly dispersed in trophozoites, immature schizonts, and mature schizonts. Malate dehydrogenase thermal aggregation assays indicated that recombinant EtsHsp20.4 had molecular chaperone activity in vitro. These results suggested that EtsHsp20.4 might be involved in sporulation in external environments and intracellular growth of the parasite in the host.

Mapping the Distribution of Cysts from the Toxic Dinoflagellate Cochlodinium polykrikoides in Bloom-Prone Estuaries by a Novel Fluorescence In Situ Hybridization Assay.

Cochlodinium polykrikoides is a cosmopolitan dinoflagellate that is notorious for causing fish-killing harmful algal blooms (HABs) across North America and Asia. While recent laboratory and ecosystem studies have definitively demonstrated that Cochlodinium forms resting cysts that may play a key role in the dynamics of its HABs, uncertainties regarding cyst morphology and detection have prohibited even a rudimentary understanding of the distribution of C. polykrikoides cysts in coastal ecosystems. Here, we report on the development of a fluorescence in situ hybridization (FISH) assay using oligonucleotide probes specific for the large subunit (LSU) ribosomal DNA (rDNA) of C. polykrikoides. The LSU rDNA-targeted FISH assay was used with epifluorescence microscopy and was iteratively refined to maximize the fluorescent reaction with C. polykrikoides and minimize cross-reactivity. The final LSU rDNA-targeted FISH assay was found to quantitatively recover cysts made by North American isolates of C. polykrikoides but not cysts formed by other common cyst-forming dinoflagellates. The method was then applied to identify and map C. polykrikoides cysts across bloom-prone estuaries. Annual cyst and vegetative cell surveys revealed that elevated densities of C. polykrikoides cysts (>100 cm(-3)) during the spring of a given year were spatially consistent with regions of dense blooms the prior summer. The identity of cysts in sediments was confirmed via independent amplification of C. polykrikoides rDNA. This study mapped C. polykrikoides cysts in a natural marine setting and indicates that the excystment of cysts formed by this harmful alga may play a key role in the development of HABs of this species.

The Toxoplasma gondii cyst wall protein CST1 is critical for cyst wall integrity and promotes bradyzoite persistence.

Toxoplasma gondii infects up to one third of the world's population. A key to the success of T. gondii as a parasite is its ability to persist for the life of its host as bradyzoites within tissue cysts. The glycosylated cyst wall is the key structural feature that facilitates persistence and oral transmission of this parasite. Because most of the antibodies and reagents that recognize the cyst wall recognize carbohydrates, identification of the components of the cyst wall has been technically challenging. We have identified CST1 (TGME49_064660) as a 250 kDa SRS (SAG1 related sequence) domain protein with a large mucin-like domain. CST1 is responsible for the Dolichos biflorus Agglutinin (DBA) lectin binding characteristic of T. gondii cysts. Deletion of CST1 results in reduced cyst number and a fragile brain cyst phenotype characterized by a thinning and disruption of the underlying region of the cyst wall. These defects are reversed by complementation of CST1. Additional complementation experiments demonstrate that the CST1-mucin domain is necessary for the formation of a normal cyst wall structure, the ability of the cyst to resist mechanical stress, and binding of DBA to the cyst wall. RNA-seq transcriptome analysis demonstrated dysregulation of bradyzoite genes within the various cst1 mutants. These results indicate that CST1 functions as a key structural component that confers essential sturdiness to the T. gondii tissue cyst critical for persistence of bradyzoite forms.

Genetic crosses and complementation reveal essential functions for the Plasmodium stage-specific actin2 in sporogonic development.

Malaria parasites have two actin isoforms, ubiquitous actin1 and specialized actin2. Actin2 is essential for late male gametogenesis, prior to egress from the host erythrocyte. Here, we examined whether the two actins fulfil overlapping functions in Plasmodium berghei. Replacement of actin2 with actin1 resulted in partial complementation of the defects in male gametogenesis and, thus, viable ookinetes were formed, able to invade the midgut epithelium and develop into oocysts. However, these remained small and their DNA was undetectable at day 8 after infection. As a consequence sporogony did not occur, resulting in a complete block of parasite transmission. Furthermore, we show that expression of actin2 is tightly controlled in female stages. The actin2 transcript is translationally repressed in female gametocytes, but translated in female gametes. The protein persists until mature ookinetes; this expression is strictly dependent on the maternally derived expression. Genetic crosses revealed that actin2 functions at an early stage of ookinete formation and that parasites lacking actin2 are unable to undergo sporogony in the mosquito midgut. Our results provide insights into the specialized role of actin2 in Plasmodium development in the mosquito and suggest that the two actin isoforms have distinct biological functions.

Genomic insights into processes driving the infection of Alexandrium tamarense by the Parasitoid Amoebophrya sp.

The regulatory circuits during infection of dinoflagellates by their parasites are largely unknown on the molecular level. Here we provide molecular insights into these infection dynamics. Alexandrium tamarense is one of the most prominent harmful algal bloom dinoflagellates. Its pathogen, the dinoflagellate parasitoid Amoebophrya sp., has been observed to infect and control the blooms of this species. We generated a data set of transcripts from three time points (0, 6, and 96 h) during the infection of this parasite-host system. Assembly of all transcript data from the parasitoid (>900,000 reads/313 Mbp with 454/Roche next-generation sequencing [NGS]) yielded 14,455 contigs, to which we mapped the raw transcript reads of each time point of the infection cycle. We show that particular surface lectins are expressed at the beginning of the infection cycle which likely mediate the attachment to the host cell. In a later phase, signal transduction-related genes together with transmembrane transport and cytoskeleton proteins point to a high integration of processes involved in host recognition, adhesion, and invasion. At the final maturation stage, cell division- and proliferation-related genes were highly expressed, reflecting the fast cell growth and nuclear division of the parasitoid. Our molecular insights into dinoflagellate parasitoid interactions point to general mechanisms also known from other eukaryotic parasites, especially from the Alveolata. These similarities indicate the presence of fundamental processes of parasitoid infection that have remained stable throughout evolution within different phyla.

Didymium xerophilum, a new myxomycete from the tropical Andes.

A new species of Didymium (Myxomycetes), D. xerophilum, is described, and some details of its life cycle are provided. The new species was collected during studies of arid areas of Argentina and Peru. It can be distinguished by the persistent funnel-shaped invagination of the peridium, the top of which appears as a deep umbilicus in closed sporothecae, and the calcareous hypothallus shared among several sporocarps. This combination of characters, with a circumscissile dehiscence of the sporotheca and a cream stalk packed with rhombic lime crystals, is unknown in other described species. Morphology was examined with scanning electron microscopy and light microscopy, and micrographs of relevant details are included here. Phylogenetic analysis with 18S rDNA sequences of different species of Didymium supports the distinct identity of this new species. Some collections of this myxomycete were made at up to 4600 m, an altitude almost unknown for this group of microorganisms.

Temporal and non-permanent division of labor during sorocarp formation in the social amoeba Acytostelium subglobosum.

Somatic cell differentiation is crucial for the development of multicellular organisms. While the development of a fruiting body in Dictyostelium discoideum represents a simple model of this process with separation of stalk cells from the spore lineage, that of Acytostelium subglobosum is not accompanied by cell type separation. This species produces acellular stalks and, seemingly, all aggregated amoebae become spores; however, it possesses homologs for the stalk-cell marker genes of D. discoideum. In this study, we analyzed the spatio-temporal expression of A. subglobosum orthologs for D. discoideum stalk- or spore-lineage markers to clarify the developmental process of A. subglobosum. We first found that the prespore vesicles, which contained spore coat proteins, started to accumulate in the tip region and were observed in the entire sorogen throughout later development, confirming that all A. subglobosum cells became spores. The expression of a stalk-lineage gene ortholog, As-ecmA, started at the mound stage and was prominent in the protruding sorogen. Although two spore-lineage gene orthologs, As-cotD1 and -cotD2, were likewise detected shortly after cell aggregation and increased in intensity until tip formation, their expression diminished in the protruding sorogen. Double-fluorescence staining of these prestalk and prespore marker genes revealed that the expression of these marker genes was mutually exclusive and that expression switching occurred in the early tip. Our results indicate that A. subglobosum cells become committed to the spore lineage first, and then, while keeping this commitment intact, participate in stalk formation. Instead of the permanent division of labor observed in D. discoideum, A. subglobosum produces fruiting bodies by all cells contributing to the formation of the stalk as well as forming spores.

A rare combination of ribonucleotide reductases in the social amoeba Dictyostelium discoideum.

Ribonucleotide reductases (RNRs) catalyze the only pathway for de novo synthesis of deoxyribonucleotides needed for DNA replication and repair. The vast majority of eukaryotes encodes only a class I RNR, but interestingly some eukaryotes, including the social amoeba Dictyostelium discoideum, encode both a class I and a class II RNR. The amino acid sequence of the D. discoideum class I RNR is similar to other eukaryotic RNRs, whereas that of its class II RNR is most similar to the monomeric class II RNRs found in Lactobacillus spp. and a few other bacteria. Here we report the first study of RNRs in a eukaryotic organism that encodes class I and class II RNRs. Both classes of RNR genes were expressed in D. discoideum cells, although the class I transcripts were more abundant and strongly enriched during mid-development compared with the class II transcript. The quaternary structure, allosteric regulation, and properties of the diiron-oxo/radical cofactor of D. discoideum class I RNR are similar to those of the mammalian RNRs. Inhibition of D. discoideum class I RNR by hydroxyurea resulted in a 90% reduction in spore formation and decreased the germination viability of the surviving spores by 75%. Class II RNR could not compensate for class I inhibition during development, and an excess of vitamin B12 coenzyme, which is essential for class II activity, did not improve spore formation. We suggest that class I is the principal RNR during D. discoideum development and growth and is important for spore formation, possibly by providing dNTPs for mitochondrial replication.

The Toxoplasma nuclear factor TgAP2XI-4 controls bradyzoite gene expression and cyst formation.

Toxoplasma gondii undergoes many phenotypic changes during its life cycle. The recent identification of AP2 transcription factors in T. gondii has provided a platform for studying the mechanisms controlling gene expression. In the present study, we report that a recombinant protein encompassing the TgAP2XI-4 AP2 domain was able to specifically bind to a DNA motif using gel retardation assays. TgAP2XI-4 protein is localized in the parasite nucleus throughout the tachyzoite life cycle in vitro, with peak expression occurring after cytokinesis. We found that the TgAP2XI-4 transcript level was higher in bradyzoite cysts isolated from brains of chronically infected mice than in the rapidly replicating tachyzoites. A knockout of the TgAP2XI-4 gene in both T. gondii virulent type I and avirulent type II strains reveals its role in modulating expression and promoter activity of genes involved in stage conversion of the rapidly replicating tachyzoites to the dormant cyst forming bradyzoites. Furthermore, mice infected with the type II KO mutants show a drastically reduced brain cyst burden. Thus, our results validate TgAP2XI-4 as a novel nuclear factor that regulates bradyzoite gene expression during parasite differentiation and cyst formation.

Identification of differentially expressed water-insoluble proteins in the encystment process of Colpoda cucullus by two-dimensional electrophoresis and LC-MS/MS analysis.

In the encystment process of the ciliate protist Colpoda cucullus, we observed that the cell total protein abundance was reduced at 12 h-1 d after the onset of encystment induction subsequent to the reduction in mRNA abundance. We analyzed the alteration of the expression levels of water-insoluble proteins by two-dimensional polyacrylamide gel electrophoresis using polyoxyethylene (20) sorbitan monooleate (Tween-80), and we identified proteins whose expression levels were altered in the encystment process by a liquid chromatography tandem mass spectrometry analysis. The expression level of a 60-kDa protein (p60; heat shock protein 60) was temporarily enhanced and that of a 55-kDa protein (p55; actin) and a 49-kDa protein (p49; actin) was enhanced in the Colpoda encystment process. In mature cysts, the expression level of p55 and p49 tended to be reduced, whereas the expression level of a 50-kDa protein (p50d; α-tubulin), a 25-kDa protein (p25; α-tubulin) and a 52-kDa protein (p52c; ß-tubulin) was enhanced.

Facultative cheater mutants reveal the genetic complexity of cooperation in social amoebae.

Cooperation is central to many major transitions in evolution, including the emergence of eukaryotic cells, multicellularity and eusociality. Cooperation can be destroyed by the spread of cheater mutants that do not cooperate but gain the benefits of cooperation from others. However, cooperation can be preserved if cheaters are facultative, cheating others but cooperating among themselves. Several cheater mutants have been studied before, but no study has attempted a genome-scale investigation of the genetic opportunities for cheating. Here we describe such a screen in a social amoeba and show that cheating is multifaceted by revealing cheater mutations in well over 100 genes of diverse types. Many of these mutants cheat facultatively, producing more than their fair share of spores in chimaeras, but cooperating normally when clonal. These findings indicate that phenotypically stable cooperative systems may nevertheless harbour genetic conflicts. The opportunities for evolutionary moves and countermoves in such conflicts may select for the involvement of multiple pathways and numerous genes.

Bradyzoite pseudokinase 1 is crucial for efficient oral infectivity of the Toxoplasma gondii tissue cyst.

The tissue cyst formed by the bradyzoite stage of Toxoplasma gondii is essential for persistent infection of the host and oral transmission. Bradyzoite pseudokinase 1 (BPK1) is a component of the cyst wall, but nothing has previously been known about its function. Here, we show that immunoprecipitation of BPK1 from in vitro bradyzoite cultures, 4 days postinfection, identifies at least four associating proteins: MAG1, MCP4, GRA8, and GRA9. To determine the role of BPK1, a strain of Toxoplasma was generated with the bpk1 locus deleted. This BPK1 knockout strain (Δbpk1) was investigated in vitro and in vivo. No defect was found in terms of in vitro cyst formation and no difference in pathogenesis or cyst burden 4 weeks postinfection (wpi) was detected after intraperitoneal (i.p.) infection with Δbpk1 tachyzoites, although the Δbpk1 cysts were significantly smaller than parental or BPK1-complemented strains at 8 wpi. Pepsin-acid treatment of 4 wpi in vivo cysts revealed that Δbpk1 parasites are significantly more sensitive to this treatment than the parental and complemented strains. Consistent with this, 4 wpi Δbpk1 cysts showed reduced ability to cause oral infection compared to the parental and complemented strains. Together, these data reveal that BPK1 plays a crucial role in the in vivo development and infectivity of Toxoplasma cysts.