Eukaryotic Pathway Research Articles

Recognition of nonproline N-terminal residues by the Pro/N-degron pathway

Eukaryotic N-degron pathways are proteolytic systems whose unifying feature is their ability to recognize proteins containing N-terminal (Nt) degradation signals called N-degrons, and to target these proteins for degradation by the 26S proteasome or autophagy. GID4, a subunit of the GID ubiquitin ligase, is the main recognition component of the proline (Pro)/N-degron pathway. GID4 targets proteins through their Nt-Pro residue or a Pro at position 2, in the presence of specific downstream sequence motifs. Here we show that human GID4 can also recognize hydrophobic Nt-residues other than Pro. One example is the sequence Nt-IGLW, bearing Nt-Ile. Nt-IGLW binds to wild-type human GID4 with a Kd of 16 μM, whereas the otherwise identical Nt-Pro-bearing sequence PGLW binds to GID4 more tightly, with a Kd of 1.9 μM. Despite this difference in affinities of GID4 for Nt-IGLW vs. Nt-PGLW, we found that the GID4-mediated Pro/N-degron pathway of the yeast Saccharomyces cerevisiae can target an Nt-IGLW-bearing protein for rapid degradation. We solved crystal structures of human GID4 bound to a peptide bearing Nt-Ile or Nt-Val. We also altered specific residues of human GID4 and measured the affinities of resulting mutant GID4s for Nt-IGLW and Nt-PGLW, thereby determining relative contributions of specific GID4 residues to the GID4-mediated recognition of Nt-Pro vs. Nt-residues other than Pro. These and related results advance the understanding of targeting by the Pro/N-degron pathway and greatly expand the substrate recognition range of the GID ubiquitin ligase in both human and yeast cells.

Origin and diversification of the cardiolipin biosynthetic pathway in the Eukarya domain.

Cardiolipin (CL) and its precursor phosphatidylglycerol (PG) are important anionic phospholipids widely distributed throughout all domains of life. They have key roles in several cellular processes by shaping membranes and modulating the activity of the proteins inserted into those membranes. They are synthesized by two main pathways, the so-called eukaryotic pathway, exclusively found in mitochondria, and the prokaryotic pathway, present in most bacteria and archaea. In the prokaryotic pathway, the first and the third reactions are catalyzed by phosphatidylglycerol phosphate synthase (Pgps) belonging to the transferase family and cardiolipin synthase (Cls) belonging to the hydrolase family, while in the eukaryotic pathway, those same reactions are catalyzed by unrelated homonymous enzymes: Pgps of the hydrolase family and Cls of the transferase family. Because of the enzymatic arrangement found in both pathways, it seems that the eukaryotic pathway evolved by convergence to the prokaryotic pathway. However, since mitochondria evolved from a bacterial endosymbiont, it would suggest that the eukaryotic pathway arose from the prokaryotic pathway. In this review, it is proposed that the eukaryote pathway evolved directly from a prokaryotic pathway by the neofunctionalization of the bacterial enzymes. Moreover, after the eukaryotic radiation, this pathway was reshaped by horizontal gene transfers or subsequent endosymbiotic processes.

Functional Analysis of Mitochondrial Acyl Carrier Proteins.

Fatty acid synthesis is a ubiquitous pathway in both prokaryotes and eukaryotes. Plants synthesize fatty acids for a variety of uses, including membrane bilayers and seed energy reserves, but also for the production of lipoic acid, which is a cofactor for some enzymes. The multistep biosynthetic

Quantitative Profiling of Arabidopsis Polar Glycerolipids under Two Types of Heat Stress.

At the cellular level, the remodelling of membrane lipids and production of heat shock proteins are the two main strategies whereby plants survive heat stress. Although many studies related to glycerolipids and HSPs under heat stress have been reported separately, detailed alterations of glycerolipids and the role of HSPs in the alterations of glycerolipids still need to be revealed. In this study, we profiled the glycerolipids of wild-type Arabidopsis and its HSP101-deficient mutant hot-1 under two types of heat stress. Our results demonstrated that the alterations of glycerolipids were very similar in wild-type Arabidopsis and hot-1 during heat stress. Although heat acclimation led to a slight decrease of glycerolipids, the decrease of glycerolipids in plants without heat acclimation is more severe under heat shock. The contents of 36:x monogalactosyl diacylglycerol (MGDG) were slightly increased, whereas that of 34:6 MGDG and 34:4 phosphatidylglycerol (PG) were severely decreased during moderate heat stress. Our findings suggested that heat acclimation could reduce the degradation of glycerolipids under heat shock. Synthesis of glycerolipids through the prokaryotic pathway was severely suppressed, whereas that through the eukaryotic pathway was slightly enhanced during moderate heat stress. In addition, HSP101 has a minor effect on the alterations of glycerolipids under heat stress.

Facile and scalable expression and purification of transcription factor IIH (TFIIH) core complex

Transcription factor IIH (TFIIH) plays essential roles in both the initiation of RNA Polymerase II-mediated transcription and the Nucleotide Excision Repair (NER) pathway in eukaryotes. In NER, the 7-subunit TFIIH Core sub-complex is responsible for the opening and extension of the DNA bubble created at the lesion site, utilizing the molecular motors XPB and XPD. Mutations in Core subunits are associated with a series of severe autosomal recessive disorders characterised by symptoms such as mild-to-extreme photosensitivity, premature ageing, physical and neurological anomalies, and in some cases an increased susceptibility to cancer. Although TFIIH Core has been successfully obtained in the past, the process has always remained challenging and laborious, involving many steps that severely hindered the amount of pure, active complex obtained. This has limited biochemical and functional studies of the NER process. Here we describe improved and simplified processes for the cloning, expression and purification of the 7-subunit TFIIH Core sub-complex. The combined use of auto-cleavable 2A-like sequences derived from the Foot-and-Mouth Disease Virus (FMDV) and the MultiBac™ cloning system, a powerful baculoviral expression vector specifically conceived for the obtaining of multi-subunit eukaryotic complexes, allowed us to obtain a single, 7-gene plasmid in a short time using regular restriction cloning strategies. Additionally, expression of the construct in High Five™ insect cells paired with a simple 5-step purification protocol allowed the extraction of a pure, active TFIIH Core sub-complex in milligram quantities.

Lipid Composition and Associated Gene Expression Patterns during Pollen Germination and Pollen Tube Growth in Olive (Olea europaea L.).

Pollen lipids are essential for sexual reproduction, but our current knowledge regarding lipid dynamics in growing pollen tubes is still very scarce. Here, we report unique lipid composition and associated gene expression patterns during olive pollen germination. Up to 376 genes involved in the biosynthesis of all lipid classes, except suberin, cutin and lipopolysaccharides, are expressed in olive pollen. The fatty acid profile of olive pollen is markedly different compared with other plant organs. Triacylglycerol (TAG), containing mostly C12–C16 saturated fatty acids, constitutes the bulk of olive pollen lipids. These compounds are partially mobilized, and the released fatty acids enter the β-oxidation pathway to yield acetyl-CoA, which is converted into sugars through the glyoxylate cycle during the course of pollen germination. Our data suggest that fatty acids are synthesized de novo and incorporated into glycerolipids by the ‘eukaryotic pathway’ in elongating pollen tubes. Phosphatidic acid is synthesized de novo in the endomembrane system during pollen germination and seems to have a central role in pollen tube lipid metabolism. The coordinated action of fatty acid desaturases FAD2–3 and FAD3B might explain the increase in linoleic and alpha-linolenic acids observed in germinating pollen. Continuous synthesis of TAG by the action of diacylglycerol acyltransferase 1 (DGAT1) enzyme, but not phosphoplipid:diacylglycerol acyltransferase (PDAT), also seems plausible. All these data allow for a better understanding of lipid metabolism during the olive reproductive process, which can impact, in the future, on the increase in olive fruit yield and, therefore, olive oil production.

The Pheromone Module SteC-MkkB-MpkB-SteD-HamE Regulates Development, Stress Responses and Secondary Metabolism in Aspergillus fumigatus.

In order for eukaryotes to efficiently detect and respond to environmental stimuli, a myriad of protein signaling pathways are utilized. An example of highly conserved signaling pathways in eukaryotes are the mitogen-activated protein kinase (MAPK) pathways. In fungi, MAPK pathways have been shown to regulate a diverse array of biological processes, such as asexual and sexual development, stress responses and the production of secondary metabolites (SMs). In the model fungus Aspergillus nidulans, a MAPK pathway known as the pheromone module is utilized to regulate both development and SM production. This signaling cascade consists of the three kinases SteC, MkkB, and MpkB, as well as the SteD adaptor protein and the HamE scaffold. In this study, homologs of each of these proteins have been identified in the opportunistic human pathogen A. fumigatus. By performing epitope tagging and mass spectrometry experiments, we have shown that these proteins form a pentameric complex, similar to what is observed in A. nidulans. This complex has been shown to assemble in the cytoplasm and MpkB enters the nucleus, where it would presumably interact with various transcription factors. Pheromone module mutant strains exhibit drastic reductions in asexual sporulation, vegetative growth rate and production of SMs, such as gliotoxin. Mutants also display increased sensitivity to cell wall and oxidative stress agents. Overall, these data provide evidence of the existence of a conserved MAP kinase signaling pathway in Aspergillus species and suggest that this pathway is critical for the regulation of fungal development and secondary metabolism.

Auto Arginine-GlcNAcylation Is Crucial for Bacterial Pathogens in Regulating Host Cell Death.

Many Gram-negative bacterial pathogens utilize the type III secretion system (T3SS) to inject virulence factors, named effectors, into host cells. These T3SS effectors manipulate host cellular signaling pathways to facilitate bacterial pathogenesis. Death receptor signaling plays an important role in eukaryotic cell death pathways. NleB from enteropathogenic Escherichia coli (EPEC) and SseK1/3 from Salmonella enterica serovar Typhimurium (S. Typhimurium) are T3SS effectors. They are defined as a family of arginine GlcNAc transferase to modify a conserved arginine residue in the death domain (DD) of the death receptor TNFR and their corresponding adaptors to hijack death receptor signaling. Here we identified that these enzymes, NleB, SseK1, and SseK3 could catalyze auto-GlcNAcylation. Residues, including Arg13/53/159/293 in NleB, Arg30/158/339 in SseK1, and Arg153/184/305/335 in SseK3 were identified as the auto-GlcNAcylation sites by mass spectrometry. Mutation of the auto-modification sites of NleB, SseK1, and SseK3 abolished or attenuated the capability of enzyme activity toward their death domain targets during infection. Loss of this ability led to the increased susceptibility of the cells to TNF- or TRAIL-induced cell death during bacterial infection. Overall, our study reveals that the auto-GlcNAcylation of NleB, SseK1, and SseK3 is crucial for their biological activity during infection.

Constraints on the expansion of paralogous protein families.

Duplication and divergence is a major mechanism by which new proteins and functions emerge in biology. Consequently, most organisms, in all domains of life, have genomes that encode large paralogous families of proteins. For recently duplicated pathways to acquire different, independent functions, the two paralogs must acquire mutations that effectively insulate them from one another. For instance, paralogous signaling proteins must acquire mutations that endow them with different interaction specificities such that they can participate in different signaling pathways without disruptive cross-talk. Although duplicated genes undoubtedly shape each other's evolution as they diverge and attain new functions, it is less clear how other paralogs impact or constrain gene duplication. Does the establishment of a new pathway by duplication and divergence require the system-wide optimization of all paralogs? The answer has profound implications for molecular evolution and our ability to engineer biological systems. Here, we discuss models, experiments, and approaches for tackling this question, and for understanding how new proteins and pathways are born.

The interplay of spatial organization and biochemistry in building blocks of cellular signalling pathways.

Biochemical pathways and networks are central to cellular information processing. While a broad range of studies have dissected multiple aspects of information processing in biochemical pathways, the effect of spatial organization remains much less understood. It is clear that space is central to intracellular organization, plays important roles in cellular information processing and has been exploited in evolution; additionally, it is being increasingly exploited in synthetic biology through the development of artificial compartments, in a variety of ways. In this paper, we dissect different aspects of the interplay between spatial organization and biochemical pathways, by focusing on basic building blocks of these pathways: covalent modification cycles and two-component systems, with enzymes which may be monofunctional or bifunctional. Our analysis of spatial organization is performed by examining a range of 'spatial designs': patterns of localization or non-localization of enzymes/substrates, theoretically and computationally. Using these well-characterized in silico systems, we analyse the following. (i) The effect of different types of spatial organization on the overall kinetics of modification, and the role of distinct modification mechanisms therein. (ii) How different information processing characteristics seen experimentally and studied from the viewpoint of kinetics are perturbed, or generated. (iii) How the activity of enzymes (bifunctional enzymes in particular) may be spatially manipulated, and the relationship between localization and activity. (iv) How transitions in spatial organization (encountered either through evolution or through the lifetime of cells, as seen in multiple model organisms) impacts the kinetic module (and pathway) behaviour, and how transitions in chemistry may be impacted by prior spatial organization. The basic insights which emerge are central to understanding the role of spatial organization in biochemical pathways in both bacteria and eukaryotes, and are of direct relevance to engineering spatial organization of pathways in bottom-up synthetic biology in cellular and cell-free systems.

Clamping, Bending, and Twisting Inter-Domain Motions in the Misfold-Recognising Portion of UDP-Glucose: Glycoprotein Glucosyl-Transferase

UDP-glucose: glycoprotein glucosyltransferase (UGGT) is the glycoprotein folding checkpoint in the eukaryotic glycoprotein secretory pathway. The enzyme detects misfolded glycoproteins in the Endoplasmic Reticulum, and flags them for retention by re-glucosylating them on an N-linked glycan. The fit of a UGGT crystal structure to a negative stain electron microscopy reconstruction of UGGT in complex with an antibody suggests that the misfold-sensing N-terminal portion of UGGT and its C-terminal catalytic domain are tightly associated. Molecular Dynamics (MD) simulations capture UGGT in so far unobserved conformational states, and principal component analysis of the MD trajectories affords a description of UGGT's overall inter-domain motions, highlighting three types of inter-domain movements: bending, twisting and clamping. These inter-domain motions modify the accessible surface area of the enzyme's central saddle. We propose to name the maximum distance between a site of misfolding on a UGGT glycoprotein substrate and an N-linked glycan that monomeric UGGT can re-glucosylate on the same glycoprotein. MD simulations estimate the Parodi limit to be around 60-70 A. Re-glucosylation assays using UGGT deletion mutants suggest that the TRXL2 domain is necessary for activity against urea-misfolded bovine thyroglobulin. Our findings support a one-size-fits-all adjustable spanner substrate recognition model, with a crucial role for the TRXL2 domain in the recruitment of misfolded substrates.

Neisseria gonorrhoeae Blocks Epithelial Exfoliation by Nitric-Oxide-Mediated Metabolic Cross Talk to Promote Colonization in Mice

Several pathogens suppress exfoliation, a key defense of epithelia against microbial colonization. Common among these pathogens, exemplified by Neisseria gonorrhoeae, is their ability to bind carcinoembryonic antigen-related cell adhesion molecules (CEACAMs). Gonococcal CEACAM engagement triggers the expression of CD105, which is necessary to block epithelial exfoliation, whereas homotypic CEACAM-CEACAM interactions or antibody-mediated CEACAM clustering does not lead to CD105 expression. Here, we show that CEACAM-associated bacteria release nitric oxide (NO) during anaerobic respiration, and membrane-permeable NO initiates a eukaryotic signaling pathway involving soluble guanylate cyclase (sGC), protein kinase G, and the transcription factor CREB to upregulate CD105 expression. A murine vaginal infection model with N.gonorrhoeae reveals this metabolic cross communication allows bacterial suppression of epithelial exfoliation to facilitate mucosal colonization. Disrupting NO-initiated responses in host cells re-establishes epithelial exfoliation and inhibits mouse genital tract colonization by N.gonorrhoeae, suggesting a host-directed approach to prevent bacterial infections.

How to study intertwined and autoregulated eukaryotic signal transduction pathways.

Mitogen-activated protein kinase (MAPK) pathways are a major means of eukaryotic cells to adapt to environmental changes, in the case of microorganisms, and to nutritional and hormonal signals, in the case of multicellular organisms. Numerous defects in such architecturally conserved pathways have been associated with different human cancers. These signaling cascades usually commence with sensors located in the plasma membrane, which through specific protein kinases activate a conserved tripartite MAPK module. Phosphorylation of their targets, that is, cytosolic proteins and/or transcription factors, then triggers the proper cellular response. In the model yeast Saccharomycescerevisiae and other fungi, the cell wall integrity pathway (CWI) has been extensively studied and its components may serve as targets for antifungal drugs of clinical and agricultural importance. Another well-known MAPK cascade, the high osmolarity glycerol (HOG) pathway, is required to cope with osmotic stress. In the past decade, it has become increasingly evident that such pathways do not act in a linear top-down fashion, but are highly regulated by internal feedback mechanisms as well as by cross-pathway interactions. The work of Jiménez-Gutiérrez et al. in this issue provides an elegant way to identify new players in these complex networks. Comment on: https://doi.org/10.1111/febs.15288.

The function of histone lysine methylation related SET domain group proteins in plants

Histone methylation, which is mediated by the histone lysine (K) methyltransferases (HKMTases), is a mechanism associated with many pathways in eukaryotes. Most HKMTases have a conserved SET (Su(var) 3-9,E(z),Trithorax) domain, while the HKMTases with SET domains are called the SET domain group (SDG) proteins. In plants, only SDG proteins can work as HKMTases. In this review, we introduced the classification of SDG family proteins in plants and the structural characteristics of each subfamily, surmise the functions of SDG family members in plant growth and development processes, including pollen and female gametophyte development, flowering, plant morphology and the responses to stresses. This review will help researchers better understand the SDG proteins and histone methylation in plants and lay a basic foundation for further studies on SDG proteins.

Rice Stripe Mosaic Virus–Encoded P4 Is a Weak Suppressor of Viral RNA Silencing and Is Required for Disease Symptom Development

Viral suppressors of RNA silencing (VSRs) are a cluster of viral proteins that have evolved to counteract eukaryotic antiviral RNA silencing pathways, thereby contributing to viral pathogenicity. In this study, we revealed that the matrix protein P4 encoded by rice stripe mosaic virus (RSMV), which is an emerging cytoplasmic rhabdovirus, is a weak RNA silencing suppressor. By conducting yeast two-hybrid, bimolecular fluorescence complementation, and subcellular colocalization assays, we proved that P4 interacts with the rice endogenous suppressor of gene silencing 3 (OsSGS3). We also determined that P4 overexpression has no effect on OsSGS3 transcription. However, P4 can promote the degradation of OsSGS3 via ubiquitination and autophagy. Additionally, a potato virus X-based expression system was used to confirm that P4 enhances the development of mosaic symptoms on Nicotiana benthamiana leaves by promoting hydrogen peroxide accumulation but not cell death. To verify whether P4 is a pathogenicity factor in host plants, we generated transgenic P4-overexpressing rice plants that exhibited disease-related developmental defects including decreased plant height and excessive tillering. Our data suggest that RSMV-encoded P4 serves as a weak VSR that inhibits antiviral RNA silencing by targeting OsSGS3.

Transcriptional analysis of deoxynivalenol-induced apoptosis of sow ovarian granulosa cell.

Litter size is one of the most important economic traits in pig production. Recent studies identified that deoxynivalenol (DON), a widespread toxin in fodder, was associated with animal prolificacy. However, the underlying mechanisms have not yet been completely elucidated. Here, we used porcine ovary granulosa cells (pGCs) as a vector to establish DON concentration-time models and performed cell morphology and transcriptome analysis to identify and analyse the effects of DON on reproductive performance in swine. The results showed that DON can induce morphological changes and apoptosis of pGCs, while inhibiting cell proliferation. Moreover, these effects of DON on pGCs were dose-dependent. After treatment of pGCs with different concentrations of DON, the percentage of cells in S phase and G2/M phase increased. RNA-seq analyses revealed 5,937 differentially expressed genes, of which 1995 were down-regulated and 3,942 were up-regulated after DON treatment. KEGG enrichment analysis indicated important metabolic pathways such as IL-17 signalling pathway, eukaryotic ribosome synthesis pathway, RNA transport pathway and RNA degradation. Based on our results, we speculate that the effects of DON are related to the DNA damage process. Our study provides novel insights and a foundation to further understand the effect of DON on swine prolificacy.

Coordination of host and symbiont gene expression reveals a metabolic tug-of-war between aphids and Buchnera

Symbioses between animals and microbes are often described as mutualistic, but are subject to tradeoffs that may manifest as shifts in host and symbiont metabolism, cellular processes, or symbiont density. In pea aphids, the bacterial symbiont Buchnera is confined to specialized aphid cells called bacteriocytes, where it produces essential amino acids needed by hosts. This relationship is dynamic; Buchnera titer varies within individual aphids and among different clonal aphid lineages, and is affected by environmental and host genetic factors. We examined how host genotypic variation relates to host and symbiont function among seven aphid clones differing in Buchnera titer. We found that bacteriocyte gene expression varies among individual aphids and among aphid clones, and that Buchnera gene expression changes in response. By comparing hosts with low and high Buchnera titer, we found that aphids and Buchnera oppositely regulate genes underlying amino acid biosynthesis and cell growth. In high-titer hosts, both bacteriocytes and symbionts show elevated expression of genes underlying energy metabolism. Several eukaryotic cell signaling pathways are differentially expressed in bacteriocytes of low- versus high-titer hosts: Cell-growth pathways are up-regulated in low-titer genotypes, while membrane trafficking, lysosomal processes, and mechanistic target of rapamycin (mTOR) and cytokine pathways are up-regulated in high-titer genotypes. Specific Buchnera functions are up-regulated within different bacteriocyte environments, with genes underlying flagellar body secretion and flagellar assembly overexpressed in low- and high-titer hosts, respectively. Overall, our results reveal allowances and demands made by both host and symbiont engaged in a metabolic "tug-of-war."

Structural Analyses on the Deamidation of N-Terminal Asn in the Human N-Degron Pathway.

The N-degron pathway is a proteolytic system in which a single N-terminal amino acid acts as a determinant of protein degradation. Especially, degradation signaling of N-terminal asparagine (Nt-Asn) in eukaryotes is initiated from its deamidation by N-terminal asparagine amidohydrolase 1 (NTAN1) into aspartate. Here, we have elucidated structural principles of deamidation by human NTAN1. NTAN1 adopts the characteristic scaffold of CNF1/YfiH-like cysteine hydrolases that features an α-β-β sandwich structure and a catalytic triad comprising Cys, His, and Ser. In vitro deamidation assays using model peptide substrates with varying lengths and sequences showed that NTAN1 prefers hydrophobic residues at the second-position. The structures of NTAN1-peptide complexes further revealed that the recognition of Nt-Asn is sufficiently organized to produce high specificity, and the side chain of the second-position residue is accommodated in a hydrophobic pocket adjacent to the active site of NTAN1. Collectively, our structural and biochemical analyses of the substrate specificity of NTAN1 contribute to understanding the structural basis of all three amidases in the eukaryotic N-degron pathway.

The role of heterotrimeric G proteins in the control of symbiosis development in legume plants

Heterotrimeric G proteins are involved in the regulation of signaling pathways in eukaryotes. Previously, the data about possible participation of heterotrimeric G proteins in the regulation of nodulation in legumes were obtained, however, specific proteins, their composition and role in this process remain poorly understood. In this work searching of the genes encoding the alpha, beta, and gamma subunits of heterotrimeric G proteins based on an analysis of the Pisum sativum L. genome was performed, as well as the dynamics of the gene expression encoding the particular subunits of G proteins in the process of symbiosis was studied. In addition, a significant effect of beta 1-subunit gene suppression by RNA interference on the nodulation process was revealed.

Trends in Symbiont-Induced Host Cellular Differentiation.

Bacteria participate in a wide diversity of symbiotic associations with eukaryotic hosts that require precise interactions for bacterial recognition and persistence. Most commonly, host-associated bacteria interfere with host gene expression to modulate the immune response to the infection. However, many of these bacteria also interfere with host cellular differentiation pathways to create a hospitable niche, resulting in the formation of novel cell types, tissues, and organs. In both of these situations, bacterial symbionts must interact with eukaryotic regulatory pathways. Here, we detail what is known about how bacterial symbionts, from pathogens to mutualists, control host cellular differentiation across the central dogma, from epigenetic chromatin modifications, to transcription and mRNA processing, to translation and protein modifications. We identify four main trends from this survey. First, mechanisms for controlling host gene expression appear to evolve from symbionts co-opting cross-talk between host signaling pathways. Second, symbiont regulatory capacity is constrained by the processes that drive reductive genome evolution in host-associated bacteria. Third, the regulatory mechanisms symbionts exhibit correlate with the cost/benefit nature of the association. And, fourth, symbiont mechanisms for interacting with host genetic regulatory elements are not bound by native bacterial capabilities. Using this knowledge, we explore how the ubiquitous intracellular Wolbachia symbiont of arthropods and nematodes may modulate host cellular differentiation to manipulate host reproduction. Our survey of the literature on how infection alters gene expression in Wolbachia and its hosts revealed that, despite their intermediate-sized genomes, different strains appear capable of a wide diversity of regulatory manipulations. Given this and Wolbachia's diversity of phenotypes and eukaryotic-like proteins, we expect that many symbiont-induced host differentiation mechanisms will be discovered in this system.