Molecular Determinants of a Symbiotic Chronic Infection

ANNUAL REVIEWS Further Annu. Rev. Genet. 2008.42:413-441. Downloaded from arjournals.annualreviews.org by University of Georgia on 03/31/09. For personal use only. Click here for quick links to Annual Reviews content online, including: • Other articles in this volume • Top cited articles • Top downloaded articles • Our comprehensive search Molecular Determinants of a Symbiotic Chronic Infection Katherine E. Gibson,1,2 Hajime Kobayashi,2 and Graham C. Walker2 Present address 1 Department of Biology, University of Massachusetts, Boston, Massachusetts 02125 2 Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; email: gwalker@mit.edu Annu. Rev. Genet. 2008. 42:413–41 Key Words The Annual Review of Genetics is online at genet.annualreviews.org Rhizobium, legume, bacterial invasion, signaling, stress response, cell cycle This article’s doi: 10.1146/annurev.genet.42.110807.091427 c 2008 by Annual Reviews. Copyright  All rights reserved 0066-4197/08/1201-0413$20.00 Abstract Rhizobial bacteria colonize legume roots for the purpose of biological nitrogen fixation. A complex series of events, coordinated by host and bacterial signal molecules, underlie the development of this symbiotic interaction. Rhizobia elicit de novo formation of a novel root organ within which they establish a chronic intracellular infection. Legumes permit rhizobia to invade these root tissues while exerting control over the infection process. Once rhizobia gain intracellular access to their host, legumes also strongly influence the process of bacterial differentiation that is required for nitrogen fixation. Even so, symbiotic rhizobia play an active role in promoting their goal of host invasion and chronic persistence by producing a variety of signal molecules that elicit changes in host gene expression. In particular, rhizobia appear to advocate for their access to the host by producing a variety of signal molecules capable of suppressing a general pathogen defense response. 413 INTRODUCTION Annu. Rev. Genet. 2008.42:413-441. Downloaded from arjournals.annualreviews.org by University of Georgia on 03/31/09. For personal use only. Although nitrogen is one of the most abundant elements present on Earth, it is also one of the most limiting for biological growth because it is largely found in the inaccessible form dinitrogen (N2 ). Biological nitrogen fixation is the process by which chemically inert N2 present in the atmosphere is enzymatically reduced to the metabolically usable form ammonia (NH3 ) through the action of nitrogenase (129). The ability to catalyze the conversion of N2 to NH3 has evolved only among microbes, including the rhizobia, cyanobacteria, azobacteria, frankia, and archaea (137). This review focuses on developmental events that underlie biological nitrogen fixation within the context of an intimate symbiosis between rhizobia, a phylogenetically diverse group of gram-negative soil bacteria within the family Rhizobiaceae, and their hosts the Leguminosae (or Fabaceae) family of flowering plants. This rhizobium-legume symbiosis is established under nitrogen-limiting soil conditions and is estimated to contribute nearly half of all current biological nitrogen fixation (68). In addition to its environmental and agricultural significance (170), this symbiosis provides a tractable model system for identifying and characterizing certain mechanisms employed by invasive bacteria during chronic host interactions as they transition from a free-living environment to their niche within the host. In fact, many rhizobial genes required for either host invasion or chronic persistence have orthologs in closely related alphaproteobacterial pathogens, such as Agrobacterium and Brucella, and often these orthologs have an effect on virulence (11, 140, 156). Moreover, plants can detect phytopathogens using receptors that are evolutionarily related to those employed by legumes to detect symbiotic rhizobia (144, 180). Thus, the broad importance of this symbiosis is ever growing. Due to the wide breadth of biological processes involved in establishing the legumerhizobium symbiosis, as well as the natural diversity in symbiotic mechanisms, we are un- 414 Gibson · Kobayashi · Walker able to review all the events that underlie this complex interaction. For instance, not discussed here are the Type III and Type IV secretion systems that play an important role in diverse rhizobial symbioses (32, 112), nor the bacterial quorum sensing systems that modulate symbiotic interactions (67, 145). In addition, it is clear that plant hormone balance plays a critical role in regulating nodule development (122). Rather, our goal is to provide the reader with molecular insight into several regulatory aspects of this symbiosis while providing reference to reviews that discuss these processes in more detail. EVOLUTION OF SYMBIOSIS The rhizobium-legume symbiosis, a relatively recent evolutionary adaptation, is thought to have evolved from the ancient arbuscular mycorrhizal symbiosis that is nearly ubiquitous throughout the plant kingdom and provides plants with the essential mineral nutrient phosphorus (73). This evolutionary relationship has been inferred based on findings that several host genes represent common requirements for the establishment of both rhizobial and mycorrhizal symbioses. Given that nearly all vascular plants interact with mycorrhizal symbionts, it remains unclear why the nitrogen-fixing symbiosis is strictly limited to legume species, with the exception of Parasponia. Current understandings of legume evolution and the appearance of nodulation indicate that the first symbiosis event involved bacterial invasion of roots via cracks in the host epidermis where lateral roots emerge (158). Subsequent to this, developmental mechanisms evolved, likely through the process of gene duplication, to craft the highly selective symbiosis described here. In particular, the emergence of a host-derived infection structure allows host control over the bacterial infection process (59). The symbiotic capacity of rhizobia is thought to have evolved in part through horizontal gene transfer events based on several observations. Within the symbiotic rhizobial Annu. Rev. Genet. 2008.42:413-441. Downloaded from arjournals.annualreviews.org by University of Georgia on 03/31/09. For personal use only. lineage, Sinorhizobium is estimated to have diverged from Bradyrhizobium approximately 500 Mya (168), which is well before the initial appearance of legume species approximately 60 Mya (158). Rhizobia also tend to have large and multipartite genomes consisting of a chromosome supplemented with one or more independent plasmids (81), which contribute to an evolutionarily dynamic genome through the process of horizontal gene transfer. Moreover, rhizobial genes involved in symbiosis are often located within chromosomal islands or on plasmids: Sinorhizobium meliloti genes involved in NF biosynthesis (nod, nol, noe) and nitrogen fixation (nif and fix) are located on pSymA whereas others required for EPS (exopolysaccharide) biosynthesis (exo) and C4 -dicarboxylic acid utilization (dct) are located on pSymB (60). Horizontal transfer of these genomic elements has been observed among bacteria within the rhizosphere and has the ability to convert a nonsymbiont into a symbiont through a single transfer event (6, 160, 161). Other than symbiosis genes, there is no significant synteny shared between the plasmids of various rhizobial species or rhizobial chromosomes (19, 188). Finally, it was recently discovered that certain betaproteobacteria are also capable of establishing nitrogen-fixing symbioses with legumes (117), and comparative phylogenetic analyses support the notion that the plasmid-borne symbiotic genes (nod and nif ) in these Burkholderia are derived from at least one horizontal gene transfer event (25). NODULE DEVELOPMENT To establish the symbiosis, free-living soil rhizobia elicit de novo formation of a specialized root organ, the nodule, from their host. The root nodule ultimately houses many thousands of individual rhizobia that catalyze nitrogen fixation. Organogenesis of the nodule occurs via a developmental program that involves dedifferentiation of quiescent (G0 ) root cortical cells into an actively dividing meristem (Figure 1a), which is functionally analogous to an animal stem cell population (159). These di- viding meristem cells form the initial nodule primordium from which a mature nodule develops (Figure 1a). A subset of these meristem cells enter a modified cell cycle program that results in endoreduplication and produces greatly enlarged cells. It is this subset of endoploid cells that are invaded and colonized by rhizobia for the purpose of nitrogen fixation (Figure 1d ). To colonize the developing nodule, bacteria present in the soil provoke root hair curling by the host (Figure 1a,b). The root hair curl traps intimately associated bacteria, which are then able to invade the nodule through a hostderived structure called the infection thread (IT) (Figure 1a,c). The IT is a tube originating at the tip of the root hair that initially forms via localized hydrolysis of the cell wall and that continues its growth via deposition of new cell wall material (59). In this way, the IT carries bacteria from the root surface toward the population of newly created endoploid cells where it undergoes significant ramification. Ultimately, bacteria within the IT are deposited into the host cell cytoplasm in a process that resembles endocytosis; they then enter a differentiation program that results in their ability to catalyze nitrogen fixation. Generally, nodules fall into two morphological classes based on their pattern of meristem growth: indeterminate, with a meristem derived from inner cortical cells, and determinate, with a meristem derived from outer cortical cells. Indeterminate and determinate nodules also differ in the relative persistence of meristem proliferation. In determinant legumes, including most Old World tropical species and Lotus japonicus, the basal meristem undergoes a limited spurt of cell division such that further nodule growth depends on an expansion in cell size rather than in cell number. Both plant and bacterial development proceeds synchronously within this particular type of nodule; an exception is senescence, which occurs within older nodules on an individual cellular basis (133). Thus, a determinate nodule contains a relatively homogenous population of developing plant and bacterial cells at any given time point, which allows precise analysis of temporal gene expression www.annualreviews.org • Molecular Determinants of a Symbiotic Chronic Infection Rhizobia: gramnegative alphaproteobacteria that engage in symbiosis with legumes and fix nitrogen Horizontal gene transfer: incorporation of genetic material from an unrelated organism (also called lateral gene transfer) Chromosomal island: section within a genome having evidence of horizontal origins NF: nodulation (Nod) factor EPS: exopolysaccharide Rhizosphere: local soil environment surrounding plant roots Synteny: preservation of gene order on chromosomes of related species Meristem: undifferentiated plant cells undergoing cell growth and division Endoreduplication: repeated genomic replication without cytokinesis Endoploid: cells that have undergone endoreduplication IT: infection thread Indeterminate nodule: nodule with a persistent meristem and a heterogenous plant and bacterial developing cell population 415 and function within both symbiotic partners (Figure 1e, f ). In contrast, legumes that form indeterminate nodules, including most temperate species such as Medicago truncatula, create a persistent meristem. In these nodules, the plant allows prolonged bacterial invasion of postmitotic (G0 ) endoploid cells that are continuously generated by the meristem. As a result, each developmental stage required to establish the symbiosis is present within a single mature indeterminate nodule and in a spatially organized manner (Figure 1a). The distinct regions found Annu. Rev. Genet. 2008.42:413-441. Downloaded from arjournals.annualreviews.org by University of Georgia on 03/31/09. For personal use only. Determinate nodule: nodule with a temporary meristem and a relatively homogenous plant and bacterial developing cell population a BACTERIAL INVASION Rhizobium b O - COCH 3 CH2 HO HO O C O -SO3H C H2-OH O O O HO NH H3C O C N CH2 O HO O OH NH H3COC n H Nod factors Flavonoids in a mature indeterminate nodule, proximal to distal from the root, are the meristem (Zone I), the invasion zone (Zone II), the interzone (Zone II/III), the nitrogen-fixing zone (Zone III), and the senescent zone (Zone IV) (172). In addition to distinct morphological features, these zones can be distinguished by patterns of host gene expression. While much research has focused on plant and bacterial physiology in Zones I–III, comparatively less is known regarding the genetic regulation and physiology of senescence in Zone IV although this process is also of critical agricultural consequence (133). I There is often strict specificity in the establishment of a nitrogen-fixing symbiosis between II OH OH ←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− O OH O HO OH Figure 1 O Root hair Root hair curl III Root hair invasion IT Nod factors EPS ROS IV Nodule primordium Cell division c d e IT f n v Bacteroid PBM 416 Gibson · Kobayashi · Walker Schematic model of nodule development. (a-b) Host flavonoids exuded into the soil trigger bacterial Nod Factor production. Nod factor is perceived by host receptors and elicits various host responses, such as root hair curling and root hair invasion. Root hair invasion also requires bacteria EPS and host ROS production. Nod factors induce mitotic cell division in the root cortex (represented in blue), leading to formation of the nodule meristem. An indeterminate nodule originates from the root inner cortex and has a persistent meristem (Zone I). The nodule also contains an invasion zone (Zone II) and a nitrogenfixing zone (Zone III). In older nodules, a senescent zone (Zone IV) develops in which both plant and bacterial cells degenerate. (c) Bacteria enter the nodule through root hairs in a structure called the infection thread (IT) that elongates toward the nodule meristem; nucleus (n), vacuole (v). (d ) At the tip of the growing IT, bacteria are endocytosed into the cytoplasm of postmitotic endoploid cells. Each bacterium is surrounded by a host-derived peribacteroid membrane (PBM) and proceeds to differentiate into the specialized symbiotic form called a bacteroid. Bacteroids establish a chronic infection of the host cytoplasm and enzymatically reduce dinitrogen to provide a source of biologically usable nitrogen to the host (Zone III). (e) In contrast to an indeterminate nodule, a determinate nodule lacks a persistent meristem and all developmental stages proceed synchronously. ( f ) Infected cells of determinate nodules typically lack vacuoles (v). Annu. Rev. Genet. 2008.42:413-441. Downloaded from arjournals.annualreviews.org by University of Georgia on 03/31/09. For personal use only. host legume species and their symbionts. For instance, the bacterium S. meliloti is compatible with species of Medicago (alfalfa), Melilotus (sweetclover), and Trigonella (fenugreek), whereas Rhizobium etli is compatible only with species of Phaseolus (bean). In contrast to these restricted host range rhizobia, some bacteria have a broad host range, like Rhizobium sp. NGR234, which is capable of nodulating 232 legumes from 112 distantly related genera (132). Based on rhizobial phylogenetic relationships, it was suggested that the restricted host range symbiosis evolved from an ancestral broad host range symbiosis (132), and perhaps the specificity engendered by narrow host range interactions creates a finely tuned and more effective symbiosis. The Host Flavonoid Signal Compatible rhizobia are uniquely capable of gaining entry and invading the host nodule based on a series of reciprocal signaling events (Figure 1b). The principal signals originating from the host and perceived by rhizobia in the soil are derived from the flavonoid family of secondary plant metabolites (Figure 2a). The roots of leguminous plants exude a diverse cocktail of flavonoids and isoflavonoids into the soil (128). Compatible bacteria in the rhizosphere can elicit quantitative increases and qualitative changes in flavonoid exudation. Exactly which flavonoid in the rhizosphere a compatible bacterium perceives can be difficult to determine since plants secrete a complex mixture. In fact, it is quite likely that the spectrum of flavonoids exuded by a legume, rather than a single flavonoid compound, provides a determinant for host specificity. At least some rhizobia are chemotactic toward compatible flavonoids, suggesting that certain aspects of host specificity are established before the bacterium and its host physically interact (20). Host Flavonoids Are Perceived by Bacterial NodDs Plant-derived flavonoids elicit a significant transcriptional response from compatible bac- teria within the rhizosphere, most of which is NodD-dependent and results in Nodulation Factor (NF) signal production (Figure 2b) (23). While expression of genes required for NF synthesis is induced by host flavonoids, expression of these same genes can be repressed in the presence of ammonia. NodD belongs to the LysR family of DNAbinding transcription factors, which have an Nterminal ligand-binding domain that regulates the activity of the associated C-terminal DNAbinding domain. The NodD ligand-binding domain is thought to function as a flavonoid receptor and, in the presence of a compatible host, NodD induces the expression of genes involved in NF biosynthesis. For example, the daidzein and genistein isoflavonoids of Glycine max (soybean) induce NF gene expression in Bradyrhizobium japonicum (Figure 2a). However, daidzein prevents NF production in the noncompatible bacterium S. meliloti, which responds positively to the flavone luteolin (Figure 2a), and does so in a NodD-dependent manner (127). Generally, the NodDs of broad host range rhizobia respond to a wider range of flavonoid species than those present in restricted host range bacteria (128). For instance, NodD1 from the broad host range symbiont Rhizobium sp. NGR234 responds positively to a structurally diverse range of compounds, including phenolics (vanillin and isovanillin) that are inhibitors for other rhizobia (97). The transfer of Rhizobium sp. NGR234 nodD1 to a restricted host range rhizobium, like S. meliloti, extends the spectrum of plants the transconjugant recognizes as a symbiotic partner (16). NodD regulates transcription by binding cisacting regulatory sequences called nod-boxes and these elements are generally found upstream of the promoters of the nod, nol, and noe genes involved in NF production (8, 82). However, interesting nuances to NodD-dependent regulation are beginning to emerge, including the identification of genes unrelated to NF biosynthesis within the NodD regulon (87, 109, 165), and a temporal progression to flavonoidinduced gene expression that implies NodD coordinates a complex regulatory hierarchy (87). www.annualreviews.org • Molecular Determinants of a Symbiotic Chronic Infection 417 a Flavenoids b Nod factor OH NodH NodL OH O HO Luteolin O - COCH3 OH CH2 O HO HO O HO H O CH2 O O HO NH H3C O C N O C HO O - SO3H C H 2 -OH O n O OH NH H3COC Daidzein NodABC Annu. Rev. Genet. 2008.42:413-441. Downloaded from arjournals.annualreviews.org by University of Georgia on 03/31/09. For personal use only. O OH NodEF O HO Genestin Figure 2 Representative signaling molecules critical for symbiosis. (a) Host flavonoids: luteolin, daidzein, and genestin. (b) The Nod factor produced by S. meliloti and biosynthetic enzymes (Nod proteins) involved in its synthesis. (c) The S. meliloti exopolysaccharide succinoglycan. ExoH is responsible for succinyl modification; succinoglycan molecular weight is controlled by ExoPTQ and two extracellular glycosylases, ExsH and ExoK. (d ) Schematic representation of S. meliloti lipopolysaccharide (LPS). LpsB is a glycosyltransferase with broad substrate specificity involved in synthesis of the LPS core. AcpXL, LpsXL, and BacA are required for the Very-LongChain Fatty Acid (C28) modification of lipid A. 418 OH O OH c Succinoglycan d LPS O-antigen O ( 4 Glc 1 β 4 Glc 1 β 6 6 4 Glc 1 β 3 Gal 1 β ) HO n β 1 P OH O O O H ExoPTQ ExsH, ExoK β 1 Glc Lipid A H NH H HO HO O O 6 LpsB O H Acetyl Glc Core OH HO H HO O H O H O NH O O O O β 1 6 Succinyl 4 Pyruvyl ExoH 3 β 1 Glc 6 CH3 CH3 CH3 CH3 AcpXL LpsXL BacA HO CH3 Gibson · Kobayashi · Walker P OH HO 3 Glc H OH Annu. Rev. Genet. 2008.42:413-441. Downloaded from arjournals.annualreviews.org by University of Georgia on 03/31/09. For personal use only. The Bacterial NF Response Host Responses to NF Bacterial NF functions as a key that opens the door to its host (128), meaning there is a high degree of stringency for NF chemical structure that determines whether the host allows bacterial invasion to proceed. NF also functions as a mitogen and modifies the plant hormone balance to elicit the primordium formation that ultimately gives rise to mature nodule tissue (Figure 1a) (37, 56, 122). It was recently shown that NF also plays a role in S. meliloti biofilm development and in a host-independent manner (58); thus, NF appears to perform a significant role in both free-living and symbiotic lifestyles. NF is a complex signaling molecule secreted from the cell as a cocktail of β-(1,4)-linked N-acetyl-d-glucosamine (GlcNAc) trimers, tetramers, or pentamers (Figure 2b) (37). The chitin backbone is modified on the nonreducing terminal residue at the C2 position by a fatty acid; however, the size and saturation-state of this lipid chain varies in a species-specific manner. NF can be further decorated with a variety of chemical substituents, including acetyl, arabinosyl, carbamoyl, fucosyl, methyl, and sulfuryl additions. In fact, a given rhizobial species will produce a mixture of NF compounds, anywhere from 2 to 60 distinct molecules, and this is especially true of broad host range bacteria (128). The diversity of NF structures produced by rhizobia derives from the combined presence of species-specific genes and allelic variation of the common nodulation genes (128). Common nod genes (including nodA, B, and C ), which are found in nearly all rhizobial species and are capable of cross-species complementation, are responsible for synthesis of the NF chitin backbone. In contrast, host-specific nod genes confer specificity for nodulation of a particular host and are involved in various modifications of the chitin backbone. The ability to synthesize and secrete NF can be transferred to Escherichia coli by introduction of the NF biosynthetic gene cluster, and this confers upon E. coli the ability to elicit several of the early NF host responses described below (178). A number of physiological responses to NF are observed when either bacteria or purified NF are applied to roots (37), and these responses have been used to position host genes within a signaling pathway (122). Purified NF is effective at eliciting most host responses at nanomolar concentrations. Initial root epidermal responses include an alkalinization of the cytosol, and a depolarization of the plasma membrane within minutes of root inoculation. These two responses appear to depend on a brief NF-induced Ca2+ -influx that precedes them by seconds, and they are closely followed by a prolonged Ca2+ -spiking response that lasts between 20 to 60 min. Purified NF is also sufficient to induce root hair deformation and root hair curling within a few hours of application. Root hair deformation likely relies on Ca2+ induced changes to the organization of the actin cytoskeleton, which produce a reorientation of cell growth. In fact, NF can accumulate within the host plasma membrane (66), and appears to provide a direct positional cue to the host such that the tip of the root hair grows toward the site of greatest NF concentration (51). NF elicits significant changes in the expression of host genes, including those induced early in nodule development that are referred to as early nodulin, or ENOD, genes (37, 122). More globally, transcriptome profiles reveal that plant genes predicted to be involved in responses to abiotic and biotic stresses, as well as cell reorganization and proliferation, are rapidly induced by rhizobia and largely in a NF-dependent manner (49, 105, 116). At the earliest postinoculation time point of 1 h, M. truncatula genes that encode functions related to abiotic stress and disease resistance are upregulated while those involved in translation and cytoskeletal organization are downregulated (105). Abiotic stress and disease resistance genes become significantly downregulated by 6 h postinoculation and this trend continues until at least 3 days postinoculation (105). As discussed below, some of this regulation may be enhanced by bacterial EPSs, cyclic β glucans www.annualreviews.org • Molecular Determinants of a Symbiotic Chronic Infection Mitogen: a substance that elicits cell division Transcriptome: complete set mRNAs expressed in a cell or cell population 419 and LPSs. Concurrent with a dramatic reorientation of root cell growth, genes involved in cytoskeletal functions and cell-wall biogenesis are induced from 1–12 h postinoculation (105). Between 12–48 h, genes involved in translation, cell growth and division, and chromosomal organization are upregulated in a manner consistent with the initiation of meristem proliferation within the root cortex (105, 116). As one might predict, increased expression of cell division genes is transient within a determinate nodule and decreases concurrent with the onset of nitrogen fixation (29, 90). The importance of NF structure for biological activity has been demonstrated through biochemical analyses of NFs produced by hostspecific nod gene mutants and the corresponding phenotypic characterization of plant responses. For instance, the first symbiotically active NF structure described was that produced by S. meliloti, which is a chitin tetramer with an N-linked C16 unsaturated fatty acid and both O-sulfuryl and O-acetyl modifications (Figure 2b) (149). An S. meliloti mutant deficient for the host-specific nodH gene lacks the O-sulfuryl substitution at the reducing terminus of its NF and correspondingly loses the capacity to nodulate its natural M. truncatula host, yet it acquires the novel ability to nodulate Vicia hirsute (139). The purified nonsulfated NF produced by a nodH mutant elicits Ca2+ -spiking from its natural host only when present at concentrations much greater than is required for wild-type NF (123, 139, 178), suggesting the sulfate modification contributes to host range specificity by modulating the affinity between NF and its host receptor. While the S. meliloti nodH gene is required to elicit initial physiological responses from M. truncatula, the host-specific nodEF (NF acylation) and nodL (NF acetylation) genes are subsequently involved in host invasion. Individual null mutants have a delayed nodulation phenotype associated with inefficient bacterial invasion, whereas the nodFL double mutant has a more severe defect in infection thread formation (5, 103). Despite the block in bacterial invasion, a nodFL double mutant (and LPS: lipopolysaccharide Annu. Rev. Genet. 2008.42:413-441. Downloaded from arjournals.annualreviews.org by University of Georgia on 03/31/09. For personal use only. LysM: lysin motif 420 Gibson · Kobayashi · Walker its purified NFs) triggers certain morphological responses from root hairs in a manner indistinguishable from the wild type. These include the early Ca2+ -spiking and root hair curling responses, as well as induction of certain ENODs and nodule primordium formation (5, 24, 178). Thus, in M. truncatula there are distinct structural requirements for early NFdependent root hair responses (i.e., NF sulfation) vs later NF-dependent infection events (i.e., NF sulfation, and acetylation or acylation). The relative ease with which plant symbiosis mutants can now be characterized molecularly has significantly expanded our understanding of the host signal transduction pathway that allows bacterial invasion based on NF perception (78, 122). Putative NFreceptors, including MtNFP and MtLYK3, belong to the lysin motif (LysM) receptor-like kinase family, which have an extracellular domain homologous to the bacterial LysM proteins that bind β-(1,4)-linked GlcNAc derived from peptidoglycan. Host responses to specific NF structures depend on the LysM domain specifically, and a one amino acid difference within this motif can alter the range of rhizobia recognized for symbiosis (134). Additional NF signaling genes have been identified that function downstream of the LysM receptor-like kinases (78, 122). For example, several dmi (does not make infections) mutants have been characterized in M. truncatula, and the affected genes have orthologs in L. japonicus. Highlighting the importance of the NF-dependent Ca2+ -spiking response, MtDMI3 encodes a putative Ca2+ -calmodulindependent protein kinase (CCaMK) (115). MtDMI1 encodes a nuclear-localized cation channel that may help modulate Ca2+ -spiking, and MtDMI2 encodes a putative receptor-like kinase (76, 119). While all of the dmi mutants display root hair deformation and rapid Ca2+ influx in response to NF, dmi1 and dmi2 mutants are unable to elicit the subsequent Ca2+ -spiking response (50, 72, 75, 159, 177, 179). In contrast, the dmi3 mutant is indistinguishable from wild type with regard to each of these physiological NF responses (177). Thus, M. truncatula Annu. Rev. Genet. 2008.42:413-441. Downloaded from arjournals.annualreviews.org by University of Georgia on 03/31/09. For personal use only. DMI1 and DMI2 appear to function downstream of MtNFP and MtLYK3, but upstream of the Ca2+ -calmodulin-dependent protein kinase encoded by DMI3. Recent reports showing that DMI3 can be genetically modified to elicit spontaneous nodule formation suggest it may be possible to intelligently engineer nonlegume species that are capable of establishing symbiotic nitrogen fixation (65, 166). Although NF signaling is a nearly universal means of establishing the rhizobium nitrogenfixing symbiosis with compatible legumes, exceptions are emerging. The recent sequencing of photosynthetic Bradyrhizobium sp. BTAi1 and ORS278, which form nitrogen-fixing nodules on the roots and stems of aquatic host, Aeschynomene sensitiva, revealed that the common nodABC genes are absent in these species (63). Moreover, a NF-deficient mutant of the closely related Bradyrhizobium sp. ORS285 forms nitrogen-fixing root and stem nodules on A. sensitiva with the same efficiency as its wild-type parental strain. Thus, the host A. sensitiva initiates nodule development in a NF-independent manner and instead may respond to the secretion of bacterial purine derivatives with cytokinin-like activity, highlighting the importance that host hormone balance plays in nodule formation (63). REACTIVE OXYGEN AND NITROGEN SPECIES As with many host-microbe interactions (39, 118, 187), the rhizobium-legume symbiosis can be associated with a host-generated release of reactive oxygen species (ROS: O·− 2 , H2 O2 , and HO· ) and reactive nitrogen species (RNS: NO· ). As discussed below, application of purified S. meliloti LPS can suppress ROS production in response to fungal elicitors in M. truncatula tissue culture cells (1, 148); however, the role this response plays in symbiosis is unclear. The application of purified S. meliloti NF to M. truncatula roots also inhibits ROS production in response to fungal elicitors (151), and correspondingly limits the expression of putative ROS-generating NADPH oxidase genes (104). The NF-mediated inhibition of ROS efflux occurs within 1h of treatment and is dependent on the putative NF receptor encoded by MtNFP but not the downstream DMI1 gene (104, 151). NF-mediated suppression of ROS production plays a causative role in the root hair deformation and curling responses to rhizobia (104). However, subsequent stages of symbiosis between M. truncatula and S. meliloti result in the long-term production of ROS and RNS in distinct areas of the nodule. While RNS are primarily associated with plant cells that have been infected with bacteria (12), ROS are associated with the plant cell wall of both infection threads and infected host cells (142, 147). This ROS efflux requires NF, arguing that ROS may play a positive role in bacterial invasion (136). The M. truncatula response to NF includes induction of the rip1 gene encoding a putative peroxidase that could be involved in hydrogen peroxide-dependent cross-linking of cell wall proteins (31). Both the induction of rip1 expression and the production of ROS require that M. truncatula has a functional DMI1 gene (136), genetically separating the early DMI1-independent decrease in ROS from this later response. The DMI1 requirement for rip1 induction is bypassed when root tissues are exposed to exogenous H2 O2 (136). Generally, free-living rhizobia are more susceptible to ROS-mediated killing than are other common soil bacteria like Bacilli and Pseudomonads (120), suggesting that symbiotic levels of ROS are probably unable to differentially prevent soil pathogens from taking advantage of ITs to invade nodules. One role for ROS production may be to promote proper IT development and growth by either cross-linking cell wall glycoproteins or degrading cell wall– associated polysaccharides to aid IT elongation. Only a small percentage of newly formed ITs actually penetrate the inner cortical cell layer, and the unsuccessful, or aborted, ITs display certain characteristics of the hypersensitive plant defense response (171), which typically includes ROS production. Thus, this ROS efflux could play a role in limiting bacterial invasion. www.annualreviews.org • Molecular Determinants of a Symbiotic Chronic Infection ROS: reactive oxygen species RNS: reactive nitrogen species 421 Annu. Rev. Genet. 2008.42:413-441. Downloaded from arjournals.annualreviews.org by University of Georgia on 03/31/09. For personal use only. Recent observations are consistent with the idea that ROS may also function as a positive signal perceived by bacteria during invasion. This is based on the finding that an S. meliloti strain overexpressing the KatB catalase has a nodule invasion defect that is primarily associated with aberrant IT growth (76). KatB is responsible for detoxifying H2 O2 and thereby limits the concentration of exogenously applied ROS within the bacterial cell (4). Bacterial overexpression of KatB likely acts as a strong catalyst for detoxifying ROS and may lower the local concentration of free oxygen radicals able to modify the IT compartment. KatB overexpression presumably also leads to significantly decreased concentrations of ROS within the IT-localized bacterial cell. The symbiosis defect associated with KatB overexpression could therefore reflect the fact that ROS functions as a cytoplasmic signal that the bacterium uses to regulate functions essential to invasion. It is unlikely this ROS signal would be perceived through the OxyR redox-sensitive regulatory protein since an S. meliloti oxyR mutant has no obvious symbiosis defect with M. truncatula (41, 107). However, the putative redox-sensitive CbrA two-component histidine kinase is required for bacterial invasion of Medicago hosts and regulates a number of genes specifically required for bacterial invasion (61, 62), making CbrA a promising candidate for a bacterial redox-sensor involved in bacterial invasion. Although ROS appears to promote rhizobial invasion, these bacteria must also be able to combat this stress in order to achieve symbiosis. A screen for ROS-sensitive S. meliloti mutants that simultaneously display aberrant symbiosis phenotypes revealed a variety of functions related to bacterial metabolism and exopolysaccharide production (41). With regard to genes specifically required to detoxify ROS, S. meliloti has three that encode catalase enzymes (katA, katB, and katC) and one encoding superoxide dismutase activity (sodB) (77, 146). Several uncharacterized genes include an extracellular peroxidase (Smc01944), a bacteriocupreinfamily superoxide dismutase (sodC), and an alkylhydroperoxidase (ahpC), each of which 422 Gibson · Kobayashi · Walker may combat ROS exposure; however, null phenotypes for these genes have not been reported (7, 60). Null mutants for either katA, katB, katC, or sodA are fully capable of establishing the symbiosis (40, 77, 152), although the katAC and katBC double mutants have decreased symbiotic proficiency (77, 152). In particular, the katBC mutant has a severe symbiosis defect that results in formation of aberrantly small nodules that are incapable of nitrogen fixation (77). Although the katB and katC genes are strongly expressed in bacteria located within growing ITs, where ROS is concentrated, the phenotype of the double mutant is subsequently revealed during bacterial uptake into the host cell cytoplasm: intracellular bacteria lack the surrounding peribacteroid membrane and undergo rapid senescence for reasons that are unclear (77). MODULATION OF THE HOST DEFENSE RESPONSE The initiation of ITs is a major checkpoint for the host in terms of deciding whether to allow bacterial invasion to proceed. In the past few years, several plant mutants affected in IT formation and growth have been isolated, and their further characterization will likely shed light on some of the mechanisms that the host uses to create and control the growth of this structure (30, 91, 106, 163, 176, 189). Rhizobial invasion of the host nodule via the IT is strongly influenced by a complex variety of bacterial polysaccharides in addition to NF, including secreted EPSs and K-antigens, secreted and periplasmic cyclic β glucans, and the outer membrane-localized LPSs (14, 57, 78, 155, 157). Similar to NF, several of these molecules exert their effects on symbiosis in a structurally dependent manner, arguing that they may function as signals between invading bacteria and their host. In fact, recent evidence suggests that the exopolysaccharide succinoglycan may help further define species-specificity in addition to NF (153). A shared and outstanding question regarding these bacterial polysaccharides is their potential role in modulating a plant defense response to bacterial invasion. However, definitive proof for such possibilities awaits the identification and characterization of specific host receptors. Annu. Rev. Genet. 2008.42:413-441. Downloaded from arjournals.annualreviews.org by University of Georgia on 03/31/09. For personal use only. Exopolysaccharide The biosynthesis of rhizobial EPS and its regulation have been most extensively studied with regard to the S. meliloti macromolecule referred to as succinoglycan, or EPS I, (Figure 2c) (15, 78, 155). S. meliloti succinoglycan is secreted as a polymer of repeating octasaccharide subunits (one galactose and seven glucose residues) modified with succinyl, acetyl, and pyruvyl substituents (Figure 2c). Polymerization of the succinoglycan monomer results in secretion of either low molecular weight (LMW) forms (monomers, dimers, and trimers) or high molecular weight (HMW) forms (polymers of several hundred subunits). Succinoglycan plays a critical role in the S. meliloti symbiosis with Medicago hosts. Succinoglycan-deficient mutants elicit nodule organogenesis by virtue of NF signaling (86), but these aberrantly small nodules are devoid of bacteria and therefore incapable of nitrogen fixation (99). These rhizobial mutants are compromised for host invasion to the extent that IT formation proceeds from only 10% of bacterially colonized root hairs and the ITs that do form terminate prematurely before they reach the nodule primordium (27). While EPSs play a passive role in protecting the bacterium from host-derived stresses within the IT (36), they are also thought to perform a signaling function from rhizobia to their host (78, 79). Part of the evidence for this is based on the observation that LMW forms of succinoglycan are more effective at promoting symbiosis than HMW forms (10, 169, 182). Moreover, an exoH mutant is unable to succinylate the succinoglycan monomer and therefore produces predominantly HMW succinoglycan (98); since this mutant is also severely compromised for symbiosis a merely protective role is not sufficient to explain all of the requirements for succinoglycan in host invasion. Increasingly, the evidence available suggests that bacterial EPSs play a role in modulating the host defense response to bacterial invasion. For example, the premature termination of bacterial infection observed with EPS-deficient mutants is associated with symptoms of a host defense response and, in particular, the production of antimicrobial phenolics and phytoalexins (119, 126). To test the hypothesis that succinoglycan promotes symbiosis as a signaling molecule, a global transcriptome analysis was performed on M. truncatula plants inoculated with succinoglycan-deficient S. meliloti at a time point just prior to IT formation (3 days postinoculation) (79). Thus, this experiment identified host genes differentially regulated in response to EPS production, rather than IT failure per se, and the largest group of genes upregulated during an EPS-deficient interaction encode putative plant defense proteins (79). As mentioned above, M. truncatula defense genes are upregulated 1 h postinoculation with wild type S. meliloti; however, expression of this same class of genes decreases by 6 h postinoculation and remains low for at least 3 days (105). Taken together, these observations suggest that a primary consequence of EPS production is the suppression of a potentially lethal host defense response, and in the absence of EPS, this unproductive response may cause a block in IT formation (27). Insight into how S. meliloti regulates production and modification of exopolysaccharides is therefore important to our understanding of the physiological requirements for host invasion. An increasingly large number of regulators modulate succinoglycan production ex planta, including ExoS, ExoR MucR, SyrA, and the more recently identified CbrA (62, 155). The first regulators of succinoglycan to be identified were the ExoS two-component histidine kinase and ExoR. ExoS is an essential gene in S. meliloti and forms a two-component signal transduction pathway with the response regulator ChvI, which is also essential and functions as a DNA-binding transcription factor (26). ExoS phosphorylates ChvI directly and thereby promotes increased exo/exs expression www.annualreviews.org • Molecular Determinants of a Symbiotic Chronic Infection 423 Annu. Rev. Genet. 2008.42:413-441. Downloaded from arjournals.annualreviews.org by University of Georgia on 03/31/09. For personal use only. and greater succinoglycan production (26). ExoR has no homology to any known regulators, however a null mutation alters the transcription of several exo biosynthetic genes (138). Early observations suggested a genetic link between exoR and exoS: extragenic suppressors of an exoR null mutant symbiosis defect were mapped near exoS and these suppressors displayed a concomitant change in exo gene transcription (125). More recent evidence indicates that ExoR is a periplasmic protein that functions upstream of the ExoS/ChvI twocomponent pathway and is hypothesized to directly repress ExoS kinase activity by binding its periplasmic sensing domain (183). An outstanding question remains as to the nature of the stimulus perceived by ExoS and/or ExoR. The two-component histidine kinase CbrA has also been identified as a regulator of succinoglycan production. Unlike the overproduction of wild-type forms of succinoglycan in exoS and exoR mutants (46), a cbrA mutant appears to overproduce predominantly LMW forms of succinoglycan, and this phenotype is correlated with increased transcription of several exo genes involved in LMW succinoglycan biosynthesis, including exoH, exoK, and exoT (62). Moreover, the cbrA mutation leads to increased expression of additional genes involved in promoting bacterial IT invasion, including the ndvA transporter of cyclic β glycans (described below) and the sinI regulator of galactoglucan (EPS II) production (61). Thus, it was proposed that CbrA coordinates multiple aspects of bacterial physiology to promote bacterial invasion of the nodule, and that the physiology of the cbrA mutant is optimized for this process. Given that CbrA contains at least one PAS domain (62), a motif that commonly monitors redox changes, CbrA may coordinate bacterial physiology in response to the high redox environment of the IT. However, since the cbrA mutant remains defective for symbiosis despite a physiology optimized for IT invasion (61), it appears that CbrA plays an additional role during subsequent bacteroid differentiation, as discussed below. 424 Gibson · Kobayashi · Walker Cyclic β Glucan Rhizobia generally produce β-(1,2)-glucans that are macrocyclic and unbranched polymers of glucose, containing anywhere from 17 to 40 residues depending on the rhizobial strain (157). The synthesis of cyclic β glucan is dependent on a glycosyltransferase encoded by ndvB (called cgs in Mesorhizobium loti ) and its secretion is dependent on the ABC-type inner membrane transporter ndvA. Bradyrhizobium species are an exception and produce branched macrocyclic glucans that contain both β-(1,3) and β-(1,6) glycosidic bonds catalyzed by glycosytransferases encoded by ndvB and ndvC. The chemical characteristics of cyclic β glucans can be modified by the addition of phosphocholine, sn-1-phosphoglycerol, succinic and methylmalonic acid substituents. Disruption of ndvB (cgs) in S. meliloti (M. loti ) blocks symbiosis at the stage of bacterial attachment to root hairs and IT invasion and thereby results in the formation of aberrantly small and empty nodules (34, 48). In contrast, a B. japonicum ndvB mutant is able to elicit normal nodule development and invade host tissues, although the resulting nodules do not fix nitrogen (47). Like succinoglycan, the cyclic β glucans may play a role in modulating a host defense response to bacterial invasion. Specifically, M. loti cyclic β glycans are required to suppress highlevel production of antimicrobial phytoalexins during symbiotic development with L. japonicus (35). Many host defense response genes are induced in a mature nodule during a wildtype symbiosis (29, 90), perhaps to provide the nutrient-rich nodule with a defense against parasites. These same genes are generally expressed at decreased levels during the ineffective symbiosis of the cgs mutant, with the striking exception of highly induced PAL expression (35). PAL encodes an enzyme predicted to participate in the synthesis of antimicrobial phenolic compounds, and consistent with increased PAL expression during the cgs mutant symbiosis, L. japonicus nodules accumulate phenolic compounds to a greater extent than is observed during a wild-type symbiosis (35). Purified B. japonicum cyclic β glucans are able to block a host defense response to fungal elicitors in the determinate legume G. max and in a structurally dependent manner (17), further suggesting these polysaccharides may be able to prevent a host defense response during rhizobial invasion. Annu. Rev. Genet. 2008.42:413-441. Downloaded from arjournals.annualreviews.org by University of Georgia on 03/31/09. For personal use only. BACTERIAL REQUIREMENTS FOR INTRACELLULAR COLONIZATION Lipopolysaccharide Throughout symbiotic development the S. meliloti cell surface is in intimate association with its host but this is particularly true of the microsymbiont within the symbiosome. Not surprisingly then, the bacterial cell surface plays an important role in promoting rhizobial intracellular adaptation, including the lipopolysaccharide (LPS) component of the gram-negative outer membrane (14, 83, 157). LPS is a complex macromolecule composed of a lipid A membrane anchor and an oligosaccharide core, which can be further modified by the addition of a variable O-antigen polysaccharide (135). Generally, host perception of LPS from pathogenic bacteria plays a significant role in defense responses to invasion via the innate immune system (130). It has therefore been of great interest to understand the specific role that rhizobial LPS plays in symbiotic development. Bacteroid LPS has increased hydrophobicity compared to that of free-living bacteria, suggesting there are LPS modifications in planta that may contribute to symbiosis (38, 84). Rhizobia produce a lipid A with unique structural characteristics that distinguish it from the potent E. coli innate defense elicitor endotoxin (135), although there is variation in certain elements among the rhizobia (14, 83). For instance, most rhizobia produce lipid A species lacking either one or both of the 1- and 4′ -phosphate groups present on the β-(1,6)glucosamine disaccharide of E. coli; Sinorhizobium lipid A is an exception to this trend as it is bisphosphorylated (Figure 2d ). In addition, rhizobial lipid A moieties are most often modified with a secondary N-linked acyloxyacyl residue composed of a C28 or C30 VeryLong-Chain Fatty Acid (VLCFA) that has the capacity to span the entire lipid bilayer of the outer membrane (Figure 2d ). This particular modification has generated much interest as a structural feature that could shield rhizobial LPS from recognition by the host innate immune system and thereby promote rhizobial invasion and persistence. In fact, the presence of VLCFA modified lipid A has been observed in several related bacteria that establish persistent host infections, including all rhizobia, agrobacteria, and brucella that have been analyzed. The acpXL and lpsXL genes encode an acyl carrier protein and an acyl transferase, respectively, that together catalyze the VLCFA secondary acylation of lipid A in rhizobia. Rhizobial acpXL and lpsXL mutants completely lack the VLCFA-modified lipid A during free-living growth but remain effective at establishing a nitrogen-fixing symbiosis (53, 150, 175), although bacteroid development is mildly perturbed and leads to decreased nitrogen-fixation (173). This weak symbiosis phenotype is likely explained by the recent discovery that acpXL mutants of R. leguminosarum b.v. viciae form bacteroids that actually contain VLCFA-modified lipid A, indicating that they express an alternative system for VLCFA modification in planta (174); thus, the role that the VLCFA modification plays in symbiosis remains to be determined. Alteration of the LPS carbohydrate content, in either the core or the O-antigen, has an aberrant effect in a variety of symbioses (83). For example, an S. meliloti lpsB mutant has a dramatically altered LPS core and is incapable of establishing a chronic host infection (21). While the mutant displays normal host IT invasion and is taken up into the host cell cytoplasm, it undergoes rapid senescence and is degraded within the symbiosome compartment, suggesting LPS plays a critical role in rhizobial adaptation and persistence within the particular environment of the host cell cytoplasm (21). www.annualreviews.org • Molecular Determinants of a Symbiotic Chronic Infection VLCFA: very-longchain fatty acid 425 Annu. Rev. Genet. 2008.42:413-441. Downloaded from arjournals.annualreviews.org by University of Georgia on 03/31/09. For personal use only. The precise function of LPS in promoting symbiosis remains unclear (14). Defects in LPS can sensitize bacteria to membrane-disrupting agents and antimicrobial peptides so that it may provide a protective barrier against environmental stress and host defense responses. However, there are indications that S. meliloti LPS may also play an active role by suppressing the release of ROS (1, 148). Specifically, M. truncatula tissue culture cells respond to yeast elicitors with an oxidative burst and the increased expression of defense genes involved in plant secondary metabolism, like PAL, and cell wall metabolism (1, 164). When tissue culture cells are exposed to elicitor in the presence of S. meliloti lipid A, this LPS component is capable of suppressing the oxidative burst and dampening the plant transcriptional response (148, 164), indicating an interaction between rhizobial LPS and its host could suppress any potential immune response to intracellular bacteria. This could be particularly important for bacteria within the symbiosome as they no longer express genes for the biosynthesis of succinoglycan (9), which appears to dampen a potential plant defense response to bacteria within the IT (78, 79). Lipid A moieties isolated from the related intracellular mammalian pathogen Brucella abortus, which also undergo VLCFA modification, are only weak elicitors of an innate immune response in mouse macrophages (93, 94). Perhaps these related alphaproteobacteria share some of the strategies that rhizobia use to evade detection by the innate immune system during chronic infection. BacA The bacA gene encodes an inner membrane protein that plays an essential role in the early stages of S. meliloti bacteroid development (64). With a bacA mutant, the early steps in symbiosis, such as formation and development of infection threads, proceed as efficiently as with wild type but the mutant lyses shortly after being endocytosed into the cytoplasm of plant cells. BacA function is also essential for the chronic infection of B. abortus (100), a mam426 Gibson · Kobayashi · Walker malian pathogen that can survive and replicate in host macrophages, highlighting the broad importance of BacA function to chronic bacterial persistence during intracellular infection (140). Generally, the loss of bacA function has been associated with an increased resistance to certain antimicrobial peptides (74, 96, 113). In E. coli, deletion of the bacA isofunctional homolog, sbmA, produces increased resistance to microcins B17 and J25, bleomycin, and Bac7, which is an antimicrobial peptide of mammalian origin (96, 113). Similarly, the bacA mutants of S. meliloti and B. abortus show increased resistance to bleomycin relative to the wild type (74, 100). Thus, BacA may play a role in the transport of modified peptides across the inner membrane (64). In fact, it was proposed that BacA functions as an importer for host-derived peptide(s) that promote bacteroid differentiation (114). Specifically, it was suggested that bacterial uptake of Nodule-specific Cysteine-Rich (NCR) peptides produced by indeterminate legumes could trigger the terminal differentiation of bacteroids (3, 114), analogous to the role defensins play in suppressing the proliferation of bacterial pathogens (190). In S. meliloti and B. abortus, BacA also affects the VLCFA modification of the lipid A component of LPS (52, 54). BacA has homology to the transmembrane domain of eukaryotic ATPbinding cassette (ABC) transporters of the ABCD family (52). ABCD transporters function in the peroxisome and this includes the human adrenoleukodystrophy protein (hALDP) that is thought to transport activated VLCFAs across the peroxisomal membrane (181). Approximately 50% of lipid A moieties isolated from S. meliloti bacA mutants (as well as B. abortus) lack VLCFAs (52). Thus, it was suggested that BacA may export VLCFAs from the cytoplasm and across the inner membrane, and perhaps the lack of VLCFA-modified lipid A causes the defects in chronic infection observed with bacA mutants (52). As mentioned above, rhizobial acpXL and lpxXL mutants completely lack VLCFA-modified lipid A during free-living growth and are able to establish Annu. Rev. Genet. 2008.42:413-441. Downloaded from arjournals.annualreviews.org by University of Georgia on 03/31/09. For personal use only. a successful symbiosis despite bacteroid morphological abnormalities (53, 150, 173, 175). However, at least R. leguminosarum acpXL bacteroids contain VLCFA-modified lipid A in planta (174), indicating an alternative system for VLCFA modification exists that may require BacA function. It remains to be determined whether BacA is directly responsible for either one or both of the proposed transport reactions. S. meliloti strains carrying 12 site-directed mutations in bacA have phenotypes intermediate to the wild-type and bacA null mutant (101), consistent with a role for BacA in multiple, nonoverlapping functions. Moreover, it was reported that functional SbmA is required for full efficiency of the tetracycline exporter TetA in E. coli (42), suggesting that BacA function could similarly affect the activity of other membrane proteins. Future studies that reconstitute BacA into liposomes, followed by detailed transport assays, will be needed to elucidate its precise function. DEVELOPMENTAL REGULATION OF THE CELL CYCLE Symbiotic regulation of the plant cell cycle and the role of endoreduplication in nodule development has been studied extensively (56, 122). In contrast, an understanding of how the bacterial cell cycle may be regulated during symbiosis is just beginning to emerge. Bacteroids within determinate hosts have the ability to dedifferentiate into free-living bacteria once they are released from a senescent nodule (114). In contrast, at least some rhizobia that form a symbiosis with indeterminate legumes undergo a terminal differentiation program that precludes viability outside the host cell cytoplasm (114). Whether a given rhizobial species undergoes terminal differentiation appears to be a decision controlled by the legume host (114). During free-living growth, and presumably within the IT, S. meliloti grows as a rod-shaped bacterium with no greater than a 2N complement of its genome (Figure 3a,b) (114), which implies that these bacteria initiate DNA replication only once per cell cycle. In contrast, one remarkable aspect of terminal bacteroid differentiation is a branched cell morphology that is accompanied by several rounds of genomic endoreduplication (Figure 3c) (114). The correlation between bacteroid endoreduplication and the inability to dedifferentiate and resume growth outside the host suggests the act of endoreduplication may be a defining event in terminal bacteroid differentiation. The indeterminate symbiosis therefore represents a novel bacterial cell cycle event, i.e., endoreduplication, which may play an important role in either intracellular persistence or efficient nitrogen fixation. Taken together, these observations imply that the S. meliloti cell cycle has at least three branch points subject to in planta regulation (Figure 3d ), and it will be of great interest to understand how the cell decides which path to choose under different host conditions. While regulatory aspects of the S. meliloti cell cycle have been little studied, that of the related Caulobacter crescentus alphaproteobacterium has been dissected in great detail and thereby serves as a powerful model on which to base future studies in S. meliloti. Briefly, in C. crescentus the CtrA response regulator collaborates with the DnaA replication initiator and GcrA transcription factor to globally control cell cycle progression (154). Throughout the cell cycle, CtrA concentrations are strictly regulated, and this plays a critical role in mediating cell cycle progression. Additionally, an elaborate two-component signal transduction pathway containing the essential response regulator DivK regulates the concentration of active CtrA-P (18). Through this multi-layered regulation of active CtrA-P concentrations, C. crescentus limits the initiation of DNA replication to once per cell cycle (Figure 4a), in contrast with E. coli and other fast-growing bacteria. The coordination between DNA replication and cell division that allows only one replication initiation event per cell cycle is also observed in S. meliloti (114). C. crescentus undergoes an asymmetric cell division during each cell cycle for the purpose of nutrient adaptation (Figure 4b), and the DivJ and PleC two-component histidine kinase www.annualreviews.org • Molecular Determinants of a Symbiotic Chronic Infection 427 ←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− G1 a Figure 3 Free-living S G2 Annu. Rev. Genet. 2008.42:413-441. Downloaded from arjournals.annualreviews.org by University of Georgia on 03/31/09. For personal use only. b Infection thread c Bacteroid differentiation S x 3˜4 d G2 G1 2 Model for in planta modulation of cell cycle 1 S 3 G0 428 Gibson · Kobayashi · Walker Schematic representation of the rhizobium cell cycle at different stages of symbiosis. (a) The S. meliloti cell cycle is modeled after that of the alphaproteobacterium Caulobacter crescentus. A cell division cycle is comprised of three distinct phases: G1 , S, and G2 . In C. crescentus, DNA replication is initiated only once per cell cycle, in S phase; however, chromosome segregation begins during S phase and continues in G2 phase. Cell division begins in G2 phase and is completed before the next DNA replication initiation event. During free-living growth, S. meliloti is thought to initiate DNA replication only once per cell cycle and divides asymmetrically to produce daughter cells of different size. In analogy to C. crescentus, the small daughter cell likely proceeds into G1 phase while the larger daughter cell directly re-enters S phase. (b) S. meliloti proliferating in the IT originate from a clonal expansion of founder cells entrapped in the tip of the root hair curl. Cells appear to lack flagella and are loosely associated with one another in a pole-to-pole manner, typically forming two or three columns with a braided appearance. Active propagation of bacteria is observed only in a limited area called the growth zone near the tip of the IT, while bacteria outside of the growth zone do not grow or divide. It seems likely that the restricted growth of bacteria enables synchronization of bacterial growth with extension of the IT. (c) Bacteria colonize the cytoplasm of plant cells located in the invasion zone (see Figure 1d ). Bacteria are surrounded by a plant-derived membrane and differentiate into a bacteroid. In S. meliloti, DNA endoreduplication occurs during bacteroid differentiation and results in dramatic cell branching and enlargement to a length of 5–10 µm; in comparison, free-living counterparts are rod-shaped cells of 1–2 µm. These terminally differentiated (G0 phase) bacteroids are unable to resume future growth. Orange lines, host plasma membrane; green lines, host cell wall. (d ) A model of the S. meliloti cell cycle in planta has three possible exits from S phase, two of which (in blue) represent an exit from the typical free-living cycle (in red ). Bacteria within the infection thread are thought to progress through the cell cycle in the same manner as free-living cells, and in particular transition from S phase into G2 phase (represented by arrow 1). Bacteria that undergo bacteroid differentiation undertake the process of endoreduplication and therefore re-enter G1 phase after the completion of S phase (represented by arrow 2); the bacteria may cycle from S to G1 multiple times during endoreduplication. Once endoreduplication is complete, the bacteroid enters a terminally differentiated state (G0 ) and is no longer able to initiate cellular growth or DNA replication (represented by arrow 3). a G1 Cell cycle S Replication initiation G2 Chromosome replication Chromosome Cell segregation separation Annu. Rev. Genet. 2008.42:413-441. Downloaded from arjournals.annualreviews.org by University of Georgia on 03/31/09. For personal use only. b Caulobacter Swarmer Stalked Predivisional Daughters “Small” “Large” Predivisional Daughters c Sinorhizobium DivK DivK〜P Figure 4 Schematic representation of cell-cycle progression in C. crescentus and S. meliloti. (a) The cell cycle is comprised of three distinct phases: G1 , S, and G2 (shown in red ). The timing of cell cycle-related events is shown in blue. (b) During G1 phase, C. crescentus is a swarmer cell, which is motile and has a polar flagellum and pili. When entering S phase, the swarmer cell ejects the flagellum, retracts the pili, and differentiates into a stalked cell. The stalked cell is uniquely competent to initiate DNA replication. After chromosome segregation in G2 phase, the cell divides asymmetrically to produce two different daughter cells: a swarmer and a stalked cell. During cell-cycle progression, cellular localization of DivK is controlled by phosphorylation: nonphosphorylated DivK is uniformly distributed in the cytoplasm and DivK∼P is localized to the cell poles. (c) Recently it was shown that S. meliloti also divides asymmetrically to form a “small” cell and a “large” cell. Localization of the DivK homolog indicates that the “small” and “large” cells are counterparts of the C. crescentus swarmer and stalked cells, respectively. Flagella are omitted from this scheme because their localization has not been examined during cell cycle progression in S. meliloti. www.annualreviews.org • Molecular Determinants of a Symbiotic Chronic Infection 429 Annu. Rev. Genet. 2008.42:413-441. Downloaded from arjournals.annualreviews.org by University of Georgia on 03/31/09. For personal use only. sensors mediate this asymmetry. Other alphaproteobacteria also undergo asymmetric cell division and have orthologs of C. crescentus cell cycle regulators present in their genomes (70), including S. meliloti (Figure 4c), B. abortus, and Agrobacterium tumefaciens. CbrA has strong homology in its C-terminal kinase domain to the DivJ and PleC kinases of DivK (70), suggesting that its primary function may be to regulate cell cycle progression in S. meliloti via regulation of DivK phosphorylation, as was observed for its B. abortus ortholog PdhS (71). In fact, during free-living growth the cbrA mutant displays a filamentous, branched morphology that coincides with a greater than 2N genomic content, indicating the mutant is unable to coordinate DNA replication with cell division (K.E. Gibson & G.C. Walker, unpublished). Perhaps CbrA plays a role in modifying the cell cycle program during symbiosis and loss of this function is primarily responsible for the in planta defects of the cbrA mutant. If this is the case, it will be of interest to determine how CbrA coordinates cell cycle progression with cell envelope physiology and succinoglycan production. It is possible that at least some of the cbrA mutant phenotypes are an indirect consequence of altered cell cycle progression given that multiple cell surface characteristics are cell cycle regulated in C. crescentus. The process of endoreduplication undoubtedly creates an intense demand for dNTPs within the developing bacteroid. The dNTPs required for DNA metabolism in all organisms are synthesized by the enzyme ribonucletide reductase (RNR) (78). S. meliloti has only one RNR encoded in its genome, NrdJ, and this is a vitamin B12 -dependent enzyme (33). As a Class II RNR that is both oxygen-independent and oxygen-insensitive (80), this enzyme likely provides a key adaptation for rhizobial persistence within the microaerobic environment of the host cell cytoplasm. The B12 biosynthetic enzyme BluB, which catalyzes formation of the lower ligand 5,6-dimethylbenzimidazole, was fortuitously found to be required for symbiosis between S. meliloti and M. sativa based on its involvement in succinoglycan biosynthesis 430 Gibson · Kobayashi · Walker (22, 162). A bluB mutant appears able to infect the host via normal IT growth, suggesting that any alteration to the succinoglycan does not affect invasion; however, bacteroids are not observed within the host cell cytoplasm and the nodules that develop are unable to fix nitrogen (22). Thus, BluB function in B12 biosynthesis is necessary for symbiosis due to the requirement for a B12 -dependent enzyme(s), for which NrdJ is one possible candidate. During the indeterminate symbiosis between Mesorhizobium huakuii and Astragalus sinicus, bacterial DNA replication is limited to those bacteria associated with the meristem in Infection Zone II and Interzone II/III (89). This observation suggests that, at least in some rhizobia, DNA replication is permanently blocked once the bacteroid completes endoreduplication. Bacterial genes involved in DNA repair are induced within mature nodules (9), suggesting that DNA integrity is maintained within bacteroids in the absence of DNA replication. Importantly, these DNA repair genes include several that encode nonhomologous endjoining (NHEJ) proteins involved in double strand break repair (88), a process also utilized by terminally differentiated cells in higher eukaryotes. Little is currently known regarding rhizobial cell division during symbiosis, although the altered morphology of bacteroids implies an underlying regulation of this process. Several S. meliloti genes involved in cell division have been characterized, for instance ftsZ1 and ftsZ2 (108, 110, 111), as well as minCDE (28). Blocking the process of cell division via overexpression of ftsZ1 or minCD causes altered cell morphology during free-living growth that is reminiscent of the branched and filamentous bacteroid (28, 95). Treatment of S. meliloti with DNA-damaging agents, which impinge on DNA replication and cell division in other bacteria, also inhibits cell division and results in branched cell morphology (95). Although the mechanisms that underlie these changes in cell morphology and how they relate to bacteroid differentiation are still unknown, ftsZ1 and ftsZ2 expression is decreased in bacteroids consistent with a block in cell division after differentiation (9, 13). Annu. Rev. Genet. 2008.42:413-441. Downloaded from arjournals.annualreviews.org by University of Georgia on 03/31/09. For personal use only. NUTRIENT EXCHANGE Invading bacteria within the IT are ultimately endocytosed by postmitotic G0 endoploid cells and remain encapsulated within a modified plasma membrane called the peribacteroid membrane (PBM) (Figure 1d, f ). The symbiosome, consisting of the PBM and its microsymbiont, is thereby formed and within this PBM-bound compartment bacteria establish a persistent, or chronic, infection of the host. In this manner, a single host cell will ultimately house hundreds of bacteria. Only after bacteria have gained access to the host cell cytoplasm do they differentiate into the morphologically distinct bacteroid form that is capable of nitrogen fixation. The in planta differentiation of rhizobia involves significant metabolic changes that promote adaptation and nitrogen fixation (121, 131). For example, rhizobia undertake respiratory chain modifications that allow energy utilization under microaerobic conditions, repress glycolysis genes, and activate C4 -dicarboxylic acid utilization pathways for carbon metabolism. Induction of nitrogenase gene expression is triggered in a FixJ-dependent manner by the microaerobic conditions of the host cell cytoplasm (55), which is in turn critical for the activity of this oxygen-sensitive enzyme (45, 92). In addition to genes required for nitrogenase activity, FixJ is responsible for induction of nearly all the bacterial genes whose expression increases in mature nodules (9), indicating that the oxygen-sensing two-component pair FixL and FixJ plays a profound role in regulating bacteroid physiology. In contrast, host gene expression in nodules colonized by the rhizobial fixJ mutant is largely indistinguishable from nodules colonized by wild-type rhizobia, suggesting that nodule morphogenesis and bacterial invasion contribute more to host gene regulation than does nitrogen fixation (9). Creation and maintenance of the host microaerobic environment is dependent on structural aspects of the nodule that form an oxygen diffusion barrier in combination with high expression levels of plant leghemoglobin, which can be 25% of total soluble protein in a nodule and helps limit the concentration of free oxygen to 3–22 nM (124, 167, 186). The host supports high nitrogenase activity by providing bacteroids with a constant flux of O2 for aerobic respiration and with energy in the form of C4 -dicarboxylic acids derived from the photosynthate sucrose (102, 141, 143). The metabolic product of the nitrogenase enzyme reaction is ammonia, and this appears to be provided to the host both directly and indirectly through its incorporation into alanine by the bacterial enzyme alanine dehydrogenase (2). Generally, nitrogen secreted from the bacteroid is assimilated by the host through its incorporation into the amino acids glutamine and glutamate by the enzymes glutamine synthetase (GS) and glutamate synthase (GOGAT), respectively (185). In determinate nodules, the large bacterially infected cells are interspersed with small noninfected cells that appear to be specialized for further nitrogen assimilation, involving conversion of amino acids (glutamine and arginine) into either ureides or amides that are exported from the nodule to the rest of the plant. PBM: peribacteroid membrane HOST SANCTIONS ON SYMBIOTIC RHIZOBIA From an evolutionary point of view, why do rhizobial bacteria maintain the large number of genes required for mutualism with their legume hosts (44)? This question is particularly relevant in light of the recent observation that bacteroids within indeterminate nodules are terminally differentiated and unable to give rise to progeny (114). However, even a mature indeterminate nodule in the soil can contain anywhere from 105 –1010 clonally related bacteria that are located within the invasion zone (Zone II; Figure 1a) and have not yet differentiated. Thus, a single symbiotic rhizobium is predicted to have greater fitness if it successfully colonizes a nodule than its nonsymbiotic cousin residing in the soil where growth can be severely limited by nutrient availability. www.annualreviews.org • Molecular Determinants of a Symbiotic Chronic Infection 431 Annu. Rev. Genet. 2008.42:413-441. Downloaded from arjournals.annualreviews.org by University of Georgia on 03/31/09. For personal use only. While there appears to be a fitness gain for rhizobia able to invade the nodule, it is also clear that the host has evolved mechanisms that prevent nonfixing rhizobia from parasitizing the legume nodule for energy. While the host controls the infection process and nodule morphology, it is the microsymbiont that largely dictates the efficiency of nitrogen fixation. Mathematical modeling suggests that if legumes treat fixing and nonfixing rhizobial strains within the nodule similarly, then nonfixing rhizobia would quickly outcompete nitrogen fixers (184). Perhaps for this reason, the host imposes effective sanctions on nonfixing rhizobial cheaters within the nodule (85, 184). So far, host sanctions have been found to take the form of severe O2 limitation to nonfixing rhizobia within the nodule, which restricts bacterial growth and viability. Thus, the legume host is capable of imposing selective pressure on rhizobia that may affect the evolution of bacterial populations in favor of nitrogen fixers (44). SUMMARY POINTS 1. There tends to be strict species-specificity between legumes and their compatible symbionts, and this specificity is defined in part by NF structure. NF elicits changes in host physiology and gene expression that lead to nodule development by virtue of a NFperception pathway involving LysM receptor-like kinases. The host specificity engendered by NF structure is dependent on the extracellular LysM domain, which likely binds NF directly. Initial IT formation and its continued growth also has NF requirements and in fact displays a greater stringency for chemical modification of the NF backbone than do earlier physiological responses. 2. NF elicits two temporally and genetically separable effects on host ROS production. During initial stages of symbiosis, NF is responsible for reducing ROS production and this promotes early morphological responses in root hairs that initiate the process of rhizobial invasion. During subsequent nodule development, a host-derived ROS efflux produced in response to NF plays a positive role in promoting bacterial IT invasion, possibly by modifying the cell wall of the IT or by providing a redox signal to bacteria that elicits IT-specific physiological adaptations. 3. A variety of rhizobial cell-surface and secreted polysaccharides appear to function as signals from the bacterium to its host. In particular, transcriptome analyses of host responses to polysaccharide-deficient bacterial mutants suggest that at least one role for these macromolecules is to suppress the expression of certain host defense response genes, such as PAL, that are involved in the production of antimicrobial compounds. Moreover, purified rhizobial LPS and cyclic β glucan are capable of inhibiting the plant defense response to fungal elicitor in a tissue culture setting. 4. S. meliloti typically initiates DNA replication only once per cell cycle during free-living growth and presumably within the host IT. However, bacteria that colonize the host cell cytoplasm undergo the novel process of endoreduplication as it differentiates into a bacteroid. Ultimately, this bacteroid becomes terminally differentiated such that no further DNA replication or cellular growth is observed and its viability becomes strictly limited to the intracellular environment of the host. These observations imply that the S. meliloti cell cycle has at least three unique branch points that are subject to in planta regulation. 432 Gibson · Kobayashi · Walker DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review. ACKNOWLEDGMENTS Annu. Rev. Genet. 2008.42:413-441. Downloaded from arjournals.annualreviews.org by University of Georgia on 03/31/09. For personal use only. We apologize to those investigators whose work we could not discuss owing to space limitations. We thank members of the Walker lab for many thoughtful discussions and for critical reading of the manuscript. This work was supported by the National Institute of Health grant GM31010 (to G.C.W.), National Cancer Institute (NCI) grant CA21615-27 (to G.C.W.) and JSPS Postdoctoral Fellowships for Research Abroad (to H.K.). G.C.W. is an American Cancer Society Research Professor. LITERATURE CITED 1. Albus U, Baier R, Holst O, Puhler A, Niehaus K. 2001. Suppression of an elicitor-induced oxidative burst in Medicago sativa cell-cultures by Sinorhizobium meliloti lipopolysaccharides. New Phytol. 151:597–606 2. Allaway D, Lodwig EM, Crompton LA, Wood M, Parsons R, et al. 2000. 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Downloaded from arjournals.annualreviews.org by University of Georgia on 03/31/09. For personal use only. Contents Volume 42, 2008 Mid-Century Controversies in Population Genetics James F. Crow ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 1 Joshua Lederberg: The Stanford Years (1958–1978) Leonore Herzenberg, Thomas Rindfleisch, and Leonard Herzenberg ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣19 How Saccharomyces Responds to Nutrients Shadia Zaman, Soyeon Im Lippman, Xin Zhao, and James R. Broach ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣27 Diatoms—From Cell Wall Biogenesis to Nanotechnology Nils Kroeger and Nicole Poulsen ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣83 Myxococcus—From Single-Cell Polarity to Complex Multicellular Patterns Dale Kaiser ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 109 The Future of QTL Mapping to Diagnose Disease in Mice in the Age of Whole-Genome Association Studies Kent W. Hunter and Nigel P.S. Crawford ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 131 Host Restriction Factors Blocking Retroviral Replication Daniel Wolf and Stephen P. Goff ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 143 Genomics and Evolution of Heritable Bacterial Symbionts Nancy A. Moran, John P. McCutcheon, and Atsushi Nakabachi ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 165 Rhomboid Proteases and Their Biological Functions Matthew Freeman ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 191 The Organization of the Bacterial Genome Eduardo P.C. Rocha ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 211 The Origins of Multicellularity and the Early History of the Genetic Toolkit for Animal Development Antonis Rokas ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 235 Individuality in Bacteria Carla J. Davidson and Michael G. Surette ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 253 vii Transposon Tn5 William S. Reznikoff ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 269 Selection on Codon Bias Ruth Hershberg and Dmitri A. Petrov ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 287 How Shelterin Protects Mammalian Telomeres Wilhelm Palm and Titia de Lange ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 301 Annu. Rev. Genet. 2008.42:413-441. Downloaded from arjournals.annualreviews.org by University of Georgia on 03/31/09. For personal use only. Design Features of a Mitotic Spindle: Balancing Tension and Compression at a Single Microtubule Kinetochore Interface in Budding Yeast David C. Bouck, Ajit P. Joglekar, and Kerry S. Bloom ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 335 Genetics of Sleep Rozi Andretic, Paul Franken, and Mehdi Tafti ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 361 Determination of the Cleavage Plane in Early C. elegans Embryos Matilde Galli and Sander van den Heuvel ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 389 Molecular Determinants of a Symbiotic Chronic Infection Kattherine E. Gibson, Hajime Kobayashi, and Graham C. Walker ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 413 Evolutionary Genetics of Genome Merger and Doubling in Plants Jeff J. Doyle, Lex E. Flagel, Andrew H. Paterson, Ryan A. Rapp, Douglas E. Soltis, Pamela S. Soltis, and Jonathan F. Wendel ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 443 The Dynamics of Photosynthesis Stephan Eberhard, Giovanni Finazzi, and Francis-André Wollman ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 463 Planar Cell Polarity Signaling: From Fly Development to Human Disease Matias Simons and Marek Mlodzik ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 517 Quorum Sensing in Staphylococci Richard P. Novick and Edward Geisinger ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 541 Weird Animal Genomes and the Evolution of Vertebrate Sex and Sex Chromosomes Jennifer A. Marshall Graves ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 565 The Take and Give Between Retrotransposable Elements and Their Hosts Arthur Beauregard, M. Joan Curcio, and Marlene Belfort ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 587 Genomic Insights into Marine Microalgae Micaela S. Parker, Thomas Mock, and E. Virginia Armbrust ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 619 The Bacteriophage DNA Packaging Motor Venigalla B. Rao and Michael Feiss ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 647 viii Contents The Genetic and Cell Biology of Wolbachia-Host Interactions Laura R. Serbus, Catharina Casper-Lindley, Frédéric Landmann, and William Sullivan ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 683 Effects of Retroviruses on Host Genome Function Patric Jern and John M. Coffin ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 709 Annu. Rev. Genet. 2008.42:413-441. Downloaded from arjournals.annualreviews.org by University of Georgia on 03/31/09. For personal use only. X Chromosome Dosage Compensation: How Mammals Keep the Balance Bernhard Payer and Jeannie T. Lee ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ ♣ 733 Errata An online log of corrections to Annual Review of Genetics articles may be found at http:// genet.annualreviews.org/errata.shtml Contents ix