molecular determinants of a symbiotic chronic infectionglycobiology/walker review.pdf · molecular...

ANRV361-GE42-19 ARI 11 October 2008 10:52

Molecular Determinantsof a Symbiotic ChronicInfectionKatherine E. Gibson,1,2 Hajime Kobayashi,2

and Graham C. Walker2

Present address 1Department of Biology, University of Massachusetts, Boston,Massachusetts 021252Department of Biology, Massachusetts Institute of Technology, Cambridge,Massachusetts 02139; email: [email protected]

Annu. Rev. Genet. 2008. 42:413–41

The Annual Review of Genetics is online atgenet.annualreviews.org

This article’s doi:10.1146/annurev.genet.42.110807.091427

Copyright c© 2008 by Annual Reviews.All rights reserved

0066-4197/08/1201-0413$20.00

Key Words

Rhizobium, legume, bacterial invasion, signaling, stress response, cellcycle

AbstractRhizobial bacteria colonize legume roots for the purpose of biologicalnitrogen fixation. A complex series of events, coordinated by host andbacterial signal molecules, underlie the development of this symbioticinteraction. Rhizobia elicit de novo formation of a novel root organwithin which they establish a chronic intracellular infection. Legumespermit rhizobia to invade these root tissues while exerting control overthe infection process. Once rhizobia gain intracellular access to theirhost, legumes also strongly influence the process of bacterial differenti-ation that is required for nitrogen fixation. Even so, symbiotic rhizobiaplay an active role in promoting their goal of host invasion and chronicpersistence by producing a variety of signal molecules that elicit changesin host gene expression. In particular, rhizobia appear to advocate fortheir access to the host by producing a variety of signal molecules ca-pable of suppressing a general pathogen defense response.

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INTRODUCTION

Although nitrogen is one of the most abun-dant elements present on Earth, it is also oneof the most limiting for biological growth be-cause it is largely found in the inaccessible formdinitrogen (N2). Biological nitrogen fixationis the process by which chemically inert N2

present in the atmosphere is enzymatically re-duced to the metabolically usable form ammo-nia (NH3) through the action of nitrogenase(129). The ability to catalyze the conversion ofN2 to NH3 has evolved only among microbes,including the rhizobia, cyanobacteria, azobac-teria, frankia, and archaea (137).

This review focuses on developmentalevents that underlie biological nitrogen fixa-tion within the context of an intimate symbio-sis between rhizobia, a phylogenetically diversegroup of gram-negative soil bacteria withinthe family Rhizobiaceae, and their hosts theLeguminosae (or Fabaceae) family of floweringplants. This rhizobium-legume symbiosis is es-tablished under nitrogen-limiting soil condi-tions and is estimated to contribute nearlyhalf of all current biological nitrogen fixa-tion (68). In addition to its environmental andagricultural significance (170), this symbiosisprovides a tractable model system for identify-ing and characterizing certain mechanisms em-ployed by invasive bacteria during chronic hostinteractions as they transition from a free-livingenvironment to their niche within the host.In fact, many rhizobial genes required for ei-ther host invasion or chronic persistence haveorthologs in closely related alphaproteobac-terial pathogens, such as Agrobacterium andBrucella, and often these orthologs have an ef-fect on virulence (11, 140, 156). Moreover,plants can detect phytopathogens using recep-tors that are evolutionarily related to thoseemployed by legumes to detect symbiotic rhi-zobia (144, 180). Thus, the broad importanceof this symbiosis is ever growing.

Due to the wide breadth of biological pro-cesses involved in establishing the legume-rhizobium symbiosis, as well as the naturaldiversity in symbiotic mechanisms, we are un-

able to review all the events that underliethis complex interaction. For instance, not dis-cussed here are the Type III and Type IV se-cretion systems that play an important role indiverse rhizobial symbioses (32, 112), nor thebacterial quorum sensing systems that modu-late symbiotic interactions (67, 145). In addi-tion, it is clear that plant hormone balance playsa critical role in regulating nodule development(122). Rather, our goal is to provide the readerwith molecular insight into several regulatoryaspects of this symbiosis while providing refer-ence to reviews that discuss these processes inmore detail.

EVOLUTION OF SYMBIOSIS

The rhizobium-legume symbiosis, a relativelyrecent evolutionary adaptation, is thought tohave evolved from the ancient arbuscular my-corrhizal symbiosis that is nearly ubiquitousthroughout the plant kingdom and providesplants with the essential mineral nutrient phos-phorus (73). This evolutionary relationship hasbeen inferred based on findings that severalhost genes represent common requirements forthe establishment of both rhizobial and mycor-rhizal symbioses. Given that nearly all vascularplants interact with mycorrhizal symbionts, itremains unclear why the nitrogen-fixing sym-biosis is strictly limited to legume species, withthe exception of Parasponia. Current under-standings of legume evolution and the appear-ance of nodulation indicate that the first sym-biosis event involved bacterial invasion of rootsvia cracks in the host epidermis where lateralroots emerge (158). Subsequent to this, devel-opmental mechanisms evolved, likely throughthe process of gene duplication, to craft thehighly selective symbiosis described here. Inparticular, the emergence of a host-derived in-fection structure allows host control over thebacterial infection process (59).

The symbiotic capacity of rhizobia isthought to have evolved in part through hor-izontal gene transfer events based on severalobservations. Within the symbiotic rhizobial

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lineage, Sinorhizobium is estimated to havediverged from Bradyrhizobium approximately500 Mya (168), which is well before the ini-tial appearance of legume species approximately60 Mya (158). Rhizobia also tend to have largeand multipartite genomes consisting of a chro-mosome supplemented with one or more in-dependent plasmids (81), which contribute toan evolutionarily dynamic genome through theprocess of horizontal gene transfer. Moreover,rhizobial genes involved in symbiosis are of-ten located within chromosomal islands or onplasmids: Sinorhizobium meliloti genes involvedin NF biosynthesis (nod, nol, noe) and nitro-gen fixation (nif and fix) are located on pSymAwhereas others required for EPS (exopolysac-charide) biosynthesis (exo) and C4-dicarboxylicacid utilization (dct) are located on pSymB (60).Horizontal transfer of these genomic elementshas been observed among bacteria within therhizosphere and has the ability to convert anonsymbiont into a symbiont through a sin-gle transfer event (6, 160, 161). Other thansymbiosis genes, there is no significant syntenyshared between the plasmids of various rhizo-bial species or rhizobial chromosomes (19, 188).Finally, it was recently discovered that certainbetaproteobacteria are also capable of estab-lishing nitrogen-fixing symbioses with legumes(117), and comparative phylogenetic analysessupport the notion that the plasmid-borne sym-biotic genes (nod and nif ) in these Burkholderiaare derived from at least one horizontal genetransfer event (25).

NODULE DEVELOPMENT

To establish the symbiosis, free-living soil rhi-zobia elicit de novo formation of a special-ized root organ, the nodule, from their host.The root nodule ultimately houses many thou-sands of individual rhizobia that catalyze ni-trogen fixation. Organogenesis of the noduleoccurs via a developmental program that in-volves dedifferentiation of quiescent (G0) rootcortical cells into an actively dividing meristem(Figure 1a), which is functionally analogous toan animal stem cell population (159). These di-

Rhizobia: gram-negativealphaproteobacteriathat engage insymbiosis withlegumes and fixnitrogen

Horizontal genetransfer:incorporation ofgenetic material froman unrelated organism(also called lateral genetransfer)

Chromosomal island:section within agenome havingevidence of horizontalorigins

NF: nodulation (Nod)factor

EPS:exopolysaccharide

Rhizosphere: localsoil environmentsurrounding plantroots

Synteny: preservationof gene order onchromosomes ofrelated species

Meristem:undifferentiated plantcells undergoing cellgrowth and division

Endoreduplication:repeated genomicreplication withoutcytokinesis

Endoploid: cells thathave undergoneendoreduplication

IT: infection thread

Indeterminatenodule: nodule with apersistent meristemand a heterogenousplant and bacterialdeveloping cellpopulation

viding meristem cells form the initial noduleprimordium from which a mature nodule de-velops (Figure 1a). A subset of these meristemcells enter a modified cell cycle program that re-sults in endoreduplication and produces greatlyenlarged cells. It is this subset of endoploid cellsthat are invaded and colonized by rhizobia forthe purpose of nitrogen fixation (Figure 1d ).

To colonize the developing nodule, bacte-ria present in the soil provoke root hair curlingby the host (Figure 1a,b). The root hair curltraps intimately associated bacteria, which arethen able to invade the nodule through a host-derived structure called the infection thread(IT) (Figure 1a,c). The IT is a tube originatingat the tip of the root hair that initially formsvia localized hydrolysis of the cell wall and thatcontinues its growth via deposition of new cellwall material (59). In this way, the IT carriesbacteria from the root surface toward the popu-lation of newly created endoploid cells where itundergoes significant ramification. Ultimately,bacteria within the IT are deposited into thehost cell cytoplasm in a process that resemblesendocytosis; they then enter a differentiationprogram that results in their ability to catalyzenitrogen fixation.

Generally, nodules fall into two morpholog-ical classes based on their pattern of meristemgrowth: indeterminate, with a meristem derivedfrom inner cortical cells, and determinate, witha meristem derived from outer cortical cells. In-determinate and determinate nodules also differin the relative persistence of meristem prolifer-ation. In determinant legumes, including mostOld World tropical species and Lotus japonicus,the basal meristem undergoes a limited spurtof cell division such that further nodule growthdepends on an expansion in cell size rather thanin cell number. Both plant and bacterial de-velopment proceeds synchronously within thisparticular type of nodule; an exception is senes-cence, which occurs within older nodules onan individual cellular basis (133). Thus, a de-terminate nodule contains a relatively homoge-nous population of developing plant and bacte-rial cells at any given time point, which allowsprecise analysis of temporal gene expression

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Determinate nodule:nodule with atemporary meristemand a relativelyhomogenous plant andbacterial developingcell population

and function within both symbiotic partners(Figure 1e, f ). In contrast, legumes that formindeterminate nodules, including most temper-ate species such as Medicago truncatula, create apersistent meristem. In these nodules, the plantallows prolonged bacterial invasion of postmi-totic (G0) endoploid cells that are continuouslygenerated by the meristem. As a result, eachdevelopmental stage required to establish thesymbiosis is present within a single mature in-determinate nodule and in a spatially organizedmanner (Figure 1a). The distinct regions found

OOH

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in a mature indeterminate nodule, proximal todistal from the root, are the meristem (Zone I),the invasion zone (Zone II), the interzone (ZoneII/III), the nitrogen-fixing zone (Zone III), andthe senescent zone (Zone IV) (172). In additionto distinct morphological features, these zonescan be distinguished by patterns of host gene ex-pression. While much research has focused onplant and bacterial physiology in Zones I–III,comparatively less is known regarding the ge-netic regulation and physiology of senescencein Zone IV although this process is also of crit-ical agricultural consequence (133).

BACTERIAL INVASION

There is often strict specificity in the establish-ment of a nitrogen-fixing symbiosis between

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 1Schematic model of nodule development. (a-b) Hostflavonoids exuded into the soil trigger bacterial NodFactor production. Nod factor is perceived by hostreceptors and elicits various host responses, such asroot hair curling and root hair invasion. Root hairinvasion also requires bacteria EPS and host ROSproduction. Nod factors induce mitotic cell divisionin the root cortex (represented in blue), leading toformation of the nodule meristem. An indeterminatenodule originates from the root inner cortex and hasa persistent meristem (Zone I). The nodule alsocontains an invasion zone (Zone II) and a nitrogen-fixing zone (Zone III). In older nodules, a senescentzone (Zone IV) develops in which both plant andbacterial cells degenerate. (c) Bacteria enter thenodule through root hairs in a structure called theinfection thread (IT) that elongates toward thenodule meristem; nucleus (n), vacuole (v). (d ) At thetip of the growing IT, bacteria are endocytosed intothe cytoplasm of postmitotic endoploid cells. Eachbacterium is surrounded by a host-derivedperibacteroid membrane (PBM) and proceeds todifferentiate into the specialized symbiotic formcalled a bacteroid. Bacteroids establish a chronicinfection of the host cytoplasm and enzymaticallyreduce dinitrogen to provide a source of biologicallyusable nitrogen to the host (Zone III). (e) In contrastto an indeterminate nodule, a determinate nodulelacks a persistent meristem and all developmentalstages proceed synchronously. ( f ) Infected cells ofdeterminate nodules typically lack vacuoles (v).


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host legume species and their symbionts. Forinstance, the bacterium S. meliloti is compatiblewith species of Medicago (alfalfa), Melilotus(sweetclover), and Trigonella (fenugreek),whereas Rhizobium etli is compatible only withspecies of Phaseolus (bean). In contrast to theserestricted host range rhizobia, some bacteriahave a broad host range, like Rhizobium sp.NGR234, which is capable of nodulating 232legumes from 112 distantly related genera(132). Based on rhizobial phylogenetic rela-tionships, it was suggested that the restrictedhost range symbiosis evolved from an ancestralbroad host range symbiosis (132), and perhapsthe specificity engendered by narrow hostrange interactions creates a finely tuned andmore effective symbiosis.

The Host Flavonoid Signal

Compatible rhizobia are uniquely capable ofgaining entry and invading the host nodulebased on a series of reciprocal signaling events(Figure 1b). The principal signals originatingfrom the host and perceived by rhizobia in thesoil are derived from the flavonoid family ofsecondary plant metabolites (Figure 2a). Theroots of leguminous plants exude a diverse cock-tail of flavonoids and isoflavonoids into the soil(128). Compatible bacteria in the rhizospherecan elicit quantitative increases and qualitativechanges in flavonoid exudation. Exactly whichflavonoid in the rhizosphere a compatible bac-terium perceives can be difficult to determinesince plants secrete a complex mixture. In fact, itis quite likely that the spectrum of flavonoids ex-uded by a legume, rather than a single flavonoidcompound, provides a determinant for hostspecificity. At least some rhizobia are chemo-tactic toward compatible flavonoids, suggestingthat certain aspects of host specificity are estab-lished before the bacterium and its host physi-cally interact (20).

Host Flavonoids Are Perceivedby Bacterial NodDs

Plant-derived flavonoids elicit a significanttranscriptional response from compatible bac-

teria within the rhizosphere, most of whichis NodD-dependent and results in NodulationFactor (NF) signal production (Figure 2b) (23).While expression of genes required for NF syn-thesis is induced by host flavonoids, expressionof these same genes can be repressed in the pres-ence of ammonia.

NodD belongs to the LysR family of DNA-binding transcription factors, which have an N-terminal ligand-binding domain that regulatesthe activity of the associated C-terminal DNA-binding domain. The NodD ligand-bindingdomain is thought to function as a flavonoidreceptor and, in the presence of a compat-ible host, NodD induces the expression ofgenes involved in NF biosynthesis. For exam-ple, the daidzein and genistein isoflavonoids ofGlycine max (soybean) induce NF gene expres-sion in Bradyrhizobium japonicum (Figure 2a).However, daidzein prevents NF production inthe noncompatible bacterium S. meliloti, whichresponds positively to the flavone luteolin(Figure 2a), and does so in a NodD-dependentmanner (127). Generally, the NodDs of broadhost range rhizobia respond to a wider rangeof flavonoid species than those present in re-stricted host range bacteria (128). For instance,NodD1 from the broad host range symbiontRhizobium sp. NGR234 responds positively toa structurally diverse range of compounds, in-cluding phenolics (vanillin and isovanillin) thatare inhibitors for other rhizobia (97). The trans-fer of Rhizobium sp. NGR234 nodD1 to a re-stricted host range rhizobium, like S. meliloti,extends the spectrum of plants the transconju-gant recognizes as a symbiotic partner (16).

NodD regulates transcription by binding cis-acting regulatory sequences called nod-boxesand these elements are generally found up-stream of the promoters of the nod, nol, and noegenes involved in NF production (8, 82). How-ever, interesting nuances to NodD-dependentregulation are beginning to emerge, includingthe identification of genes unrelated to NFbiosynthesis within the NodD regulon (87, 109,165), and a temporal progression to flavonoid-induced gene expression that implies NodD co-ordinates a complex regulatory hierarchy (87).


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a Flavenoids b Nod factor

c Succinoglycan

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Figure 2Representativesignaling moleculescritical for symbiosis.(a) Host flavonoids:luteolin, daidzein, andgenestin. (b) The Nodfactor produced byS. meliloti andbiosynthetic enzymes(Nod proteins)involved in itssynthesis. (c) TheS. melilotiexopolysaccharidesuccinoglycan. ExoHis responsible forsuccinyl modification;succinoglycanmolecular weight iscontrolled by ExoPTQand two extracellularglycosylases, ExsH andExoK. (d ) Schematicrepresentation ofS. melilotilipopolysaccharide(LPS). LpsB is aglycosyltransferasewith broad substratespecificity involved insynthesis of the LPScore. AcpXL, LpsXL,and BacA are requiredfor the Very-Long-Chain Fatty Acid(C28) modification oflipid A.


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The Bacterial NF Response

Bacterial NF functions as a key that opens thedoor to its host (128), meaning there is a highdegree of stringency for NF chemical structurethat determines whether the host allows bacte-rial invasion to proceed. NF also functions as amitogen and modifies the plant hormone bal-ance to elicit the primordium formation thatultimately gives rise to mature nodule tissue(Figure 1a) (37, 56, 122). It was recently shownthat NF also plays a role in S. meliloti biofilm de-velopment and in a host-independent manner(58); thus, NF appears to perform a significantrole in both free-living and symbiotic lifestyles.

NF is a complex signaling molecule secretedfrom the cell as a cocktail of β-(1,4)-linkedN-acetyl-d-glucosamine (GlcNAc) trimers,tetramers, or pentamers (Figure 2b) (37). Thechitin backbone is modified on the nonreducingterminal residue at the C2 position by a fattyacid; however, the size and saturation-state ofthis lipid chain varies in a species-specific man-ner. NF can be further decorated with a va-riety of chemical substituents, including acetyl,arabinosyl, carbamoyl, fucosyl, methyl, and sul-furyl additions. In fact, a given rhizobial specieswill produce a mixture of NF compounds, any-where from 2 to 60 distinct molecules, and thisis especially true of broad host range bacteria(128).

The diversity of NF structures produced byrhizobia derives from the combined presenceof species-specific genes and allelic variation ofthe common nodulation genes (128). Commonnod genes (including nodA, B, and C ), whichare found in nearly all rhizobial species and arecapable of cross-species complementation, areresponsible for synthesis of the NF chitin back-bone. In contrast, host-specific nod genes con-fer specificity for nodulation of a particular hostand are involved in various modifications of thechitin backbone. The ability to synthesize andsecrete NF can be transferred to Escherichia coliby introduction of the NF biosynthetic genecluster, and this confers upon E. coli the abilityto elicit several of the early NF host responsesdescribed below (178).

Mitogen: a substancethat elicits cell division

Transcriptome:complete set mRNAsexpressed in a cell orcell population

Host Responses to NF

A number of physiological responses to NF areobserved when either bacteria or purified NFare applied to roots (37), and these responseshave been used to position host genes within asignaling pathway (122). Purified NF is effec-tive at eliciting most host responses at nanomo-lar concentrations. Initial root epidermalresponses include an alkalinization of the cy-tosol, and a depolarization of the plasma mem-brane within minutes of root inoculation. Thesetwo responses appear to depend on a briefNF-induced Ca2+-influx that precedes them byseconds, and they are closely followed by aprolonged Ca2+-spiking response that lasts be-tween 20 to 60 min. Purified NF is also suffi-cient to induce root hair deformation and roothair curling within a few hours of application.Root hair deformation likely relies on Ca2+-induced changes to the organization of the actincytoskeleton, which produce a reorientation ofcell growth. In fact, NF can accumulate withinthe host plasma membrane (66), and appears toprovide a direct positional cue to the host suchthat the tip of the root hair grows toward thesite of greatest NF concentration (51).

NF elicits significant changes in the ex-pression of host genes, including those in-duced early in nodule development that are re-ferred to as early nodulin, or ENOD, genes (37,122). More globally, transcriptome profiles re-veal that plant genes predicted to be involvedin responses to abiotic and biotic stresses, aswell as cell reorganization and proliferation,are rapidly induced by rhizobia and largely ina NF-dependent manner (49, 105, 116). Atthe earliest postinoculation time point of 1 h,M. truncatula genes that encode functions re-lated to abiotic stress and disease resistance areupregulated while those involved in translationand cytoskeletal organization are downregu-lated (105). Abiotic stress and disease resistancegenes become significantly downregulated by6 h postinoculation and this trend continues un-til at least 3 days postinoculation (105). As dis-cussed below, some of this regulation may beenhanced by bacterial EPSs, cyclic β glucans


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LPS:lipopolysaccharide

LysM: lysin motif

and LPSs. Concurrent with a dramatic reori-entation of root cell growth, genes involved incytoskeletal functions and cell-wall biogenesisare induced from 1–12 h postinoculation (105).Between 12–48 h, genes involved in translation,cell growth and division, and chromosomal or-ganization are upregulated in a manner consis-tent with the initiation of meristem prolifera-tion within the root cortex (105, 116). As onemight predict, increased expression of cell di-vision genes is transient within a determinatenodule and decreases concurrent with the on-set of nitrogen fixation (29, 90).

The importance of NF structure for bio-logical activity has been demonstrated throughbiochemical analyses of NFs produced by host-specific nod gene mutants and the correspond-ing phenotypic characterization of plant re-sponses. For instance, the first symbioticallyactive NF structure described was that pro-duced by S. meliloti, which is a chitin tetramerwith an N-linked C16 unsaturated fatty acidand both O-sulfuryl and O-acetyl modifications(Figure 2b) (149). An S. meliloti mutant defi-cient for the host-specific nodH gene lacks theO-sulfuryl substitution at the reducing termi-nus of its NF and correspondingly loses thecapacity to nodulate its natural M. truncatulahost, yet it acquires the novel ability to nodulateVicia hirsute (139). The purified nonsulfated NFproduced by a nodH mutant elicits Ca2+-spikingfrom its natural host only when present at con-centrations much greater than is required forwild-type NF (123, 139, 178), suggesting thesulfate modification contributes to host rangespecificity by modulating the affinity betweenNF and its host receptor.

While the S. meliloti nodH gene is requiredto elicit initial physiological responses fromM. truncatula, the host-specific nodEF (NF acy-lation) and nodL (NF acetylation) genes are sub-sequently involved in host invasion. Individualnull mutants have a delayed nodulation phe-notype associated with inefficient bacterial in-vasion, whereas the nodFL double mutant hasa more severe defect in infection thread for-mation (5, 103). Despite the block in bac-terial invasion, a nodFL double mutant (and

its purified NFs) triggers certain morpholog-ical responses from root hairs in a mannerindistinguishable from the wild type. Theseinclude the early Ca2+-spiking and root haircurling responses, as well as induction of cer-tain ENODs and nodule primordium formation(5, 24, 178). Thus, in M. truncatula there aredistinct structural requirements for early NF-dependent root hair responses (i.e., NF sulfa-tion) vs later NF-dependent infection events(i.e., NF sulfation, and acetylation or acylation).

The relative ease with which plant symbio-sis mutants can now be characterized molec-ularly has significantly expanded our un-derstanding of the host signal transductionpathway that allows bacterial invasion basedon NF perception (78, 122). Putative NF-receptors, including MtNFP and MtLYK3,belong to the lysin motif (LysM) receptor-likekinase family, which have an extracellular do-main homologous to the bacterial LysM pro-teins that bind β-(1,4)-linked GlcNAc derivedfrom peptidoglycan. Host responses to specificNF structures depend on the LysM domainspecifically, and a one amino acid differencewithin this motif can alter the range of rhizobiarecognized for symbiosis (134).

Additional NF signaling genes have beenidentified that function downstream of theLysM receptor-like kinases (78, 122). For ex-ample, several dmi (does not make infections)mutants have been characterized in M. trun-catula, and the affected genes have orthologsin L. japonicus. Highlighting the importanceof the NF-dependent Ca2+-spiking response,MtDMI3 encodes a putative Ca2+-calmodulin-dependent protein kinase (CCaMK) (115).MtDMI1 encodes a nuclear-localized cationchannel that may help modulate Ca2+-spiking,and MtDMI2 encodes a putative receptor-likekinase (76, 119). While all of the dmi mutantsdisplay root hair deformation and rapid Ca2+-influx in response to NF, dmi1 and dmi2 mutantsare unable to elicit the subsequent Ca2+-spikingresponse (50, 72, 75, 159, 177, 179). In contrast,the dmi3 mutant is indistinguishable from wildtype with regard to each of these physiolog-ical NF responses (177). Thus, M. truncatula


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DMI1 and DMI2 appear to function down-stream of MtNFP and MtLYK3, but upstreamof the Ca2+-calmodulin-dependent protein ki-nase encoded by DMI3. Recent reports show-ing that DMI3 can be genetically modified toelicit spontaneous nodule formation suggest itmay be possible to intelligently engineer non-legume species that are capable of establishingsymbiotic nitrogen fixation (65, 166).

Although NF signaling is a nearly universalmeans of establishing the rhizobium nitrogen-fixing symbiosis with compatible legumes, ex-ceptions are emerging. The recent sequenc-ing of photosynthetic Bradyrhizobium sp. BTAi1and ORS278, which form nitrogen-fixing nod-ules on the roots and stems of aquatic host,Aeschynomene sensitiva, revealed that the com-mon nodABC genes are absent in these species(63). Moreover, a NF-deficient mutant of theclosely related Bradyrhizobium sp. ORS285forms nitrogen-fixing root and stem noduleson A. sensitiva with the same efficiency asits wild-type parental strain. Thus, the hostA. sensitiva initiates nodule development ina NF-independent manner and instead mayrespond to the secretion of bacterial purinederivatives with cytokinin-like activity, high-lighting the importance that host hormone bal-ance plays in nodule formation (63).

REACTIVE OXYGENAND NITROGEN SPECIES

As with many host-microbe interactions (39,118, 187), the rhizobium-legume symbiosis canbe associated with a host-generated releaseof reactive oxygen species (ROS: O·−

2 , H2O2,and HO·) and reactive nitrogen species (RNS:NO·). As discussed below, application of puri-fied S. meliloti LPS can suppress ROS produc-tion in response to fungal elicitors in M. trun-catula tissue culture cells (1, 148); however, therole this response plays in symbiosis is unclear.The application of purified S. meliloti NF toM. truncatula roots also inhibits ROS produc-tion in response to fungal elicitors (151), andcorrespondingly limits the expression of puta-tive ROS-generating NADPH oxidase genes

ROS: reactive oxygenspecies

RNS: reactivenitrogen species

(104). The NF-mediated inhibition of ROS ef-flux occurs within 1h of treatment and is de-pendent on the putative NF receptor encodedby MtNFP but not the downstream DMI1 gene(104, 151). NF-mediated suppression of ROSproduction plays a causative role in the root hairdeformation and curling responses to rhizobia(104).

However, subsequent stages of symbiosis be-tween M. truncatula and S. meliloti result in thelong-term production of ROS and RNS in dis-tinct areas of the nodule. While RNS are pri-marily associated with plant cells that have beeninfected with bacteria (12), ROS are associatedwith the plant cell wall of both infection threadsand infected host cells (142, 147). This ROSefflux requires NF, arguing that ROS may playa positive role in bacterial invasion (136). TheM. truncatula response to NF includes in-duction of the rip1 gene encoding a putativeperoxidase that could be involved in hydro-gen peroxide-dependent cross-linking of cellwall proteins (31). Both the induction of rip1expression and the production of ROS re-quire that M. truncatula has a functional DMI1gene (136), genetically separating the earlyDMI1-independent decrease in ROS from thislater response. The DMI1 requirement for rip1induction is bypassed when root tissues are ex-posed to exogenous H2O2 (136).

Generally, free-living rhizobia are more sus-ceptible to ROS-mediated killing than are othercommon soil bacteria like Bacilli and Pseudomon-ads (120), suggesting that symbiotic levels ofROS are probably unable to differentially pre-vent soil pathogens from taking advantage ofITs to invade nodules. One role for ROS pro-duction may be to promote proper IT de-velopment and growth by either cross-linkingcell wall glycoproteins or degrading cell wall–associated polysaccharides to aid IT elongation.Only a small percentage of newly formed ITs ac-tually penetrate the inner cortical cell layer, andthe unsuccessful, or aborted, ITs display certaincharacteristics of the hypersensitive plant de-fense response (171), which typically includesROS production. Thus, this ROS efflux couldplay a role in limiting bacterial invasion.


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Recent observations are consistent with theidea that ROS may also function as a positivesignal perceived by bacteria during invasion.This is based on the finding that an S. melilotistrain overexpressing the KatB catalase has anodule invasion defect that is primarily asso-ciated with aberrant IT growth (76). KatB isresponsible for detoxifying H2O2 and therebylimits the concentration of exogenously appliedROS within the bacterial cell (4). Bacterial over-expression of KatB likely acts as a strong cat-alyst for detoxifying ROS and may lower thelocal concentration of free oxygen radicals ableto modify the IT compartment. KatB overex-pression presumably also leads to significantlydecreased concentrations of ROS within theIT-localized bacterial cell. The symbiosis de-fect associated with KatB overexpression couldtherefore reflect the fact that ROS functionsas a cytoplasmic signal that the bacterium usesto regulate functions essential to invasion. It isunlikely this ROS signal would be perceivedthrough the OxyR redox-sensitive regulatoryprotein since an S. meliloti oxyR mutant hasno obvious symbiosis defect with M. truncatula(41, 107). However, the putative redox-sensitiveCbrA two-component histidine kinase is re-quired for bacterial invasion of Medicago hostsand regulates a number of genes specificallyrequired for bacterial invasion (61, 62), mak-ing CbrA a promising candidate for a bacterialredox-sensor involved in bacterial invasion.

Although ROS appears to promote rhizo-bial invasion, these bacteria must also be able tocombat this stress in order to achieve symbiosis.A screen for ROS-sensitive S. meliloti mutantsthat simultaneously display aberrant symbiosisphenotypes revealed a variety of functions re-lated to bacterial metabolism and exopolysac-charide production (41). With regard to genesspecifically required to detoxify ROS, S. melilotihas three that encode catalase enzymes (katA,katB, and katC) and one encoding superox-ide dismutase activity (sodB) (77, 146). Severaluncharacterized genes include an extracellu-lar peroxidase (Smc01944), a bacteriocuprein-family superoxide dismutase (sodC), and analkylhydroperoxidase (ahpC), each of which

may combat ROS exposure; however, null phe-notypes for these genes have not been reported(7, 60). Null mutants for either katA, katB,katC, or sodA are fully capable of establishingthe symbiosis (40, 77, 152), although the katACand katBC double mutants have decreased sym-biotic proficiency (77, 152). In particular, thekatBC mutant has a severe symbiosis defect thatresults in formation of aberrantly small nodulesthat are incapable of nitrogen fixation (77). Al-though the katB and katC genes are stronglyexpressed in bacteria located within growingITs, where ROS is concentrated, the phenotypeof the double mutant is subsequently revealedduring bacterial uptake into the host cell cyto-plasm: intracellular bacteria lack the surround-ing peribacteroid membrane and undergorapid senescence for reasons that are unclear(77).

MODULATION OF THE HOSTDEFENSE RESPONSE

The initiation of ITs is a major checkpoint forthe host in terms of deciding whether to allowbacterial invasion to proceed. In the past fewyears, several plant mutants affected in IT for-mation and growth have been isolated, and theirfurther characterization will likely shed light onsome of the mechanisms that the host uses tocreate and control the growth of this structure(30, 91, 106, 163, 176, 189).

Rhizobial invasion of the host nodule via theIT is strongly influenced by a complex vari-ety of bacterial polysaccharides in addition toNF, including secreted EPSs and K-antigens,secreted and periplasmic cyclic β glucans, andthe outer membrane-localized LPSs (14, 57,78, 155, 157). Similar to NF, several of thesemolecules exert their effects on symbiosis ina structurally dependent manner, arguing thatthey may function as signals between invadingbacteria and their host. In fact, recent evidencesuggests that the exopolysaccharide succinogly-can may help further define species-specificityin addition to NF (153). A shared and outstand-ing question regarding these bacterial polysac-charides is their potential role in modulating


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a plant defense response to bacterial invasion.However, definitive proof for such possibilitiesawaits the identification and characterization ofspecific host receptors.

Exopolysaccharide

The biosynthesis of rhizobial EPS and its regu-lation have been most extensively studied withregard to the S. meliloti macromolecule referredto as succinoglycan, or EPS I, (Figure 2c) (15,78, 155). S. meliloti succinoglycan is secretedas a polymer of repeating octasaccharide sub-units (one galactose and seven glucose residues)modified with succinyl, acetyl, and pyruvylsubstituents (Figure 2c). Polymerization ofthe succinoglycan monomer results in secre-tion of either low molecular weight (LMW)forms (monomers, dimers, and trimers) or highmolecular weight (HMW) forms (polymers ofseveral hundred subunits).

Succinoglycan plays a critical role in theS. meliloti symbiosis with Medicago hosts.Succinoglycan-deficient mutants elicit noduleorganogenesis by virtue of NF signaling (86),but these aberrantly small nodules are devoidof bacteria and therefore incapable of nitrogenfixation (99). These rhizobial mutants are com-promised for host invasion to the extent thatIT formation proceeds from only 10% of bac-terially colonized root hairs and the ITs that doform terminate prematurely before they reachthe nodule primordium (27). While EPSs playa passive role in protecting the bacterium fromhost-derived stresses within the IT (36), theyare also thought to perform a signaling functionfrom rhizobia to their host (78, 79). Part of theevidence for this is based on the observation thatLMW forms of succinoglycan are more effec-tive at promoting symbiosis than HMW forms(10, 169, 182). Moreover, an exoH mutant is un-able to succinylate the succinoglycan monomerand therefore produces predominantly HMWsuccinoglycan (98); since this mutant is alsoseverely compromised for symbiosis a merelyprotective role is not sufficient to explain allof the requirements for succinoglycan in hostinvasion.

Increasingly, the evidence available suggeststhat bacterial EPSs play a role in modulatingthe host defense response to bacterial invasion.For example, the premature termination of bac-terial infection observed with EPS-deficientmutants is associated with symptoms of a hostdefense response and, in particular, the pro-duction of antimicrobial phenolics and phy-toalexins (119, 126). To test the hypothesis thatsuccinoglycan promotes symbiosis as a signal-ing molecule, a global transcriptome analysiswas performed on M. truncatula plants inoc-ulated with succinoglycan-deficient S. melilotiat a time point just prior to IT formation(3 days postinoculation) (79). Thus, this exper-iment identified host genes differentially reg-ulated in response to EPS production, ratherthan IT failure per se, and the largest groupof genes upregulated during an EPS-deficientinteraction encode putative plant defense pro-teins (79). As mentioned above, M. truncatuladefense genes are upregulated 1 h postinocula-tion with wild type S. meliloti; however, expres-sion of this same class of genes decreases by6 h postinoculation and remains low for at least3 days (105). Taken together, these observationssuggest that a primary consequence of EPS pro-duction is the suppression of a potentially lethalhost defense response, and in the absence ofEPS, this unproductive response may cause ablock in IT formation (27).

Insight into how S. meliloti regulates pro-duction and modification of exopolysaccharidesis therefore important to our understandingof the physiological requirements for host in-vasion. An increasingly large number of reg-ulators modulate succinoglycan production explanta, including ExoS, ExoR MucR, SyrA, andthe more recently identified CbrA (62, 155).

The first regulators of succinoglycan to beidentified were the ExoS two-component histi-dine kinase and ExoR. ExoS is an essential genein S. meliloti and forms a two-component signaltransduction pathway with the response reg-ulator ChvI, which is also essential and func-tions as a DNA-binding transcription factor(26). ExoS phosphorylates ChvI directly andthereby promotes increased exo/exs expression


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and greater succinoglycan production (26).ExoR has no homology to any known reg-ulators, however a null mutation alters thetranscription of several exo biosynthetic genes(138). Early observations suggested a geneticlink between exoR and exoS: extragenic sup-pressors of an exoR null mutant symbiosis de-fect were mapped near exoS and these sup-pressors displayed a concomitant change in exogene transcription (125). More recent evidenceindicates that ExoR is a periplasmic proteinthat functions upstream of the ExoS/ChvI two-component pathway and is hypothesized to di-rectly repress ExoS kinase activity by bind-ing its periplasmic sensing domain (183). Anoutstanding question remains as to the na-ture of the stimulus perceived by ExoS and/orExoR.

The two-component histidine kinase CbrAhas also been identified as a regulator of suc-cinoglycan production. Unlike the overproduc-tion of wild-type forms of succinoglycan in exoSand exoR mutants (46), a cbrA mutant appearsto overproduce predominantly LMW forms ofsuccinoglycan, and this phenotype is correlatedwith increased transcription of several exo genesinvolved in LMW succinoglycan biosynthesis,including exoH, exoK, and exoT (62). Moreover,the cbrA mutation leads to increased expres-sion of additional genes involved in promot-ing bacterial IT invasion, including the ndvAtransporter of cyclic β glycans (described be-low) and the sinI regulator of galactoglucan(EPS II) production (61). Thus, it was proposedthat CbrA coordinates multiple aspects of bac-terial physiology to promote bacterial invasionof the nodule, and that the physiology of thecbrA mutant is optimized for this process. Giventhat CbrA contains at least one PAS domain(62), a motif that commonly monitors redoxchanges, CbrA may coordinate bacterial phys-iology in response to the high redox environ-ment of the IT. However, since the cbrA mutantremains defective for symbiosis despite a physi-ology optimized for IT invasion (61), it appearsthat CbrA plays an additional role during sub-sequent bacteroid differentiation, as discussedbelow.

Cyclic β Glucan

Rhizobia generally produce β-(1,2)-glucansthat are macrocyclic and unbranched polymersof glucose, containing anywhere from 17 to40 residues depending on the rhizobial strain(157). The synthesis of cyclic β glucan is de-pendent on a glycosyltransferase encoded byndvB (called cgs in Mesorhizobium loti ) and itssecretion is dependent on the ABC-type in-ner membrane transporter ndvA. Bradyrhizo-bium species are an exception and producebranched macrocyclic glucans that contain bothβ-(1,3) and β-(1,6) glycosidic bonds catalyzedby glycosytransferases encoded by ndvB andndvC. The chemical characteristics of cyclicβ glucans can be modified by the additionof phosphocholine, sn-1-phosphoglycerol, suc-cinic and methylmalonic acid substituents. Dis-ruption of ndvB (cgs) in S. meliloti (M. loti )blocks symbiosis at the stage of bacterial attach-ment to root hairs and IT invasion and therebyresults in the formation of aberrantly small andempty nodules (34, 48). In contrast, a B. japon-icum ndvB mutant is able to elicit normal noduledevelopment and invade host tissues, althoughthe resulting nodules do not fix nitrogen (47).

Like succinoglycan, the cyclic β glucans mayplay a role in modulating a host defense re-sponse to bacterial invasion. Specifically, M. loticyclic β glycans are required to suppress high-level production of antimicrobial phytoalexinsduring symbiotic development with L. japon-icus (35). Many host defense response genesare induced in a mature nodule during a wild-type symbiosis (29, 90), perhaps to providethe nutrient-rich nodule with a defense againstparasites. These same genes are generallyexpressed at decreased levels during the ineffec-tive symbiosis of the cgs mutant, with the strik-ing exception of highly induced PAL expres-sion (35). PAL encodes an enzyme predictedto participate in the synthesis of antimicro-bial phenolic compounds, and consistent withincreased PAL expression during the cgs mu-tant symbiosis, L. japonicus nodules accumulatephenolic compounds to a greater extent thanis observed during a wild-type symbiosis (35).


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Purified B. japonicum cyclic β glucans are ableto block a host defense response to fungal elic-itors in the determinate legume G. max and ina structurally dependent manner (17), furthersuggesting these polysaccharides may be ableto prevent a host defense response during rhi-zobial invasion.

BACTERIAL REQUIREMENTSFOR INTRACELLULARCOLONIZATION

Lipopolysaccharide

Throughout symbiotic development the S.meliloti cell surface is in intimate associationwith its host but this is particularly true ofthe microsymbiont within the symbiosome.Not surprisingly then, the bacterial cell sur-face plays an important role in promoting rhi-zobial intracellular adaptation, including thelipopolysaccharide (LPS) component of thegram-negative outer membrane (14, 83, 157).LPS is a complex macromolecule composed ofa lipid A membrane anchor and an oligosac-charide core, which can be further modified bythe addition of a variable O-antigen polysac-charide (135). Generally, host perception ofLPS from pathogenic bacteria plays a signifi-cant role in defense responses to invasion viathe innate immune system (130). It has there-fore been of great interest to understand thespecific role that rhizobial LPS plays in symbi-otic development. Bacteroid LPS has increasedhydrophobicity compared to that of free-livingbacteria, suggesting there are LPS modifica-tions in planta that may contribute to symbiosis(38, 84).

Rhizobia produce a lipid A with uniquestructural characteristics that distinguish itfrom the potent E. coli innate defense elicitorendotoxin (135), although there is variation incertain elements among the rhizobia (14, 83).For instance, most rhizobia produce lipid Aspecies lacking either one or both of the 1- and4′-phosphate groups present on the β-(1,6)-glucosamine disaccharide of E. coli; Sinorhizo-bium lipid A is an exception to this trend as

VLCFA: very-long-chain fatty acid

it is bisphosphorylated (Figure 2d ). In addi-tion, rhizobial lipid A moieties are most oftenmodified with a secondary N-linked acyloxya-cyl residue composed of a C28 or C30 Very-Long-Chain Fatty Acid (VLCFA) that has thecapacity to span the entire lipid bilayer of theouter membrane (Figure 2d ). This particu-lar modification has generated much interestas a structural feature that could shield rhizo-bial LPS from recognition by the host innateimmune system and thereby promote rhizobialinvasion and persistence. In fact, the presence ofVLCFA modified lipid A has been observed inseveral related bacteria that establish persistenthost infections, including all rhizobia, agrobac-teria, and brucella that have been analyzed.

The acpXL and lpsXL genes encode an acylcarrier protein and an acyl transferase, re-spectively, that together catalyze the VLCFAsecondary acylation of lipid A in rhizobia.Rhizobial acpXL and lpsXL mutants com-pletely lack the VLCFA-modified lipid A dur-ing free-living growth but remain effective atestablishing a nitrogen-fixing symbiosis (53,150, 175), although bacteroid developmentis mildly perturbed and leads to decreasednitrogen-fixation (173). This weak symbiosisphenotype is likely explained by the recent dis-covery that acpXL mutants of R. leguminosarumb.v. viciae form bacteroids that actually con-tain VLCFA-modified lipid A, indicating thatthey express an alternative system for VLCFAmodification in planta (174); thus, the role thatthe VLCFA modification plays in symbiosis re-mains to be determined.

Alteration of the LPS carbohydrate content,in either the core or the O-antigen, has an aber-rant effect in a variety of symbioses (83). Forexample, an S. meliloti lpsB mutant has a dramat-ically altered LPS core and is incapable of estab-lishing a chronic host infection (21). While themutant displays normal host IT invasion and istaken up into the host cell cytoplasm, it under-goes rapid senescence and is degraded withinthe symbiosome compartment, suggesting LPSplays a critical role in rhizobial adaptation andpersistence within the particular environmentof the host cell cytoplasm (21).


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The precise function of LPS in promotingsymbiosis remains unclear (14). Defects in LPScan sensitize bacteria to membrane-disruptingagents and antimicrobial peptides so that itmay provide a protective barrier against envi-ronmental stress and host defense responses.However, there are indications that S. melilotiLPS may also play an active role by suppress-ing the release of ROS (1, 148). Specifically, M.truncatula tissue culture cells respond to yeastelicitors with an oxidative burst and the in-creased expression of defense genes involved inplant secondary metabolism, like PAL, and cellwall metabolism (1, 164). When tissue culturecells are exposed to elicitor in the presence ofS. meliloti lipid A, this LPS component is capa-ble of suppressing the oxidative burst and damp-ening the plant transcriptional response (148,164), indicating an interaction between rhizo-bial LPS and its host could suppress any poten-tial immune response to intracellular bacteria.This could be particularly important for bac-teria within the symbiosome as they no longerexpress genes for the biosynthesis of succino-glycan (9), which appears to dampen a poten-tial plant defense response to bacteria withinthe IT (78, 79). Lipid A moieties isolated fromthe related intracellular mammalian pathogenBrucella abortus, which also undergo VLCFAmodification, are only weak elicitors of an in-nate immune response in mouse macrophages(93, 94). Perhaps these related alphaproteobac-teria share some of the strategies that rhizobiause to evade detection by the innate immunesystem during chronic infection.

BacA

The bacA gene encodes an inner membraneprotein that plays an essential role in the earlystages of S. meliloti bacteroid development (64).With a bacA mutant, the early steps in symbio-sis, such as formation and development of in-fection threads, proceed as efficiently as withwild type but the mutant lyses shortly after be-ing endocytosed into the cytoplasm of plantcells. BacA function is also essential for thechronic infection of B. abortus (100), a mam-

malian pathogen that can survive and replicatein host macrophages, highlighting the broadimportance of BacA function to chronic bac-terial persistence during intracellular infection(140).

Generally, the loss of bacA function has beenassociated with an increased resistance to cer-tain antimicrobial peptides (74, 96, 113). InE. coli, deletion of the bacA isofunctional ho-molog, sbmA, produces increased resistance tomicrocins B17 and J25, bleomycin, and Bac7,which is an antimicrobial peptide of mammalianorigin (96, 113). Similarly, the bacA mutants ofS. meliloti and B. abortus show increased resis-tance to bleomycin relative to the wild type (74,100). Thus, BacA may play a role in the trans-port of modified peptides across the inner mem-brane (64). In fact, it was proposed that BacAfunctions as an importer for host-derived pep-tide(s) that promote bacteroid differentiation(114). Specifically, it was suggested that bac-terial uptake of Nodule-specific Cysteine-Rich(NCR) peptides produced by indeterminatelegumes could trigger the terminal differenti-ation of bacteroids (3, 114), analogous to therole defensins play in suppressing the prolifer-ation of bacterial pathogens (190).

In S. meliloti and B. abortus, BacA also affectsthe VLCFA modification of the lipid A com-ponent of LPS (52, 54). BacA has homology tothe transmembrane domain of eukaryotic ATP-binding cassette (ABC) transporters of theABCD family (52). ABCD transporters func-tion in the peroxisome and this includes the hu-man adrenoleukodystrophy protein (hALDP)that is thought to transport activated VL-CFAs across the peroxisomal membrane (181).Approximately 50% of lipid A moieties iso-lated from S. meliloti bacA mutants (as well asB. abortus) lack VLCFAs (52). Thus, it was sug-gested that BacA may export VLCFAs fromthe cytoplasm and across the inner membrane,and perhaps the lack of VLCFA-modified lipidA causes the defects in chronic infection ob-served with bacA mutants (52). As mentionedabove, rhizobial acpXL and lpxXL mutants com-pletely lack VLCFA-modified lipid A duringfree-living growth and are able to establish


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a successful symbiosis despite bacteroid mor-phological abnormalities (53, 150, 173, 175).However, at least R. leguminosarum acpXL bac-teroids contain VLCFA-modified lipid A inplanta (174), indicating an alternative systemfor VLCFA modification exists that may requireBacA function.

It remains to be determined whether BacA isdirectly responsible for either one or both of theproposed transport reactions. S. meliloti strainscarrying 12 site-directed mutations in bacA havephenotypes intermediate to the wild-type andbacA null mutant (101), consistent with a rolefor BacA in multiple, nonoverlapping functions.Moreover, it was reported that functional SbmAis required for full efficiency of the tetracyclineexporter TetA in E. coli (42), suggesting thatBacA function could similarly affect the activ-ity of other membrane proteins. Future studiesthat reconstitute BacA into liposomes, followedby detailed transport assays, will be needed toelucidate its precise function.

DEVELOPMENTAL REGULATIONOF THE CELL CYCLE

Symbiotic regulation of the plant cell cycle andthe role of endoreduplication in nodule devel-opment has been studied extensively (56, 122).In contrast, an understanding of how the bacte-rial cell cycle may be regulated during symbiosisis just beginning to emerge. Bacteroids withindeterminate hosts have the ability to dediffer-entiate into free-living bacteria once they arereleased from a senescent nodule (114). In con-trast, at least some rhizobia that form a sym-biosis with indeterminate legumes undergo aterminal differentiation program that precludesviability outside the host cell cytoplasm (114).Whether a given rhizobial species undergoesterminal differentiation appears to be a decisioncontrolled by the legume host (114).

During free-living growth, and presumablywithin the IT, S. meliloti grows as a rod-shapedbacterium with no greater than a 2N comple-ment of its genome (Figure 3a,b) (114), whichimplies that these bacteria initiate DNA repli-cation only once per cell cycle. In contrast, one

remarkable aspect of terminal bacteroid differ-entiation is a branched cell morphology thatis accompanied by several rounds of genomicendoreduplication (Figure 3c) (114). The cor-relation between bacteroid endoreduplicationand the inability to dedifferentiate and resumegrowth outside the host suggests the act ofendoreduplication may be a defining event interminal bacteroid differentiation. The indeter-minate symbiosis therefore represents a novelbacterial cell cycle event, i.e., endoreduplica-tion, which may play an important role in ei-ther intracellular persistence or efficient nitro-gen fixation. Taken together, these observationsimply that the S. meliloti cell cycle has at leastthree branch points subject to in planta regula-tion (Figure 3d ), and it will be of great interestto understand how the cell decides which pathto choose under different host conditions.

While regulatory aspects of the S. meliloticell cycle have been little studied, that of therelated Caulobacter crescentus alphaproteobac-terium has been dissected in great detail andthereby serves as a powerful model on whichto base future studies in S. meliloti. Briefly, inC. crescentus the CtrA response regulator col-laborates with the DnaA replication initiatorand GcrA transcription factor to globally con-trol cell cycle progression (154). Throughoutthe cell cycle, CtrA concentrations are strictlyregulated, and this plays a critical role in me-diating cell cycle progression. Additionally, anelaborate two-component signal transductionpathway containing the essential response regu-lator DivK regulates the concentration of activeCtrA-P (18). Through this multi-layered regu-lation of active CtrA-P concentrations, C. cres-centus limits the initiation of DNA replication toonce per cell cycle (Figure 4a), in contrast withE. coli and other fast-growing bacteria. The co-ordination between DNA replication and celldivision that allows only one replication ini-tiation event per cell cycle is also observed inS. meliloti (114).

C. crescentus undergoes an asymmetric celldivision during each cell cycle for the purposeof nutrient adaptation (Figure 4b), and theDivJ and PleC two-component histidine kinase


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Infection thread

Bacteroid differentiation

G1

G2

S

Free-living

a

b

c

S x 34

G1G2

S

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d

Model for in plantamodulation of cell cycle

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←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 3Schematic representation of the rhizobium cell cycleat different stages of symbiosis. (a) The S. meliloticell cycle is modeled after that of thealphaproteobacterium Caulobacter crescentus. A celldivision cycle is comprised of three distinct phases:G1, S, and G2. In C. crescentus, DNA replication isinitiated only once per cell cycle, in S phase;however, chromosome segregation begins during Sphase and continues in G2 phase. Cell divisionbegins in G2 phase and is completed before the nextDNA replication initiation event. During free-livinggrowth, S. meliloti is thought to initiate DNAreplication only once per cell cycle and dividesasymmetrically to produce daughter cells ofdifferent size. In analogy to C. crescentus, the smalldaughter cell likely proceeds into G1 phase whilethe larger daughter cell directly re-enters S phase.(b) S. meliloti proliferating in the IT originate from aclonal expansion of founder cells entrapped in thetip of the root hair curl. Cells appear to lack flagellaand are loosely associated with one another in apole-to-pole manner, typically forming two or threecolumns with a braided appearance. Activepropagation of bacteria is observed only in a limitedarea called the growth zone near the tip of the IT,while bacteria outside of the growth zone do notgrow or divide. It seems likely that the restrictedgrowth of bacteria enables synchronization ofbacterial growth with extension of the IT. (c)Bacteria colonize the cytoplasm of plant cells locatedin the invasion zone (see Figure 1d ). Bacteria aresurrounded by a plant-derived membrane anddifferentiate into a bacteroid. In S. meliloti, DNAendoreduplication occurs during bacteroiddifferentiation and results in dramatic cell branchingand enlargement to a length of 5–10 μm; incomparison, free-living counterparts are rod-shapedcells of 1–2 μm. These terminally differentiated (G0phase) bacteroids are unable to resume futuregrowth. Orange lines, host plasma membrane; greenlines, host cell wall. (d ) A model of the S. meliloti cellcycle in planta has three possible exits from S phase,two of which (in blue) represent an exit from thetypical free-living cycle (in red ). Bacteria within theinfection thread are thought to progress through thecell cycle in the same manner as free-living cells, andin particular transition from S phase into G2 phase(represented by arrow 1). Bacteria that undergobacteroid differentiation undertake the process ofendoreduplication and therefore re-enter G1 phaseafter the completion of S phase (represented byarrow 2); the bacteria may cycle from S to G1multiple times during endoreduplication. Onceendoreduplication is complete, the bacteroid entersa terminally differentiated state (G0) and is nolonger able to initiate cellular growth or DNAreplication (represented by arrow 3).


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Caulobacter

Sinorhizobium

Replication initiation

Chromosome replication

Chromosome segregation

Cell separation

G1 G2S

Swarmer Stalked Predivisional Daughters

“Small” “Large” Predivisional Daughters

Cell cycle

a

b

c

DivK DivK～P

Figure 4Schematic representation of cell-cycle progression in C. crescentus and S. meliloti. (a) The cell cycle iscomprised of three distinct phases: G1, S, and G2 (shown in red ). The timing of cell cycle-related events isshown in blue. (b) During G1 phase, C. crescentus is a swarmer cell, which is motile and has a polar flagellumand pili. When entering S phase, the swarmer cell ejects the flagellum, retracts the pili, and differentiatesinto a stalked cell. The stalked cell is uniquely competent to initiate DNA replication. After chromosomesegregation in G2 phase, the cell divides asymmetrically to produce two different daughter cells: a swarmerand a stalked cell. During cell-cycle progression, cellular localization of DivK is controlled byphosphorylation: nonphosphorylated DivK is uniformly distributed in the cytoplasm and DivK∼P islocalized 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” cellsare counterparts of the C. crescentus swarmer and stalked cells, respectively. Flagella are omitted from thisscheme because their localization has not been examined during cell cycle progression in S. meliloti.


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sensors mediate this asymmetry. Other al-phaproteobacteria also undergo asymmetriccell division and have orthologs of C. crescentuscell cycle regulators present in their genomes(70), including S. meliloti (Figure 4c), B. abor-tus, and Agrobacterium tumefaciens. CbrA hasstrong homology in its C-terminal kinase do-main to the DivJ and PleC kinases of DivK (70),suggesting that its primary function may be toregulate cell cycle progression in S. meliloti viaregulation of DivK phosphorylation, as was ob-served for its B. abortus ortholog PdhS (71). Infact, during free-living growth the cbrA mu-tant displays a filamentous, branched morphol-ogy that coincides with a greater than 2N ge-nomic content, indicating the mutant is un-able to coordinate DNA replication with celldivision (K.E. Gibson & G.C. Walker, unpub-lished). Perhaps CbrA plays a role in modify-ing the cell cycle program during symbiosis andloss of this function is primarily responsible forthe in planta defects of the cbrA mutant. If thisis the case, it will be of interest to determinehow CbrA coordinates cell cycle progressionwith cell envelope physiology and succinogly-can production. It is possible that at least someof the cbrA mutant phenotypes are an indirectconsequence of altered cell cycle progressiongiven that multiple cell surface characteristicsare cell cycle regulated in C. crescentus.

The process of endoreduplication undoubt-edly creates an intense demand for dNTPswithin the developing bacteroid. The dNTPsrequired for DNA metabolism in all organismsare synthesized by the enzyme ribonucletidereductase (RNR) (78). S. meliloti has only oneRNR encoded in its genome, NrdJ, and thisis a vitamin B12-dependent enzyme (33). As aClass II RNR that is both oxygen-independentand oxygen-insensitive (80), this enzyme likelyprovides a key adaptation for rhizobial persis-tence within the microaerobic environment ofthe host cell cytoplasm. The B12 biosyntheticenzyme BluB, which catalyzes formation of thelower ligand 5,6-dimethylbenzimidazole, wasfortuitously found to be required for symbio-sis between S. meliloti and M. sativa based onits involvement in succinoglycan biosynthesis

(22, 162). A bluB mutant appears able to infectthe host via normal IT growth, suggesting thatany alteration to the succinoglycan does not af-fect invasion; however, bacteroids are not ob-served within the host cell cytoplasm and thenodules that develop are unable to fix nitrogen(22). Thus, BluB function in B12 biosynthesis isnecessary for symbiosis due to the requirementfor a B12-dependent enzyme(s), for which NrdJis one possible candidate.

During the indeterminate symbiosis be-tween Mesorhizobium huakuii and Astragalussinicus, bacterial DNA replication is limited tothose bacteria associated with the meristem inInfection Zone II and Interzone II/III (89). Thisobservation suggests that, at least in some rhi-zobia, DNA replication is permanently blockedonce the bacteroid completes endoreduplica-tion. Bacterial genes involved in DNA repairare induced within mature nodules (9), suggest-ing that DNA integrity is maintained withinbacteroids in the absence of DNA replication.Importantly, these DNA repair genes includeseveral that encode nonhomologous end-joining (NHEJ) proteins involved in doublestrand break repair (88), a process also utilizedby terminally differentiated cells in higher eu-karyotes.

Little is currently known regarding rhizo-bial cell division during symbiosis, althoughthe altered morphology of bacteroids impliesan underlying regulation of this process. Sev-eral S. meliloti genes involved in cell divisionhave been characterized, for instance ftsZ1 andftsZ2 (108, 110, 111), as well as minCDE (28).Blocking the process of cell division via over-expression of ftsZ1 or minCD causes alteredcell morphology during free-living growth thatis reminiscent of the branched and filamen-tous bacteroid (28, 95). Treatment of S. melilotiwith DNA-damaging agents, which impingeon DNA replication and cell division in otherbacteria, also inhibits cell division and resultsin branched cell morphology (95). Althoughthe mechanisms that underlie these changes incell morphology and how they relate to bac-teroid differentiation are still unknown, ftsZ1and ftsZ2 expression is decreased in bacteroids


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consistent with a block in cell division after dif-ferentiation (9, 13).

NUTRIENT EXCHANGE

Invading bacteria within the IT are ultimatelyendocytosed by postmitotic G0 endoploid cellsand remain encapsulated within a modifiedplasma membrane called the peribacteroidmembrane (PBM) (Figure 1d, f ). The sym-biosome, consisting of the PBM and its mi-crosymbiont, is thereby formed and within thisPBM-bound compartment bacteria establish apersistent, or chronic, infection of the host. Inthis manner, a single host cell will ultimatelyhouse hundreds of bacteria. Only after bacte-ria have gained access to the host cell cyto-plasm do they differentiate into the morpho-logically distinct bacteroid form that is capableof nitrogen fixation. The in planta differentia-tion of rhizobia involves significant metabolicchanges that promote adaptation and nitrogenfixation (121, 131). For example, rhizobia un-dertake respiratory chain modifications that al-low energy utilization under microaerobic con-ditions, repress glycolysis genes, and activateC4-dicarboxylic acid utilization pathways forcarbon metabolism. Induction of nitrogenasegene expression is triggered in a FixJ-dependentmanner by the microaerobic conditions of thehost cell cytoplasm (55), which is in turn criti-cal for the activity of this oxygen-sensitive en-zyme (45, 92). In addition to genes required fornitrogenase activity, FixJ is responsible for in-duction of nearly all the bacterial genes whoseexpression increases in mature nodules (9), indi-cating that the oxygen-sensing two-componentpair FixL and FixJ plays a profound role in reg-ulating bacteroid physiology. In contrast, hostgene expression in nodules colonized by the rhi-zobial fixJ mutant is largely indistinguishablefrom nodules colonized by wild-type rhizobia,suggesting that nodule morphogenesis and bac-terial invasion contribute more to host generegulation than does nitrogen fixation (9).

Creation and maintenance of the host mi-croaerobic environment is dependent on struc-tural aspects of the nodule that form an oxygen

PBM: peribacteroidmembrane

diffusion barrier in combination with high ex-pression levels of plant leghemoglobin, whichcan be 25% of total soluble protein in a nod-ule and helps limit the concentration of freeoxygen to 3–22 nM (124, 167, 186). The hostsupports high nitrogenase activity by providingbacteroids with a constant flux of O2 for aero-bic respiration and with energy in the form ofC4-dicarboxylic acids derived from the photo-synthate sucrose (102, 141, 143). The metabolicproduct of the nitrogenase enzyme reaction isammonia, and this appears to be provided tothe host both directly and indirectly throughits incorporation into alanine by the bacterialenzyme alanine dehydrogenase (2). Generally,nitrogen secreted from the bacteroid is assimi-lated by the host through its incorporation intothe amino acids glutamine and glutamate by theenzymes glutamine synthetase (GS) and gluta-mate synthase (GOGAT), respectively (185). Indeterminate nodules, the large bacterially in-fected cells are interspersed with small non-infected cells that appear to be specialized forfurther nitrogen assimilation, involving conver-sion of amino acids (glutamine and arginine)into either ureides or amides that are exportedfrom the nodule to the rest of the plant.

HOST SANCTIONS ONSYMBIOTIC RHIZOBIA

From an evolutionary point of view, why do rhi-zobial bacteria maintain the large number ofgenes required for mutualism with their legumehosts (44)? This question is particularly rele-vant in light of the recent observation that bac-teroids within indeterminate nodules are ter-minally differentiated and unable to give rise toprogeny (114). However, even a mature inde-terminate nodule in the soil can contain any-where from 105–1010 clonally related bacteriathat are located within the invasion zone (ZoneII; Figure 1a) and have not yet differentiated.Thus, a single symbiotic rhizobium is predictedto have greater fitness if it successfully colonizesa nodule than its nonsymbiotic cousin residingin the soil where growth can be severely limitedby nutrient availability.


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While there appears to be a fitness gain forrhizobia able to invade the nodule, it is also clearthat the host has evolved mechanisms that pre-vent nonfixing rhizobia from parasitizing thelegume nodule for energy. While the host con-trols the infection process and nodule morphol-ogy, it is the microsymbiont that largely dictatesthe efficiency of nitrogen fixation. Mathemati-cal modeling suggests that if legumes treat fix-ing and nonfixing rhizobial strains within thenodule similarly, then nonfixing rhizobia would

quickly outcompete nitrogen fixers (184). Per-haps for this reason, the host imposes effectivesanctions on nonfixing rhizobial cheaters withinthe nodule (85, 184). So far, host sanctions havebeen found to take the form of severe O2 limi-tation to nonfixing rhizobia within the nodule,which restricts bacterial growth and viability.Thus, the legume host is capable of imposingselective pressure on rhizobia that may affectthe evolution of bacterial populations in favorof nitrogen fixers (44).

SUMMARY POINTS

1. There tends to be strict species-specificity between legumes and their compatible sym-bionts, and this specificity is defined in part by NF structure. NF elicits changes in hostphysiology and gene expression that lead to nodule development by virtue of a NF-perception pathway involving LysM receptor-like kinases. The host specificity engen-dered by NF structure is dependent on the extracellular LysM domain, which likely bindsNF directly. Initial IT formation and its continued growth also has NF requirements andin fact displays a greater stringency for chemical modification of the NF backbone thando 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 andthis promotes early morphological responses in root hairs that initiate the process ofrhizobial invasion. During subsequent nodule development, a host-derived ROS effluxproduced 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 bacteriathat elicits IT-specific physiological adaptations.

3. A variety of rhizobial cell-surface and secreted polysaccharides appear to function assignals from the bacterium to its host. In particular, transcriptome analyses of host re-sponses to polysaccharide-deficient bacterial mutants suggest that at least one role forthese 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 defenseresponse to fungal elicitor in a tissue culture setting.

4. S. meliloti typically initiates DNA replication only once per cell cycle during free-livinggrowth and presumably within the host IT. However, bacteria that colonize the hostcell cytoplasm undergo the novel process of endoreduplication as it differentiates intoa bacteroid. Ultimately, this bacteroid becomes terminally differentiated such that nofurther DNA replication or cellular growth is observed and its viability becomes strictlylimited to the intracellular environment of the host. These observations imply that theS. meliloti cell cycle has at least three unique branch points that are subject to in plantaregulation.


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DISCLOSURE STATEMENT

The authors are not aware of any biases that might be perceived as affecting the objectivity of thisreview.

ACKNOWLEDGMENTS

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 ofthe manuscript. This work was supported by the National Institute of Health grant GM31010 (toG.C.W.), National Cancer Institute (NCI) grant CA21615-27 (to G.C.W.) and JSPS PostdoctoralFellowships for Research Abroad (to H.K.). G.C.W. is an American Cancer Society ResearchProfessor.

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AR361-FM ARI 3 October 2008 13:37

Annual Review ofGenetics

Volume 42, 2008Contents

Mid-Century Controversies in Population GeneticsJames F. Crow � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Joshua Lederberg: The Stanford Years (1958–1978)Leonore Herzenberg, Thomas Rindfleisch, and Leonard Herzenberg � � � � � � � � � � � � � � � � � � � � � � �19

How Saccharomyces Responds to NutrientsShadia Zaman, Soyeon Im Lippman, Xin Zhao, and James R. Broach � � � � � � � � � � � � � � � � � � � �27

Diatoms—From Cell Wall Biogenesis to NanotechnologyNils Kroeger and Nicole Poulsen � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �83

Myxococcus—From Single-Cell Polarity to ComplexMulticellular PatternsDale Kaiser � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 109

The Future of QTL Mapping to Diagnose Disease in Mice in the Ageof Whole-Genome Association StudiesKent W. Hunter and Nigel P.S. Crawford � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 131

Host Restriction Factors Blocking Retroviral ReplicationDaniel Wolf and Stephen P. Goff � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 143

Genomics and Evolution of Heritable Bacterial SymbiontsNancy A. Moran, John P. McCutcheon, and Atsushi Nakabachi � � � � � � � � � � � � � � � � � � � � � � � � � 165

Rhomboid Proteases and Their Biological FunctionsMatthew Freeman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 191

The Organization of the Bacterial GenomeEduardo P.C. Rocha � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 211

The Origins of Multicellularity and the Early History of the GeneticToolkit for Animal DevelopmentAntonis Rokas � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 235

Individuality in BacteriaCarla J. Davidson and Michael G. Surette � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 253

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Transposon Tn5William S. Reznikoff � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 269

Selection on Codon BiasRuth Hershberg and Dmitri A. Petrov � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 287

How Shelterin Protects Mammalian TelomeresWilhelm Palm and Titia de Lange � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 301

Design Features of a Mitotic Spindle: Balancing Tension andCompression at a Single Microtubule Kinetochore Interface inBudding YeastDavid C. Bouck, Ajit P. Joglekar, and Kerry S. Bloom � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 335

Genetics of SleepRozi Andretic, Paul Franken, and Mehdi Tafti � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 361

Determination of the Cleavage Plane in Early C. elegans EmbryosMatilde Galli and Sander van den Heuvel � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 389

Molecular Determinants of a Symbiotic Chronic InfectionKattherine E. Gibson, Hajime Kobayashi, and Graham C. Walker � � � � � � � � � � � � � � � � � � � � � � 413

Evolutionary Genetics of Genome Merger and Doubling in PlantsJeff 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 PhotosynthesisStephan Eberhard, Giovanni Finazzi, and Francis-Andre Wollman � � � � � � � � � � � � � � � � � � � � 463

Planar Cell Polarity Signaling: From Fly Development to HumanDiseaseMatias Simons and Marek Mlodzik � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 517

Quorum Sensing in StaphylococciRichard P. Novick and Edward Geisinger � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 541

Weird Animal Genomes and the Evolution of Vertebrate Sex and SexChromosomesJennifer A. Marshall Graves � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 565

The Take and Give Between Retrotransposable Elementsand Their HostsArthur Beauregard, M. Joan Curcio, and Marlene Belfort � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 587

Genomic Insights into Marine MicroalgaeMicaela S. Parker, Thomas Mock, and E. Virginia Armbrust � � � � � � � � � � � � � � � � � � � � � � � � � � � 619

The Bacteriophage DNA Packaging MotorVenigalla B. Rao and Michael Feiss � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 647

viii Contents

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The Genetic and Cell Biology of Wolbachia-Host InteractionsLaura R. Serbus, Catharina Casper-Lindley, Frederic Landmann,and William Sullivan � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 683

Effects of Retroviruses on Host Genome FunctionPatric Jern and John M. Coffin � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 709

X Chromosome Dosage Compensation: How MammalsKeep the BalanceBernhard 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

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