Structure and Functions of the Bacterial Microbiota of Plants · PDF fileThis process refers...

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Structure and Functions of theBacterial Microbiota of PlantsDavide Bulgarelli,1 Klaus Schlaeppi,1

Stijn Spaepen,1,2 Emiel Ver Loren van Themaat,1

and Paul Schulze-Lefert1

1Max Planck Institute for Plant Breeding Research, D-50829 Cologne, Germany;email: [email protected] of Microbial and Plant Genetics, KU Leuven, B-3001 Heverlee, Belgium

Annu. Rev. Plant Biol. 2013. 64:807–38

First published online as a Review in Advance onJanuary 30, 2013

The Annual Review of Plant Biology is online atplant.annualreviews.org

This article’s doi:10.1146/annurev-arplant-050312-120106

Copyright c© 2013 by Annual Reviews.All rights reserved

Keywords

rhizosphere, endophyte, phyllosphere, plant growth–promotingrhizobacteria, rhizodeposition, plant innate immunity

Abstract

Plants host distinct bacterial communities on and inside various plantorgans, of which those associated with roots and the leaf surface are bestcharacterized. The phylogenetic composition of these communities isdefined by relatively few bacterial phyla, including Actinobacteria, Bac-teroidetes, Firmicutes, and Proteobacteria. A synthesis of available datasuggests a two-step selection process by which the bacterial microbiotaof roots is differentiated from the surrounding soil biome. Rhizode-position appears to fuel an initial substrate-driven community shift inthe rhizosphere, which converges with host genotype–dependent fine-tuning of microbiota profiles in the selection of root endophyte assem-blages. Substrate-driven selection also underlies the establishment ofphyllosphere communities but takes place solely at the immediate leafsurface. Both the leaf and root microbiota contain bacteria that provideindirect pathogen protection, but root microbiota members appear toserve additional host functions through the acquisition of nutrients fromsoil for plant growth. Thus, the plant microbiota emerges as a funda-mental trait that includes mutualism enabled through diverse biochem-ical mechanisms, as revealed by studies on plant growth–promoting andplant health–promoting bacteria.

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Contents

INTRODUCTION . . . . . . . . . . . . . . . . . . 808THE HOST AS DRIVER FOR

THE ESTABLISHMENT OFRHIZOBACTERIALASSEMBLAGES . . . . . . . . . . . . . . . . . . 809Rhizodeposition Mediating

Substrate-Driven CommunityShifts of the Soil Biome . . . . . . . . . 809

Host Genotype–DependentFine-Tuning of the RootMicrobiota . . . . . . . . . . . . . . . . . . . . . 814

A Two-Step Selection Modelfor Root MicrobiotaDifferentiation . . . . . . . . . . . . . . . . . . 816

DISEASE-SUPPRESSIVE SOILS:A DERIVED BIOLOGICALPHENOMENON OF THEROOT MICROBIOTA. . . . . . . . . . . . 817

MICROBIOTA AND PLANTDOMESTICATION . . . . . . . . . . . . . . 818

BACTERIAL COMMUNITIESOF THE PHYLLOSPHERE . . . . . . 819Factors Explaining Community

Composition . . . . . . . . . . . . . . . . . . . 820Source of Inoculum . . . . . . . . . . . . . . . . 821Microbial Traits for Adaptation to

the Phyllosphere Environment . . 822Microbial Assemblies of Other Plant

Organs . . . . . . . . . . . . . . . . . . . . . . . . . 823PLANT GROWTH–PROMOTING

MICROORGANISMS . . . . . . . . . . . . 823Plant Growth Promotion . . . . . . . . . . . 824Biological Control . . . . . . . . . . . . . . . . . 828Plant Growth–Promoting

Rhizobacteria in a CommunityContext . . . . . . . . . . . . . . . . . . . . . . . . 829

The Rhizosphere: A Future Modelfor Molecular PrinciplesUnderlying Niche Formation. . . . 830

INTRODUCTION

In the past 30 years, molecular-genetic analysisof binary plant-pathogen interactions has un-covered the logic of the plant innate immune

system, explaining how plants recognize non-and modified-self molecular structures to trig-ger immune responses and how host-adaptedpathogens subvert the immune response tocause disease (74). In parallel work, the cellularsignaling pathways underlying binary mutual-istic interactions between legumes and nodule-forming rhizobial and mycorrhizal associationswere defined and shown to overlap, suggestingthat molecular components of these legume-specific networks have nonlegume counterpartsin all flowering plants (100). However, these bi-nary parasitic and mutualistic interactions, all ofwhich are linked to the appearance of macro-scopically visible disease symptoms or benefi-cial infection structures such as root nodules,are merely extreme outcomes of a continuum ofinterorganismal associations. In nature, healthyplants host a remarkable diversity of microbesknown as the plant microbiota (22, 30, 78, 85,89). These microbial assemblies appear to besymptomless at first glance, possibly represent-ing a continuum of symbiosis ranging fromcommensalistic to mutualistic interactions. Thelatter provide often-overlooked host servicessuch as indirect pathogen protection and nutri-ent acquisition from soil for plant growth (seebelow). Thus, the plant microbiota emerges asa novel trait that extends the capacity of plantsto adapt to the environment.

Advances in understanding the molecularbasis of fundamental traits are often driven bynew technologies. Next-generation sequencingtechnologies and corresponding bioinformatictools have begun to transform plant microbiotaresearch (see sidebar, Metagenomics: Sequenc-ing and Computational Methodologies). TheseDNA sequencing technologies have for the firsttime enabled systematic culture-independentsurveys of the microbiota, revealing its taxo-nomic structure and the relative abundance ofcommunity members.

A habitat is a specific place occupied by acommunity of organisms for growth and re-production. Thus, plant organs colonized bymicrobial communities with a distinctive phy-logenetic structure represent different habitats.A niche is defined as the totality of biological

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Mutualism: arelationship betweentwo organisms that ismutually beneficial

Parasitism: arelationship betweentwo organisms inwhich one benefits andthe other is harmed

Commensalism: arelationship betweentwo organisms inwhich one benefitswithout affecting theother

Symbiosis: a closebiological relationshipamong two or moreindividuals of differentspecies

and environmental factors that affect a speciesin a habitat, and includes how a species uses thisenvironment (132). Thus, the establishment ofa population of a particular microbiota memberin a community context on a plant organ can beregarded as niche colonization.

Here we present a critical appraisal of plantmicrobiota research, with a focus on plant-associated bacterial communities. Plants alsohost fungal and eukaryotic communities, which,although they can be of critical importance, arenot the focus here. We discuss common anddistinctive features of root and leaf microbiota,present a two-step selection model by whichthe bacterial root microbiota is recruited fromthe surrounding soil biome, and show how thismodel can help to explain derived biologicalphenomena such as soil suppressiveness. Fi-nally, we discuss a range of biochemical mech-anisms underlying rhizobacterial plant growthpromotion and pathogen protection and pro-pose that these exemplify traits encoded by themicrobiome.

THE HOST AS DRIVER FORTHE ESTABLISHMENT OFRHIZOBACTERIALASSEMBLAGES

Soil represents one of the richest microbialecosystems on Earth (50). However, at high tax-onomic rank, a few bacterial phyla—includingAcidobacteria, Actinobacteria, Bacteroidetes,Chloroflexi, Firmicutes, and Proteobacteria—recapitulate most of the diversity of contrastingsoil biomes (46). Table 1 compiles publishedcultivation-independent surveys of the bacte-rial rhizosphere and endosphere microbiota re-trieved from different plant species grown indifferent soils, and indicates that these twoecological habitats are formed by soil biomecommunity shifts that give rise to a distinctivephylogenetic structure with a few dominatingphyla. The underrepresentation of Acidobac-teria members and increased proportion ofProteobacteria and Actinobacteria members inthe rhizosphere and endosphere suggest that acombination of edaphic and plant host–derived

METAGENOMICS: SEQUENCING ANDCOMPUTATIONAL METHODOLOGIES

Questions on community composition (“Who is there?”), func-tion (“What can they do?”), and activity (“What do they do?”)can be addressed by sequencing 16S rRNA, DNA, or mRNAfrom environmental samples. The continual reductions in costand increases in production speed and read length of sequencingtechnologies have made high-resolution community profiling astandard laboratory routine (81). Reports on communities closeto or inside plants show medium taxonomical complexity (seeTable 2 below), resulting in saturated 16S rRNA profiles: Nonew phylotypes are found by increasing sequencing depth using agiven primer (56). Capturing all functions (genes) in a communityis more complicated owing to the high diversity within plant-associated phylotypes (86), contamination with host material,and natural sequencing bias toward the few dominating species ortranscripts. The increasing size of generated data sets in compar-ative functional metagenomics (“How do they differ?”) comes,however, with significant costs in computational infrastructure(81) and challenges computational methodology to improve in ef-ficiency, accuracy, and reproducibility (57). Notable efforts of theopen-source community enabling cloud compatibility includeCloVR (Cloud Virtual Resource) (4), MG-RAST (Metage-nomics Rapid Annotation Using Subsystem Technology) (143),and QIIME (Quantitative Insights into Microbial Ecology) (23).

factors shape the bacterial microbiota compo-sition close to and inside plant roots.

Rhizodeposition MediatingSubstrate-Driven Community Shiftsof the Soil Biome

One potential molecular mechanism underly-ing the formation of a distinctive rhizospheremicrobiota from soil biomes is rhizodeposition.This process refers to intertwined plant devel-opmental and secretory activities in the rootsystem. Rhizodermis cells secrete a wide rangeof compounds, including organic acid ions,inorganic ions, phytosiderophores, sugars, vi-tamins, amino acids, purines, and nucleosides,and the root cap produces polysaccharide mu-cilage (28). Rhizodeposition also refers to the

www.annualreviews.org • Plant-Associated Bacterial Microbiota 809

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Table 1 Bacterial phyla dominating rhizosphere and endophyte bacterial assemblages

Host Species Rhizosphere Endosphere Dominating phyla ReferenceWild oat (Avena fatua)a � Actinobacteria

FirmicutesProteobacteria

29

Oak (Quercus sp.) � AcidobacteriaActinobacteriaProteobacteria

127

Poplar (Populus deltoides) � AcidobacteriaProteobacteria

58

� ProteobacteriaCultivated potato(Solanum tuberosum)a

� ActinobacteriaBacteroidetesFirmicutesProteobacteria

138

Cultivated potato(Solanum tuberosum)

� ActinobacteriaProteobacteria

65

Cultivated potato(Solanum tuberosum)

� ActinobacteriaBacteroidetesProteobacteria

90

Sugar beet (Beta vulgaris)a � ActinobacteriaFirmicutesProteobacteria

95

Cultivated maize (Zea mays)b � Proteobacteria 19Cultivated rice (Oryza sativa)c � Actinobacteria

Proteobacteria78

Cultivated rice (Oryza sativa)d � FirmicutesProteobacteria

120

Thale cress (Arabidopsis thaliana) � AcidobacteriaPlanctomycetesProteobacteria

22

� ActinobacteriaBacteroidetesProteobacteria

Thale cress (Arabidopsis thaliana) � AcidobacteriaActinobacteriaBacteroidetesProteobacteria

89

� ActinobacteriaBacteroidetesFirmicutesProteobacteria

aData generated with PhyloChip.bData generated with a custom-designed 16S rRNA gene microarray.cData generated from whole-metagenome shotgun and 16S rRNA gene clone libraries.dData generated from Sanger sequencing of endophyte-metagenome shotgun clones.


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Microbiota: the set ofmicroorganisms of aparticular habitat

Soil biome: all soiltype–dependentmicroorganisms in aparticular habitat,including nematodesand protists

release of a specialized cell population, calledroot cap border cells, into the rhizosphere (32).Root cap border cells are particularly attractivecandidates for contributors to the rhizosphereeffect because this cell population typicallyremains alive after desquamation from theroot corpus into soil (59). For example, rootcap border cells of maize remain viable in therhizosphere for a week or longer (133). As aconsequence, rhizosphere soil collected forroot microbiota studies is inevitably “contami-nated” with live and dead root cap border cells(Figure 1). Rhizodeposits account for ∼11%of net photosynthetically fixed carbon and10–16% of total plant nitrogen, although thesevalues vary greatly depending on plant speciesand plant age (73). The net sequestration of or-ganic carbon and nitrogen by roots is thoughtto stimulate soil microbial multiplication in thevicinity of root tissues because (a) most knownsoil bacteria are organotrophs, i.e., they derivethe energy for growth from organic substrates,and (b) the accessibility and availability oforganic compounds are limited in most soils (3,31). In the following we discuss experimentalresults that have provided the first molecular in-sights into the presumed role of rhizodepositionin the establishment of a rhizosphere micro-biota distinct from that of the surrounding soil.

Arabidopsis roots release border-like cells(BLCs) from the root caps into the exteriorenvironment (134). These cells were namedBLCs because their desquamation from theroot tip involves the release of organized cellfiles rather than individual cells, as in manyother plant species. Ultrastructural analysisof Arabidopsis BLCs revealed numerous Golgistacks and Golgi vesicles in the cytoplasm,suggesting that these cells have a high secretoryactivity in the rhizosphere (134). Pharmacolog-ical interference of plant cell wall proteoglycanfunctions by application of 3,4-dehydroproline[which inhibits the O-glycosylation of cellwall proteoglycans, including arabinogalactanproteins (AGPs)] or the β-glucosyl Yarivreagent (which binds and precipitates AGPs)resulted in reduced adhesion of the Rhizobiumsp. YAS34 strain to Arabidopsis BLCs and the

Bacteria

Living root cap border cell

Decaying root cap border cells

Organic compounds released by rhizodeposition

Soil

Rhizosphere

Endosphere

Figure 1Niche differentiation at the root-soil interface. From outside to inside, thehabitats are the soil, rhizosphere, and endosphere. Rhizodeposits generatedfrom root cap border cells and the rhizodermis provoke a shift in the soil biome.Cellular disjunction of the root surface during lateral root emergence providesa potential entry gate for the rhizosphere microbiota into the root interior.

rhizodermis in a gnotobiotic test system (134).This points to a potential function of BLC-and rhizoplane-derived cell wall proteoglycansin the attachment of Rhizobium to root cells.One caveat in the interpretation of these exper-iments is that Rhizobium sp. YAS34 was orig-inally isolated from the sunflower rhizosphere(134), and it is not known whether this strainis an indigenous member of the Arabidopsis


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Microbiome: the setof genomes of themicroorganisms in aparticular habitat

Rhizosphere: theregion of soilsurrounding plantroots in which thechemistry andmicrobiology areinfluenced by theroots’ growth,respiration, andnutrient exchange

Endosphere: themicrobial habitatinside plant organs

Edaphic factors: soilproperties thatinfluence biologicalactivity

Rhizosphere effect:enhanced bacterialactivity in therhizosphere

Operationaltaxonomic unit(OTU): a terminalnode in a phylogeneticanalysis

root microbiota. The identification of severalindigenous Rhizobium species in the Arabidopsisroot microbiota (22, 89) should make it possibleto examine the proposed function of AGPs inthe attachment of host-adapted Rhizobium toa nonleguminous root system. The supportingevidence for a link between host-released AGPsand bacterial attachment is the identificationof an AGP in a high-molecular-weight fractionfrom pea root exudates, which is sufficientto induce biofilm formation of Rhizobiumleguminosarum on an artificial glass surface(146). The biofilm on glass is thought to mimicin vivo Rhizobium biofilm formation on rootsand root hairs of nonleguminous plants (115,146).

Genetic support for a function of AGPsin root colonization beyond Rhizobium comesfrom the characterization of an Arabidopsis mu-tant, rat1, that is resistant to transformationby Agrobacterium tumefaciens (52). Arabidop-sis RAT1 encodes a lysine-rich root-expressedAGP, and rat1 mutant plants show reducedAgrobacterium binding to both the rhizoplaneand root hairs, indicating that RAT1 is neededfor an initial binding step during Agrobac-terium root infection. Agrobacterium is a defin-able genus of the family Rhizobiaceae (45), im-plying a conserved function of root AGPs inthe attachment of at least a subset of soil-bornebacteria. Further experimentation is needed todetermine how broadly important AGPs are forthe indigenous microbiota and whether geneticdepletion of Arabidopsis root AGPs can be com-pensated for by particular rhizobacteria duringmicrobiota differentiation.

Evidence for a dynamic root microbiotastructure along the longitudinal axis of the rootsystem was obtained by PhyloChip analysis ofwild oat (Avena fatua) (29). In these exper-iments, rhizosphere prokaryotic communities(without root tissue) were separately studiedat and close to the root tip (0–4 cm from theroot tip), in the root hair zone (4–8 cm fromthe root tip), and at the mature roots (8–16 cmfrom the root tip). Of the 1,917 taxa detected,∼8% (147 taxa) displayed root zone–dependentenrichment (29). In addition, the highest live

bacterial counts were in rhizosphere soil col-lected from the root tip and root hairs, thenext highest were in the rhizosphere of the ma-ture root zone, and the lowest were in bulk soil(29). Thus, if rhizodeposits are causally linkedto the formation of a rhizosphere-specificbacterial microbiota, then the observed rootzone–dependent enrichment of subsets of thiscommunity should reflect local differences inamounts and/or composition of metabolites re-leased along the longitudinal axes of roots. Forexample, the differentiation and release of rootcap border cells only at the root tip (Figure 1)is consistent with a dynamic substructure ofthe microbiota along the longitudinal rootaxis.

Plant genes encoding membrane-residentATP-binding cassette (ABC) transporterproteins are plausible candidates for genes me-diating the export of small molecules from rootcells into the rhizosphere. Mutants of sevenArabidopsis ABC transporter–encoding genesthat are highly expressed in roots were grownin Arabidopsis-accustomed soil over two gener-ations, and the microbiota of their roots withattached soil was compared with that of wild-type plants by automated ribosomal intergenicspacer analysis (ARISA) (6). One ABC trans-porter mutant, abcg30, exhibited differences inARISA profile compared with the wild type, andthis correlated with an altered metabolic profilein abcg30-derived root exudates collected from21-day-old seedlings grown in liquid media.NMR spectroscopy of exudates collected fromliquid media–grown abcg30 plants showed thatthey contained elevated levels of phenolicsand reduced amounts of sugars. Reassessmentof the root microbiota profiles of singlenonreplicated samples of wild-type and abcg30plants by low-pass 16S rRNA gene pyrose-quencing suggested an increased abundance ofpotentially beneficial bacteria in abcg30 mutantroots. However, this conclusion is based onlow-abundance operational taxonomic units(OTUs), and their significance is difficult toassess because the variation of low-abundanceOTUs between full factorial replicates in suchexperiments is generally high (22, 89).


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To test a potential driver role of commonroot exudates in soil biome community shifts,Eilers et al. (41) simulated exudation by addingthe low-molecular-weight carbon substratesglucose, glycine, or citric acid to microcosmscontaining three soils derived from grassland,hardwood forest, and coniferous forest. The ad-dition of each substrate altered the soil commu-nity composition in a soil type–dependent andcarbon substrate–dependent manner. Across alltreatments and all tested soil types, the observedcommunity shifts resulted mainly from an in-crease in the relative abundance of the subphylaBetaproteobacteria and Gammaproteobacteriaand the phylum Actinobacteria, suggesting theexistence of specific bacterial taxa that preferen-tially respond to organic carbon substrate addi-tion. The community shifts in soil are unlikelyto be the result of indirect pH shifts becausethe low-molecular-weight carbon substrate so-lutions were equally adjusted to pH 7 (41).

Although these experiments ignore thefacts that in vivo soil bacteria are exposedto a mixture of exudate molecules and thatonly a single relatively high test substrateconcentration was examined, it is a strikingcoincidence that in the rhizosphere and/or rootendosphere compartments of different plantspecies, Actinobacteria and Proteobacteriabecome almost invariably enriched (Table 1).Thus, it is possible that the establishment ofa distinctive rhizosphere bacterial communityis at least partly the result of substrate-drivencommunity shifts fueled by the secretion ofphotoassimilates from root cells. Supportingevidence for this hypothesis comes frommolecular-genetic work on the chemotaxis ofthe soil-borne pathogenic bacterium Ralstoniasolanacearum, which invades host plants viaroots (148). R. solanacearum is specificallyattracted by diverse amino acids, organic acids,and root exudates from its host plant, tomato.R. solanacearum mutants lacking either cheAor cheW, two key regulatory components ofbacterial chemotaxis, were found to be fullynonchemotactic but retained otherwise normalswimming behavior. The nonchemotacticmutants reached the same population size as

the wild type in a soil soak assay but exhibitedreduced virulence. Importantly, the chemotaxismutants were as virulent as the wild-type strainwhen inoculated directly into the plant stem(148), which is consistent with the idea thatbacterial chemotaxis makes a contributionto the early phase of host colonizationthrough the perception of root exudates inthe rhizosphere. Similarly, a nonchemotacticcheA mutant of the plant growth–promotingbacterium Pseudomonas fluorescens resulted instrongly reduced competitive tomato rootcolonization ability and identified malic acidand citric acid as major chemoattractants forthis microbe in the tomato rhizosphere (34).By applying distinct approaches such as invitro chemotactic assays, transcriptome studieson bacterial gene expression, and colonizationassays with wild-type and benzoxazinoid-deficient corn mutant plants, a recent studydemonstrated that another plant growth–promoting bacterium, Pseudomonas putida, isrecruited to plant roots by chemotaxis towardthe benzoxazinoid secondary metabolites (99).Although these studies provide compellingevidence for a role of root exudates in attractingindividual rhizobacteria, it remains to be shownwhether this function is retained in a commu-nity context and whether a mixture of exudatemolecules is sufficient to enforce the charac-teristic taxonomic community differentiationseen in the root microbiota (Table 1).

Direct evidence for a net carbon flux fromplants to rhizobacterial communities can be ob-tained by stable-isotope probing (SIP) tech-niques. SIP in combination with microbiotaDNA profiling (DNA-SIP) offers unprece-dented opportunities to obtain deeper insightsinto interorganismal carbon flow at the plant-soil interface (24). The rhizosphere representsan ideal experimental system for SIP-basedcommunity profiling because the labeled sub-strate, typically 13CO2, can be added to create adefined atmosphere in phytotrons in which at-mospheric 13CO2 is converted into organic car-bon by the Calvin-Benson cycle in green leaves.Long-distance transport of organic 13C fromshoot to root, its release, and its subsequent


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capture by the root microbiota can be measuredby purifying labeled (“heavy”) 13C rhizobacte-rial chromosomal DNA from the nonlabeled(“light”) DNA fraction using CsCl density gra-dient centrifugation. A key advantage of DNA-SIP is that the label serves as a selective tagfor active bacteria within the root microbiotawhose growth is stimulated by root exudation.

In combination with low-resolution micro-bial ribotyping, SIP has been utilized to explorethe potential effect of altered glucosinolatemetabolism on bacterial and fungal commu-nities in the Arabidopsis rhizosphere (20). Glu-cosinolates are a class of Capparales-specificphytochemicals previously shown to haveantimicrobial activity in plant-microbe andplant-insect interactions (11). Microbial DNAsamples of the rhizosphere and roots of a trans-genic Arabidopsis line expressing the sorghumcytochrome P450–encoding gene CYP79A1,which is known to produce high levels of theexogenous tyrosine-derived p-hydroxybenzylglucosinolate (7), were inspected by low-resolution denaturing gradient gel elec-trophoresis (DGGE) fingerprinting following13CO2 labeling and compared with wild-typeArabidopsis plants (20). Although glucosinolateproducts were undetectable in rhizospheresoil, Alphaproteobacteria (mainly Rhizobiales)and fungal communities discriminated boththe active rhizosphere and root endophytecommunities of the CYP79A1-expressing linefrom those of the wild type. However, thesefindings do not provide clues about whetherendogenous Arabidopsis glucosinolates con-tribute to the establishment of the Arabidopsisroot microbiota. Despite these limitations,DNA-SIP and advances of techniques that areable to trace the fate of minerals and organiccompounds in situ, such as nanometer-scalesecondary ion mass spectroscopy (69), promiseto generate high-resolution quantitative mapsof nutrient flow at the root-soil interface (25).

Host Genotype–DependentFine-Tuning of the Root Microbiota

Using high-resolution 16S rRNA gene pyrose-quencing of bulk soil, rhizosphere, and root

compartments collected from eight Arabidop-sis ecotypes grown in two soil types, Lund-berg et al. (89) identified among 778 measur-able OTUs a total of 12 OTUs exhibiting hostgenotype–dependent quantitative enrichmentin the root endophyte compartment. Utilizinga similar sequence-based 16S ribotyping plat-form but two other natural soils, Bulgarelli et al.(22) identified only one OTU of the bacterialroot endophyte community that showed sig-nificantly different quantitative enrichment be-tween the two Arabidopsis ecotypes tested. Inboth studies, soil type and the respective soilbacterial biomes had a greater influence thanthe host genotype on the composition of rootendophyte communities. Thus, a significant butweak host genotype–dependent effect acts inthe selection of Arabidopsis root-inhabiting bac-terial communities. Micallef et al. (96) reporteddifferences in both the composition and relativeabundance of rhizosphere community mem-bers among eight tested Arabidopsis ecotypes byapplying terminal restriction fragment lengthpolymorphism (T-RFLP) and ARISA. How-ever, it remains unclear how many differentialT-RFLP peaks result from sampling inaccura-cies (the rhizosphere was collected using scalpelblades) and how many genotype-specific signalsare reproducible in replicate experiments, i.e.,using independent soil samples collected fromthe same field plot.

Other than the Arabidopsis reports (22, 89,96), few studies have explored the magnitudeof host genotype–dependent variation onbacterial root microbiota profiles. UtilizingPhyloChip, a high-density 16S rRNA geneprobe array that can detect up to 8,741 knownOTUs, Weinert et al. (138) examined the rootmicrobiota of three cultivars of field-grownpotato plants (rhizosphere plus root-inhabitingbacterial communities) in two different soils. Ofthe 2,432 OTUs detected, 9% showed a quan-titative cultivar dependence in one of the soilstested and 4% showed a dependence in both.The host genotype–dependent OTUs belongmainly to the phyla Actinobacteria, Chloroflexi,Firmicutes, and Proteobacteria (138). Consis-tent with a previous DGGE analysis of the same


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biological material (137), a greater number ofOTUs (28%) differentiated the root micro-biota of these potato plants grown in differentsoils (138). Thus, similar to the Arabidopsisstudies (22, 89), soil type influences the potatoroot microbiota profiles to a greater extentthan host genotype does. Although these datawere obtained from a single growing seasonand are limited by the preselected PhyloChipprobe set, the conclusions are well supportedby statistical analysis. Unfortunately, the lackof data on the biomes of the correspondingunplanted soils precludes numerical informa-tion on the expected potato root rhizosphereeffect.

Two other reports of field experiments withpotato plants illustrate the difficulties in infer-ring general conclusions on the potato micro-biota when different sampling methods are em-ployed (65, 66). These experiments involved acomparison of bulk soil and rhizosphere com-partments of six potato cultivars grown in twosoil types. Bacterial 16S rRNA gene pyrose-quencing revealed members of the phyla Acti-nobacteria and Alphaproteobacteria as dom-inant taxa in both the unplanted soil biomeand rhizosphere communities (65). Based onthe relative abundance of 16S rRNA gene se-quences assigned to bacterial genera, the struc-ture of the rhizosphere assemblages was dif-ferent from that of the unplanted bulk soilbiomes at each of three tested developmen-tal stages (young leaf development, florescence,and senescence) (65). However, a significanthost genotype–dependent rhizosphere effectwas detected only in young potato plants. Thisdifferentiation occurred mainly on the axis thatexplained ∼5% of the observed variation in aprincipal components analysis and is thereforeweak. In addition, hierarchical clustering of therelative abundance of the major bacterial classesand phyla from the same samples did not reveala clear host development–dependent or hostgenotype–dependent effect on rhizosphere mi-crobial profiles.

Although currently available bacterialroot microbiota studies suggest a weak hostgenotype–dependent effect, it is important

to mention that the resolution power ofsequence-based 16S rRNA ribotyping isinherently limited to the species level or highertaxonomic ranks. However, subspecific geneticvariation in pathogenic microorganisms,including bacteria, has a key role in hostgenotype–dependent colonization (119). Thus,if subspecies genetic variation of microbiotamembers contributes to host colonizationsuccess, the actual host genotype–dependenteffect cannot be determined with availablecommunity fingerprinting technologies.

When wooden splinters from two treespecies were incubated in the same natural soilsused to define the Arabidopsis root-inhabitingbacterial microbiota, approximately 40% ofthe root-inhabiting OTUs also colonized thisdead plant material (22). This microbiota sub-community consists largely of Proteobacteriaand is not specific to Arabidopsis, and probablyrepresents saprophytic bacteria that populatethe roots of any plant species, including decay-ing plant litter. Notably, the other 60% of theArabidopsis root microbiota is dominated byActinobacteria, followed by Proteobacteria andBacteroidetes (22). For a deeper interpretationof these findings, it is relevant that upon termi-nation of root primary growth (i.e., when rootsreach their maximal length), secondary growthensues, characterized by root thickening andthe appearance of secondary phloem andsecondary xylem. The latter tissue is typicallyresponsible for the woody appearance of ma-ture root systems and results from the apoptoticdeath of cell files, which leaves behind largeamounts of lignified cell wall cellulose microfib-rils (97). Within a few weeks after germinationof Arabidopsis seeds, part of the primary root(rhizodermis, cortex, and endodermis) isreplaced by new cells during secondary thick-ening (38). Thus, woody material is an integralpart of mature root systems utilized for mostroot microbiota studies. In this wider context itis possible that Actinobacteria, Proteobacteria,and Bacteroidetes represent early root endo-phytes and that the Actinobacteria members areoutcompeted during root secondary growth.Alternatively, there is no dynamic succession


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of root microbiota members during root de-velopment, but woody parts and metabolicallyactive root cells are instantly populated bydistinct bacterial subcommunities. Irrespectiveof these alternative niche-filling mechanismsof live and dead root cells, the detection ofpotentially saprobic bacteria in the roots of liveArabidopsis plants could point to their activity inthe decomposition of organic matter after plantdeath.

A Two-Step Selection Model for RootMicrobiota Differentiation

A comparison of the bacterial and fungal rootmicrobiota of mature poplar (Populus deltoides)trees growing at two natural sites revealed strik-ingly different endophyte community compo-sitions compared with the surrounding rhizo-sphere (58). 16S rRNA gene pyrosequencing ofthe bacterial endophyte communities displayedan order-of-magnitude reduction in richness(number of OTUs identified by rarefactionanalysis) and were dominated by members ofProteobacteria (>80% of retrieved OTUs). Incontrast, Acidobacteria dominated rhizosphereassemblages and was underrepresented in theroot-inhabiting communities (58). Fungal rhi-zosphere and endophyte samples had similaramounts of Pezizomycotina, whereas Agari-comycotina was more abundant in the root en-dosphere. It is possible that undersampling ofthe endophyte compartment partly contributedto the marked differences in the communitystructures of the rhizosphere and endosphere.It is also possible that complex interactions be-tween the fungal and bacterial microbiota con-tributed to the rhizosphere and endospherecommunity differentiation, because poplar isunusual among higher plants in that it engagesin symbiotic interactions with both endomy-corrhizal Glomeromycota fungi and ectomy-corrhizal Ascomycotina and Basidiomycotinafungi. With these limitations and the peculiarpoplar biology aspect in mind, this shows thatthe root interior represents a microbial habi-tat on its own and is not fortuitously filled byrhizosphere members.

Qualitatively similar observations were re-ported in two independent studies on the bac-terial root microbiota of Arabidopsis (22, 89). Inboth studies, the microbiota inhabiting root tis-sues is markedly differentiated from the one thatpopulates the rhizosphere or unplanted soil.This was evident from both a reduced richness(estimated through rarefaction curves) of theroot-inhabiting communities and concomitantincreases in the abundance of Actinobacteria,Bacteroidetes, and Proteobacteria. In contrast,Acidobacteria members that dominate both un-planted soil and the rhizosphere were virtuallyexcluded from the root endosphere (22, 89). Animportant finding of these two studies is that theendosphere taxonomic profiles are remarkablysimilar (mainly Actinobacteria, Bacteroidetes,and Proteobacteria), although the plants weregrown in four different soils on two continents.In addition, the rhizosphere community dif-ferentiation in comparison with bulk soil wasweak in all four tested soils (22, 89). Theseresults argue strongly against fortuitous nichefilling of the endosphere from rhizosphere as-semblages and predict the existence of differ-ent host-controlled mechanisms underlying thedifferentiation of rhizosphere and endospherecommunities.

A collective synthesis of the available litera-ture suggests a two-step selection process, grad-ually differentiating the root microbiota fromthe surrounding soil biome. In this model, rhi-zodeposition fuels an initial substrate-drivencommunity shift in the rhizosphere, whichconverges with host genotype–dependent fine-tuning of microbiota profiles during endophytemicrobiota differentiation (Figure 2). Accord-ingly, substrate-driven selection in the rhizo-sphere is expected to persist in the endosphere.Note that the magnitude of selection in the rhi-zosphere compared with that of the endospherecan vary greatly in different plant species andas a function of the host genotype (Table 1).Likewise, the magnitude of rhizosphere com-munity differences relative to that of the soilbiome can vary in different soil types. One pre-diction of this model is the existence of geneticadaptation of the host to different soil types;


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Edaphic factors

Rhizodeposits and cell wall features

Host genotype factors

Bacterial phylaAcidobacteria

Actinobacteria

Proteobacteria

Bacteroidetes and Firmicutes

Figure 2A two-step selection model for root microbiotadifferentiation. Edaphic factors determine thestructure of bacterial communities in soil biomes. Inthe first differentiation step, rhizodeposits and hostcell wall features promote the growth oforganotrophic bacteria, thereby initiating a soilbiome community shift. In the second step,convergent host genotype–dependent selectionclose to and within the root corpus fine-tunescommunity profiles thriving on the rhizoplane andwithin plant roots.

i.e., optimal plant growth depends on specificcombinations of host genotype–dependent andsoil type–dependent bacterial start inoculum.

Among numerous factors that could explainthe observation of distinctive rhizosphere andendophyte communities within the root mi-crobiota, the innate immune system is a primecandidate for the selection of a distinctive rootendophyte microbiota. Plants have evolved anelaborate innate immune system consisting oftwo classes of immune receptors that detectthe presence of nonself molecules both insideand on the surface of host cells (74). Nonselfrecognition activates powerful immune re-sponses to terminate microbial multiplicationof pathogens. The identification of an increas-ing number of pattern recognition receptorson the plant cell surface during the past decadeis intuitively difficult to reconcile with the

colonization of the root interior by soil-derivedendophytic microbial communities becausethis class of immune receptors detects a widearray of microbe-associated molecular pat-terns (MAMPs) (17). Characterized MAMPsrecognized by cognate cell surface receptorsare epitopes derived from bacterial flagellin,the translation elongation factor Tu, bacteriallipopolysaccharide, or fungal chitin (13, 17).The discovery of root endophyte communitieswith a defined taxonomic structure could bereconciled with the current framework of plantinnate immunity if these microbes are capableof immune response interception, as has beendemonstrated for pathogenic microbes (18).It is also conceivable that endophytes evolvedeffective MAMP camouflage mechanisms toescape immune receptor detection. Finally,it is possible that the innate immune systemis activated upon endophyte colonizationbut this limits endophyte multiplication atmicrobial titers that are well below those ofpathogenic bacteria causing disease symptoms.Clearly, future experimentation is neededto discriminate between these three models,which are not necessarily mutually exclusive.

DISEASE-SUPPRESSIVE SOILS:A DERIVED BIOLOGICALPHENOMENON OF THE ROOTMICROBIOTA

Disease-suppressive soils are those in whichlittle or no disease occurs under conditionsthat are favorable for disease development(77). Disease suppressiveness can be a naturalproperty of certain soils and persist in theabsence of cultivation or can be induced aftera monoculture of the same crop species forseveral years, followed by a severe diseaseoutbreak (14). In both cases, the bacterialsoil biome plays a critical role: Pathogeninoculation of pasteurized suppressive soilsinvariably leads to reestablishment of thehost disease (95). Whereas natural diseasesuppression is not limited to a particular plantspecies and affects a broad range of pathogens,induced disease suppression arises from specific


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host-pathogen combinations, suggesting a pri-mary role for the root microbiota in controllingthe disease (14). Host plants protected againstsoil-borne pathogens through soil disease sup-pressiveness do not appear to harbor crypticdisease-resistance genes against the respectivepathogens (77), supporting the idea that thistrait is delegated to the root microbiota.

PhyloChip analysis of the rhizospheremicrobiota of sugar beet plants grown onsoils either suppressive of or conducive toRhizoctonia solani, a major fungal pathogen ofsugar beet, revealed that the two conditionsdid not significantly alter the detected numberof bacterial taxa and that both soil types weredominated by members of Actinobacteria,Firmicutes, and Proteobacteria (95). Likewise,heat treatments, amendment of conducivesoil with a small amount (10%) of suppressivesoil, and suppressive soil inoculation with R.solani did not provoke a significant effect in thediversity of the analyzed rhizosphere bacterialmicrobiota (95). These data suggest that dis-ease suppression might arise from factors otherthan the mere presence or absence of certainbacteria. Consistent with this, Bray-Curtisdissimilarity matrix analysis calculated on therelative abundance of the identified bacterialand archaeal OTUs discriminated the rhizo-sphere microbiota of the suppressive soils fromthat of the conducive soils (95). Burkholderi-aceae, Lactobacillaceae, Pseudomonadaceae,and Xanthomonadales were identified as themost dynamic in the data set—i.e., these taxawere responsive to all soil conditions tested.In contrast, Actinobacteria members largelyaccounted for the observed differentiationamong the tested suppressive soil, suppressivesoil inoculated with R. solani, and conducivesoil (95).

Interestingly, the enrichment of Actinobac-teria in the Arabidopsis root microbiota dependson metabolically active root cells (22), andthe wide range of antimicrobial compoundssecreted by members of this bacterial phylum(10) suggests a possible role of such compoundsin indirectly protecting sugar beet against thesoil-borne fungal pathogen. In a parallel

experimental attempt, a culture-dependentisolation of bacteria from the suppressive soilwas conducted and yielded a disproportion-ate amount of pseudomonads (95). SeveralPseudomonas strains were identified that are sig-nificantly enriched in the disease-suppressivesoil compared with conducive soils. This isnotable because the relative abundance of Pseu-domonas producing the antibiotic compound2,4-diacetylphloroglucinol (2,4-DAPG) cor-relates with the control of another soil-bornefungal pathogen, Gaeumannomyces graminis, insoil suppressive of the “take-all disease” causedby this fungus in cereals (107). Only one of theisolated Pseudomonas strains from the R. solanidisease-suppressive soil conferred protectionagainst the pathogenic fungus in a plantbioassay, indicating that more bacteria thanthe above-mentioned Actinobacteria mightcontribute to the observed soil suppressiveness.However, random transposon mutagenesisof this Pseudomonas strain revealed that somemutants have the competence to colonize therhizosphere at similar levels compared withthe wild-type strain but fail to confer diseaseprotection (95). This suggests that Pseudomonasrhizosphere colonization competence canbe uncoupled from disease suppression (95).Although the exact molecular mechanisms trig-gering the establishment of disease-suppressivesoils remain largely obscure, the ensuingsoil biome community shift(s) initiated bysevere disease outbreak likely contribute to thephenomenon of soil suppressiveness.

MICROBIOTA AND PLANTDOMESTICATION

Since its inception ∼10,000 years ago, plantdomestication has produced a large num-ber of cultivated plants from wild ancestorsthrough continuous anthropogenic selectionto meet the food and feed demand of humansocieties (106). Domestication has progres-sively homogenized plant genotypes, therebyeroding natural genetic variability present innondomesticated ancestors (37). Because hostgenotype–dependent selection is an essential


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Teosinte: the wildancestor of cultivatedmaize

Phyllosphere: themicrobial habitatdefined by the surfaceof aboveground plantorgans

component of the two-step selection model forroot microbiota (Figure 2), the hypothesis thatdomestication has inadvertently affected mi-crobiota profiles seems reasonable. It has beenpostulated that old cultivated forms and theirwild ancestors were generally exposed to moremarginal soils before the invention of syntheticfertilizer–driven agricultural production, andtheir gene pools might have a different adaptivecapacity to engage in probiotic associationswith rhizosphere microbes compared with thegene pools of present-day cultivars (144). Theroot microbiota might therefore represent anuntapped trait for future rational plant breed-ing through the selection of host genotypesthat capture an optimal microbiota from agiven soil type to reduce synthetic fertilizerinputs. Experimental data testing this idea,although unfortunately sparse, are discussedbelow.

To test the hypothesis that crop evolution-ary history shaped how the five main geneticgroups of maize interact with soil bacteria in therhizosphere, Bouffaud et al. (19) examined theirrhizobacterial community composition using a16S rRNA taxonomic microarray that targets19 bacterial phyla. Differences in the com-munity composition of 21-day-old seedlingsgrown in the same European soil were foundin the abundance of certain Betaproteobacteriaand Burkholderia members and were subse-quently validated by quantitative polymerasechain reaction (PCR). However, most of thecommunity structures were common to thefive genetic groups, which is reminiscent of aweak host genotype–dependent activity in thecomposition of the Arabidopsis root bacterialassemblages (89). Notably, the few differencesin community profiles did not correlate withgenetic distances between the five tested maizegroups or individual lines. These data suggestthat the genetic structure of maize that aroseduring crop diversification, but not the extentof maize diversification in itself, influencesthe selection of rhizobacterial communities inmaize seedlings. It remains to be seen whetherthis also applies to the maize root endosphereand can be replicated using 16S rRNA gene

sequencing technology that targets all knownbacterial phyla.

T-RFLP analysis of the microbiota re-trieved from surface-sterilized seeds and stemsof 14 genotypes of corn cultivars, landraces, andancestors (teosinte) revealed distinct seed endo-sphere profiles of plants grown in distinct pe-doclimatic regions (72). Once these plants weregrown for one generation in the same soil andunder the same climatic conditions, endophytediversity disappeared, suggesting the existenceof a seed-heritable core endosphere micro-biota across the genus Zea. However, becausethe applied seed surface sterilization methoddoes not destroy microbial DNA detectableby PCR, it remains possible that the observedT-RFLP profiles represent surface-attachedbacteria rather than Zea seed endophytes.

BACTERIAL COMMUNITIESOF THE PHYLLOSPHERE

Microbial communities dwell on and in aerialplant organs. The term phyllosphere refersto aboveground plant surfaces as a habitat formicrobes. The bulk of this surface is providedby green leaves, and it is thought to representone of the largest microbial habitats on Earth.Compared with fungi and archaea, bacteria arethe most prevalent phyllosphere-colonizingmicrobes, with bacterial titers averaging ap-proximately 106–107 microbial cells per squarecentimeter of leaf area (85). Phyllospheremicroorganisms are exposed to acute fluctua-tions in temperature, humidity, and UV lightirradiation and face limited access to nutrients(62). This differs from the comparatively weakand buffered fluctuations of abiotic conditionsprevailing in the rhizosphere. Phyllospheremicrobial communities impact global carbonand nitrogen cycles and provide microbialservices to the host, e.g., indirect pathogenprotection (85, 141).

In comparison with diverse microbialenvironments such as coastal seawater habitatsand farm soil, the phyllosphere represents anenvironment of reduced bacterial complexity(30). This is similar to other host-associated


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Table 2 Bacterial phyla dominating phyllosphere bacterial assemblages

Host species Dominating phyla ReferenceSoybean (Glycine max)a Actinobacteria

BacteroidetesProteobacteria

30

56 tree speciesb ActinobacteriaBacteroidetesFirmicutesProteobacteria

107

Cultivated rice (Oryza sativa)a ActinobacteriaProteobacteria

78

Cultivated lettuce (Lactuca sativa)b BacteroidetesFirmicutesProteobacteria

108

Cultivated spinach (Spinacia oleracea)b ActinobacteriaProteobacteria

87

Salt cedar (Tamarix sp.)b ActinobacteriaFirmicutesProteobacteria

47

Several plant speciesc FirmicutesProteobacteria

141

aData generated with shotgun metagenomics and 16S rRNA clone libraries.bData generated with pyrosequencing of 16S rRNA gene amplicons.cData generated with 16S rRNA gene clone libraries.

habitats, including the rhizosphere and thevertebrate gut microbiota. Thus, relatively fewbacterial phyla define the phylogenetic struc-ture of phyllosphere communities (Table 2):Actinobacteria, Bacteroidetes, Firmicutes,and Proteobacteria, of which the last oftendominates the phyllosphere community. A fewbacterial genera, including Pseudomonas, Sph-ingomonas, Methylobacterium, Bacillus, Massilia,Arthrobacter, and Pantoea, appear to composethe core of phyllosphere communities. DNAand protein samples collected from leaf sur-faces of field-grown soybean and clover aswell as from a wild population of Arabidopsisthaliana identified Sphingomonas spp. andMethylobacterium spp. as the most commoncommunity members, both belonging to theclass of Alphaproteobacteria (30).

Members of the genus Sphingomonas maycontribute to plant health, as evidenced by thesuppression of disease symptoms and reducedgrowth of the foliar pathogen Pseudomonassyringae pv. tomato strain DC3000 on A. thaliana

under laboratory conditions (68). Interestingly,this beneficial service to the host was not ob-served in sphingomonads isolated from air,dust, or water, indicating that the capacity forbiocontrol is unique to plant-adapted strains.Using a forward-genetic in planta screen, 10mutants of Sphingomonas sp. Fr1 were identi-fied that have intermediate disease-suppressivecapabilities (but retain leaf colonization compe-tence) and map to seven genomic regions (135).This points to the existence of several parallelmolecular mechanisms, each contributing par-tially to the disease-suppression trait of Sphin-gomonas sp. Fr1.

Factors Explaining CommunityComposition

Phyllosphere communities for 10 treespecies, all located within the same 35-hectare area, have been surveyed using apyrosequencing-based approach (110). Thevariation found between individual trees ofthe same species (intraspecific variation) and


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between distinct species (interspecific varia-tion) was investigated. In addition, bacterialphyllosphere variation was tested betweensamples collected from the same individual(intraindividual variation). The bacterialcommunity variation was largest betweensamples from different tree species and lowerat the intraspecific and intraindividual levels.However, interspecific variation, measured inUniFrac distances, was only slightly higher(∼0.70) compared with those found amonginterspecific and interindividual samples(∼0.66 and ∼0.64, respectively). This indicatesthat the phyllosphere bacterial microbiotadisplays considerable variability in communitycomposition even in replicate samples from thesame host plant. When intraspecies communityvariability was tested as a function of geo-graphic distance by comparing phyllospherecommunities from Pinus ponderosa sampledfrom several locations around the globe, min-imal geographic differentiation was observed(110). These observations support the notionthat the host plant species is a determinantfor the structure of the phyllosphere com-munity. Root-associated bacterial assemblies,in contrast, are defined largely by soil type(i.e., the bacterial start inoculum present inthe surrounding soil biome), and the hostgenotype is responsible for the fine-tuning ofcommunity structure during the establishmentof the root endophyte microbiota (see thetwo-step selection model shown in Figure 2).

In this context, it is relevant that thecharacterization of the leaf microbiota is stillfragmentary; there have been no studies di-rectly comparing epiphytic and endophytic leafmicrobiota profiles by culture-independentmethods. Similarly, only a handful of stud-ies have systematically compared leaf- withroot-associated communities collected fromthe same plant individuals (78). Fundamentalphysiological differences need to be takeninto account when comparing rhizosphereand phyllosphere communities. Unlike rootexudation, in which significant amounts of pho-toassimilates are released into the rhizospherespace, there is no evidence for an equivalent

mechanism that releases large quantities ofsoluble organic compounds to the leaf surface.Thus, instead of a gradual substrate-drivencommunity shift of the soil biome initiatedat a distance from the root corpus in therhizosphere, the selection of phyllospherecommunities appears to take place solely atthe immediate leaf surface. Although the leafcuticle and plant cell wall molecules in princi-ple provide ample organic matter for bacterialgrowth, soluble organic compounds are scarceon the leaf surface (85). These differences inthe abundance of organic substrates on leaf androot surfaces might at least partly enable thedifferentiation of distinctive phyllosphere andrhizosphere communities through a commonprinciple, substrate-driven selection (see belowevidence for distinctive molecular adaptationstrategies of phyllosphere bacteria).

Source of Inoculum

The defined phylogenetic structure of thelow-complexity phyllosphere communitiesprompts questions regarding the source of itsstart inoculum. Intuitively, one might think ofair and its aerosols, which flow around leavesand are known to transport bacteria. However,the typical bacterial titer in air as determinedby different methods ranges from 101 to 105

cells per cubic meter (43), which is orders ofmagnitude lower than the typical titer in soil,which ranges from 106 to 109 cells per gram(142). In addition, aerosol-associated bacteriatypically have a mixed and variable origin,ranging from marine and soil to plant and an-imal sources, and must survive in an extremelynutrient-poor environment exposed to UVlight. Notably, abundant sequences assigned toSphingomonas and Pseudomonas were identifiedin clone libraries of several aerosol samples,indicating that air presents one route of trans-mission for these genera (43). Neighboringplants and plant debris constitute anotherimportant immigration source, as these bacte-ria have already adapted to the phyllosphere.For a Mediterranean site, Vokou et al. (136)determined the relatedness between airborne


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Bacterial phylaActinobacteria

Proteobacteria

Bacteroidetes and Firmicutes

Soil~106–109 g–1

Leaf area~106–107 cm–2

Other sources

Rhizosphere~106–109 g–1

Root endosphere~104–108 g–1

AtmosphereAtmosphere~10~101–10–105 m–3–3

Atmosphere~101–105 m–3

Figure 3Bacterial titers and phyllosphere community composition. Numbers ofbacterial cells in phyllosphere, atmosphere, rhizosphere, and root and soilbacterial communities were taken from References 43, 85, 123, and 142,respectively. An exact comparative enumeration of bacteria in thesemicrohabitats based on a literature search is not possible, as the experimentalsetups differ in many variables, such as plant species, cultivation media,sampling unit (e.g., fresh or dry root weight), and bacterial diversity(colony-forming units based on reisolation of a single strain versus communitycolony-forming units). The taxonomic structure of phyllosphere communitiesis dominated by Actinobacteria ( purple), Bacteroidetes and Firmicutes ( yellow),and Proteobacteria (red ) (see Table 2). Open and solid arrows representinoculation routes for the phyllosphere and root microbiota, respectively.

bacterial populations and the phyllospherebacterial communities of nine perennial plantspecies using DGGE followed by cloningand sequencing of dominant bands. Only 2 of28 taxa were present in both the air and thephyllosphere, whereas 8 and 18 unique bandswere detected, respectively, documenting thehigh degree of dissimilarity between these twomicrobial habitats. Another case study useda metaproteogenomics approach to comparethe phyllosphere communities of paddy-field-grown rice (Oryza sativa) plants with the corre-sponding rhizosphere communities in relationto the flooding water of the paddy field (78).Hierarchical cluster analysis of the proteomecomposition indicated a closer relatednessbetween phyllosphere and water communitiesthan between phyllosphere and rhizospherecommunities, possibly pointing to a water-based start inoculum for the phyllosphere ofpaddy rice. In comparison with the root micro-biota, the origin of the bacterial phyllospheremicrobiota appears to be much more variableand remains ill defined (Figure 3).

Microbial Traits for Adaptationto the Phyllosphere Environment

Remarkable insights into phyllosphericlifestyles of bacteria have been obtained bycombining metagenomic and metaproteomicapproaches. Delmotte et al. (30) identifiedmicrobial proteins, which appear to reflectdifferential adaptation strategies to the leafenvironment of two abundant phyllospherecolonizers, Sphingomonas spp. and Methy-lobacterium spp. For example, Methylobacteriumexpresses proteins allowing the use of methanol,a by-product of plant cell wall metabolism,as its carbon and energy source (126), indi-cating that these bacteria adapt via a specificmethylotrophic one-carbon metabolism to thephyllosphere. In contrast, multiple transportproteins, including TonB-dependent recep-tors, were found from Sphingomonas spp.,suggesting the exploitation of a large substratespectrum, which could serve as an alternativeadaptation strategy to scavenge diverse plant


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PGPRs: plantgrowth–promotingrhizobacteria

metabolites present in low amounts on the leafsurface for bacterial growth.

Microbial Assemblies of OtherPlant Organs

In comparison with rhizosphere and green leafhabitats, little is known about microbial envi-ronments in other plant organs. Limited infor-mation is available on bacterial communitiesthriving on flowers, fruits, and seeds. Highlyabundant Enterobacteriaceae species were iso-lated on synthetic media from petal-associatedbacterial communities of Saponaria officinalisand Lotus corniculatus, and the composition ofthese communities was clearly different fromthose found on green leaves (75). Similarly,based on cultivation-dependent methods, bac-teria are the most abundant colonizers of flow-ers, fruits, and seeds. For example, bacteria ofStyrian oil pumpkin flowers and fruits reachdensities of 107 and 104 cells per gram of tis-sue, respectively, whereas seed-associated bac-teria reach at most 102 colony-forming units(CFU) (49). In grapevine, the lowest bacterialtiters were observed in seeds compared withthe titers in flowers and berries, but all threemicroenvironments harbor at least three or-ders of magnitude less bacteria than the rhi-zosphere (27). It has been speculated that bac-terial communities of flowers and seeds serveas reservoir for biocontrol bacteria with an-tagonistic functions against microbial pumpkindiseases.

PLANT GROWTH–PROMOTINGMICROORGANISMS

Plant growth–promoting microorganisms aremainly soil- and rhizosphere-derived organ-isms that are able to colonize plant roots insignificant numbers (105–107 CFU per gramof fresh root) and influence plant growth in apositive manner under certain environmentaland soil conditions (123). Most molecular re-search has focused on rhizobacteria, also clas-sified as plant growth–promoting rhizobacteria(PGPRs). The best-studied examples belong to

diverse genera, such as Azospirillum, Gluconace-tobacter, Pseudomonas, and Rhizobium, althoughsome gram-positive genera are also well stud-ied (e.g., Bacillus and Paenibacillus). There hasalways been a bias in the isolation of PGPRs to-ward diazotrophic bacteria owing to the histori-cal assumption that biological nitrogen fixationis an important mechanism for plant growthpromotion (see discussion below). Other mech-anisms for direct plant growth promotion werelater described and elucidated, such as phyto-hormone production, nutrient solubilization,and nitrogen metabolism. Indirect mechanismsof plant growth promotion are related mainlyto the suppression of (soil-borne) pathogenicand deleterious microorganisms by exclusionand antagonism, and these mechanisms are at-tributed more to general plant health than toplant growth promotion.

Although thousands of plant growth–promoting bacterial strains have been isolatedduring the past few decades, the exact mode ofaction from inoculation of a potentially ben-eficial microorganism until the final outcome(yield increase) is still very much a black box.This likely reflects our sparse knowledge ofmolecular processes that control yield in anagricultural context. First of all, PGPR researchhas been lacking model organisms, which wouldallow better comparisons of data between dif-ferent laboratories. Second, molecular andsystems approaches are underused, althoughin recent years a catch-up operation to im-plement state-of-the-art technologies (mainlyfor Pseudomonas and Azospirillum) has beenemployed. In addition, mechanistic insightswere mostly inferred from artificial laboratorysetups, hampering extrapolation to agriculturalsettings. Although field trials have shown thatPGPRs have the potential to increase plantyield under certain environmental and soilconditions, most results are not reproducibleunder other conditions, raising questions aboutthe wide-scale application of these microor-ganisms. A prominent factor affecting fieldtrials is the influence of the indigenous fieldbiome and how it can influence the outcome ofinoculation experiments. Thus, in the context


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of an indigenous soil biome and distinctiverhizosphere and endosphere microbiota, seedcoating with individual PGPRs for subsequentrhizosphere colonization is essentially a nichecompetition experiment in which resourcepartitioning, competitive exclusion, or coexis-tence determines the effectiveness of a givenPGPR. In this respect, the term rhizospherecompetence is commonly used to refer to thesurvival and colonization potential of PGPRs,although ultimately this phenomenon needsto be formalized in the wider context of nichetheory (132).

Bacterial traits such as motility, chemotaxis,attachment, growth, and stress resistance con-tribute to the overall competence of a bacteriumto survive in the rhizosphere and successfullycolonize plant tissues. Rhizosphere competenceis mostly overlooked when identifying PGPRsowing to the quest for mechanistic insights inthe growth-promoting effect. However, effec-tive rhizosphere competence can be a key fac-tor for the successful application of PGPRs.One such important trait is flagellar motility,because it is fundamental for a directed move-ment toward the plant root and the initial adhe-sion phase. Pseudomonas fluorescens, Pseudomonasputida, and Azospirillum brasilense mutants lack-ing (polar) flagella are severely affected in (oreven incapable of) plant root colonization (35,130). Also, at the community level, an overrep-resentation of clusters of orthologous groups ofproteins related to flagellar biosynthesis is ob-served in the rhizosphere compared with bulksoil (8), confirming the importance of this traitin rhizosphere competence. Another importantcompetence trait is chemotaxis (the movementof cells toward or away from certain chemi-cals), and clusters of orthologous groups of pro-teins related to chemotaxis are overrepresentedbut less diverse in the rhizosphere comparedwith bulk soil, indicating the selection of a sub-group by plant roots (8, 21). It will be inter-esting to see whether future whole-genome in-formation on entire rhizosphere communitiesallows us to define a core set of physiologicalfunctions needed for rhizosphere colonization(competence).

Plant Growth Promotion

Once established on or in the plant (rhizo-sphere, rhizoplane, or endosphere), rhizobac-teria can influence plant growth via differentmolecular mechanisms. We first discuss bio-chemical mechanisms employed by rhizobacte-ria for the mobilization and provision of plantnutrients and then review mechanisms of rhi-zobacterial interference with plant hormonelevels for plant growth promotion. Finally, wedescribe how bacterium-derived volatile or-ganic chemicals and signal molecules that reg-ulate population-dependent bacterial behaviorimpact plant growth.

Biological nitrogen fixation. Biological ni-trogen fixation is the process to reduce gaseousdinitrogen to ammonia by the nitrogenaseenzyme complex (N2 + 8H+ + 16 ATP →2NH3 + H2 + 16ADP + 16Pi) and is wellknown in rhizobia-legume symbiosis (account-ing for up to 460 kg fixed N hectare−1 year−1).This process is not restricted to symbioticmicroorganisms; biological nitrogen-fixingcapacity in vitro has also been demonstrated forrhizosphere and endophyte bacteria. However,in most plant-bacteria systems the nitrogenattributed to the plant is less than 10 kgfixed N hectare−1 year−1. Significant nitrogenfixation under field conditions has been shownin sugarcane and rice, mostly using the 15Nnatural abundance technique, with Braziliansugarcane varieties having at least 40 kg fixedN hectare−1 year−1 (128).

The use of nitrogen-fixing-defective mutantstrains (Nif−, defective in an essential nitrogen-fixation gene) has in a few cases provided directevidence that biological nitrogen fixation is re-sponsible for growth promotion, such as in theinteraction of Azoarcus sp. BH72 with Kallargrass and the interaction of Klebsiella pneumoniae342 with wheat. The mode of transfer of fixednitrogen, transferred directly from atmosphericdinitrogen or indirectly via death and mineral-ization, is unknown (64, 67). It must be notedthat both bacteria are validated root endo-phytic bacteria, retrieved in high numbers from


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interior plant tissues upon inoculation [up to7 × 107 CFU per gram of dry root for Azoarcus(111) and 6 × 108 CFU per gram of fresh rootfor wheat (67)]. In less intimate interactions,such as rhizosphere- and rhizoplane-colonizingbacteria, nitrogen fixation does not contributesignificantly to plant growth promotion and canbe seen as a survival strategy of bacteria underlow nitrogen levels.

Nitrogen metabolism. Nitrogen is generallyconsidered one of the major limiting nutrientsin plant growth. Available genome sequencesof rhizosphere and endophyte bacteria revealin most cases a versatile carbon and nitrogenmetabolism. One specific conversion in the ni-trogen cycle has been studied intensively: dis-similatory nitrate reduction or denitrification,by which nitrate (NO3

−) is reduced to nitrite(NO2

−) as an alternative respiratory pathway.Nitrite can further be converted to nitrogen ox-ides (N2O and NO) or ammonia. Its role is plantgrowth promotion is related mainly to the lattercompounds. NO is a potent signaling moleculein plants, altering root growth and prolifera-tion in an auxin-dependent manner (79). In A.brasilense, neither inoculation of a mutant strainproducing only 5% of the wild-type NO level(by a mutation in a key gene for periplasmic ni-trate reductase) nor inoculation of the wild-typestrain in combination with an NO scavengerwas able to induce (lateral) root formation, aswas observed for inoculation with the wild-typestrain alone (98).

Another example of how nitrogen cyclingcan contribute to plant growth is the tritrophicinteraction between certain endophytic fungi,insects, and plants. Behie et al. (12) recentlyshowed that the fungus Metarhizium is able totransfer insect-derived nitrogen to the plant viahyphae by parasitizing and killing soil-borne in-sects. Using 15N-injected larvae, they showedthat in this interaction, 12–48% of the plantnitrogen content is insect derived. The asso-ciation between plant and fungus is probablymutualistic, because a plant carbon transporter,allowing the exchange of carbon, is required forsuccessful colonization (44).

Phosphorus solubilization. Strategies toimprove phosphorus availability/uptake cancontribute significantly to plant growth, be-cause less than 5% of the phosphorus content ofsoils is bioavailable to plants. Microorganismswith the capacity to solubilize mineral phos-phorus are abundant in most soils (up to 40%of the culturable population) and can be easilydetermined by plating on a solidified mediumwith incorporation of an insoluble phosphorusform (e.g., hydroxyapatite). Halo formationaround colonies indicates the phosphorus solu-bilization capacity of these strains. Well-knownisolates belong to Bacillus, Pseudomonas, or Peni-cillium genera. Mineralization/solubilizationis achieved by the production of organicacids (such as acetate, succinate, citrate,and gluconate) or phosphatases, liberatingorthophosphate from inorganic and organicphosphorus pools. Several genes involved inphosphorus solubilization have been foundand characterized (112). However, owing tothe lack of in-depth studies, it is difficult todifferentiate between direct microbial solubi-lization and indirect plant root stimulation bymicrobes allowing better nutrient uptake.

In this context, arbuscular mycorrhizal sym-bioses have an important role in phosphorus nu-trition under phosphorus-deficient conditions.Many studies have demonstrated that arbuscu-lar mycorrhizae can be seen as extensions of theplant root system, exploiting the soil for phos-phorus. In this sense they can be compared withroot hairs, but they forage a broader space (inthe centimeter range) around the roots. A sub-set of plant phosphate transporters are specifi-cally expressed in root cortical cells containingarbuscular mycorrhizal feeding structures (ar-buscules) and are needed for efficient transportof fungus-derived phosphate across a symbioticmembrane (periarbuscular membrane) into thehost cytoplasm (70, 109, 147).

Siderophore production. Similar to phos-phorus, iron is abundant in soil, but it is not veryavailable to plants owing to the low solubilityof Fe3+ oxides. Plants have developed differentstrategies to counteract this low availability. In


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the first strategy (the reduction strategy, foundmainly in dicots and nongraminaceous mono-cots), protons and organic acids are releasedto decrease the soil pH, thereby increasingiron availability. In the second strategy (thechelation strategy, found mainly in grasses),plant roots release low-molecular-weightiron-chelating molecules (e.g., mugineic acid).These siderophores can efficiently bind ironand are then taken up by root cells (71).

Like plants, microorganisms can releaseorganic acids and a broad range of siderophoresunder iron-limiting conditions. In this way,a complex competition for iron occurs in therhizosphere between different microorganismsand between microorganisms and plants.Because bacterial siderophores generally have ahigher affinity for iron than phytosiderophoresdo, the outcome of this competition can beunfavorable for plants. In addition, microbescan even degrade several phytosiderophores.Therefore, more in-depth studies are neededto estimate the importance of siderophoreproduction in plant growth promotion(83, 91).

In one specific, well-documented case, bac-terial siderophores can indirectly contributeto plant growth promotion. Most soil-derivedfluorescent pseudomonads can efficientlyscavenge iron via siderophore production(e.g., pyoverdine). In this way, they antagonizesome fungal plant pathogens (e.g., Fusariumoxysporum) and restrict their growth in therhizosphere, thereby enhancing plant healthindirectly (40).

Phytohormone biosynthesis and in-terference. Phytohormones are chemicalcompounds that promote and influence plantgrowth and development. Phytohormones areoften divided into five major classes—auxins,cytokinins, gibberellins, abscisic acid, andethylene—although more recently othercompounds with hormonal activity havebeen identified, such as strigolactones andbrassinosteroids (116). In the growth mediumof many soil- and plant-associated bacteria,phytohormonal production is frequentlyobserved, in multiple cases even including dif-

ferent compounds produced by a single strain(104). However, the extent to which thesecontribute to plant growth promotion has notbeen proven for all compounds. Therefore, werestrict this part to two well-documented cases:auxin production and 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity.

Auxin production. The family of moleculeswith auxin activity is involved in many aspectsof plant growth and development. The mostabundant member is indole-3-acetic acid (IAA).Although this molecule was isolated and iden-tified decades ago (for a historical perspective,see 1), the major pathway for IAA biosynthesiswas just recently discovered (92, 125, 145). Themain precursor for biosynthesis is tryptophan,but multiple biosynthesis pathways have beendescribed in plant-associated microorganisms,including pathogens. Auxin production is acommon feature of many soil- and plant-associated bacteria. The best-characterizedauxin biosynthesis routes in bacteria aredesignated the indole-3-acetamide (IAM) andindole-3-pyruvate (IPyA) pathways. In thefirst pathway, well known from pathogenicbacteria, tryptophan is converted to IAM by atryptophan monooxygenase; in a second step,IAA is formed by conversion of IAM by an IAMhydrolase. In the second pathway, found mainlyin beneficial bacteria, tryptophan is transami-nated to IPyA; in a second, rate-limiting step,IPyA is decarboxylated by an IPyA decar-boxylase or phenylpyruvate decarboxylase toindole-3-acetaldehyde, which is finally oxi-dized to IAA (spontaneously or by an aldehydeoxidase/dehydrogenase). The observation thatmany PGPRs could produce IAA, in combina-tion with inoculation experiments with mutantstrains altered in auxin production, has led tothe conclusion that auxin production is a majorplant growth–promoting trait (124). For A.brasilense, a direct link between IAA productionand altered root morphology was demonstratedin wheat inoculation experiments: A mutantstrain defective in the IPyA decarboxylase couldnot induce the same morphological changes(36). In greenhouse experiments with wheatunder suboptimal nitrogen fertilization, plants


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inoculated with the wild-type strain had ahigher yield than control plants or plantsinoculated with the mutant strain (122). It washypothesized that bacterial auxin productionleads to root proliferation, resulting in a highertotal root surface, which allows the plant toabsorb more nutrients and water from the soil(80).

In some fungal species, such as Tricho-derma, Piriformospora, and nonpathogenicFusarium species, auxin plays a role in plantgrowth stimulation. In most examples, fungalauxin biosynthesis is involved in this growthpromotion (60). However, this is not thecase for Piriformospora indica: In that species,auxin biosynthesis is necessary only for rootcolonization in the biotrophic phase (61).However, P. indica induces a higher auxinconcentration inside Chinese cabbage via anexuded compound of the fungus (82).

The regulation of auxin biosynthesis hasbeen extensively studied for many bacteria. Themain regulatory factors are environmental fac-tors (such as carbon limitation, pH, and ma-trix potential) and plant factors (such as specificcompounds or surfaces) (for a review, see 124).An interesting and rather unusual regulationhas been observed for A. brasilense: The expres-sion of the key gene (encoding IPyA decarboxy-lase) is induced by IAA itself (positive-feedbackregulation) (129). Important genetic factorsregulating auxin biosynthesis in gammapro-teobacteria include RpoS (a general regula-tor in response to stress and starvation) andthe two-component system GacS/GacA (in-volved in competitiveness). The expression ofkey genes therefore shows a typical stationary-phase-dependent expression (76, 102).

Plant growth is not stimulated only by IAAproduction. In particular cases, IAA degrada-tion by microorganisms can also stimulate rootelongation, as illustrated by the interaction ofP. putida 1290 with radish plants. The sourceof IAA could be the plants themselves or otherIAA-producing microorganisms. Thus, IAA de-graders can have a function in the rhizospherein auxin homeostasis by elevating or reducinglocal auxin concentrations (84).

ACC deaminase activity. The phytohormoneethylene was first described as a fruit-ripeninghormone but is now known to have a muchbroader role in other processes, such assenescence, abscission, and pathogen-defensesignaling. Under diverse stresses, ethylenebiosynthesis is induced, thereby inhibitingroot growth and plant growth (2). Somemicroorganisms can interfere with ethylenebiosynthesis by expression of the enzyme ACCdeaminase, encoded by the acdS gene. This en-zyme converts the ethylene precursor ACC toα-ketobutyrate and ammonia. These microor-ganisms can enhance plant growth by metabo-lizing ACC exuded by plant roots. Because theACC concentration outside the roots decreases,ACC exudation increases and ethylene biosyn-thesis inside the plant stalls owing to the lack ofprecursor. This attenuates ethylene-dependentinhibitory responses and therefore increasesplant growth, especially under stress conditions(54, 55). In addition, ACC deaminase activitycan be enhanced by microbial auxin productionbecause the auxin induces the biosynthesis ofACC synthase in the plant, thereby increas-ing the biosynthesis of ACC (5). It will beinteresting to examine whether rhizobacteriaproducing AcdS and auxin frequently co-occurin indigenous root microbiota and whethertheir co-occurrence enhances plant growthpromotion. The importance of ACC deaminaseactivity in plant growth promotion has beenextensively studied not only by using mutants inacdS but also by overexpressing acdS in plants.acdS-expressing microorganisms and plants areable to alleviate the growth inhibition inducedby ethylene synthesis under stress conditions,such as flooding, drought, toxic compounds,and pathogen attack (for reviews, see 53, 55).

Interference with quorum sensing. QS isa key mechanism to regulate gene expressionin a population-dependent manner by theaccumulation of signal molecules. At a certainthreshold concentration (quorum), a regulatoris triggered allowing downstream regulation ofgene expression. The N-acylhomoserine lac-tone (AHL)–based system is well documented


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in gram-negative bacteria and allows them tocoordinate their behavior at a population level.Hence, QS is involved in several important pro-cesses, such as virulence, biofilm maturation,symbiosis, and survival (48). QS is not restrictedto prokaryotes; it can also be an interkingdomsignal, a well-developed concept in bacteria-vertebrate interactions (63). A few reports alsomention a role in bacteria-plant interactions.In Medicago truncatula, AHL can alter proteinexpression, especially proteins involved in plantdefense. However, the plant response dependson the AHL structure and the specific tissue(93). In tomato, AHL-producing bacteriainduce systemic resistance against Alternaria ina salicylic-acid- and ethylene-dependent man-ner, pointing toward a role for AHL in the bio-control of pathogens (118). Schikora et al. (117)recently showed that the mitogen-activatedprotein kinase AtMPK6 is required for AHL-dependent defense responses in A. thaliana.Plants can actively interfere with QS sensingby producing QS-mimicking compounds.Although the exact role of these compoundsis not known, they have been proposed todepress the virulence of pathogenic bacteria orimprove symbiosis (51, 105). In terms of plantgrowth promotion, one report demonstratedthe capacity of AHLs to modify the root archi-tecture of Arabidopsis, similar to a classical auxinresponse; however, auxin signaling pathwaysare not involved in the AHL response (101).

Volatile compounds. Direct contact betweenmicroorganisms and the plant is not always nec-essary for growth promotion. Some microbesrelease volatile organic compounds (VOCs)with a growth-promoting capacity. The best-documented case is Bacillus subtilis, whichproduces the active compounds 3-hydroxy-2-butanone (acetoin) and 2,3-butanediol. Aknockout mutant in the biosynthesis path-way for both compounds demonstrated the di-rect involvement of VOCs in growth promo-tion (114). Later, the production of VOCswith growth-promoting activity was shown forother bacterial species/genera, such as Bacil-lus amyloliquefaciens, Pseudomonas chlororaphis,

Burkholderia, and Serratia, and the spectrum ofcompounds was broadened toward 1-hexanol,indole, and pentadecane (16, 114). In B. subtilis,VOC production also induces systemic resis-tance (113). VOC-mediated signaling in plantsis highly complex because almost all hormonalpathways have been shown to be involved in thesignaling. A clear role for auxin homeostasis andsignaling has been demonstrated using a mi-croarray approach and reporter lines (113, 150).

VOC production by fungi is known to beinvolved in self and interspecies recognition(140). The role of VOCs in plant interactions isless well documented, although in a few exam-ples fungal ethylene emission can alter the rootarchitecture of the host plant.

Biological Control

Biological control, or biocontrol, is the pro-cess of suppressing deleterious/pathogenic liv-ing organisms by using other living organisms.In this review, we restrict the discussion tomicroorganisms that can suppress pathogenicmicroorganisms directly or indirectly, therebyconferring plant protection. Biocontrol hasbeen extensively studied not only under lab-oratory conditions but also in field situations,leading to several commercial products. Mostproducts are based on Bacillus and Trichodermastrains owing to (seed) formulation issues, al-though Pseudomonas-based products have alsobeen commercialized in recent years (15). Thesuccess of Bacillus strains in commercializationis based on the extensive knowledge of themodes of action and applicabilities of thesestrains both in laboratory settings and in green-house and field experiments. In addition, manyantimicrobial molecules involved in pathogensuppression have been isolated and character-ized (15, 42, 103).

Biosynthesis of antimicrobial compounds.Microorganisms can synthesize a wide range ofcompounds with antimicrobial activity. Thesecompounds can be derived from the secondarymetabolism or are (modified) proteinaceousmolecules derived from ribosomal synthesisor nonribosomal peptide synthesis. The


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production of antimicrobial compounds hasbeen extensively studied in pseudomonads,bacilli, and Trichoderma species, includingthe identification of biosynthesis pathwaysand their regulation. Most commercial bio-control products contain strains belongingto these groups. Well-known and character-ized compounds are phenazines, 2,4-DAPG,pyoluteorin, pyrrolnitrin, cyclic lipopeptidesurfactants, zwittermycin A, and bacteriocins(15, 42, 103, 139). The heterocyclic nitrogen-containing phenazines have a broad antimi-crobial spectrum and have been identified inPseudomonas, Burkholderia, Streptomyces, andBrevibacterium species. The biosynthesis startsfrom the branch-point molecule chorismic acidand involves the conserved phz gene operon. Asfor most antimicrobial compounds, biosynthe-sis is regulated by two-component regulatorysystems and environmental conditions (39, 94).Although biocontrol strains do not directly pro-mote plant growth, they can influence PGPRsthat directly stimulate plant growth, as illus-trated for a 2,4-DAPG-producing P. fluorescensstrain that enhances the phytostimulatoryeffect of A. brasilense by altering the expressionof genes involved in plant growth promotion.The authors of this study speculated that2,4-DAPG is a signal molecule that coevolvedin complex plant-microbe interactions (26).

Induced systemic resistance. Inoculation ofplants with nonpathogenic bacteria can in-duce resistance against a broad spectrum ofpathogenic organisms in both below- andaboveground parts. This induced systemic re-sistance (ISR) depends mainly on jasmonateand ethylene signaling. In this way, plants areprimed to react more quickly and stronglyto a pathogen attack. ISR has been observedfor many microorganisms and their cellu-lar derivative determinants (so-called MAMPs,such as flagella, cell envelope components, andsiderophores) (33, 149). Well-characterizedISR-inducing microbes include several Pseu-domonas, Bacillus, and Serratia species and Tri-choderma harzianum. Most plant responses havebeen studied in A. thaliana, but ISR has also

been observed in bean, radish, rice, tobacco, andtomato (33, 88, 121).

Plant Growth–PromotingRhizobacteria in a CommunityContext

Above, we discussed the role of PGPRs in theirinteractions with plants and which mechanismscan be responsible for the observed (positive)plant responses (Figure 4). Many studies apply-ing strains impaired in a particular mechanism

Ch

emo

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s

Fe3+

ISR Rhizospherecompetence

Bacterialmechanisms

Competition- Nutrients

- Niche

- Antimicrobial compounds

Plant cell

Pi

ACC

IAA

α-KB + NH3

N2

Trp

NH4+

VOCs

ACC

Sys

tem

ic s

ign

al

Ethylene

Bacterialcell

Figure 4Biochemical mechanisms by which rhizobacteria mediate plant growthpromotion and plant health. Bacterial rhizosphere competence is illustrated bythe polar flagellum and chemotaxis. Several plant growth–promoting traitsdiscussed in this review are depicted: 1-aminocyclopropane-1-carboxylate(ACC) deaminase activity (lowering plant ethylene levels), auxin[indole-3-acetic acid (IAA)] biosynthesis, biological nitrogen fixation, volatileorganic compound (VOC) production, phosphorus solubilization (by thesecretion of organic acids or phosphatases, represented by the star) andsiderophore production (represented by the cross). Additional abbreviations:ISR, induced systemic resistance; α-KB, α-ketobutyrate; Pi, inorganicphosphate.


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Syntrophicinteractions:interactions in whichone species lives offthe products ofanother species

have shown that a partial plant growth promo-tion can be observed upon inoculation. This hasled to the proposition that multiple mechanismsencoded in a single organism work together,also known as the additive hypothesis (9). How-ever, direct proof of this has not been pro-vided. Another important challenge for furtherresearch is the identification of syntrophic in-teractions. Combined strains have already beenapplied, but an extensive quest for mutualisticcombinations can strengthen the plant growth–promoting effect and thus the outcome and re-producibility of field experiments/applications.In this context, it is also worth mentioningthat most PGPR research does not take intoaccount the community level that is alreadypresent in the rhizosphere. This complex envi-ronment is also a major factor in the outcomesof experiments. Therefore, syntrophic combi-nations may allow a more stable plant growth–promoting effect in a community context.

The Rhizosphere: A Future Model forMolecular Principles UnderlyingNiche Formation

The available amount of nutrients in the soil islimiting. Although the rhizosphere is nutrientrich in comparison with bulk soil owing to

rhizodeposition and root exudates, competi-tion between microorganisms determines theoutcome of the interaction with the plant. Asindicated above, microorganisms are attractedto the roots by chemotaxis toward root exu-dates (mainly sugar, amino acids, and organicacids). Once in contact with the root, themicroorganisms can (firmly) attach to the rootand occupy potential binding sites for otherorganisms, including pathogens (i.e., nichecompetition). Several studies have shown thatmost organisms are preferentially attached atnutrient-rich niches on the roots, such as placeswhere lateral roots emerge, root hair zones, andjunctions between epidermal cells (88, 131).

Owing to nutrient limitation, strains that areable to efficiently scavenge available nutrientsand/or possess a versatile metabolism (allowingthem to use a broad spectrum of carbon and ni-trogen sources) have a competitive advantageover other environmental inhabitants. Withthe increasing availability of genome sequencesand expression data for rhizosphere and plant-associated microorganisms, insights into themetabolic fluxes and conversions are expectedto increase in the coming years. Some specificcases of nutrient competition for phosphorusand iron acquisition have already been discussedabove.

SUMMARY POINTS

1. Members of the phyla Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteriadominate root rhizosphere and endosphere bacterial assemblages.

2. Root-derived rhizodeposits provide organic substrates that drive the differentiation ofthe soil biome in the rhizosphere to give rise to host genotype–individualized endospherebacterial communities.

3. Phyllosphere communities are dominated by the same few bacterial phyla as rhizospherecommunities. However, the source of phyllosphere inocula and assembly cues remainsessentially unknown.

4. The promotion of plant growth and plant health by microorganisms has been describedfor many decades. Multiple (direct or indirect) molecular mechanisms are responsiblefor this growth promotion, although a general framework for PGPRs is still missing.


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FUTURE ISSUES

1. In-depth functional analysis of plant microbiota requires the development of referenceplants and the definition of minimal experimental standards to maximize the comparisonand integration of data generated by different laboratories. Likewise, the PGPR fieldrequires model organisms to further explore the diversity of plant growth–promotingstrategies, especially as the community context has been overlooked as a factor modulatingthe experimental outcome.

2. The development of open-access and indexed bacterial culture collections representingall microbiota members as defined by deep culture-independent 16S rRNA ribotypesequences will provide essential future tools. This will enable systematic analysis of syn-thetic communities for plant growth and plant health functions under defined nutrientconditions in the laboratory. In parallel, whole-genome sequencing of such collectionswill allow functional insights into host-microbiota interactions at a much deeper resolu-tion than 16S rRNA-based ribotyping approaches provide.

3. The development of model systems and functional assays with synthetic communitieswill deconvolute ecosystem complexity, permit the definition of molecular principlesunderlying niche filling and niche competition, and aid in the identification of syntrophiccommunity interactions.

4. Comparisons of the rhizosphere, root endosphere, and phyllosphere on the same plantmaterial for a systematic assessment of community members, especially by applyingmetagenomic approaches, will reveal bacterial traits for adaptation to these habitats.

5. Host-microbiota biology offers the possibility to test niche adaptation theory and presentsa framework to test evolutionary transitions from commensalistic to mutualistic orpathogenic lifestyles of community members.

DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings thatmight be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

We are thankful to Julia Vorholt, Marc Nishimura, and Derek Lundberg for valuable comments onthe manuscript. K.S. and S.S. are supported by the Swiss National Science Foundation (PBFRP3-133544) and by a postdoctoral fellowship and mobility grant from the Research Foundation Flan-ders (FWO-Vlaanderen), respectively. P. S.-L. is supported by the ERC advanced grant ROOT-MICROBIOTA and the Max Planck Society.

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PP64-frontmatter ARI 25 March 2013 10:21

Annual Review ofPlant Biology

Volume 64, 2013Contents

Benefits of an Inclusive US Education SystemElisabeth Gantt � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Plants, Diet, and HealthCathie Martin, Yang Zhang, Chiara Tonelli, and Katia Petroni � � � � � � � � � � � � � � � � � � � � � � � � �19

A Bountiful Harvest: Genomic Insights into Crop DomesticationPhenotypesKenneth M. Olsen and Jonathan F. Wendel � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �47

Progress Toward Understanding Heterosis in Crop PlantsPatrick S. Schnable and Nathan M. Springer � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �71

Tapping the Promise of Genomics in Species with Complex,Nonmodel GenomesCandice N. Hirsch and C. Robin Buell � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �89

Understanding Reproductive Isolation Based on the Rice ModelYidan Ouyang and Qifa Zhang � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 111

Classification and Comparison of Small RNAs from PlantsMichael J. Axtell � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 137

Plant Protein InteractomesPascal Braun, Sebastien Aubourg, Jelle Van Leene, Geert De Jaeger,

and Claire Lurin � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 161

Seed-Development Programs: A Systems Biology–Based ComparisonBetween Dicots and MonocotsNese Sreenivasulu and Ulrich Wobus � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 189

Fruit Development and RipeningGraham B. Seymour, Lars Østergaard, Natalie H. Chapman, Sandra Knapp,

and Cathie Martin � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 219

Growth Mechanisms in Tip-Growing Plant CellsCaleb M. Rounds and Magdalena Bezanilla � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 243

Future Scenarios for Plant PhenotypingFabio Fiorani and Ulrich Schurr � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 267

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Microgenomics: Genome-Scale, Cell-Specific Monitoring of MultipleGene Regulation TiersJ. Bailey-Serres � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 293

Plant Genome Engineering with Sequence-Specific NucleasesDaniel F. Voytas � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 327

Smaller, Faster, Brighter: Advances in Optical Imagingof Living Plant CellsSidney L. Shaw and David W. Ehrhardt � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 351

Phytochrome Cytoplasmic SignalingJon Hughes � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 377

Photoreceptor Signaling Networks in Plant Responses to ShadeJorge J. Casal � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 403

ROS-Mediated Lipid Peroxidation and RES-Activated SignalingEdward E. Farmer and Martin J. Mueller � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 429

Potassium Transport and Signaling in Higher PlantsYi Wang and Wei-Hua Wu � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 451

Endoplasmic Reticulum Stress Responses in PlantsStephen H. Howell � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 477

Membrane Microdomains, Rafts, and Detergent-Resistant Membranesin Plants and FungiJan Malinsky, Miroslava Opekarova, Guido Grossmann, and Widmar Tanner � � � � � � � 501

The EndodermisNiko Geldner � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 531

Intracellular Signaling from Plastid to NucleusWei Chi, Xuwu Sun, and Lixin Zhang � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 559

The Number, Speed, and Impact of Plastid Endosymbioses inEukaryotic EvolutionPatrick J. Keeling � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 583

Photosystem II Assembly: From Cyanobacteria to PlantsJorg Nickelsen and Birgit Rengstl � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 609

Unraveling the Heater: New Insights into the Structure of theAlternative OxidaseAnthony L. Moore, Tomoo Shiba, Luke Young, Shigeharu Harada, Kiyoshi Kita,

and Kikukatsu Ito � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 637

Network Analysis of the MVA and MEP Pathways for IsoprenoidSynthesisEva Vranova, Diana Coman, and Wilhelm Gruissem � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 665

vi Contents

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Toward Cool C4 CropsStephen P. Long and Ashley K. Spence � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 701

The Spatial Organization of Metabolism Within the Plant CellLee J. Sweetlove and Alisdair R. Fernie � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 723

Evolving Views of Pectin BiosynthesisMelani A. Atmodjo, Zhangying Hao, and Debra Mohnen � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 747

Transport and Metabolism in Legume-Rhizobia SymbiosesMichael Udvardi and Philip S. Poole � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 781

Structure and Functions of the Bacterial Microbiota of PlantsDavide Bulgarelli, Klaus Schlaeppi, Stijn Spaepen, Emiel Ver Loren van Themaat,

and Paul Schulze-Lefert � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 807

Systemic Acquired Resistance: Turning Local Infectioninto Global DefenseZheng Qing Fu and Xinnian Dong � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 839

Indexes

Cumulative Index of Contributing Authors, Volumes 55–64 � � � � � � � � � � � � � � � � � � � � � � � � � � � 865

Cumulative Index of Article Titles, Volumes 55–64 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 871

Errata

An online log of corrections to Annual Review of Plant Biology articles may be found athttp://www.annualreviews.org/errata/arplant

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