innate immunity effectors and virulence factors in symbiosis

Available online at www.sciencedirect.com

Innate immunity effectors and virulence factors in symbiosisAttila Kereszt1,3, Peter Mergaert2, Gergely Maroti1,3 and Eva Kondorosi1,2,4

Rhizobium–legume symbiosis has been considered as a

mutually favorable relationship for both partners. However, in

certain phylogenetic groups of legumes, the plant directs the

bacterial symbiont into an irreversible terminal differentiation.

This is mediated by the actions of hundreds of symbiosis-

specific plant peptides resembling antimicrobial peptides, the

effectors of innate immunity. The bacterial BacA protein,

associated in animal pathogenic bacteria with the maintenance

of chronic intracellular infections, is also required for terminal

differentiation of rhizobia. Thus, a virulence factor of

pathogenesis and effectors of the innate immunity were

adapted in symbiosis for the benefit of the plant partner.

Addresses1 Institute for Plant Genomics, Human Biotechnology and Bioenergy,

Bay Zoltan Foundation for Applied Research, Derkovits fasor 2, Szeged,

Hungary2 Institut des Sciences du Vegetal, Centre National de la Recherche

Scientifique, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France3 Karoly Robert College, Matrai ut 36, Gyongyos, Hungary4 Institute of Biochemistry, Biological Research Center of the Hungarian

Academy of Sciences, Temesvari korut 62, Szeged, Hungary

Corresponding author: Kondorosi, Eva ([email protected])

Current Opinion in Microbiology 2011, 14:76–81

This review comes from a themed issue on

Host-microbe interactions: bacteria

Edited by Brett Finlay and Ulla Bonas

Available online 5th January 2011

1369-5274/$ – see front matter

# 2010 Elsevier Ltd. All rights reserved.

DOI 10.1016/j.mib.2010.12.002

IntroductionLeguminous plants (soybean, alfalfa, pea, bean, etc.),

forming symbiotic associations with soil bacteria of the

Rhizobiaceae family (termed rhizobia), play an important

role in the global nitrogen cycle by the reduction of

atmospheric nitrogen, the most abundant nitrogen source

on Earth. Nitrogen fixation is performed by the rhizobia

inside the cells of de novo formed plant organs, the

nodules, which usually develop on roots and occasionally

also on stems. Nodule organogenesis is initiated by bac-

terial signal molecules (Nod factors) which induce the

dedifferentiation and division of root cortical cells. Cell

proliferation leads to the formation of the nodule primor-

dium which then differentiates into the nitrogen-fixing

root nodule [1]. Nodule growth and differentiation result

from the combined action of two processes, cell divisions

in the meristem giving rise to more nodule cells and


endoreduplication-driven cell enlargement of Rhizo-bium-infected symbiotic cells [2].

Bacteria invade the root and later the nodule tissues via

trans-cellular infection threads (ITs) initiated in the root

hairs or at cracks in the root epidermis [3,4]. A key step of

nodulation is the release of bacteria from the ITs into the

cytoplasm of nodule cells via an endocytosis-like process,

resulting in organelle-like structures called symbiosomes

containing one or more bacteria enclosed by a peribacter-

oid membrane (PBM) of plant origin [5��]. After their

release into the plant cytoplasm, symbiosomes divide and

differentiate into their mature form. Ultimately, the

cytoplasm of infected cells is completely filled with

symbiosomes (Figure 1). Within the developing symbio-

somes, bacteria differentiate into their nitrogen-fixing

form termed bacteroids. This bacterial differentiation

involves drastic changes in the metabolism and gene

expression required for the adaptation of bacteria to

the nodule cell environment and for the demands of

nitrogen fixation [6–11]. The physiological differentiation

is primarily regulated by the rhizobial two-component

regulator FixLJ which senses the low oxygen concen-

tration that is prevalent in nodules [11]. In certain

legumes, such as pea, vetch or alfalfa (legumes of the

Inverted Repeat-Lacking Clade, termed IRLC), the bac-

teroid development is in addition accompanied by strik-

ing morphological and cytological changes that were

described as early as in the 19th century [12] and charac-

terized in more detail 100 years later [13]. We will discuss

here the molecular mechanism and biological meaning of

bacteroid differentiation and show that the effectors of

the host and bacteria in symbiosis bear similarities to

those of pathogenic–host interactions.

Bacteroid differentiation: the host plant rulesThe morphological changes characteristic for bacteroids

in the IRLC legumes involve elongation of the bacteria

from the free-living size of 1–2 mm to 5–10 mm (Figure 1)

and often the formation of Y-shaped branched cells that

are packaged individually into PBMs. These bacteroids

have a highly amplified genome content that is condensed

into multiple nucleoids of variable size [13–16]. The

polyploidy of these bacteroids and the induction of bac-

teroid-like cells by genetic or physiological interference

with the rhizobial cell cycle [17–20] suggest that the

bacterial cell cycle is modified when the rhizobia differ-

entiate into bacteroids, resulting in multiple rounds of

DNA replication without cytokinesis. Moreover, the

membrane-integrity of these bacteroids is also strongly

affected as indicated by detergent-sensitivity or the slow

uptake of propidium iodide, a membrane-integrity dye

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http://dx.doi.org/10.1016/j.mib.2010.12.002

Innate immunity effectors and virulence factors in symbiosis Kereszt et al. 77

Glossary of terms and abbreviations

Rhizobia: Gram-negative alpha-proteobacteria that establish

symbiosis with legumes to reduce atmospheric nitrogen gas.

IRLC: the Inverted Repeat Lacking Clade of legumes is characterized

by the loss of one copy of the approximately 25 kb large inverted

repeat encoding a duplicate set of ribosomal RNA genes in the

chloroplast genome.

Nod factors: Signaling molecules of rhizobia that induce the nodule

developmental program in the host legumes at picomolar

concentrations. Structurally the Nod factors are lipo-

chitooligosaccharides composed of a backbone of three to five b-1,4-

linked N-acetylglucosamines with an unsaturated acyl chain on the

non-reducing end and various substitutions at the terminal sugar

residues. The modification of the core Nod factor structure is the basis

of host-specificity, that is different plants recognize and respond to

different Nod factors.

IT: Infection threads are trans-cellular tubes that form initially by

localized cell wall hydrolysis and membrane invagination and

continue their growth via the deposition of newly synthesized cell-wall

material and extracellular matrix. They keep the growing and dividing

rhizobia topologically outside of the plant cells and lead them into the

newly formed nodule cells.

Bacteroid: The symbiotic form of rhizobia present in plant cells that

perform the reduction of nitrogen gas into ammonia, a compound

which can be metabolized by the eukaryotic partner.

PBM: The peribacteroid membranes envelop bacteroids in the

infected nodule cells and form structural and functional interface

between the legume plant and its symbiont, that is they separate

bacteria from the plant cytoplasm and mediate metabolite exchange

between the partners. PBM is of plant origin and has a mixed, plasma

membrane and endosome identity.

Symbiosome: An organelle-like structure in the infected nodule cells

which is composed of the peribacteroid membrane and one (in IRLC

legumes) or more (in non-IRLC legumes) enveloped bacteroids.

AMPs: Antimicrobial peptides are natural antibiotics of

proteinaceous nature, present in nearly all organisms, from bacteria to

plants and animals.

NCRs: Nodule-specific Cysteine-Rich peptides are secreted, 25–60

amino acid long peptides characterized by four or six conserved

cysteines that are only produced in the nodule cells. They are similar

to a group of AMPs termed defensins which are the effectors of innate

immunity in plants and animals.

Figure 1

A B

E F

C D

Current Opinion in Microbiology

Terminally and reversibly differentiated bacteroids. Strongly elongated

bacteroids in M. truncatula nodules (a)–(c) and rod-shaped bacteroids in

soybean nodules (d)–(f). (a,d) Symbiotic cells entirely filled with

bacteroids. (b,e) Isolated bacteroids stained with DAPI. (c,d) Electron

microscopy images of bacteroids. Note the accumulation of extensive

PHB granules (white inclusions) in the reversibly differentiated

bacteroids (f) but not in the terminally differentiated bacteroids (e). Scale

bars are 10 mm in a–d and 1 mm in e,f. The image in e is taken from [13].

probe, which does not enter intact cells [16,21,22]. Bac-

teroids isolated from the nodules of IRLC legumes do not

form colonies, as they have lost their reproductive

capacity. Therefore, bacteroid differentiation is irrevers-

ible and terminal. The morphological and cytological

changes are independent of the process of nitrogen fix-

ation itself since mutants in the fixLJ regulatory genes or

in the nitrogenase-encoding genes also exhibit terminal

bacteroid differentiation [23�].

Another unusual type of terminal differentiation occurs in

legumes like peanut and related Aeschynomene species

where the bacteroids are also strongly enlarged but

spherical [24,25]. Nevertheless, terminal bacteroid differ-

entiation is not a general feature of rhizobia. In legumes

such as soybean, cowpea, bean or lotus, the size and

morphology of bacteroids are similar to those of the

free-living bacteria and multiple bacteroids can be found

in a symbiosome (Figure 1). These latter bacteroids are

reversibly differentiated as they maintain their cell

division capacity and generate offspring.


Comparing nearly isogenic, recombinant or natural broad-

host range rhizobial strains capable of nodulating plants

from different legume clades (e.g. pea and lotus, vetch

and bean, peanut and cowpea) revealed that the differ-

entiation fate of the bacteroids was dependent on the host

plant: bacteroids exhibited terminal differentiation in one

plant species, and reversible differentiation in the other

one [16,24]. Therefore, the bacteroid type is controlled by

the host plant, not by the rhizobial genetic repertoire.

Bacteroid morphology — as an indicator of terminal or

reversible bacteroid differentiation — was investigated in

an evolutionary context [25]. Mapping the bacteroid type

on the legume phylogenetic tree and ancestral state re-

construction indicate that the reversible fate is the ances-

tral state of bacteroids whereas terminal bacteroid


78 Host-microbe interactions: bacteria

differentiation has evolved independently in the IRLC,

and other (Dalbergioid, Genistoid, Millettioid, and Mir-

belioid) legume lineages. It is likely that each of these

different lineages evolved their own individual mechan-

isms for terminal differentiation of their rhizobial endo-

symbionts.

The multiple origin of terminal bacteroid differentiation

strongly suggests that it provides a significant benefit to

the plant. The symbiotic performance of two broad-host

range Rhizobium strains in different legumes with term-

inal and reversible bacteroid differentiation, respect-

ively, showed indeed that the terminally differentiated

bacteroids were more efficient, sustaining more plant

growth per investment in nodule production and requir-

ing less respiration (energy) for a given nitrogenase

activity [26��].

Identification of defensin-like molecules asplant factors governing bacteroiddifferentiation in IRLC legumesThe identification of plant factors required for terminal

bacteroid differentiation in the IRLC legumes was based

on the assumptions that the corresponding genes firstly,

should be induced during nodule formation and

expressed in the symbiotic cells and secondly, should

be conserved in IRLC legumes but not in legumes with

reversible bacteroid differentiation. Transcriptome

analysis using microarray data as well as data-mining in

EST databases [23�,27,28] identified nodule-specific

cysteine-rich (NCR) peptides as likely candidates. The

NCR gene family in the IRLC model legume Medicagotruncatula encodes more than 400 different peptides

which are most similar to defensin-like antimicrobial

peptides (AMPs) [27,28]. Homologs have been found

in other IRLC legumes but not in species forming

nodules with reversibly differentiated bacteroids such

as Lotus japonicus, bean and soybean with sequenced

genomes and/or available transcriptome data. The M.truncatula NCR genes are strictly nodule-specific. Tran-

scriptome analysis of nodules obtained with a large col-

lection of symbiotic mutants of M. truncatula and its

bacterial partner Sinorhizobium meliloti revealed that

NCR gene expression was linked to symbiotic cell for-

mation [23�]. Moreover, for the tested examples, the

expression of NCR genes was restricted to the Rhizo-bium-infected plant cells, where different subsets of

NCR genes were activated during distinct developmental

stages [27].

Several lines of evidence confirmed that the NCR pep-

tides are the factors that induce terminal bacteroid

differentiation [29��]. NCRs are targeted to and accumu-

late in high amounts in symbiosomes and bacteroids.

Like most known symbiosome proteins, NCRs have a

signal peptide characteristic for the family and enter into

the secretory pathway that is extremely prominent in the


symbiotic cells [23�] and necessary for symbiosome for-

mation (Figure 2) [30�,31�]. The M. truncatula dnf1mutant, deficient in a nodule-specific subunit of the

signal peptidase complex of the secretory pathway, forms

nonfunctional nodules [31�]. In this mutant, the NCRs

are not targeted to the bacteroids — they are blocked in

the endoplasmic reticulum — and there is no bacteroid

differentiation [29��]. On the other hand, expression of

NCR genes in L. japonicus, in which bacteroids are rever-

sibly differentiated and the NCR genes are absent, was

sufficient to induce features of terminal bacteroid differ-

entiation: symbiosomes contained single and remarkably

elongated bacteroids [29��]. The in vitro responses of S.meliloti toward pure NCRs, notably, high permeability of

the membrane, inhibition of bacterial proliferation,

DNA accumulation and cell elongation, were similar

to the characteristics of the bacteroids. Fluorescently

labeled NCR peptides were localized on the cell envel-

ope and later at the bacterial cell division plane, in line

with their possible role in cytokinesis inhibition, while

permitting DNA replication and cell elongation in bac-

teroids [29��].

AMPs are known as effector molecules of innate immu-

nity in both the animal and plant kingdoms with a major

role to fight microbial infections through their strong

antimicrobial activity [32] (G Maroti et al., unpublished

data). The involvement of AMPs in symbiotic plant–bacterium interactions was therefore unanticipated. On

the other hand, the features of terminally differentiated

bacteroids such as the increased membrane permeability

and the inhibition of bacterial cell division are known

activities of AMPs.

BacA, a bacterial function required forterminal bacteroid differentiation in IRLClegumesBacA is an integral membrane protein in bacteria that

belongs to the ATP binding cassette (ABC) superfamily

of membrane transporters. In those rhizobia that nodu-

late IRLC legumes, the BacA protein is essential for

bacteroid differentiation and for the formation of func-

tional nodules [33,34,35�]. bacA mutants induce nodule

formation and the bacteria are released from infection

threads into the symbiotic nodule cells but they fail to

differentiate and undergo immediate senescence

[33,35�]. In roots inoculated with wild-type bacteria

there are two waves of transcriptional changes during

nodule development [23�]. In contrast, the second

transcriptome switch is not activated in nodules

induced by the bacA mutant indicating that BacA-

mediated bacteroid differentiation is essential for the

completion of the nodule developmental program. In

striking contrast, the bacA gene is dispensable for

symbiosis and bacteroid development in those rhizobia

that nodulate legumes with reversible bacteroid differ-

entiation [35�,36].



Figure 2

Rhizobium

symbiosome

3

2

1

infe

ctio

n th

read

golgi

NCR

BacA

SPCDNF1

ER

SPC

bacteroid

cell wall

cytosol of a symbiotic cell

Signal Peptide -xn-C-x5-C-xn-C-xn-C-x4-C-x-C-xn-

nucleus

Current Opinion in Microbiology

The possible role of the BacA protein, the secretory pathway and the NCR peptides in the terminal bacteroid differentiation. The plant cell is infected

via budding-off symbiosomes from the infection threads. Each symbiosome carries one bacterium. Rhizobia differentiate stepwise to elongated,

polyploid bacteroids [16]. This bacteroid differentiation is mediated by different sets of NCR peptides (red) that are targeted to symbiosomes via the

secretory pathway (green) [29��]. A nodule-specific component of the secretory pathway, SPC (blue; defective in the dnf1-1 mutant [31�]) is essential

for trafficking NCR peptides to the symbiosomes and for bacteroid differentiation (1). The rhizobial BacA protein is not needed for the release of

symbiosomes from infection threads but required for bacteroid differentiation [33,35�]. Its activity may change the identity of the symbiosome

membrane to fuse with NCR containing vesicles (2) or modify the bacterial cell envelop which might affect the susceptibility of the bacteria to NCRs (3).

Inset: The structure of the NCR preprotein. The N-terminal signal peptide (green) directs the preproteins to the lumen of the ER. Signal peptides are

inserted in the ER membrane where the signal peptide is removed by SPC releasing a mature NCR peptide in the ER lumen that is subsequently

transported to its correct destination. DNF1 is a nodule-specific subunit of the SPC. The conserved cysteines in mature NCR are indicated; � marks a

variable amino acid.

Under free-living conditions, the bacA mutant displays

pleiotropic phenotypes associated with an altered cell

envelope such as reduction in the very-long-chain fatty

acid content of LPS, increased sensitivity against deter-

gents and low-level resistance toward the glycopeptide

antibiotic bleomycin [37,38]. Moreover, since the BacA

protein promotes uptake of the AMP Bac7, the mutant

became completely insensitive to this peptide [39].

However, these phenotypes can be uncoupled from

the symbiotic phenotype and thus none of these pheno-

types on their own can account for the essential role of

BacA in terminal bacteroid differentiation [39–41]. Most

likely, BacA is crucial for the alteration of cell envelope

during bacteroid development as reflected by the upre-

gulation of a large number of genes encoding cell mem-

brane components in the bacA mutant isolated from

nodules [35�].

The composition of the bacterial surface is an important

determinant of bacterial sensitivity toward AMPs [42].

The BacA function is only needed in the IRLC legumes


that challenge their bacteroids with NCR peptides, which

suggests that the BacA protein affects NCR-mediated

bacteroid differentiation. The bacA mutation, through its

effects on the bacterial cell surface might provoke an

insensitivity or hypersensitivity toward the NCR pep-

tides. Both possibilities could explain why bacA mutants

cannot differentiate properly. Alternatively, BacA could

contribute to the PBM composition which may permit the

fusion of NCR-containing secretory vesicles with the

symbiosomes. In such a scenario, the absence of bacteroid

differentiation in bacA mutants would result from the lack

of peptides transported to the symbiomes, similarly to

what happens in the dnf1 mutant.

BacA is conserved in many bacteria. Intriguingly, it is

important for the pathogenicity of bacteria such as Bru-cella abortis [43] and Mycobacterium tuberculosis [44], which

both establish intracellular infections in macrophages. In

this environment, these pathogens almost certainly

encounter AMPs as it has been shown for Salmonellainfections of macrophages [45].


80 Host-microbe interactions: bacteria

ConclusionsRecent studies offered the first insights into the molecular

mechanisms of terminal bacteroid differentiation in

IRLC legumes, implying the symbiotic exocytotic

machinery for protein transport to symbiosomes, the role

of NCR AMPs and the rhizobial BacA protein (Figure 2).

Arguments based on phylogenetic analysis and physio-

logical measurements of symbiotic performance indicate

that terminally differentiated bacteroids perform better

and give a higher benefit to the plant. This occurs on the

expense of the microsymbionts locked within the nodule

cells with an altered physiology that does not allow life explanta. Moreover, terminally differentiated bacteroids

with their weakened membranes and without the possib-

ility to escape from the nodule might be more efficiently

digested during nodule senescence thereby providing

more nutrients for the plant than bacteroids with revers-

ible differentiation.

Still, many questions remain or have been raised as a

result of these studies. For example, why do terminally

differentiated bacteroids perform better than reversibly

differentiated bacteroids? Possibly, large polyploid bac-

teroids can sustain a higher metabolic activity, in a similar

way as it is believed for polyploid eukaryotic cells [2]. The

nutrient exchange between the host and the bacteroid

might be more efficient when symbiosomes contain a

single bacteroid and not multiple ones as in the case of

reversibly differentiated bacteroids. The AMP-like NCR

peptides affect membrane integrity and inhibit bacterial

divisions. However, the extreme sequence divergence of

the NCR peptides suggests that NCRs might have

diverse activities, protecting the viability of bacteroids

or affecting directly the bacteroid metabolism. The sto-

rage compound polyhydroxybutyrate (PHB) accumulates

in the reversibly (Figure 1) but not in the terminally

differentiated bacteroids [26��,46,47]. PHB storage

represents a waste for the plant as it drives the energy

away from the nitrogen fixation. This is avoided in

terminally differentiated bacteroids where the PHB syn-

thesis might be actively repressed. At present it is

unknown whether NCRs are involved, directly or

indirectly, in the inhibition of PHB production. It will

be of great importance to identify the molecular targets of

individual NCR peptides and the affected pathways in

the bacteroids. Another exciting question is related to the

precise mechanism of BacA action. Moreover, we wish to

understand the mechanism and biological significance of

terminal bacteroid differentiation also in other legume

lineages and to compare the independent evolutionary

strategies for terminal bacteroid differentiation.

AcknowledgementsWork in our laboratories is supported by the French Agence Nationale de laRecherche, grant ANR-09-BLAN-0396-01 and the Hungarian NationalOffice for Research and Technology, grants OMFB-00441/2007 andOMFB-00128/2010. We thank Agnes Ullmann and Pal Venetianer for theircomments on the manuscript.


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http://dx.doi.org/10.1104/pp.110.163436

innate immunity effectors and virulence factors in symbiosis

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