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Antonie van Leeuwenhoek 81: 373–383, 2002.
© 2002 Kluwer Academic Publishers. Printed in the Netherlands.
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Microbe–plant interactions: principles and mechanisms
Ben J.J. Lugtenberg∗ , Thomas F.C. Chin-A-Woeng & Guido V. Bloemberg
Leiden University, Institute of Molecular Plant Sciences, Clusius Laboratory, Wassenaarseweg 64, 2333 AL Leiden,
The Netherlands (∗ Author for correspondence)
Key words: anti-fungal metabolites, biofertilizers, biopesticides, colonization, genomics, induced systemic resistance, pathogens, phase variation, phytostimulators, quorum sensing, recognition, regulation, rhizoremediators,
two-component systems, Type 4 pili, Type III secretion systems, Type IV secretion systems
Abstract
The present status of research on the molecular basis of microbe–plant interactions is discussed. Principles and
mechanisms which play a role in the interactions of microbial pathogens, biofertilizers, phytostimulators, rhizoremediators and biocontrol agents with the plants are treated. Special emphasis is given to colonization, phase
variation, two-component systems, quorum sensing, complex regulation of the syntheses of extracellular enzymes
and secondary metabolites, Type 4 pili and Type III and Type IV secretion systems.
Abbreviations: ACC – 1-aminocyclopropane-1-carboxylic acid; AFM – anti-fungal metabolite; AHL – Nacylhomoserine lactone; CLSM – confocal laser scanning microscopy; DFI – differential fluorescensce induction;
EPS – extracellular polysaccharide; ISR – induced systemic resistance; IVET – in vivo expression technology; LPS
– lipopolysaccharide; ORF – open reading frame; OT – optical trapping; QS – quorum sensing; PC – phosphatidyl
choline; PCN – phenazine-1-carboxamide; Phl – 2,4-diacetyl phloroglucinol; SAR – systemic acquired resistance;
TTSS – type III secretion system
The challenge of microbiology in the beginning of
the 21st century
In the second half of the twentieth century a major focus of microbiology was on trying to understand the
behaviour of model microbes under laboratory conditions. The challenges for the next decades include
understanding of the behaviour of microbes in their
natural and often complex habitats, such as the rhizosphere and the phyllosphere. A microbe seems largely
to be driven by principles which also govern our own
behaviour: a microbe wants to survive, also under hostile conditions, and if conditions are more favourable
it will eat and proliferate. In order to understand a microbe’s behaviour in a certain habitat, it is essential
to know which genes are expressed, what their functions are, how the gene products interact, how these
processes depend on biotic and abiotic conditions, and
towards which behaviour traits this leads. In theory,
the use of the new technology often designated as ‘Genomics’ can answer these questions. However, before
becoming over-optimistic it is good to realize that even
from the best-known organism Escherichia coli K-12,
biochemical function has not yet been established for
one third of the 4288 ORFs.
Presently, the DNA of several microbes and plants
which are studied in microbe-plant model systems has
been sequenced or will soon be sequenced. On the
other hand, sequencing is still expensive and the sequences of many interesting organisms will not soon
be available. For example, although the opportunistic
human pathogen Pseudomonas aeruginosa PAO-1 has
been sequenced (5570 ORFs; Stover et al. 2000), it
is unlikely that the genomic sequences of the many
Pseudomonas strains which are important plant pathogens, phytostimulators, bioremediators or biocontrol
agents will soon be available. Based on comparative
genomics between E. coli strains it can be predicted
that their genomes not only differ in a few ‘islands’,
but that two Pseudomonas strains will differ in hundreds of segments larger than 50 bp. Also the genomes
of fungi and plants are being sequenced, a process
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which delivers less functional information since only
approx. 1.5% represents ORFs.
Other novel technologies which will also be useful
to study microbial behaviour are the use of autofluorescent proteins in combination with CLSM (confocal
laser scanning microscopy), atomic force microscopy,
single molecule detection, and DFI (differential fluorescence induction) combined with OT (optical trapping) (Allaway et al. 2001). Considering the massive
amounts of information that will be gathered with the
help of these expensive technologies, networking and
cost-sharing will be key words in the next decades.
Microbe–plant interactions: general
considerations
Based on their effects on the plant, microbes interacting with plants can be classified as pathogenic, saprophytic and beneficial. Pathogens can attack leaves,
stems or roots. An interesting new research field is the
interaction between water-borne microbes and underwater plants. Saprophytes, which live on dead plant
material, will not be further treated in this overview although they play a crucial role in important processes
such as the cycling of elements and composting.
Beneficial microbes are often used as inoculants
(Bloemberg & Lugtenberg 2001). They can be classified according to the goal of their application: biofertilizers (such as rhizobia, which have been applied
commercially for over a century), phytostimulators
(such as auxin-producing, root-elongating Azospirillum), rhizoremediators (pollutant degraders which use
root exudate as their carbon source), and biopesticides.
None of the numerous microbe-plant interactions
is completely understood. Therefore, we have not
chosen for summing up a huge number of facts per
interesting microbe but we will restrict ourselves to
a number of newly discovered principles and mechanisms, and discuss a limited number of interactions
between microbes and plants. By now it is clear that
microbes in their interactions with plants, no matter
whether the microbe is beneficial or pathogenic, often use the same mechanisms, although in different
combinations and for different purposes. Similarly, it
is clear that microbes in their interaction with plants
use similar strategies as in their interactions with other
eukaryotes such as fungi and humans (Lugtenberg &
Dekker 1999; Chin-A-Woeng et al. 2000; Lugtenberg
et al. 2001). It is thus good to realise that, although
one uses the term microbe–plant interactions, the real-
ity is that in the rhizosphere and in the phyllosphere
microbes also interact with each other.
Plant recognition by microbes
Recognition is considered to be the initial key event
in the response of plants to microbes. Recognition can
occur through physical interaction – such as through
adhesins, fimbriae, flagella, and Type III and Type
IV secretion systems – or through signalling by small
molecules.
It has been estimated that between 30 and 40 genes
are involved in the biosynthesis and functioning of
type 4 pili in P. aeruginosa (Darzins & Russell 1997).
In this bacterium type 4 pili are involved in the initial
contact between the bacteria and the epithelial cell surface, in a unique type of locomotion called twitching
motility, and in biofilm formation (O’Toole & Kolter
1998). Although type 4 pili are present in various plant
pathogens, their role in attachment to plant surfaces
is poorly documented. In the case of Xanthomonas
campestris pv. hyacinthi type 4 pili were shown to
be involved in attachment to hyacinth leaves whereas
P. syringae pv. phaseolicola attaches to stomata via
its type 4 pili (Romantschuck & Bamford 1986). Camacho (2000) obtained evidence for a role of type 4
pili of P. fluorescens WCS365 in tomato root colonization. Dörr et al. (1998) have indicated a role for type 4
pili in attachment of Azoarcus to grass roots as wel as
to fungal mycelium. The pilA promoter of Azoarcus is
induced upon starvation in carbon-free medium (Plessl
et al. 2000).
One of the interesting and sophisticated mechanism used by bacterial pathogens to attack eukaryotic
host cells is a specialized protein secretion system,
the TTSS, that delivers bacterial virulence proteins
(‘effector proteins’) directly into the host cell. TTSSs share four important features (He 1988; Galán
& Collmer 1999). (i) They are widespread among
plant and animal bacterial pathogens, and mutations
affecting Type III protein secretion often eliminate
bacterial virulence completely. More recent evidence
suggests also roles of TTSSs in symbiosis and biological control (see further on). (ii) Many of their genes
share sequence similarities with those of the flagellar assembly machiney and flagellum-like structures,
leading to the speculation that TTSSs are evolutionary derived from the flagellar assembly apparatus. (iii)
Type III secretion is activated in vivo upon contact
with the host cell. (iv). The TTSS is Sec-independent.
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The TTSS of Yersinia spp. is the best-studied example
(Cornelis & Wolf-Watz 1997).
Proteinaceous pertussus toxins are injected into human cells by a Type IV secretion system. Another
member of this system is used for the translocation of single stranded T-DNA-protein complexes by
Agrobacterium tumefaciens. The channel is composed
of 11 VirB proteins and the VirD4 coupling factor.
Transfer of the VirE2 and VirF proteins into plant
cells can also occur independently of T-DNA transport
(Vergunst et al. 2000). DNA of broad host range conjugative plasmids is also transferred through similar
structures.
A second Type IV secretion system has been
detected (Wood et al. 2001) in the genome of A.
tumefaciens, which is composed of 4 replicons: one
linear chromosome, one circular chromosome, the
tumor-inducing (Ti) plasmid, and a cryptic plasmid.
Plant surface colonization by microbes
Colonization of plant tissues can be visualized by
using CLSM analysis of autofluorescent proteins expressed in the bacterial and/or fungal partners (Spellig
et al. 1996; Bloemberg et al. 1997; Tombolini et al.
1999; Lagopodi et al. 2002). Using different forms of
autofluorescent proteins, Bloemberg et al. (2000) were
able to visualize three different bacterial populations
simultaneously. Using two differently colored populations of the same P. fluorescens WCS 365 bacterium,
it was concluded that, after inoculation of seedlings,
occasionally single bacteria reach a position at a lower
part of the root where they can divide without being
frequently visited by a new invader (Bloemberg et al.
2000).
It is generally assumed that the colonization of
plant tissue is a crucial step in pathogenesis as well
as for beneficial effects of microbes on the plant. The
plant surface contains many microbes which, however,
are not homogeneously distributed. Rather, deserted
areas as well as areas with many microbes, often
present as microcolonies or biofilms, are present. Such
microcolonies are ideal places for bacteria to communicate with each other, e.g. by QS (quorum sensing).
Many genes and traits are involved in colonization (for
a review, see Lugtenberg et al. 2001). The results of
colonization studies show that successful colonization
strategies include: (i) good defense of the cell’s interior, (ii) efficient uptake of low concentrations of
nutrients, including those provided by other organ-
isms, and (iii) weakening or destruction of target and
competing organisms. In this respect it should be noted
that little research has been carried out on microbemicrobe interactions in the rhizosphere. Investigations
towards the molecular basis of bacterium–fungus interactions have already started (Smith et al. 1999;
Lee & Cooksey 2000). The recent discovery that
Pseudomonas biocontrol bacteria not only colonize
the plant root but also hyphae of the pathogenic fungus
Fusarium oxysporum f.sp. radicis-lycopersici (Lagopodi et al. 2002) indicates that such interactions may
be important. Interestingly, many colonization genes
are shared by plant-beneficial bacteria and humanpathogenic bacteria, indicating that colonization of
eukaryotic tissue by bacteria has started early in evolution (Lugtenberg & Dekkers 1999; Lugtenberg et al.
2001).
Using colonization mutants, it was shown that
colonization is crucial for biocontrol of tomato foot
and root rot by the PCN (phenazine-1-carboxamide)
producing P. chlororaphis strain PCL1391 (Chin-AWoeng et al. 2000) but not, or to a lower extent, for
biocontrol of this disease by the ISR inducing P. fluorescens strain WCS365 (Dekkers et al. 2000). Interestingly, P. putida mutants impaired in seed colonization
have recently been described (Espinosa-Urgel et al.
2000). In this respect it is interesting to note that,
after seed inoculation, growth of B. cereus in the spermosphere of tomato depends on the tomato genotype
(Smith et al. 1999; Simon et al. 2001).
IVET (in vivo expression technology) is a powerful
tool to identify promoters and genes that are expressed
specifically under certain conditions, e.g. when a bacterium interacts with a plant (Rainey 1999) or a fungus
(Lee & Cooksey 2000). Considering the high number
of colonization genes, a reasonable understanding of
the mechanisms of root, seed and leaf colonization can
only be obtained if the number of bacterial genes to
be tested for a role in colonization can be reduced by
preselection. IVET and Genomics of microbe-plant interactions are promising tools for such a preselection.
Genomics is also a good approach to unravel which
plant genes play a role in colonization.
Phase variation
Phase variation results in subpopulations within a bacterial ‘pure culture’ which differ in the make up of
their bacterial cell surface. This phenomenon occurs
in many pathogens and is assumed to be a strategy to
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escape the host’s immune system. It also appeared to
play a role in root colonization (Dekkers et al. 1998).
In a bacterial culture subpopulations with different degrees of rhizosphere competence can apparently exist
next to each other. One of the mechanisms of phase
variation is based on the action of a site-specific recombinase of the λ-integrase gene family which can
either delete or reverse DNA fragments. Such DNA
rearrangements will largely contribute to the flexibility of bacterial genomes (e.g. Flores et al. 2000).
It is important to note that phase variation can affect important (putative) root colonization traits, such
as Type 4 fimbriae, and flagellum-mediated swimming, swarming and twitching, as well as chemotaxis
(Déziel et al. 2001).
Synthesis of extracellular enzymes and secondary
metabolites by microbes
Two-component systems are present in many gramnegative bacteria. Usually, a sensor kinase in the cytoplasmic membrane autophosphorylates upon activation by an environmental stimulus and subsequently
phosphorylates a response regulator protein in the
cytosol which then acts as a transcription factor. The
GacA/GacS system is the best studied example but its
environmental stimulus has still not been identified.
It should be noted that GacS was formerly known
as LemA. Regulation in P. aeruginosa is extremely
complex with an extraordinary number of putative
two-component systems: 55 sensors, 89 response regulators and 14 sensor-response regulator hybrids, far
more than found in other genomes (Stover et al. 2000).
Some bacteria can communicate with cells of their
own population and sometimes with other bacteria
through exchange of soluble signal molecules. Of
these, the N-acyl homoserine lactones (AHLs) are the
best studied examples, but other types of intercellular signal compounds exist. These include cyclic
dipeptides and quinolones in P. aeruginosa (Holden
et al. 1999; Pesci et al. 1999), two unidentified
low molecular weight compounds in Xanthomonas
campestris (Poplawsky et al. 1998), and a volatile
fatty acyl methyl ester in Ralstonia solanacearum
(Flavier et al. 1997). In gram-positive bacteria γ butyrolactones are used for intercellular communication. Interestingly, one of the AHL QS molecules has previously been described as a bacteriocin
(Schripsema et al. 1996).
AHLs are involved in interactions between bacteria
and eukaryotes such as conjugation between Agrobacterium and plant cells, and production of AFMs and
other antibiotics and of extracellular enzymes, all of
which are produced to attack competitors or foreign
cells. Interestingly, also plants secrete substances that
mimic bacterial AHL activities (Teplitski et al. 2000).
It appeared that halogenated furanones produced
by a marine red algae are able to disrupt AHLregulated behaviours (Givskov et al. 1996; Kjelleberg
et al. 1997), presumably by competitive inhibition for
the AHL-receptor protein (Manefield et al. 1999). Molecules which inhibit the action of AHLs, if applied
to pathogens, have promise (as lead compounds) for
disease control. Tobacco has been genetically modified to secrete AHLs, which appeared to affect bacteria
present in its rhizosphere (Fray et al. 1999).
Extracellular enzymes and secondary metabolites,
such as AHLs and AFMs (anti-fungal metabolites), are
very important for the interactions of bacteria in the
rhizosphere. The regulation of the production of these
molecules is extremely complex, in terms of regulatory genes as well as regulatory environmental factors.
Regulatory components involve rpoS, the gacS/gacA
two-component system, a regulatory (PrrB) RNA molecule (Aarons et al. 2000), as well as an RNA
binding protein, RsmA, which is a repressor of secondary metabolism. The latter molecule is involved in
a GacA-dependent switch from primary to secondary
metabolism, acting at the level of translation (Blumer
et al. 1999).
Quorum sensing plays a role in the synthesis of
several exoenzymes and AFMs such as phenazines
(Pierson et al. 1994; Chin-A-Woeng et al. 2001). A
two component gacA/gacS system has been described
which interacts with the phzl/phzR quorum sensing
system (Chancey et al. 1999; Chin-A-Woeng et al.
2001).
The production of AFMs is often limited. The
product of phlR acts as a suppressor of Phl (2,4 diacetyl phloroglucinol) production (Delany et al. 2000),
whereas psrA limits PCN production (Chin-A-Woeng,
unpublished), and synthesis of pyoluteorin is repressed
by the serine protease Lon (Whistler et al. 2000).
The production of Phl, like that of auxin in Azospirillum (Vande Broek et al. 1999) is subject to autoinduction. This autoinduction is countered by the plant
metabolite salicylate, by the bacterial metabolite pyoluteorin and by fusaric acid, a toxin produced by
the pathogenic fungus Fusarium (Schneider-Keel et al.
2000).
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Environmental factors which influence the biosynthesis of secondary metabolites by pseudomonads
include ions, sugars and glycerol (Duffy & Défago
1999; Chin-A-Woeng 2000). P. putida PCL1444 was
selected from an environmental sample as the strain
which had the best combination of naphthalene degradation and grass root colonization. Its behaviour
appeared to be determined to a large extent by grass
exudates components: it grew fastest on the major
grass exudates components glucose and succinic acid
and expression of its naphthalene degradation operons
was highest in the presence of these carbon sources
(Kuiper 2001). These results strongly indicate that by
selecting for the traits fast growth and good colonization, one in fact selects for efficient utilization of
exudate.
Plant pathogens
Fungi are the most important plant pathogens followed
by viruses and bacteria. Ordinarily, the plant rejects
the pathogen: most plants are not normally colonised
and parasitized by the pathogen. They are ‘non-host
plants’ for these pathogens. This phenomenon is called
‘basic resistance’ or ‘basic incompatibility’. Successful parasitism depends on the production of the right
combination of pathogenicity factors to attack a particular pathogen. In such a case ‘basic compatibility’
exists between these partners. Basic compatibility is a
highly specific phenomenon between a particular plant
species and the appropriate pathogen species or forma
specialis.
An elicitor is a microbial signal which is recognized by a plant receptor. Subsequent transduction
of the signal can result in a defense response by
which the plant may ward off the attack. Most flagellin molecules e.g. from Bacillus, Escherichia and
Pseudomonas, but not from Rhizobium and Agrobacterium, share a stretch of approximately 20 amino
acids in their N-terminal domain which act as general
elicitors (Felix et al. 1999). Other general elicitors are
β-glucans, chitin fragments (including Nod factors!)
and ergosterol. Plants recognise these substances with
extremely high sensitivity and specificity (Felix et al.
1999). Many of these general elicitors occur in pathogens as well as in saprophytes and symbionts. It is
therefore unlikely that recognition of these molecules
induces an all-out defense in the plant. Rather, it
has been speculated that these chemoperception systems might have an orienting role, allowing the plant
to ‘smell’ the presence of microbes and of non-self
organisms in its environments (Boller 1995).
One of the defense strategies which the host plant
can deploy is the ‘hypersensitive response’ (HR) or
‘hypersensitive cell death’. The injured plant cell dies
very rapidly, causing necrosis of immediately adjacent
tissue (Programmed Cell Death; Lam et al. 2001). This
strategy cuts the pathogen off from its nutrients. It is
unable to spread and multiply and will finally die.
Phytopathogenic bacteria often contain a cluster of
conserved genes, the hrp cluster, which is essential for
the induction of the hypersensitive response in nonhost plants as well as for pathogenesis in susceptible
host plants. The majority of these hrp genes encode
elements of a TTSS for proteins (Lindgren 1997; Van
Gijsegem et al. 1993) which may be evolutionary
derived from the flagellar structure (Macmab 1999).
Some hrp gene products, including a protein called
harpin, are assumed to be secreted through the type III
‘syringe’, similar to how flagellin subunits are secreted
through the hollow cylinder in the flagellum’s basal
body and its filament. In the case of the TTSS these
secreted hrp gene products, presently often designated
as ‘effector molecules’, are assumed to be injected
into the host cell. Delivery of the Avr (avirulence)
gene product, followed by recognition in the plant
cell, is essential for development of plant resistance
(e.g. Bonas 2000). In the human pathogen Yersinia
enterocolitica several virulence factors, including a
virulence-associated phospholipase are known to be
exported through the TTSS (Cornelis & Wolf-Watz
1997; Young et al. 1999).
The tomato pathogen Cladosporium fulvum grows
within the intracellular spaces of the leaves, where
it secretes in susceptible cultivars, as well as during
infection of resistant plants, a polypeptide product of
the Avr9 gene which causes HR only in race-specific
resistant cultivars which carry the cf9 resistance gene.
The 28 amino acid polypeptide encoded by Avr9 acts
as a race-specific elicitor. This and a similar peptide
encoded by Avr4, are used for unravelling the mechanism of plant resistance (see article by P. de Wit and
colleagues in this volume). Like many plant resistance
genes, the Cf9 and Cf4 resistance genes contain many
glycosylated leucine-rich repeats which are believed
to interact, directly or indirectly, with other proteins,
carbohydrates or ligands.
Important bacterial virulence factors include exotoxins such as coronatine (Liyanage et al. 1995) and
the biosurfactant toxin syringomycin (Hutchison &
Gross 1997). The latter toxin seems to act through the
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formation of ion channels in plant plasma membranes
which initiate a cascade of intracellular signalling
events. Pectate lyases are important virulence factors
of soft-rotting erwinias (Keen 1996).
Interestingly, P. aeruginosa strain PA14 is infectious both for the plant A. thaliana and for mouse. Several different mutations reduce pathogenicity in both
hosts, indicating that they encode virulence factors for
both plants and animals (Rahme et al. 1995).
Biofertilizers
Biofertilization accounts for approximately 65% of the
nitrogen supply of crops worldwide. Rhizobiaceae and
Mycorrhizae are the best-known examples of biofertilization. The former microbes are applied to increase
crop yield from leguminous plants, such as soybean,
alfalfa and clover grown in nitrogen-poor soil (Spaink
et al. 1998). The Mycorrhizae are used for reforestation of fir plants and act by extending the rhizosphere,
thereby enormously increasing the effective root system of the plant (Bonfante & Perotto 1995; Smith
& Gianninazzi-Pearson 1998). In addition, rather recently several endophytic nitrogen fixers were discovered. Azoarcus is able to fix nitrogen in Kaller
grass and rice (Reinhold-Hurek & Hurek 1998; Steenhoudt & Vanderleyden 2000) whereas Herbaspirillum
and Acetobacter fix N2 in sugar cane (Okon et al.
1998).
Roots of leguminous plants secrete flavonoids
which induce Rhizobium bacteria to form nodules in
a host-specific way. Some of these flavonoids are able
to activate the NodD regulatory protein which results
in activation of nod operon which encodes the biosynthesis of extremely host-specific Nod-metabolites
(reviewed in Spaink et al. 2000). This host-specificity
is an excellent example of evolutionary fine-tuning of
interactions between microbes and plants. The bacteria enter the plant through infection threads, change
into bacteroids in which form they convert atmospheric N2 into ammonia. This N-source can be used
by the plant. To complete the symbiosis, the plant supplies the bacteroids with a dicarboxylic acid carbon
source.
Bacterial components which play a role in the
recognition of the plant in various stages of the nodulation process include the Nod-factors, EPSs (extracellular polysaccharides), LPSs (lipopolysaccharides),
K-antigens (which are structurally very distinct from
LPSs) and periplasmatic cyclic glucans (Spaink et al.
2000). Some of these molecules are modified during
the nodulation process (Goosen-de Roo et al. 1991;
Lugtenberg 1998). The exact role of the PSs (polysaccharides) is hard to unravel since many mutants in
the biosyntheses of these macromolecules are pleiotrophic whereas non-pleiotrophic mutations are sometimes complemented by other PSs. A typicality of
the LPS of rhizobia is the presence of very long
chain HO-fatty acids, such as 27-OH-C28:0 (Hollingsworth & Carlson 1989), which can also be present in
Nod-factors (Spaink 2000).
The membrane phospholipid PC (phosphatidylcholine) is a major lipid of eukaryotic membranes, but found in only few prokaryotes, such as
Bradyrhizobium japonicum, in which 52% of the total
phospholipid can consist of PC. A group lead by Otto
Geiger and Isabel Lopez-Lara discovered a new PC
biosynthetic pathway in which choline (present in the
rhizosphere) and CDP-diacylglyceride are condensed
(Sohlenkamp et al. 2000). B. japonicum mutants unable to synthesize PC are inefficient in their symbiosis
with the soybean host plant (Minder et al. 2001). The
genomes of two Rhizobiaceae have been sequenced.
Interesting features are the following. (1) The presence of genes homologous to those encoding TTSSs
(‘machines’) which are used by pathogenic bacteria to
deliver virulence factors into host cells. Recent results show that the rhizobial TTSS secretes specific
proteins and is involved in the establishment of the
symbiosis. In some cases secreted proteins negatively
affect the nodulation process, perhaps by suppressing
plant defense responses (Viprey et al. 1998; Marie et
al. 2001; Spaink et al. 2001). (2) The presence of a
502 kb chromosomally-integrated ‘symbiosis island’
in Mesorhizobium loti R7A. This island is integrated
in a phe-tRNA gene in a process mediated by a P4
integrase of which the gene is located on one end of the
island. The island can be transferred by conjugation to
non-symbiotic mesorhizobia which converts them to
Lotus symbionts. The island of strain R7A contains a
Type IV secretion system similar to the VirB/VirD4
pilus from A. tumefaciens whereas a similar M. loti
symbiosis island encodes a TTSS (Ronson et al. 2001).
Natera et al. (2000) analysed proteomes of the
symbiosis between Sinorhizobium meliloti strain 1021
and the legume Melilotus alba (white sweetclover)
with the aim of characterizing symbiosis proteins.
They reported that hundreds of proteins were present
in altered amounts in the symbiotic vs. the nonsymbiotic situation.
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Vesicular-arbuscular Mycorrhizae occur in over
80% of all terrestrial plant species (Bonfante & Perotto
1995). These fungi are obligate symbionts and colonize the cortex of the root in order to obtain carbon
from their host plant, while assisting the plant with
the uptake of mineral nutrients, predominantly phosphate, from the soil. In many soils the bioavailable
phosphate is present in low concentrations which may
be limiting for growth (Harrison et al. 1996). The
arbuscular mycorrhizal fungus Gigaspora margarita
harbors a resident population of as many as 250 000
endosymbiotic Burkholderia cells in the cytoplasm of
one cell (Ruiz-Lozano & Bonfante 2000).
Phytostimulators
Some bacteria belonging to the genus Azospirillum
promote plant growth as free-living organisms. A
polar flagellum, of which the flagellin was shown
to be a glycoprotein, is involved in attachment of
Azospirillum cells to plant roots. Azospirillum fixes
atmospheric nitrogen but the observed growth promotion may rather be related to plant growth promoters
produced by the bacterium than by its nitrogen fixing
capacity. Indeed, three types of plant growth promoting substances have been detected in the supernatant
fluids of Azospirillum cultures, namely auxins, cytokinins and gibberellines. Of these, the auxin IAA
(indole-3-acetic acid) is quantitatively the most important one. Three pathways are known to convert
tryptophan into IAA. Inoculation experiments with
Azospirillum mutants altered in IAA production support the view that after Azospirillum inoculation IAA
causes increased rooting, which in turn enhances
mineral uptake (Steenhoudt & Vanderleyden 2000).
Similar results were obtained with a plant growth
promoting P. fluorescens strain, which converts exogenous tryptophan into IAA. The strain promotes
maturation of radish in greenhouse experiments,
most likely due to the high concentration of tryptophan measured in radish exudate (G. Bloemberg, N.
Makarova, T. Azarova, C. Mulders, L. Kravchenko,
I.A. Tikhonovich and B. Lugtenberg, unpublished).
Rhizoremediators
Most environmental pollutants can be degraded by
selected microbes. The process stops when the microbe is starved of food. In order to ascertain that
such microbes can have access to the best food source
available in soil, namely root exudates, Kuiper et al.
(2001) have described an enrichment method for the
isolation of microbes which combine the properties
of: (i) degradation of a selected pollutant, and (ii)
excellent root colonization. They have termed this process ‘rhizoremediation’ instead of phytoremediation
to emphasize the roles of the root exudates and the
rhizosphere competent microbe.
Biocontrol agents or biopesticides
Preparations of many microbes are commercially
available as biological control agents to protect plants
against fungi. The major applied organisms are the
bacteria Bacillus, Pseudomonas and Streptomyces and
the fungi Trichoderma, Gliocladium and Fusarium.
The mechanisms used by these biocontrol agents include the following. (i). Niche exclusion [e.g. by
the frost damage causing leaf colonizing P. syringae (Lindow 1995)]. (ii). Competition for nutrients
[e.g. for carbon by Fusarium (Alabouvette 1986) and
for Fe3+ siderophore complexes by P. putida strain
WCS358 (Bakker et al. 1990)]. (iii). Production of
chitinase by Serratia marcescens (Ordentlich et al.
1987) and by Trichoderma (Sivan & Chet 1989). (iv).
Production of AFMs (antifungal metabolites), such
as Phl (2,4-diacetyl phloroglucinol) [e.g. by pseudomonads (Keel et al. 1990; Bangera & Tomashow
1996), phenazine derivatives (Tomashow & Weller
1988; Pierson et al. 1995; Chin-A-Woeng et al.
1998; Delany et al. 2001), zwittermycin (Stohl et al.
1996), pyoluteorin (Nowak-Thompson et al. 1999),
and pyrrolnitrin (Kirnes et al. 1998). Recently it has
been discovered that certain biosurfactants also act
as AFMs (Stanghellini & Miller 1997; Nielsen et
al. 2000; Thrane et al. 2000). (v). ISR (Induced
Systemic Resistance) is triggered by certain nonpathogenic Pseudomonas rhizobacteria which make
the plant extremely reactive towards a range of pathogens, including leaf pathogens (van Peer et al. 1991;
M’piga et al. 1997). Flagella, LPS (lipopolysaccharide) and siderophores have been implied as bacterial
components involved in causing ISR (van Loon et al.
1998).
ISR differs from SAR (systemic acquired resistance), which is induced upon pathogen infection.
Simultaneous activation of SAR and ISR resulted in
an additive effect on the level of induced protection
of Arabidopsis thaliana against P. syringae pv. tomato
380
(van Wees et al. 2000). Ethylene signalling plays a role
in P. fluorescens WCS417r mediated ISR functions.
Introduction of the ACC deaminase gene, encoding
the cytoplasmic enzyme ACC deaminase, which converts ACC (1-aminocyclopropane-1-carboxylic acid, a
precursor of ethylene) to ammonia and α-ketobutyrate,
into P. fluorescens strain CHAO improved its ability to
protect cucumber against Pythium damping off. It was
proposed that ACC deaminase decreases the level of
pathogen-induced plant ethylene and therefore eliminates the pathogen-induced inhibitory effect of ethylene
on root elongation (Wang et al. 2000).
We would like to finish the overview with a striking
similarity in the strategies of two Pseudomonas strains
to kill eukaryotes. P. aeruginosa kills the nematode C.
elegans and this killing requires colonization as well
as the synthesis of the phenazine-derivative pyocyanine (Mahajan et al. 1999), whereas P. chlororaphis
kills fungi, including Fusarium and this killing is accompanied by colonization of hyphae (Lagopodi et al.
2002) and, if present, the root surface (Chin-A-Woeng
et al. 2000) as well as by phenazine-1-carboxamide
production (Chin-A-Woeng et al. 1999).
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