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Indole-3-acetic acid inmicrobial andmicroorganism-plantsignalingStijn Spaepen, Jos Vanderleyden & Roseline Remans
Department of Microbial and Molecular Systems, Centre of Microbial and Plant Genetics, Heverlee, Belgium
Correspondence: Jos Vanderleyden,
Department of Microbial and Molecular
Systems, Centre of Microbial and Plant
Genetics, K.U. Leuven, Kasteelpark Arenberg
20, 3001 Heverlee, Belgium. Tel.: 132
16321631; fax: 132 16321966; e-mail:
jozef.vanderleyden@biw.kuleuven.be
Received 4 December 2006; revised 3 March
2007; accepted 12 March 2007.
First published online 18 May 2007.
DOI:10.1111/j.1574-6976.2007.00072.x
Editor: Fritz Unden
Keywords
indole-3-acetic acid; plant–microorganism
interactions; microbial signaling.
Abstract
Diverse bacterial species possess the ability to produce the auxin phytohormone
indole-3-acetic acid (IAA). Different biosynthesis pathways have been identified
and redundancy for IAA biosynthesis is widespread among plant-associated
bacteria. Interactions between IAA-producing bacteria and plants lead to diverse
outcomes on the plant side, varying from pathogenesis to phytostimulation.
Reviewing the role of bacterial IAA in different microorganism–plant interactions
highlights the fact that bacteria use this phytohormone to interact with plants as
part of their colonization strategy, including phytostimulation and circumvention
of basal plant defense mechanisms. Moreover, several recent reports indicate that
IAA can also be a signaling molecule in bacteria and therefore can have a direct
effect on bacterial physiology. This review discusses past and recent data, and
emerging views on IAA, a well-known phytohormone, as a microbial metabolic
and signaling molecule.
Introduction
In 1880 Charles Darwin proposed that some plant growth
responses are regulated by ‘a matter which transmits its
effects from one part of the plant to another’ (Darwin &
Darwin, 1880). Several decades later, this ‘matter,’ termed
auxin (from the Greek ‘auxein’ which means ‘to grow’), was
identified as indole-3-acetic acid (IAA) (Kogl & Koster-
mans, 1934; Went & Thimann, 1937). IAA has since been
implicated in virtually all aspects of plant growth and
development (reviewed by Woodward & Bartel, 2005 and
Teale et al., 2006).
Intriguingly, the discovery of IAA as a plant growth
regulator coincided with the first indication of the molecular
mechanisms involved in tumorigenesis induced by Agrobac-
terium. Agrobacterium-induced tumors were shown to be
sources of IAA (Link & Eggers, 1941) and capable of growth
in the absence of plant growth regulators, which are
normally required to incite growth of callus from sterile
plant tissues (White & Braun, 1941). It was later found that
not only plants but also microorganisms including bacteria
and fungi are able to synthesize IAA (Kaper & Veldstra,
1958; Gruen, 1959; Perley & Stowe, 1966; Libbert et al., 1970;
Arshad & Frankenberger, 1991; Costacurta & Vanderleyden,
1995).
In recent years, advancement in understanding the IAA
signaling pathway in plants has been truly spectacular. The
role of IAA in bacteria has not thus far been investigated in
such detail. Undoubtedly, the advancement in plant IAA
signaling has also intensified research on the various aspects
of bacterial IAA synthesis, including its role in bacteria–
plant interactions.
As more bacterial species have been analyzed, the role
of auxins in plant–microorganism interactions appears
diverse. Molecular studies on the biochemical pathways of
bacterial IAA synthesis and their regulation have provided
some clues on the possible outcomes of the interactions
between plants and IAA-producing bacteria, varying from
pathogenesis to phytostimulation. Recently, a number of
studies have clearly shown that IAA can be a signaling
molecule in microorganisms, in both IAA-producing and
IAA-nonproducing species. These findings raise new intri-
guing questions on the role of IAA in bacteria and their
interaction with plants.
IAA biosynthesis pathways in bacteria
With the analysis of additional bacterial species, different
bacterial pathways to synthesize IAA have been identified. A
high degree of similarity between IAA biosynthesis pathways
FEMS Microbiol Rev 31 (2007) 425–448 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
in plants and bacteria was observed. Here an overview of
bacterial IAA biosynthesis pathways is given (Fig. 1), and the
current status of related genes, proteins and intermediate
metabolites is discussed. Where relevant, comparisons with
plant IAA biosynthesis are made.
Tryptophan has been identified as a main precursor for
IAA biosynthesis pathways in bacteria. The identification of
intermediates led to the identification of five different path-
ways using tryptophan as a precursor for IAA.
Indole-3-acetamide pathway
The indole-3-acetamide (IAM) pathway is the best charac-
terized pathway in bacteria. In this two-step pathway
tryptophan is first converted to IAM by the enzyme trypto-
phan-2-monooxygenase (IaaM), encoded by the iaaM gene.
In the second step IAM is converted to IAA by an IAM
hydrolase (IaaH), encoded by iaaH. The genes iaaM and
iaaH have been cloned and characterized from various
bacteria, such as Agrobacterium tumefaciens, Pseudomonas
syringae, Pantoea agglomerans, Rhizobium and Bradyrhizo-
bium (Sekine et al., 1989; Clark et al., 1993; Morris, 1995;
Theunis et al., 2004). The IAM-related genes have been
detected on the chromosome in different Pseudomonas
species as well as on plasmids such as pPATH of
Pa. agglomerans (Glickmann et al., 1998; Manulis et al.,
1998).
The IAM pathway was described previously as a bacterial-
specific pathway, as no evidence for this pathway could be
found in plants. However, with an improved, highly sensi-
tive method for the analysis of IAM using a combination of
HPLC and GC-MS/MS techniques it was proven beyond
doubt that IAM is an endogenous compound of Arabidopsis
thaliana (Pollmann et al., 2002). Experiments described by
Piotrowski et al. (2001) and Pollmann et al. (2003) further
support the operation of the IAM pathway in Arabidopsis.
Indole-3-pyruvate pathway
The indole-3-pyruvate (IPyA) pathway is thought to be a
major pathway for IAA biosynthesis in plants. However,
the key genes/enzymes have not been identified yet in plants.
In bacteria, IAA production via the IPyA pathway has
been described in a broad range of bacteria, such as the
pythopathogenic bacterium Pa. agglomerans, the beneficial
bacteria Bradyrhizobium, Azospirillum, Rhizobium and
Enterobacter cloacae, and cyanobacteria. The first step in
this pathway is the conversion of tryptophan to IPyA by
an aminotransferase (transamination). In the rate-limiting
step, IPyA is decarboxylated to indole-3-acetaldehyde
(IAAld) by indole-3-pyruvate decarboxylase (IPDC). In the
last step IAAld is oxidized in IAA (Fig. 1). The gene,
encoding for the key enzyme, IPDC, has been isolated and
characterized from Azospirillum brasilense, En. cloacae,
Fig. 1. Overview of the different pathways to synthesize IAA in bacteria. The intermediate referring to the name of the pathway or the pathway itself is
underlined with a dashed line. IAAld, indole-3-acetaldehyde; IAM, indole-3-acetamide; IPDC, indole-3-pyruvate decarboxylase; Trp, tryptophan.
FEMS Microbiol Rev 31 (2007) 425–448c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
426 S. Spaepen et al.
Pseudomonas putida and Pa. agglomerans (Koga et al., 1991;
Costacurta et al., 1994; Brandl & Lindow, 1996; Patten &
Glick, 2002b). In Azospirillum lipoferum, the ipdC gene is
located on the chromosome (Blaha et al., 2005) but in most
cases the genome localization of this gene has not been
determined. In these organisms, insertional inactivation of
the pathway resulted in a lower IAA production, up to 90%
reduction in Az. brasilense (Prinsen et al., 1993), indicating
the importance of the IPyA pathway in auxin production.
However, no mutants completely abolished in IAA bio-
synthesis could be constructed, indicating redundancy for
IAA biosynthesis pathways.
Tryptamine pathway
In bacteria, the tryptamine (TAM) pathway has been
identified in Bacillus cereus by identification of tryptophan
decarboxylase activity (Perley & Stowe, 1966) and in Azos-
pirillum by detection of the conversion of exogenous trypta-
mine to IAA (Hartmann et al., 1983). In plants tryptamine
was identified as an endogenous compound and genes
encoding for tryptophan decarboxylases (catalyzing the
decarboxylation of tryptophan to tryptamine) have been
cloned and characterized from different plants, indicating
an IAA biosynthetic pathway via tryptamine in plants. The
rate-limiting step for this pathway in plants is probably
catalyzed by a flavin monooxygenase-like protein (YUCCA)
(conversion of tryptamine to N-hydroxyl-tryptamine). The
presence of the intermediates, which are downstream of
N-hydroxyl-tryptamine (presumably indole-3-acetaldoxime
and indole-3-acetaldehyde), still needs to be confirmed (Bak
et al., 2001; Zhao et al., 2001). The last step of this pathway
in bacteria is different to that in plants: in bacteria TAM is
directly converted to IAAld by an amine oxidase (Hartmann
et al., 1983).
Tryptophan side-chain oxidase pathway
Tryptophan side-chain oxidase (TSO) activity has only been
demonstrated in Pseudomonas fluorescens CHA0. In this
pathway tryptophan is directly converted to IAAld bypass-
ing IPyA, which can be oxidized to IAA (Oberhansli et al.,
1991). There are no indications that this pathway exists in
plants.
Indole-3-acetonitrile pathway
The biosynthesis of IAA via indole-3-acetonitrile (IAN) has
been extensively studied in plants in recent years. The last
step in this pathway – the conversion of IAN to IAA by a
nitrilase – was identified by Bartling et al. (1992); the steps
leading to the formation of IAN from tryptophan are still a
matter of debate. Recently, two pathways were suggested for
this formation: one via indolic glucosinolates (glucobrassi-
cin) and another via indole-3-acetaldoxime (Bak et al., 2001;
Zhao et al., 2001). A tryptophan-independent pathway for
the biosynthesis of IAN in plants has been suggested, but not
further examined (Normanly et al., 1993; Bartling et al.,
1994). In bacteria such as Alcaligenes faecalis (Nagasawa
et al., 1990; Kobayashi et al., 1993) nitrilases have been
detected with specificity for indole-3-acetonitrile. In
Ag. tumefaciens and Rhizobium spp., nitrile hydratase and
amidase activity could be identified, indicating the conver-
sion of IAN to IAA via IAM (Kobayashi et al., 1995).
Tryptophan-independent pathway
Analysis of knock-out mutants of Arabidopsis thaliana for
tryptophan biosynthesis (defective in tryptophan synthase
alpha and beta) revealed increased levels of IAA conjugates,
which led to the proposal of a tryptophan-independent
pathway for the biosynthesis of IAA (Last et al., 1991;
Normanly et al., 1993). This pathway branches from in-
dole-3-glycerolphosphate or indole. However, no enzyme of
this pathway has been characterized. The importance (and
existence) of the tryptophan-independent pathway has been
questioned (Muller & Weiler, 2000).
A bacterial tryptophan-independent pathway could be
demonstrated in Az. brasilense by feeding experiments with
labeled precursors. This pathway is predominant in case no
tryptophan is supplied to the medium: 90% of the IAA is
synthesized via the tryptophan-independent pathway, while
0.1% is produced via the IAM pathway (Prinsen et al., 1993).
As no specific enzymes of this pathway have yet been
identified, the existence of this pathway is currently being
reexamined.
Having described these pathways individually, it should
be noted that some bacteria possess more than one pathway.
In Pa. agglomerans for instance genes for the IAM as well as
for the IPyA pathway have been identified (Manulis et al.,
1998). Our present knowledge on IAA biosynthesis in
bacteria dates back to the end of the last century, as reflected
by the list of references. The use of improved analytical
techniques for the detection and quantitation of intermedi-
ates as well as the rapid progress in functional genomics will
undoubtedly provide a more detailed knowledge on the
different IAA biosynthesis pathways present in bacteria.
IAA conjugation and storage
In plants most IAA is found in a conjugated form; only a
small amount of free IAA is present. The role of these
conjugates is diverse: IAA conjugates are involved in trans-
port, storage and protection of IAA from enzymatic degra-
dation. Furthermore, conjugates can control IAA levels in
the cell (homeostatic mechanism) and these compounds can
allow the catabolism of IAA (Cohen & Bandurski, 1982;
Seidel et al., 2006).
FEMS Microbiol Rev 31 (2007) 425–448 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
427Microbial auxin signaling
The only characterized bacterial gene involved in IAA
conjugation is the indole-3-acetic acid-lysine synthetase
(iaaL) from Pseudomonas savastanoi pv. savastanoi. The
gene product converts IAA to IAA-lysine via an ATP-
dependent formation of an amide bond between the carbox-
yl group of IAA and the epsilon amino acid group of lysine.
It is presumed that IAA is released through hydrolysis from
its conjugated form inside the plant tissue by plant enzymes
(Glass & Kosuge, 1986).
In bacteria some intermediates of IAA biosynthesis can be
converted to storage compounds, e.g. the reduction of
indole-3-pyruvate and indole-3-acetaldehyde to indole-3-
lactic acid (ILA) and indole-3-ethanol or tryptophol respec-
tively. The physiological function of these compounds
remains unknown. ILA is inactive as a phytohormone but
it can compete for auxin-binding sites in plants with IAA
(Sprunck et al., 1995).
Genome-wide screening for key genes inIAA biosynthesis
It has been suggested that 80% of rhizosphere bacteria
produce IAA (Patten & Glick, 1996; Khalid et al., 2004).
However, studies on the identification and characterization
of the key genes or proteins involved in IAA biosynthesis are
few and have mostly been directed to one specific gene or
protein of a biosynthetic pathway. Although a vast collection
of IAA-producing strains are available, the identification of
genes or proteins is restricted to a small group of ‘model
organisms,’ e.g. Ag. tumefaciens, Azospirillum, Pa. agglomer-
ans and En. cloacae. Even in these model organisms,
attempts to generate mutants, completely impaired in IAA
production, failed, indicating that IAA is synthesized via
multiple pathways present in a single bacterial species.
To extend the overview of bacteria with the capacity to
produce IAA, publicly available genomes were analyzed
in silico for the presence of IAA biosynthetic genes. In a first
stage well-characterized genes or proteins were selected to
function as bait in a BLAST search (see Table 1). The genes or
proteins involved in the tryptophan-independent and TSO
pathway are unknown and therefore these pathways could
not be searched for. Additionally, the IAN pathway was
excluded from the analysis due to the lack of characterized
genes or proteins of this pathway in bacteria. In the second
phase of the BLAST algorithm (Altschul et al., 1997), the
translated nucleotide sequence or protein sequence of the
baits were used to identify similar proteins in the sequenced
genomes. BLAST results are summarized and interpreted in
Table 2, which shows the distribution of the different path-
ways among the annotated genomes. Of the 369 genomes
sequenced (sequences retrieved from NCBI on 12 September
2006), 57 genome sequences (15.4%) contain genes neces-
sary to synthesize IAA from tryptophan. The presence of
IAA biosynthetic genes in the genome does not imply that
the bacterium is capable of producing IAA: functional
analysis of the genes is still needed to confirm a possible role
in IAA production. The uncoupling between the presence of
IAA biosynthetic genes and IAA biosynthesis is illustrated by
the anaerobic aromatic-degrading denitrifying bacterium
Azoarcus strain EbN1. The genome encodes a phenylpyr-
uvate decarboxylase and other genes that form part of the
IPyA pathway (Rabus et al., 2005). The bacterium, however,
does not produce auxins (neither IAA nor phenylacetic acid)
as such. The IAA biosynthesis genes are involved in the
Ehrlich pathway (degradation of amino acids via transami-
nation, decarboxylation and dehydrogenation), leading to
phenylacetic acid, which can be further catalyzed into
benzoyl-coenzyme A (CoA) or into 3-ketoadipyl-CoA via
an aerobic degradation. Benzoyl-CoA is a central molecule
that can be further metabolized (via aerobic b-oxidation to
acetyl-CoA and succinyl-CoA, which can enter the tricar-
boxylic acid cycle; Rabus, 2005).
Among bacteria for which the genome is sequenced and
available at NCBI, genes encoding for the IPyA pathway are
most abundant, present in 89.5% of the putative IAA-
producing strains. Even human pathogens possess genes
encoding for IAA biosynthesis via IPyA. However, as de-
scribed above for Azoarcus, it is likely that the genes in
Table 1. Selected proteins as bait for BLAST search
Pathway Gene Organism Accession no.
Cut-off BLAST
E value
IAM Tryptophan monooxygenase Agrobacterium tumefaciens C58 AAD30489 1e-60
IAM Indole-3-acetamide hydrolase Agrobacterium tumefaciens C58 AAD30488 1e-50
IPyA Indole-3-pyruvate decarboxylase Enterobacter cloacae P23234 1e-75
IPyA Phenylpyruvate decarboxylase Azospirillum brasilense Sp245 P51852 1e-75
IPyA/TAM Indole-3-acetaldehyde dehydrogenase Ustilago maydis FB1� AAC49575 1e-75
TAM Tryptophan decarboxylase Catharanthus roseus� CAA47898 1e-50
TAM Copper amine/tyramine oxidase Klebsiella aerogenes P49250 1e-75
�Member of the Eukaryota; sequence data for the gene/protein are not available from Bacteria.
IAM, indole-3-acetamide; IPyA, indole-3-pyruvate; TAM, tryptamine.
FEMS Microbiol Rev 31 (2007) 425–448c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
428 S. Spaepen et al.
human pathogenic strains are not involved in IAA produc-
tion. They are part of the Ehrlich pathway, leading to
molecules that can enter different metabolic cycles as energy
source. Similarly, ruminal bacteria and anaerobic bacteria
from the human large intestine are able to metabolize
tryptophan and phenylalanine to indolic and phenolic
compounds such as IAA and phenylacetic acid (Amin &
Onodera, 1997; Smith & Macfarlane, 1997; Mohammed
et al., 2003).
In plant-associated bacteria, both the IAM and the IPyA
pathway are distributed among the sequenced genomes.
Phytopathogenic organisms tend to use the IAM pathway
to produce IAA, whereas beneficial bacteria tend to use the
IPyA pathway. As only five genomic sequences of plant-
associated bacteria that have been annotated encode for one
or more possible IAA biosynthetic pathways, this tendency
needs further confirmation. The capacity to produce IAA is
not as widespread as expected in plant-associated bacteria:
19.2% of the sequenced strains have one or more IAA
biosynthetic pathways encoded in their genome. However,
these data are biased owing to a high number of sequenced
genomes of plant-associated Pseudomonas and Xanthomo-
nas strains (46.2% of the plant-associated bacteria, for
which the genome sequence is publicly available). Only the
genome sequence of Ps. syringae pv. syringae B728a encodes
for a possible IAA biosynthetic pathway.
A remarkable observation is the distribution of the TAM
pathway (identified in 8.8% of the putative IAA-producing
strains): genes encoding enzymes of the TAM pathway were
found in cyanobacteria, and soil and plant-associated bac-
teria (Rhodopseudomonas and Mesorhizobium). Via feeding
experiments, the intermediate TAM was identified in Azos-
pirillum and Bacillus cereus (Hartmann et al., 1983). Pro-
teins similar to tryptophan decarboxylase, involved in the
first step of the TAM pathway, have not yet been identified
and characterized in bacteria.
The production of IAA via IPyA has been described for
cyanobacteria (Sergeeva et al., 2002). As cyanobacteria are
the progenitors of chloroplasts, the evolutionary link be-
tween plant and bacterial IAA biosynthesis becomes rele-
vant. In three cyanobacteria species possible key genes,
involved in IAA biosynthesis, could be determined: both
genes for the IPyA pathway, which is considered as a major
IAA biosynthesis pathway detected in plants, as for the TAM
pathway have been identified.
Factors that influence bacterial IAAbiosynthesis
Reports describing factors that alter the level of IAA
biosynthesis and/or the expression of IAA biosynthesis genes
in bacteria are numerous. However, it is not yet possible to
integrate these factors into a comprehensive regulatory
scheme of IAA biosynthesis in bacteria. This is partly due
to the diversity of IAA expression regulation across IAA
biosynthesis pathways and across bacteria. Furthermore,
there is a lack of integrated studies following IAA expression
in different bacteria and different environments.
This part of the review gives an overview of the different
factors that have been described thus far to modulate IAA
biosynthesis and/or expression of genes involved in IAA
biosynthesis and of the remaining gaps in our understand-
ing of the regulation of IAA biosynthesis in bacteria.
Environmental factors modulating IAAbiosynthesis
A first class of factors influencing IAA biosynthesis in
diverse bacteria is related to environmental stress, including
acidic pH, osmotic and matrix stress, and carbon limitation.
In Az. brasilense IAA production and expression of the key
gene ipdC have been shown to be increased under carbon
limitation, during reduction in growth rate and under acidic
pH (Ona et al., 2003, 2005; Vande Broek et al., 2005).
Interestingly, carbon limitation and reduction in growth
rate are both associated with the entry in the stationary
phase, raising the question of the importance of population
growth status in IAA biosynthesis and, vice versa, the
importance of IAA in cell population behavior. The con-
certed action of these two factors is in agreement with the
observation that overproduction of the stress-related
Table 2. Distribution of IAA biosynthetic pathways in publicly available
annotated genomes of bacteria, including cyanobacteria
Pathway IAM IPyA TAM
Plant-associated bacteria 2/5 2/5 1/5
Plant pathogens 2/2 0/2 0/2
Beneficial bacteria 0/3 2/3 1/3
Soil bacteria� 2/5 3/5 1/5
Human pathogens 0/25 25/25 0/25
Cyanobacteria� 0/3 2/3 2/3
Others� 0/19 19/19 1/19
Total� 4/57 51/57 5/57
The number of publicly available genome sequences was 369 (NCBI
database accession on 12 September 2006). Using the BLAST algorithm,
the baits of Table 1 were used to identify similar genes, using the cut-off
value indicated in Table 1. A biosynthetic pathway was present in a
genome sequence when the E value of all biosynthetic genes leading
from tryptophan to IAA was below the threshold. Using this method,
possible IAA biosynthetic pathways could be identified in 57 bacterial
species.�The total number of IAA biosynthetic pathways is higher than the
number of bacterial species due to redundancy in Burkholderia xenover-
ans LB400, Rhodopseudomonas palustris HaA2 and the cyanobacterium
Anabaena variabilis ATCC 29413. All three organisms have the IPyA
pathway in common, in addition to another pathway.
IAM, indole-3-acetamide; IPyA, indole-3-pyruvate; TAM, tryptamine.
FEMS Microbiol Rev 31 (2007) 425–448 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
429Microbial auxin signaling
stationary-phase sigma factor RpoS enhanced IAA produc-
tion in En. cloacae and Ps. putida (Saleh & Glick, 2001;
Patten & Glick, 2002a).
The pH inducibility of ipdC gene expression in
Az. brasilense seems to be independent of other regulating
environmental factors. This was shown using different
deletions and mutations in the promoter of ipdC (Vande
Broek et al., 2005). An alternative sigma factor, which is
acid-regulated, was suggested to be responsible in passing on
the pH response to ipdC expression (Ona et al., 2003; Vande
Broek et al., 2005). In contrast to Azospirillum, the expres-
sion of the ipdC gene of Pa. agglomerans is not regulated by
pH. In this bacterial species the ipdC gene was shown to be
under control of other environmental stresses: expression
increased 18-fold under low solute and matrix potentials
(Brandl & Lindow, 1997). This effect was not observed for
the expression of ipdC from Az. brasilense (Vande Broek
et al., 2005).
Despite the diversity in stress factors modulating IAA
expression in different bacteria, a common feature can be
identified. The expression of genes involved in IAA bio-
synthesis is fine-tuned to encounter environmental stresses
associated with the soil and plant environment. An acidic
pH is typical for the rhizosphere environment due to proton
extrusion through membranes of root cells. Matrix potential
may be an important condition encountered in the environ-
ment of epiphytic bacteria on plant surfaces such as
Pa. agglomerans.
A second class of factors altering bacterial IAA produc-
tion contains plant extracts or specific compounds and/or
the presence of plant surfaces. In the symbiotic bacterium
Rhizobium sp. strain NGR234, flavonoids, which are pro-
duced by the host plant and accumulate in the rhizosphere,
stimulate IAA production in a NodD1-, NodD2- and
SyrM2-dependent manner. NodD1, NodD2 and SyrM2
form a regulatory cascade that coordinates the expression
of genes under influence of flavonoids (Prinsen et al., 1991;
Theunis et al., 2004). In Xanthomonas axonopodis IAA
production is increased in the presence of plant leaf extracts
from Citrus cinensis. The question as to which specific
compound is responsible for this induction remains unclear
(Costacurta et al., 1998). Similarly, in the phytopathogenic
gram-positive bacterium Rhodococcus fascians IAA produc-
tion is induced by plant extracts. Interestingly, only extracts
from plant tissue infected with R. fascians, the so-called
leavy galls, could upregulate IAA production in R. fascians.
An extract from a mock-inoculated plant had no effect on
IAA biosynthesis (Vandeputte et al., 2005).
The expression of the ipdC gene of Pa. agglomerans is very
low in broth culture and independent from carbon source,
pH, tryptophan availability and O2 availability, but increases
dramatically when the bacterium is grown on plant surfaces,
indicating that a signal from the plant surface is involved
in transcriptional regulation and subsequent IAA pro-
duction. The environmental cues altering ipdC expression
in Pa. agglomerans allow the bacterium to modify its
habitat and exploit plant surfaces (Brandl & Lindow, 1997).
A later study on the spatial pattern of ipdC expression
on plant leaves revealed a heterogeneous distribution of
expression along the surface environment, suggesting that
the leaf surface significantly varies over a small scale (Brandl
et al., 2001). These results indicate that IAA production
by bacteria can be remarkably different within a micro-
environment.
An important molecule that can alter the level of IAA
synthesis is the amino acid tryptophan, identified as the
main precursor for IAA and thus expected to play a role in
modulating the level of IAA biosynthesis. The application of
exogenous tryptophan was shown to increase strongly the
IAA production in various bacteria, for example Azospir-
illum, Pa. agglomerans, Ps. putida and Rhizobium (Prinsen
et al., 1993; Brandl & Lindow, 1996; Patten & Glick, 2002b;
Theunis et al., 2004). In both Az. brasilense Sp7 as in
Ps. putida GR12-2 the presence of tryptophan increased the
expression level of the key enzyme ipdC, three- to four-fold
and five-fold, respectively (Zimmer et al., 1998; Patten &
Glick, 2002b). In the rhizosphere tryptophan can originate
from two sources: released from degrading root and micro-
bial cells and from root exudates. Support for the first source
is the observation that IAA is produced during the late
exponential and stationary phase in Azospirillum (Omay
et al., 1993). The amount of tryptophan in plant root
exudates can vary strongly among plant species (Kravchen-
ko et al., 2004). Although the amount of tryptophan in root
exudates is rather low (Kravchenko et al., 1991), exogenous
tryptophan can be efficiently absorbed by bacteria. As the
Km value, an indicator of the affinity of the transporter for
tryptophan, is low, the affinity of bacteria for tryptophan is
high, which makes them good scavengers for tryptophan.
This results in efficient transport into the cells, even at low
concentration (Marlow & Kosuge, 1972).
Besides the role of the precursor tryptophan in the
regulation of IAA production, IAA biosynthesis can also be
modulated by the end-product IAA and by its intermediates.
In Ps. savastanoi pv. savastanoi the activity of the first
enzyme in the IAM pathway, IaaM, is controlled through
feedback inhibition by IAM and IAA (Hutcheson & Kosuge,
1985). Curiously, while tryptophan stimulates IAA produc-
tion in Azospirillum, anthranilate, a precursor for trypto-
phan, reduces IAA synthesis. By this mechanism IAA
biosynthesis is fine-tuned because tryptophan inhibits an-
thranilate formation by a negative feedback regulation on
the anthranilate synthase, resulting in an indirect induction
of IAA production (Hartmann & Zimmer, 1994).
Interestingly, nitrile hydratase, which catalyzes the first
step in the conversion of IAN to IAA in Ag. tumefaciens, is
FEMS Microbiol Rev 31 (2007) 425–448c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
430 S. Spaepen et al.
induced by its product IAM, indicating a positive feedback
regulation in IAA biosynthesis (Kobayashi et al., 1995).
Another positive feedback mechanism has been described
for Az. brasilense Sp245 by Vande Broek et al. (1999). The
level of IAA production by Az. brasilense increases during
growth, with highest levels of IAA production during the
stationary phase (Prinsen et al., 1993). The expression of the
key gene for IAA production in the presence of significant
amounts of tryptophan, the indole-3-pyruvate decarboxy-
lase gene, increases with the cell density and reaches its
maximum at stationary phase. Cell density-dependent in-
duction of genes can be mediated by small diffusible signal
molecules. Surprisingly, in Az. brasilense Sp245 IAA itself is
responsible for the increase in expression by induction of the
ipdC gene (Vande Broek et al., 1999). Other auxins such as
naphataleneacetic acid and phenylacetic acid (Somers et al.,
2005) also upregulate the expression. Compounds that had
no effect on the expression were IAA conjugates, indole-3-
butyric acid and tryptophan, although IAA production is
enhanced by tryptophan. In contrast to negative control of
the transcription of biosynthetic genes by the end-product,
the positive feedback regulation or autoinduction by IAA is
rather unusual (Vande Broek et al., 1999). The ipdC gene of
Az. brasilense was the first discovered bacterial gene that is
induced by auxins, indicating that proteins responsible for
IAA perception and signal transduction are present in
bacteria.
Genetic factors
Different genetic factors have been described to affect the
level of IAA biosynthesis in bacteria. Firstly, the location of
auxin biosynthesis genes in the genome, either plasmid or
chromosomal, has been shown to modulate the level of IAA
production. Plasmids are generally present in various copies
in the bacterial cell, providing a higher number of IAA
biosynthesis genes that can be transcribed as compared with
IAA biosynthesis genes that are located on the chromosome
(Brandl & Lindow, 1996; Patten & Glick, 1996). In
Ps. savastanoi pv. savastanoi the biosynthetic genes are
located on a plasmid, whereas in Ps. syringae pv. syringae
the homologous genes are encoded on the chromosomal
DNA. In the latter species, a much smaller amount of IAA is
produced. When this Pseudomonas strain is equipped with a
low-copy plasmid, encoding the IAA operon, IAA produc-
tion is increased four-fold (Mazzola & White, 1994), in-
dicating the importance of either the location or the copy
number or both of the encoding genes.
Secondly, the mode of expression, constitutive vs. in-
duced, of IAA biosynthesis genes was observed to differ
across biosynthesis pathways and across bacterial species. In
Ag. tumefaciens and Agrobacterium rhizogenes the region of
the Ti plasmid containing iaaM and iaaH is transferred and
integrated into the plant genome. The genes are expressed
under control of strong constitutive promoters, resulting
in the production of high levels of IAA inside the plant
tissue (reviewed by Costacurta & Vanderleyden, 1995). In
Ps. fluorescens CHA0 the IPyA pathway is presumably
constitutive, while the TSO pathway is only active during
the stationary phase (Oberhansli et al., 1991).
Furthermore, two transcriptional regulators, RpoS –
which regulates the transcription of genes in response to
stress conditions and starvation – and the two-component
system GacS/GacA – which controls the expression of genes
of which many are induced during a late logarithmic growth
phase and have a role in maintaining the competitiveness of
the bacterium in the rhizosphere – have been shown to
interact with the expression level of IAA biosynthesis genes.
The ipdC genes show a typical stationary-phase-dependent
expression. IAA production starts in the late logarithmic to
early stationary phase and the promoter region of some
ipdC genes contains a sequence similar to the consensus
sequence recognized by RpoS. In Ps. putida GR12-2 and
Pa. agglomerans ipdC expression is regulated by RpoS
(Brandl et al., 2001; Patten & Glick, 2002a): the ipdC
promoter region contains elements similar to the RpoS
recognition sequence, and mutants, carrying constitutively
expressed rpoS on a plasmid, produce IAA earlier at con-
sistently elevated levels as compared with wild-type cells
(Patten & Glick, 2002a). In Azospirillum species RpoS is not
present (RpoS is not detected in Alphaproteobacteria). In
this case alternative sigma factors, RpoN and possibly RpoH,
regulate IAA expression (Gysegom, 2005).
In Pseudomonas chlororaphis, the GacS/GacA two-com-
ponent system acts as a negative regulator of the trypto-
phan-dependent IAA biosynthesis (Haas & Keel, 2003; Kang
et al., 2006). The involvement of RpoS and GacS in IAA
production was further confirmed by overexpression of the
rpoS and gacS genes of Ps. fluorescens in two En. cloacae
strains (Saleh & Glick, 2001).
Bacterial IAA in plant--microorganisminteractions
With the analysis of further bacterial species, the role of
bacterial IAA in plant–microorganism interactions appears
to be diverse. Previously, bacterial auxin production was
mainly associated with pathogenesis, specifically with bac-
terial gall formation. However, it became apparent that
many of the phytopathogenic (not only gall-inducing) as
well as plant growth-promoting bacteria have the ability to
synthesize IAA. The broad distribution of IAA biosynthesis
genes across bacteria, the existence of different metabolic
pathways and the diversity in outcomes on the plant side
when IAA-synthesizing bacteria interact with plants, evoke
the question as to why bacteria produce IAA. Except for a
FEMS Microbiol Rev 31 (2007) 425–448 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
431Microbial auxin signaling
few cases, the link between IAA synthesis and plant, pheno-
type has not been demonstrated or at least remains ambig-
uous. This part of the review aims to give an insight into
bacterial IAA production as one of different strategies to
seduce the plant partner.
Factors steering the outcome of the interactionbetween IAA-producing bacteria and plants
IAA is the main auxin in plants, controlling many important
physiological processes including cell enlargement and
division, tissue differentiation, and responses to light and
gravity (Taiz & Zeiger, 1998; Woodward & Bartel, 2005;
Teale et al., 2006). Bacterial IAA producers interacting with
plants have the potential to interfere with any of these
processes by changing in a spatiotemporal way the plant
auxin pool. The impact of exogenous auxin on plant
development ranges from positive to negative effects. The
consequence for the plant is usually a function of (1) the
amount of IAA produced that is available to the plant and
(2) the sensitivity of the plant tissue to changes in IAA
concentration.
The optimal IAA concentration range for a given plant
phenotype may be extremely narrow, as demonstrated by
the isolation of a plant growth-promoting Ps. putida strain
producing IAA and of a plant growth-inhibiting IAA over-
producer mutant producing only four times the amount of
IAA synthesized by the wild-type strain (Xie et al., 1996;
Persello-Cartieaux et al., 2003). Similarly, inoculation with
Pseudomonas thivervalensis, an IAA-producing strain, re-
sulted in reproducible morphological changes of Arabidopsis
roots without effects on plant growth when inoculated at
a concentration of 105 CFU mL�1, and in inhibition of
plant growth when inoculated at concentrations above
106 CFU mL�1 (Persello-Cartieaux et al., 2001). It cannot,
however, be excluded that other factors (e.g. metabolites)
than auxin production contribute to the inhibitory effect of
highly concentrated inoculants. The use of mutant and
transgenic strains and the analysis of inoculant supernatants
are necessary to distinguish further the role of different
bacterial factors in the diverse outcome on the plant side
upon inoculation with increasing concentrations of bacterial
cells.
The actual concentration of bacterial IAA available to
the plant is, in part, contingent upon the physical relation-
ship between the two organisms. Whereas bacterial phyto-
pathogens infect their host plants and, in the peculiar case
of agrobacteria transform plant cells, beneficial bacteria
(symbionts excepted) appear to exert their effect predomi-
nantly while colonizing the external surface of a plant
(Patten & Glick, 1996). However, as more plant tissues are
analyzed for the presence of bacteria, an increasing number
of IAA-producing plant growth-promoting rhizobacteria
(PGPR) are detected inside the plant tissue (Rosenblueth &
Martinez-Romero, 2006).
The pathway used by the plant-associated bacterial strain
to synthesize IAA may further affect the outcome on the
plant side. Phytopathogenetic symptoms are mostly linked
to the IAM pathway, which is generally believed to be a
specific microbial pathway (see above). However, as men-
tioned above, there is growing evidence for the presence of
an IAM pathway in Arabidopsis thaliana (Piotrowski et al.,
2001; Pollmann et al., 2002, 2003, 2006). As plants may not
generally possess the metabolic intermediates or pools of
this pathway, they may not be able to maintain IAA at
nontoxic or physiologically appropriate levels in their tissues
by feedback regulation (Persello-Cartieaux et al., 2003). In
contrast, the biosynthesis of IAA via indole-3-pyruvic acid
and indole-3-acetaldehyde is predominant in higher plants
and is observed in pathogenic as well as in nonpathogenic
bacteria. Most beneficial bacteria produce IAA via the IPyA
pathway. This view of an IAM route linked to pathogenesis
and an IPyA pathway involved in epiphytic and rhizosphere
fitness of the bacteria has been supported through the
generation of mutants of Pa. agglomerans pv. gypsophilae, a
gall-inducing bacterium, in which the different biosynthetic
pathways were disrupted individually or in combination.
Inactivation of the IAM pathway caused the largest reduc-
tion in gall size whereas inactivation of the IPyA pathway
caused a minor, nonsignificant decrease in pathogenicity.
The reverse effect was observed for epiphytic fitness: inacti-
vation of the IAM pathway did not affect colonization
capacity whereas inactivation of the IPyA pathway did
(Manulis et al., 1998). The difference in function of the two
IAA biosynthesis pathways in Pa. agglomerans was asso-
ciated with differences in expression profiles of the corre-
sponding genes. The ipdC gene showed enhanced expression
during colonization while iaaM was upregulated during
later phases of the plant–microorganism interactions, in-
cluding gall formation. However, it was also reported that
cranberry stem gall is induced by multiple opportunistic
bacteria, including Pa. agglomerans strains, which produce
IAA exclusively through the IPyA pathway. These data
indicate that pathogenesis is not necessarily associated with
the IAM pathway for IAA biosynthesis (Vasanthakumar &
McManus, 2004). Furthermore, some nonpathogenic sym-
biotic bacteria including Rhizobium synthesize IAA mainly
through the IAM pathway (Theunis et al., 2004).
In addition to the amount of bacterial auxin produced,
the contrasting effects of IAA on plant development are
linked to the sensitivity of the host itself. The study of the
effect of auxin-producing bacterial strains on wheat (Kucey,
1988), blackcurrant and sourcherry softwood (Dubeikovsky
et al., 1993) highlights the cultivar specificity of the response
of plants to bacteria. More extremely, auxin-resistant mu-
tants of Arabidopsis are insensitive to Ps. thivervalensis
FEMS Microbiol Rev 31 (2007) 425–448c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
432 S. Spaepen et al.
colonization while the same inoculation on wild-type plants
induces changes in root morphology (Persello-Cartieaux
et al., 2001). The host plant can also take an active part in
the regulation of microbial IAA biosynthesis. Transcription
of ipdC in Pa. agglomerans pv. gypsophilae is induced in
response to bean and tobacco compounds, the structure of
which is not yet known (Brandl & Lindow, 1997). As the
ability to produce auxins from tryptophan is common
among soil organisms, it is tempting to assume that bacterial
auxin is in part dependent upon plant-exuded tryptophan
(Persello-Cartieaux et al., 2003). Bar and Okon proposed
that IAA biosynthesis may contribute to bacterial survival in
the rhizosphere by detoxification of plant-exuded trypto-
phan. They observed that addition of 10 mM (1.4 mg mL�1)
or more of tryptophan strongly inhibited growth of Az.
brasilense Sp7. Instability of tryptophan, variation in exuda-
tion across plant species and environments, and the lack of
comprehensive studies on exudation in the rhizosphere
complicate and currently impede linking this observation
of microbial growth inhibition by tryptophan (Bar & Okon,
1992, 1993) with actual rhizosphere activities.
IAA in gall formation
Tumor- and gall-inducing bacteria were the first plant-
associated bacteria in which IAA biosynthesis pathways were
studied because of the suspected role of microbially released
auxins in disturbing plant morphology and development
(Morris, 1995). Agrobacterium tumefaciens, Ag. rhizogenes,
Ps. savastanoi and Pa. agglomerans pv. gypsophilae all possess
the IAM pathway, and the DNA sequence of the iaaM gene
suggests a common evolutionary origin (Morris, 1995). In
all of these bacteria, IAA is involved in pathogenesis.
However, depending on the bacterial species, the mechan-
ism of either gall or tumor formation and the role of IAA
therein differ. Tumor formation induced by Ag. tumefaciens
involves transfer of T-DNA from the bacteria into the host
genome of infected cells. This T-DNA possesses genes (iaaM
and iaaH) encoding the IAM pathway for IAA biosynthesis
(Zambryski, 1992; Zupan et al., 2000). The overproduction
of auxin and cytokinin by the transformed plant cells results
in the typical crown gall or tumor. Other transferred genes
encode enzymes involved in the synthesis of amino acid and
sugar derivatives, the opines, which the strain of Agrobacter-
ium that induces the tumor can use as a source of carbon,
nitrogen and energy. Removing the Ti phytohormone
biosynthesis genes from Agrobacterium abolishes tumor
formation, demonstrating that the IAA as well as the
cytokinins synthesized by the enzymes encoded by the
transferred bacterial oncogenes are essential for tumor
formation. It was proposed by Aloni et al. (1995) that the
high IAA concentrations induced by the enzymes encoded
by the T-DNA genes iaaM and iaaH stimulate ethylene
synthesis in the crown gall. The tumor-induced ethylene
would be the controlling signal that induces narrowing of
the vessels in the host adjacent to the tumor, which, in turn,
substantially limits water and nutrient supply to the shoot
organs above the tumor (Aloni et al., 1995).
The mechanism of gall formation by Pa. agglomerans
pv. gypsophilae on Gypsophila is fundamentally different
from that by Ag. tumefaciens. Gall formation by Pa. agglom-
erans pv. gypsophilae does not involve transfer of DNA into
host cells (Clark et al., 1989); therefore, it requires the
constant presence of living bacteria, in contrast to the
situation with Ag. tumefaciens. Pathogenic and nonpatho-
genic strains of Pa. agglomerans possess chromosomal genes
encoding the IPyA pathway for IAA biosynthesis, whereas
only pathogenic strains carry the genes encoding the IAM
pathway on the pPATHPag plasmid (Clark et al., 1993;
Brandl & Lindow, 1996; Manulis et al., 1998). Inactivation
of the IPyA pathway in Pa. agglomerans pv. gypsophilae did
not affect gall formation significantly, while mutations in
genes of the IAM pathway substantially reduced the gall size
but did not eliminate gall initiation (Clark et al., 1993;
Manulis et al., 1998). This demonstrates that in Pa. agglom-
erans pv. gypsophilae bacterial IAA synthesized by the IAM
pathway contributes to obtain optimal gall size but is not
essential for gall induction. It has been shown that the
translocation of effectors of the type III secretion system
(TTSS) into the host cells is the key factor for gall induction
by Pa. agglomerans pv. gypsophilae (Mor et al., 2001; Manulis
& Barash, 2003; Nizan-Koren et al., 2003). Similar to tumor
development induced by Ag. tumefaciens, an increase in
ethylene is associated with bacterial IAA production in gall
formation induced by Pa. agglomerans pv. gypsophilae
(Chalupowicz et al., 2006).
IAA in plant growth-promoting rhizobacteria
For various PGPR, it has been demonstrated that enhanced
root proliferation is related to bacterial IAA biosynthesis.
Studies with Azospirillum mutants altered in IAA produc-
tion support the view that increased rooting is caused by
Azospirillum IAA synthesis (Dobbelaere et al., 1999). This
increased rooting enhances plant mineral uptake and root
exudation, which in turn stimulates bacterial colonization
and thus amplifies the inoculation effect (Dobbelaere et al.,
1999; Lambrecht et al., 2000; Steenhoudt & Vanderleyden,
2000). Furthermore, the dose–response curve of roots to
cultures with increasing concentrations of Azospirillum
nicely fits the dose–response curve of roots to increasing
concentrations of IAA (Fallik et al., 1994). However, in
several studies it was shown that Azospirillum IAA
biosynthesis alone cannot account for the overall plant
growth-promoting effect observed. Therefore, the ‘additive
hypothesis’ was suggested to explain growth and yield
FEMS Microbiol Rev 31 (2007) 425–448 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
433Microbial auxin signaling
promotion with Azospirillum, postulating that growth pro-
motion is the result of multiple mechanisms (phytohor-
mone biosynthesis, nitrogen fixation, among others)
working together (Bashan & Holguin, 1997).
Similarly, the capacity of Ps. putida GR12-2 to stimulate
root elongation was shown to be related to its production of
IAA but production of IAA alone does not account for
growth promotion, as evidenced by a study with IAA-
overproducing mutants (Xie et al., 1996). In their study
transposon mutagenesis was used to isolate seven mutants
that overproduced IAA in comparison with the wild-type.
Interestingly, six of the seven mutants, including one which
produced IAA at three times the rate of GR12-2, elicited root
elongation at a level statistically equivalent to GR12-2. This
finding demonstrates that for GR12-2 inoculation there is
not a direct relationship between the level of IAA produced
and the magnitude of root elongation. One mutant that
produced IAA at four times the rate of GR12-2 lost root
elongation activity. This was explained by the interaction of
IAA with the enzyme 1-aminocyclopropane-1-carboxylate
(ACC) synthase. Large amounts of IAA produced by bacter-
ia together with endogenously produced plant IAA activate
ACC synthase, leading to production of ACC, a precursor
for ethylene. Ethylene is an inhibitor of root growth espe-
cially reducing the primary root length (Glick et al., 1998;
Glick, 2005). A key feature of GR12-2 is that the bacterium
produces ACC deaminase that converts ACC to ammonia
and a-ketobutyrate. This conversion lowers the pool of ACC
in the root environment, thereby reducing the amount of
ethylene and consequently its inhibition of root growth. The
bacterium takes advantage of this situation using ACC as a
source of nitrogen. Thus, a balanced interplay of different
factors including bacterial IAA biosynthesis rather than IAA
production per se is needed to stimulate plant growth (Xie
et al., 1996).
IAA in Rhizobium --legume symbiosis
Most Rhizobium species have been shown to produce IAA
via different pathways (Badenochjones et al., 1983; Theunis
et al., 2004), and many studies indicate that changes in auxin
balance in the host plant are a prerequisite for nodule
organogenesis (Mathesius et al., 1998). Auxins are involved
in multiple processes including cell division, differentiation
and vascular bundle formation. These three events are also
necessary for nodule formation as such. It seems likely that
auxins play a role in nodulation. Nevertheless, the exact role
of IAA in the different stages of Rhizobium–plant symbiosis
remains unclear.
A number of experiments suggest that rhizobia, in
particular Rhizobium Nod factors (lipo-chitin oligosacchar-
ides, which are produced by rhizobia upon triggering nod
gene expression by plant-derived flavonoids) interfere with
auxin transport. Moreover, the application of synthetic
polar auxin transport (PAT) inhibitors, which interfere with
the hormone balance in the root, can induce pseudonodule
structures on the root (Allen et al., 1953; Hirsch et al., 1989;
Wu et al., 1996). Direct measurements of auxin transport
using radiolabeled auxin showed that rhizobia locally inhibit
acropetal auxin transport capacity in vetch (Vicia sativa)
roots within 24 h after inoculation (Boot et al., 1999). In
addition, the expression of an auxin-responsive promoter
(GH3) was reduced acropetally from the inoculation site,
between 12 and 24 h following rhizobia inoculation or
addition of Nod factors (Mathesius et al., 1998). This was
followed by an apparent increase in auxin accumulation at
the site of nodule initiation in the inner cortex. By contrast,
in the determinate legume Lotus japonicus, no auxin trans-
port inhibition could be measured after inoculation (Pacios-
Bras et al., 2003). However, an increase of GH3 expression
was still located in the nodule initials, suggesting that high
auxin levels are required for nodule initiation. Moreover, it
has been suggested that the expression of the AUX-1-like
protein LAX in developing Medicago truncatula nodule
primordia is important for a continuous flow of auxin into
the forming primordium (de Billy et al., 2001). It is therefore
likely that auxin transport regulation is part of the process
leading to nodule initiation.
Very recently, it has been demonstrated that a cytokinin
receptor (HIT1 gene in L. japonicus) is involved in the Nod
factor signaling cascade in Lotus as well as in Medicago
(Murray et al., 2007; Tirichine et al., 2007). As the effect of
cytokinins is dependent on the auxin/cytokinin balance,
these findings may give a new dimension to the role of auxin
in nodulation.
Other observations suggest that microbially released IAA
could play a role in rhizobia–plant symbiosis. It was
demonstrated that the specific nod inducers, flavonoids, also
stimulate the production of IAA by Rhizobium (Prinsen
et al., 1991). Moreover, in Rhizobium sp. NGR234 this
flavonoid induction is dependent on the transcriptional
regulators NodD1 and NodD2 and the presence of the nod-
box NB15 upstream of the y4wEFG operon required for IAA
synthesis (Theunis et al., 2004). The link between Nod
factors as symbiotic signaling molecules and rhizobial IAA
biosynthesis points to a role for IAA in the Rhizobium–bean
symbiosis. Knocking-out the flavonoid-dependent IAA bio-
synthesis in NGR234 did not affect nodulation significantly
on Vigna and Tephrosia, although preliminary results with
Lablab have shown NGR234 to be Fix1 while the IAA�
mutants are Fix� (Theunis, 2005).
In addition, root nodules have been shown to contain
more IAA than nonnodulated roots (Dullaart & Duba, 1970;
Badenochjones et al., 1983; Basu & Ghosh, 1998; Theunis,
2005; Ghosh & Basu, 2006), and auxins could be important
for maintaining a functional root nodule (Badenochjones
FEMS Microbiol Rev 31 (2007) 425–448c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
434 S. Spaepen et al.
et al., 1983). In nodules induced by low IAA-producing
mutants of Rhizobium sp. NGR234, the IAA content is
lower than in nodules induced by the wild-type strain,
indicating that at least part of the IAA in nodules derives
from the bacteria (Theunis, 2005). Bacteroids of plants
inoculated with mutant Bradyrhizobium japonicum strains
overproducing IAA contain high amounts of IAA in com-
parison with wild-type bacteroids (Hunter, 1989), support-
ing the hypothesis that increased IAA biosynthesis in
nodules is of prokaryotic origin. Moreover, introduction of
a second synthesis pathway for auxin synthesis in rhizobia
led to larger nodules, an increase in nodule acetylene
reduction ability and a delay in nodule senescence (Camer-
ini et al., 2004). Similarly, a mutant of Br. japonicum that
produces 30-fold more IAA than the wild-type strain
showed higher nodulation efficiency (Kaneshiro & Kwolek,
1985).
The application of exogenous IAA can enhance nodula-
tion on Medicago and Phaseolus vulgaris when added at very
low concentrations (up to 10�8 M) to the medium, while
high concentrations inhibit nodulation (Plazinski & Rolfe,
1985; van Noorden et al., 2006). Combined application of
Rhizobium and Azospirillum can also increase nodulation as
demonstrated in several studies (Okon & Itzigsohn, 1995;
Burdman et al., 1996). Similar to the IAA effect on nodula-
tion, the effect of Azospirillum is dependent on the concen-
tration of the inoculum. Better root development induced
by Azospirillum IAA production could explain the increased
nodulation but also additional factors, such as changes in
flavonoid metabolism, were suggested to be involved (Burd-
man et al., 1996).
IAA in phytopathogenesis, other than gall- ortumor-inducing pathogens
Besides gall- and tumor-inducing plant pathogens, other
phytopathogens like various Ps. syringae species also synthe-
size IAA (Glickmann et al., 1998; Buell et al., 2003).
Infection can result in necrotic lesions often surrounded by
chlorotic halos. Here, the role of IAA in disease development
is not clear. In many bacterial pathogens, the hrp-gene-
encoded type III secretion system that directly translocates
effector proteins into the eukaryotic host cells is key to
pathogenesis and the development of disease symptoms (Jin
et al., 2003; He et al., 2004). Recently, a link between the
TTSS and phytohormone production in the plant pathogen
Ralstonia solanacearum was established through a host
responsive regulator of the TTSS activation cascade, HrpG
(Valls et al., 2006). It was shown that HrpG controls, in
addition to that of the TTSS, the expression of a previously
undescribed TTSS-independent pathway that includes a
number of other virulence determinants and genes probably
involved in adaptation to life in the host and which includes
IAA and ethylene biosynthesis genes. These results provide a
new, integrated view of plant bacterial pathogenicity, in
which a common regulator activates synchronously upon
infection the TTSS, other virulence determinants and a
number of adaptive functions which act cooperatively to
cause disease. In Ps. syringae the presence of a functional
Hrp promoter upstream of the iaaL gene involved in IAA
biosynthesis further supports the role for IAA production in
virulence (Fouts et al., 2002). However, the function of
bacterial IAA in pathogenesis and disease development
remains unclear. New intriguing insights have come from
studies related to plant defense.
IAA in plant defense
Plants respond to bacterial infection using a two-branched
innate immune system. The first branch, the basal defense,
recognizes and responds to molecules common to many
classes of microorganisms, including nonpathogens. The
interaction between pathogen/microbial-associated molecu-
lar patterns (PAMPs/MAMPs) and the plant pathogen
recognition receptors (PRRs) play a key role in activation of
the plant basal defense response (Abramovitch et al., 2006).
The second branch, the hypersensitive response (HR)-based
response, responds to pathogen virulence factors, either
directly or through their effects on host targets. This second,
later response is mediated by plant disease resistance genes
(R genes) (for recent reviews see Abramovitch et al., 2006;
Chisholm et al., 2006; Jones & Dangl, 2006). Navarro et al.
(2006) demonstrated a link between auxin signaling in plants
and resistance to bacterial pathogens. Bacterial PAMPs
downregulate auxin signaling in Arabidopsis by targeting
auxin receptor transcripts, as part of a plant-induced im-
mune response. It has been observed previously that bacterial
quorum sensing (QS) molecules such as N-acylhomoserine
lactones downregulate some auxin-induced genes (Mathe-
sius et al., 2003), raising the question of whether other
bacterial molecules recognized by the plant also decrease
auxin signaling. The results of Navarro et al. (2006) suggest
that lowering plant auxin signaling can increase resistance to
bacterial pathogens. A possible mechanism is the expression
of auxin-repressed plant defense genes (online supplemen-
tary material to Navarro et al., 2006). They further showed
that exogenous application of auxin enhances susceptibility
to the bacterial pathogen. These findings allow us to
hypothesize that bacterial IAA production may contribute
to circumvent the host defense system by derepressing auxin
signaling. In this way, IAA biosynthesis may play an im-
portant role in bacterial resistance and colonization on the
plant (Remans et al., 2006; see Fig. 2).
Different reports supporting this hypothesis for interac-
tions between plants and pathogenic as well as beneficial
bacteria can be cited.
FEMS Microbiol Rev 31 (2007) 425–448 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
435Microbial auxin signaling
Firstly, it has been observed in various studies, including
high-throughput analyses, that plant-colonizing bacteria
upregulate auxin signaling in plants. An overview of these
reports is given in Table 3. For some of the bacterial strains
used in these studies, it has been described that they possess
IAA biosynthesis genes. However, it has not yet been proven
that bacterial IAA effectively contributes to modulation of
auxin signaling as observed in the plant. Thilmony et al.
(2006) reported that the upregulation of IAA signaling in
Arabidopsis upon colonization of Ps. syringae DC3000 is
partially dependent on the TTSS and the phytotoxin cor-
otanine, which contributes to virulence and disease devel-
opment. This may be linked to the presence of a functional
Hrp promoter upstream of the iaaL gene involved in IAA
production in Ps. syringae, which suggests that expression of
IAA biosynthesis genes is dependent on the Hrp system
(Fouts et al., 2002). However, the use of an IAA mutant
strain is essential to unravel the role of bacterial IAA in
modulating plant IAA signaling upon colonization.
Secondly, it has been observed previously that auxins
interfere with parts of the host defense system. Auxins and
cytokinins are able to block several pathogenesis-related
(PR) enzymes, including b-glucanase (Mohnen et al., 1985;
Jouanneau et al., 1991) and chitinase (Shinshi et al., 1987) at
the mRNA level. Furthermore, comparing gene expression
profiles of Arabidopsis plants treated with IAA vs. nontreated
control plants using the Affymetrix ATH1 Gene Chip
revealed that some proteins of the disease resistance respon-
sive family are downregulated in IAA-treated plants (Red-
man et al., 2004).
Another plant defense strategy is the HR, characterized
by necrosis of plant cells in the inoculated area. In this way,
the bacteria are limited to the area originally inoculated
and do not spread beyond its edges. The HR is thought to
function in the limitation of the growth of the microorgan-
ism and is therefore associated with disease resistance
(Klement, 1982). Robinette & Matthysse (1990) reported
that bacterial auxin of Ps. savastanoi is required to block
the HR in tobacco leaves induced by Ps. syringae pv.
phaseolicola.
Interestingly, the role of bacterial IAA in induced system
resistance (ISR) elicited by some PGPR has been investigated
in several studies but in none of the reported studies did it
appear that IAA was involved in ISR (Oberhansli et al., 1991;
Beyeler et al., 1999; Cartieaux et al., 2003; Suzuki et al.,
2003). Verhagen et al. (2004) showed that ISR in Arabidopsis
Fig. 2. IAA in pathogenic and beneficial microorganism–plant interactions. A model suggested for the role of bacterial IAA production in
microorganism–plant interactions. Signaling taking place in the plant is indicated in gray boxes; signaling taking place in bacterial cells is indicated in
white boxes. Full lines indicate demonstrated links; dashed lines indicate hypothesized links. AHL, N-acyl homoserine lactone; IAA, indole-3-acetic acid;
MAMP, microbial-associated molecular pattern; PAMP, pathogen-associated molecular pattern; QS, quorum sensing; TIR, transport inhibitor response.
FEMS Microbiol Rev 31 (2007) 425–448c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
436 S. Spaepen et al.
induced by Ps. fluorescens WCS417r is not associated with
changes in the expression of genes encoding PR proteins.
Taking these reports together, they show consistency for the
link between auxin signaling and PR protein expression.
Thirdly, it has been shown that bacterial IAA biosynthesis
contributes to colonization capacity and fitness on the host.
A low IAA-producing mutant of Ps. fluorescens HP72 is
reduced in colonization ability on bentgrass roots as com-
pared with the wild-type (Suzuki et al., 2003). Similarly,
inactivation of the IPyA pathway in Pa. agglomerans
pv. gypsophilae caused a 14-fold reduction in the population
of Pa. agglomerans pv. gypsophilae on bean plants. In-
activation of the IAM pathway did not affect the coloniza-
tion ability (Manulis et al., 1998). An iaaM� mutant of
Ps. syringae pv. syringae retained the ability to colonize the
bean phylloplane but the role of the IPyA pathway in
colonization was not studied in this strain (Mazzola &
White, 1994). Taken these results together, they suggest that
the IPyA biosynthesis pathway rather than the IAM pathway
contributes to colonization capacity.
Studies of bacterial gene expression further support the
importance of bacterial IAA biosynthesis during plant
colonization, demonstrating that the ipdC gene in
Pa. agglomerans pv. gypsophilae is strongly induced during
plant colonization (Manulis et al., 1998; Brandl et al., 2001),
while the iaaM gene is expressed during later stages in the
interaction with the plant (Manulis et al., 1998). Erwinia
chrysanthemi 3937 in interaction with spinach leaves shows
an upregulation of iaaM expression (Yang et al., 2004).
Examining the influence of sugarbeet exudates on the
transcriptome of Ps. aeruginosa PAO1, no specific IAA
biosynthesis genes were found to be induced. However, a
tryptophan permease with tryptophan being a precursor for
IAA was strongly upregulated, independently from the
sugarbeet variety (Mark et al., 2005).
It is logical to postulate that bacteria use IAA as part of
their colonization strategy by stimulating proliferation of
plant tissues and thus enhanced colonization surface and
exudation nutrients for bacterial growth (Fig. 2). The link
between plant defense and auxin signaling gives an extra
dimension to the role of bacterial IAA in colonization
ability.
Taking the reports described above together, auxin signal-
ing seems to interfere with the basal defence (PAMP
recognition) as well as with the R-mediated defence re-
sponse (HR, activation of PR proteins). As proposed by
Abramovitch et al. (2006), the distinctions that are made
between basal and R-protein-mediated defenses might need
to be revised given that the PRR–PAMP interaction shares
many properties with avirulence (Avr)–R protein interac-
tions. In addition, the basal and R-protein-mediated defense
pathways might share some common signaling components
or physical defense mechanisms. For example, HR-likeTab
le3.
Exam
ple
sof
modula
tion
inpla
nt
auxi
nsi
gnal
ing
upon
colo
niz
atio
nby
pla
nt-
asso
ciat
edbac
teria
(pat
hogen
icas
wel
las
PGPR
)
Bac
terial
stra
inuse
dH
ost
pla
nt
Met
hodolo
gy
IAA
-rel
ated
signal
ing
induce
dupon
bac
terial
colo
niz
atio
nRef
eren
ce
Xan
tom
onas
cam
pes
tris
pv.
cam
pes
tris
(pat
hogen
)A
rabid
opsi
sM
easu
rem
ent
of
horm
one
leve
lsin
pla
nt
tiss
ue
upon
inocu
lation
Acc
um
ula
tion
of
IAA
inA
rabid
opsi
sle
aves
upon
inocu
lation
O’D
onnel
let
al.(2
003)
Pseu
dom
onas
syringae
pv.
tom
ato
DC
3000
(pat
hogen
)
Ara
bid
opsi
sSi
multan
eous
anal
ysis
of
phyt
ohorm
ones
,phyt
oto
xins
and
vola
tile
com
pounds
inpla
nts
Acc
um
ula
tion
of
IAA
inA
rabid
opsi
sle
aves
toget
her
with
accu
mula
tion
of
coro
tanin
e,sa
licyl
icac
id,ja
smonic
acid
and
absc
isic
acid
inA
rabid
opsi
sle
aves
upon
inocu
lation
Schm
elz
etal
.(2
003)
Pseu
dom
onas
syringae
pv.
tom
ato
DC
3000
(pat
hogen
)
Ara
bid
opsi
sA
rabid
opsi
sA
TH1
Aff
ymet
rix
Gen
eC
hip
inco
mbin
atio
nw
ith
Pst
wild
-typ
ean
dm
uta
nt
stra
ins
and
E.co
liw
ild-t
ype
and
muta
nt
stra
ins
Diffe
rential
expre
ssio
nof
ato
talo
f44
auxi
n-r
elat
edgen
esupon
inocu
lation.
Take
nto
get
her
the
resu
lts
sugges
tth
atPs
tis
impac
ting
host
auxi
nsi
gnal
ing,
pote
ntial
lyder
epre
ssin
gth
epat
hw
ay,al
tering
auxi
nm
ove
men
tan
dac
tiva
ting
bio
synth
esis
of
the
horm
one.
Thilm
ony
etal
.(2
006)
Pseu
dom
onas
thiv
erva
len
sis
stra
inM
LG45
(PG
PR)
Ara
bid
opsi
sA
rabid
opsi
sY
2001
AFG
CD
NA
mic
roar
ray
inco
mbin
atio
nw
ith
MLG
45
wild
-typ
e(N
ewm
anet
al.,
1994;Sc
hen
ket
al.,
2000;W
hite
etal
.,2000)
Upre
gula
tion
of
ase
tof
auxi
nre
sponse
-rel
ated
gen
es,in
cludin
gxy
logly
can
endo-1
,4-b
-Dglu
canas
epre
curs
or
(At2
g30270),
aputa
tive
auxi
n-r
egula
ted
pro
tein
(At2
g33830)an
dhea
t-,au
xin-,
ethyl
ene-
and
woundin
g-induce
dsm
allp
rote
in(A
t4g02380)upon
inocu
lation
Car
tiea
ux
etal
.(2
003)
Pseu
dom
onas
fluore
scen
sFP
T9601-T
5(P
GPR
)A
rabid
opsi
sA
rabid
opsi
sA
TH1
Aff
ymet
rix
Gen
eC
hip
inco
mbin
atio
nw
ith
FPT9
601-T
5w
ild-t
ype
Upre
gula
tion
of
ase
tof
auxi
nre
sponse
-rel
ated
gen
es,in
cludin
gxy
logly
can
endo-1
,4-b
-Dglu
canas
epre
curs
or
(At2
g30270),
aputa
tive
auxi
n-r
egula
ted
pro
tein
(At2
g33830)an
dhea
t-,au
xin-,
ethyl
ene-
and
woundin
g-induce
dsm
allp
rote
in(A
t4g02380)upon
inocu
lation
Wan
get
al.
(2005)
Sinorh
izobiu
mm
elilo
tiRm
1021
Med
icag
oM
edic
ago
Aff
ymet
rix
gen
ech
ipin
com
bin
atio
nw
ith
Rm
1021
wild
-typ
ean
dm
uta
nt
stra
ins
Upre
gula
tion
of
auxi
n-r
esponsi
vepro
tein
TC40969_R
C_a
tin
nodule
sco
mpar
edw
ith
roots
Bar
net
tet
al.
(2004)
Pseu
dom
onas
fluore
scen
sW
CS4
17r
Ara
bid
opsi
sA
ffym
etrix
Gen
eChip
conta
inin
gpro
be
sets
for
c.8000
Ara
bid
opsi
sgen
es(Z
hu
&W
ang,2000)
Upre
gula
tion
of
two
auxi
n-induce
dpro
tein
s(1
5017_a
tan
d16751_a
t)Ver
hag
enet
al.
(2004)
IAA
,in
dole
-3-a
cetic
acid
;PG
PR,pla
nt-
gro
wth
pro
moting
rhiz
obac
terium
;Ps
t,Ps
eudom
onas
syringae
pv.
tom
ato.
FEMS Microbiol Rev 31 (2007) 425–448 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
437Microbial auxin signaling
programmed cell death has been reported in response to
PAMPs, and shared mitogen-activated protein kinase
(MAPK) cascades are associated with both PRR and
R-gene-mediated signaling. Further research is needed to
unravel the role of bacterial auxin synthesis in the different
dimensions of plant defense (Abramovitch et al., 2006).
Interestingly, some similarity between auxin signaling in
bacteria–plant interactions, in which IAA is produced by
both partners and seems to play a role in circumvention of
the host defense, can be found with signaling by bacterial QS
molecules in bacteria–host interactions. Well-described QS
signaling molecules such as N-acyl homoserine lactones
(AHLs) exhibit structural similarities to many eukaryotic
hormones and a growing number of reports have documen-
ted apparent biological effects of AHLs on eukaryotic cells.
In bacteria–animal interactions, QS signals appear to be
used by bacteria to prevent the host immune system from
mounting an effective defense and thus from establishing a
productive infection in the host (Shiner et al., 2005). The
reverse communication conduit also appears to be open as
mammalian hormones can interact with components of the
bacterial QS machinery (reviewed by Shiner et al., 2005).
The growing number of examples of interkingdom signaling
molecules evokes the question regarding the role of IAA as a
signaling molecule in bacteria.
IAA as a signaling molecule in bacteria
In addition to the hypothesis that bacterial IAA contributes
to circumvent the host defense by derepressing the auxin
signaling in the plant, IAA also can have a direct effect on
bacterial survival and its resistance to plant defense (Remans
et al., 2006; see Fig. 2). Evidence has been accumulating that
some microorganisms, independent of their ability to pro-
duce IAA, make use of auxin as a signaling molecule steering
microbial behavior.
Examples of IAA as a signaling molecule inmicroorganisms
Research on the regulation of the IAA synthesis in Azospir-
illum resulted in new insights that IAA regulates expression
of genes involved in IAA synthesis by a positive feedback
mechanism (Vande Broek et al., 1999; Lambrecht et al.,
2000), and that a motif known as an auxin responsive
element from plant studies, AuxRe, is present in the up-
stream region of the Azospirillum gene ipdC known to be
regulated by IAA (Lambrecht et al., 1999; Vande Broek et al.,
2005). Comparative analysis shows a striking similarity, in
terms of the mechanism of gene regulation, with indole
signaling in Escherichia coli, postulated to be a mechanism of
QS signaling (Wang et al., 2001; Domka et al., 2006; Walters
& Sperandio, 2006). Wang et al. demonstrated that indole
activates transcription of tnaAB, gabT and astD. Activation
of the tnaAB operon is predicted to induce more indole
production. astD and gabT are involved in pathways that
degrade amino acids to pyruvate or succinate. These results
led the authors to speculate that signaling by indole may
have a role in adaptation of bacterial cells to a nutrient-poor
environment where amino acid catabolism is an important
energy source (Wang et al., 2001). Other targets of indole-
mediated signaling were found recently indicating a role for
indole signaling in biofilm formation (Domka et al., 2006)
and in the stable maintenance of multicopy plasmids (Chant
& Summers, 2007). These findings on indole signaling and
the similarity with IAA signaling in Azospirillum pose
questions regarding targets of IAA signaling in Azospirillum.
The signaling role of IAA has been further demonstrated
in other microbial species. In Ag. tumefaciens IAA inhibits
vir gene expression by competing with the inducing pheno-
lic compound acetosyringone for interaction with VirA (Liu
& Nester, 2006). The authors postulate this vir gene inhibi-
tion by IAA as a putative negative feedback system upon
increased IAA production by transformed plant cells. How-
ever, it is not known whether the amount of exuded IAA
from the transformed plant tissue is within the concentra-
tion range to downregulate vir gene expression.
In Ps. syringae pv. syringae, IAA was shown to be involved
in the expression of syringomycin synthesis which is re-
quired for full virulence of Ps. syringae pv. syringae strains on
stone fruits (Xu & Gross, 1988a, b). IAA� mutants of
Ps. syringae pv. syringae were significantly reduced in
syringomycin production (Mazzola & White, 1994).
Recently, the use of an iaaM deletion mutant of Erwinia
chrysanthemi 3937 indicated a positive role of IAA biosynth-
esis on TTSS and exoenzymes through the Gac-Rsm
posttranscriptional regulatory pathway. Compared with
wild-type Er. chrysanthemi 3937, the expression level of an
oligogalacturonate lyase, ogl, and three endo-pectate lyases,
pelD, pelI and pelL, was reduced in the iaaM mutant. In
addition, the transcription of TTSS genes, dspE (a putative
TTSS effector) and hrpN (TTSS hairpin), was found to be
diminished in the iaaM mutant of Ee. chrysanthemi 3937
(Yang et al., 2007).
Looking at the unicellular eukaryote Saccharomyces cere-
visiae, it is worth mentioning that addition of IAA to the
culture medium provoked invasive growth and differential
gene expression (Prusty et al., 2004). Among the genes
induced by IAA, a gene involved in adhesion, FLO11, was
detected, suggesting that FLO11 activation by elevated con-
centrations of IAA that occur at plant wound sites might be
crucial for feral yeast cells to infect wound sites in plants
(Verstrepen & Klis, 2006). Similarly, activation of adhesins in
animal pathogens occurs when the cells perceive an oppor-
tunity for infection, enabling cells to adhere to the appro-
priate tissue and establish a colony or biofilm of infectious
cells (Verstrepen et al., 2004; Domergue et al., 2005).
FEMS Microbiol Rev 31 (2007) 425–448c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
438 S. Spaepen et al.
In E. coli evidence that IAA might be a signal able to
coordinate of bacterial behavior enhancing protection
against damage by adverse conditions was recently described
by Bianco et al. (2006a). They showed that IAA induces the
expression of genes related to survival under stress condi-
tions. Here, it has to be taken into account that auxin
molecules can interact with cell-wall peroxidases, inducing
the formation of reactive oxygen species (ROS) within the
cell wall (Kawano et al., 2001). Concerning the differential
gene expression in E. coli upon IAA addition, it is not clear
whether these genes are induced upon IAA as a signaling
molecule or upon IAA as a molecule provoking a stress
condition by induction of ROS. Unfortunately, only indole
and not acetic acid, neither a ROS-inducing compound,
were included as a control to study IAA-altered gene
expression, leaving doubt regarding whether the expression
profile presented is IAA specific. A subsequent report of
Bianco et al. (2006b) further supported the observation that
IAA is able to induce changes in gene expression in E. coli. In
their study, it was observed that genes involved in the central
metabolic pathways such as the tricarboxylic acid cycle
(TCA), glyoxylate shunt and amino acid biosynthesis (leu-
cine, isoleucine, valine and proline) were upregulated by
IAA, whereas the fermentative adhE gene was downregu-
lated. Data on differential gene expression upon addition of
appropriate control metabolites are missing, preventing the
distinction between IAA-specific induced genes and genes
altered in expression by structurally related metabolites.
For cyanobacteria it has been shown that IAA triggers
differentiation of cyanobacterial hormogenia (Bunt, 1961),
although direct evidence for IAA signaling in cyanobacteria
has not been described. In view of the fact that the
progenitors of extant cyanobacteria were ancestors of plas-
tids, the possible evolutionary link between IAA signaling in
plants and cyanobacteria is worth examining. Interestingly,
IAA plays a key role in modulating the level of the bacterial
alarmone guanosine 50-diphosphate 30-diphosphate
(ppGpp) in the chloroplasts of plant cells. In bacteria ppGpp
mediates the ‘stringent control’ upon stress conditions
(reviewed by Braeken et al., 2006). Takahashi et al. (2004)
detected ppGpp for the first time in the chloroplasts of plant
cells. They further showed that plant hormones including
jasmonic acid, abscisic acid and ethylene modulate levels of
ppGpp in plants while IAA blocks the effect of other plant
hormones on ppGpp. These data point towards research
into the role of IAA and other plant hormones in modula-
tion of ppGpp levels in bacterial cells.
Finally, it was reported by Liu & Nester (2006) that high
concentrations of IAA (200 mM) inhibit growth of many
plant-associated bacteria but not the growth of bacteria
that occupy other ecological niches. This emphasizes the
role of IAA as a signaling molecule in microorganism–plant
interactions.
The findings representing IAA as a signaling molecule in
bacteria shed new light on the role of IAA in microorga-
nism–plant interactions.
IAA transport and reception in bacteria
A crucial issue and major challenge in studying IAA as a
signaling molecule in bacteria is the mechanism of IAA
reception and transport in bacteria. As yet, no receptor nor a
membrane transport system for IAA has been described in
bacteria. The search for auxin receptors and transport
systems has mainly focused on plants, with recent break-
throughs including the discovery of the Arabidopsis auxin
receptor TIR1 (Dharmasiri et al., 2005; Kepinski & Leyser,
2005) and mechanisms of auxin transport (for a recent
review see Kramer & Bennett, 2006). Recent discoveries
indicating that IAA can act also as a signaling molecule in
bacteria have given attention to the question of IAA
receptors and transport systems in bacteria. The capacity of
some bacteria such as Pseudomonas and Bradyrhizobium to
degrade IAA (Proctor, 1958; Tsubokura et al., 1961; Mino,
1970; Egebo et al., 1991; Jensen et al., 1995; Olesen &
Jochimsen, 1996; Leveau & Lindow, 2005) indicates that
the uptake of IAA by bacteria does occur. Transport of IAA
over plant cell membranes occurs through a combination of
membrane diffusion and carrier-mediated transport (re-
viewed by Kramer & Bennett, 2006). IAA can enter a plant
cell either in its protonated form (IAAH) by membrane
diffusion or in its anionic form (IAA�) by the action of a
proton-driven auxin influx carrier system. Once inside the
cell, IAA is predominantly in anionic form, necessitating
carrier-mediated transport to exit the plant cell (Delbarre
et al., 1996).
Membrane diffusion of IAAH is often argued to be the
predominant flux of auxin into plant cells (Bean et al., 1968;
Gutknecht & Walter, 1980). Also in bacteria, the ability of
protonated auxin to diffuse across the bacterial lipid mem-
brane needs to be considered. IAA is a weak acid with a
dissociation constant (pK) of 4.8 (Delbarre et al., 1996). It
thus partitions between the anion IAA� and the protonated
IAAH according to the environmental pH. In neutral or
basic conditions, IAA�will dominate (99.4% ionized at pH
7), whereas in strongly acidic compartments IAAH dom-
inates (99.8% protonated at pH 2). The rhizosphere is
generally considered to be a weakly acid environment (Hin-
singer et al., 2003), and therefore it is expected that a
considerable part of IAA is in its protonated form in the
rhizosphere. This leads to the hypothesis that IAA can enter
the bacterial cell by membrane diffusion, dependent on the
permeability of the bacterial membrane and the environ-
mental pH.
Interestingly, studies on IAA degradation report IAA
degradation capacity at a pH of 7.0–7.1. This indicates that
FEMS Microbiol Rev 31 (2007) 425–448 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
439Microbial auxin signaling
IAA� can enter the bacterial cell, suggesting the existence of
a proton-driven auxin influx carrier (Proctor, 1958; Tsubo-
kura et al., 1961; Mino, 1970; Egebo et al., 1991; Jensen et al.,
1995; Olesen & Jochimsen, 1996; Leveau & Lindow, 2005).
For the synthetic auxin 2,4-dichlorophenoxyacetate (2,4-D)
an active transport system has been described in several
2,4-D-degrading bacteria, for example Ralstonia, Bradyrhi-
zobium and Delftia (Leveau et al., 1998; Kitagawa et al., 2002;
Muller & Hoffmann, 2006). 2,4-D uptake was shown to be
an energy-dependent process involving a transport protein
encoded by a gene (tfdK in Ralstonia and Delftia, cadK in
Bradyrhizobium) that shows similarity to genes encoding the
major facilitator superfamily transporters. In Delftia acid-
ovorans, it was further observed that the presence of the
uncoupler carbonylcyanide m-chlorophenylhydrazone led
to strong inhibition of 2,4-D uptake, suggesting proton
symport as the likely active mechanism (Muller & Hoff-
mann, 2006). This shows analogy to active IAA import in
plant cells, which is driven by proton influx (Li et al., 2005).
Interruption of the gene tdfK encoding a 2,4-D transport
protein in Ralstonia eutropha decimated 2,4-D uptake rates
but did not abolish uptake completely, indicating that
alternative mechanisms for 2,4-D uptake (e.g. membrane
diffusion) exist (Leveau et al., 1998).
Transfer of cadRABKC genes, involved in 2,4-D degrada-
tion in Bradyrhizobium sp. strain HW13, to a nondegrader
Sinorhizobium meliloti Rm1021 enabled this strain to de-
grade 2,4-D. Of note here is the observation that function-
ality of the gene cadK encoding the transport system was not
necessary to turn S. meliloti Rm1021 into a 2,4-D degrader.
These data suggest that the wild-type strain of S. meliloti
Rm1021 possesses a mechanism for 2,4-D uptake despite its
inability to degrade 2,4-D (Kitagawa et al., 2002).
The question as to what extent 2,4-D transport systems
are comparable with IAA transport in bacteria remains
unanswered. Structural differences between 2,4-D and IAA
can limit the extrapolation of the 2,4-D transport system to
IAA transport. Recently, two articles were published tackling
the structure–activity relationships of auxin-like molecules
(Ferro et al., 2006, 2007). Using a computational approach
(Molecular Quantum Similarity Measures) and subsequent
statistical analysis to identify structural similarity groups,
the authors showed that IAA and 2,4-D share the same
quantum spatial regions, indicating structural similarity.
However, differences between IAA and 2,4-D have been
observed in binding activity to plant auxin transporters/
receptors (Venis & Napier, 1995; Napier et al., 2002;
Kepinski & Leyser, 2005; Badescu & Napier, 2006).
Ongoing plant research might provide other relevant
information on putative IAA transporters and receptors in
bacteria. Advancement in understanding IAA signaling
pathways in plants has been truly spectacular over the past
5 years (for recent reviews on auxin receptors see Badescu &
Napier, 2006; Parry & Estelle, 2006; and on auxin transport
see Fleming, 2006; Kramer & Bennett, 2006). Back-to-back
papers identified the F-box protein transport inhibitor
response 1 (TIR1) as a plant receptor for auxin (Dharmasiri
et al., 2005; Kepinski & Leyser, 2005). TIR1 belongs to a
small protein family which is part of the Arabidopsis F-box
family. Currently, three other TIR1-related F-box protein/
receptors (AFB1, AFB2, AFB3) have been identified as auxin
receptors in Arabidopsis (Dharmasiri et al., 2005; Parry &
Estelle, 2006). Exploiting the NCBI database using the BLAST
algorithm to search for homology between the amino acid
sequence of TIR1 (Dharmasiri et al., 2005) and bacterial
proteins, no strong homology was found. However, a
putative F-box-like protein was detected in Candidatus
Protochlamydia amoebophila UWE25 (see supplementary
Table S1).
TIR1 functions as part of a molecular complex SCFTIR1
that attaches the cell’s garbage tag, ubiquitin, to proteins
destined to be recycled. Auxin, by glomming onto TIR1,
helps the SCFTIR1 complex to ubiquitinate Aux/IAA pro-
teins. When the Aux/IAA proteins are broken down, the
genes they repress turn on. Interestingly, similarity for the
functionality of the SCFTIR receptor system can be found
with another indole receptor, the tryptophan receptor of
bacteria. However, the TIR1 pathway has extra layers of
control built in. Tryptophan binds to the bacterial Trp
repressor protein to induce a conformational change that
allows it to bind directly to the trp operator sequence and so
control gene expression (Ramesh et al., 1994). Arguably, a
switching error in such a system will be immediately
amplified. The activity of TIR1 is one step removed from
transcriptional regulation by the ubiquitination system,
allowing small errors to be buffered (Dharmasiri et al.,
2005; Parry & Estelle, 2006).
The TIR1-like signaling cannot account for all auxin
responses in plants (reviewed by Badescu & Napier, 2006).
Regulation through proteolysis and transcriptional activity,
occurring upon TIR1-auxin signaling, takes time; it is
inevitably much slower than membrane depolarization, for
example. Therefore, rapid auxin responses in plants are
unlikely to be TIR1-mediated and another type of auxin
receptor is expected to be involved in rapid auxin-mediated
responses (Badescu & Napier, 2006). Detailed auxin binding
data have been reported for only one other protein, the
auxin-binding protein 1 (ABP1) (Venis & Napier, 1995;
Napier et al., 2002). Besides its strong ability to bind auxin,
the physiological role of ABP1 remains unclear (Napier
et al., 2002). The majority of ABP1 is localized in the
endoplasmatic reticulum, where the pH is too high for
auxin binding. However, some ABP1 is also found on the
plasma membrane, and ABP1 antibody experiments have
implicated this pool in auxin-mediated cell expansion.
Exploiting the NCBI database using BLAST software, some
FEMS Microbiol Rev 31 (2007) 425–448c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
440 S. Spaepen et al.
homology was found between ABP1 and putative auxin-
binding proteins in various bacteria, for example S. meliloti,
Shewanella amazonensis, Ps. syringae pv. syringae, Ps. syrin-
gae pv. phaseolicola and Ps. syringae pv. tomato (Table S1). It
will be interesting to unravel the role of these putative auxin-
binding-like proteins in bacteria.
In addition, genetic studies in Arabidopsis have led to the
identification of AUX1, pGlycoprotein (PGP, an ABC trans-
porter) and PIN classes as IAA influx and efflux facilitators
(Parry et al., 2001; Paponov et al., 2005; Geisler & Murphy,
2006; see review by Kramer and Bennett, 2006). In a broad
set of bacteria, e.g. various Pseudomonas species (Ps. fluor-
escens, Ps. syringae and Ps. putida), Bradyrhizobium, Mesor-
hizobium, Sinorhizobium and Rhizobium species, proteins
showing strong homology (E values between 4e-90 and 1e-
58) in amino acid sequence with the plant PGP transporter
were detected (Table S1). As PGP is an ABC transporter, the
homology between PGP and bacterial proteins is not
surprising given that ABC transporters are common trans-
port systems in bacteria. Determination of the specificity of
the bacterial ABC transporters that are homologous to the
plant auxin transporter PGP is required to unravel their
putative role in IAA transport in bacteria.
In addition, some degree of homology (Table S1) in
amino acid sequence was found between the plant auxin
transport facilitator PIN and bacterial proteins predicted as
auxin efflux carriers in various species belonging to, among
others, Ralstonia, Burkholderia and Mesorhizobium, which
are 2,4-D degraders (Leveau et al., 1998; Kitagawa et al.,
2002).
Taking these data together, it can be suggested that certain
similarity in IAA reception and transport/IAA signaling
between plants and bacteria may exist.
Conclusions and perspectives
Over 120 years after Darwin’s description of IAA as ‘a matter
which transmits its effects from one part of the plant to
another,’ plant and microbial research results allow us to
identify IAA as a multivalent signaling molecule and as ‘a
matter which transmits its effects within plants and among
plants and bacterial cells.’ Emerging views on IAA as a
common language between bacteria and plants pave the
way for future research on IAA signaling in microorganisms.
A recent editorial in Science (Vogel, 2006) emphasized the
importance of the advancement in unraveling IAA signaling
pathways in plant communication. The moment to extend
this knowledge to other kingdoms seems highly favorable.
Current postgenomic studies already indicate a role for IAA
signaling in bacteria and microorganism–plant interactions.
However, dedicated functional genomic studies are needed
to unravel the function and mechanism of IAA signaling in
bacteria and during the different stages of microorganism–
plant interactions. The increasing amount of genomic data
will further broaden insight into the distribution and
evolution of the capacity to participate in IAA signaling
among organisms. It is clear that not only plants and
bacteria will take part in the IAA conversations, but that
researchers in plant science and microbiology will undoubt-
edly continue talking about this intriguing molecule.
Acknowledgements
S.S. is financed in part by the Fund for Scientific Research
Flanders (G.0085.03) and in part by the Belgian Govern-
ment (IUAP P5/03). R.R. is a recipient of a predoctoral
fellowship from the ‘Vlaamse Interuniversitaire Raad
(VLIR)’. We acknowledge financial support from the K.U.
Leuven (CoE SymBioSys).
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Supplementarymaterial
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Table S1. BLAST homology results: IAA receptor and
transport protein.
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448 S. Spaepen et al.
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