Genin S, Boucher C.. Lessons learned from the genome analysis of Ralstonia solanacearum. Annu Rev Phytopathol 42: 107

Abstract

Ralstonia solanacearum is a devastating plant pathogen with a global distribution and an unusually wide host range. This bacterium can also be free-living as a saprophyte in water or in the soil in the absence of host plants. The availability of the complete genome sequence from strain GMI1000 provided the basis for an integrative analysis of the molecular traits determining the adaptation of the bacterium to various environmental niches and pathogenicity toward plants. This review summarizes current knowledge and speculates on some key bacterial functions, including metabolic versatility, resistance to metals, complex and extensive systems for motility and attachment to external surfaces, and multiple protein secretion systems. Genome sequence analysis provides clues about the evolution of essential virulence genes such as those encoding the Type III secretion system and related pathogenicity effectors. It also provided insights into possible mechanisms contributing to the rapid adaptation of the bacterium to its environment in general and to its interaction with plants in particular.

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10.1146/annurev.phyto.42.011204.104301
Annu. Rev. Phytopathol. 2004. 42:107–34
doi: 10.1146/annurev.phyto.42.011204.104301
Copyright c
2004 by Annual Reviews. All rights reserved
First published online as a Review in Advance on March 24, 2004
LESSONS LEARNED FROM THE GENOME ANALYSIS
OF RALSTONIA SOLANACEARUM
St´
ephane Genin and Christian Boucher
Laboratoire Interactions Plantes-Microorganismes, CNRS-INRA, Castanet-Tolosan,
France; email: sgenin@toulouse.inra.fr, boucher@toulouse.inra.fr
KeyWords bacterial wilt, pathogenicity, protein secretion systems, lateral gene
transfer, soil bacterium
■Abstract Ralstonia solanacearum is a devastating plant pathogen with a global
distribution and an unusually wide host range. This bacterium can also be free-living as
asaprophyte in water or in the soil in the absence of host plants. The availability of the
complete genome sequence from strain GMI1000 provided the basis for an integrative
analysis of the molecular traits determining the adaptation of the bacterium to various
environmental niches and pathogenicity toward plants. This review summarizes cur-
rent knowledge and speculates on some key bacterial functions, including metabolic
versatility, resistance to metals, complex and extensive systems for motility and attach-
ment to external surfaces, and multiple protein secretion systems. Genome sequence
analysis provides clues about the evolution of essential virulence genes such as those
encoding the Type III secretion system and related pathogenicity effectors. It also pro-
vided insights into possible mechanisms contributing to the rapid adaptation of the
bacterium to its environment in general and to its interaction with plants in particular.
INTRODUCTION
Ralstonia solanacearum,asoilborne pathogen in the Proteobacteria βsubdivi-
sion, causes lethal wilting disease of more than 200 plants worldwide. This very
wide host range covers both monocots and dicots, extending from annual plants to
trees and shrubs (38). The bacterium enters plant roots, invades the xylem vessels,
and spreads rapidly to aerial parts of the plant through the vascular system. The
vascular dysfunction induced by this extensive colonization causes wilting symp-
toms and eventual plant death. The importance of the disease lies in the pathogen’s
wide geographic distribution in warm and tropical climates (38). Recently, this
geographical spectrum has been extended to more temperate countries in Europe
and North America as the result of the dissemination of strains adapted to cooler
environmental conditions.
While biochemical, genetic, and molecular approaches allowed the identifi-
cation of a general array of virulence factors (reviewed in 33, 70), the recent
0066-4286/04/0908-0107$14.00 107
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108 GENIN BOUCHER
completion of the genome sequence of strain GMI1000 (68) represents a sig-
nificant advance in opening the way to an exhaustive analysis of the molecular
complexity governing both pathogenicity and the organism’s versatility. This re-
view focuses on diverse functions related to the ability of the bacterium to interact
and respond to the environment, and more specifically, to functions contributing
to the infection and colonization of plants. However, several traits that may poten-
tially condition pathogenicity, such as the production of phytohormones (33) or
multiple quorum-sensing systems (32, 33, 70), are not discussed in this review.
GENOME STRUCTURE
ABipartite Genome
Sequencing of strain GMI1000 established that the genome of this organism is
organized in two large circular replicons of 3.7 and 2.1 megabases, respectively,
referred to as the chromosome and the megaplasmid (68). R. solanacearum is one
of the very first organisms for which such a bipartite genomic organization has
been documented. This organization is specific not only to this particular strain
but also appears to be characteristic of R. solanacearum in that the presence of a
megaplasmid had been established in most, though not all, strains of the species
that have been investigated for this character (67). More recently, this organization
has been further confirmed using pulsed field gel electrophoresis, and these experi-
ments established little size variation for the two replicons between different strains
(C. Boucher, unpublished data). The largest replicon exhibits characteristic fea-
tures of a bacterial chromosome. It carries the rnpA,dnaA,dnaN, and gyrAB genes,
as well as a DnaA-binding box, that are characteristic of the origin of replication
of bacterial chromosomes; moreover, all the essential housekeeping genes of the
bacterium are located on the largest replicon. By contrast, the origin of replication
of the smaller replicon is flanked by the repA gene and by several RepA-binding
boxes. Furthermore, this replicon does not carry vital genes that are not also present
on the chromosome. The smaller replicon therefore clearly originates from a plas-
mid (68). However, using criteria as diverse as average base composition, codon
usage, it is clear that the two replicons in this bacterium have coevolved over a long
time span (17). This long coevolution resulted in the recovery by the megaplasmid
of several duplications of essential housekeeping genes such as one copy of rDNA,
two of tRNA, and one copy of the elongation factor G. Genetic colonization of the
megaplasmid by housekeeping functions is also illustrated by the presence on this
replicon of 55 genes that encode enzymes controlling the biosynthesis of amino
acids, nucleotides, and cofactors. Although the corresponding genes have been
maintained on the chromosome in some cases, in many instances they have been
lost with the result that the two replicons are now needed for the bacteria to be
able to grow prototrophically on minimal culture medium. This suggests that with
R. solanacearum,weare facing an example of evolution whereby an originally
dispensable plasmid gradually evolves to become an indispensable component of
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RALSTONIA SOLANACEARUM GENOME 109
the genome. The driving force underlying such an evolution is not clear. However,
combining the large number of plasmid-borne genes coding for adaptation to di-
verse environments together with genes controlling basic metabolic processes on
the same replicon is an efficient way to ensure the maintenance of a large set of
dispensable genes and thereby increase the repertoire of “permanent” genes.
Although the presence of small plasmids, with a size below 100 kb, has been
reported in several strains of R. solanacearum (53), the presence of such genetic
elements is the exception rather than the rule (C. Boucher, unpublished data). A
different strategy has been developed by this bacterium to acquire genetic infor-
mation that may be transiently required under particular environmental conditions
(see below).
AMosaic Structure
As mentioned above, the two replicons, because of their long coevolution, have
a similar base composition with an average G +C content near 67%. However,
important local variations, ranging from 50% to 70%, are observed in more than
50 stretches of DNA that together represent 7% of the total genome. These GC-
biased regions are disseminated over the two replicons and are often associated with
tRNAs or mobile genetic elements such as insertion sequences and bacteriophages
(68). Most of these regions individually span several kilobases and carry genes that
differ in codon usage from the general codon usage utilized by the bacterium. Taken
together, these features are highly indicative of these regions having been acquired
through lateral gene transfer (LGT) and of the probability that they could play an
important role in the rapid adaptation of the bacterium to the change of ecological
niche. Such a hypothesis is also consistent with the ability of the bacterium to be
naturally transformed by acquisition of genetic material from the environment and
insertion of the incoming DNA into its genome (7, 10).
AMETABOLICALLY VERSATILE SAPROPHYTIC
SOIL BACTERIUM
Although generally considered as a plant pathogen, R. solanacearum behaves pri-
marily as an saprophytic bacterium able to survive for long periods of time in
various natural habitats such as surface waters and different types of soils. It thus
requires specific functions to enable it to use a variety of organic substrates as en-
ergy sources and to cope with toxic compounds that are often present in soils. The
bacterium therefore contains a large repertoire of catabolic genes, genes involved
in the detoxification of noxious compounds, and has also developed sophisticated
chemotaxis and adhesion mechanisms to facilitate efficient colonization and main-
tenance in specific ecological niches.
Although the catabolic pathways encoded in R. solanacearum have not yet been
comprehensively analyzed, the cataloguing of 176 genes involved in catabolic
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110 GENIN BOUCHER
TABLE 1 Putative degradative genes identified in Ralstonia solanacearum together with the
corresponding substrates and end-products
Gene reference Replicon Substrate End-products
RSc1337 to RSc1342 Chromosome Aromatic/aliphatic Phenol +sulfite
sulfonate
RSc2753 Chromosome Benzoate, toluate Catechol (2-hydro-1,2
dihydroxybenzoate)
RSc2242 Chromosome 4-hydroxybenzoate Protocatechuate
RSc2871 Chromosome Hippurate Benzoate +glycine
RSc0626, RSc0627 Chromosome Isoquinoline Isoquinolin-1(2H)-one
RSc1822 Chromosome Maleate Fumarate
RSc3355, RSc3356 Chromosome Phenylalanine Tyrosine
RSc1631 to RSc1633 Chromosome 3-polyhydroxybutyric Polyoxyalkalonates
acid
RScc1441, RSc1442 Chromosome Protocatechuate Succinate, acetate
(3,4-dihydroxybenzoate)
RSc1085 to RSc1092 Chromosome Naphtalene Fumarate, pyruvate
RSp0222, RSp0223 Megaplasmid Vanillin (4-Hydroxy-3- Protocatechuate
methoxybenzaldehyde) (3–4-dihydroxybenzoate)
RSp0058 Megaplasmid Stachydrine Proline
activities (http://histone.toulouse.inra.fr/bioinfo/annotation/iANT/bacteria/ralsto/
index.html) shows that this organism is particularly well-equipped and closely
resembles the two other soilborne bacteria Pseudomonas aeruginosa (74) and
Pseudomonas putida (55). These genes provide a means to utilize a wide range
of substrates including amino acids, sugars and fatty acids as well as different
substrates listed in Table 1. Several of the aromatic compounds predicted to be
degraded by R. solanacearum (ferulate, vanillate, hydroxybenzoates, and protocat-
echuates) are derivatives of lignin, released during the process of lignin degradation
in the soil. Therefore, after the plant has been killed by the pathogen, it continues
to provide an environmentally favorable ecological niche for the development of
R. solanacearum in the soil.
The bacterium also produces several extracellular hydrolytic enzymes includ-
ing a pectinase (RSp0138), polygalacturonases (PglA, PehB, PehC), proteases, and
glucanases (RSp0162, RSp0583) (reviewed in 70), which generate low-molecular-
weight substrates that can be assimilated by the bacteria (see 34). GMI1000 en-
codes at least 54 hydrolases and 16 transferases with unknown substrate specificity,
some of which may be important in enlarging the catabolic ability of the bacterium.
The metabolic versatility of R. solanacearum is also exemplified by a large reper-
toire of 83 ABC transporters devoted to import substrates that are then metabolized
or to export toxic compounds out of the cell.
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RALSTONIA SOLANACEARUM GENOME 111
RESISTANCE TO METAL AND DRUGS
In a recent extensive analysis of metal resistance genes present in R. solanacearum,
Mergeay and colleagues (51) identified eight putative metal resistance gene clus-
ters on the megaplasmid and one on the chromosome that share homology with ars
(arsenite, arsenate), chr (chromate), cop [Cu(II)], czc [Cd(II), Zn(II), Cu(II)], cnr
[Co(II),Ni(II)], mer [Hg(II) and organomercurials], pbr [Pb(II)] and sil [Ag(I)]
genes from R. metallidurans.This organism taxonomically closely related to
R. solanacearum is also a soil organism that is particularly adapted to polluted
soils. The metal resistance genes include three P1-ATPases that confer resistance
to lead (RSp0319), copper (RSc3348), and to a nondefined cation (RSc1274). The
corresponding proteins act to pump out these cations when the intracellular con-
centration raises above homeostatic limits. Additional genes encode constituents
of cation efflux systems comprising members of the HME-RND family of pro-
teins, which are specialized in the efflux of heavy metals (56). These genes are
organized in seven clusters that are all present on the megaplasmid (Table 2). No
functional analysis of these genes has been undertaken to date, and in some cases
it is difficult to predict the identity of the metal(s) handled by each individual
system. In addition, it is not clear whether all these systems are actually func-
tional since some gene clusters are not complete compared to their counterpart in
R. metallidurans.Nevertheless, although R. solanacearum is not as intensively pre-
pared as R. metallidurans for metal resistance, it is clearly overequipped compared
to the vast majority of other bacteria that have been sequenced.
In addition to bona fide metal resistance genes, R. solanacearum also harbors a
set of “orphan” genes coding for five ArsR and three MerR-related regulatory pro-
teins. These proteins that bind metal cations modulate expression of certain genes
in response to variation in intracellular cation concentrations (56). The presence of
these genes in R. solanacearum provides additional evidence that this bacterium
is adapted to cope with environments such as soils where metals may be found in
a wide range of concentrations.
Finally, R. solanacearum also harbors nine additional CzcA paralogues that
belong to the HME family (Table 2), which is predicted to control efflux of various
noxious compounds from bacteria. These genes, together with two β-lactamase
genes, might also play an important role during the life of the bacterium in the
soil since they provide an efficient means of resistance toward toxic compounds
produced by many soil-dwelling microorganisms.
PROTEIN SECRETION SYSTEMS
R. solanacearum appears to be outstandingly well-equipped for protein secretion.
Protein secretion systems are of major importance for the bacterium in free-living
conditions, as they are essential tools for interacting with and exploiting the en-
vironment. Moreover, they represent a unifying theme among most, if not all,
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112 GENIN BOUCHER
TABLE 2 List of genes governing metal and drug resistance in Ralstonia solanacearum
GMI1000
Replicon Gene reference Possible resistance to
Metal resistance associated genes
P1-ATPase systems
Megaplasmid RSp0319 to RSp0320 Lead
Chromosome RSc3348 Copper
Chromosome RSc1274 Unknown
HME-RDN dependent systems
Megaplasmid RSp0488 to RSp0493 Cobalt, zinc, cadmium
Megaplasmid RSp0528 to RSp0530 Chromate
Megaplasmid RSp0654 to RSp0660 Copper
Megaplasmid RSp0926 to RSp0928 Silver
Megaplasmid RSp1039 to RSp1044 Cobalt, nickel
Orphan merR
Megaplasmid RSp0137
Chromosome RSc0280
Chromosome RSc1584
Chromosome RSc2322
Chromosome RSc3347
Orphan arsR
Megaplasmid RSp1318
Megaplasmid RSp1349
Chromosome RSp0993
Chromosome RSc1543
Chromosome RSc1960
Other resistances
HME dependent systems
Megaplasmid RSp0312 Drugs
Megaplasmid RSp0670 Drugs
Megaplasmid RSp0818 Drugs
Megaplasmid RSp1113 Drugs
Megaplasmid RSp1198 Drugs
Megaplasmid RSp1199 Drugs
Megaplasmid RSp1457, RSp1458 Drugs
Megaplasmid RSp1594 to RSp1599 Drugs
Chromosome RSc1653, RSc1654 Drugs
Chromosome RSc3205 to RSc3208 Drug
β-lactamases
Megaplasmid RSp0030 Antibiotic
Chromosome RSc0258 Antibiotic
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RALSTONIA SOLANACEARUM GENOME 113
pathogenic bacteria. Although secreted virulence proteins serve many divergent
functions, Gram-negative bacteria have developed only a limited number of se-
cretion systems by which such proteins pass through their outer membrane. Re-
markably, R. solanacearum possesses genetic information for all six major protein
secretion pathways that have been characterized for Gram-negative bacteria (8,
20, 27, 39, 40, 69). To date, only two of these protein secretion systems have been
experimentally studied and both were shown to be essential for R. solanacearum
pathogenicity: the Type II and the Type III secretion systems. Mutants defective
in either system are severely impaired in colonization ability and multiplication
in planta (9, 43). However, mutants defective in individual exported proteins have
subtle or no virulence phenotypes, suggesting that these secreted proteins are
collectively important for disease and that many of them are likely functionally
redundant.
Multiple Type II Secretion Systems
The Type II secretion system is an extension of the general secretory pathway that
drives translocation of diverse exoproteins across the outer membrane of many
Gram-negative bacteria (69). Twelve genes predicted as coding for this pathway
(gspC-N) were identified in strain GMI1000. None of the gsp genes encodes a
putative prepilin peptidase. In fact, only one such gene in the GMI1000 genome,
pilD,isrequired for the synthesis of Type IV pili (49). Similarly, in P. aeruginosa,
the PilD/XcpA prepilin peptidase was shown to be used both for Type IV pilus
assembly and Type II secretion process (69). Interestingly, the order of the secretion
genes differs from that of other species: (a) the gspDEF genes are located on the
right-hand border of the cluster, and (b) instead of being upstream of the gspD
gene, gspC is located on the left-hand border of the cluster upstream of gspG,in
the opposite orientation from that of the rest of the genes in the cluster.
The analysis of culture supernatants of R. solanacearum Type II secretion-
defective mutants revealed that at least six major exoproteins transit through this
pathway, including plant cell wall–degrading enzymes (a pectinase, an endoglu-
canase, and two polygalacturonases, PglA and PehB), and Tek, a 28-kD protein
of unknown function (43). Mutants unable to secrete Type II secretion-dependent
exoproteins completely lost the ability to cause disease symptoms and to effi-
ciently colonize plant stems (43), whereas individual mutations in various plant
cell wall–degrading enzymes result only in minor virulence phenotypes (34, 70).
This strongly suggests that this group of exoproteins is required for infection and
killing of host plants.
Recent studies on Type II secretion in other bacterial species revealed that,
surprisingly, the Gsp pathway may be in fact the tree that prevents us from seeing
the forest. P. aeruginosa and P. putida were both shown to possess a second, dis-
tinct, Gsp-related pathway involved in the secretion of the alkaline phosphatase
LasA (5) or a manganese-oxidizing enzyme (26), respectively. R. solanacearum
was shown to contain three gene clusters (two on the megaplasmid and one on the
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114 GENIN BOUCHER
chromosome) very similar to the xcm cluster in P. putida, all unlike the “ortho-
dox” gsp cluster (26). These Gsp-related clusters contain gspE,gspD (encoding
secretin), and gspG homologues but lack, for example, homologues of gspL and
M(26). Substrates of these Gsp-related pathways are unknown in R. solanacearum
but these secretion systems may well be dedicated to specific substrates. R. solana-
cearum,P. putida, and P. aeruginosa are ubiquitous bacteria found in various en-
vironments such as soil or water, which suggests that the presence of multiple
functional Type II secretion systems in these organisms is related to their high
potential for adaptation.
The Type III Secretion System (TTSS)
hrp GENES AND TTSS-EFFECTORS The TTSS of Gram-negative plant pathogens
has provoked great interest for over 15 years because it plays a major role in
the pathogenicity of several important pathogens that differ in host range and
lifestyle. TTSS-defective mutants of R. solanacearum are unable to cause disease
symptoms on host plants or to induce the defensive hypersensitive response on
nonhost (resistant) plants. This completely nonpathogenic phenotype illustrates
the collective importance of the effector proteins that are injected into plant cells
by the system (13, 20). The TTSS is encoded by the hrp gene cluster that spans
a23-kb region on the megaplasmid (82). However, hrp genes are not essential
to the plant root invasion process because Type III secretion mutants retain the
ability to invade the vascular system of naturally infected tomato plants, although
their respective population levels remain very low compared to those reached by
the wild-type strain (80, 83). This impaired growth of hrp mutants in planta is
presumably a consequence of the low availability of nutrients and/or general plant
defense responses. It also suggests that TTSS-effectors can suppress these host de-
fense responses, as recently demonstrated for the P. syringae effector AvrPto (37a),
and somehow promote disease development by ensuring the rapid multiplication
of the bacterium during the early stages of root infection.
REPERTOIRE OF TTSS-EFFECTORS IN STRAIN GMI1000 The availability of complete
genome sequences marked the beginning of a new era of research that combines
experimental and computational approaches to come up with the complete in-
ventory of TTSS-effectors (19). In R. solanacearum,alarge pool of candidate
TTSS-effectors was identified through a regulation-based approach aimed at es-
tablishing an exhaustive list of genes belonging to the HrpB regulon. HrpB, an
AraC family regulator, is believed to bind to the “hrpII box” promoter sequence,
a 25-base pair DNA motif (TTCGn16TTCG) required for the HrpB-dependent
activation of TTSS-secretion genes and the popABC effector operon (21). Among
the 96 promoters identified through a genome-wide search of the hrpII box in
GMI1000, 43/58 were experimentally confirmed as being controlled by HrpB
(22). Interestingly, several of these HrpB-regulated genes encode proteins carry-
ing conserved domains likely to be involved in the interaction with host plant cell
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RALSTONIA SOLANACEARUM GENOME 115
targets (different types of leucine-rich-repeats, ankyrin, or pentatricopeptide re-
peats) (22). The fact that such TTSS-effector domains are found only in the plant
kingdom (such as pentatricopeptide repeats) or show the best relatedness to plant
proteins raises the possibility that these effectors may act directly as agonists or an-
tagonists of plant cellular components. Preliminary transcriptomic analyses using
microarrays representative of the whole GMI1000 genome indicate that the HrpB
regulon comprises ∼130 genes (A. Occhialini, S. Genin & C. Boucher, unpub-
lished results). In light of these data, and based on the analysis of the N-terminal
amino acid sequence features predictive of GMI1000 TTSS-substrates (22), we
estimate that R. solanacearum might possess up to 60–80 TTSS-effectors.
To date, nine GMI1000 effector proteins are known to transit through the
TTSS. Direct evidence for translocation into plant cells using the adenylate cy-
clase reporter fusion system (15, 73) was reported for the avirulence gene product
PopP2 (see below) and the RipA, RipB, RipG, and RipT effectors (22). RipA
and RipG both belong to multigenic families (5–7 members) that are unique to
R. solanacearum. PopP2 and RipT, although unrelated in sequence, possess the
catalytic triad characteristic of classes of cysteine proteases related to Yersinia sp.
YopP/J and YopT, respectively (24, 72). RipB harbors a motif homologous to that
found in several N-ribohydrolases (84), suggesting that it could promote, within
plant cells, the hydrolysis of a nucleoside-containing molecule (22). Elucidation
of TTSS-effector function will therefore require identification of the host cellular
components that they specifically target in order to promote pathogenesis.
TTSS-EFFECTORS RECOGNIZED BY THE PLANT SURVEILLANCE SYSTEM Because re-
sistance of solanaceous crops to R. solanacearum based on single race-specific
resistance genes had never been reported in the literature, it was surprising to dis-
cover in the GMI1000 genome several genes homologous to typical avr genes in
Pseudomonas syringae/Xanthomonas sp. (68). A recent study did indeed confirm
that resistance to bacterial wilt can occur in a classical gene-for-gene manner,
involving the TTSS-effector popP2, which encodes a member of the AvrRxv/
YopP protein family, and the Arabidopsis RRS1-R gene (24) partners. RRS1-R
is a member of the class of TIR-NBS-LRR resistance proteins and possesses
a C-terminal extension with a WRKY domain (25). This WRKY domain is a
conserved DNA-binding module that interacts with a nucleotide target sequence,
termed the W-box, which is prominent in many pathogen-responsive plant pro-
moters (30). PopP2 contains functional nuclear localization signals (NLS) respon-
sible for the translocation of the effector molecule into the plant nucleus (24).
RRS1-R interacts physically with PopP2 in yeast two-hybrid assays and both pro-
teins were shown to colocalize in the nucleus, but the nuclear translocation of
RRS1-R is dependent upon the NLS of PopP2 (24). The simplest model for the
PopP2/RRS1-R interaction postulates that PopP2, a putative cysteine protease, has
an unidentified host nuclear target. The cytoplasmic RRS1-R protein may act as
a sentinel that recognizes PopP2 and, upon this interaction, reaches the nucleus
as a PopP2/RRS1-R complex. This interaction could also convert RRS1-R into
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116 GENIN BOUCHER
an active form in order to activate the transcription of a battery of plant defense
genes.
Also identified are two additional GMI1000 avirulence genes, popP1 and
avrA, that are recognized by resistant petunia lines and tobacco species, respec-
tively (14, 47). However, the corresponding resistance genes remain to be
identified.
Type IV Secretion Genes: A Conjugative Transposon
The chromosome in R. solanacearum carries a gene cluster (RSc2574 to RSc2588)
that contains most of the genes required to express a Type IV secretion system
related to those found on IncP or Ti plasmids (27). This gene cluster is part of a 45-
kb conjugative transposon, a term defining chromosomal conjugative elements that,
unlike conjugative plasmids, cannot be isolated as circular replicative molecules
(12). Transfer of these conjugative elements allows the one-step acquisition of
novel functions that can be advantageous for the bacteria such as antibiotic or
heavy metals resistance, catabolism of various substrates, or bacteriocin synthesis
(12).
In addition to a complete Type IV secretion gene cluster, the GMI1000 conjuga-
tive transposon also contains some plasmid-related genes (repA,parAB) and an
orthologue of the TraG motor protein responsible for DNA transfer during conju-
gation (27). Transposition is most likely initiated by an integrase tyrosine recombi-
nase encoded by the int gene and involves a site-specific excision/integration pro-
cess (79). Although its mobility has not yet been demonstrated in R. solanacearum,
this element belongs to a new family of broad host range conjugative elements
found in various soil proteobacteria. This family includes the Ralstonia sp. Tn4371
element carrying biphenyl-degrading genes (79) and the 611-kb symbiosis island
of Mesorhizobium loti MAFF303099 (42). Homologous elements were also found
in the genome sequence of Azotobacter vinelandii and in the plant pathogen Er-
winia chrysanthemi 3937 (79), and several of these large integrated mobile (or
potentially mobile) elements have also been named “genomic islands.”
Interestingly, comparison of gene clusters revealed that these transposable el-
ements have a clear modular structure made up of very similar operons (transfer
and replication/partition genes) that are separated by a set of completely unre-
lated ORFs (12, 79). In Tn4371, these ORFs are the 13 bph genes involved in the
catabolism of biphenyl, and in M. loti, the corresponding part is composed of a
200-kb region of the symbiotic island. In GMI1000, this part of the conjugative
element is composed of 13 genes, most of which are of unknown function. How-
ever, we identified four genes predicted to encode a carboxylesterase, a glutathione
S-transferase, and two limonene-1,2-epoxide hydrolases, suggesting that this gene
cluster could be involved in detoxification or catabolism of secondary metabolites.
Limonene (4-isopropenyl-1-methylcyclohexene) is the most widespread terpene
in nature and is formed by over 300 plants (28). Although the precise physiological
roles of most plant monoterpenes have not been defined, it is generally agreed that
they play roles in defense and plant–plant communication (71). Enzymes such as
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RALSTONIA SOLANACEARUM GENOME 117
limonene-1,2-epoxide hydrolases may be involved in the defense of the bacterium
against toxic epoxides produced by plants. However, no experimental evidence
is yet available in support of a role for these genes as a “fitness island” or an
“ecological island” (36).
Other Protein Secretion Systems
Putative fitness/virulence determinants exported outside of the bacterial cell by
three other secretion mechanisms and presumably important for environment/host
interactions were found in strain GMI1000. The first group is composed of three
RTX-like toxin genes (RSp0294, RSp0295, and RSp1180), which may be ex-
ported via a Type I secretion system as suggested by the presence of hlyBD-
related neighboring genes (8). RTX-like bacteriocin genes appear to be widespread
among several Gram-negative plant pathogens (Xylella fastidiosa,Xanthomonas
sp., Agrobacterium) and may play a role in determining the ecological success of
these microorganisms (82a).
Another class of proteins in R. solanacearum (hemagglutinin-related proteins)
is likely to be secreted by two-partners secretion (TPS) families (40). A TPS system
consists of two proteins, an outer-membrane transporter protein and a protein
secreted by the transporter. Genes encoding the transporter and substrate protein
are usually found in the operon or within the same locus (40). At least ten GMI1000
hemagglutinin/adhesin-related proteins (RSc0887, RSc1775, RSc3183, RSc3188,
RSp0540, RSp1073, RSp1444, RSp1536, RSp1539, and RSp1545) are predicted
to be secreted via the TPS pathway: These are large proteins with masses ranging
between 132 and 353 kDa, and many homologues in other bacterial species are
involved in attachment tovarious surfaces (see below) and, in some cases, virulence
(40).
Two other hemagglutinin-related proteins (RSc0115 and RSc3162) possess the
features of autotransporter proteins (also called Type V secretion system) (39).
Autotransporters are characterized by the presence of a Sec-dependent N-terminal
signal peptide and a C-terminal 14 or 15 β-strand domain that forms a pore in the
outer membrane through which the mature protein passes to the cell surface (39).
Finally, two Tad (tight adherence)-related secretion systems (RSc0649-0661 and
RSp1082-1092) homologous to those originally found in Actinobacillus actino-
mycetemcomitans and involved in the export and assembly of bundled pili (61; see
below) were also found in the GMI1000 genome.
MOTILITY AND BACTERIAL ADHESION
There is in GMI1000 an abundance of genes encoding attachment factors and/or
extracellular structures that promote motility. In addition to the main polar flag-
ella responsible for swimming motility (78), R. solanacearum produces Type
IV pili (Tfp) that determine the so-called “twitching motility” (49), a form of
flagella-independent translocation of bacteria over solid surfaces. In GMI1000,
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118 GENIN BOUCHER
there are >20 chromosomal genes organized in four unlinked operons that are
candidates for the synthesis and function of Tfp. Tfp are composed primarily of
a single pilin protein, PilA, assembled into a flexuous polar filament (44). In fact,
Tfp play multiple roles in the biology of R. solanacearum: The pilA mutant is im-
paired for twitching motility but it is also no longer competent for transformation
(i.e., the natural ability to take up DNA from its milieu) and does not exhibit polar
attachment to plant cells (44) as typically observed during cocultivation of bacteria
with plant cell suspensions (2). Moreover, a pilA mutant is reduced in virulence in
tomato plants (44). A likely explanation for the fact that Type IV piliation is more
important for virulence of R. solanacearum than it appears to be for Xanthomonas
campestris or P. syringae (60, 65) is that contact with and invasion of unwounded
tomato roots by R. solanacearum added into the soil is a more demanding pro-
cess than that when the foliar pathogens are sprayed directly onto leaf surfaces
(49). Piliation probably contributes to pathogenesis both during invasion of roots
by promoting adherence to and colonization of root surfaces, migration to wound
sites and when the bacterium is inside the plant. Interestingly, biogenesis of Tfp
pili in R. solanacearum is subjected to a stringent regulatory control dependent on
PhcA, a master regulator that is itself controlled by a unique autoinduction system
(for a review, see 70).
Analysis of the GMI1000 genome provides strong presumptions that
R. solanacearum produces multiple pili/fimbriae structures. In addition to Tfp,
some of the recently identified Type II secretion systems (see above) are predicted
to produce Type II pseudopili, bundled type of pili that confer increased adhesive
capabilities in the case of P. aeruginosa (29). GMI1000 also possess two dis-
tinct tad-related gene clusters (RSc0649-0661 and RSp1082-1092), widespread
genomic islands in many pathogenic or soil Proteobacteria (61). tad genes in Acti-
nobacillus actinomycetemcomitans were shown to produce another type of bundled
pili that mediate adherence and are required for tenacious biofilm formation (61).
There is indeed suspicion that R. solanacearum is able to form biofilms in the
plant on host xylem vessel walls; these specialized aggregates may protect the
pathogen from host defenses and help bacterial survival during latent infections
and saprophytic life.
More than 15 ORFs in GMI1000 code for large proteins containing variable in-
ternal repeats typically found in many bacterial nonfimbrial adhesin/hemagglutinin
molecules. One ORF (RSp1620) encodes a protein similar to various bacterial
adhesins that promote tight adhesion to mammalian cells; in plant pathogenic
bacteria, similar adhesins were also shown to be involved in aggregation and at-
tachment to host surfaces (66) but their contribution to virulence was minor (64)
or detectable only on specific plant species using in vitro pathogenicity assays
(66). Because adhesin-like molecules appear to be widely distributed among var-
ious nonpathogenic soil Proteobacteria, it seems plausible that most of these cell
surface-associated determinants are general attachment factors useful in the soil
environment or for plant/rhizosphere interactions. A similar hypothesis can be
proposed for three sugar-binding lectins recently identified in GMI1000 and that
were characterized biochemically (75, 76; A. Imberty, personal communication).
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RALSTONIA SOLANACEARUM GENOME 119
EVOLUTION OF VIRULENCE
The sequencing and the subsequent comparison of complete bacterial genomes
has dramatically changed our vision on the notion of bacterial species and the
processes that govern evolution of these organisms. Microbial genomes are in
a constant state of flux, whereby new genes are acquired by horizontal transfer
and preexisting genes are lost by mutation (59). The mutational mechanisms pos-
sessed by pathogenic bacteria can lead, in certain circumstances, to rapid adaptive
changes. These changes often include pathogenicity traits such as phase variation
or adaptation of the parasite to new hosts (52). In the second part of this review, we
present some features of the GM1000 genome that indicate the possible contribu-
tion of such processes to the rapid adaptation of the bacterium to its environment
in general and to interaction with plants in particular.
Evolution of hrp Gene Clusters
A high degree of similarity exists between TTSS proteins and flagellar proteins, and
it has often been suggested that TTSS genes evolved from genes encoding flagellar
proteins (13, 86). However, a recent phylogenetic analysis indicates that although
TTSS genes and the flagellar export mechanism share a common ancestor, they
each evolved independently (35). Surprisingly, Gophna and coworkers concluded
that the divergence between TTSS and flagella is very ancient, probably before
the appearance of the first multicellular eukaryotes on the evolutionary stage (35).
This finding raises the probability that the historical roles played by ancestral TTSS
proteins might have been radically different from the host-bacterial interactions
mediated by current Type III secretion systems.
Most TTSS gene clusters from plant pathogens are located within pathogenicity
islands (PAI) and therefore present the features of DNA regions that are mobile (6)
or plausibly acquired from LGT (3, 4, 13). Although a precise definition of a PAI
is elusive, these regions are usually characterized by a G +C content that differs
from the rest of the genome and by the presence of insertion sequence elements,
tRNA genes, and/or genes for integrases and transposases (37). For example, the
Hrp PAI in P. syringae was shown to be composed of a tripartite mosaic structure
in which the TTSS structural genes are surrounded by regions that carry effector
genes (3). The right-hand region appears to be relatively conserved in size and
gene content among the P. syringae species and was named CEL (for conserved
effector locus), whereas the left-hand region was highly variable and was named
EEL (exchangeable effector locus) (3).
Long Coevolution in R. solanacearum of the hrp Gene Cluster
with the Core Genome
Unlike most other plant pathogens, several observations support the view that
the GMI1000 Hrp region is ancient and was not acquired from a recent LGT
event. First, the GMI1000 hrp cluster and surrounding regions do not exhibit the
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120 GENIN BOUCHER
classic compositional features of PAI: There is no bordering tRNA, no major G +
C content variation over an 80-kb region encompassing the hrp gene cluster and
flanking regions, and the single transposable element found in this region is lo-
cated 36 kb upstream of the hrp cluster left border. This apparent lack of evi-
dence for a LGT event is especially striking when comparing the GMI1000 TTSS
gene cluster with the related ones in other pathogens. Based on similarities in
hrp gene organization and regulation, plant pathogenic bacteria have been clas-
sified into two groups, group I (Erwinia amylovora and P. syringae) and group
II (R. solanacearum and species of Xanthomonas). Figure 1 shows a compar-
ison of TTSS genes and surrounding regions from several xanthomonads, R.
solanacearum GMI1000, and Burkholderia pseudomallei,ahuman pathogen that
possesses a TTSS homologous to those of phytopathogens of group II (63). In
all these pathogens, the TTSS core gene cluster is remarkably well conserved in
terms of colinearity and overall sequence similarity, the only exceptions being:
(a) the regulatory hrpB gene, which is absent in all hrp clusters known to date in
xanthomonads and (b) the last gene of the transcription unit 4, which is dissimi-
lar between species (hrpX/hrpD6/ORF3). Recent phylogenetic studies support the
presumption that LGT of the TTSS gene cluster occurred between these species
and further suggest that Xanthomonas was more likely to be the recipient than the
donor (35).
As for P. syringae, the TTSS gene cluster region from Xanthomonas species
and B. pseudomallei satisfies the general criteria for a PAI (presence of bordering
tRNA and/or mobile genetic elements, several low G +C content genes). Ex-
amination of these upstream regions shows obvious examples of a “recent” LGT
event, such as the xopD gene in X. campestris pv. vesicatoria, which carries a hrp
box in its promoter, suggesting acquisition from a plant pathogen from Group I
(57). Another example is provided by the insertion in X. campestris pv. campestris
of a set of five genes (including two transposases and two TTSS-effector homo-
logues) within a conserved Xanthomonas ORF that borders the left-hand side of
the Hrp PAI (Figure 1). In comparison, the only data available on the variability
of the hrp flanking regions in R. solanacearum relate to the sequence analysis
of the left-hand border of another strain and reveal a high level of conservation:
The PopABC effector proteins, as deduced from the sequence in strain OE1-1 (race
1, biovar 1) are 95% to 99% homologous to those of GMI1000 (race 1, biovar 3)
(41). Such a high level of identity tends therefore to suggest that the hrp region in
R. solanacearum is ancient and stabilized.
The Hrp Region in R. solanacearum:AClustering
of Different Classes of Genes Activated in Planta?
A major difference in the Hrp region between R. solanacearum and xanthomonads
lies in the linkage of a set of TTSS regulatory genes to the TTSS core secretion
genes in GMI1000. These regulatory genes, in particular those encoding the PrhA-
I-R module, are involved in the strong activation of hrp genes when bacteria are
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RALSTONIA SOLANACEARUM GENOME 121
in contact with the plant cell wall (2, 11). A peculiarity of the PrhAIR plant sens-
ing/signal transduction module is that the corresponding genes are not organized
in the operon, unlike other similar signal transfer systems described to date: prhA
lies in the left border of the hrp gene cluster and prhIR are located on the right-hand
side of the cluster (Figure 1). This observation raised speculation that the TTSS
core secretion genes could have been recruited into the pre-existing operon of a
primeval pathogen already possessing a plant detection system (11). Alternatively,
the prhA and prhIR genes may have been recruited separately, but the former of
these two hypotheses seems more likely, both statistically (it requires one event)
and conceptually (the ability to detect plant cells would precede the recruitment
of a pathogenicity effector injection system).
In the close vicinity to the GMI1000 TTSS gene cluster are also found genes that
encode proteins secreted by the Type II secretion system, such as PglA, an endo-
polygalacturonase (PG) (see Figure 1), and PehC, an exo-PG that lies 25 kb to the
right-hand of the hrp gene cluster together with genes involved in the uptake and
metabolism of galacturonate, the main degradation product of pectin by PGs (34).
Another gene found upstream of pglA (RSp0881) also possesses structural features
found in some plant cell wall–degrading enzymes. The composite GMI1000 Hrp
region therefore appears to regroup TTSS-secretion, regulation and effector genes,
as well as other virulence genes, all activated during the plant infection process.
This clustering of genes responsible for related, but not identical, functions is
also in agreement with the hypothesis that the Hrp region in R. solanacearum is
probably more ancestral than previously thought. As proposed by Lawrence &
Roth in their selfish operon model (48), natural selection may have driven such
clustering of genes that mutually affect host fitness via relatively ancient and
multistep acquisition processes. Genomic sequences of hrp-flanking regions in
other strains of R. solanacearum will soon provide complementary information
on the evolutionary status of the Hrp region.
Evolution of TTSS-Effector Genes
TWO MAIN CLASSES OF TTSS-EFFECTOR GENES To date, the pool of GMI1000
TTSS-effector genes comprises at least 46 genes (9 TTSS-substrates +35 hrpB-
regulated genes encoding products with N-terminal features predictive of TTSS-
exported proteins) (22). When looking at the distribution of these effector genes
in the genome, several are found, not surprisingly, in the vicinity of the hrp gene
cluster (Figure 1), but most appear to be scattered on both replicons, although they
are more important in frequency on the megaplasmid (26 genes) than on the chro-
mosome (20 genes). TTSS-effector genes can be divided into two classes: those
probably acquired by LGT (low G +C content, unusual codon usage, association
with mobile genetic elements or bacteriophage sequences) and those, in contrast,
that look like “core genome” genes (i.e., ancestral genes or acquired through an-
cient LGT). Surprisingly, the first category comprises fewer genes (7/46) includ-
ing popP1 and popP2, both avrRxv/yopP family members (24, 47), ripT (yopT
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122 GENIN BOUCHER
family) (22), RSp0572 (hopPtoH homologue), and RSp0213 (related to Pantoea
agglomerans pthG). As observed for the TTSS-associated genes in the hrp region,
a large proportion of effector genes (39/46) have apparently been subjected to a
long coevolution with the rest of the genome. There are in the GMI1000 genome
many examples of nonclustered TTSS-effectors found as single genetic units bor-
dered by typical housekeeping genes (such as, among others, RSc1349, RSp0323,
RSp1388 or RSc3369, an avrPphE family member). We hypothesize that this sub-
group represents the pool of ancient TTSS-effectors in R. solanacearum that were
mobilized to promote disease, presumably by suppressing primeval plant defense
mechanisms in response to pathogen-associated molecular patterns (58). How-
ever, the determination of ancestry of an effector gene is difficult to establish and,
again, this problem will be simplified when genome sequences for other strains of
R. solanacearum become available and allow an in-depth phylogenetic analysis
for each gene.
Approximately 20 GMI1000 TTSS-effectors belong to effector families that
are common in the epiphytic plant pathogens P. syringae and Xanthomonas sp.
(Table 3). In some cases (such as PopP2 or RipT), the level of identity with pro-
teins from other species is rather low (21–27%), suggesting that, despite a prob-
able similar biochemical activity (cysteine protease), the biological substrates of
these effectors and consequently their cellular effect on host cells might be very
different. In other cases, the higher level of gene product conservation between
species suggests a more conserved biological role. Among the most conserved and
widespread TTSS-effectors are the HopPtoG and HopPtoQ-homologues; despite
a wide difference in the G +C content of the respective genes in P. syringae and
R. solanacearum (18%), these exhibit a high degree of identity at the amino acid
level (>45%) over the complete protein sequence (Table 3). This observation al-
lows us to speculate that such effectors have been subjected to convergent evolution
toward an identical or similar host cell target.
Although the number of complete genome sequences of plant pathogens is
still relatively scarce and allows only preliminary conclusions to be drawn about
comparative genomics, it seems probable that several TTSS-effector gene families
are unique to a given species. For example, R. solanacearum GMI1000 possesses
>20 candidate TTSS-effectors that exhibit no homology to other known bacterial
proteins (22), inferring that the evolutionary driving force resulted in the spreading
via LGT and the stabilization of only a subgroup of TTSS-effector genes in recip-
ient species. The preservation of such genes may be favored in recipient strains
if they provide a valuable and short-term adaptive function, such as the specific
suppression of a host defense response triggered by the recognition of another
TTSS-effector (1, 10a, 31, 81). Another speculative view would be to consider the
high number of TTSS-effector genes maintained in R. solanacearum as a poten-
tial reservoir of genetic resources that can be mobilized to create novel effector
specificity either through mutational changes or by generating chimeric proteins.
An example of the latter case in the GMI1000 genome is provided by RSp0213,
which presents a modular structure with an N-terminal domain related to the
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RALSTONIA SOLANACEARUM GENOME 123
TABLE 3 Common TTSS-effector families in R. solanacearum GMI1000 and four epiphytic plant pathogens
R.s
Effector GMI1000 XcvaXccbXaccPstd
family mGC% =67emGC% =64 mGC% =65 mGC% =65 mGC% =58
AvrPphD RSp0304 XopB — — HopPtoD1
GC% =64 I =33%f,L=92gI=36%, L =78
GC% =56 GC% =55
AvrPphE RSc3369 nk∗XCC1629 XAC3224 AvrPphE
GC% =68 I =27%, L =47 I =25%, L =70 I =26%, L =83
GC% =62 GC% =60 GC% =54
XCC1246 XAC0286
I=28%, L =33 I =26%, L =71
GC% =50 GC% =64
XACb0011
I=25%, L =43
GC% =61
AvrPphF RSp0822 nk — — HopPtoF
GC% =69 I =36%, L =56
GC% =48
RSp0323 nk XCC3600 — HopPtoG
GC% =62 I =46%, L =91 I =53%, L =92
GC% =57 GC% =44
RSc3290 nk XCC3258 — HopPtoH
GC% =59 I =36%, L =100 I =45%, L =100
GC% =51 GC% =47
RSp0572
GC% =58
(Continued)
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124 GENIN BOUCHER
TABLE 3 (Continued)
R.s
Effector GMI1000 XcvaXccbXaccPstd
family mGC% =67emGC% =64 mGC% =65 mGC% =65 mGC% =58
RSp1281 nk XCC1089 — HolPtoR
GC% =71 I =26%, L =84 I =26%, L =85
GC% =65 GC% =59
RipB nk XCC1072 XAC4333 HopPtoQ
GC% =70 I =47%, L =82 I =47%, L =79 I =47%, L =84
GC% =63 GC% =68 GC% =51
AvrRxv/YopP PopP1 AvrXv4#XCC3731 — —
GC% =57 I =57%, L =100 I =23%, L =70
GC% =48 GC% =60
AvrRxv/YopP PopP2 XopJ XCC3731 — —
GC% =60 I =27%, L =60 I =21%, L =36
GC% =57 GC% =60
AvrBs3 RSc1815 AvrBs3#— PthA2†—
GC% =66 I =49%, L =70 I =47%, L% =74
GC% =67 GC% =66.5
LRR-PopC PopC HpaG — HpaF —
GC% =64 I =34%, L =30 I =38%, L% =37
RSp0842 GC% =64 GC% =63.8
GC% =72
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RALSTONIA SOLANACEARUM GENOME 125
HLK RSc1386 nk XCC1247 XAC1208 —
GC% =69 I =34%, L =93 I =31%, L =92
RSp0160 GC% =57 GC% =62
GC% =68
RSp0215
GC% =68
HopPtoA RSp1277 nk——HopPtoA1
GC% =69 I =36%, L =63
GC% =61
HopPtoA2
I=35%, L =63
GC% =57
RSp1239 XopC — — —
GC% =67 I =46%, L =40
GC% =47
Abbreviations: (a)Xanthomonas campestris pv. vesicatoria 85–10, (b)X. campestris pv. campestris ATCC 33913, (c)Xanthomonas axonopodis pv. citri 306,
(d)Pseudomonas syringae pv. tomato DC3000. (e)mean genomic G +C content, ( f) identity level (BLAST result), (g)%of the length of the GMI1000 protein sequence aligned
with the query sequence. ∗nk, not known. †Three other pthA-related genes located on plasmids are present in this strain. #Genes cloned from X.c.v. strains 91–118 (avrRxv4)or
71–21 (avrBs3).
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126 GENIN BOUCHER
P. agglomerans PthG protein N termini and a C-terminal domain that does not
resemble any of the proteins previously identified.
THE AvrBS3 EFFECTOR FAMILY A striking illustration on the evolution of TTSS-
effector genes is provided by the AvrBS3 family members (reviewed in 85). The
GMI1000 RSc1815 gene encodes the first member of the avrBs3 gene family
found to date outside of the genus Xanthomonas (68). AvrBs3 gene family mem-
bers are very similar in structure but differ in the number and order of a series
of nearly identical 102-bp direct repeats in the center of the open reading frame.
The RSc1815 gene product possesses 17.5 internal repeats in which the struc-
ture specifically differs from that of all other members of the AvrBs3 family on
two main points: (a) Each repeat encodes a 35-amino acid segment (instead of
34), and (b) although the first half of each amino acid repeat is identical to the
AvrBs3-type, the second half appears to have evolved divergently, as reflected by
a high degree of sequence dissimilarity (Figure 2). This observation has two im-
plications: First, it strongly suggests that the RSc1815 repeats arose by successive
duplications of an initial variant repeat rather than as the result of a recent gene
transfer process from Xanthomonas species; second, it shows that AvrBs3 internal
repeats have a modular structure, which may have important consequences for
structure/function of these repeats. Interestingly, the dissimilar C-terminal halves
of the AvrBs3/RSc1815 repeats share conserved leucine residues arranged in a
manner reminiscent of leucine-rich-repeat motifs (LxxLxxxxxL) (Figure 2). Fi-
nally, the RSc1815 gene product possesses potential nuclear localization signals
but unlike most other members of the AvrBs3 family, does not contain in its C-
terminal domain the acidic domain that is presumed to function in plant cell nuclei
as a transcription activation domain (77, 85). Disruption of the GMI1000 RSc1815
gene did not result in significant change during interactions with the plants tested
(22).
Mutation, Elimination and (Reversible)
Inactivation of Virulence Genes
GENE INACTIVATION An important concept emerging from the genome analyses
of bacterial pathogens is that deletion of genes could serve as means of bac-
terial adaptation (59). In the case of plant pathogens, evolutionary models pre-
dict that without any virulence function of the Avr factor, the selection pressure
on the pathogen imposed by the plant population that acquires the matching R
gene will result in selection for loss or mutated versions of the avr gene. Ex-
amination of the GMI1000 genome provides multiple instances of evidence of
potential virulence genes that have been either transiently inactivated or lost. For
example, homologues of avr genes (RSc0227/0228 and RSc0582, homologous
to avrRpm1 and avrD, respectively) of P. syringae have frameshift(s) in their
coding sequence. Another mode of inactivation, but potentially reversible, is de-
termined by transposable elements: This is the case, for example, for two genes
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RALSTONIA SOLANACEARUM GENOME 127
encoding RTX-like toxins (RSc0102/0104 and RSc0246/0249) and two genes en-
coding probable TTSS-effectors (RSc3241/3243, corresponding to popP3,athird
avrRxv/yopP family member, and RSp1218/1215, predicted to encode a protein
with ankyrin-repeats). Interestingly, a functional version of popP3 has been iden-
tified in nine other strains of R. solanacearum, showing that transposons could
contribute to turning pathogenicity-related genes on and off. A similar situation
was found for the popP1 gene that is active in GMI1000 but inactivated in 2 of the
other 13 strains investigated (46).
Reversible inactivation of a virulence gene can be nicely illustrated with the
mechanisms regulating phase variation in R. solanacearum through the genetic
alteration of the phcA gene. phcA encodes a LysR-type transcriptional regulator that
controls several major physiological processes (including pathogenicity) during
the bacterium’s life cycle (reviewed in 70). The activity of PhcA is controlled
by an atypical quorum sensing signal molecule (32) but the so-called phenotypic
conversion from a wild-type pathogenic to a nonpathogenic and highly motile form
can also be the consequence of a broad array of mutational events within the phcA
gene (insertion of transposable elements, deletions, base substitution or insertion of
tandem duplications) (62). In some cases, reversion events from the nonpathogenic
to the pathogenic form is specifically detected in planta, thus suggesting that the
rate of phenotypic switching can be modulated by environmental factors (62).
CONTINGENCY LOCI There is multiple proof that pathogenic bacteria have evolved
mechanisms for increasing the frequency of random variations in those genes that
are involved in critical interactions with their hosts (52, 54). Having elevated mu-
tation rates in a specific subset of genes may be highly advantageous, allowing
certain phenotypic traits to respond rapidly, by natural selection, to unpredictable
changes in the environment. In certain pathogenic prokaryotes, such mutational
mechanisms are dependent on the presence of functional and hypervariable mi-
crosatellites (also called contingency loci), which are prone to high mutation rate
through the process of replication slippage (50, 54). The term of “adaptive evo-
lution” is used to describe this ability of bacteria to have high variability (high
mutation rate) in certain contingency genes, but low variability (normal mutation
rates) in “normal” housekeeping genes (54).
We have identified in the GMI1000 genome several candidate contingency loci
that harbor different classes of simple sequences repeats (SSR). One of these loci
corresponds to one of the two tad gene clusters that direct the biosynthesis of a
type of bundled pili possibly involved in biofilm formation (61, see above). The
tad operon promoter is characterized by a relative abundance of GCG nucleotide
triplets and contains 6 contiguous GCG repeats 42 base pairs upstream of the
start codon of the first gene of the operon (RSp1087). This promoter structure is
reminiscent of those determining the production of phase-variant fimbriae in other
bacterial pathogens and which are controlled by a repeat sequence tract (50, 54).
SSR were also found associated to two TTSS-effector genes. In one, RSp1218
(which also carries the insertion of a tranposable element), 10 tandem repeats of
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128 GENIN BOUCHER
a 9-nucleotide element are found between the hrpII box control element and the
translation start codon, suggesting that these SSR could alter or modulate gene
expression. In the second case, RSp0215 (HLK effector family), 6 tandem repeats
of a 9-nucleotide element are found within the coding sequence. The biological
significance of such contingency repeats in a TTSS-effector gene is unclear and
needs to be tested experimentally. However, an attractive hypothesis, based on the
fact that forward and back mutation rates can approach equality at contingency loci,
is that these mutations cause the genes that carry SSR to switch rapidly between
functional and nonfunctional states (52). Such a mechanism could be used by the
pathogen to counter host defense mechanisms at various stages of the infection
process.
Finally, note that bacteria are not clonal populations in nature. The combina-
tion of LGT events, gene inactivation processes, and the creation of variation by
mutation in the recurrent generation of diversity may therefore create a more dy-
namic distribution of functional virulence factors in the bacterial population than
what can be deduced from a snapshot of a genome sequence. R. solanacearum
GMI1000 is an unusual wide host range pathogen and in this regard such gene
acquisition/inactivation mechanisms could lead to the natural selection for indi-
viduals that acquired or combined genes that confer the higher fitness on a given
host.
CONCLUDING REMARKS
As highlighted by Mergeay et al. (51), the genus Ralstonia is remarkably diverse
in terms of ecological niches and specialization or capabilities: plant pathogenesis
(R. solanacearum), nodulation and nitrogen fixation in tropical legumes (R. tai-
wanensis) (16), nosocomial infection (R. picketti,gilardii,paucula,respiraculi)
(18), hydrogenotrophy (R. eutropha), colonization of metal-rich biotopes (R. met-
allidurans), and degradation of recalcitrant aromatic compounds and man-made
chemicals (R. oxalitica,eutropha,basilensis). The genomic era is now opening
up the possibility of identifying the distinct set of genes, acquired or lost dur-
ing evolution, that are involved in the specialization of these microorganisms to
radically different environmental niches. The first information should soon be
available from comparative genomics as other Ralstonia sequencing projects near
completion (R. metallidurans,eutropha). In addition, the use of DNA microarrays
should theoretically provide a means to identify a core of common genes present
in infectious (pathogens or symbionts) Ralstonia and to study the distribution of
pathogenicity-specific genes within various species.
Also important will be the genome sequence analysis of other strains of
R. solanacearum to estimate the extent of genomic diversity within the species,
adiversity that is presumably rather high considering the ecological versatility
of the bacterium and its natural ability for competence. This will constitute a first
step in establishing a catalogue of virulence genes (such as TTSS-effectors) within
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RALSTONIA SOLANACEARUM GENOME 129
the species and in studying their distribution among a representative ensemble of
R. solanacearum strains.
With >15 regulatory genes identified to date as playing a role in the control of
pathogenicity (33, 70), R. solanacearum is one of the best known plant pathogenic
bacteria in terms of regulation of pathogenicity. Transcriptomic analyses using
such regulatory mutants should identify groups of target genes contributing to
pathogenicity. All together, these genome-based approaches will lead to a far better
global picture of the multiple determinants contributing to bacterial wilt disease.
The Annual Review of Phytopathology is online at http://phyto.annualreviews.org
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RALSTONIA SOLANACEARUM GENOME C-1
Figure 1 Schematic of the hrp gene clusters and flanking regions in plant
pathogens from group II (R. solanacearum GMI1000, Rs, and Xanthomonas
species) and the bps gene cluster in Burkholderia pseudomallei (Bps). Abbreviations
of Xanthomonas species and pathovars: Xanthomonas campestris pv. vesicatoria
(Xcv) and pv. campestris (Xcc); Xanthomonas axonopodis pv. campestris (Xac) and
pv. glycines (Xag). The conserved hrp/bps gene clusters are symbolized by red
boxes containing genes (small red arrows) organized in four transcriptional units. In
the flanking regions, large arrows represent genes with the direction of transcription.
Red indicates genes encoding TTSS-substrates or TTSS-associated proteins; the
blue label is for TTSS regulatory genes. Purple indicates two genes encoding known
or probable substrates of the General Secretion Pathway (PglA: endo-polygalactur-
onase). Transposable elements are shown in green (Tnp: transposase; green triangles
figure transposase remnants; the green rectangle indicates an insertion sequence).
Black arrows correspond to genes encoding unknown hypothetical proteins (empty
arrows) or proteins with an assigned cellular function (filled arrows). Arrows filled
with identical motifs indicate that the corresponding genes exhibit significant
homology or similar functional role between species. R. solanacearum brg genes
correspond to experimentally demonstrated hrpB-regulated genes. The dotted line
delineates the insertion of a block of five genes in Xcc within the sequence of a gene
homologous to Xac/Xcv XAC418/ORF1 genes, respectively (XCC1246: avrPphE-
homolog, XCC1247: encodes a protein related to the GMI1000 HLK family of
TTSS-effectors). HrpY/HrpE1/HrpE: major subunit of the Hrp pilus, HrpF: TTSS-
translocon protein. XCC1242/3 corresponds to a pseudogene, PopC/HpaF/HpaG
belong to a family of LRR-containing proteins. Data compiled from References 13,
22, 23, 45, 63.
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C-2 GENIN ■BOUCHER
Figure 2 AvrBs3 family members contain a series of 2 ×17 conserved amino acid
tandem repeats. Comparison of the amino acid repeat units (here numbers 3 to 7) of
Xanthomonas campestris pv. vesicatoria AvrBs3 (positions 360–529) and R. solana-
cearum GMI1000 RSc1815 (positions 393–567). Residues colored in red are con-
served between the two types of repeats; in blue or green, the residues are conserved
only in the AvrBs3 or RSc1815 repeats, respectively. The asterisks denote the vari-
able twelfth and thirteenth positions of the AvrBs3 repeat units (85), which also cor-
respond to amino acid variation positions in RSc1815.
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P1: LDI
July 1, 2004 23:3 Annual Reviews AR221-FM
Annual Review of Phytopathology
Volume 42, 2004
CONTENTS
FRONTISPIECE,Anne K. Vidaver x
THE ACCIDENTAL PLANT PATHOLOGIST,Anne K. Vidaver 1
TOBACCO MOSAIC VIRUS:AMODEL SYSTEM FOR PLANT BIOLOGY,
Karen-Beth G. Scholthof 13
ASSESSMENT AND MANAGEMENT OF SOIL MICROBIAL COMMUNITY
STRUCTURE FOR DISEASE SUPPRESSION,Mark Mazzola 35
ANALYSIS OF DISEASE PROGRESS AS A BASIS FOR EVALUATING DISEASE
MANAGEMENT PRACTICES,M.J. Jeger 61
EVOLUTION OF PLANT PARASITISM AMONG NEMATODES,J.G. Baldwin,
S.A. Nadler, and B.J. Adams 83
LESSONS LEARNED FROM THE GENOME ANALYSIS OF RALSTONIA
SOLANACEARUM,St´
ephane Genin and Christian Boucher 107
MANAGEMENT AND RESISTANCE IN WHEAT AND BARLEY TO FUSARIUM
HEAD BLIGHT,Guihua Bai and Gregory Shaner 135
COMPARATIVE GENOMICS ANALYSES OF CITRUS-ASSOCIATED BACTERIA,
Leandro M. Moreira, Robson F. de Souza, Nalvo F. Almeida Jr.,
Jo˜
ao C. Setubal, Julio Cezar F. Oliveira, Luiz R. Furlan,
Jesus A. Ferro, and Ana C.R. da Silva 163
SYSTEMIC ACQUIRED RESISTANCE,W.E. Durrant and X. Dong 185
MOLECULAR ASPECTS OF PLANT VIRUS TRANSMISSION BY OLPIDIUM AND
PLASMODIOPHORID VECTORS,D’Ann Rochon, Kishore Kakani,
Marjorie Robbins, and Ron Reade 211
MICROBIAL DIVERSITY IN SOIL:SELECTION OF MICROBIAL POPULATIONS BY
PLANT AND SOIL TYPE AND IMPLICATIONS FOR DISEASE SUPPRESSIVENESS,
P. Garbeva, J.A. van Veen, and J.D. van Elsas 243
MICROBIAL DYNAMICS AND INTERACTIONS IN THE SPERMOSPHERE,
Eric B. Nelson 271
BIOLOGICAL CONTROL OF CHESTNUT BLIGHT WITH HYPOVIRULENCE:
AC
RITICAL ANALYSIS,Michael G. Milgroom and Paolo Cortesi 311
INTEGRATED APPROACHES FOR DETECTION OF PLANT PATHOGENIC
BACTERIA AND DIAGNOSIS OF BACTERIAL DISEASES,
Anne M. Alvarez 339
v
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P1: LDI
July 1, 2004 23:3 Annual Reviews AR221-FM
vi CONTENTS
NEMATODE MOLECULAR DIAGNOSTICS:FROM BANDS TO BARCODES,
Tom Powers 367
TYPE III SECRETION SYSTEM EFFECTOR PROTEINS:DOUBLE AGENTS IN
BACTERIAL DISEASE AND PLANT DEFENSE,Allan Collmer
and James R. Alfano 385
PLANT VIRUS SATELLITE AND DEFECTIVE INTERFERING RNAS:NEW
PARADIGMS FOR A NEW CENTURY,Anne E. Simon, Marilyn J. Roossinck,
and Zolt´
an Havelda 415
CHEMICAL BIOLOGY OF MULTI-HOST/PATHOGEN INTERACTIONS:CHEMICAL
PERCEPTION AND METABOLIC COMPLEMENTATION,Andrew G. Palmer,
Rong Gao, Justin Maresh, W. Kaya Erbil, and David G. Lynn 439
INDEX
Subject Index 465
ERRATA
An online log of corrections to Annual Review of Phytopathology chapters
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