From Genes To Genomes

MOLECULAR PLANT PATHOLOGY (2003) 4(1), 1730
Pathogen profile
Blackwell Science, Ltd
Soft rot erwiniae
Soft rot erwiniae: from genes to genomes

I A N K . TO T H * , KE N N E T H S. B E L L , M A R I A C. H O L E VA A N D PA U L R . J. B I R C H
Plant-Pathogen Interactions Programme, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK
SUMMARY
The soft rot erwiniae, Erwinia carotovora ssp. atroseptica (Eca), E.
carotovora ssp. carotovora (Ecc) and E. chrysanthemi (Ech) are
major bacterial pathogens of potato and other crops world-wide.
We currently understand much about how these bacteria attack
plants and protect themselves against plant defences. However,
the processes underlying the establishment of infection, differences in host range and their ability to survive when not causing
disease, largely remain a mystery. This review will focus on our
current knowledge of pathogenesis in these organisms and discuss how modern genomic approaches, including complete
genome sequencing of Eca and Ech, may open the door to a new
understanding of the potential subtlety and complexity of soft rot
erwiniae and their interactions with plants.
Taxonomy: The soft rot erwiniae are members of the Enterobacteriaceae, along with other plant pathogens such as Erwinia
amylovora and human pathogens such as Escherichia coli ,
Salmonella spp. and Yersinia spp. Although the genus name Erwinia
is most often used to describe the group, an alternative genus name
Pectobacterium was recently proposed for the soft rot species.
Host Range: Ech mainly affects crops and other plants in
tropical and subtropical regions and has a wide host range that
includes potato and the important model host African violet
(Saintpaulia ionantha). Ecc affects crops and other plants in subtropical and temperate regions and has probably the widest host
range, which also includes potato. Eca, on the other hand, has a
host range limited almost exclusively to potato in temperate
regions only.
Disease Symptoms: Soft rot erwiniae cause general tissue
maceration, termed soft rot disease, through the production of
plant cell wall degrading enzymes. Environmental factors such as
temperature, low oxygen concentration and free water play an
essential role in disease development. On potato, and possibly
other plants, disease symptoms may differ, e.g. blackleg disease
is associated more with Eca and Ech than with Ecc.
Useful Websites: http://www.scri.sari.ac.uk/TiPP/Erwinia.htm,
http://www.ahabs.wisc.edu:16080/pernalab/erwinia/index.htm,
*Correspondence: E-mail: itoth@scri.sari.ac.uk
2003 BLACKWELL PUBLISHING LTD
http://www.tigr.org/tdb/mdb/mdbinprogress.html,
http://www.sanger.ac.uk/Projects/E_carotovora/.
I N T RO D U C T I O N
The soft rot erwiniae are pathogens of many plant species, affecting crops in temperate to tropical regions world-wide. Eca has a
narrow host range restricted almost exclusively to potato in temperate regions. Ech is more frequent in subtropical and tropical
climates and has a host range that includes carnation, leopold
lily, maize, pineapple, potato and African violet (Saintpaulia ionantha), the latter of which has been used extensively as a model
system for research. Ech also causes disease on certain crops and
other plants in temperate regions, e.g. dahlia and potato. Ecc
mainly affects crops in subtropical and temperate regions and
has probably the widest host range, including Brussels sprout,
carrot, celery, cucumber, capsicum, turnip, chicory and potato.
However, many other crops are rotted by these pathogens postharvest (for reviews see Prombelon and Kelman, 1980; Prombelon and Salmond, 1995). Ech and Ecc are phenotypically and
genetically more diverse than Eca and, in some cases, different
groups of Ecc and Ech can be related to geographical location
and, in the case of Ech, host range (Avrova et al., 2002; Boccara
et al., 1991; Nassar et al., 1994a, 1996; Smith and Bartz, 1990;
Toth et al., 1999a).
When not causing disease, the soft rot erwiniae appear to
undergo endophytic, epiphytic and saprophytic lifestyles in
plants, on plant surfaces and in the soil and ground water, respectively (Prombelon and Kelman, 1980; Prombelon and Salmond,
1995). However, little is known about these alternative life-styles.
For example, during the period when Erwinia cells are present in
intercellular spaces within the plant, but before infection is initiated, a period that can last for several months, does the pathogen
lie dormant and unrecognized by the plant or is there a dynamic
process of bacterial cell growth countered by plant defences? Do
the erwiniae attach to plant cells or is infection initiated from
free-living bacteria? Ecc is virtually ubiquitous in temperate soils,
while Eca is often difficult to isolate: Does the wider host range
of Ecc and its greater genetic diversity assist in this survival, and
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I. K. TOTH et al.
is it able to compete better with other micro-organisms through

the production of anti-microbial compounds?
Pathogenicity determinants of these bacteria have been
studied for over 80 years, but in the last 20 years, with the impact
of molecular approaches, significant progress has been made
in understanding disease processes. However, more questions
remain unanswered. For example, what are the early signalling
events between pathogen and plant that allow the disease process to begin? Do soft rot erwiniae translocate proteins into plant
cells that interfere with the resistance process (virulence genes)
and are these proteins recognized by non-hosts to trigger the
hypersensitive response (HR), a ubiquitous, localized, programmed cell death that prevents further spread of the pathogen? This article provides an overview of our current knowledge
on soft rot erwinia pathogenicity, but we also look to the future
and genomics, which may provide new insights into our understanding of many aspects of the biology of these pathogens.
TA X O N O M Y
The genus Erwinia was first described in 1917 to encapsulate all
members of the Enterobacteriaceae that cause disease on plants,
irrespective of their relatedness to other members of the family
(Prombelon, 1990). Over the years this has caused many
nomenclatural difficulties and has led to the relocation of various
species into other genera, notably E. stewartii to Pantoea stewartii (Mergaert et al., 1993), E. herbicola to Pantoea agglomerans
(Gavini et al., 1989), E. dissolvens to Enterobacter dissolvens
(Brenner et al., 1986) and E. salicis to Brenneria salicis (Hauben
et al., 1998). It has also been suggested by Hauben et al. (1998),
on the basis of 16S rDNA sequence analysis, that the soft rot
erwinias be renamed Pectobacterium carotovorum ssp. atrosepticum (for Eca), Pectobacterium carotovorum ssp. carotovorum
(for Ecc) and Pectobacterium chrysanthemi (for Ech), supporting
an earlier proposal by Waldee (1945) to rename the group similarly. However, at present Pectobacterium has not been widely
adopted by the Erwinia research community.
T H E D I S E A S E P RO C E S S
The soft rot erwinias are found on plant surfaces and in soil where
they may enter the plant via wound sites or through natural
openings on the plant surface, e.g. lenticels. Once inside the plant
they reside in the vascular tissue and intercellular spaces of
suberized or thin-walled parenchymatous tissues (as found in
lenticels and wounds) where they remain until environmental
conditions, including free water, oxygen availability and temperature, become suitable for disease development (Prombelon
and Kelman, 1980; Prombelon and Salmond, 1995).
Free water is essential for optimal disease development, even
in suitable temperature and oxygen-limiting conditions, and may
have several functions. As motility has been linked to virulence in

Eca (Mulholland et al., 1993) and also appears to be co-regulated
with other virulence factors in Eca and Ech (Condemine et al.,
1999; Harris et al., 1998; Shih et al., 1999), free water may allow
bacterial cells to move more easily through plant tissue. An
increase in free water may also lead to a decrease in available
oxygen, creating a micro-aerobic or anaerobic environment
within the plant. This has little effect on the pathogens ability to
grow, but has a major effect on limiting oxygen-dependent
defences within the plant (Bolwell and Wojtaszek, 1997). It may
also lead to an increase in the turgidity of plant cells, with oxygen
deficiency affecting cell membrane integrity, together leading to
solute leakage and increased susceptibility to decay (Prombelon
and Lowe, 1975). In addition to free water and oxygen depletion,
temperature is an important factor in disease development, and
can influence which of the soft rot erwiniae cause disease. For
example, Prombelon et al. (1987a) showed that a soil temperature of 20 C was an important transition point, above which Eca,
and below which Ech, were not apparently pathogenic. The abilities of the soft rot erwiniae to grow at different temperatures is
also clearly demonstrated in vitro, where it is used to differentiate
the pathogens, i.e. at 27 C all three pathogens will grow, at
33.5 C only Ecc and Ech will grow and at 37 C only Ech will
grow (Prombelon et al., 1987b). However, in addition to differences in growth, a tight thermal regulation on the production of
cell wall degrading enzymes (exoenzymes) has been demonstrated (Lanham et al., 1991; Nguyen et al., 2002).
The big guns: plant cell wall degrading enzymes
The main weapon in the soft rot erwinia arsenal is the coordinated production of high levels of multiple exoenzymes, including pectinases, cellulases and proteases, which break down plant
cell walls and release nutrients for bacterial growth (for reviews
on exoenzymes see Barras et al., 1994; Prombelon, 2002; Py
et al., 1998; Thomson et al., 1999). Cellulases, which exhibit
mainly endoglucanase activity, break down cellulose in the primary and secondary cell walls of the host plant. There are at least
two cellulases in both Ech (CelZ, Y) and Ecc (CelV, S) and, while
not essential for pathogenicity, they do appear to act in synergy
with other exoenzymes of various classes to attack the plant
(Boccara et al., 1994; Boyer et al., 1984, 1987; Me et al., 1995;
Saarilahti et al., 1990; Walker et al., 1994). Several proteases in
Ech, and at least one in Ecc have also been described (Dahler
et al., 1990; Kysti et al., 1991). These may act either to provide
amino acids for biosynthesis of microbial proteins or degradation
of host proteins associated with resistance (Heilbronn and Lyon,
1990; Kysti et al., 1991) but, like cellulases, appear to play only
a minor role in pathogenesis (Marits et al., 1999).
Pectinases are the main exoenzymes involved in disease development. These exoenzymes break down and utilize pectins in the
MOLECULAR PLANT PATHOLOGY (2003) 4(1), 1730 2003 BLACKWELL PUBLISHING LTD
Soft rot erwiniae
middle lamella and plant cell walls, causing tissue collapse, cell
damage and cell leakage (Barras et al., 1994; Prombelon,
2002). Many of these pectinasespectate lyase (Pel), pectin
lyase (Pnl), pectin methyl esterase (Pme) and polygalacturonase
(Peh)exist in multiple forms (isoenzymes) encoded by independent genes that, in some cases at least, are clustered and
appear to be derived from successive rounds of gene duplication
(Barras et al., 1987; McMillan et al., 1994). While the exact
nature of this diversity is not known, there is some evidence to
support both a wide substrate diversity and independent regulation (Nachin and Barras, 2000). Pectate lyases (Pels) are the main
pectinases in pathogenesis and, as with other exoenzymes, their
number varies between species, subspecies and strains. There are
generally five major Pels in two families (Pel A, D, E and Pel B, C)
and at least four secondary Pels (Pel I, L, Z and X) in Ech, four
major Pels (Pel A, B, C and D) and other minor Pels in Ecc, and
three major pels in Eca (Pel A, B and C) (Barras et al., 1987; Beaulieu
et al., 1993; Hinton et al., 1989; Kelemu and Collmer, 1993; Lei
et al., 1985; McMillan et al., 1994; van Gijsegem, 1989; for review
see Thomson et al., 1999). The secondary Pels are only induced
in planta and regulated separately from those produced in minimal
medium containing pectate (Beaulieu et al., 1993; Kelemu and
Collmer, 1993), and although they have a low enzymic activity,
appear to have an important role in either infection or host specificity (Lowkowska et al., 1995; Pissavin et al., 1996; Shevchik
et al., 1997). Additional isoenzymes of Pel, Pnl, Pme and Peh are
also induced in minimal medium in the presence of both pectate
and pectin (McMillan et al., 1994).
Although the production of pectinases per se is essential for
pathogenicity, not all isoenzymes are required in all situations.
For example, in Ech the Pel A, D, E family plays a larger role in
pathogenicity than the Pel B, C family (Boccara et al., 1988).
Beaulieu et al. (1993) also showed in Ech that different pectinases
were required for virulence on different hosts, e.g. a mutation
in pelD reduced virulence in chicory leaves but not potato tubers,
while a mutation in pelE actually increased virulence on both
hosts.
When to arm and when to fireregulation and
secretion of exoenzymes
Although exoenzymes are not unique to the soft rot erwiniae,
being found in many saprophytes (Prombelon and Salmond,
1995), the ability of these erwiniae to co-ordinately produce large
amounts of these exoenzymes and target them extracellularly at
critical stages of infection makes them formidable pathogens.
This is accomplished through sophisticated regulatory networks
and secretion systems within the pathogen. It is not surprising
therefore that a considerable body of information is available on
both the regulation and secretion of these exoenzymes and other
pathogenicity factors.
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Regulation
Virulence determinants in the soft rot erwiniae are controlled by
complex, sometimes interrelated, regulatory networks, which act
either positively or negatively on one (targeted regulation) or several
(global regulation) determinants. They are stimulated by factors
such as oxygen and nitrogen availability, temperature, osmolarity,
iron deprivation, growth phase, catabolite repression, plant degradation intermediates, plant extracts, DNA-damaging agents and
very likely other factors yet to be identified. Newly identified regulatory proteins, and a better understanding of existing ones (e.g.
see Cui et al., 2001; Hyytiainen et al., 2001; Marits et al., 2002; Nachin
and Barras, 2000; Nasser and Reverchon, 2002; Nguyen et al.,
2002; Shih et al., 1999) continue to be added to the list of known
regulators that have been reviewed extensively elsewhere (for
reviews see Harris et al., 1998; Hugouvieux-Cotte-Pattat et al.,
1996; Thomson et al., 1999). Two examples of regulation that
illustrate the way in which soft rot erwiniae perceive their environment prior to and during infection are discussed below.
Regulation by degradation intermediates. The action of pectinases on pectin and pectate in the middle lamella and cell walls
leads to a range of breakdown products. It was recognized early
in the study of exoenzyme regulation that within these breakdown
products there were substances involved in the induction of exoenzymes, acting as a positive feedback mechanism to accelerate
exoenzyme production to higher levels within the plant (Collmer
and Bateman, 1981; Hugouvieux-Cotte-Pattat and Robert-Baudouy,
1987). The breakdown intermediates 5-keto-4-deoxyuronate (DKI),
2,5-diketo-3-deoxygluconate (DKII) and 2-keto-3-deoxygluconate
(KDG) were subsequently found to be responsible by interacting
with a transcriptional repressor, KdgR (Chatterjee et al., 1985;
Condemine et al., 1986; Nasser et al., 1994b; Reverchon et al.,
1989). In the absence of infection, KdgR binds to a conserved
binding site in the operators of a number of different genes
involved in pectinolysis, including the pectate lyases, but also
other exoenzymes such as cellulases and proteases, genes involved in Type II (outT ) secretion and genes encoding proteins
secreted by the Type III system (Condemine and Robert-Baudouy,
1987; Condemine et al., 1992; Hugouvieux-Cotte-Pattat and
Robert-Baudouy, 1989; Liu et al., 1999; Nasser et al., 1994b). As
the disease process begins and more breakdown intermediates
are formed, they interact with KdgR, causing it to dissociate from
its binding site and leading to induction/de-repression of the
pathogenicity determinants. This coordinated production of
exoenzymes and other pathogenicity factors at a precise stage in
the infection process is necessary to overcome plant host defences
and lead to disease development (Hugouvieux-Cotte-Pattat
et al., 1996; Thomson et al., 1999).
Cell density-dependant regulation (quorum sensing). The genes expI
and carI, homologues of luxI in Vibrio (Photobacterium) fischeri ,
2003 BLACKWELL PUBLISHING LTD MOLECULAR PLANT PATHOLOGY (2003) 4(1), 1730
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I. K. TOTH et al.
Fig. 1 (a) Comparison of healthy potato plant (left) and plant infected with Erwinia carotovora ssp. atroseptica (right) showing severe wilting and stem rot due to
blackleg disease (see base of stem). (b) African violet (Saintpaulia ionantha) leaf infected with Erwinia chrysanthemi. (c) Growth of soft rot erwinia on crystal violet
pectate (CVP) medium showing characteristic cavities formed by the production of exoenzymes. (d) In planta virulence screening assay by stab inoculation of Erwinia
carotovora subsp. atroseptica into potato stems showing increasing severity from left to right.
produce a small diffusible molecule called N-(3-oxohexanoyl)- Lhomoserine lactone (OHHL), which is constitutively expressed by
soft rot erwiniae at low basal levels (Jones et al., 1993; Pirhonen
et al., 1991; for reviews see Hugouvieux-Cotte-Pattat et al.,
1996; Loh et al., 2002; Miller and Bassler, 2001; Thomson et al.,
1999). As the bacterial population increases within the plant to
reach a high cell density, thought to be around 106 cells/mL,
OHHL reaches a critical level within the population, sufficient to
fully activate the genes expR and carR. These transcriptional activators, in turn, induce the production of exoenzymes and the
antibiotic carbapenem, respectively (and likely other pathogenicity factors) but also have an auto-inducing effect on the expI
and carI genes themselves, again accelerating the production of
pathogenicity factors (McGowan et al., 1995; Nasser et al., 1998;
Reverchon et al., 1998). Interestingly, OHHL is unstable at low
alkaline pH, which may explain why an early response of plants
to soft rot erwinia attack is to alkalize the site of infection to a
pH of > 8.2 (Byers et al., 2002).
Secretion
The rapid induction of exoenzymes and other pathogenicity factors within the bacterial cell is of little consequence unless they
can be efficiently targeted to the extracellular environment. To
accomplish this, soft rot erwiniae have a number of secretion systems (Types I, II and III) all of which have very different mechanisms that appear to be conserved between different bacterial
species both within and outside the Erwinia genus. The Type I system secretes protease from the cytoplasm to the extracellular
space in a single step but, while this system has been studied in
detail in Ech, it appears to have a relatively minor role in pathogenicity (Dahler et al., 1990; Delepelaire and Wandersman, 1990;
Ltoff et al., 1990). The Type II system, on the other hand, is
essential for pathogenicity and secretes pathogenicity determinants such as pectinases and cellulases in a two-step mechanism.
The first step is a sec-dependent protein export system that
exports proteins to the periplasm. The second step, controlled by
a 15 gene out cluster, includes the formation of a structure that
Soft rot erwiniae
spans the periplasmic compartment and outer membrane and

channels proteins, recognized by a signal sequence, to the outside of the cell. The system has been studied extensively in Ecc
and Ech and is present in Eca (Andro et al., 1984; Ji et al., 1987;
Murata et al., 1990; Thurn and Chatterjee, 1985; for reviews see
Russel, 1998; Sandkvist, 2001; Thomson et al., 1999). However,
despite a high level of interspecies amino acid identity within
the soft rot erwiniae, out genes from Ecc do not complement
mutations in equivalent genes in Ech and vice versa, suggesting
a degree of species-specificity (Py et al., 1991). Regulation of the
Type II system is, at least in part, under the control of KdgR and
may also operate under a quorum-sensing mechanism (Condemine
and Robert-Baudouy, 1995; Condemine et al., 1992).
The Type III secretion systeman indicator of subtlety?
The Type III secretion system (TTSS) in the soft rot erwiniae is not
involved in the secretion of exoenzymes but it may still play a crucial role in the plant interaction and, as such, is currently under
intense scrutiny within the research community (for reviews see
Collmer and Beer, 1998; Galan and Collmer, 1999; Hueck, 1998;
Mudgett and Staskawicz, 1998). The TTSS in Gram-negative bacteria, often referred to as the hrp (hypersensitive response and
pathogenicity) system in phytopathogens, translocates effector
proteins into host plant cells to assist in bacterial virulence and
can elicit an HR on non-host plants (Lahaye and Bonas, 2001).
However, little is known about the role of these effectors once
inside the plant cell.
E. amylovora (Ea) has the best characterized TTSS in the
Erwinia genus, and mutations in the Ea hrp cluster lead to
reduced virulence and loss of HR (Barny et al., 1990; Collmer and
Beer, 1998; Eastgate, 2000). An Ea Type III secretion effector
(TTSE), HrpN (harpin), when expressed in E. coli, and purified also
elicits an HR (Wei et al., 1992). However, when the hrpN gene is
mutated in different Ea strains, different virulence and HR phenotypes are seen, suggesting a degree of strain-specificity (Barny
et al., 1995; Wei et al., 1992). The Ea hrp cluster also includes a
second HR-inducing TTSE gene, hrpW, with an accompanying
chaperone (Gaudriault et al., 1998; Kim and Beer, 1998), and
adjacent to the cluster is the disease specific operon dspEF (dspAB ).
DspE is also secreted by the Hrp system (with DspF acting as its
chaperone) and is required for pathogenicity but not the HR
(Bogdanove et al., 1998a,b; Gaudriault et al., 1997). DspE belongs
to the AvrRxb/YopJ family of TTSEs, which are thought to act as
transcriptional regulators that repress the host defence response
(Lahaye and Bonas, 2001).
Hrp gene clusters have been identified in Ech (Ham et al.,
1998), Ecc (Rantakari et al., 2001) and Eca (Bell et al., 2002) but
their structural organization differs. In Ech, the hrpN and hrpC
operons are flanked by hecAB and plcA whilst the TTSEs hrpW
and dspE and their chaperones are not found at this locus (Kim
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and Beer, 1998; Kim et al., 1998). In contrast, the organization in

Eca is more similar to that in Ea: plcA is not found at this locus,
hrpW is present (with chaperone) and dspEF are also present
adjacent to the hrp cluster. Eca does however, have hecAB genes
similar to those of Ech, outside the hrp cluster (next to dspEF )
(Bell et al., 2002) and these have not been reported in Ea. The
ability of the TTSS in Ech to deliver TTSEs has been demonstrated using the Hrp-dependent avirulence protein AvrB from
P. syringae ( Ham et al., 1998). Additionally, in Ech, the use of a
multiple pel mutant ( pelABCE ), deficient in exoenzyme action,
elicits an HR on tobacco, hrpN /pelABCE double mutants do not,
while a single Ech hrpN mutant shows reduced virulence on
chicory (Bauer et al., 1994; Bauer et al., 1995). Recent studies with
improved bioassays, including a lower bacterial inoculum and
a number of susceptible varieties of African violet, have added
further weight to the perceived importance of the hrp cluster in
Ech pathogenesis (Yang et al., 2002). Mutants in hrpG and hrcC
are greatly reduced in virulence on certain cultivars but produce
significant disease on others and are indistinguishable from the
wild-type on potato tubers. A hrpN mutant shows delayed symptoms on Africa violet but when deleted in five major pel genes is
non-pathogenic, suggesting that the presence of pectic enzymes
may be sufficient to mask the effects of some mutations. On
tobacco, hrpG and hrcC mutants do not produce an HR, while a
hrpN mutant gives a reduced HR (Yang et al., 2002). Unlike Ech,
Ecc does not normally elicit an HR on tobacco but it can do so
when HrpN production is de-repressed (Cui et al., 1996). However, hrpN mutants retain their wild-type ability to macerate
celery petioles (Mukherjee et al., 1997). Nevertheless, a role for
the TTSS in Ecc pathogenicity is proposed by Rantakari et al.
(2001) who found that Ecc growth during the early stages of
infection of Arabidopsis is reduced in a hrcC mutant, although
the mechanism and the effector protein(s) have yet to be
determined. The role of the Eca hrp cluster in either pathogenicity
or HR has not yet been determined but, together with further
work on Ech and Ecc, is likely to be the focus of detailed study in
the coming years.
Iron acquisition and protection from plant defences
Another process that is crucial for pathogenesis is iron uptake,
which was first linked to pathogenicity in Ech through the isolation of cell surface mutants (Expert and Toussaint, 1985; for
review see Expert, 1999). Ech produces the siderophores chrysobactin and achromobactin in order to acquire iron from the ironpoor environment of the plant apoplast. Mutants defective in
chrysobactin-mediated iron transport remain localized within
African violet leaves, suggesting a role in bacterial spread
throughout the plant (Enard et al., 1988). Mutants deficient in
the biosynthesis of achromobactin, however, fail to spread from
the site of inoculation altogether, and could be involved at the
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I. K. TOTH et al.
very onset of infection (Enard and Expert, pers. comm.). In Ech a

number of pectate lyase genes are also regulated by iron, adding
to the complexity of exoenzyme production and also clearly demonstrating the precision with which these pathogens initiate disease (Sauvage and Expert, 1994). Ecc produces both chrysobactin
and aerobactin but no role in disease development has yet been
demonstrated for either siderophore (Bull et al., 1996; Franza
et al., 1991; Ishimaru and Loper, 1992), and in Eca, novel sequences
similar to genes involved in iron acquisition have been identified
and are currently under investigation (Bell et al., unpublished data).
Although soft rot generally occurs in anaerobic conditions
when the host response is impaired (Prombelon and Kelman,
1980), the oxidative burst is an important form of host defence
against the soft rot erwiniae (Bolwell and Wojtaszek, 1997). Several virulence-associated genes have been identified that protect
against damage by this mechanism, including some that utilize
iron. For example, the suf operon in Ech is thought to be involved
in incorporating iron into antioxidant defence in the form of Fe-S
clusters, and mutants in genes from this operon show an
increased susceptibility to oxidative stress and reduced virulence (Nachin et al., 2001). Similarly, the iron-containing flavohaemoprotein HmpX, mutants of which show reduced virulence,
may defend against reactive oxygen damage and is also required
for Pel synthesis in microaerobic conditions (Favey et al., 1995).
Other genes in Ech involved in defence against the oxidative
burst include sodA (superoxide dismutase), which may negate
the effects of superoxide anions (O 2 ) by their conversion to H2O2
(Santos et al., 2001), msrA (methionine sulphoxide reductase),
which encodes a protein that repairs oxidized proteins (El Hassouni
et al., 1999), and ind genes that, in some Ech strains, encode
the production of the blue pigment indigoidine that also confers
increased resistance to oxidative stress (Reverchon et al., 2002).
In all cases, mutations in these genes reduce virulence.
In addition to oxidative stress, plants produce antimicrobial
peptides, and the sap operon (sensitivity to antimicrobial peptides)
in Ech, homologous to a similar system in Salmonella typhimurium,
defends against such peptides. SapA, a periplasmic component of
the system, binds the antimicrobial peptide and transports it to
the cytoplasm, where it is degraded. A mutation in sapA shows
reduced virulence on a level similar to that observed in a pel /
hrp double mutant (Lopez-Solanilla et al., 1998, 2001).
THE BACTERIAL CELL SURFACE

The bacterial cell surface is the first line of defence against any
attempt by the host to prevent infection, and it is therefore not
surprising that some genes required for full pathogenicity were
first identified as cell surface mutants. For example, the siderophoredependant iron assimilation system in Ech was revealed by analysis of mutants with defective cell surface composition (Expert
and Toussaint, 1985) (see above). An rffG- mutant in Eca, isolated
on the basis of altered phage resistance and reduced virulence,

has a pleiotropic phenotype, including cell surface defects. It
shows alterations in the synthesis of enterobacterial common
antigen, outer membrane proteins, lipopolysaccharide (LPS) and
flagella, as well as reduced enzyme production, lack of motility
and an increased sensitivity to surface-active agents (Toth et al.,
1999b). Other phage resistant mutants of Ech show a structural
change to the LPS core region, are reduced in virulence but unaffected in exoenzyme production and other phenotypes (Schoonejans
et al., 1987). The eps genes of Ech, which are required for the
synthesis of extracellular polysaccharide (EPS), and apparently
involved in LPS synthesis, are also required for full virulence.
These genes are directly linked to exoenzyme production and are
under the control of the exoenzyme repressor PecT (Condemine
et al., 1999). Although no precise roles for the above mutations
or the cell surface components they synthesize have yet been
determined, they may be involved in protection against host
defences or attachment to host cells (see below).
Osmoregulated periplasmic glucans (OPGs), which are cell
envelope components of all Gram-negative bacteria, are also
essential for the in planta growth of Ech (Page et al., 2001).
Mutants in two OPG synthesis genes ( opgGH ) lack OPG and are
completely non-virulent. Like rffG and eps, they show a pleiotropic phenotype, in this case exhibiting reduced exoenzyme synthesis, excess exopolysaccharide synthesis and reduced motility,
and while reduced exoenzyme synthesis is expected to contribute
to loss of virulence, co-inoculation experiments with mutant and
wild-type strains have shown that the OPGs themselves are
essential for growth in planta.
The importance of the cell surface in attachment of the soft rot
erwiniae to host plant cells during pathogenesis is not clear, yet
in other enterobacterial pathogens this process is essential for
successful infection (Cao et al., 2001). In Ech the outer membrane protein intimin, which is also found in E. coli, allows Ech to
bind to animal cells (Duarte et al., 2000) but any role for intimin
in binding to plant cells during infection has yet to be determined.
The strongest evidence for attachment comes from Wallace and
Prombelon (1992), who showed that Ecc cell binding to potato
leaf surfaces is reduced by treatment with a haemagglutinin
inhibitor, suggesting a role for haemagglutinins in such binding.
A region of the Eca genome containing sequences similar to haemagglutinin or adhesin-like genes in various animal and plant
pathogens has recently been identified (Bell et al., 2002) and two
genes of this sort have also been found in Ech, although their role
in binding has not yet been established (Kim et al., 1998).
Competition in the disease environment
With successful release of nutrients during infection comes
competition and scavenging from other opportunistic microorganisms and, indeed, other pectolytic and non-pectolytic bacteria
Soft rot erwiniae
are often isolated from diseased plant tissues (Prombelon and

Salmond, 1995). The soft rot erwiniae may compete with these
bacteria by producing antibiotics. For example, some strains of
Ecc produce carbepenem (Parker et al., 1982), an antibiotic that is
co-regulated with pathogenicity factors (including exoenzymes)
through quorum sensing (see above). In this way, during exponential growth within the plant prior to disease initiation, the
rapid onset of exoenzyme production is accompanied by antibiotic production (Byers et al., 2002; McGowan et al., 1995), which
may prevent other bacteria profiting from the nutrients released.
The pab gene in Ech may also be involved in antibiotic biosynthesis,
with the gene contiguous to it ( ybiT) appearing to encode an
ABC transporter involved in antibiotic resistance to this or other
antibiotics. When tested in potato tubers or chicory leaves , ybiT
mutants retain full virulence but, in the presence of the wild-type
strain or selected saprophytic bacteria, show a reduced ability to
compete (Llama-Palacios et al., 2002).
G E N E EX P RE S S I O N D U R I N G P L A N T E R W I N I A
I N T E RA C T I O N S
Interactions between plants and pathogens involve complex recognition events that lead to signalling cascades and the regulation
of numerous genes that are required for the interaction. Recent
years have seen the development of new technologies for investigating changes in gene expression during infection (Birch and
Kamoun, 2000), and several of these have been applied to gain
an insight into host defence responses to Eca and Ecc and into
changes in gene expression in Ech.
The plant response
Many exoenzymes produced by the soft rot erwiniae trigger plant
defences, probably through the release of elicitor-active cell wall
fragments (Davis et al., 1984; Palva et al., 1993). Indeed, E. carotovora culture filtrates containing these enzymes, and oligogalacturonides (OGAs) derived from enzymatic breakdown of pectin,
up-regulate a variety of defence genes in the non-host plant Arabidopsis thaliana (Norman et al., 1999; Norman-Setterblad et al.,
2000; Vidal et al., 1997, 1998). More recently, a technique called
suppression subtractive hybridization (SSH) has been used to
generate a cDNA library enriched for sequences up-regulated
1 h after infiltration of potato leaves with Eca (Dellagi et al.,
2000a,b). Amongst the sequences recovered was a potato gene
encoding a WRKY DNA binding protein that was shown to be upregulated by culture filtrate from sonicated, recombinant E. coli
containing either pelB or pelD, again associating plant defence
responses with cell wall elicitor activity (Dellagi et al., 2000b). In
an independent study, Ecc and OGAs were both shown to upregulate novel receptor-like protein kinase genes in potato that
were again isolated using SSH (Montesano et al., 2001).
23
Recent work by Asai et al. (2002) describes a signalling cascade

involved in innate immunity in Arabidopsis, which may explain
the role of the above induced plant factors in resistance to the soft
rot erwiniae. The innate immune system, a first line of defence against
infectious disease, functions to detect pathogen-associated
molecular patterns (PAMPs). For example, flagellin represents a
PAMP in bacterial pathogens of both animals and plants (and is
conserved in soft rot erwiniae). In plants, a receptor-like kinase,
FLS2, mediates the innate immune response to flagellin (GomezGomez and Boller, 2002) and, following recognition of flagellin
by FLS2, the plant response is mediated by a MAP kinase signalling cascade and WRKY22/29 transcription factors. Constitutive
activation of this pathway provides resistance to pathogens (Asai
et al., 2002) and it is likely that additional PAMPs (or other pathogenderived signals) may converge into a conserved MAP kinase
signalling cascade. Additional PAMPs could, for example, include
OGAs as these are products of pectin breakdown generated by
plant pathogens. The WRKY transcription factor and receptor-like
kinases up-regulated by soft rot erwiniae (see above) may thus be
components of an innate immune response to OGAs in potato; an
area currently under investigation (P. Birch, pers. comm.).
Gene expression in the pathogens
While in vitro studies have proved invaluable for identifying pathogenicity determinants and their regulators, more subtle interactions may be missed in the absence of direct contact with
the plant or plant material. To address this, Beaulieu and Van
Gijsegem (1990) studied gene expression in Ech in the presence
of plant extract using a promotorless antibiotic resistance gene in
phage Mu. Some mutants were found to be affected in pectate
lyase (pelA) production, iron assimilation and galacturonate
catabolism, the importance of the latter only coming to light
through this approach (Beaulieu and Van Gijsegem, 1990). In an
attempt to determine whether plant induction was host specific,
Beaulieu and Van Gijsegem (1992) then tested these and other
reduced virulence mutants on other plant species. While most
plant-inducible mutants showed similar reductions in virulence
on all plants tested, some differences were observed, e.g. one
mutant was virulent on pea plantlets but exhibited reduced virulence on African violet and Witloof chicory leaves. More recently, a
number of studies have shown the induction of secondary pectate
lyases following in-planta gene expression (Beaulieu et al., 1993;
Kelemu and Collmer, 1993see above). However, while the above
approaches have proved effective in identifying novel genes inducible
by plant extract, they still fall short of a true interaction with the
living host since, for example, active plant regulatory and biochemical processes are essential for events such as HR elicitation
by HrpNEa (He et al., 1994). With this in mind, novel technologies
for profiling differential gene expression in soft-rot erwiniae at
different stages of infection are being developed or adopted.
24
I. K. TOTH et al.
Isolation of differentially expressed genes from both host and

pathogen during their interaction is dependent on the method of
cDNA synthesis. Studies in Plant Response (see above) use an
oligo-dT primer to synthesize cDNA, which anneals to the poly A
tail at the 3 end of eukaryotic mRNAs. Prokaryotic mRNAs lack
3 poly A tails, and thus bacterial cDNA cannot be synthesized
by this method. However, using a mixture of 11-mer primers
designed to anneal to conserved regions in the 3 ends of enterobacterial genes, representative cDNAs were synthesized from Eca
and Ecc and differential gene expression was profiled using
cDNA-amplified fragment length polymorphism (cDNA-AFLP)
(Dellagi et al., 2000c). This approach offers the potential to distinguish differentially expressed bacterial and plant genes during
Erwinia plant interactions by using different strategies for cDNA
synthesis. The two cDNA populations may then be compared
using cDNA-AFLP profiling.
DNA microarray technology has recently been applied to study
differential gene expression in Ech during its interaction with
African violet (Okinaka et al., 2002). An array consisting of
5000 randomly selected 2.53.5 kb genomic clones was
synthesized and screened with cDNAs produced from cultured
bacterial cells and from infected plant tissue. Clones containing
differentially expressed genes were sequenced, and those found
to be up-regulated in planta included genes encoding virulence
factors, iron scavengers, transporters and proteins involved in
protection from plant response mechanisms, as well as a number
of novel genes as yet unpublished. It was found that many of
these differentially expressed genes did not appear to directly
damage the host, but might aid survival in planta. Even with
these approaches, however, studying the plantErwinia interaction transcriptome has involved challenging the plant with large
inoculum levels of the pathogen. Under these conditions, pathogen
levels may be above those required for the activation of exoenzyme
production through quorum sensing or other regulation mechanisms. As a consequence, the early more subtle interactions may
be missed. The effect of reducing quantities of inoculum, in which
exoenzymes are at a basal level of production, more in keeping
with the early stages of natural infection, has yet to be studied.
It will be interesting to see in the coming years whether microarrays and PCR-based technologies such as SSH and cDNA-AFLP
are sensitive enough to detect changes in both host and pathogen
gene expression during the natural onset of infection.
GENOMICS
Genome sequencing and comparative genomics
Whole genome sequencing has already profoundly influenced
the direction of research for a number of microbes, and Erwinia
will be no exception. Genome sequencing projects have been
initiated recently for both Eca strain SCRI1043 (http://
www.scri.sari.ac.uk/TiPP/Erwinia.htm; http://www.sanger.ac.uk/
Projects/E_carotovora/) and Ech strain 3937 (http://
www.ahabs.wisc.edu:16080/pernalab/erwinia/index.htm; http://
www.tigr.org/tdb/mdb/mdbinprogress.html). In both cases, high
throughput sequencing of random small insert clones has been
completed (to approximately eightfold genome coverage) and is
being followed by assembly and gap closure by directed sequencing. Preliminary analysis of random shotgun sequence data suggests that both genomes are approximately 5 Mb (Julian Parkhill
and Nicole Perna, pers. comms.). The complete sequences of both
genomes are due for release in 2003. They will serve as blueprints
for future research into all aspects of these pathogens biology,
particularly in the search for effectors and elicitors involved in
pathogenicity and host-specificity.
As a prelude to the complete sequencing of Eca and Ech, partial (sample) sequencing of both genomes was undertaken. Bell
et al. (2002) targeted a selected region of the Eca genome. Two
large overlapping fragments of cloned genomic DNA (spanning
200 kb) from a Bacterial Artificial Chromosome (BAC) library
were partially sequenced to reveal the same complement of 28
hrp genes as found in Ea (see above). In addition, sequences
flanking the hrp cluster included orthologues of known or putative pathogenicity operons from other Erwinia species, such as
dspAB (Ea), hecAB and pecSM (E. chrysanthemi ), sequences similar to X. fastidiosa haemagglutinin-like genes, and sequences
similar to rhizobacterial opine catabolism genes. BAC end
sequences from other loci around the Eca genome showed similarity to more genes of interest, including those involved in iron
acquisition and phytotoxin synthesis in Pseudomonas spp. (Bell
et al., unpublished data). In Ech, a random sample of 1777
genomic sequences revealed genes encoding exoenzymes, regulatory and Hrp proteins. However, it also revealed sequences
similar to genes involved in the synthesis of phytohormones and
phytotoxins, and in opine catabolism (N. Perna and F. Blattner, pers.
comm.). Only 61% of the Ech sequences showed a strong degree
of similarity to E. coli, suggesting that as much as 2.0 Mb of the
genome might carry genes specific to its plant pathogenic life-style.
Both of these limited sequencing efforts imply that Eca and Ech may
have hitherto unsuspected traits that could be relevant to disease,
and indeed to life in the absence of disease, and it will be
interesting to see what emerges from whole genome sequencing.
The availability of two complete soft rot erwinia genome
sequences will allow a thorough comparison of their many
shared genes. For example, it is clear that there are similarities
in the genes that encode, regulate and facilitate the export of
exoenzymes (see above). However this comparison will also
reveal genetic differences between the species in terms of exoenzymes, Type III effectors and other pathogenicity factors. This will
be invaluable in understanding their biology more fully, including
the molecular bases for differences in host-range and disease
symptoms. Comparisons of the soft rot erwiniae genomes with
Soft rot erwiniae
those of other enterobacterial pathogens (e.g. Blattner et al.,

1997; McClelland et al., 2001; Parkhill et al., 2001a,b) will shed
light on the evolution of this bacterial family. The picture currently
emerging is of a shared enterobacterial chromosomal backbone
derived from a common ancestor but with extensive horizontal
gene transfer (that may confer novel traits upon the recipients),
as well as gene loss by deletion or decay into non-functional
pseudogenes (that may reflect changes in the life-style of the
bacteria, such as restricting host range) (Parkhill et al., 2001a,b;
Perna et al., 2001a,b). The availability of an increasing number of
annotated and relatively well-characterized enterobacterial
genomes should allow these similarities and differences to be
defined and functions readily assigned to many of the genes
in the soft rot erwiniae genomes. Many common features are
shared between plant and animal pathogens, e.g. regulatory
genes, secretion systems, attachment mechanisms and defences
from host oxidative bursts (Cao et al., 2001), and comparative
genomics will help to elucidate the functions of such genes in the
soft rot erwiniae. Besides those genes involved in pathogenesis,
new insights into nutrient utilization, possible starvation mechanisms and other environment-associated processes may also be
revealed. On the other hand, genes found in the soft rot erwiniae
but not in other enterobacteria (many of which are animal pathogens) may be involved in plant pathogenesis or other plantassociated life-style, especially where similar genes are found in
other plant-associated bacteria.
Several complete plant pathogen and plant symbiont genome
sequences have already been published (da Silva et al., 2002;
Galibert et al., 2001; Goodner et al., 2001; Kaneko et al., 2000;
Salanoubat et al., 2002; Simpson et al., 2000; Wood et al., 2001)
and several more are well advanced <http://www.tigr.org/tdb/
mdb/mdbinprogress.html>. This has lead to the identification of
many candidate pathogenicity determinants in both poorly and
well-studied phytopathogens. Searching soft rot erwiniae
genome sequences for homologies to known pathogenicity
genes and the use of bioinformatic approaches to identify conserved regulatory motifs may identify novel targets for research,
i.e. in the latter case leading to the identification of pathogenicity
regulons under common transcriptional control (Collmer et al.,
2001; Salanoubat et al., 2002). For example, as soft rot erwiniae
are known to possess both kdgR and hrp box promoter
sequences (Liu et al., 1999; Rantakari et al., 2001; Reverchon
et al., 1989), complete genome sequences should reveal all
genes that possess these promoter motifs and perhaps identify
novel conserved motifs, helping to unravel the complex cascades
that lead to disease development and host resistance.
Functional genomics
With a complete genome sequence, various high throughput
approaches are available to investigate gene function. DNA
25
microarrays will allow us to measure temporal changes in the

expression of all genes during adaptations to, for example, physiological conditions (including anaerobiosis and responses to
other environmental stresses), pathogenesis (including the different stages of disease processes on different hosts) or different
life-styles (including saprophytic, epiphytic and endophytic). This
approach has already proved valuable for Ech (see above) despite
the lack of a complete and defined gene set for the array (Okinaka
et al., 2002). Where changes under the conditions described are at
the translational, rather than transcriptional level (e.g. temperature),
parallel analyses of the proteome can be made. Moreover, proteomics is a way to unequivocally identify membrane-associated
or secreted proteins, which are most likely to interact with the plant.
Following the identification of candidate genes, good systems
for gene knock-out are essential to further investigate their function. The soft rot erwiniae are well suited to such functional studies
due to their genetic amenability (Thomson et al., 1999) and, to
facilitate functional genomic studies in Eca, a pooled mutation
grid has been constructed (Bell et al., unpublished data), allowing rapid PCR screening for mutations in any given gene. In this
way, and with appropriate bioassays, numerous gene targets
derived from the above approaches may be assessed.
CONCLUSIONS
For over 20 years the use of molecular biology has led to significant advances in our understanding of pathogenicity in the soft
rot erwiniae. The large-scale coordinated production and targeting of exoenzymes clearly has a major impact on disease development. However, as we investigate further, other more subtle
molecular processes are implicated in interactions with the plant,
including cell-to-cell attachment, defence against plant responses and the possibility of protein delivery directly into the
host cell through a Type III secretion system. The soft rot erwiniae
are now entering the genomics era and we will soon have the full
catalogue of genes that these organisms possess. This information, together with new methods for analysing gene expression
in planta, the analysis and comparison of whole genome
sequences, and novel approaches to high throughput gene functional analyses, is certain to reveal more of the biology of these
pathogens, their survival in the environment, and the nature of
their interactions with both host and non-host plants.
ACKNOWLEDGEMENTS
Following the sad death of Prof. Noel Keen we would like to
dedicate this review to him for his drive and enthusiasm in
obtaining funds for the E. chrysanthemi genome sequencing
project and for many unparalleled years of creative thought and
quality scientific achievements in the field of plantpathogen
interactions. We would like to thank SEERAD for their financial
26
I. K. TOTH et al.
support, NATO for funding the PhD studentship of M.C.H., and

D. Expert for supplying the photograph of E. chrysanthemi infected
African violet.
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MOLECULAR PLANT PATHOLOGY (2003) 4(1), 1730

Soft rot erwiniae

Soft rot erwiniae: from genes to genomes

2003 BLACKWELL PUBLISHING LTD

is it able to compete better with other micro-organisms through

have several functions. As motility has been linked to virulence in

Soft rot erwiniae

Soft rot erwiniae

spans the periplasmic compartment and outer membrane and

and Beer, 1998; Kim et al., 1998). In contrast, the organization in

very onset of infection (Enard and Expert, pers. comm.). In Ech a

THE BACTERIAL CELL SURFACE

on the basis of altered phage resistance and reduced virulence,

Soft rot erwiniae

are often isolated from diseased plant tissues (Prombelon and

Recent work by Asai et al. (2002) describes a signalling cascade

Isolation of differentially expressed genes from both host and

Soft rot erwiniae

those of other enterobacterial pathogens (e.g. Blattner et al.,

microarrays will allow us to measure temporal changes in the

support, NATO for funding the PhD studentship of M.C.H., and

Soft rot erwiniae

mutants deficient in oligogalacturonide lyase. Proc. Natl Acad. Sci. USA,

Heilbronn, J. and Lyon, G.D. (1990) The ineffectuality of potato protease

Ltoff, S., Delepelaire, P. and Wandersman, C. (1990) Protease

Soft rot erwiniae

profiling of Erwinia chrysanthemi 3937 genes that are regulated during

of the soft-rot pathogen Erwinia carotovora: partial characterisation of

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