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MOLECULAR PLANT PATHOLOGY (2003) 4 (1 ) , 17–30

© 2003 BLACKWELL PUBLISHING LTD 17

BlackwellScience,Ltd

Pathogen profileSoftrot erwiniae

Soft rot erwiniae: from genes to genomes

IAN K . TOTH*, KENNETH S . BELL , MARIA C . HOLEVA AND PAUL R . J . B IRCH

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, differ-

ences 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 dis-

cuss 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 Entero-

bacteriaceae

, 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 sub-

tropical 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,

http://www.tigr.org/tdb/mdb/mdbinprogress.html,

http://www.sanger.ac.uk/Projects/E_carotovora/.

INTRODUCTION

The soft rot erwiniae are pathogens of many plant species, affect-ing crops in temperate to tropical regions world-wide. Eca

has a

narrow host range restricted almost exclusively to potato in tem-

perate 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 ion-

antha

), 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 post-harvest (for reviews see Pérombelon and Kelman, 1980; Pérom-

belon 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, respec-

tively (Pérombelon and Kelman, 1980; Pérombelon 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 initi-

ated, 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

*Correspondence: E-mail: [email protected]



18

I. K. TOTH et al.

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(2003) 4

( 1 ) , 17–30 © 2003 BLACKWELL PUBL ISH ING LTD

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 proc-

ess 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, pro-

grammed cell death that prevents further spread of the patho-

gen? 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 under-

standing of many aspects of the biology of these pathogens.

TAXONOMY

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

(Pérombelon, 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 stew-

artii

(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. atrosep-

ticum

(for Eca

), Pectobacterium carotovorum

ssp. carotovorum

(for Ecc

) and Pectobacterium chrysanthemi

(for Ech

), supporting

an earlier proposal by Waldee (1945) to rename the group sim-

ilarly. However, at present ‘

Pectobacterium

’ has not been widely

adopted by the ‘

Erwinia

’ research community.

THE DISEASE PROCESS

The soft rot erwinias are found on plant surfaces and in soil where

they may enter the plant via wound sites or through naturalopenings 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 temper-

ature, become suitable for disease development (Pérombelon

and Kelman, 1980; Pérombelon 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 pathogen’s 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 (Pérombelon

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, Pérombelon et al

. (1987a) showed that a soil temper-

ature of 20°

C was an important transition point, above which Eca

,and below which Ech

, were not apparently pathogenic. The abil-

ities 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 (Pérombelon et al.

, 1987b). However, in addition to differ-

ences in growth, a tight thermal regulation on the production of

cell wall degrading enzymes (exoenzymes) has been demon-

strated (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 co-

ordinated production of high levels of multiple exoenzymes, includ-

ing 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; Pérombelon, 2002; Py

et al

., 1998; Thomson et al

., 1999). Cellulases, which exhibit

mainly endoglucanase activity, break down cellulose in the prim-

ary 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; Mäe 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; Kyöstiö 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; Kyöstiö 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 devel-

opment. These exoenzymes break down and utilize pectins in the



20 I. K. TOTH et al.

MOLECULAR PLANT PATHOLOGY (2003) 4 ( 1 ) , 17–30 © 2003 BLACKWELL PUBL ISH ING LTD

produce a small diffusible molecule called N-(3-oxohexanoyl)-L-

homoserine 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 10 6 cells/mL,

OHHL reaches a critical level within the population, sufficient to

fully activate the genes expR and carR . These transcriptional act-ivators, in turn, induce the production of exoenzymes and the

antibiotic carbapenem, respectively (and likely other pathogeni-

city 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 fac-

tors 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 sys-

tems (Types I, II and III) all of which have very different mechan-

isms that appear to be conserved between different bacterial

species both within and outside the Erwinia genus. The Type I sys-

tem secretes protease from the cytoplasm to the extracellularspace in a single step but, while this system has been studied in

detail in Ech , it appears to have a relatively minor role in patho-

genicity (Dahler et al ., 1990; Delepelaire and Wandersman, 1990;

Létoffé et al ., 1990). The Type II system, on the other hand, is

essential for pathogenicity and secretes pathogenicity deter-

minants 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

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.



Soft rot erwiniae 21

© 2003 BLACKWELL PUBLISHING LTD MOLECULAR PLANT PATHOLOGY (2003) 4 (1 ) , 17–30

spans the periplasmic compartment and outer membrane and

channels proteins, recognized by a signal sequence, to the out-

side 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 system—an 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 cru-cial 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 bac-

teria, 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 toreduced 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 pheno-

types 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

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 demon-

strated 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 producesignificant disease on others and are indistinguishable from the

wild-type on potato tubers. A hrpN mutant shows delayed symp-

toms 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). How-

ever, 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 iso-lation of cell surface mutants (Expert and Toussaint, 1985; for

review see Expert, 1999). Ech produces the siderophores chryso-

bactin and achromobactin in order to acquire iron from the iron-

poor 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





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 demon-

strating the precision with which these pathogens initiate dis-

ease (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 (Pérombelon and Kelman,

1980), the oxidative burst is an important form of host defence

against the soft rot erwiniae (Bolwell and Wojtaszek, 1997). Sev-

eral 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-Sclusters, and mutants in genes from this operon show an

increased susceptibility to oxidative stress and reduced viru-

lence (Nachin et al ., 2001). Similarly, the iron-containing flavo-

haemoprotein 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 (O2– ) 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, encodethe 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 siderophore-

dependant iron assimilation system in Ech was revealed by ana-

lysis 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 unaf-

fected 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 pleio-

tropic phenotype, in this case exhibiting reduced exoenzyme syn-

thesis, 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 roterwiniae 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 mem-

brane 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

Pérombelon (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 hae-

magglutinin or adhesin-like genes in various animal and plantpathogens 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 micro-

organisms and, indeed, other pectolytic and non-pectolytic bacteria





are often isolated from diseased plant tissues (Pérombelon 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 expon-

ential growth within the plant prior to disease initiation, the

rapid onset of exoenzyme production is accompanied by anti-

biotic 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).

GENE EXPRESSION DURING PLANT–ERWINIA

INTERACTIONS

Interactions between plants and pathogens involve complex recogni-

tion 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 invest-

igating 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. caro-

tovora culture filtrates containing these enzymes, and oligogalac-

turonides (OGAs) derived from enzymatic breakdown of pectin,

up-regulate a variety of defence genes in the non-host plant Ara-

bidopsis 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-regulated1 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 up-

regulated 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 up-

regulate novel receptor-like protein kinase genes in potato that

were again isolated using SSH (Montesano et al ., 2001).

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 (Gomez-

Gomez and Boller, 2002) and, following recognition of flagellin

by FLS2, the plant response is mediated by a MAP kinase signal-

ling 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 pathogen-

derived 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-likekinases 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 patho-

genicity determinants and their regulators, more subtle inter-

actions 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 inphage 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 virul-

ence on African violet and Witloof chicory leaves. More recently, a

number of studies have shown the induction of secondary pectatelyases following in-planta gene expression (Beaulieu et al ., 1993;

Kelemu and Collmer, 1993—see 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 bio-

chemical 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.





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 entero-

bacterial 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 dis-

tinguish 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 withAfrican violet (Okinaka et al ., 2002). An array consisting of

≈ 5000 randomly selected 2.5–3.5 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 plant–Erwinia inter-action 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 mecha-

nisms. 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 microar-

rays 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 sequen-

cing. Preliminary analysis of random shotgun sequence data sug-

gests 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, par-

tial (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 put-

ative pathogenicity operons from other Erwinia species, such as

dspAB (Ea ), hecAB and pecSM (E. chrysanthemi ), sequences sim-

ilar 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 simi-

larity 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 1777genomic sequences revealed genes encoding exoenzymes, regu-

latory 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 genomesequences 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 exo-

enzymes, 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





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 thesoft rot erwiniae. Besides those genes involved in pathogenesis,

new insights into nutrient utilization, possible starvation mechan-

isms 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 path-

ogens) may be involved in plant pathogenesis or other plant-

associated 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 con-

served 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; Reverchonet 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

microarrays will allow us to measure temporal changes in the

expression of all genes during adaptations to, for example, phys-

iological conditions (including anaerobiosis and responses to

other environmental stresses), pathogenesis (including the differ-

ent 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, pro-

teomics 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 func-

tion. 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 ‘mutationgrid’ has been constructed (Bell et al., unpublished data), allow-

ing 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 signi-

ficant advances in our understanding of pathogenicity in the soft

rot erwiniae. The large-scale coordinated production and target-

ing of exoenzymes clearly has a major impact on disease devel-

opment. However, as we investigate further, other more subtlemolecular processes are implicated in interactions with the plant,

including cell-to-cell attachment, defence against plant re-

sponses 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 informa-

tion, 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 func-

tional 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 plant–pathogen

interactions. We would like to thank SEERAD for their financial





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