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Update on Pathogen Recognition
Guarding the Goods. New Insights into the Central Alarm
System of Plants1
Roger W. Innes*
Department of Biology, Indiana University, Bloomington, Indiana 47405–7107
The discovery that one’s kitchen has been invaded
by mice is often made indirectly. The holes chewed in
the muesli bag and the teeth marks on the corn flakes
box are a dead give away. Although you have not seen
the mouse, you deploy your defensive weapons, and if
successful, succeed in protecting your valuable goods
from the invasion, hopefully before all of your food
has been eaten. Recent research results indicate that
plants also make use of such indirect surveillance
systems to protect themselves from being consumed
by pathogens. Rather than wait for a direct observation
of the pest, plants appear to activate their defenses as
soon as pathogen-induced damage is detected. As I
describe in this Update, recent work in Arabidopsis has
provided compelling evidence that such indirect surveillance may be the rule, rather than the exception,
when it comes to plant alarm systems.
PATHOGEN RECOGNITION AND GENE-FOR-GENE
INTERACTIONS
The idea that plants may detect pathogens by indirect mechanisms is still relatively new (van der
Biezen and Jones, 1998b) and goes against dogma that
has pervaded the plant pathology field for decades.
This dogma posited that plants detected pathogens
using receptors that directly bound pathogen-derived
elicitor molecules (Gabriel and Rolfe, 1990). This
receptor-ligand model grew out of classical genetic
work performed by H.H. Flor over 50 years ago (Flor,
1956). Flor studied interactions between flax (Linum
usitatissimum) and flax rust. He observed that the difference between flax varieties that were resistant versus
those that were susceptible to specific rust strains
could usually be attributed to a single dominant resistance (R) gene present in the resistant plant. Likewise,
the difference between pathogen strains that were
avirulent versus virulent on specific flax varieties
could usually be attributed to a single dominant avirulence (avr) gene in the avirulent strain. Resistance was
observed only when a specific R gene in flax was
matched with a corresponding avr gene in the pathogen. Based on his genetic analyses, Flor likened such
1
This work was supported by the National Institute of General
Medical Sciences (grant no. R01GM46451).
* E-mail [email protected]; fax 812–855–6082.
www.plantphysiol.org/cgi/doi/10.1104/pp.104.040410.
plant-pathogen interactions to antibody-antigen interactions in mammals, leading to speculation that Avr
genes either encoded the antigens or encoded enzymes
that produced the antigens. Such gene-for-gene
interactions have been subsequently described in
numerous crop and wild plant species, including
Arabidopsis.
MOLECULAR ANALYSES OF PLANT R GENES
Over 40 R genes with recognition specificity for
specific pathogen strains have been isolated from 10
plant species, including both monocots and dicots (for
review, see Martin et al., 2003). The proteins encoded
by these genes can be grouped into four general classes
based on predicted structures: transmembrane receptor kinases, transmembrane receptors without a kinase
domain, cytosolic kinases, and cytosolic proteins that
contain a putative nucleotide binding site (NB). With
the exception of the cytosolic kinase class, all of these
proteins contain Leu rich repeats (LRRs). In other
proteins, LRRs are known to participate in protein to
protein interactions, including binding of peptide
ligands by receptors (Kobe and Kajava, 2001). Careful
sequence analysis of R gene families revealed that the
putative ligand binding surface of LRR domains in
many families are undergoing diversifying selection
(Parniske et al., 1997; Meyers et al., 1998), meaning that
mutations that give rise to amino acid substitutions
outnumber silent mutations. This observation is consistent with idea that the LRR domains of R proteins
might directly bind pathogen-derived proteins.
Of the four classes of plant R genes, the NB-LRR
class is by far the largest. R genes in this class have
been shown to confer resistance to viral, bacterial,
oomycete, and fungal pathogens, and even to nematodes and aphids (Martin et al., 2003). Ten Arabidopsis
R genes of known function have been cloned and all
belong to the NB-LRR class. Analysis of the complete
Arabidopsis genomic sequence revealed 149 genes
containing both an NB and LRR domain (Meyers
et al., 2003). NB-LRR genes can be further subdivided
into two major subclasses based on their N-terminal
domains. TIR-NB-LRR genes have similarity to Toll
and Interleukin 1 receptors, while the non-TIR class
typically contains a coiled-coil (CC) domain (Meyers
et al., 2003). Phylogenetic trees based on only the
NB domain separate the TIR and non-TIR classes
from each other, indicating that these classes do not
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695
Innes
recombine (Meyers et al., 1999). Even with the addition
of transmembrane receptor-type R genes, Arabidopsis
is unlikely to contain more than 250 functional R
genes. An interesting question to ponder is how Arabidopsis can detect the multitude of potentially infectious pathogens with less than 250 R genes. If the above
indirect surveillance hypothesis is correct, plants do
not need to detect a multitude of specific pathogen
molecules but only the damage caused by them.
NB-LRR proteins lack obvious transmembrane domains or signal peptides and are presumed to be
located in the plant cell cytoplasm. A cytoplasmic
location for plant R proteins came as a surprise,
initially, as most models had predicted them to be cell
surface receptors. However, it was soon established
that plant bacterial pathogens use a type III secretion
system (TTSS) to translocate avirulence proteins into
host cells (Alfano and Collmer, 1996). Several groups
subsequently showed that expression of bacterial
avirulence genes in plant cells, either transiently or
in stable transgenic plants, induces cell death in an R
gene-specific manner. This programmed cell death
response is essentially the same as that induced by
avirulent pathogens and is referred to as the hypersensitive response (HR). The HR is believed to be the
endpoint of a complex suite of physiological changes
that limit pathogen growth by producing toxic metabolites and depriving the pathogen of nutrients and
water.
Most recently, similar experiments with Avr proteins from Flor’s flax rust pathogen have revealed that
these are also detected from inside plant cells (Dodds
et al., 2004), indicating that at least some fungal
pathogens also translocate Avr/virulence proteins
across host cell membranes. This latter work is particularly noteworthy, because coexpression of the flax
NB-LRR protein L6 and the fungal Avr protein
AvrL567 in tobacco (Nicotiana tabacum) induced an
HR-like response, while expression of either protein
alone did not. This result implies that the flax L6
protein can activate the cell death program in tobacco
in an Avr-dependent manner. This represents the first
example of an NB-LRR protein retaining functional
specificity across plant families.
THE BIOLOGICAL FUNCTIONS OF BACTERIAL
AVIRULENCE PROTEINS
Why do bacterial pathogens translocate avirulence
proteins into plant cells? We now know that avirulence
proteins simply represent a small subset of the cocktail
of proteins that are injected into host cells via the type
III secretion system (Buell et al., 2003). These proteins,
which are commonly referred to as TTSS effector
proteins, are collectively essential to pathogenesis
as mutations that block secretion abrogate disease
(Alfano and Collmer, 1996). However, we are only
just beginning to gain insights into the functions of
specific TTSS effectors, which appear to vary widely
696
between bacterial strains (Innes, 2003). The AvrRpt2
protein of Pseudomonas syringae has been shown to
suppress defense responses in Arabidopsis and enhance the virulence of P. syringae (Chen et al., 2000).
Several other TTSS effectors from P. syringae have
recently been shown to suppress defense responses
induced by other TTSS effectors resident in the same
bacterium (i.e. mutation of a given TTSS effector can
render a P. syringae strain avirulent on a normally
susceptible host; Abramovitch et al., 2003; Bretz et al.,
2003; Espinosa et al., 2003). One of these, HopPtoD,
contains a Tyr phosphatase domain, and mutations
that eliminate phosphatase activity block the HR
suppression activity (Bretz et al., 2003; Espinosa et al.,
2003). The targets of HopPtoD have not yet been identified. The XopD protein of Xanthomonas campestris
has recently been shown to possess SUMO isopeptidase activity and to be nuclear localized, suggesting
that it may target nuclear proteins and remove SUMO
groups, which are ubiquitin-like peptides that modify
the localization and/or stability of proteins (Hotson
et al., 2003). As with HopPtoD, the specific targets of
XopD, and how it contributes to virulence, are not
known. The P. syringae AvrPphB protein functions as
Cys protease, and furthermore, protease activity is
required for induction of the HR in both Arabidopsis
and tobacco (Shao et al., 2002; Shao et al., 2003; Zhu
et al., 2004), suggesting that it is the enzymatic activity
of AvrPphB, rather than the protein by itself, that
activates resistance. Whether and how AvrPphB contributes to the virulence of P. syringae remains to be
determined.
AN ALTERNATIVE MODEL FOR HOW R PROTEINS
MEDIATE PATHOGEN RECOGNITION
The realization that plants have a limited repertoire
of R genes, and that pathogen avirulence proteins are
effectors that target host cell processes led to the
development of a new model for R protein function
dubbed the guard hypothesis (van der Biezen and
Jones, 1998b). The authors suggested that NB-LRR
genes function to guard the targets of pathogen
virulence proteins. According to this model, pathogen
effectors target host cell proteins in order to suppress
defense responses or elicit susceptible responses (e.g.
leakage of water and nutrients). NB-LRR proteins
evolved as a counter-defense and function to monitor
the status of the effector targets. For example, effectors
could cause an allosteric change in their targets that
promote binding by the NB-LRR protein, which in
turn triggers the HR. Since the potential targets of
pathogen virulence proteins are probably quite limited, this model would explain how a plant can detect
thousands of pathogens with only few hundred R
genes.
Evidence in support of the guard model has been
mounting in the last 2 years, particularly for the
recognition of P. syringae avirulence proteins. For
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Pathogen Perception by Plants
example, recognition of the AvrPto protein by tomato
requires the Pto gene, which encodes a protein kinase,
and Prf, which encodes an NB-LRR protein. According
to the guard model, Pto would represent the target and
Prf the guard. Consistent with this, AvrPto physically
interacts with Pto in a yeast (Saccharomyces cerevisiae)
two-hybrid assay (Tang et al., 1996). More importantly,
mutations have been identified in Pto that cause it to
induce an HR-like response in tomato (Lycopersicon
esculentum) in the absence of AvrPto, and this response
is dependent on Prf (Rathjen et al., 1999). These Pto
mutations may mimic the allosteric change induced by
AvrPto.
A second example that supports the guard model
is the interaction of the P. syringae effectors AvrB and
AvrRpm1 with the Arabidopsis RIN4 protein. RIN4
interacts with these effectors in both yeast two-hybrid
assays and coimmunoprecipitation assays (Mackey
et al., 2002). Expression of AvrB and AvrRpm1 inside
Arabidopsis cells induces phosphorylation of RIN4
(Mackey et al., 2002). RIN4 also physically interacts
with RPM1, which is the NB-LRR protein that mediates recognition of both AvrB and AvrRpm1. Thus,
RIN4 may represent the target of AvrB and AvrRpm1,
while RPM1 guards RIN4. Intriguingly, a third P.
syringae avirulence protein, AvrRpt2, induces the
elimination of RIN4 (Axtell and Staskawicz, 2003;
Mackey et al., 2003), suggesting that RIN4 is the target
of at least three TTSS effectors. Recognition of AvrRpt2
is mediated by the NB-LRR protein RPS2, not RPM1,
but RPS2 also physically interacts with RIN4 (Axtell
and Staskawicz, 2003; Mackey et al., 2003), suggesting
that RIN4 is guarded by at least two different R
proteins. RPS2 appears to be activated by loss of
RIN4, as a RIN4 knockout mutation is lethal in plants
containing a functional RPS2 gene but has no obvious
phenotype in plants lacking RPS2 function. It is not
clear how AvrB, AvrRpm1, or AvrRpt2 induce changes
in RIN4, or why RIN4 is a target. It is also not clear
how RIN4 modifications activate RPM1 and RPS2.
The strongest data in support of the guard model
comes from work on the P. syringae effector protein
AvrPphB. Similar to the AvrPto story described above,
recognition of AvrPphB by Arabidopsis requires both
an NB-LRR protein, RPS5, and a protein kinase, PBS1
(Warren et al., 1999). Deletion of RPS5 or PBS1 blocks
AvrPphB recognition, but these deletions have no
effect on other Arabidopsis R genes, nor do they cause
any morphological phenotypes. Significantly, PBS1 is
cleaved by AvrPphB within the activation loop of
PBS1, and such cleavage is required for activation of
RPS5-mediated resistance (Shao et al., 2003). Thus,
RPS5 is thought to function as a surveillance protein
that senses specific damage to PBS1.
Although the above data strongly support an indirect mechanism of pathogen recognition by NB-LRR
proteins, there are now two reports of physical associations between plant NB-LRR proteins and pathogen
proteins. The first involves the Pi-ta protein from rice
(Oryza sativa) and the AVR-Pita protein from the
fungus Magnaportha grisea (Jia et al., 2000). Both yeast
two-hybrid assays and an in vitro filter-binding assay
demonstrated a physical interaction between the
C-terminal Leu rich domain of Pi-ta and AVR-Pita.
Interestingly, AVR-Pita is homologous to known metalloproteases, and mutations in the putative catalytic
site of AVR-Pita obviate its physical interactions with
Pi-ta, suggesting that Pi-ta might in fact be a substrate
of AVR-Pita. Perhaps there is a second NB-LRR protein
that guards Pi-ta.
The second example is the RRS1 protein of Arabidopsis, which has been shown to interact with the
PopP2 protein of Ralstonia solanacearum using a yeast
split ubiquitin yeast two-hybrid assay (Deslandes et al.,
2003). In addition, RRS1 and PopP2 colocalize to the
nucleus when transiently coexpressed in Arabidopsis
protoplasts. Nuclear localization of RRS1 is dependent
on a nuclear localization signal present in PopP2,
suggesting that PopP2 drags RRS1 into the nucleus.
Interpretation of these data is complicated, however,
by the presence of a WRKY transcriptional activator
domain on the C terminus of RRS1 and the putative
SUMO-protease activity of PopP2. As with the interaction between Pi-ta and AVR-Pita, RRS1 might
represent a target of PopP2, rather than being a specific
receptor for PopP2.
SIMILARITIES BETWEEN PLANT NB-LRR
PROTEINS AND ANIMAL PROTEINS THAT
MEDIATE IMMUNE RESPONSES
Clues to how NB-LRR proteins may activate defense
responses have come from comparison to animal
proteins. The NB domain of plant NB-LRR proteins
shares significant similarity to several animal proteins
known to regulate programmed cell death and immune responses (van der Biezen and Jones, 1998a;
Inohara and Nunez, 2003). Because the region of
similarity extends beyond the nucleotide binding
pocket, this domain has been referred to as the NBARC domain, for nucleotide binding and similarity to
Apaf-1, R genes and Ced-4 (van der Biezen and Jones,
1998a). More recently, several mammalian NB-ARC
proteins that have C-terminal LRR domains have been
identified that are structurally quite similar to plant
NB-LRR proteins (Inohara and Nunez, 2003). These
proteins differ from plant NB-LRR domains at their N
termini, with two of the best characterized members,
NOD1 and NOD2, having caspase recruitment domains in this position. Significantly, NOD1 and NOD2
both function in the mammalian innate immune
system and are activated by bacterial peptidoglycan
fragments (Girardin et al., 2003).
Because human NOD proteins appear to function in
pathogen perception and are structurally similar to
plant NB-LRR proteins, an understanding of how
plant NB-LRR proteins are regulated may enhance
our understanding of how NOD proteins are regulated, and vice versa. Work on the human NOD1 and
Apaf-1 proteins has led to development of the induced
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Innes
proximity model for NOD protein activation (for
review, see Inohara and Nunez, 2003). In this model,
the NB-ARC domains mediate homo-oligomerization (NOD stands for nucleotide oligomerization domain), but such oligomerization is inhibited by the
C-terminal domains (WD-40 repeats in the case of
Apaf-1 and LRRs in the case of NOD1). Oligomerization
of Apaf-1 and NOD1 brings together downstream signaling proteins that are physically associated with the
N-terminal caspase recruitment domains. The induced
proximity of the downstream signaling proteins then
leads to activation of apoptotic and inflammatory
pathways.
In support of this model, deletion of the C-terminal
domains of either Apaf-1 or NOD1 promotes selfoligomerization and activation of downstream responses (Inohara and Nunez, 2003). Full-length Apaf-1
can be induced to oligomerize in vitro by the addition of
cytochrome C (Hu et al., 1999). Apaf-1 binds cytochrome C, and this binding requires both the WD-40
repeat domain and the NB-ARC domain, as well as
ATP hydrolysis (Hu et al., 1999). Based on these data, it
is hypothesized that the WD-40 repeat domain contains
a ligand binding pocket that is unmasked only upon
a conformational change induced by ATP hydrolysis.
Binding of ligand to the WD-40 repeat domain then
induces a conformational change that enables self-oligomerization via the NB-ARC domain. This general
model is consistent with current data on the NOD1
and NOD2 proteins (Inohara and Nunez, 2003),
although the putative NOD1 and NOD2 ligands
(peptidoglycan fragments) have not been shown to bind
to these proteins.
REGULATION OF PLANT NB-LRR
PROTEIN FUNCTION BY
INTRAMOLECULAR INTERACTIONS
It is not yet clear whether the NB-ARC domains
of plant NB-LRR proteins also mediate selfoligomerization. The best insights to date come from
work on the Rx protein of potato (Solanum tuberosum),
which belongs to the CC-NB-LRR subclass (Moffett
et al., 2002). Rx mediates resistance to potato virus X
(PVX) and is specifically activated by the PVX coat
protein. Although a physical interaction between the
coat protein and Rx has not been shown, the coat protein
somehow regulates intramolecular interactions among
the CC, NB-ARC, and LRR domains of Rx; the CCNB-ARC domain coimmunoprecipitates with the LRR
domain when these domains are expressed from separate constructs, and this physical association is disrupted by coexpression of the PVX coat protein
(Moffett et al., 2002). Furthermore, such coexpression
reconstitutes coat protein-dependent activation of the
HR. Importantly, expression of the CC-NB-ARC domain by itself does not induce an HR, unless highly
overexpressed, and even then only in certain genotypes of tobacco (Bendahmane et al., 2002). Likewise,
698
expression of the LRR domain by itself does not induce
an HR. Thus, induction of the HR requires coexpression of the CC-NB-ARC, LRR, and coat proteins, but
paradoxically, none of these proteins appear to be
stably associated when all three are coexpressed. One
plausible explanation for this result is that the coat
protein does not directly interact with Rx, but in fact
induces a change in a yet-to-be identified host protein,
which in turn triggers Rx conformational changes.
Intramolecular interactions between the various
domains of NB-LRR proteins are also indicated by
work on the tomato Mi-1.1 protein. By creating domain swaps between Mi-1.1 and its close paralog
Mi-1.2, Hwang and Williamson (2003) demonstrated
that a mismatch between the N-terminal region and
the LRR region resulted in a protein that constitutively
activated an HR-like response. They further showed
that a single amino acid substitution in the LRR region
of Mi-1.2 could also cause a constitutive HR and that
this activity could be suppressed by overexpression of
the N-terminal region of Mi-1.1. These data suggest
that interactions between the N-terminal domain and
LRR domain may be important for regulation of HR
activation.
WHAT IS THE ROLE OF NUCLEOTIDE BINDING
IN NB-LRR PROTEIN FUNCTION?
The nucleotide binding domain of plant NB-LRR
proteins is highly conserved and mutations in this
domain invariably inactivate NB-LRR signaling (Tao
et al., 2000; Tornero et al., 2002). Given its central role
in NB-LRR function, surprisingly little is known about
the role of nucleotide binding in NB-LRR activation. In
fact, nucleotide binding has been demonstrated unequivocally for only two plant NB-LRR proteins, I-2
and Mi-1, both from tomato (Tameling et al., 2002). The
NB domains of these two proteins were expressed in
Escherichia coli and shown in a filter-binding assay to
specifically bind ATP rather than other nucleoside
triphosphates. Significantly, these recombinant proteins also hydrolyzed ATP, suggesting that nucleotide
hydrolysis may represent a key step in plant NB-LRR
protein activation.
This hypothesis is supported by the work on the
mammalian Apaf-1 protein described above. The
binding of Apaf-1 to its ligand cytochrome C and
subsequent oligomerization requires dATP or ATP,
and substitution of the nonhydrolyzable analog ATPgS prevents both steps (Hu et al., 1999). This observation indicates that ATP hydrolysis rather than simple
ATP binding is required for Apaf-1 to bind ligand and
form oligomers. If ATP hydrolysis is similarly required
for plant NB-LRR protein function, it would suggest
that conformational changes associated with NB-LRR
activation may only be observed in the presence of
dATP/ATP.
Although it is not yet clear how ATP hydrolysis
regulates ligand binding by Apaf-1 or other NB-ARC
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Pathogen Perception by Plants
containing proteins, analogies to GTP binding proteins
would suggest that hydrolysis causes a conformational
change, and further, that nucleotide exchange between
ADP and ATP may be an important aspect of regulating signaling. Figure 1 presents a model for how ATP
hydrolysis and nucleotide exchange might be incorporated into the regulation of plant NB-LRR protein
function. Although speculative, this model makes
several testable predictions now that potential ligands
for RPM1, RPS2, and RPS5 have been identified. In
particular, it predicts that modification of the ligand (a
host protein) by the pathogen virulence protein induces ATP hydrolysis by the NB-LRR protein, causing
a conformational change in the NB-LRR protein that
allows oligomerization. Inactivation of signaling
would occur when ADP dissociates. The inactive
NB-LRR protein would then have to bind another
molecule of ATP before it would be able to function
once again.
OTHER COMPONENTS OF THE PLANT
ALARM SYSTEM
A recent flurry of papers has established that many
NB-LRR proteins interact with cytosolic HSP90 proteins (Hubert et al., 2003; Lu et al., 2003; Takahashi
et al., 2003). These interactions appear to be important
for either the folding of NB-LRR proteins and/or the
formation of a stable NB-LRR protein complex, as
mutation or silencing of HSP90s causes a sharp reduction in NB-LRR protein accumulation (Hubert
et al., 2003; Lu et al., 2003).
HSP90s may be assisted in the putative assembly
of NB-LRR resistance protein complexes by two other
conserved proteins, RAR1 and SGT1. RAR1 interacts
with HSP90, and mutations in the Arabidopsis RAR1
gene cause a loss of detectable RPM1 protein (Tornero
et al., 2002; Hubert et al., 2003). SGT1 interacts with
both HSP90 and RAR1 (Takahashi et al., 2003), but
appears to play a different role from RAR1, as silencing
of SGT1 in Nicotiana benthamiana does not affect
accumulation of the NB-LRR protein Rx, despite
blocking Rx-mediated resistance (Lu et al., 2003).
Arabidopsis contains two SGT1 genes (Austin et al.,
2002). Mutations in SGT1b compromise resistance
mediated by a specific subset of NB-LRR proteins that
only partially overlaps with those affected by rar1
mutations. Knockouts of both SGT1 genes in Arabidopsis appear to be lethal as homozygous double
mutants have not been recovered. SGT1 has also been
shown to interact with an SCF ubiquitin ligase complex and the COP9 signalosome (Azevedo et al., 2002)
and thus may participate in multiple signaling pathways in plants via regulation of protein degradation.
For a more detailed discussion of these papers see the
recent dispatch by Schulze-Lefert (2004).
CONCLUSIONS
Although much progress has been made in the last 2
years, there still remain large gaps in our understanding of the plant alarm system. Evidence is mounting
that many, and perhaps most, pathogens are detected
indirectly via the enzymatic activity of their virulence
proteins. However, we still lack compelling evidence
Figure 1. An induced proximity model based on data from the human Apaf-1 protein and consistent with current data on plant
NB-LRR proteins. A pathogen virulence protein modifies a host target protein, which then causes a conformational change in the
NB-LRR protein and ATP hydrolysis. This enables oligomerization of the NB-LRR protein via the NB-ARC domain (blue oval),
which activates resistance via inducing proximity of an unidentified activator protein. ADP release causes disassociation of the
complex. ATP must bind to restart the cycle. Specificity for the modified host protein is determined by both the N-terminal
activator binding domain (blue square) and the LRR domain (hatched rectangle). These domains interact with each other in the
absence of ligand. The host target protein is shown interacting with the NB-LRR protein prior to modification based on data on
the Arabidopsis RIN4 protein (see text).
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Innes
that a modified host protein directly activates NB-LRR
signaling. An even more glaring gap in our current
understanding is how plant NB-LRR proteins activate
defense responses. Presumably these proteins physically associate with one or more downstream activator
proteins, but we currently have very little information
as to what these activators might be. Filling these gaps
will undoubtedly keep many laboratories busy for
some time to come. At this point we have a partial
parts list for the alarm system and have some information on how each of the parts work, but have yet
to sort out the wiring.
ACKNOWLEDGMENTS
I thank Dr. Jeff Dangl, University of North Carolina, and Dr. Jeff Ellis,
CSIRO, Canberra, Australia, for sharing papers in press, and the members of
my laboratory for stimulating discussions and comments on this manuscript.
Received February 3, 2004; returned for revision February 14, 2004; accepted
February 16, 2004.
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