Download Prospects for understanding avirulence gene function Frank F White

291
Prospects for understanding avirulence gene function
Frank F White*, Bing Yang† and Lowell B Johnson‡
Avirulence genes are originally defined by their negative impact
on the ability of a pathogen to infect their host plant. Many
avirulence genes are now known to represent a subset of
virulence factors involved in the mediation of the
host–pathogen interaction. Characterization of avirulence
genes has revealed that they encode an amazing assortment of
proteins and belong to several gene families. Although the
biochemical functions of the avirulence gene products are
unknown, studies are beginning to reveal the features and
interesting relationships between the avirulence and virulence
activities of the proteins. Identification of critical virulence
factors and elucidation of their functions promises to provide
insight into plant defense mechanisms, and new and improved
strategies for the control of plant disease.
type-III secretory pathways have been reviewed extensively [5], and are remarkable for their capacity to deliver
virulence and avirulence proteins into the host cells
(reviewed in [6]). Although not all avirulence and virulence gene products are necessarily delivered via the Hrp
pathway, this review will emphasize the intrinsic value of
Hrp-associated bacterial avirulence gene products and the
features of these proteins that might be relevant to their
functions within the host cell.
Avirulence genes as virulence genes
Introduction
The number of known dual-acting bacterial avirulence
genes has grown steadily since the identification of avrBs2
from Xanthomonas campestris pv. vesicatoria (Table 1) [7].
The virulence effects of these genes usually affect the
population of the bacteria in the infected tissue and are
often discernible as causing changes in lesion size, number, or appearance (Figure 1). Mutations in avrBs2 resulted
in impaired ability of Xanthomonas to grow on pepper cultivars that lacked the Bs2 gene for resistance [7]. The
avrBs2 gene is found in many species of Xanthomonas and
the polypeptide that it encodes has a sequence related to
those of agrocinopine synthase and glycerol phosphodiesterase, suggesting that this polypeptide has an enzymatic
role in the pathogenicity of many members of the genus
[8]. The fact that the AvrBs2 functioned upon expression
in the plant host cells suggests that the protein is secreted
from the bacterium and, therefore, likely to function within the host cell [9••]. A variety of phosphorylatedlipid-derived signaling molecules in plants are potential
substrates for AvrBs2 [10].
Avirulence genes cause a plant pathogen or pest to elicit a
resistance response in a host plant. The genes occur in
viruses, bacteria, fungi, nematodes and insects. Related
genes have also been identified in species of Rhizobium
suggesting their involvement in symbiotic interactions [1].
Avirulence genes are defined by corresponding resistance
(R) genes of which a relatively large number have now
been cloned (reviewed in [2]). The resistance response is
typically accompanied by a hypersensitive reaction (HR),
which is a form of programmed cell death; a burst of
superoxide production; and the expression of defense
genes (reviewed in [3,4]). The lack of a clear biological
function attributable to many avirulence genes has been
puzzling. This puzzlement has steadily given way to the
view that many avirulence genes have dual functions, having a role in virulence as well as avirulence, and thus
function as important mediators of the interaction
between pathogen and host. This concept of avirulence
gene products as virulence factors, although not new, was
greatly advanced by studies of the Hrp (HR and pathogenicity) pathway, a type-III secretory pathway, and by
comparisons to the closely related type-III secretion pathways of animal pathosystems. Both plant and animal
One of the difficulties in assessing the intrinsic properties
of some bacterial avirulence genes is that they have a pronounced effect on virulence only when present in the
appropriate species, strain or pathovar, or in a pathovar on
a particular host species or variety. The virulence effects of
avrA and avrE have been observed in only one strain of
Pseudomonas syringae pv. tomato, PT23 [11]. The avrE locus
consists of two coding sequences and both polypeptides
are required for virulence and avirulence activity. AvrF, the
product of the open reading frame that is downstream of
AvrE, has the characteristics of a chaperone and may be
involved in the secretion of AvrE [12]. Homologs of avrE
were originally identified as the critical virulence factors
DspE (also known as DspA) and DspF (also known as DspB)
in Erwinia amylovora; yet these homologs were dispensable for the elicitation of a HR on nonhost species, hence
their identification as disease specific (Dsp) loci [12,13].
The DspEF locus also functions as an avirulence locus
when transferred to P. syringae pv. glycinea [12]. The
polypeptide responsible for avirulence activity has yet to
be characterized. The virulence effect of avrPto was
observed after its transfer to strain T1 of P. syringae pv.
Addresses
Department of Plant Pathology, Kansas State University,
4024 Throckmorton Hall, Manhattan, Kansas 66506, USA
*e-mail: [email protected]
† e-mail: [email protected]
‡ e-mail: [email protected]
Current Opinion in Plant Biology 2000, 3:291–298
1369-5266/00/$ — see front matter
© 2000 Elsevier Science Ltd. All rights reserved.
Abbreviations
HR
hypersensitive reaction
Hrp
HR and pathogenicity
NLS
nuclear localization signals
R
resistance
292
Biotic interactions
Figure 1
(a)
(b)
Current Opinion in Plant Biology
tomato (Figure 1) [14•,15•]. AvrRpm1 (also called
avrPmaA1.RPM1) is required for full virulence of P. syringae
pv. maculicola on Arabidopsis [16], yet loss of a virtually
identical allele had no apparent effect in P. syringae pv. pisi
on pea [17]. AvrRpt2 has a virulence effect in strain
DC3000 of P. syringae pv. tomato on the No-0 land race of
Arabidopsis but not on land race Col-0 (which does not
have a functional RPS2) (B Kunkel, personal communication). Interestingly, avrRpt2 has also been found to have
deleterious effects when expressed in Col-0, indicating
that the encoded protein may affect both Arabidopsis land
races, but its effects in enhancing the virulence of the bacterium are observable only on No-0 ([18]; B Kunkel,
personal communication). Interestingly, avrRpt2 increased
the size of the bacterial population of strain ES4326 of
P. syringae pv. maculicola on the rps2 mutant of Col-0
(J Greenberg, personal communication).
The intrinsic value in virulence of an avirulence gene may
also require environmental conditions or epidemiological
factors that are not included in the virulence assay. The
avrb6 from X. campestris pv. malvacearum does not alter the
size of the bacterial population within inoculated leaves but
results in the release of greater numbers of bacteria to the
leaf surface [19]. Thus, the release and spread, or invasiveness, of the pathogen, which are often not assayed, may be
as important as the size of the pathogen population [20].
Avrb6 is a member of the avrBs3 avirulence gene family of
which there are many members, including several dual-acting genes (reviewed in [21]). For example, the retention of
multiple avrBs3 family members within strains of the cotton pathogen X. campestris pv. malvacearum and the rice
pathogen X. oryzae pv. oryzae may facilitate the generation
of additional virulence alleles by recombination [22–24].
Members without apparent virulence activity may represent defeated virulence factors or virulence factors with
specificity for other host species. Additional alleles may
allow a strain to have a broader host range or more rapid
adaptation to the deployment of R genes in intensively
Symptoms of the dual-acting avirulence genes
avrPto and pthXO1 in compatible interactions.
(a) Tomato leaves infected with P. syringae
pv. tomato T1 with (right) and without (left)
avrPto. (b) Rice leaves infected with X. oryzae
pv. oryzae. Inoculations from left to right:
strain PXO99A; PXO99AhrpC–
(nonpathogenic); PXO99Amx2 (containing a
mutation in pthXO1, an uncharacterized dualacting avrBs3 family member); and
PXO99Amx2 with pthXO1. Dark areas on
leaves represent water-soaked lesions. Watersoaked appearance, caused by the release of
water from the infected cells, is a common
symptom of foliar necrotizing bacterial
pathogens. The tomato leaves were dipinoculated. Rice leaves were inoculated using
needle-less syringes.
managed crop species such as cotton and rice, which are
grown in environments conducive to bacterial disease.
Avirulence genes and general host defense
responses
A working model of the role of some avirulence gene
products can be derived from the events in the invasion
process of Yersinia spp. (and related bacteria) that use
type-III secretory systems to enhance virulence and invasiveness. Bacterial lipoproteins and lipopolysaccharides of
Yersinia, among other compounds, trigger host proinflammatory responses. The bacteria, for their part, secrete a
variety of factors via a type-III pathway that interrupt the
signaling processes. Some of the affected host pathway
components have been identified including MAP
(i.e. mitogen-activated protein) kinases, GTP-binding
proteins and actin filaments, which play a role in cell organization and molecular trafficking (reviewed in [25•]). A
variety of compounds produced by microorganisms also
elicit defense gene expression in plants (reviewed in [26]).
The elicitors are produced by a broad range of bacteria
including virulent strains. The response is therefore considered nonspecific or general resistance in contrast to the
specific avirulence-gene-dependent responses. MAP
kinases that are activated by general elicitors have been
identified in plants [27,28•], and at least one GTP-binding
factor is associated with programmed cell death [29]. The
virulence function of some avirulence proteins may be
analogous to that of the type-III-dependent factors of
pathogenic bacteria, that is, to interfere with signal and
structural pathways of the general defense responses.
Hrp-dependent interference with the defense gene
expression of the host has been shown to occur, although
the individual factors involved remain to be identified
(reviewed in [30]).
Genetic approaches are underway to identify the genes
involved in general defense responses to virulent
pathogens. Three genes, EDS1, PAD4, and NDR1, that
Prospects for understanding avirulence gene function White, Yang and Johnson
293
Table 1
Bacterial avirulence genes with virulence or virulence-associated properties.
Gene or protein
Organism or virus
Features, possible function
and localization in host*
Related proteins or alleles†
avrBs2
X. c pv. vesicatoria
Cytoplasmic
avrXa7
X. o. pv. oryzae
avrb6
pthN
pthA
avrRxv
X. c. pv. malvacearum
X. c. pv. malvacearum
X. citri
X. c. pv. malvacearum
NLS, AD
DNA binding, nucleus
Nucleus
Nucleus
Cell hyperplasia, nucleus
MAP kinase binding?
Agrocinopine synthase,
glycerol-phosphodiesterase
avrBs3 family
avrRpm1
avrPto
avrE
MYM, inner membrane?
MYM, kinase binding, inner membrane
Unknown
avrA
avrRpt2
P. s. pv. maculicula
P. s. pv. tomato (T1)
P. s. pv. tomato (PT23)
E. amylovora
P. s. pv. syringae
E. amylovora
P. s. pv. tomato (PT23)
P. s. pv. tomato
avrPphF
virPphA
P. s. pv. phaseolicola
P. s. pv. phaseolicola
Unknown
Unknown, inhibits HR
avrF
Reference(s)
[8]
DspA (DspE) (Ea)
[21,23]
[55,56•]
[22,63]
[64]
[65•]
[35,36,66]
[38,39,41]
[16,67]
[14•,15•]
[12,13,68]
Chaperone
DspB (DspF) (Ea)
[12,13]
Cytoplasmic
Cytoplasmic
AvrA (Psg), AvrBs1
[68,69]
[70] (a)
avrBs3 family
avrBs3 family
avrBs3 family
AvrBsT, Orf5 (Pss), YopJ (Yps),
YopP (Ye), AvrA (St), y410 (Rl)
AvrPpiA1 (Psp)
[71•]
[71•]
*AD, transcription activation domain; MYM, myristoylation motif; NLS
nuclear localization signal motif. †Species indicated in parentheses.
Ea, E. amylovora; Psg, P. syringae pv. glycinea; Psph, Psp, P.
syringae pv. pisi; Pss, P. syringae pv. syringae; St, Salmonella
typhimurium; Ye, Y. enterocolitica; and Yps, Y. pseudotuberculosis.
(a) B Kunkel, J Greenberg, personal communications.
allow the enhanced growth of virulent pathogens have
been cloned and characterized [31,32••,33]. EDS1 and
PAD4 have sequence similarity to lipases, whereas
NDR1 is predicted to be a membrane protein and no
potential biochemical function has been assigned to it.
PAD4 and EDS1 could well begin a phospholipid signaling cascade. It would be interesting to determine
whether the activity of some avirulence genes, particularly avrBs2, is affected on plants in which these genes have
been mutated. Biochemical approaches to characterizing
defense responses to pathogens are also underway. A conserved segment of bacterial flagellin was recently
demonstrated to be a potent elicitor of defense gene
expression. The active peptides are present in a variety of
pathogenic bacteria and perceived at the nanomolar level
by a broad range of plant species [34••]. Biochemical
analyses should help to identify the signaling complexes
that may be targeted by the individual virulence factors
from bacterial pathogens.
X. campestris pv. vesicatoria (avrBsT) [36], P. syringae pv.
syringae [37•], Erwinia amylovora [12] and Rhizobium
leguminosarum [38]. Although avrRxv has not been shown
to be required for virulence, the protein product of avrRxv
has sequence relatedness to the virulence factor YopJ from
Yersinia pseudotuberculosis (Figure 2). YopJ has additional
homologs in Y. pestis (YopP) and Salmonella typhimurium
(AvrA). YopJ, YopP and AvrA are secreted by type-III systems and required for virulence [39–41]. Recently, YopJ
has been shown to bind MAPKK1 and IKKβ (an inhibitor
of NFκb kinase kinase), which are involved in the activation of NFκb during an inflammatory response to infection
by Yersinia [42••]. It is tempting to speculate, therefore,
that AvrRxv and related proteins from plant pathogenic
bacteria interact with MAP kinases that are otherwise activated by general defense signaling mechanisms.
Shared virulence factors of plant and animal
pathogenic bacteria
Signal pathways that are conserved in both plant and animal cells may be responsible for the broad membership of
the avrRxv/yopJ gene family. Members of this family are
found in a diverse collection of Gram negative pathogenic
and symbiotic bacteria. The original avrRxv gene was
identified after its transfer from X. campestris pv. vesicatoria
to X. campestris pv. phaseoli, thereby converting X. campestris
pv. phaseoli into an incompatible strain on some bean cultivars [35]. Related genes have been identified in
Avirulence gene products are targeted to cellular
organelles
The products of two groups of avirulence genes with virulence effects appear to be targeted to specific organelles
within the host cell. Members of the first group are identified by the presence of a myristoylation motif, which, if
functional, would localize the protein to the cytoplasmic
surfaces of membranes (Figure 3). The motif in AvrPto is
required for avirulence and virulence activity (X Tang,
personal communication). Replacement of the glycine at
the second position (G2A) destroyed all AvrPto activity,
yet the modified protein was still secreted and capable of
interaction with Pto, the corresponding R-gene product
in the yeast two-hybrid system. Upon expression in the
294
Biotic interactions
Figure 2
Yopj
AvrA(St)
AvrRxv
AvrBsT
Y4LO
Consensus
Figure 3
50
64
120
98
66
***
SRM DVEVMPALVIQANNKYPEMNLNLVTSPL
EET DLEMMPFLVAQANKKYPELNLKFVMSVH
RLMDIENLPHLVRSYDNRLNNLNLRSFDTPG
TKADVENKYYLAHAYNERFPELHLSCHDSAQ
LSLDIRNLPLLAASYNRRYPDLDLRHMDSPA
Dve mp L
n kypem L
s
80
94
150
128
96
Current Opinion in Plant Biology
AvrPto
AvrPto
AvrRpm1
AvrRpm1
AvrB
AvrB
AvrC
AvrC
Pto
Pto
p60Src
p60Src
*
MGNICVGGSRMAH
MGCVSSTSRSTGY
MGCVSSKSTTVLS
MGNVCFRPSRSHV
MGSKYSKATNSIN
MGSSKSKPKDPSQ
Current Opinion in Plant Biology
Alignment of the amino-terminal region of members of the AvrRxv
family. The alignment shows that the residues of the amino-terminal
portion of the protein, which are the products of five genes of the
avrRxv family, are conserved. The region includes part of the putative
SH2 domain of YopJ [40]. Replacement mutation of DVE (***) in YopJ
causes the loss of YopJ activity.
plants, wild-type AvrPto was found almost exclusively in
the membrane fraction of the host cells, whereas the G2A
mutant was present in the soluble fraction (X Tang,
personal communication). Curiously, Pto also has a myristoylation site (Figure 2). Replacement of the glycine at
position two in Pto, however, had no effect on its ability
to confer resistance in tomato [43]. One possible caveat
of this work was that the mutant Pto gene was expressed
using the strong CaMV35S promoter, which may not
result in expression patterns that are representative of
those under the endogenous promoter. Tests using the
wild-type promoter are in progress (G Martin, personal
communication). Rpm1, the R-gene product corresponding to AvrRpm1, is associated with the inner plasma
membrane [44]. AvrRpm1 has a myristoylation motif and
presumably is also targeted to the inner plasma membrane surface. AvrB and AvrC, which are related to each
other by sequence similarity [45], also have myristoylation motifs, although their significance is unknown [46].
AvrB is also recognized by Rpm1 [47]. The functions of
AvrPto and AvrRpm1 in virulence are unknown. Pto is a
serine/threonine kinase, and AvrPto is known to bind Pto
in resistance signaling [48,49]. AvrPto might, therefore,
be a kinase binding protein. Targeting the avirulence
proteins to the inner membrane would be consistent with
a possible role for them in interfering with a receptorlinked signaling pathway.
Potential myristoylation motifs in avirulence proteins. The asterisk
indicates the glycine at the number two position, which is the site of
myristoylation. Replacement of G with A in AvrPto eliminates both
virulence and avirulence activity. The amino-terminal sequences of Pto
and pSrc60, a previously characterized myristoylated proto-oncogene
from chicken [72], are shown for comparison.
Family members differ in the number and the order of the
repeats as identified by the variable region (Figure 4).
Critical, yet unknown, structural features that are
involved in specificity for avirulence and virulence lie
within the repeat domains [19,24,52]. Functional nuclear
localization signals (NLS) are present in the carboxyl-coding portion of the prototype gene product [53], and are
required for the avirulence activity of avrBs3 and avrXa10
[54,55]. The NLS-coding regions of avrXa7 are also
required avirulence and virulence activities, both of which
can be restored by the addition of the SV40 T-antigen
motif (B Yang et al., unpublished results). The function of
the avrBs3-like gene products upon reaching the nucleus
is unknown. Curiously, the proteins have a transcriptional
activation domain in the carboxy terminus, which is
required for the avirulence activities of AvrXa10, AvrXa7
and AvrBs3 [55,56•]. AvrXa7 also requires the activation
domain for virulence activity and has recently been shown
to be a double-stranded DNA binding protein (B Yang et
al., unpublished results). Thus, AvrXa7 and, by inference,
the other members of the family have features consistent
with those of transcription factors and possibly act by
altering the transcription of one or more genes. The highly conserved nature of the members of the family may
reflect a conserved target in the diverse host species.
Conclusions and further speculations
The second group of targeted bacterial avirulence proteins are encoded by the avrBs3 family members [50,51].
These proteins, which are restricted to the genus
Xanthomonas, appear to be targeted to host nuclei. The
family consists of a large number of near-identical genes
with varying R-gene specificities in a variety of host
plants, and some members are dual functional (Table 1).
Within the middle third of the protein is a repeated
sequence of 34 amino acids that varies in copy number
from 14 copies in Avrb6 from X. campestris pv. malvacearum
to 25 copies in AvrXa7 (reviewed in [51]). The repeats are
highly conserved with the most consistent variation present at codons 12 and 13 of the individual repeat units.
Understanding the role of avirulence genes may provide
some practical benefits for crop protection. Identification
of virulence factors and their functional domains should
allow geneticists and genetic engineers to focus on strategies that are directed at the critical factors of pathogens.
The plants themselves may already have provided some of
the tools. Over time, the process of adaptation between
pathogen and host may have led to recognition of critical
virulence factors or critical domains within the factors by
host plants, forcing the pathogen, in a sense, to decide
between triggering a resistance reaction or converting to a
less virulent strain. The longer the time needed for the
pathogen to adapt to an R-gene product, the greater the
Prospects for understanding avirulence gene function White, Yang and Johnson
durability of resistance. Particularly successful adaptations
by plants may even alter the host range of a pathogen
species. The possibility of selecting R genes that correspond to important virulence factors of a pathogen is
already in the process of being tested. Laugé et al. [57]
were able to identify an R gene (Cf-ECP2), which corresponds to the virulence factor ECP2 from Cladosporium
fulvum, by expressing the protein in the PVX vector. ECP2
is present in all strains of C. fulvum and, therefore, may be
critical to the capacity of the fungus to cause significant
disease. Whether the Cf-ECP2 gene recognizes an important domain of ECP2, and how readily ECP2 can convert
to an unrecognized yet active form, remains to be shown.
The Bs2 gene corresponds to the widely occurring and
dual-acting avrBs2 and represents an R gene that could
potentially be deployed against a variety of Xanthomonas
disease complexes. The gene was recently cloned, and its
effectiveness in a variety of crop species can now be tested [9••]. Some of the potential difficulties of this approach
are perhaps indicated by the fact that Bs2 has only been
shown to be effective in Solanaceous species. Nevertheless,
only a few heterologous expressions have been performed,
and, if highly restricted, the gene activity in other species
may be improved after the limiting factors of Bs2 activity
in heterologous species are better understood.
This review has emphasized the possible role of avirulence
genes in altering the host’s pathogen surveillance system.
The mode-of-action by which virulence is enhanced has
not, however, been determined for any bacterial avirulence
gene product. Some virulence factors may act on physiological and homeostatic processes other than defense
mechanisms of the host cells. The secretion of auxin by bacteria, for example, has been proposed to stimulate the
release of nutrients from host plant tissues and to assist in
bacterial growth in the phyllosphere [58]. The behaviors of
some avirulence proteins raise interesting questions regarding their function in virulence and resistance. Do the
apparent requirements for specific localization motifs for virulence indicate that the proteins are targeted to specific
sites, possibly defense signaling complexes, within the host?
Does the simultaneous localization requirements for avirulence activity indicate that the host R-gene products are
either similarly targeted or exist as components or guardians
of the host complexes [59]? Other evidence fuels the speculation that the virulence and avirulence activities of some
genes may be intimately linked. Recent preliminary reports
indicate that the flagellin receptor, which is associated with
general resistance, is a serine/threonine receptor kinase
related to the product of the R gene Xa21 [60]. Mutations
EDS1 and NDR1 affect both general resistance (i.e. allow
the greater growth of virulent strains) and R-gene induced
resistance [61,62]. Rapid progress in determining the biochemical functions of bacterial virulence factors is expected
thanks to the wealth of information provided by the analysis
of animal pathosystems and the development of Arabidopsis
as a model system. Understanding the function of virulence
factors will provide important insights toward understanding
295
Figure 4
(a)
B P
S
S
B
NLS A B C
AD
(b)
Repeat
AvrXa10
AvrBs3
Avrb6
PthA
PthN
1
NI
HD
HD
NI
NI
2
HG
NG
NI
NG
HD
3
NI
NS
NG
NI
HD
4
HG
NG
HD
NI
NI
5
NI
NI
HD
NG
HD
6
NI
NI
NI
HD
NI
7
NN
NI
HD
NG
NG
8
HD
HD
NI
HD
NI
9 10 11 12 13 14 15 16 17 18
NI HD NN HG NS NG HD NG
HD NG NS NS HD HD HD NG HD NG
NS HD HD HD NN NG
NG NG NG NG NS HD HD NG NG
NN HD NI NG NG NN
Current Opinion in Plant Biology
(a) Map of the prototype gene in the avrBs3 family. The central repeat
domain is represented by series of open boxes. Black boxes designate
nuclear localization motif sequences A, B, and C, and the
transcriptional activation domain. The conserved restriction sites B,
BamHI; P, PstI; and S, SphI are shown. (b) Organization of 34-aminoacid repeat units in selected members of the avrBs3 family. Each
individual repeat unit is represented by the amino-acid residues at
positions 12 and 13.
the mechanisms of plant defense, and practical strategies
aimed at limiting crop losses caused by plant disease.
Update
Evidence for the myristoylation of AvrRpm1 and its
requirement for the proper functioning of the protein in
virulence and avirulence has been recently published [73].
A similar requirement for the avirulence activity of AvrB
and AvrPphB was also shown [74]. Evidence has also been
presented that the cysteine residue adjacent to the acylated glycine residue may be subject to palmitoylation.
Alterations of C3 in AvrRpm1 decreased the efficacy of the
protein, although not to the extent of the alteration of
G2 [73]. The AvrPphB myristoylation site is only exposed
after a proteolytic cleavage at K62/G63 [74]. Expression
and localization of the proteins within the plant demonstrated that the AvrRpm1 and AvrB are found in the cell
plasma membrane [73].
Acknowledgements
The authors would like to thank Brad Porter, Xiaoyan Tang and Jian-Min
Zhou for helpful discussions, and the colleagues who supplied preprints and
unpublished information. The expert assistance of Ms. Diana Pavlasko was
used in the preparation of the manuscript. This is publication number
00-363-J from the Kansas Agriculture Experiment Station.
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