MOLECULAR PLANT PATHOLOGY (2005) 6(1), 79–83
DOI: 10.1111/J.1364-3703.2004.00265.X
Blackwell Publishing, Ltd.
Short communication
Coronatine and salicylic acid: the battle between Arabidopsis and
Pseudomonas for phytohormone control
A N N A B L O C K 1 , E R I C S C H M E L Z 2 , J E F F R E Y B. J O N E S 3 A N D H A R RY J. K L E E 1 *
1
Plant Molecular and Cellular Biology Program, Horticultural Sciences Department, PO Box 110690, University of Florida, Gainesville, FL 32611-0690, USA
United States Department of Agriculture–Agricultural Research Service, Center for Medical Agricultural & Veterinary Entomology, 1700 SW 23rd Drive, Gainesville,
FL 32608, USA
3
Department of Plant Pathology, University of Florida, Gainesville, FL 32611-0680, USA
2
S U M M A RY
The phytotoxin coronatine is a jasmonate mimic produced by
Pseudomonas syringae pv. tomato (Pst ). Coronatine acts as a
virulence factor in Arabidopsis and mutants insensitive to coronatine are resistant to Pst and have higher levels of salicylic acid
(SA). In this work we used the SA-deficient lines NahG and sid22 to determine if coronatine acts directly as a virulence factor or
indirectly by SA suppression. Using coronatine-deficient Pst mutants
we demonstrated that the lack of coronatine compromises Pst
virulence in both wild-type and SA-deficient Arabidopsis. Thus,
the action of coronatine is not due to SA suppression. Rather, SAindependent jasmonate-responses are the most likely mechanism
for its action.
INTRODUCTION
The phytotoxin coronatine is an important virulence factor for
the virulent bacterial pathogen Pseudomonas syringae pv. tomato
(Pst). Coronatine is believed to mimic the active jasmonate 12-oxophytodienoic acid (OPDA) that is a precursor to jasmonic acid (JA)
(Weiler et al., 1994). Tn5 transposon insertions into Pst DC3000
produced the coronatine-deficient mutants Pst DC3661 and
Pst DC3118 (Moore et al., 1989). These mutants have reduced
virulence on Arabidopsis thaliana in dip inoculations but not in
injection inoculations. However, the disease symptoms in both
forms of inoculation are reduced as compared with wild-type
(Mittal and Davis, 1995).
The evidence that coronatine is a virulence factor that acts via
stimulating jasmonate responses is reinforced by studies with the
Arabidopsis coronatine-insensitive mutant coi1 (Feys et al., 1994).
*Correspondence: Tel.: +1 352 3928249; fax: +1 352 8462063; e-mail: [email protected]
© 2005 BLACKWELL PUBLISHING LTD
COI1 is a part of an E3 ubiguitin-ligase involved in a jasmonate
response pathway (Xie et al., 1998). The coi1 mutants have
increased resistance to Pst , are jasmonate insensitive and
have increased levels of salicylic acid (SA) upon infection.
Jasmonates are a group of active compounds produced from
linolenic acid, which include OPDA, JA and methyl jasmonate
(MeJA). Several Arabidopsis mutants that cannot produce
or sense jasmonates have normal susceptibility to Pst. These
mutants include the fad3-3 fad7-2 fad8 triple mutant and the
jasmonate signalling mutant jar1-1 (Kloek et al., 2001).
Studies in tomato show that coronatine acts by stimulating
jasmonate response pathways rather than by inducing jasmonate
biosynthesis and that MeJA application can restore virulence to
coronatine-deficient Pst (Zhao et al., 2003). In tomato, jasmonates
also influence SA production as they are necessary for SA induction
in response to infection with Xanthomonas campestris pv. vesicatoria
(O’Donnell et al., 2003). This illustrates the strong impact jasmonates
can have on SA levels. Coronatine is an interesting virulence factor,
as it appears to act by usurping host phytohormone-signalling
pathways. Studying its action not only provides information
on how Pst is a successful pathogen but also increases the understanding of phytohormone signalling and action.
Coronatine stimulates jasmonate responses and in Arabidopsis
jasmonates are believed to be antagonistic to SA (Glazebrook et al.,
2003). Furthermore, SA-deficient Arabidopsis plants have higher
levels of JA than wild-type in response to infection with Pst (Heck
et al., 2003; Spoel et al., 2003). It remains unclear whether the
action of coronatine in Arabidopsis derives solely from stimulation
of the jasmonate pathway or if it is an indirect mechanism acting
via SA suppression.
The involvement of SA in coronatine action is an important
question as SA controls basal resistance of Arabidopsis to Pst.
Arabidopsis mutants that show enhanced susceptibility to
virulent pathogens often accumulate less SA than their wildtype parents following infection (Zhou et al., 1998). Conversely,
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increased SA in the coi1 mutant may account for its resistance to
Pst. The removal of SA has been accomplished by introduction of
the nahG transgene encoding a Pseudomonas putida salicylate
hydroxylase (EC 1.14.13.1), which converts SA into catechol
(Yamamoto et al., 1965). These NahG plants have enhanced
susceptibility to several pathogens, including Pst (Delaney et al.,
1994; Lawton et al., 1995). An Arabidopsis SA biosynthesis mutant,
sid2-2, that fails to accumulate SA in response to pathogens has
also been identified. This line contains a loss of function mutation
in the SA biosynthesis enzyme isochorismate synthase 1 (EC 5.4.4.2),
which converts chorismate to isochorismate (Wildermuth et al.,
2001). sid2-2 also has enhanced susceptibility to Pst (Nawrath
and Metraux, 1999).
The role of SA in Arabidopsis basal resistance and the proposed
antagonism between SA and jasmonates suggests that the
activation of jasmonate-responses by coronatine could suppress
SA. This hypothesis is supported by studies performed on the
coi1-20 mutant expressing the NahG transgene that shows the
enhanced susceptibility of NahG rather than the resistance of
coi1-20 (Kloek et al., 2001). However, the coi1-20 NahG line is
compromised in both SA and JA signalling and therefore coronatine action independent of SA cannot be discounted.
The aim of this study was to determine if the action of coronatine
as a virulence factor is dependent upon the antagonistic relationship between SA and jasmonates. In other words, is coronatine
action SA-dependent? As a first step in determining the role of
coronatine in relation to SA, the amount of coronatine present
in Columbia, sid2-2 and NahG plants following dip inoculation
with 107 cfu/mL of Pst DC3000 was determined as described in
Schmelz et al. (2004) (Fig. 1a). NahG plants accumulated significantly higher levels of coronatine than Columbia and sid2-2. The
higher coronatine levels in NahG may be due to the accumulation
of catechol in this line as NahG demonstrates several altered
defence responses that are not due to SA-deficiency (Heck et al.,
2003; Van Wees and Glazebrook, 2003). As discussed below, these
data also show that coronatine production does not directly
correlate with bacterial populations.
As coronatine is believed to act via jasmonate-signalling, a
study of coronatine action cannot ignore endogenous jasmonate
production. Therefore, JA (Fig. 1b) and OPDA (Fig. 1c) levels in
Pst-infected Columbia, sid2-2 and NahG Arabidopsis were determined as described in Schmelz et al. (2004). These data show that
JA and OPDA were induced in all lines in response to Pst and that
NahG and sid2-2 accumulated more jasmonates than Columbia.
Fig. 1 Coronatine and jasmonate production in SA-deficient Arabidopsis.
Wild-type Arabidopsis (Columbia) and the SA-deficient lines sid2-2 and NahG
were dip inoculated with 1 × 107 cfu/mL of virulent Pst DC3000. The levels of
(a) coronatine, (b) JA and (c) OPDA were determined in the different lines. Bars
= standard error (n = 3). (d) The mRNA expression levels of PDF1.2 and VSP
were determined in plants mock inoculated and inoculated with Pst DC3000
at 48 hpi.
MOLECULAR PLANT PATHOLOGY (2005) 6(1), 79–83 © 2005 BLACKWELL PUBLISHING LTD
Coronatine and salicylic acid
Fig. 2 Bacterial growth and cell death in SA-deficient Arabidopsis infected
with coronatine-deficient Pst. Wild-type (Columbia) Arabidopsis and the SAdeficient lines sid2-2 and NahG were dip inoculated with 1 × 107 cfu/mL of
virulent Pst DC3000 or coronatine-deficient Pst DC3661. (a) Wild-type and
coronatine-deficient Pst populations were measured in all three lines at 96 hpi.
Bars equal standard error (n = 5). (b) Cell death in all lines inoculated with
wild-type or coronatine-deficient Pst was determined in the form of percentage
ion leakage by measuring conductance at 96 hpi. 100% ion leakage is defined
as conductance produced by the same tissue when boiled. Bars = standard
error (n = 10).
As coronatine and jasmonates are believed to stimulate similar
responses, markers for jasmonate signalling could quantify their
combined action. Two common markers for jasmonate responses
are the plant defensin gene PDF1.2 and vegetative storage
protein (VSP ). Both PDF1.2 and VSP are induced in response to MeJA
feeding and their expression is higher in NahG than Columbia in
response to Pst DC3000 infection (Spoel et al., 2003). Expression
of PDF1.2 and VSP by RNA gel blot analysis demonstrated higher
induction of PDF1.2 by Pst DC3000 in NahG than Columbia
with expression in sid2-2 intermediate between that of NahG
and Columbia at 48 h post infection (hpi). VSP, on the other
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hand, is induced to a similar level in all lines at this time (Fig. 1d).
This suggests that the combined levels of coronatine and jasmonates correlate with the expression of PDF1.2 better than VSP.
The action of coronatine may be jasmonate response-mediated
SA suppression. To test this hypothesis, wild-type Pst DC3000 and
the coronatine-deficient mutant Pst DC3661 were used to determine if the reduced virulence of coronatine-deficient Pst was
SA-dependent. NahG and sid2-2 had higher bacterial populations
than Columbia due to the role of SA in maintaining basal resistance. It is interesting to note that coronatine production does not
have a direct relationship to bacterial populations, as sid2-2 had
higher populations of Pst than Columbia but similar coronatine
levels. NahG, by contrast, contained more coronatine than sid2-2
yet had similar bacterial populations. This indicates that Pst may
require a bacterial population threshold to be met, or specific host
factors to be released to trigger increased coronatine production.
Infection with coronatine-deficient Pst resulted in higher bacterial
populations in NahG and sid2-2 than in Columbia (Fig. 2a). However,
coronatine deficiency reduced bacterial populations in SA-deficient
as well as in wild-type Arabidopsis. If the action of coronatine is
achieved by the suppression of SA synthesis, then SA-deficient
Arabidopsis lines would be unaffected by coronatine deficiency.
The more severe symptom development and higher bacteria populations of wild-type than coronatine-deficient Pst in SA-deficient
lines demonstrates that the action of coronatine as a virulence
factor is not SA-dependent.
Cell death in response to Pst as measured by ion leakage was
higher in SA-deficient lines than in Columbia (Fig. 2b). However,
the reduced virulence of coronatine-deficient Pst is accompanied
by reduced cell death in all lines. Visible symptom development
also reflected the differences in bacterial populations. There was
more chlorotic development in SA-deficient Arabidopsis than in
Columbia in response to Pst (Fig. 3). However, chlorotic development was more pronounced in all lines in response to wild-type
than to coronatine-deficient Pst. These effects of coronatine deficiency were confirmed with an additional coronatine-deficient
mutant Pst DC3118 and the SA signalling mutant npr1-1 (data
not shown).
These results demonstrate that the action of coronatine as a
virulence factor is not SA-dependent. By stimulating jasmonate
responses, however, coronatine may still lead to SA suppression.
To determine if the production of coronatine by Pst influenced the
levels of SA and jasmonates within the plants, SA, JA and OPDA
levels were measured at 48 hpi in Columbia, sid2-2 and NahG
plants that had been mock inoculated or inoculated with Pst
DC3000 or Pst DC3661 (Fig. 4). SA production is negligible in
sid2-2 and NahG plants inoculated with either Pst DC3000 or Pst
DC3661 (Fig. 4a). However, SA production is higher due to inoculation with Pst DC3000 than Pst DC3661 in Columbia, indicating
that coronatine does not suppress SA. The levels of JA (Fig. 4b)
and OPDA (Fig. 4c) are reduced in all lines inoculated with Pst
© 2005 BLACKWELL PUBLISHING LTD MOLECULAR PLANT PATHOLOGY (2005) 6(1), 79–83
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A. BLOCK et al.
Fig. 3 Symptom development in SA-deficient Arabidopsis infected with
coronatine-deficient Pst. Wild-type (Columbia) Arabidopsis and the SAdeficient lines sid2-2 and NahG were dip inoculated with 1 × 107 cfu/mL of
virulent Pst DC3000 or coronatine-deficient Pst DC3661. This photograph was
taken at 96 hpi and is representative of four independent biological replicates.
DC3661 as compared with those inoculated with Pst DC3000.
These data suggest that coronatine has the ability to stimulate
both SA and jasmonates. Whether this stimulation is direct or
simply a consequence of the higher pathogen levels in these
plants remains to be determined.
What is clear is that upon infection with Pst, Arabidopsis
produces both SA and jasmonates to control its defence responses.
Pst can influence these responses to its favour by using coronatine
to stimulate jasmonate responses. However, removing coronatine
results in less disease whether or not SA is produced. This
suggests that jasmonates play a larger SA-independent role in
controlling the disease response than previously suspected.
AC K N O W L E D G E M E N T S
This work was supported in part by a grant to H.J.K. from
the National Science Foundation (IBN0091064) and the Florida
Agricultural Experiment Station. This is FAES Journal series
number R-10568.
Fig. 4 Phytohormone induction in response to Pst DC3000 and Pst DC3661.
Wild-type (Columbia) Arabidopsis and the SA-deficient lines sid2-2 and NahG
were mock inoculated or inoculated with 1 × 107 cfu/mL of virulent Pst
DC3000 or coronatine-deficient Pst DC3661. The levels of (a) salicylic acid,
(b) jasmonic acid and (c) OPDA due to mock inoculation or at 48 hpi were
determined in the different lines. Bars equal standard error (n = 3).
MOLECULAR PLANT PATHOLOGY (2005) 6(1), 79–83 © 2005 BLACKWELL PUBLISHING LTD
Coronatine and salicylic acid
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© 2005 BLACKWELL PUBLISHING LTD MOLECULAR PLANT PATHOLOGY (2005) 6(1), 79–83