MPMI Vol. 20, No. 7, 2007, pp. 759–768. doi:10.1094 / MPMI -20-7-0759.

© 2007 The American Phytopathological Society

Rhizobacteria-Induced Priming in Arabidopsis

Is Dependent on Ethylene, Jasmonic Acid, and NPR1

Il-Pyung Ahn,1 Sang-Woo Lee,2 and Seok-Cheol Suh1

1
National Institute of Agricultural Biotechnology, Rural Development Administration, Suwon, 441-100, Korea; 2Gyeonggi
Province Agricultural Research and Extension Services, Hwaseong, 445-972, Korea
Submitted 12 May 2006. Accepted 29 January 2007.

A nonpathogenic rhizobacterium, Pseudomonas putida responsible for the broad spectrum of disease protection. A se-
LSW17S, elicited systemic protection against Fusarium ries of elegant research projects has been performed to dissect
wilt and pith necrosis caused by Fusarium oxysporum f. sp. the signaling pathways and plant genes essential for induced
lycopersici and P. corrugata in tomato (Lycopersicon escu- systemic resistance (ISR), triggered by a strain of Pseudomo-
lentum L.). LSW17S also confers disease resistance against nas fluorescens, using an Arabidopsis-based pathosystem
P. syringae pv. tomato DC3000 (DC3000) on Arabidopsis (Pieterse et al. 1996, 1998, 2000; Ton et al. 2001; van Wees et
ecotype Col-0. To investigate mechanisms underlying dis- al. 2000; Verhagen et al. 2004). Rhizobacteria enhance disease
ease protection, expression patterns of defense-related resistance through activation of specific defense mechanisms;
genes PR1, PR2, PR5, and PDF1.2 and cellular defense re- for example, cell wall reinforcements (Benhamou et al. 1996),
sponses such as hydrogen peroxide accumulation and cal- accumulation of defense-related materials and enzymes
lose deposition were investigated. LSW17S treatment ex- (Benhamou et al. 1998; Chen et al. 2000), secondary metabo-
hibited the typical phenomena of priming. Strong and lite production (Ongena et al. 2000), and impediment of infec-
faster transcription of defense-related genes was induced tion processes of pathogens such as inhibition of sporangial
and hydrogen peroxide or callose were accumulated in and zoospore germination (Yan et al. 2002). Lipopolysaccha-
Arabidopsis treated with LSW17S and infected with rides, siderophores, or salicylic acid from rhizobacteria also
DC3000. In contrast, individual actions of LSW17S and are indispensable for successful disease protection (Buysens et
DC3000 did not elicit rapid molecular and cellular defense al. 1996; De Meyer et al. 1999; Ramamoorthy et al. 2001).
responses. Priming by LSW17S was translocated systemi- One of the most interesting results described in previous lit-
cally and retained for more than 10 days. Treatment with erature is the relationship between expression of defense-related
LSW17S reduced pathogen proliferation in Arabidopsis responses and disease protection by ISR. Elevation of defense-
ecotype Col-0 expressing bacterial NahG; however, npr1, related gene transcriptions has been assumed as molecular evi-
etr1, and jar1 mutations impaired inhibition of pathogen dence of whether or not resistance is induced (Friedrich et al.
growth. Cellular and molecular priming responses support 1996; Gaffney et al. 1993; Gorlach et al. 1996; Kim et al.
these results. In sum, LSW17S primes Arabidopsis for 2004; Park and Kloepper 2000). However, some agents do not
NPR1-, ethylene-, and jasmonic acid-dependent disease re- trigger cellular and molecular defense responses per se even
sistance, and efficient molecular and cellular defense re- though they confer obvious disease resistance on the susceptible
sponses. plants (Conrath et al. 2001; Graham and Graham 1994; Pieterse
et al. 2001; Ryu et al. 2003; Ton et al. 2005). Discrepancy be-
Additional keyword: induced systemic resistance. tween disease protection and transcription of defense-related
genes has been an interesting theme in ISR. Subsequent patho-
gen challenge on the plants pretreated with the above agents
Plants have well-organized defense systems that decipher entailed augmented defense-related responses such as fortified
pathogen signals and induce appropriate defenses. Timely rec- defense-related gene expression, accumulation of active oxy-
ognition of pathogen attack and prompt expression of pertinent gen species (AOS), callose deposition, and papillae formation
defense responses are the key differences between compatible singly or in combination. This facet brings up the rapid and
(susceptible) and incompatible (resistant) interactions (Bennett efficient activation of defense systems in immunized animals
et al. 2005; Lu et al. 2004; McDowell and Dangl 2000; and humans infected by a pathogen (Cho et al. 2002; Ludewig
Umemura et al. 2003; Yang et al. 1997). The resistance (R) et al. 1998). The enhanced capacity to express pertinent defense
gene product of a plant acts as a receptor for the corresponding mechanisms is termed “elicitation competency” (Graham and
pathogen’s avirulence (Avr) gene product and their interaction Graham 1994) or “priming” (Conrath et al. 2001, 2002). Agents
sequentially triggers defense-related responses. Subsequent inducing priming do not activate the plant’s defense responses,
defense responses have many features in common with those but rather enhance the plant’s ability to suppress future patho-
of systemic resistance induced by certain rhizobacteria. gen attacks (Conrath et al. 2002; Jakab et al. 2001, 2005; Pozo
Rhizobacteria protect susceptible plants from multiple- et al. 2004; Ton et al. 2005; Zimmerli et al. 2000). In this re-
pathogen infection. Moreover, they promote plant growth and spect, primed plants share many characteristics with resistant
provide other benefits for environmental safety. There is ample plants harboring R genes. There is evidence that priming is not
research elucidating bacterial factors and plant mechanisms an uncommon phenomenon in induced resistance. For exam-
ple, low doses of plant defense activators such as benzo-
Corresponding author: I.-P. Ahn; Telephone: 82-31-299-1719; Fax: 82-31- (1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH),
299-1692; E-mail: jinhyung@[Link] β-aminobutyric acid (BABA), 2,6-dichloroisonicotinic acid

Vol. 20, No. 7, 2007 / 759

(DCINA), salicylic acid, and methyl jasmonate are capable of tured parsley cells or cucumber hypocotyls (Conrath et al.
priming a susceptible host (Conrath et al. 2002; Faize et al. 2001; Kauss et al. 1999).
2004; Si-Ammour et al. 2003). Priming is one of the most effi- In previous research, we showed the effect of P. putida
cient types of induced resistance because the metabolic invest- LSW17S as a biocontrol agent inhibiting multiple fungal and
ment of the plant for the constitutive activation of the defense bacterial diseases in Solanaceous crops (Lee et al. 2005). Treat-
system is reduced or prevented. Molecular research on priming ment with LSW17S protected tomato (Lycopersicon esculentum
by rhizobacteria has focused on identifying plant genes through L.) against Fusarium wilt and its culture filtrate inhibited co-
analyses of gene expression before and after pathogen chal- nidial germination of plant pathogens (Lee 2004). Although
lenge in the rhizobacteria-treated plants (Verhagen et al. 2004), this in vitro effect should be one of the mechanisms of disease
screening of gene-trap lines, and map-based cloning. resistance, impairment of conidial germination alone is insuffi-
Virulent pathogens induce rapid cellular defense responses cient to explain the broad spectrum of systemic resistance con-
such as production of AOS, callose and phenolic compound ferred by this specific rhizobacterium.
deposition, and augmented expression of defense-related genes The aims of this study were to characterize the defense-
in plants treated with biological control agents or plant defense related cellular and molecular responses of plants treated with
activators (Faize et al. 2004; Jeun et al. 2004; Pare et al. 2005; strain LSW17S, and to investigate the relationship between
D.-S. Park et al. 2002; K. Park et al. 2000; Ryu et al. 2000; resistance and priming. To accomplish these goals, the effects
Yedidia et al. 1999). In addition to arresting pathogen prolif- of pathogen inoculation on the expression of defense-related
eration in planta, AOS is involved in cell wall reinforcement genes and induction of cellular defense responses such as AOS
(Olivain et al. 2003) and acts as signaling molecules mediating accumulation and callose deposition were analyzed in Arabi-
systemic resistance (Alvarez et al. 1998; Bolwell et al. 1995, dopsis ecotype Col-0 and four of its defense signaling mutants.
1998; Chamnongpol et al. 1998; Tenhaken et al. 1995;
Wojtaszek 1997). Callose is a fluorescent β-1,3-glucan com- RESULTS
plex and its rapid deposition is an indicator of defense-related
responses (Devadas et al. 2002; Flors et al. 2005; Ton and P. putida LSW17S protects Arabidopsis
Mauch-Mani 2004). In the absence of elicitors or subsequent against bacterial infection.
stress, methyl jasmonate, salicylic acid, or DCINA alone did The bioprotection capacity of P. putida LSW17S was assessed
not induce AOS elevation or callose accumulation in the cul- in Arabidopsis thaliana ecotype Col-0, which is susceptible to P.

Fig. 1. Responses of Arabidopsis to Pseudomonas syringae pv. tomato DC3000 (DC3000) infection after P. putida LSW17S treatment. A, Necrotic lesions
normally present on Arabidopsis ecotype Col-0 due to DC3000 are suppressed in LSW17S-treated leaves. Col-0 plants treated with Escherichia coli DH5α
also were inoculated 5 days later with DC3000 or DC3000 expressing avrRpm1 (DC3000[avrRpm1]) at 108 CFU/ml in 0.85% NaCl, Tween 20 at 250 μg/ml
as control. Representative plant from each treatment was photographed 5 days postinoculation (dpi). B, Quantification of systemic resistance against
DC3000 infection in Arabidopsis. Plants were treated with LSW17S (+), 1.2 mM benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH; +), or
DH5α (+) in 0.85% NaCl or 0.85% NaCl only (–). Leaves were sprayed with 0.85% NaCl and Tween 20 at 250 μg/ml (mock, –) or inoculated with
DC3000(avrRpm1) (+) or DC 3000 (+) 5 days later. Data are means (disease index of the percentage of symptomatic leaves relative to control plants [100%])
with standard errors from 25 plants. Ratings were performed 5 dpi. Different letters indicate statistically significant differences among treatments (Duncan’s
multiple range tests; P < 0.05). Inset: time course of bacterial growth in the leaves treated with mock or LSW17S and inoculated with DC3000 or
DC3000(avrRpm1). Data points are means (CFU per 0.1 g) with standard errors from 25 randomly selected leaves. All experiments were done more than
three times and similar results were obtained.

760 / Molecular Plant-Microbe Interactions

syringae pv. tomato DC3000 (DC3000). Avirulent DC 3000 ex- attained a maximum level at 24 hpi. In the incompatible interac-
pressing avrRpm1 (DC3000[avrRpm1]) also was inoculated tion, faster and stronger transcriptions of these genes were ob-
onto Col-0 as a resistant control. Plants pretreated with either served within 6 hpi. Rapid and fortified transcriptions of the four
ISR-activating bacterial strain LSW17S or non-ISR-activating genes tested were observed at 6 to 48 hpi in Arabidopsis treated
strain Escherichia coli DH5α and challenged with DC3000 5 with LSW17S and challenged with virulent DC3000. Mock
days later are shown in Figure 1A. Yellowing and water-soaking treatment did not affect mRNA synthesis of defense-related
symptoms were observed on the leaves of mock- or DH5α- genes significantly (data not shown).
treated plants, whereas leaves of LSW17S-treated plants did not
exhibit typical disease progression. Minute brown spots were Persistence and dose dependency of LSW17S effects.
observed 18 h after inoculation. No further symptom of the dis- We investigated the duration of disease resistance by
ease was observed. Similar perturbation was observed on the LSW17S in Arabidopsis. Arabidopsis was inoculated with
leaves inoculated with avirulent DC3000(avrRpm1). In addition, DC3000 at 3, 5, and 10 days after LSW17S treatment.
BTH and LSW17S treatments protected Arabidopsis approxi- LSW17S treatment inhibited disease progression caused by
mately 60 and 58%, respectively (Fig. 1B). Pathogen prolifera- DC3000 by 60, 51,, and 64% in Arabidopsis at 3, 5, and 10
tion also was inhibited significantly by LSW17S treatment. On days after treatment, respectively (Fig. 3A). Small, dark-brown
the other hand, DH5α did not affect disease progression. lesions were observed on the inoculated leaves at 18 hpi. Ro-
bust transcriptions of PR1 and PDF1.2 genes were observed at
Defense-related gene expression 6 hpi in all LSW17S-treated plants (Fig. 3B). In addition,
by LSW17S and pathogen challenge. LSW17S significantly reduced disease progression at 106 and
To investigate the mechanisms of disease protection conferred 108 CFU/g final densities (Fig. 4A and B). Disease progression
by LSW17S, transcript accumulations of three pathogenesis- also was inhibited significantly by both treatments. The fresh
related (PR) genes and PDF1.2 were examined. LSW17S treat- weight of plants increased with the 106 CFU/g LSW17S treat-
ment alone did not induce PR1 and PDF1.2 mRNA accumula- ment, but the difference in fresh weight of LSW17S- and
tions in Arabidopsis (Fig. 2). However, expressions of all four mock-treated plants was not significant.
defense-related genes were observed at 12 to 24 h postinocula-
tion (hpi) in the compatible interaction. Transcript accumulation

Fig. 2. Effect of LSW17S and pathogen challenge on the expression of PR

and PDF1.2 genes. Arabidopsis plants (n = 25) were mock treated (0.85%
NaCl) or treated with LSW17S in 0.85% NaCl and challenged 5 days later
with virulent DC3000 or avirulent DC3000(avrRpm1); dpi and dpt = days
postinoculation and days posttreatment, respectively. A, Transcriptions of Fig. 3. Treatment of LSW17S suppresses bacterial infection of Arabidopsis
PR1 and PDF1.2 genes in Arabidopsis treated with LSW17S. RNAs from up to 10 days through the induction of resistance responses. The Arabi-
LSW17S-treated Arabidopsis roots (Local) and from leaves (Systemic) dopsis ecotype Col-0 was inoculated with Pseudomonas syringae pv. to-
that were harvested at indicated time points. The blots were hybridized mato DC3000 at 3, 5, and 10 days after LSW17S treatment. A, Bacterial
with Arabidopsis PR1 and PDF1.2 probes labeled with [32P]-dCTP. Equal disease progression in mock-, DH5α-, and LSW17S-treated Arabidopsis
sample loading was confirmed by ethidium-bromide staining of the rRNA following DC3000 infection at 3, 5, and 10 days after each treatment. Each
in the gel. B, Transcription of PR genes and PDF1.2 gene in Arabidopsis bar represents the mean of disease index with standard error from 20 to 25
treated with LSW17S and challenged with virulent pathogen. Arabidopsis plants. B, Transcriptions of PR1/PDF1.2 genes in Arabidopsis leaves in-
was mock and LSW17S treated Arabidopsis and challenged with virulent oculated with DC3000 at 3, 5, and 10 days after mock (0.85% NaCl), Es-
(DC3000) or avirulent DC3000(avrRpm1) pathogens. RNA was isolated cherichia coli DH5α, or LSW17S treatments. Total RNA was extracted
from Arabidopsis leaves harvested at the indicated time points after inocu- from the leaves of 25 plants 6 h postinoculation, separated by denaturing
lation, separated by denaturing gel electrophoresis, and transferred to nylon gel electrophoresis, and transferred to nylon membranes. The blots were
membranes. The blots were hybridized with Arabidopsis PR1, PR2, PR5, hybridized with Arabidopsis PR1 and PDF1.2 probes labeled with [32P]-
and PDF1.2 probes labeled with [32P]-dCTP. Equal sample loading was dCTP. Equal sample loading was confirmed by ethidium-bromide staining
confirmed by ethidium-bromide staining of the rRNA in the gel. of the rRNA in the gel.

Vol. 20, No. 7, 2007 / 761

Production of hydrogen peroxide and callose induction. transgenic Col-0, and the three mutants were treated with
The effects of LSW17S and DC3000 challenge on hydrogen LSW17S 5 days prior to pathogen inoculation. LSW17S treat-
peroxide accumulation and callose induction at 6 and 24 hpi ment protected NahG (a line expressing bacterial NahG)
were determined. Both responses provide reliable evidence of plants from DC3000 invasion. However, it did not confer
whether or not plants are primed. Hydrogen peroxide and cal- npr1 (a mutant that does not accumulate PR1 in response to
lose production were absent in LSW17S- or mock-treated salicylic acid), etr1 (an altered perception of ethylene mu-
Arabidopsis (Fig. 5). In contrast, these defense-related re- tant), and jar1 (a mutant that displays reduced sensitivity to
sponses were highly regulated within the leaf tissue or trichome methyl jasmonate) with disease resistance (Fig. 6A). There-
of plants treated with LSW17S and recovered 6 and 24 h after fore, the disease-protecting effect induced by LSW17S is de-
DC3000 challenge. Inoculation was performed 5 days after pendent on ethylene, jasmonic acid, and NPR1. LSW17S was
LSW17S treatment. The level of defense responses was similar recovered from the rhizosphere of all plant lines without sig-
with Arabidopsis challenged with avirulent pathogen DC3000 nificant differences (Fig. 6B); therefore, the loss of disease-
(avrRpm1). protecting capacity in npr1, etr1, and jar1 is not due to poor
colonization. In addition, streptomycin-resistant bacteria were
Priming by LSW17S is dependent absent from leaf extracts of all the plant lines. These results
on ethylene, jasmonic acid, and NPR1 gene. indicate that priming by LSW17S is translocated systemically
To analyze the mode of action of LSW17S on the resis- (data not shown).
tance of Arabidopsis against DC3000, the ecotype Col-0, one To investigate the mode of action of priming by LSW17S at
the molecular and cellular levels, we examined the transcrip-
tion of PR1/PDF1.2 via reverse-transcription polymerase
chain reaction (RT-PCR) and production of hydrogen peroxide
or callose induction in the LSW17S-treated Col-0, transgenic
Col-0 expressing NahG, and its mutants with or without the
subsequent challenge of DC3000. LSW17S treatment did not
affect the assessed molecular and cellular responses (Figs. 6C
and D). Faster and fortified inductions of hydrogen peroxide at
6 hpi and callose at 24 hpi were evident in the Col-0 and NahG
treated with LSW17S and challenged with DC3000. The npr1,
etr1, and jar1 mutations impaired the defense responses. Tran-
scriptions of PR1 and PDF1.2 also were induced at 6 hpi in
the Col-0 plants pretreated with LSW17S. Hence, LSW17S
alone did not induce expression of both genes. PR1 transcrip-
tion was not observed in NahG; however, LSW17S and
DC3000 induced PDF1.2 expression in the same plants at 6
hpi. Transcription of both genes was almost completely abol-
ished by the npr1, etr1, and jar1 mutations.

DISCUSSION
We previously found that tomato (L. esculentum L.) wilt dis-
ease, caused by Fusarium oxysporum f. sp. lycopersici, is sig-
nificantly inhibited if soil is drenched with P. putida LSW17S
(Lee 2004). Additional investigations revealed that the same
treatment also protects eggplant, tomato, and red pepper from
bacterial wilt, and that LSW17S inhibits conidial germination
and mycelial growth of several plant-pathogenic fungi. To fur-
ther examine the protection conferred by LSW17S and its un-
derlying resistance mechanisms, the effects of LSW17S on the
interaction between A. thaliana and a virulent bacterial patho-
gen, P. syringae pv. tomato DC3000, were investigated.

P. putida LSW17S confers systemic resistance

in Arabidopsis.
Our results here clearly demonstrate that LSW17S protects
Arabidopsis from infection by DC3000. Repeated challenge
tests corroborated this disease-protecting capacity. Although
Fig. 4. Growth and pathogen proliferation in Arabidopsis Col-0 plants previous study has shown that LSW17S inhibits the conidial
treated with increasing concentrations of LSW17S. Bacterization with
germination of plant-pathogenic fungus and protects tomato
LSW17S and Pseudomonas syringae pv. tomato DC3000 challenge were
performed, except the 106 concentration of LSW17S. A, Growth of Arabi- against F. oxysporum infection, neither LSW17S nor its culture
dopsis plants treated with increasing concentration of LSW17S. Arabidop- filtrate arrests DC3000 growth in vitro (data not shown). Control
sis did not show any distinctive alterations given the different concentra- of multiple diseases in numerous host species in the absence of
tions of LSW17S. B, Quantification of plant growth and disease inhibition direct pathogen inhibition is a typical characteristic of systemic
by increasing concentration of LSW17S 10 days after treatment. The dis- resistance induced by rhizobacteria (Jetiyanon et al. 2003;
ease index was evaluated as described in Figure 1. Each bar represents the
Kloepper et al. 2004; Krause et al. 2003; Raupach and Kloepper
mean of fresh weight and disease index with standard error from 25 plants.
Different letters indicate statistically significant differences between treat- 1998; Zhang et al. 2004). Therefore, we concluded that
ments (Duncan’s multiple range tests; P < 0.05). All experiments were LSW17S induces systemic resistance in Arabidopsis against
done more than three times and similar results were obtained. DC3000 infection.

762 / Molecular Plant-Microbe Interactions

Priming by LSW17S. of defense-related genes, a relationship between them is not
Following LSW17S treatment, when the plants were chal- obligated. Similar to the present results, viral infection has
lenged with DC3000, mRNA synthesis of PR1 genes and been found to trigger augmented expression of defense-related
PDF1.2 gene was accelerated and potentiated (Fig. 2). However, genes in tobacco treated with rhizobacteria conferring systemic
neither LSW17S treatment nor DC3000 challenge alone induced resistance (Ahn et al. 2002). Expression of the tobacco PR-1a
the transcription activation. Although resistance-inducing gene has been triggered by endophytic bacterium (Park and
agents protect disease progression and upregulate expression Kloepper 2000). On the other hand, a P. fluorescens strain did

Fig. 5. Effect of priming by LSW17S on hydrogen peroxide accumulation and callose deposition in Arabidopsis. Four-week-old Arabidopsis plants were
grown in horticultural soil mix supplemented with LSW17S (+) at 108 CFU/g in 0.85% NaCl or with 0.85% NaCl only (–). Five days later, leaves of the
plants were sprayed with Tween 20 (–) at 250 μg/ml, inoculated with virulent Pseudomonas syringae pv. tomato DC3000 (108 CFU/ml) (DC3000; +) or with
avirulent P. syringae pv. tomato DC3000 expressing avrRpm1 (108 CFU/ml) (DC3000[avrRpm1]; +). Accumulation of hydrogen peroxide and callose depo-
sition was seen under light and epifluorescence microscope as described in Materials and Methods. Bars = 50 μm. All experiments were done more than
three times and similar results were obtained.

Vol. 20, No. 7, 2007 / 763

not affect transcription of PR genes and the PDF1.2 gene in rhizobacteria is host or strain specific. Microarray analyses
Arabidopsis in spite of its obvious disease protection capacity also confirmed these results (Cartieaux et al. 2003; Kloepper et
regardless of the pathogen infection (Pieterse et al. 1996; van al. 2004; Verhagen et al. 2004; Wang et al. 2005).
Wees et al. 1999). These results and our data demonstrate that Priming by LSW17S persists for a long period. LSW17S-
defense-related gene expressions are not reliable evidence in induced disease resistance lasted up to 10 days (Fig. 3A). In
determining whether a plant is in a resistant or armed state, addition, pathogen challenge triggered accelerated expression
suggesting that the mode of action of systemic resistance by of PR1 and PDF1.2 in Arabidopsis pretreated with LSW17S.

Fig. 6. Systemic resistance and cellular and molecular defense responses of LSW17S-treated Arabidopsis Col-0, NahG, npr1, etr1, and jar1 against Pseudo-
monas syringae pv. tomato DC3000. A, Numbers of DC3000 in leaves of the Arabidopsis thaliana ecotype Col-0 and signaling mutants of the same ecotype
treated with LSW17S 5 days prior to bacterial inoculation. Different letters indicate statistically significant differences between mock- and LSW17S-treated
plants (Duncan’s multiple range tests; P < 0.05). Open bars, mock-treated Arabidopsis leaves; solid bars, LSW17S-treated Arabidopsis leaves. B, Colonization of
Arabidopsis by LSW17S. Arabidopsis roots were harvested five days after LSW17S treatment and the number of colony-forming units of streptomycin-
resistant LSW17S was estimated as described in Materials and Methods. Data are means with standard errors from 20 to 25 plants. Different letters indicate
statistically significant differences between treatments (Duncan’s multiple range tests; P < 0.05). C, Hydrogen peroxide accumulation and callose deposition
in Arabidopsis. Treatment and observation were conducted. D, Analysis of PR1 and PDF1.2 gene expression by reverse-transcription polymerase chain
reaction (RT-PCR) in Arabidopsis five days after LSW17S treatment and 6 hpi with DC3000. First-strand cDNA was synthesized using 50 ng of total RNA
isolated from leaves of the indicated plant and treatment combinations and anchored oligo-dT. PCR product was obtained after amplification of 0.5 μl of
first-strand cDNA and each gene-specific primer. The products were analyzed by using gene-specific probes.

764 / Molecular Plant-Microbe Interactions

Long-lasting priming indicates the successful colonization and vealed significantly different responses. Augmented expression
successful interaction between host and LSW17S. In the previ- of PR genes was observed in Arabidopsis treated with
ous research, we also found stable colonization by LSW17S LSW17S and then infected with DC3000, whereas WCS417r
on and within tomato roots (Lee et al. 2005). Persistence of treatment did not induce PR gene transcription regardless of
rhizobacteria-induced systemic resistance against plant-patho- the pathogen infection (van Wees et al. 1999; Verhagen et al.
genic fungi, bacteria, and nematodes has been described in 2004). Our results also showed that, in LSW17S-primed Arabi-
tomato (Yan et al. 2002), Arabidopsis (van Wees et al. 1997), dopsis, pathogen challenge triggered higher hydrogen peroxide
and potato (Hallmann et al. 2001). LSW17S at 106 CFU/g pro- accumulation and callose deposition. However, WCS417r did
moted Arabidopsis growth through several repeated experi- not trigger transcription of Arabidopsis genes related to pro-
ments, but the increase in fresh weight was not significant. duction of AOS within leaves regardless of the subsequent
LSW17S treatment inhibited fungal and bacterial diseases of pathogen challenge. Although this result does not absolutely
tomato and significantly increased growth of Solanaceous exclude the possibility that reactive oxygen species could play
crops (Lee et al. 2005). Hence, growth-promoting activity in a role in WCS417r-mediated ISR, there has been no report
LSW17S is plant species specific. Taken together, LSW17S about the effects of WCS417r on the production of AOS within
should be a good candidate for biological control of multiple Arabidopsis.
potent diseases in Cruciferous and Solanaceous crops. In summary, LSW17S primes and induces cellular and mo-
The effects of LSW17S on cellular defense responses were lecular defense mechanisms in Arabidopsis against pathogens.
investigated to determine the relationship between priming and Bacterization with LSW17S primes the plant into a highly
systemic resistance induced by LSW17S. No discrete hydro- competent state for a long time in the absence of detectable
gen peroxide accumulation was detected in the LSW17S- or physiological variations. Priming has physiological and ge-
mock-treated leaves (Fig. 5). However, strong hydrogen perox- netic importance, and it is one of the most economical and
ide accumulation was observed in the leaf tissues and effective modes of resistance because it prevents dispensable
trichomes treated with DC3000 5 days later. Similarly, higher metabolic consumption in plants. Plants divert this metabolic
peroxidase activity was observed in tomato and pigeon pea investment for growth and other fitness-related processes
treated and inoculated with rhizobacteria and pathogens (Podile (Bostock 2005; Heil 2001; Purrington 2000). Recently, costs
and Laxmi 1998; Silva et al. 2004). Formation of callose also and benefits of priming were analyzed and compared with
was investigated because hydrogen peroxide is one of the sec- those of systemic acquired resistance by BTH or constitutive
ondary signaling messengers regulating defense responses. activation of resistance by cpr1 mutation (van Hulten et al.
LSW17S treatment and subsequent pathogen challenge were 2006). Plant growth and seed production in primed Arabidop-
required for callose deposition. Similarly, newly formed cal- sis are superior even when disease occurs. Along with conven-
lose was accumulated within and around the infection sites in tional antibiotics, previously developed plant defense activators,
roots of pea treated with rhizobacteria (Benhamou et al. 1996, biocontrol organisms, and improved seed varieties, LSW17S
1998). These results and our findings support the concept that could be a novel disease control strategy that is environmentally
primed Arabidopsis treated with LSW17S is in a surveillance friendly.
state extremely sensitive to pathogen challenge. In addition,
priming by LSW17S and R-gene-dependent resistance share MATERIALS AND METHODS
several cellular and molecular defense mechanisms, including
hydrogen peroxide accumulation, callose deposition, and ex- Plants and pathogen challenge.
pression patterns of defense-related genes. Seed of A. thaliana ecotype Col-0, Col-0 expressing bacterial
the NahG gene, and its signaling mutants npr1, etr1, and jar1
Priming by LSW17S is dependent were obtained from The Arabidopsis Information Resource.
on ethylene, jasmonic acid, and NPR1. Arabidopsis plants were grown in a growth chamber at 22ºC,
To investigate defense-signaling pathways regulated by 65 to 70% relative humidity, and 16 h of illumination daily.
LSW17S, disease-inhibiting activities, hydrogen peroxide ac- Four-week-old A. thaliana plants were used for bacterial and
cumulation, callose deposition, and PR1/PDF1.2 transcriptions chemical treatments.
were assessed in the Arabidopsis signaling mutants that failed The DC3000 strain of P. syringae pv. tomato containing
to respond to salicylic acid, ethylene, and jasmonic acid (Fig. 6). pVSP61 (DC3000) and pVSP61 carrying avrRpm1 (DC 3000
The pathogen proliferation in planta clearly was reduced in [avrRpm1]) were cultivated on King’s medium B (Difco Labo-
LSW17S-treated Col-0 and bacterial NahG-transgenic plants; ratories, Detroit) containing kanamycin at 50 μg/ml for 2 days at
however, this inhibitory effect was not evident in npr1, etr1, 28ºC (Bisgrove et al. 1994; Grant et al. 1995). To inoculate
and jar1 plants in spite of the successful colonization of Arabidopsis, bacterial cells were retrieved from the medium
LSW17S (Fig. 6A and B). This is additional potent evidence with 0.85% NaCl solution supplemented with Tween 20 at 250
that disease protection by LSW17S is based on plant-mediated μg/ml, and the concentration was adjusted to 1 × 108 CFU/ml. In
ISR and not on antibiosis. In addition, rapid and accelerated all, 25 Arabidopsis plants were inoculated 5 days after transplan-
expressions of both PR1 and PDF1.2 were triggered by patho- tation into LSW17S-supplemented horticultural soil mix. Plants
gen challenge in the Col-0 plants pretreated with LSW17S were inoculated by spraying bacterial suspension until all the
(Figs. 2B and 3B), and npr1, etr1, and jar1 mutations impaired leaves were covered with fine droplets. The inoculated plants
PR1/PDF1.2 transcriptions by priming (Fig. 6D). These results were kept in a dew chamber for 16 h at 25ºC and 100% relative
indicate that the concealed resistance state conferred by humidity, then transferred to a growth chamber. Disease severity
LSW17S extends its effects through ethylene, jasmonic acid, was estimated as described previously (Pieterse et al. 1996) with
and NPR1. Induction of systemic resistance within NahG- some modifications. Five days after inoculation, disease severity
transgenic plants and impairment of this resistance by npr1, was assessed by determining the percentage of leaves per plant
etr1, and jar1 have been described in Arabidopsis treated with with typical yellowing or water-soaking symptoms (25 plants
a P. fluorescens WCS417r and another rhizobacterium (Pieterse per treatment). Moreover, CFU within 1 g (fresh weight) of
et al. 1996; Ryu et al. 2003). Although defense signaling in our Arabidopsis leaves from each of five plants per treatment
study is not novel, comparative analyses on defense mechanisms through plating dilutions on King’s B medium containing kana-
for systemic resistance between LSW17S and WCS417r re- mycin at 50 μg/ml was estimated.

Vol. 20, No. 7, 2007 / 765

Treatment of microorganisms and chemicals. cDNA was synthesized from 50 ng of total RNA of the leaves
P. putida LSW17S and E. coli DH5α kept at –70ºC as 15% recovered 5 days after bacterization at 6 hpi, using the Re-
glycerol stock were grown on tryptic soy agar (TSA; Difco verse-iT first-strand synthesis kit and anchored oligo-dT as
Laboratories) at 28ºC for 48 h. For bacterial treatment of indicated by the manufacturer’s instructions (ABGene, Epsom,
Arabidopsis plants, cell suspensions of LSW17S and DH5α U.K.). Independent PCR using equal aliquots (0.5 μl) of cDNA
were retrieved from the colonies on TSA with sterilized 0.85% samples was performed using PR1/PDF1.2-specific primers
NaCl solution and the concentration of cell suspension was described (Vieira Dos Santos et al. 2003). Each PCR product
adjusted with the same solution. Arabidopsis plants were was blotted and Southern analyses were performed using PR1
transplanted into cylindrical plastic pots (10 cm in diameter by or PDF1.2 probe labeled with [32P]-dCTP. The tubulin gene
8 cm in height) containing horticultural soil amended with (TUB) was amplified as a quantitative control (Lee et al.
bacterial suspension 5 days prior to pathogen challenge, unless 2000).
otherwise indicated. To minimize physical damage by trans-
planting, Arabidopsis and soil were transferred into the pots at ACKNOWLEDGMENTS
the same time. DH5α and increasing concentrations of
LSW17S were applied to Arabidopsis. The whole plants were This research was supported by grants from the National Institute of
recovered and their fresh weights were measured 10 days after Agricultural Biology to I.-P. Ahn and a grant to S.-C. Suh from the Crop
treatment. Pots with growing Arabidopsis were dipped into 1.2 Functional Genomics Center of the 21st Century Frontier Research Pro-
gram (CG2211) funded by the Ministry of Science and Technology and
mM solutions of BTH (Sigma-Aldrich, St. Louis) for 30 min 5
the Rural Development Administration of the Korean government. We
days prior to pathogen challenge. There were five replicates deeply thank M. E. M. Orden for editing this article.
for each treatment on a total of 25 plants per treatment.

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