Priming of Induced Plant Defense Responses

UWE CONRATH
1
Plant Biochemistry & Molecular Biology Group, Department of Plant
Physiology, RWTH Aachen University, Aachen 52056, Germany
I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362
II. Types of IR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362
A. Systemic Acquired Resistance (SAR) .. .. .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. 362
B. Resistance Induced by Benecial Microorganisms . .. .. ... .. .. .. .. .. .. .. .. 363
C. Resistance Induced by Chemicals .. .. ... .. .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. 364
D. Resistance Induced by Wounding . .. ... .. .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. 365
E. Resistance Induced by Modications of Primary Metabolism.. .. .. .. .. 366
III. Priming is a Mechanism of IR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
A. History .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. 367
B. Elucidation of Priming in Parsley Cell Cultures .. .. .. .. ... .. .. .. .. .. .. .. .. 367
C. Priming in SAR.. .. .. ... .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. 369
D. Priming Induced by Benecial Microorganisms .. .. .. .. ... .. .. .. .. .. .. .. .. 372
E. Priming in BABA-IR .. .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. 374
F. Priming in Wound-Induced Resistance .. .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. 376
G. Priming by Modications in Primary Metabolism. .. .. ... .. .. .. .. .. .. .. .. 378
IV. Relevance of Priming in Plant Production. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379
A. Costs and Benets of Priming .. .. .. .. ... .. .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. 379
B. Exploiting Priming in Greenhouse and Field . .. .. .. .. .. ... .. .. .. .. .. .. .. .. 380
V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384
1
Corresponding author: Email: uwe.conrath@bio3.rwth-aachen.de
Advances in Botanical Research, Vol. 51 0065-2296/09 $35.00
Copyright 2009, Elsevier Ltd. All rights reserved. DOI: 10.1016/S0065-2296(09)51009-9
ABSTRACT
Upon infection by a pathogen, colonization of the roots by certain benecial
microbes, or after treatment with various chemicals, plants can establish a unique
physiological situation which is called the primed state of the plant. In the primed
condition, plants respond faster and/or more strongly with the activation of defense
responses when subsequently challenged by microbial pathogens, herbivorous insects,
or abiotic stresses. The potentiated activation of defense responses in primed plants is
frequently associated with enhanced resistance to the challenging stress. Although
priming has been known as a component of various induced resistance phenomena
for decades, most of the progress in the understanding of the phenomenon has been
made over the past decade. Here, I summarize the current knowledge of priming, its
role in various forms of induced resistance, and its relevance for plant protection in
the greenhouse and in the eld.
I. INTRODUCTION
Most plants are sessile organisms unable to escape unfavorable changes in
their environment. To counter harmful conditions in their surroundings,
plants therefore have evolved elaborate mechanisms to sense stress and
adapt to it by rapid, dynamic, and complex alterations in their physiology.
At best, the physiological switch leads to enhanced resistance against a given
stressor without causing major tness costs. A thorough understanding of
the molecular and physiological basis of induced resistance (IR) to disease
will lead to a better understanding of signal perception and transduction in
plants and allow for effective and sustainable pest management in the eld.
II. TYPES OF IR
A. SYSTEMIC ACQUIRED RESISTANCE (SAR)
When a plant becomes infected by a pathogen, it can develop resistance to a
broad and distinctive spectrum of pathogens (Durrant and Dong, 2004;
Ryals et al., 1996). The pathogen-induced resistance can be established in
the tissue surrounding the site of initial infection and also in the distant,
uninfected parts of the plant. The former type of IR has been called loca-
lized acquired resistance (LAR), while the latter was named systemic
acquired resistance (SAR) (Hammerschmidt, 2009; Ross, 1961a,b). It has
been known for many years that both LAR and SAR are frequently
associated with the accumulation of so-called pathogenesis-related (PR)
proteins (Van Loon et al., 2006). Some of these proteins have antimicrobial
activity and, therefore, may contribute tothe resistance (VanLoonet al., 2006;
362 U. CONRATH
see also Section III.A). The identity of the long-distance signal(s) that travel(s)
fromthe site of primary infection to the remote parts of the plant to induce PR
gene expression and SAR, however, is still unclear (Conrath, 2006; Durrant
and Dong, 2004, Grant and Lamb, 2006; Heil and Ton, 2008; Ryals et al.,
1996). In the early 1990s, studies with transgenic tobacco and Arabidopsis
thaliana plants that constitutively accumulate a salicylic acid(SA) hydroxylase
of bacterial origin clearly demonstrated that the plant hormone SAis required
in the distal tissue for SARto be expressed (Delaney et al., 1994; Gaffney et al.,
1993; Vernooij et al., 1994). More recent work with Arabidopsis mutants
affected in either the biosynthesis of SA or in SA signaling conrmed this
conclusion (Dong, 2001). While the important role of SA in the development
of SAR was without any controversy, it has remained unclear whether SA is
the long-distance signal that travels from the site of primary pathogen
infection throughout the plant to induce SAR (Champigny and Cameron,
2009). Some ndings argued in favor of SA as the long-distance signal
(Shulaev et al., 1995; Yalpani et al., 1991) while others argued against it
(Meuwly et al., 1995; Rasmussen et al., 1991; Smith-Becker et al., 1998;
Vernooij et al., 1994).
Over the past few years, several other signaling molecules have emerged as
possible candidates for the endogenous long-distance signal for SAR(reviewed
by Vlot et al., 2008). These include methyl salicylate (MeSA), which is the
methyl ester of SA (Park et al., 2007), lipid-derived signaling molecules
(Maldonado et al., 2002; Nandi et al., 2004), including jasmonic acid (JA)
(Truman et al., 2007) and azelaic acid (Jung et al., 2009), peptides (Xia et al.,
2004), and reactive oxygen species (ROS) (Alvarez et al., 1998). Together,
these ndings argue that a complex and possibly variable combination of
systemic signals may be required to fully induce the bona de SAR response.
B. RESISTANCE INDUCED BY BENEFICIAL MICROORGANISMS
1. Induced systemic resistance (ISR)
Colonization of plant roots by selected strains of nonpathogenic plant
growth-promoting rhizobacteria (PGPR), such as various species of the
genera Pseudomonas (Ahn et al., 2007a; Van Loon, 2007; Van Loon et al.,
1998), Bacillus (Kloepper et al., 2004), or Bradyrhizobium (Cartieaux et al.,
2008), can induce a distinct broad-spectrum resistance response in both
below- and above-ground parts of the plant. This type of IR was named
rhizobacteria-mediated ISR (De Vleesschauwer and Ho fte, 2009;
Van Loon, 2007; Van Loon et al., 1998). In contrast to SAR, ISR is not
associated with PR gene expression or SA accumulation (Pieterse et al.,
1996). It rather requires responsiveness to the plant hormones JA and
PRIMING PLANT DEFENSE 363
ethylene (ET) (Pieterse et al., 1998). Thus, although having similar
phenotypes, SAR and ISR recruit different, yet partly overlapping sets of
plant hormones for their establishment.
2. Resistance induced by symbiotic fungi
Interactions of plants with benecial microorganisms other than those causing
ISR can also result in systemic, broad-spectrum resistance. The symbiosis
between barley roots and the endophytic basidiomycete Piriformospora indica,
for example, confers systemic resistance to various root and leaf pathogens
(Waller et al., 2005). These include the necrotrophic root-rot fungus Fusarium
culmorum and the biotrophic fungus Blumeria graminis f. sp. hordei (Waller
et al., 2005). The signaling mechanism by which Pi. indica induces resistance to
these twopathogens inbarleyis not known, but it seems tobe independent of SA
and JA while being associated with the activation of the glutathioneascorbate
cycle, indicating an increase in antioxidative capacity in Pi. indica-elicited IR
(Waller et al., 2005). Systemic resistance induced by the endophytic fungus
Trichoderma asperellum T34 protected Arabidopsis against a wide range of
pathogens through engagement of the same signaling components as used in
Pseudomonas uorescens (strainWCS417r)-mediatedISR(Segarra et al., 2009).
IR to pathogens was also reported for plants whose roots have been
colonized by mycorrhizal symbionts (Pozo and Azco n-Aguilar, 2007; Pozo
et al., 2005). For example, transcript proling of the shoots of Medicago
truncatula plants whose roots had been colonized by the arbuscular
mycorrhizal fungus Glomus intraradices revealed both systemic expression
of various defense-associated genes and establishment of an IR response to
the bacterium Xanthomonas campestris pv. alfalfae (Liu et al., 2007).
C. RESISTANCE INDUCED BY CHEMICALS
1. Synthetic SA analogs
Besides pathogens and benecial microorganisms, many different inorganic
and organic compounds have been reported to induce resistance in plants
(Kuc, 2001). These include some synthetic SAanalogs. 2,6-Dichloroisonicotinic
acid and its methyl ester (both are referred to as INA) were the rst synthetic
compounds reported to activate a phenocopy of bona-de SAR (Kessmann
et al., 1994). Some years later, benzo(1,2,3)thiadiazole-7-carbothioic acid
S-methyl ester (BTH; synonym: acibenzolar S-methyl, ASM) was introduced
as another synthetic and highly potent activator of SAR (Friedrich et al., 1996;
Gorlach et al., 1996; Lawton et al., 1996). SA, INA(methyl ester), and BTHare
assumed to activate SAR via a same signaling pathway (Ryals et al., 1996).
364 U. CONRATH
2. -Aminobutyric acid
Various amino acids have been shown to induce resistance in plants (Kuc,
2001). Among these, the nonprotein amino acid -aminobutyric acid
(BABA) has received a lot of attention. The compound was shown to be a
potent inducer of resistance against abiotic stress (Jakab et al., 2005;
Zimmerli et al., 2008), nematodes (Oka et al., 1999), insects (Hodge et al.,
2005), and microbial pathogens (Cohen, 2002; Jakab et al., 2001). In
Arabidopsis, the BABA-IR to the oomycete Hyaloperonospora arabidopsidis
does not require SA accumulation (Zimmerli et al., 2000), while BABA-
induced protection against the bacterium Pseudomonas syringae pv. tomato
strain DC3000 (Pst) (Zimmerli et al., 2000) and the gray-mold fungus Botry-
tis cinerea (Zimmerli et al., 2001) is SA-dependent. Research on the induction
by BABA of resistance to Plectosphaerella cucumerina revealed that, in this
case, the induction does not require ET or SA-mediated signaling. It rather
depends on the plant hormone abscisic acid (ABA) (Ton and Mauch-Mani,
2004). In sum, the BABA-IR response in Arabidopsis seems to ward off
various types of pathogens by recruiting distinct signaling pathways.
D. RESISTANCE INDUCED BY WOUNDING
It is generally assumed that physical injury can make living plant tissue prone
to pathogen invasion. However, over the past few years it has become
increasingly clear that wounding, whether caused by mechanical damage or
feeding by herbivorous insects, can also serve as an effective stimulus for the
induction of local and systemic resistance to microbial pathogens or herbiv-
orous insects (Chassot et al., 2007, 2008; Francia et al., 2007; Green and
Ryan, 1972; Ryan, 1990). This wound-induced resistance (WIR) involves
direct activation of many genes, including those encoding protease inhibi-
tors. These proteins can inactivate enzymes with important roles in either
disease symptom development or digestion of plant tissue in the insect gut
(Green and Ryan, 1972; Ryan, 1990). Genetic studies have shown that
several compounds of the octadecanoid pathway (e.g., JA) can act as endog-
enous mediators for wound-induced defense gene activation and resistance
against herbivorous insects (Howe, 2004). However, it is also known that an
extensive number of wound-activated genes can be induced via a signaling
pathway that is independent of JA (Reymond et al., 2000), indicating a
complex nature of the wound response in plants.
Systemin, the rst plant peptide for which a signaling function has been
demonstrated, is involved in the wound-induced activation of protease in-
hibitor (PIN) genes in tomato (Pearce et al., 1991; Ryan, 1992) and other
species of the Solanaceae family, such as potato, bell pepper, and black
PRIMING PLANT DEFENSE 365
nightshade (Constabel, 1999; Constabel et al., 1998). At wound sites, the
18-amino acid peptide systemin is processed from a 200-amino acid precur-
sor protein, called prosystemin, and transported throughout the plant
(Narvaez-Vasquez et al., 1995). Because protease inhibitor gene expression
was constitutive in transgenic tomato plants that overexpressed the
PROSYSTEMIN gene, it was assumed that systemin is the endogenous
signal that would move within the plant to mediate the systemic wound
response (McGurl et al., 1994). However, a series of grafting experiments
with the suppressed in prosystemin-mediated responses-1 (spr1) mutant of
tomato, which is defective in systemin perception, revealed that JA, rather
than systemin, is responsible for systemic signaling of the wound response
(Lee and Howe, 2003). Systemin is needed locally in the damaged tissue but
not in the systemic, undamaged tissue of wounded plants for signal trans-
duction. Thus, it seems now that wound-induced systemin locally contributes
to the biosynthesis of JA, which regulates the production of, or acts as, a
mobile wound signal that is transported throughout the plant to trigger the
systemic wound response (Lee and Howe, 2003).
E. RESISTANCE INDUCED BY MODIFICATIONS OF PRIMARY METABOLISM
While it is obvious that exposure of plants to biotic or abiotic stresses can
affect photosynthesis, partitioning of assimilates, and source-sink relationships
(Schwachtje and Baldwin, 2008), little is known about the impact of primary
plant metabolism on IR in plants. One frequently reported resistance pheno-
type in plants is the so-called high-sugar resistance. This type of IR is
associated with elevated levels of soluble carbohydrates which result from
certain alterations in primary metabolism (Horsfall and Dimond, 1957). The
concept of high-sugar resistance has been supported by various studies
demonstrating that application of sugar to various plant tissues, or provoking
the accumulation of sugar in transgenic plants, can lead to activation of
various PRgenes (Herbers et al., 1996a,b; Johnson and Ryan, 1990). Similarly,
tubers of transgenic potato plants with decreased activity of the plastid ATP/
ADP transporter AATP1(St) not only display reduced starch content, but they
also have altered levels of primary metabolites, such as glucose and other
carbohydrates (Geigenberger et al., 2001; Tjaden et al., 1998). The alterations
in primary metabolism coincide with an IR response to the soft-rot causing
bacterium Pectobacterium atrosepticum (Linke et al., 2002) and the fungal
pathogen Alternaria solani (Conrath et al., 2003) in the tubers, and with IR
to the late blight-causing oomycete Phytophthora infestans in the leaves
(Conrath et al., 2003). Thus, certain alterations in plant primary metabolism
can cause tissue-specic resistance to a variety of biotic challenges.
366 U. CONRATH
III. PRIMING IS A MECHANISM OF IR
A. HISTORY
For many years, IR in plants has been suggested to be on the basis of the
direct activation of defense responses in systemic tissue of pathogen-infected
plants. In case of SAR, these directly induced responses in the systemic tissue
include the accumulation of PR-proteins (Durrant and Dong, 2004; Ryals
et al., 1996). Many PR-proteins display antimicrobial activity presumably
through hydrolytic activities on cell walls of potential microbial pathogens
and contact toxicity, and maybe also as compounds involved in plant defense
signaling (Van Loon et al., 2006). However, as the expression of cloned genes
for PR-proteins in transgenic plants does not generally lead to enhanced
resistance against diverse pathogens, the actual contribution of PR-proteins
to IR appears to be minor (Van Loon, 2000).
As research on IR had focused primarily on the role of PR-proteins and
other directly induced defense-related compounds, it has not been widely
appreciated that the enhanced defensive capacity characteristic of IR is also
associated with a sensitized state in which the plant responds more rapidly
and/or more robustly with the activation of defense responses after exposure
to a biotic or abiotic stressor (Conrath and Go llner, 2008; Conrath et al.,
2002, 2006; Kuc, 1987) (Fig. 1). The state of enhanced capacity to activate
stress-induced defense responses has been called the primed (or sensi-
tized) state of the plant. As a matter of fact, as early as the 1980s, Kuc (1987)
had already argued that priming would be an important component of SAR.
Yet, although priming could be a unifying mechanism for the different types
of IR in plants, the phenomenon did not attract much attention at the time
(Van Loon, 2000). In the 1990s, an important role of priming in SAR was
supported by the nding that there is close correlation between the capability
of various chemicals to activate resistance against tobacco mosaic virus
(TMV) in tobacco (Conrath et al., 1995) and their capacity to prime for
enhanced PHENYLALANINE AMMONIA-LYASE (PAL) gene expression
induced by microbe-associated molecular pattern (MAMP) elicitor treat-
ment in cultured parsley cells (Katz et al., 1998; Thulke and Conrath,
1998), or upon infection of Arabidopsis plants with Pst (Kohler et al., 2002).
B. ELUCIDATION OF PRIMING IN PARSLEY CELL CULTURES
Only in the early 1990s, Uwe Conrath and associates provided the rst
biochemical analyses of the priming phenomenon in plants (Conrath et al.,
2002, 2006). In their initial studies, the authors employed a model system of a
PRIMING PLANT DEFENSE 367
parsley cell culture and a MAMP elicitor from Phytophthora sojae to eluci-
date molecular aspects of priming and the associated amplication of
MAMP elicitor-induced defense responses. They showed that pretreatment
with low concentrations of compounds that would not directly induce cellu-
lar defense responses but did induce SAR in plants, such as SA, INA, or
BTH, primed parsley cells in a time-dependent manner for stronger activa-
tion of various cellular defense responses by low MAMP elicitor doses that
would not per se signicantly induce these defenses directly. These reactions
were also induced by the low doses of the MAMP elicitor in nonprimed
parsley cells, but to a much lesser extent. The analyses encompassed the so-
called oxidative burst (a term describing the production of various ROS),
rapid changes in ion transport across the plasma membrane, the synthesis
and secretion of antimicrobial secondary metabolites (phytoalexins), various
cell wall phenolics, a lignin-like polymer, as well as the expression of various
defense-related genes (Katz et al., 1998; Kauss and Jeblick, 1995; Kauss
et al., 1992a; 1993; Thulke and Conrath, 1998). In subsequent studies using
the parsley cells, it was demonstrated that the effect of the SAR inducers on
defense gene activation depended on the dose of the inducer applied and the
gene that was assayed (Katz et al., 1998; Thulke and Conrath, 1998). While
some defense genes were found to be directly responsive already to moderate
concentrations of SA or BTH, others were not induced at all. This second set
of defense genes rather displayed very strong activation after a priming
treatment of the cells with moderate concentrations of SA or BTH followed
A
(no SAR)
(SAR)
B
Fig. 1. Priming for more robust induction of defense gene expression and reduced
lesion formation are part of the SAR response of plants. Leaves of tobacco (Nicotiana
tabacumcv. Samsun NN) plants transformed with a chimeric gene that is composed of
the promoter of a PR-10 gene of Asparagus ofcinalis fused to the coding sequence of
the GUS reporter gene, were inltrated with either buffer as the control (A), or with
Ps. syringae pv. syringae to induce SAR (B). Seven days later, systemic tissue (photo)
of each plant was infected with lesion-causing TMV and after another 7 days histo-
chemically assayed for GUS reporter gene activity (blue) and lesion development.
Bar 1 cm. Photograph courtesy of Luis Mur (University of Wales, UK).
368 U. CONRATH
by challenge treatment with a very low, suboptimal dose of the MAMP
elicitor (Katz et al., 1998; Thulke and Conrath, 1998). These results attested
to a dual role for inducers of SAR in the activation of plant defense
responses: on the one hand, moderate doses of SA or BTH directly activated
a specic set of defense genes, while, on the other hand, they primed cells for
enhanced expression of a different set of defense genes when the cells were
subjected to challenge treatment with the MAMP elicitor.
C. PRIMING IN SAR
1. Tobacco
In 1996, the group of John Draper reported the rst molecular analysis of the
priming phenomenon in intact plants (Mur et al., 1996). The authors demon-
strated that a soil-drench pretreatment with SA of transgenic tobacco plants
expressingeitherachimericgenecomposedof thepromoter of anAsparagus PR-
10 gene and the coding sequence of the reporter gene -GLUCURONIDASE
(GUS), or the promoter of the defense-relatedPAL3 gene fusedtoGUS, didnot
signicantly induce gene activation (Mur et al., 1996). However, after infection
with Ps. syringae pv. syringae or after wounding, activation of the reporter
gene was much stronger in the SA-pretreated plants than it was in plants that
had not been pretreated with SA (Mur et al., 1996). A few years later, the same
groupdemonstratedthat thelossof resistancetoavirulent bacterial pathogens in
SAhydroxylase-expressing transgenic tobacco was associated with an attenua-
tion of the SA-potentiated oxidative burst (Mur et al., 2000). Together, these
ndings conrmed, at the molecular level, that priming is a part of IR in whole
plants.
2. Arabidopsis
In 1999, Van Wees et al. demonstrated that induction of SAR by infection of
Arabidopsis leaves with avirulent Pst
avrRpt2
resulted in priming of the systemic
tissue, exhibited as elevated expression levels of PR genes. Pretreatment with
BTH likewise primed Arabidopsis for more robust induction of the PAL gene
by Pst (Kohler et al., 2002). Together, these two reports allowed for in-depth
molecular and genetic studies of the priming phenomenon in plants. Thus, the
Arabidopsis mutant enhanced disease resistance-1 (edr1) constitutively displays
systemically enhanced resistance to Pst as well as to the fungus Erisyphe
cichoracearum (Frye and Innes, 1998). It is interesting that edr1 differs
from other known Arabidopsis mutants with constitutively enhanced
disease resistance in that it does not display constitutive activation of the
PR-1 and -2 genes, although their transcripts accumulated to higher levels
after pathogen attack (Frye and Innes, 1998). This observation and the nding
PRIMING PLANT DEFENSE 369
that edr1, when compared to the wild type, displays more robust induction of
defense responses upon pathogen infection strongly suggest an engagement of
the EDR1 protein in priming.
In response to infection with avirulent pathogens the Arabidopsis nonex-
presser of PR genes-1 (npr1) mutant (also named noninducible immunity-1
(nim1) or salicylic-acid insensitive-1 (sai1)) accumulates SA levels that are
similar to those found in the wild type. However, the npr1 mutant is unable to
express biologically or chemically induced SAR (Cao et al., 1994; Delaney
et al., 1995, Shah et al., 1997). Upon pretreatment with BTH the enhanced
activation of Pst-induced PAL gene expression is absent in the npr1 mutant,
while PAL gene expression per se is not abolished in this mutant (Kohler
et al., 2002). This result demonstrates that a functional NPR1 gene is
required for this priming in Arabidopsis.
In a converse manner, the constitutive expresser of PR genes-1 (cpr1) and
cpr5 mutants of Arabidopsis both express SAR in the absence of pretreatment
with activators of SAR (Bowling et al., 1994, 1997). This is probably because
the cpr1 and cpr5 mutants are both in a state of enhanced defense readiness
that resembles SAR in that both express defense responses in addition to
exhibiting the primed state. Not only is a set of defense genes constitutively
expressed (Bowling et al., 1994, 1997), but both these mutants are also
sensitized for higher PAL gene activation upon infection by Pst (Kohler
et al., 2002). Thus, it is likely that because of the enhanced SA levels in
cpr1 and cpr5 (Bowling et al., 1994, 1997) these plants are permanently
primed. Because of permanent priming, cpr1 and cpr5 might be able to
quickly and strongly activate various cellular defense reactions upon attack
by pathogens. In this context it is worth mentioning that the constitutively
enhanced disease resistance of another Arabidopsis mutant referred to as
cpr5-2 has been ascribed to the boosted activation of the PR-1 defense gene
in these plants (Boch et al., 1998).
Since priming leads to more robust induction of defense responses and
ultimately to resistance, it can be anticipated that the phenomenon includes
improved perception and/or amplication of the defense response-inducing
signal from the pathogen (Conrath et al., 2006). It has been proposed that
priming is associated with increased accumulation, and/or posttranslational
modication, of inactive cellular signaling proteins that play an important
role in signal amplication (Conrath et al., 2006). Subsequent exposure to
stress could activate, or modulate, these dormant signaling proteins,
thereby initiating signal amplication leading to faster and/or stronger acti-
vation of defense responses and IR (Conrath et al., 2006). However, the
identity of these hypothetical proteins has remained obscure. Recently, two
members of the mitogen-activated protein kinase (MAPK) family of
370 U. CONRATH
signaling enzymes, MPK3 and MPK6, were found to accumulate upon
priming in Arabidopsis without displaying enzyme activity (Beckers et al.,
2009). Possibly because of the enhanced level of inactive MPK3 and MPK6
in primed plants, the capacity for subsequent signaling is increased, and upon
stimulation by biotic or abiotic stresses MPK3 and MPK6 activity is aug-
mented, associated with enhanced induction of defense responses and IR
(Beckers et al., 2009). These ndings argue that pre-stress deposition of the
signaling components MPK3 and MPK6 is a critical step in priming plants
for full induction of defense responses during IR. Future genetic and
biochemical analysis will probably yield more candidates for cellular com-
ponents with an important role in priming for enhanced disease resistance.
3. Other species
Observations similar to those made with the parsley cell culture and tobacco
and Arabidopsis plants have been reported for many other plant species. For
instance, pretreatment with physiological concentrations of SA had negligi-
ble effects on soybean cell suspension cultures. However, when the
SA-pretreated cells were challenged with an avirulent strain of Ps. syringae
pv. glycinea, the activation of defense genes, the oxidative burst, and cell
death were markedly enhanced (Shirasu et al., 1997). In a similar manner,
BTH primed Agastache rugosa suspension cells for augmented production of
rosmarinic acid upon induction by a yeast extract elicitor (Kim et al., 2001).
BTH also sensitized sunower plants for increased production and excretion
of phytoalexins and phenolic compounds upon infection with the sunower
rust fungus Puccinia helianthi (Prats et al., 2002), and it also primed cucum-
ber plants for enhanced activation of various defense genes as well as resis-
tance to Colletotrichum orbiculare (Cools and Ishii, 2002). In addition, BTH
sensitized cowpea (Vigna unguiculata) plants for rapid, transient increases in
PAL and chalcone isomerase activity, potentiated accumulation of the iso-
avonoid phytoalexins kievitone and phaseollidin, and resistance to damp-
ing-off disease caused by Colletotrichum destructivum (Latunde-Dada and
Lucas, 2001). Furthermore, treatment with SA primed intact Asparagus
ofcinalis plants for faster and stronger induction by Fusarium oxysporum
f. sp. asparagi of peroxidase or PAL activity, and for quicker and more
robust lignin deposition associated with enhanced resistance to F. oxysporum
f. sp. asparagi (He and Wolyn, 2005). Moreover, vitamin B
1
(thiamine) was
shown to prime rice, Arabidopsis, and several vegetable crop plants for faster
and stronger activation of defense-related genes and IR to various fungal,
bacterial, and viral pathogens (Ahn et al., 2005). In Arabidopsis the vitamin
B
1
-induced priming required hydrogen peroxide and the NPR1 gene (Ahn
et al., 2007b). In cucumber plants, infection by the anthracnose-causing
PRIMING PLANT DEFENSE 371
fungus Colletotrichum lagenarium induced resistance to further infection by
the same pathogen. This SAR response was associated with enhanced
deposition of a lignin-like polymer in cell wall sites under fungal appressoria
(Hammerschmidt and Kuc, 1982), indicative of priming of cell wall
reinforcement in this species.
D. PRIMING INDUCED BY BENEFICIAL MICROORGANISMS
1. Priming in ISR
Most of the studies that investigated priming by benecial microorganisms
made use of ISR-eliciting PGPR. The rst evidence that priming of plant
defense responses is involved in ISR came from experiments with carnation
(Dianthus caryophyllus). Inoculation with F. oxysporum f. sp. dianthi of
carnation plants displaying ISR led to a faster rise in phytoalexin levels
than in noninduced control plants (Van Peer et al., 1991). In a similar
manner, Bacillus pumilus (strain SE34) induced systemic resistance against
the root-rot fungus F. oxysporum f. sp. pisi in bean (Benhamou et al., 1996).
Upon challenge infection with the same fungus, the walls of root cells
were rapidly strengthened at sites of attempted fungal penetration through
apposition of callose and phenolic material (Benhamou et al., 1996).
In Arabidopsis, priming associated with the systemic resistance induced by
root-colonizing Ps. uorescens strain WCS417r has been studied at the molec-
ular level. Although WCS417r-elicited ISR is effective against a broad and
distinctive sprectrum of pathogens, it is not associated with the activation of
genes encoding PR-proteins (Pieterse et al., 1996). Analyses of the Arabidopsis
transcriptome have shown that locally in the colonized roots, WCS417r bacte-
ria induce the expression of 94 genes (Leon-Kloosterziel et al., 2005; Verhagen
et al., 2004). However, in the systemic leaves, no signicant alteration in gene
expression was observed. Thus, WCS417r-elicited ISR is not associated with
obvious changes in gene expression in distant leaves (Verhagen et al., 2004). In
Arabidopsis expressing WCS417r-mediated ISR, 81 genes showed enhanced
expression upon infection of the leaves with Pst, indicating that these plants
were primed to respond in a faster and/or more robust manner to pathogen
attack (Van Wees et al., 1999; Verhagen et al., 2004). Most of the genes with
potentiated induction have been described as being regulated by either JA or
ET, or both. The ndings conrmed earlier results demonstrating that coloni-
zation of the roots by WCS417r primed Arabidopsis for augmented induction
of the JA- and/or ET-responsive genes VEGETATIVE STORAGE
PROTEIN-2 (VSP2), PLANT DEFENSIN-1.2 (PDF1.2), HEVEIN-LIKE
PROTEIN (HEL), and ACC (1-Aminocyclopropane-1-carboxylic acid)
OXIDASE (ACO) (Hase et al., 2003; Van Wees et al., 1999). In contrast to
372 U. CONRATH
gene expression, signicant alterations in the production of either JA or ET
have not beenobservedinthe plants exhibiting ISR(Pieterse et al., 2000). These
observations argue that the state of ISR is based on an enhanced sensitivity to
these plant hormones rather than just on an increase in their production
(Pieterse et al., 2000).
Two recent studies by Corne Pieterse and associates suggest that the Ps.
uorescens WCS417r-induced priming and the associated ISR of Arabidopsis
require the transcription factor MYB72 in the roots (Van der Ent et al., 2008)
and the helix-loop-helix transcription factor MYC2 in the leaves (Pozo et al.,
2008). MYC2 plays a role not only in the regulation of JA-responsive genes
(Berger et al., 1996), but also in ABA and drought signaling (Abe et al., 2003).
Studies with other PGPR on different plant species generally conrm that
ISR is associated with primed expression of defense genes (reviewed by
Van Wees et al., 2008). Ryu et al. (2004) demonstrated that some plant-growth
promoting Bacillus spp. can prime plants by the release of volatile organic
compounds (VOCs). Bacillus subtilis GB03 produces the green leaf volatiles
3-hydroxy-2-butanone and (2R,3R)-()-2,3-butanediol, which canprime Ara-
bidopsis for augmented defense responses against herbivore attack or patho-
gen infection. In that case, a signaling pathway that is independent of SA, JA,
and the NPR1 gene, yet requiring ET, is triggered (Ryu et al., 2004).
2. Priming in benecial interactions other than ISR
In addition to SAR and ISR, the primed state is a common feature also of
resistance responses that are induced by benecial microorganisms other than
PGPR. For example, colonization of tomato roots by mycorrhizal fungi
protected the plant systemically against Phytophthora parasitica with no
detectable accumulation of PR-proteins before pathogen assault. Only after
Ph. parasitica attack, mycorrhizal plants accumulated signicantly more PR-
1a and basic -glucanases than non-mycorrhizal plants (Cordier et al., 1998;
Pozo et al., 1999, 2002). Ultrastructural studies revealed that plants
with established mycorrhizal symbiosis also displayed pectin-rich, callose-
containing cell wall depositions at the sites of attempted pathogen infection,
whereas non-mycorrhizal plants did not (Cordier et al., 1998; Pozo et al., 1999,
2002). Certain plant growth-promoting fungi can similarly induce priming in
plants. For example, challenge inoculation with the leaf pathogen Ps. syringae
pv. lachrymans of cucumber plants that had been preinoculated with the plant
growth-promoting fungus Trichoderma asperellum (strain T203) led to aug-
mented PR gene expression (Shoresh et al., 2005). Also, like SA, infection by a
nonpathogenic isolate of the fungus F. oxysporum primed As. ofcinalis plants
for faster and stronger induction of defense reactions and enhanced resistance
to F. oxysporum f. sp. asparagi (He et al., 2002).
PRIMING PLANT DEFENSE 373
3. Priming by bacterial lipopolysaccharides
Unspecic, so-called basal defense responses can be induced in plants and
animals by various MAMP elicitors. Among other molecules, these include
small peptides, lipooligosaccharides and lipopolysaccharides (LPS)
(Newman et al., 2007). The latter are ubiquitous, indispensable components
at the cell surface of Gram-negative bacteria. They have diverse effects in
plants (Newman et al., 2007). These include prevention of the hypersensitive
response, synthesis of nitric oxide, phosphorylation of MAPKs, and priming
for more effective induction of various defense responses (Erbs and
Newman, 2003; Newman et al., 2001, 2007). In pepper leaves, for example,
the genes coding for the PR-protein P6 (an ortholog of PR-1 of tobacco), and
acidic and basic -glucanase were not or only weakly induced by LPS from
Salmonella enterica serovar minnesota or X. campestris pv. campestris,
respectively (Newman et al., 2000, 2002). However, pretreatment of the pep-
per leaves withthe LPS augmented the expression of the genes encoding the P6
protein and acidic and basic -glucanase following infection with X. campes-
tris pv. campestris (avirulent) or X. campestris pv. vesicatoria (virulent)
(Newman et al., 2002, 2007). The authors also reported that LPS pretreatment
led to faster and more robust induction of coumaroyl tyramine and feruloyl
tyramine, which have antimicrobial activity and serve to strengthen the cell
wall upon inoculation with X. campestris pv. campestris. Yet, LPS treatment
alone did not induce either coumaroyl tyramine or feruloyl tyramine produc-
tion (Newman et al., 2007). Thus, at least in plants LPS-activated IR seems to
be mediated by LPS-induced priming for enhanced activation of defense
responses upon challenge infection (Newman et al., 2002, 2007). The molecu-
lar mechanism of the LPS-induced priming, however, is still unclear.
It is noteworthy that the effects of various benecial bacteria on plants,
such as activation of ISR by PGPR, at least in some cases are believed to be a
consequence of LPS perception (Leeman et al., 1995a). Thus, it would be
interesting to know whether ISR is mediated by LPS-induced priming for
enhanced activation of defense responses and resistance.
E. PRIMING IN BABA-IR
1. Biotic stress
Research on the mechanism(s) of BABA-IRin Arabidopsis has shown that this
type of IR, just like SAR and ISR, is frequently associated with priming for
various pathogen-induced defense responses. For example, the induction of
the PR-1 gene is faster when BABA-pretreated Arabidopsis plants are
challenged with Pst than when non-pretreated plants (Zimmerli et al.,
2000). As a matter of fact, the induction kinetics of the PR-1 gene by Pst in
374 U. CONRATH
BABA-primed Arabidopsis plants are very similar to the induction kinetics of
PR-1 gene expression in the resistance response to the avirulent strain
Pst
avrRpt2
(Zimmerli et al., 2000). This priming of PR-1 expression by BABA
is dependent of NPR1, indicating that a pathway similar to SA signaling in
SAR is activated by BABA (Zimmerli et al., 2000).
In Arabidopsis BABA-IRto H. arabidopsidis coincided with fast and robust
deposition of callose-containing papillae (Zimmerli et al., 2000). This correla-
tion between BABA-IR and augmented papillae formation was intensively
studied using the interaction of Arabidopsis with the two necrotrophic fungi
Alternaria brassicicola and Pl. cucumerina. The use of various Arabidopsis
mutants indicated that neither the phytoalexin camalexin, nor SA-, JA-, or
ET-dependent defense responses seem to play a critical role in BABA-IR to
these two necrotrophic pathogens (Ton and Mauch-Mani, 2004).
Cytological investigations at sites of attempted penetration by A. brassici-
cola and Pl. cucumerina demonstrated that the formation of callose-rich
papillae was increased in attacked epidermal cells of BABA-pretreated plants
(Ton and Mauch-Mani, 2004). Pharmacological studies with 2-deoxy-D-
glucose, an inhibitor of callose synthesis, indicated that in BABA-pretreated
plants the enhanced callose deposition plays a key role in the induction of
resistance to A. brassicicola (Ton and Mauch-Mani, 2004). In a consistent
manner, the callose-decient powdery mildew-resistant-4-1 (pmr4-1) mutant
of Arabidopsis was completely blocked in BABA-IR to Pl. cucumerina.
Priming for enhanced papillae formation after fungal infection was absent
in the Arabidopsis mutants abscisic acid-insensitive-4-1 (abi4-1) and abi1-5
(Ton and Mauch-Mani, 2004). In addition, exogenous application of
ABA mimicked the effect of BABA with respect to increased formation of
callose-rich papillae and resistance to fungal ingress.
Priming for enhanced callose deposition by BABA involves the augmented
transport of vesicles through the Golgi apparatus to release precursors at the
plasma membrane. Inthis process, different soluble N-ethylmaleimide-sensitive
fusion protein attachment protein receptor (SNARE) proteins are involved.
SNAREproteins have also been linked to disease-resistance mechanisms in the
plant cell wall. Mutations in the SNARE genes PENETRATION-1 (PEN1)
(encoding syntaxin) and REQUIRED FOR mlo RESISTANCE-2 (ROR2)
(Collins et al., 2003; Lipka et al., 2005) caused a partial loss of resistance at
the level of the plant cell wall, resulting in a loss of basal resistance to the
nonadapted parasites B. graminis f. sp. hordei and Erysiphe pisi that in nature
colonize barley and pea, respectively. Both, PEN1 and ROR2 are thought to
play a role in cellular targeting of vesicles carrying phytoalexins or callose
synthase II to sites of attempted fungal penetration. Together, these ndings
suggest a prominent role of ABA in the accelerated formation of callose-rich
PRIMING PLANT DEFENSE 375
papillae through enhanced ABA-dependent transcription, or augmented
induction of SNARE genes upon pathogen attack (Leyman et al., 1999;
Zhu et al., 2002).
Indications for interaction-dependent complexity of the BABA-induced
priming mechanism(s) have been provided by Hamiduzzaman et al. (2005).
Using BABA as the inducer of resistance to downy mildew (Plasmopara
viticola) in grapevine (Vitis vinifera) the authors showed that in this interac-
tion the primed deposition of callose and phenylpropanoid-derived phenolics
is associated with resistance to the pathogen but is dependent on JA rather
than ABA (Hamiduzzaman et al., 2005).
2. Abiotic stress
It is known that SA and its derivative acetyl-SA (aspirin) can protect various
plants from abiotic stresses, such as chilling, heat, drought, and wounding
(Janda et al., 1999; Kohler et al., 2002; Senaratna et al., 2000). However,
much more information is available for the BABA-induced protection from
abiotic stress. For example, BABA is known to protect Arabidopsis from heat
(Zimmerli et al., 2008), drought, and salt stress (Jakab et al., 2005). The BABA-
induced tolerance to the latter two abiotic stresses correlated with primed
expression of SA- and ABA-responsive genes upon exposure of the BABA-
pretreated plants to drought or salt (Jakab et al., 2005). Arabidopsis mutants
with defects in ABA signaling could not be protected by BABA, while BABA-
pretreated SA-signaling mutants showed a resistance response that was similar
to wild-type plants. In the wild type, pretreatment with BABA did not lead to
ABA accumulation. Rather, the production of this plant hormone was more
rapidly induced after exposure of the plants to osmotic stress. The accelerated
ABA production resulted in more robust expression of ABA-responsive genes
and quicker closure of stomata (Jakab et al., 2005). These ndings demon-
strated that BABA-induced tolerance to osmotic stress is based on priming for
enhancedadaptationresponses rather thanondirect activationof stress defense
responses. The latter is commonly observed during acclimation treatment
in which plants are gradually exposed to an increasingly stressful situation.
Together, ABA seems to have a crucial role in BABA-induced priming for
stronger responses to both biotic and abiotic stresses, at least in Arabidopsis.
F. PRIMING IN WOUND-INDUCED RESISTANCE
1. Priming in IR to herbivores
In 1972, Green and Ryan were the rst to report that insect feeding on potato
and tomato plants activates local and systemic accumulation of proteinase
inhibitors that disrupt the activity of digestive proteases in the insect gut
376 U. CONRATH
(Green and Ryan, 1972). Wound-induced local and systemic resistance to
pathogenic microorganisms or herbivorous insects is illustrated by a recent
study of Chassot et al. (2008), who demonstrated that wounding leaves
either by squeezing with a pair of forceps or by puncturing holes with a
needle induces resistance to the gray-mold fungus B. cinerea in Arabidopsis.
This WIR seems to require neither SA, nor JA or ET. It rather needs glutathi-
one and the phytoalexin camalexin (Chassot et al., 2008). The wound stimulus
per se did not lead to camalexin production. Wounding rather primed Arabi-
dopsis for enhanced camalexin biosynthesis after B. cinerea inoculation
(Chassot et al., 2008). These observations not only revealed that glutathione
and camalexin play a likely key role in WIR of Arabidopsis to B. cinerea, but
also demonstrated that priming is a likely mechanism of wound-induced
pathogen resistance, at least in the ArabidopsisB. cinerea interaction.
Earlier studies with cell cultures and the wound signals MeJA and systemin
had already suggested that priming represents the basis of WIRto pathogenic
microbes and insects. For example, pretreatment with MeJA primed parsley
suspension cells for enhanced secretion of phytoalexins and augmented
incorporation of hydroxycinnamic acid esters and lignin-like polymers
into the cell wall (Kauss et al., 1992b). In a similar manner, pre-exposure of
cultured tomato cells to the wound-signaling peptide systemin, but not an
inactive systemin analog, led to a time-dependent enhancement of the oxida-
tive burst that was subsequently induced by oligogalacturonides (Stennis
et al., 1998).
In response to wounding or herbivore attack, plants often release extra-
oral nectar or VOCs. While some of these serve to attract parasitic or
predatory natural enemies of the herbivores (Pare and Tumlinson, 1999),
others have a role in the activation of resistance in the same (Heil and Silva
Bueno, 2007) or even nearby, unharmed plants (Baldwin and Schultz, 1983;
Heil and Kost, 2006). During the past years, there was strong evidence that
VOC-mediated IR is mediated by priming (see Section III.D.1). In a pioneer-
ing study, Engelberth et al. (2004) showed that maize seedlings when exposed
to certain volatiles from neighboring plants and subsequently challenged by a
combination of mechanical damage and exposure to regurgitant of caterpil-
lars of the beet armyworm (Spodoptera exigua), show higher production of
volatile sesquiterpenes and JA when compared to triggered plants not ex-
posed to the volatiles before. In a follow-up study, it was shown that the
VOC-induced priming for augmented induction of defense genes and emis-
sion of aromatic and terpenoid volatiles in maize correlates with reduced
caterpillar feeding and enhanced attraction of the parasitoid wasp Cotesia
marginiventris (Ton et al., 2006).
PRIMING PLANT DEFENSE 377
Further work revealed that the green leaf volatile cis-3-hexenyl acetate
serves as a wound signal that primes leaves of hybrid poplar saplings for
higher concentrations of JA and linoleic acid following gypsy moth (Lyman-
tria dispar) feeding, and for improved oxylipin signaling, terpene volatile
release, and defense responses (Frost et al., 2008). In eld studies, Heil and
Kost (2006) demonstrated that Lima bean plants respond to leaf damage
with the secretion of extraoral nectar, and this response was much higher in
plants that had been exposed before to a complex articial volatile blend that
mimics the herbivore-induced bouquet of Lima bean plants both quantita-
tively and qualitatively. In sum, these ndings strongly suggest that VOCs
play a key role in priming during the wound response of plants.
2. Priming between plant species
Over the past few years, it has become increasingly clear that priming can be
the result of plantplant communication in the wild when VOCs serve as
priming-inducing signals even between plant individuals of different species.
Kessler et al. (2006) reported that VOCs from clipped sagebrush (Artemesia
tridentata) prime nearby wild tobacco (Nicotiana attenuata) plants for
quicker production of trypsin inhibitors, and this was associated with lower
herbivore damage and higher mortality rate of young tobacco hornworm
(Manduca sexta) caterpillars (Kessler et al., 2006). Using tobacco as the
model plant Shulaev et al. (1997) proposed that MeSA, which is synthesized
from SA and acts by being reconverted into SA, may function as an airborne
signal that activates disease resistance and the expression of defense genes in
healthy tissues of infected plants and also in neighboring plants. However,
whether the resistance induced by MeSA is mediated by priming or whether
it is due to direct activation of defense responses has not been addressed in
these studies. In any case, it seems that plants can use chemical signals in their
environment to assess the risk of herbivory and integrate this information to
adjust and ne-tune their overall defense strategy.
G. PRIMING BY MODIFICATIONS IN PRIMARY METABOLISM
Although application of sugar to various plant tissues, or provoking the
accumulation of sugar in transgenic plants, can lead to activation of various
PR genes (Herbers et al., 1996a,b; Johnson and Ryan, 1990), the high-sugar
concept of resistance (Horsfall and Dimond, 1957) has been called into
question by recent work which demonstrated that expression of a yeast
invertase in the cytoplasm of potato tuber cells leads to decreased levels of
starch and enhanced levels of glucose, yet to drastic susceptibility to the soft-
rot pathogen Pe. atrosepticum (Conrath et al., 2003). In addition, the
378 U. CONRATH
enhanced resistance to Ph. infestans in leaves of transgenic potato plants with
decreased activity of the plastid ATP/ADP transporter was not associated
with obvious changes in carbohydrate accumulation, in contrast to the
enhanced disease resistance in the tubers (Conrath et al., 2003). These results
suggested that the resistance of plant tissue with elevated levels of
carbohydrates is not due to enhanced sugar levels.
This suggestion is supported by ndings demonstrating that increased
glucose levels are not associated with constitutive expression of PR genes in
potato tubers with reduced activity of the plastid ATP/ADP transporter
(Conrath et al., 2003). Detailed analyses on the timing and extent of defense
responses in these same plants provided an alternative explanation for the IR
phenotype observed. Upon exposure of leaf or tuber tissue to culture super-
natants of Pe. atrosepticum or pep13, a 13-amino acid MAMP elicitor from
oomycete cell walls, there was enhanced activation of defense responses,
including defense gene activation and the oxidative burst (Linke et al.,
2002). Thus, the IR of transgenic plants with reduced ATP/ADP transporter
activity seems to be mediated by metabolic priming for enhanced induction
of defense responses rather than by the associated elevation in carbohydrate
levels in these plants. A correlation between elevated sucrose levels and
priming of defense responses was reported recently also for rice overexpres-
sing the PRms gene from maize, which encodes a PR-1 type protein
(Casacuberta et al., 1991). In these plants elevated levels of sucrose were
associated with quicker and more robust induction of defense responses
during pathogen infection and broad-spectrum disease resistance (Go mez-
Ariza et al., 2007).
IV. RELEVANCE OF PRIMING IN PLANT
PRODUCTION
A. COSTS AND BENEFITS OF PRIMING
The direct stimulation of defense responses by external application of high
doses of SA or JA, or by the action of resistance genes reduces certain tness
characters, such as growth and fruit or seed set, under pathogen-free condi-
tions (Agrawal et al., 1999; Baldwin, 1998; Cipollini, 2002; Heidel et al., 2004,
Heil et al., 2000, Korves and Bergelson, 2004; Tian et al., 2003; Van Dam and
Baldwin, 2001). Also, plants transformed with genes encoding SA biosyn-
thetic enzymes (Mauch et al., 2001), or gain-of-resistance mutations in
Arabidopsis such as cpr1, cpr5 and cpr6, which all contain constitutively
elevated levels of SA, permanently express PR genes, have a dwarf
PRIMING PLANT DEFENSE 379
phenotype, which is associated with reduced tness (Bowling et al., 1994,
1997). These observations were also made in the eld. Heidel et al. (2004)
demonstrated that Arabidopsis mutants blocked in SA-inducible defenses, as
well as mutants showing constitutive expression of these defense responses,
all were affected in growth and seed set. The authors argued that optimal
tness would be reached at a certain level of resistance that balances tness
and defense (Heidel et al., 2004). Similar conclusions were made from re-
search on the costs of JA-inducible defenses, which seem to be affordable
only when the plant is actually exposed to herbivore attack (Agrawal et al.,
1999; Baldwin, 1998).
The dilemma between resistance and costs of defense can probably be
overcome by priming. In a recent study by Van Hulten et al. (2006), the
costs and benets of priming in Arabidopsis were determined and compared
to those of the direct activation of defense. Application of low doses of
BABA induced the primed state, caused minor growth reduction, and had
no obvious effect on seed production. In contrast, direct induction of defense
responses by high doses of either BABA or BTH strongly affected both these
tness parameters (Van Hulten et al., 2006). The effects in primed plants,
although minor, could be due to enhanced expression of genes for signaling
compounds with an important role in priming (Maleck et al., 2000;
Van Hulten et al., 2006) (see Section III.C.2). Consequently, it was suggested
that priming causes less tness costs than the direct induction of defense
(Van Hulten et al., 2006). Intriguingly, when attacked by pathogens, primed
plants showed even higher tness than non-primed ones. Thus, in pathogen-
free environments, the low tness costs of priming seem to be outweighed by
its benets after pathogen attack (Van Hulten et al., 2006).
B. EXPLOITING PRIMING IN GREENHOUSE AND FIELD
Many natural and synthetic compounds can prime plants (summarized in
Conrath et al., 2006) (Fig. 2). In addition to those mentioned above, these
include Brotomax, a commercial product that contains aluminum lignosul-
phonate (Fuster et al., 1995; Ortun o et al., 1997). Brotomax, BABA, and
some other priming-inducing compounds were shown to be potent inducers
of stress tolerance in the greenhouse and in the eld (Cohen, 2002). Foliar
application of BABA, for example, protected eld-grown grape against
Pl. viticola and suppressed disease symptoms induced by Ph. infestans on
potato and tomato plants in the eld. BABA also protected melon from
sudden wilt disease by Monosporascus cannonballus (Cohen, 2002), and
peanut from Cercosporidium personatum in both greenhouse and eld trials
(Cohen, 2002). As the compound also displays synergistic interactions with
380 U. CONRATH
Beneficial
microorganisms
Natural and
synthetic
compounds
Pathogen attack
Natural and
synthetic
compounds
Expression of
induced resistance
(reduced disease
symptoms)
Priming
Wounding
Primed plant with
enhanced defense
capacity
Pathogen
attack
Nave plant with
normal defense
capacity
Pathogen
attack
Dramatically
diseased
plant
Nave plant with normal
defense capacity is subject
to a priming-inducing treatment
Fig. 2. (continued )
certain fungicides (Cohen, 2002) BABA has a high potential to become a part
of sustainable disease management in the eld, even though at higher doses
the compound induces female sterility (Kocsis and Jakab, 2008).
INA was the rst synthetic compound shown to induce both priming of
defense responses in the lab (Kauss et al., 1992a) and SAR to fungal and
bacterial diseases on various crops in greenhouses and in the eld (Kessmann
et al., 1994). However, INA and SA were insufciently tolerated by some
crops, which prevented their practical use as plant protection compounds
(Ryals et al., 1996). The SAR inducer BTH, which primes plant cell cultures
and intact plants for defense responses (see Section II.C.1) was shown to
protect various crops against many diseases in the eld (Ryals et al., 1996).
In contrast to INA and SA, BTH was sufciently tolerated by most crops.
Therefore, the compound became attractive for practical agronomic use.
In 1996, BTH was introduced as a plant activator (Ruess et al., 1996) with
the trade names Bion

, Actigard

, or Boost

. However, BTHs economic

success was only limited because it has protective rather than curative activity.
Thus, to serve as a protectant the compound must be applied some time before
apotential pathogenattack, andeventhenis seldomas effective as commercial
fungicides. Because of this restricted activity, BTHwas not sufciently accept-
ed by farmers who were in favor of using curative standard fungicides.
Because of the repudiation of BTH, it became an opportune time to
identify plant-protecting compounds combining both direct action on the
pathogen and priming-inducing activity in the plant. Some strobilurin fungi-
cides seem to team both these activities (for a review on strobilurins, see
Sauter, 2007). In the laboratory, for example, the strobilurin fungicide
Pyraclostrobin (trade names: Headline

, Cabrio

) primed the tobacco

cultivar Xanthi-nc for faster accumulation of PR-1 proteins after infection
with TMV or the wildre pathogen Ps. syringae pv. tabaci. The Pyraclostro-
bin-induced priming for enhanced PR-1 accumulation after pathogen attack
was associated with IR to disease (Herms et al., 2002). IR to viral and
bacterial pathogens in Pyraclostrobin-treated plants was also observed in
various crops and ornamental plants both in the greenhouse and eld
Fig. 2. Ways to induce priming and resistance to biotic and/or abiotic stress in
plants. Spray or drench pretreatment of plants with certain natural or synthetic
compounds (SA, some of its analogs, BABA, LPS, and others), wounding, or
colonization of the roots by benecial microorganisms (mycorrhizal fungi, growth-
promoting fungi and rhizobacteria), causes a primed state. In the primed condition,
plants are able to respond with more robust and/or faster induction of defense
responses upon exposure to pathogen attack. Ultimately, priming causes a reduction
of disease symptoms through enhanced resistance (upper row) which is not seen in
non-primed plants (lower row).
382 U. CONRATH
(Koehle et al., 2003, 2006). In the eld the Pyraclostrobin-induced priming
was associatedwithinducedtolerance alsotoabiotic stresses, including drought
(www.agweb.com/aims/les/HeadlineAdvantage.pdf). Together, the ndings
with Pyraclostrobin suggest that this compound, in addition to exerting direct
antifungal activity, may also protect plants by priming their defense responses
to biotic, as well as abiotic stresses. This conclusion is consistent with
an earlier report demonstrating that another commercial fungicide,
Oryzemate

, enhanced the resistance to TMV in tobacco (Koganezawa et al.,

1998) and to a bacterial and an oomycete pathogen in Arabidopsis (Yoshioka
et al., 2001). Oryzemate

contains probenazole as the active ingredient which is

metabolized to saccharin in treated plants. The latter compound seems to be
responsible for the induction of priming in Oryzemate

-treated plants (Siegrist

et al., 1998).
In Arabidopsis plants with impaired disease resistance signaling, such as
SA-decient NahG plants or the npr1 mutant, the fungicides metalaxyl,
fosetyl-Al, and Cu(OH)
2
are much less effective than they are in plants
with an intact disease resistance signaling pathway (Molina et al., 1998).
This nding suggests that these substances possess some resistance-inducing
activity besides their fungicidal properties. The fungicide-mediated resistance
could be on the basis of priming because fungicide application alone does not
lead to any obvious changes in gene induction in Arabidopsis. A role for
NPR1 in priming by fungicide or chemical inducer action is also supported
by the fact that NPR1 overexpressing Arabidopsis plants display both poten-
tiated disease resistance and enhanced efcacy of fungicides (Friedrich et al.,
2001).
Similar observations have recently been made in lab and eld trials
with the insecticide Imidacloprid. One of its major degradation products,
6-chloronicotinic acid, has a structure very similar to INA. It is supposed to
cause the so-called stress shield effect on crops by priming them for
augmented expression of defense genes, enhancing their tolerance to biotic
and abiotic stresses, and increasing plant growth and yield (Thielert, 2006).
Various associations of plants with benecial microbes in the soil have also
been demonstrated to induce the primed state and/or resistance to above-
ground pathogens and abiotic stresses in the greenhouse and eld. For
example, growth-promoting Ps. uorescens (strain WCS417r) induces prim-
ing and ISR in plants (see Sections II.B.1 and III.D.1) and Ps. uorescens
(strain WCS374) was demonstrated to suppress Fusarium wilt disease
and improve yield in greenhouse-grown radish (Leeman et al., 1995b).
Furthermore, seed treatment of various crops and ornamental plants with
a mixture of endo- and ectomycorrhizal fungi (available as MycoGrow
TM
Micronized Endo/Ecto Seed Mix) enhanced their growth and induced
PRIMING PLANT DEFENSE 383
resistance in the greenhouse as well as under eld conditions (www.fungi.
com/index.html). Similarly, Pi. indica systemically primes barley against
biotic and abiotic challenges and increases growth and yield of Spilanthes
calva and Withania somnifera in the eld (Rai et al., 2001).
V. CONCLUSIONS
Over the past decade it has become increasingly clear that priming is a
complex mechanism that is part of various types of IR in plants. Priming
allows plants to activate defense responses more quickly and/or effectively
when exposed to biotic or abiotic stress. Because of its advantageous eco-
nomic features, priming represents an ecologically important adaptation to
withstand environmental challenges. The phenomenon can contribute to new
concepts for disease control as priming provides broad-spectrum resistance
without signicantly affecting growth and fruit or seed set. Priming offers a
smart, effective, and realistic option for effective plant protection, especially
when combined with conventional pesticides. Taking advantage of the natu-
ral, broad-spectrum defense capacity of plants in the eld will be facilitated
by a better understanding of the molecular, physiological, and ecological
aspects of priming, which represents an exciting challenge for future research.
ACKNOWLEDGMENTS
Research on priming in my laboratory is supported by the Peter and Traudl
Engelhorn Foundation and, in part, by BASF and the German Science
Foundation (DFG).
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