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273

Functional analysis of plant disease resistance genes and their
downstream effectors
Gregory B Martin
Plant disease resistance (R) genes encode proteins that both
determine recognition of specific pathogen-derived avirulence
(Avr) proteins and initiate signal transduction pathways leading
to complex defense responses. Recent developments suggest
that recognition specificity of R proteins is determined by
either a protein kinase domain or by a region consisting of
leucine-rich repeats. R genes conferring resistance to
bacterial, viral, and fungal pathogens appear to use multiple
signaling pathways, some of which involve distinct proteins and
others which converge upon common downstream effectors.
Manipulation of R genes and their signaling pathways by
transgenic expression is a promising strategy to improve
disease resistance in plants.
Addresses
Boyce Thompson Institute for Plant Research, and Department of Plant
Pathology, Cornell University, Tower Road, Ithaca, NY 14853-1801,

USA; e-mail: gbm7@cornell.edu
Current Opinion in Plant Biology 1999, 2:273–279
http://biomednet.com/elecref/1369526600200273
© Elsevier Science Ltd ISSN 1369-5266
Abbreviations
Avr
avirulence
HR
hypersensitive response
IL-1R
interleukin-1 receptor
JA
jasmonic acid
LRR
leucine-rich repeat
LZ
leucine zipper
MAPK mitogen-activated protein kinase
NBS
nucleotide binding site

PR
pathogenesis-related
R
resistance
SA
salicylic acid
SIPK
salicylic acid induced protein kinase
TIR
Toll/Interleukin-1 receptor
WIPK
wound-induced protein kinase

Introduction
Disease resistance (R) genes in plants often determine the
recognition of specific pathogens that express a corresponding avirulence (avr) gene. Over 20 R genes with
recognition-specificity for defined avr genes have been
isolated from seven plant species, including both monocots
and dicots. These genes are effective against bacterial,
viral, and fungal pathogens and in one interesting case, the

Mi gene from tomato, against both nematodes and an aphid
species [1–3]. There is great diversity in the lifestyles and
pathogenic mechanisms of disease-causing organisms and
it was, therefore, somewhat surprising that R genes were
found to encode proteins with certain common motifs.
Five classes of R genes are now recognized: intracellular
protein kinases; receptor-like protein kinases with an
extracellular leucine-rich repeat (LRR) domain; intracellular LRR proteins with a nucleotide binding site (NBS) and

a leucine zipper (LZ) motif; intracellular NBS–LRR proteins with a region with similarity to the Toll and
interleukin-1 receptor (TIR) proteins; and LRR proteins
that encode membrane-bound extracellular proteins.
Despite these significant insights into R gene structure,
much remains to be elucidated about the molecular mechanisms by which R proteins recognize pathogens and
transduce this information in the plant cell to initiate
defense responses. For example, although R proteins
were anticipated to play a direct role in recognizing
pathogen-derived ligands, this only has been reported for
one R protein [4,5]. Similarly, the cloning of a protein
kinase R gene, Pto, and an LRR gene, Prf, from the same

disease resistance pathway indicated that members of
these two classes might represent different steps in a signaling pathway common to other R gene pathways [6,7].
No clear evidence has emerged, however, to enable the
placement of these two classes of proteins in a single pathway. Finally, the shared motifs among R proteins
suggested that they might utilize common downstream
signaling components to activate similar defense responses. Evidence both for and against such convergence has
been reported in the past year. In this review, I examine
recent papers that are beginning to shed light on these
and other central questions regarding the role of R genes
in initiating disease resistance.

Recognition specificity of R proteins
A hallmark of R genes is their recognition-specificity for
corresponding avr genes. Because of the extracellular life
style of most plant pathogens, it was expected that R proteins would encode extracellular receptor-like proteins.
This might be the case for some R proteins (e.g., Xa21, Cf
proteins); however, in the majority of cases R genes appear
to encode intracellular proteins. The discovery of bacterial
phytopathogenic type III secretion systems that would
allow direct injection of pathogen proteins into the plant

cell suggested that recognition might occur inside the
plant cell. Transient expression of many Avr proteins
inside plant cells has confirmed this speculation (e.g., [8]).
Do R proteins then serve as primary receptors for Avr proteins? If so, what features of the R protein mediate this
recognition? Because LRRs are known to play a role in
protein–protein interactions there has been much speculation that these regions are directly involved in recognition
of Avr proteins. In support of this view, LRRs have been
shown to have considerable variation among members
within clustered gene families and there is now extensive
genome sequence data to indicate that unequal crossing
over, gene conversion, and divergent selection on genes
within families has occurred [9–14]. In a few cases, it
is known that this variation in LRRs directly correlates

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with new recognition specificities [15,16••]. In addition, a
truncated member of the Xa21 family that contains the

LRRs but not the kinase domain still confers resistance to
specific avirulent strains of Xanthomonas [17•]. Still, these
observations are only indirect evidence for the role of
LRRs in Avr protein binding. Recently, the product of the
rice blast R gene Pi-ta has been shown to interact with the
AVR-Pita protein in the yeast two-hybrid system (B Valent,
personal communication). However, it is unclear whether
this binding directly involves an LRR region. Thus,
despite a large amount of supporting evidence a role of
LRRs in recognition remains unproven.
Evidence for the direct interaction of an R and Avr protein
has come from work with the Pto–avrPto system. Pto confers resistance to Pseudomonas syringae strains that express
the AvrPto protein, and encodes a serine–threonine protein kinase that lacks any obvious receptor-like domain.
Nevertheless, two laboratories have demonstrated that Pto
interacts specifically with the AvrPto protein in the yeast
two-hybrid system and that mutations which disrupt this
interaction lead to loss of recognition in the plant cell [4,5].
Pto recognition specificity for AvrPto was initially found to
require a small region in the activation loop of the kinase
and recent results demonstrate that a threonine at position

204 plays a key role in the interaction with AvrPto [18•].
Interestingly a threonine at this position is conserved in a
large number of protein kinases and it has been proposed
that phosphorylation of this residue might lead to a conformational change of the kinase which allows AvrPto
binding. Despite this appealing explanation for recognition specificity in the Pto–AvrPto system, many questions
remain about how such binding might affect the activity of
the Pto kinase and other components of the pathway.
One hypothesis for how the Pto–AvrPto interaction stimulates disease resistance derives from the recent report that
overexpression of Pto activates a variety of defense
responses targeted against diverse pathogens [19••]. The
authors speculate that basal Pto kinase activity might
maintain a low level of pathogen defense responses even
when AvrPto is not present, and that an increase in Pto
abundance simply increases these responses [19••]. In a
further extension of this model it is possible that interaction with AvrPto might increase Pto kinase activity or it
might reduce degradation of Pto and thereby increase its
abundance in the plant cell under attack by the avirulent
pathogen. In this scenario the Prf, an LRR protein that is
required for Pto function, would play a role downstream
and not in recognition. Another intriguing suggestion has

been made that AvrPto protein, acting as a virulence factor,
might have evolved to bind and hence disrupt Pto activity
and that Prf evolved later to recognize the Pto–AvrPto
complex [20]. In this model, formation of the Pto–AvrPtoPrf complex initiates active resistance against the
Pseudomonas pathogen [21]. However, there have been no
reports to date that Prf interacts with AvrPto, Pto, or the
AvrPto–Pto complex. Nevertheless, further speculation on

the basis of this model might suggest that formation of a
Pto–AvrPto–Prf complex directs Pto kinase activity to substrates involved in resistance to Pseudomonas, or simply
stimulates activity towards substrates that Pto phosphorylates even in the absence of AvrPto. Several proteins have
been identified that interact with Pto in the absence of
AvrPto and two of these have been found to be phosphorylated by the kinase ([22–24]; Y Gu and G Martin,
unpublished data). Additional biochemical experiments
will be required to examine whether AvrPto or Prf affect
Pto phosphorylation activity.
In light of the paucity of data about the function of Prf (and
other LRR proteins) several other possibilities are still tenable. Among these are a role for Prf in the transfer of
AvrPto into the plant cell, a role for Prf (either alone or in
concert with other proteins) in localizing Pto in the plant

cell, and a role as a component in downstream signaling
leading to activation of resistance against P. syringae and
perhaps other pathogens. This latter role is consistent with
the recent demonstration that overexpression of Prf in
tomato leads to constitutive activation of some defense
responses associated with systemic acquired resistance but
not to the hypersensitive response (HR) [25•]. A downstream role for LRR genes is also supported by the recent
report that a mutation in the RPS5 bacterial LRR R gene
suppressed resistance to several avirulent Pseudomonas
strains and to strains of the downy mildew fungus ([26••];
see below).

Localization of R proteins in the plant cell
If R proteins are directly involved in recognition of Avr
proteins, then it is possible they are localized to the plasma
membrane to intercept the incoming pathogen protein.
Unfortunately, there is little information to date about the
localization of most R proteins in the plant cell. In the best
studied case, using an epitope-tagging strategy, the RPM1
protein (of the LZ–NBS–LRR class) was found to be

enriched in the plasma membrane fraction and was removable by treatments that release peripherally-associated
membrane proteins [27••]. This localization contrasts with
that predicted by the analysis of RPM1 structural motifs
and points to the need in this field for similar cell biology
studies of other R proteins. A plasma-membrane localization for the RPM1 protein supports a direct role in
recognition for RPM1 although interaction of RPM1 with
either of its corresponding Avr proteins, AvrB and
AvrRpm1, has not been observed (J Dangl, personal communication). The lack of a signal peptide or
transmembrane domain in RPM1, similar to R proteins in
several other classes, suggests that these proteins might be
tethered to membranes via an interaction with other proteins. Interestingly, a two-hybrid screen has uncovered an
RPM1-interacting protein that is a good candidate for fulfilling such a role (J Dangl, personal communication).
Characterization of the RPM1 protein in vivo also revealed
that it is rapidly degraded after its corresponding Avr

Functional analysis of plant diesease resistance genes and their downstream effectors Martin

proteins are expressed in the plant cell and also after
expression of other Avr proteins recognized by different
R genes that were present in the plant [27••]. This intriguing result suggests that protein degradation pathways

might play a significant previously unanticipated role in
limiting cellular damage during the HR. Protein degradation mediated by proteosomes is a well-established
mechanism to regulate signaling pathways in mammals
and its role in plant hormone signal transduction has been
reviewed recently [28]. Although proteosomes have not
been directly implicated in R gene signaling, a protein that
interacts with the Pto kinase in the yeast two-hybrid system shares significant similarity with the alpha subunit of
the human proteosome [29].
Additional evidence, albeit indirect, for R protein localization has been reported for the Pto kinase whose amino
terminus contains a sequence (MGSKYSK) that shares
similarity with a consensus myristylation motif [30]. In
mammalian systems myristylation plays important roles in
the subcellular localization of various kinases and phosphatases by targeting them to cellular membranes. An
early report indicated that the myristylation motif of the
Fen kinase, which is closely related to Pto, is required for
its function in a transient assay [31]. However, site-directed
mutagenesis of the critical glycine residue in this motif did
not affect the ability of the Pto kinase to confer resistance
to avrPto-expressing Pseudomonas in stable transgenic
plants of tomato [30]. Although Pto was expressed in these
experiments via the constitutive CaMV 35S promoter, neither RNA nor protein blots revealed detectable levels of
Pto in the transgenic plants. Thus it seems unlikely that
overexpression obscured an effect of loss of the myristylation motif in Pto. Recently, cell fractionation studies using
a Pto-specific antibody have found that Pto is present in
the soluble fraction (X He, G Sessa, and G Martin, unpublished data). Thus, it appears likely that Pto is localized to
the cytoplasm.

Involvement of R proteins in signal transduction
There have been significant developments in our understanding of signaling steps that lie downstream of R genes
although much still remains to be learned. The similarity
of R proteins to components of the Drosophila Toll and
mammalian IL-1R (interleukin-1 receptor) pathways has
been previously noted [32•], and the complexity of these
pathways is perhaps a harbinger of what is to be discovered
downstream of R proteins. Despite the structural similarities between the TIR-like R proteins and Toll/IL-1R, and
between Pto and the IL-1R-associated kinase (IRAK) and
the Drosophila kinase Pelle, it still remains to be determined if mechanistic similarities exist. It is worth noting
that extensive searches of publicly available plant
expressed sequence tag databases have not uncovered
genes with obvious similarities to mammalian genes
encoding MyD88, IL-1R accessory protein, TRAF6,
NF-κB and other components downstream of the IL-1
receptor (M D’Ascenzo and G Martin, unpublished data),

275

and it seems likely that plants have evolved several unique
proteins to fulfill signaling roles downstream of R genes.
Nevertheless, as in mammalian systems, a role for reactive
oxygen intermediates and nitric oxide in plant defense signaling has been established and there is now evidence in
plants for other mammalian-like second messengers of
nitric oxide signaling including cyclic GMP and cyclic
ADP-ribose [33,34]. The role of nitric oxide and other signaling molecules is reviewed in another article in this issue
(see P Bolwell this issue, pp 287–294). In addition, in common with IL-1R signaling, mitogen-activated protein
kinases (MAPKs) have been identified that are activated
by R gene pathways ([35•,36•], see below). Here, I will
focus on signaling events that are being revealed by the
functional analysis of R genes and by recently isolated
genes that appear to function downstream of R genes.
Role of the NBS–LRR motifs in signaling

With the exception of the Xa21 protein, which contains a
protein kinase domain, the primary amino acid sequence
of other LRR–NBS R proteins does not suggest an obvious
mechanism for the initiation of signal transduction. The
most thought-provoking proposition to be put forth [20]
about the function of NBS-like R proteins is based on the
noted similarity between them and the CED-4 and APAF1 proteins, which activate proteases involved in
mammalian apoptosis [37]. Heterodimerization between
CED-4 and Apaf-1 and their respective proteases, CED-3
and caspase-9, results from the interaction between homologous domains present in the amino terminal portions of
the proteins [37]. By analogy, it has been proposed that
NBS–LRR proteins might rely on their TIR or LZ regions
to form heterodimers with downstream proteins [20]. In
this model, the LRRs confer recognition specificity,
whereas the NBS serves to activate downstream effectors.
Indirect, but significant, evidence that LRRs play a direct
role in downstream signaling comes from studies of the
R gene RPS5, where a mutation affecting the third
LRR was found to suppress resistance conferred by multiple R genes [26••]. Interestingly, overexpression of the
wild-type RPS5 allele in the mutant background failed to
completely restore resistance mediated by several of these
R genes. On the basis of these results, it was proposed that
the LRR mutation might increase binding to a pathway
component shared by multiple R genes and interfere with
essential signaling. This is the first compelling evidence
that LRRs might perform a downstream signaling function. Curiously, a mutation in another carboxy-terminal
LRR specifically affected recognition of the corresponding
AvrPphB protein, but not resistance mediated by other
R genes [26••]. Thus, the LRR region of RPS5 protein
appears to function in both recognition and signaling similar to the protein kinase domain of Pto.
Signaling via the Pto kinase

Pto and Xa21, by virtue of being protein kinases, offer the
best opportunities to elucidate the role of R proteins in

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Biotic interactions

signal transduction via protein phosphorylation. Both Xa21
and Pto are active protein kinases (P Ronald, personal
communication; [24]) and, at least for Pto, kinase activity
appears to be required for its role in disease resistance. In
in vitro experiments, Pto was shown to undergo intramolecular autophosphorylation on several sites [24]. By analogy
with similar work with mammalian kinases, it is likely that
at least some of these phosphorylated sites are important
for Pto function in vivo. Identification of the phosphorylated residues is underway and will eventually lead to an
examination of their role in disease resistance (G Sessa,
M D’Ascenzo, and G Martin, unpublished data).
Pto interacts with several plant proteins in the yeast twohybrid system and to date two of these, Pti1 (a
serine–threonine kinase) and Pti4 (a DNA binding protein) have been found to be specific phosphorylation
substrates for Pto ([22]; Y Gu and G Martin, unpublished
data). Phosphorylation of Pti1 by Pto occurs on one major
site and several minor sites. The major site, interestingly,
is the same site that is autophosphorylated by Pti1 [24].
The possible role(s) of these phosphorylated residues in
disease resistance is being examined by site-directed
mutagenesis and transgenic analysis (G Sessa and
G Martin, unpublished data). Previous work has indicated
that Pti1 is involved in development of the HR [22].
However, Pti1 is a member of a gene family and antisense
(loss-of-function) experiments to examine its role have
been inconclusive (J Zhou, personal communication).
Thus, one of the best approaches to determine a role for
Pti1 might be the development of constitutive-active proteins by altering key phosphorylation sites.
The Pti4/5/6 proteins resemble the ethylene response element binding proteins (EREBPs) originally isolated from
tobacco [23]. Like the EREBPs, Pti4/5/6 bind the GCC box
cis element that is present in the promoter of many pathogenesis-related (PR) genes and other genes that are often
expressed in plants upon exposure to ethylene or pathogens.
Thus, the identification of the Pti4/5/6 genes defines the first
direct link between an R gene and expression of defenserelated genes. As expected for transcription factors, each of
the Pti4/5/6 proteins contains putative nuclear-localization
sequences and the proteins are localized to the nucleus (YT Loh, Y Gu, and G Martin, unpublished data). Using
purified proteins, Pti4 has been found to be phosphorylated
in vitro by Pto on at least four sites. Experiments are underway to examine the possible role of these phosphorylation
events in nuclear localization, binding to the GCC box, or in
transactivation of target gene expression (Y Gu and
G Martin, unpublished data). Although the Pti4/5/6 proteins
share sequence similarity they also contain highly divergent
regions [23]. In addition, the Pti4/5/6 genes are regulated differently in response to ethylene, jasmonic acid (JA), salicylic
acid (SA), wounding, and pathogens (Y Gu and G Martin,
unpublished data; KV Thara and J Zhou, personal communication). Thus, it seems possible that their proteins serve to
integrate different signals that induce common target genes.

Characterization of the Pti1/4/5/6 genes suggests that multiple pathways diverge directly from the Pto kinase.
Because Pti1 overexpression accelerates the HR, we have
proposed that one pathway leads to the HR and perhaps to
other defense responses [22]. Additional, distinct pathways
appear to be mediated by the interaction of Pto with the
putative transcription factors Pti4/5/6 [23]. The Pti1/4/5/6
genes were identified by a yeast two-hybrid screen and to
date no mutations in these genes have been identified in
tomato. Thus additional evidence for their role in disease
resistance is being sought by transgenic analyses and biochemical approaches (G Sessa, Y Gu and G Martin,
unpublished data).

Restricted conservation of signaling pathways
In light of the shared motifs among R proteins from very
diverse plant species it might be expected that downstream signaling pathways contain similar, or at least
compatible, components. If this is the case, then R genes
isolated from one species might be used to engineer new
disease resistance specificities into other economically
important species. R genes isolated from tomato have been
previously shown to function in tobacco and vice versa
[38,39]. Recently, the Cf-9 gene from tomato has been
shown to function in both potato and tobacco [40].
However, there have been no reports of Arabidopsis
R genes functioning in Solanaceous species or vice versa, or
any other successful transfers outside of a single plant family. Whether this restricted cross-taxa function is due to the
absence or incompatibility of other essential recognition or
signaling components remains to be determined.

Signaling components acting downstream of
R genes
Successes using mutant screens in Arabidopsis to identify
genes, other than R genes, that play a role in disease resistance have been recently reviewed [41]. Most of the loci
defined by these approaches do not appear to play a role in
R-gene specific pathways. One exception to this is the
NPR1/NIM1/SAI1 locus, mutations of which lead to loss of
SA-dependent PR gene expression. Isolation of the
NPR1/NIM1 gene revealed a protein with ankyrin-like
repeats that might play a role in protein–protein interactions [42,43]. NPR1/NIM1 has been previously implicated
in R gene signaling but recent characterization of an allele,
npr1-4, showed no difference in pathogen growth in an
incompatible interaction involving the avrRpt2/RPS2
genes [44]. Examination of all the nor alleles under identical conditions will be necessary to resolve the role of
NIM1/NPR1/SAI1 in R gene-mediated resitance. An
observable effect on resistance to avirulent pathogens
been seen in six additional enhanced disease susceptibility (eds) mutants [44], the pad4 mutant that is defective in
synthesis of camalexin [45], or in the dnd1 mutant that
retains gene-for-gene resistance despite a much reduced
HR [46]. However, two Arabidopsis loci, defined by mutations ndr1 and eds1, do play a definitive role in
R gene-mediated signaling and their characterization has

Functional analysis of plant diesease resistance genes and their downstream effectors Martin

led to a significant development in our understanding of
downstream pathways mediated by these genes.
Arabidopsis plants with mutations at the ndr1 and eds1 loci
are affected in resistance to both bacterial and fungal
pathogens mediated by several different R genes [47••].
Mutations at the eds1 locus lead to loss of resistance to
Pseudomonas mediated by the R gene RPS4 and to
Peronospora parasitica mediated by the RPP2/4/5/21 genes.
Mutations at the ndr1 locus suppress resistance to
Pseudomonas mediated by the R genes RPS2, RPM1, and
RPS5 [47••]. In addition, resistance to P. parasitica conferred by the RPP4 and RPP5 genes is partially
compromised in ndr1 mutants suggesting some level of
cross-talk involving both NDR1 and EDS1. The significance of these observations is that the R genes suppressed
by the eds1 mutation are of the TIR–NBS–LRR class,
whereas those suppressed by ndr1 are of the
LZ–NBS–LRR class. This exciting development thus
defines two distinct pathways that lie downstream of these
different R gene classes and provide important new tools
to understand the role of the TIR and LZ domains in
defense signaling. The NDR1 gene was cloned previously
to these developments and found to encode a novel protein whose features indicate it might be associated with a
membrane; its role remains unclear [48].
Isolation of the EDS1 gene by transposon tagging was
reported recently [49••]. EDS1 encodes a protein whose
amino terminus has similarity to eukaryotic lipases,
although the role of this possible activity in disease resistance must still be resolved. EDS1 is expressed in
healthy tissue and its transcript abundance increases 2–3
fold in response to SA or an avirulent bacterial pathogen.
Interestingly, however, exogenous application of JA,
which is derived from fatty acid intermediates, does not
induce EDS1 and is not able to ameliorate the loss of disease resistance in the eds1 mutant [49••]. Despite the
lack of response to JA, it remains possible that the EDS1
protein plays a role in modifying an intermediate
involved in JA production. Alternatively, EDS1 might
participate in another unknown signaling pathway
involving lipid intermediates. An understanding of the
role of the EDS1 protein will require biochemical analysis of its function, determination of its substrates, and
perhaps identification of additional proteins that are
involved in its activity.

Role of MAPKs in R gene signaling
A key role for protein phosphorylation in R gene signaling
is clearly implicated by the isolation of the Pto, Xa21, and
Pti1 kinases. However, there are relatively few studies that
have examined protein phosphorylation events further
downstream of R genes. Two recent studies now provide
compelling evidence for the activation of MAPKs by
R gene pathways and, in addition, suggest that these kinases are convergence points for many different defense
responses in plants [35•,36•].

277

In tobacco, two previously identified MAPKs, a 48 kD salicylic acid induced protein kinase (SIPK) and a 44 kD
wound-induced protein kinase (WIPK), were found to be
activated during infection by tobacco mosaic virus [35•]).
Activation of WIPK occurred only in tobacco plants containing the tobacco mosaic virus R gene N indicating that
WIPK might play a role in gene-for-gene mediated
defense against virus infection. Activation of WIPK
required both serine–threonine and tyrosine phosphorylation and also an increase in its mRNA and protein levels.
Although many defense responses mediated by the N gene
are dependent on SA, the activation of WIPK was not
affected in plants lacking this signaling molecule [35•].
Additional evidence that SIPK and WIPK lie downstream
of R genes comes from studies with the Cf-9 R gene and its
corresponding gene Avr9. Treatment of tobacco suspension cells expressing Cf-9 with the Avr9 peptide resulted in
rapid activation of both SIPK and WIPK [36•]. Similarly to
the observations with the N gene system, the WIPK transcript was found to increase in an Avr9-dependent fashion
in Cf-9-containing tobacco cells. Activation of WIPK and
SIPK was not perturbed by inhibitors of active oxygen
species synthesis. Thus, similarly to the SA-independent
pathway seen with the N gene system, the activation of
WIPK and SIPK by Cf-9 appears to be via a parallel pathway that is independent of at least some other signaling
pathways downstream of Cf-9. Because WIPK and SIPK
are known to be activated by many stimuli including nonspecific elicitors, wounding, and SA, these reports indicate
that some R gene pathways converge on components
shared with general stress-responsive pathways. Despite
the clear involvement of the N and Cf-9 genes in activating
MAPKs, it remains to be determined whether these kinases are directly involved in disease resistance and, if so,
what role they play.

Potential for manipulation of R gene signaling
components
One rationale for studying R genes and their downstream
signaling components is that it might be possible to manipulate expression of these genes in economically important
plant species in order to improve disease resistance.
Although the field is still very much in its infancy, there
already have been several promising reports to indicate
such a strategy is feasible. Overexpression of Prf in tomato
by integration of several copies of the gene under control
of its own promoter led to increased levels of SA and
PR genes and to increased resistance to three bacterial and
one viral pathogen [25•]. Unlike plant mutants that lead to
constitutive systemic acquired resistance, the overexpression of Prf caused no detrimental effects on plant growth
or fruit production.
Overexpression of Pto under control of the CaMV 35S promoter in three independent transgenic tomato lines elicited
an array of defense response including microscopic cell
death, SA accumulation, and PR gene expression [19••].

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The transgenic lines displayed increased resistance to a
Pseudomonas strain lacking avrPto, to virulent Xanthomonas
strains, and to the fungal pathogen Cladosporium fulvum. By
using a different approach, Pto has been shown to also limit
the systemic spread of potato virus X in tomato if the virus
is engineered to express the avrPto gene [50]. Thus
defense responses activated by Pto are effective against
bacterial, viral, and fungal pathogens. It remains to be
determined if broad-spectrum resistance mediated by Pto
(or Prf) will be effective under field conditions.
Overexpression of signaling components that lie downstream of R genes is another possible strategy to increase
disease resistance. In the first successful example of this
approach, expression of the NPR1 gene in Arabidopsis was
manipulated to increase the level of its protein to 1.5- to 3fold more than in wild-type plants [51•]. Plants with
moderately increased levels of NPR1 protein exhibited
significant increases in resistance to Pseudomonas and
Peronospora pathogens. Defense-related genes in NPR1overexpressing plants were found to be expressed more
strongly but not more rapidly during pathogen infection
[51•]. Manipulation of downstream components such as
NPR1 potentially allows activation of only certain defense
pathways. This might avoid agronomic problems associated with constitutive activation of some R gene-mediated
pathways such as those leading to the HR and could lead
to a better understanding of the contributions of specific
pathways to disease resistance.

References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:

• of special interest
•• of outstanding interest

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Parniske M, Wulff BB, Bonnema G, Thomas CM, Jones DA, Jones
JDG: Homologues of the Cf-9 disease resistance gene (Hcr9s)
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Dissection of the Fusarium I2 gene cluster in tomato reveals six
homologs and one active gene copy. Plant Cell 1998, 10:10551068.
15. Thomas CM, Dixon MS, Parniske M, Golstein C, Jones JDG: Genetic
and molecular analysis of tomato Cf genes for resistance to
Cladosporium fulvum. Phil Trans R Soc Lond 1998, 353:14131424.
16. Ellis JG, Lawrence GJ, Luck JE, Dodds PN: Identification of regions
•• in alleles of the flax rust resistance gene L that determine
differences in gene-for-gene specificity. Plant Cell 1999,
11:495-506.
The first detailed characterization of recognition specificity with the TIR–LRR
class of R genes. Shows that both the TIR and the LRR regions play a role
in recognition of pathogen strains.
17.
•

Wang GL, Ruan DL, Song WY, Sideris S, Chen L, Pi LY, Zhang S,
Zhang Z, Fauquet C, Gaut BS et al.: Xa21D encodes a receptor-like
molecule with a leucine-rich repeat domain that determines racespecific recognition and is subject to adaptive evolution. Plant
Cell 1998, 10:765-779.
Reports that the LRR region of Xa21D determines recognition specificity.
This intriguing result indicates protein kinase activity of Xa21 is not
required for recognition and raises questions of how signaling is initiated
in this system.

18. Frederick RD, Thilmony RL, Sessa G, Martin GB: Recognition
•
specificity for the bacterial avirulence protein AvrPto is
determined by Thr-204 in the activation loop of the tomato Pto
kinase. Mol Cell 1998, 2:241-245.
This detailed examination of Pto recognition specificity demonstrates the
importance of specific amino acids in the kinase activation loop and suggests that simple mutations might give rise to new R gene specificities.

1.

Milligan SB, Bodeau J, Yaghoobi J, Kaloshian I, Zabel P, Williamson VM:
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2.

Rossi M, Goggin FL, Milligan SB, Kaloshian I, Ullman DE, Williamson VM:
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Vos P, Simons G, Jesse T, Wijbrandi J, Heinen L, Hogers R, Frijters A,
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19. Tang X, Xie M, Kim YJ, Zhou J, Klessig DF, Martin GB:
•• Overexpression of Pto activates defense responses and confers
broad resistance. Plant Cell 1999, 11:15-30.
The first report that overexpression of a specific R gene can broaden its
effectiveness to previously virulent pathogens. Demonstrates the potential
for engineering R genes for increased, durable disease resistance.

4.

Scofield SR, Tobias CM, Rathjen JP, Chang JH, Lavelle DT,
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20. van der Biezen EA, Jones JDG: The NB–ARC domain: a novel
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Tang X, Frederick RD, Zhou J, Halterman DA, Jia Y, Martin GB:
Initiation of plant disease resistance by physical interaction of
AvrPto and Pto kinase. Science 1996, 274:2060-2063.

21. van der Biezen EA, Jones JDG: Plant disease-resistance proteins
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Martin GB, Brommonschenkel SH, Chunwongse J, Frary A, Ganal
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Science 1993, 262:1432-1436.

22. Zhou J, Loh Y-T, Bressan RA, Martin GB: The tomato gene Pti1
encodes a serine/threonine kinase that is phosphorylated by Pto
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7.

Salmeron JM, Oldroyd GED, Rommens CMT, Scofield SR, Kim H-S,
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the leucine-rich repeat class of plant disease resistance genes
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23. Zhou J, Tang X, Martin GB: The Pto kinase conferring resistance to
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8.

de Feyter R, McFadden H, Dennis L: Five avirulence genes from
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24. Sessa G, D’Ascenzo M, Loh YT, Martin GB: Biochemical properties
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Functional analysis of plant diesease resistance genes and their downstream effectors Martin

25. Oldroyd GED, Staskawicz BJ: Genetically engineered broad
•
spectrum disease resistance in tomato. Proc Natl Acad Sci USA
1998, 95:10300-10305.
The first report that overexpression of an LRR/NBS-like gene can activate
various defense responses and confer broadened resistance. Because Prfoverexpressing plants did not exhibit an HR (unlike overexpressed Pto
[19••]), the authors suggest that engineering components potentially downstream of R genes might cause fewer detrimental effects to the plant.
26. Warren RF, Henk A, Mowery P, Holub E, Innes RW: A mutation
•• within the leucine-rich repeat domain of the Arabidopsis disease
resistance gene RPS5 partially suppresses multiple bacterial and
downy mildew resistance genes. Plant Cell 1998, 10:1439-1452.
A well-written paper that reports the cloning and functional analysis of the
NBS–LRR-like gene RPS5. Careful plant pathology work and genetic analysis suggested that LRRs play a role in both recognition and signaling.
Represents a first significant insight into the possible function of LRRs.
Boyes DC, Nam J, Dangl JL: The Arabidopsis thaliana RPM1
disease resistance gene product is a peripheral plasma
membrane protein that is degraded coincident with the
hypersensitive response. Proc Natl Acad Sci USA 1998,
95:15849-15854.
Groundbreaking research on the localization of an LZ–NBS–LRR protein.
The observation that RPM1 is rapidly degraded upon pathogen recognition
suggests a mechanism for the plant cell to tightly regulate the HR and highlights the need for additional cell biology research in the disease resistance
field.
27.
••

28. del Pozo JC, Estelle M: Function of the ubiquitin-proteosome
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29. Zhou J, Tang X, Frederick R, Martin G: Pathogen recognition and
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30. Loh YT, Zhou J, Martin GB: The myristylation motif of Pto is not
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32. O’Neill LA, Greene C: Signal transduction pathways activated by
•
the IL-1 receptor family: ancient signaling machinery in mammals,
insects, and plants. J Leukoc Biol 1998, 63:650-657.
An excellent review from a ‘mammalian’ perspective about the conservation
of TIR-like signaling components among divergent taxa.
33. Delledonne M, Xia Y, Dixon RA, Lamb C: Nitric oxide functions as a
signal in plant disease resistance. Nature 1998, 394:585-588.
34. Durner J, Wendehenne D, Klessig DF: Defense gene induction in
tobacco by nitric oxide, cyclic GMP, and cyclic ADP-ribose. Proc
Natl Acad Sci USA 1998, 95:10328-10333.
35. Zhang S, Klessig DF: Resistance gene N-mediated de novo
•
synthesis and activation of a tobacco mitogen-activated protein
kinase by tobacco mosaic virus infection. Proc Natl Acad Sci USA
1998, 95:7433-7438.
The first report that MAPKs are activated by an R gene-mediated signaling
pathway. Because the corresponding MAPK genes are cloned, this study
opens the way to elucidating their possible role in disease resistance.
36. Romeis T, Piedras P, Zhang S, Klessig DF, Hirt H, Jones JD: Rapid avr
•
9- and Cf-9-dependent activation of MAP kinases in tobacco cell
cultures and leaves: convergence of resistance gene, elicitor,
wound, and salicylate responses. Plant Cell 1999, 11:273-288.
Nicely extends the work reported in [35•] to show that multiple pathways
including one initiated by the Cf-9 gene converge upon two MAPKs. The Cf9 pathway which activates the MAPKs is distinct from one initiating production of active oxygen species thus supporting the observation that parallel
pathways lie downstream of R genes.

37.

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38. Thilmony RT, Chen Z, Bressan RA, Martin GB: Expression of the
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39. Whitham S, McCormick S, Baker B: The N gene of tobacco confers
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40. Hammond-Kosack KE, Tang S, Harrison K, Jones JD: The tomato
Cf-9 disease resistance gene functions in tobacco and potato to
confer responsiveness to the fungal avirulence gene product
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41. Innes RW: Genetic dissection of R gene signal transduction
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42. Ryals J, Weymann K, Lawton K, Friedrich L, Ellis D, Steiner HY,
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NIM1 protein shows homology to the mammalian transcription
factor inhibitor I kappa B. Plant Cell 1997, 9:425-439.
43. Cao H, Glazebrook J, Clarke JD, Volko S, Dong X: The Arabidopsis
NPR1 gene that controls systemic acquired resistance encodes a
novel protein containing ankyrin repeats. Cell 1997, 88:57-63.
44. Volko SM, Boller T, Ausubel FM: Isolation of new Arabidopsis
mutants with enhanced disease susceptibility to Pseudomonas
syringae by direct screening. Genetics 1998, 149:537-548.
45. Zhou N, Tootle TL, Tsui F, Klessig DF, Glazebrook J: PAD4 functions
upstream from salicylic acid to control defense responses in
Arabidopsis. Plant Cell 1998, 10:1021-1030.
46. Yu IC, Parker J, Bent AF: Gene-for-gene disease resistance without
the hypersensitive response in Arabidopsis dnd1 mutant. Proc
Natl Acad Sci USA 1998, 95:7819-7824.
47.
••

Aarts N, Metz M, Holub E, Staskawicz BJ, Daniels MJ, Parker JE:
Different requirements for EDS1 and NDR1 by disease resistance
genes define at least two R gene-mediated signaling pathways in
Arabidopsis. Proc Natl Acad Sci USA 1998, 95:10306-10311.
Represents a major breakthrough in our understanding of R gene signaling
by demonstrating the existence of distinct pathways downstream of different
classes of R genes. The eds1 and ndr1 mutants will be broadly useful for
delineating signaling pathways mediated by Arabidopsis R genes and other
defense-related genes.
48. Century KS, Shapiro AD, Repetti PP, Dahlbeck D, Holub E,
Staskawicz BJ: NDR1, a pathogen-induced component required
for Arabidopsis disease resistance. Science 1997, 278:1963-1965.
49. Falk A, Feys BJ, Frost LN, Jones JDG, Daniels MJ, Parker JE: EDS1,
•• an essential component of R gene-mediated disease resistance
in Arabidopsis has homology to eukaryotic lipases. Proc Natl Acad
Sci USA 1999, 96:3292-3297.
The isolation of EDS1 (and NDR1 in 1997 [48]) are major steps towards
understanding downstream signaling. However, the uncertain role of either
of these proteins reinforces the need for innovative biochemical analysis in
the disease resistance field.
50. Tobias CM, Oldroyd GE, Chang JH, Staskawicz BJ: Plants
expressing the Pto disease resistance gene confer resistance to
recombinant PVX containing the avirulence gene avrPto. Plant J
1999, 17:41-50.
51. Cao H, Li X, Dong X: Generation of broad-spectrum disease
•
resistance by overexpression of an essential regulatory gene in
systemic acquired resistance. Proc Natl Acad Sci USA 1998,
95:6531-6536.
The first report that increased expression of a downstream signaling gene is
a promising strategy to produce Arabidopsis plants with improved disease
resistance. The conservation of NPR1-like genes in other plant species raises the possibility that this approach can be extended to crop plants.