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Exploiting the full potential of disease-resistance genes for
agricultural use
Caius M Rommens* and Ganesh M Kishore†
Effective and sustained control of fungal pathogens and
nematodes is an important issue for all agricultural systems.
Global losses caused by pathogens are estimated to be
12% of the potential crop production [1], despite the
continued release of new resistant cultivars and pesticides.
Furthermore, fungi are continually becoming resistant to
existing resistance genes and fungicides, and a few of the
pesticides are being withdrawn from the market for
environmental reasons. In addition to reducing crop yield,
fungal diseases often lower crop quality by producing toxins
that affect humans and human health. Additional methods of
disease control are therefore highly desirable. Breeding
programs based on plant disease-resistance genes are being
optimized by incorporating molecular marker techniques and
biotechnology. These efforts can be expected to result in the
first launches of new disease-resistant crops within the next
five years.
Addresses
Monsanto Company, 700 Chesterfield Parkway, St Louis, MO 63198, USA
*e-mail: [email protected]
† e-mail: [email protected]
Current Opinion in Biotechnology 2000, 11:120–125
0958-1669/00/$ — see front matter
© 2000 Elsevier Science Ltd. All rights reserved.
Abbreviations
AFP
antifungal protein
avr
avirulence
HR
hypersensitive necrotic response
LRR
leucine rich repeat
NBS
nucleotide-binding site
R
resistance
SAR
systemic acquired broad-spectrum resistance
Introduction
The most important class of genes that has been used by
breeders for disease control are the plant resistance (R)
genes: single determinants of an effective and specific
disease resistance that can often be characterized by
localized necrosis at attempted infection sites. Studies
on the utility of hundreds of R-genes for almost a century resulted in the accumulation of a wealth of knowledge
on both the potential and limitations of R-genes.
Although originally believed to provide durable resistance [2], only a few exceptional R-genes proved able to
control pathogen for an extended period of time. The
limited durability of single R-genes for many of the agronomically most important diseases, including wheat
stem rust and rice blast, made it necessary to continue
the discovery and introgression of new R-genes [3]. This
process is time-consuming and laborious, especially if Rgenes are tightly linked to undesirable traits and/or
difficult to score for phenotype. An example of such an
R-gene is the soybean rhg1 gene for control of cyst nematodes, which behaves as a quantitative trait locus (QTL)
and was difficult to separate genetically from a reduced
yield phenotype [4]. R-gene programs for control of certain diseases, such as potato late blight, even had to be
abandoned because R-genes proved too unreliable:
Phytophthora infestans, causal agent of late blight, overcame the resistance provided by all eleven R-genes that
had been introgressed from the wild species Solanum
demissum into cultivated potato for control of this important fungal disease [5].
Over the past decade, many aspects of R-genes have been
molecularly characterized [6•]. In this review, we will
describe how a current understanding of these genes and
the resistance mediated by them makes it possible to
address important issues related to their discovery, transfer
and durability. It will also discuss the development and
implementation of strategies to optimize R-gene-mediated
resistance through genetic engineering.
Efficiency of R-gene discovery and transfer
The introgression of R-genes into elite cultivars via traditional breeding can take up to 15–20 years. This
process is considerably accelerated, however, by using
molecular markers generated via random fragment
length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR)
and single nucleotide polymorphism (SNP) techniques.
These techniques make it possible to screen segregating
populations molecularly rather than for disease phenotypes, which is time-consuming and labor-intensive.
Marker-assisted breeding programs have been estimated
to reduce the time-to-market by 50–70% [7,8]. An example of a multidisciplinary effort to identify and
characterize important traits including disease resistances using modern technologies is the North American
Barley Genome Mapping Project (www.css.orst.edu/barley/nabgmp/nabgmp.htm). Similar efforts are ongoing in
all major crops.
Application of molecular markers has resulted in the isolation of almost twenty R-genes from genetically
well-characterized plant species through map-based
cloning over the past five years [6•]. Molecular analyses
showed that most R-genes isolated to date encode proteins with an amino-terminal nucleotide-binding site
(NBS) and a carboxy-terminal leucine rich repeat (LRR).
R-genes were proven to maintain their activity upon
transfer within — and in some cases even across — plant
species [9,10,11••]. The ability to isolate and transfer
R-genes eliminates the issue of retention of unwanted
and genetically linked germplasm, an important problem
Exploiting the full potential of disease-resistance genes for agricultural use Rommens and Kishore
associated with classical breeding. Genome sequencing
and genetic mapping experiments demonstrated that Rgenes are generally organized in tightly linked clusters
[12••]. Thus, new technologies in the transfer of large
DNA fragments [13] can be used to transfer multiple Rgenes simultaneously. This may enhance the durability
of resistance as it was shown that tightly linked R-genes
could act synergistically [14].
Analysis of expressed sequence tag (EST) libraries
demonstrated that plants such as Arabidopsis and soybean express hundreds of potential R-genes [15]
(AL Balmuth, CM Rommens, unpublished data). Many
such genes have already been mapped to genetically
characterized resistance loci in a variety of plant systems,
including Arabidopsis, potato, soybean, lettuce, maize
and wheat (e.g. see [16,17]). Obviously, the optimized
discovery of R-genes in both domesticated and exotic
germplasm will be of paramount importance in the
future, and these genes can be rapidly transferred into
advanced commercial germplasm based on the molecular
techniques described above.
Durability of R-genes
Exceptional R-genes have proven to provide durable disease control. Genes such as Bs2 in pepper and Xa21 in
rice are important examples that reveal the full potential
of R-genes [18,19]. The durability of Bs2 and Xa21 is a
consequence of their ability to recognize ‘avirulence’
(avr) proteins secreted by most or all races of the bacterial pathogens Xanthomonas campestris and X. oryzae,
respectively. Interestingly, the avr protein recognized by
Bs2 is not only produced by pathovars of X. campestris
that infect pepper but also by pathovars that infect hosts
such as tomato, brassica and citrus [18]. As Bs2 was
recently cloned, its utility may be greatly extended by
transferring it across species boundaries. Expression of
Bs2 in tomato has already been shown to result in resistance against bacterial spot disease caused by
X. campestris pv. vesicatoria [11••]. Other durable R-genes
that have not yet been cloned but may act in a similar
way to Bs2 and Xa21 are the barley Rpg1 gene for control
of stem rust [20] and the Lr34 gene, which protects
wheat against leaf rusts [21].
Can we develop screens to identify broad-spectrum
R-genes such as Bs2 and Xa21 more efficiently? One of
the most promising new screens developed to date is
based on the identification of broad-spectrum R-genes
that recognize ubiquitous proteins secreted by pathogens
and required for their pathogenesis [22••]. The protein
used to validate the efficacy of this screen was the ECP2
protein secreted by the tomato pathogen Cladosporium
fulvum. ECP2 is essential for full pathogenicity and is
produced by all strains of a worldwide collection of C. fulvum. By transiently expressing ECP2 in a variety of
tomato germplasm, plants were identified that responded
with a hypersensitive necrotic response (HR). These
121
plants were genetically analyzed and shown to carry a single dominant gene for ECP2-dependent HR, named
Cf-ECP2. This new plant gene was confirmed to act as a
new R-gene against C. fulvum and is expected to provide
durable disease control.
One of the most interesting R-genes is the barley mlo gene,
which confers resistance to all races of Erysiphe graminis in
barley. Resistance mediated by mlo is more durable than
that provided by most other R-genes because it does not
require activation by specific avr determinants [23].
Defense responses in barley mlo plants are constitutively
potentiated and lead to the rapid formation of subcellular
cell wall appositions, termed papilla, upon infection with
E. graminis. Most fungal penetration attempts are arrested
in these appositions, which were shown to accumulate the
antifungal compound p-coumaroyl-hydroxyagmatine [24•].
The utility of recessive mlo resistance is likely to be
extended by antisense suppression of the dominant Mlo
gene in wheat or any other plant species that is highly susceptible to Erysiphe sp.
Another durable resistance gene that lacks homology with
NBS/LRR genes is the tomato Asc gene, which provides
control of Alternaria alternata f. sp. lycopersici (AAL)
through insensitivity to the AAL toxin [25]. The isolation
of this gene was reported at the International Symposium
on Molecular Plant–Microbe Interactions meeting by
Jacques Hille and co-workers (University of Groningen,
The Netherlands). The Asc gene was shown to share
homology with the longevity gene Lag1. An understanding
of Asc resistance may provide a strategy for detoxification
of fumonisin, a toxin produced by the important corn
pathogen Fusarium moniliforme with a similar mode of
action to the AAL toxin. Fumonisin is a worldwide contaminant in food and feed, and imposes a health risk to both
humans and animals [26].
Even more effective R-genes may be isolated from resistant plant species that are sexually incompatible with
susceptible plants, named ‘non-hosts’. Although this
type of resistance can not be characterized genetically,
indirect evidence suggests that it may in some cases be
controlled by extremely durable R-genes. For example,
the non-host resistance of tobacco against the potato
pathogen Phytophthora infestans is correlated with the
ability of tobacco to respond hypersensitively to an elicitor of this pathogen, indicating the direct involvement
of R-genes [27••]. The product of a tobacco gene that
shares similarities with R-genes was indeed recently
reported to interact with a 10 kDa peptide elicitor produced by P. infestans, named INF1, in the yeast
two-hybrid system (summarized in the meeting report of
the International Symposium on Molecular Plant–
Microbe Interactions meeting [28]). A second factor
required for active non-host responses is a 100 kDa parsley plasma membrane protein that binds to a cell-wall
glycoprotein elicitor of the soybean pathogen
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Plant biotechnology
Phytophthora sojae. This parsley elicitor receptor is currently being isolated biochemically [29]. The transfer of
non-host genes such as the tobacco and parsley elicitor
receptors to susceptible hosts may have a tremendous
impact on the ability to control Phytophthora sp. and other
aggressive pathogens.
Engineering R-genes and downstream
responses
R-gene disease control programs can be further refined
by optimizing the activity of isolated R-genes before
reintroducing them into the plant. For example, the
activity of the wild-type tomato Pto gene is limited to
certain races of Pseudomonas syringae pv. tomato (Pst) that
contain the avr gene avrPto. Replacement of the weak
endogenous promoter of Pto with the strong promoter of
cauliflower mosaic virus resulted in not only a further
increased resistance to Pst(avrPto) but also a partial control of unrelated pathogens, such as Xanthomonas
campestris and Cladosporium fulvum [30•]. Studies to
probe the function of various R-gene domains by creating recombinant flax R-genes demonstrated that the
LRR region might be involved in R-gene specificity
[31••]. Importantly, exchange of an LRR region resulted
in one case in recognition of a different spectrum of
pathogens than that of the originally used R-genes [31••].
Further studies on the various domains of R-genes may
make it possible to optimize the efficacy and durability
of R-genes.
Efforts to engineer broad-spectrum resistance are not
limited to R-genes but also include approaches around
the plant defense responses elicited by these R-genes:
the rapid and localized HR and the subsequent establishment of the systemic acquired broad-spectrum
resistance (SAR) response. One elegant strategy aims to
alter regulation of the HR in such a way that this
response is induced by both virulent and avirulent
pathogens [P1]. The bottleneck of this approach is the
identification of promoters that respond tightly, rapidly
and in a cell-autonomous manner to infection. This
study may greatly benefit from novel tools in genomics,
which allow the identification of genes (and thus promoters) that only respond to very specific signals. A
variant of the above strategy is to generate a lesion mimic
phenotype via either mutant selection [32] or regulated
expression of genes that trigger the HR [33].
efficacy of either Npr1 or Npr1 homologs in crops.
Preliminary results indicate that overexpression of Npr1
in rice does lead to increased resistance against both
Xanthomonas (reported at the International Symposium
on Molecular Plant–Microbe Interactions meeting by
Pamela Ronald and co-workers, UC Davis) and
Magnaportha (N Srivastava, KMM Swords, personal communication). Overexpression of an Npr1 homolog in
wheat, however, resulted in suppression of pathogenesisrelated genes and enhanced disease susceptibility
(OV Bougri, CM Rommens, unpublished data).
Another signaling gene that has been evaluated for its utility to enhance disease resistance levels is the Myb1 gene,
which is induced by tobacco mosaic virus (TMV) in resistant tobacco plants, and encodes a transcription factor that
binds to a promoter element of the pathogenesis-related
gene PR1a [36]. Modification of Myb1 expression levels in
transgenic tobacco plants was shown to increase resistance
against both a viral (TMV) and a fungal (Rhizoctonia solani)
pathogen [P2].
Two additional highly interesting genes were identified
through mutant screens in Arabidopsis but have not yet
been isolated. These genes, named cpr6 and Ssi1, trigger
not only genes associated with SAR but also genes that
act in a second response pathway activated by jasmonic
acid [37•,38••]. Thus, both cpr6 and Ssi1 genes act as
switches modulating cross-talk between different
defense pathways. The robust resistance controlled by
cpr6 and Ssi1 is unfortunately linked to severe stunting,
and application of this technology for agriculture may
require optimization.
The very recently cloned Arabidopsis Pad4 gene may
prove exceptionally interesting for the development of
broad-spectrum disease resistance [39••]. The interesting aspect of Pad4 is that inactivation of this gene leads
to extreme susceptibility against a wide variety of
pathogens, including Erysiphe orontii, Peronospora parasitica and Pseudomonas syringae [40,41]. Thus,
overexpression of Pad4 in transgenic plants may enhance
disease-resistance signaling. The Pad4 gene has been
hypothesized to act by amplifying weak signals in disease-resistance responses through a positive feedback
cycle with salicylic acid [39••].
Overexpressing defense genes
Another effort is focused on key genes of the SAR
response. One of these key genes is the Npr1 gene,
which encodes a putative transcriptional regulator [34].
Overexpression of Npr1 enhances disease resistance levels against a broad variety of pathogens in Arabidopsis
[35••]. Importantly, this resistance is not associated with
any adverse plant phenotypes, such as stunting or undesired cell death [35••]. These findings make Npr1 an
extremely interesting candidate for agricultural
application. Experiments have been initiated to test the
Many of the genes induced by plant disease-resistance
responses encode proteins with direct antifungal activity
in vitro [42–45]. One of the most important and straightforward strategies in enhancing disease resistance is
based on the identification and expression of such antifungal proteins (AFPs). This is an extension of the
paradigm that has worked extremely well for insect control genes based on insecticidal proteins from Bacillus
thuringienesis. Reported AFP classes include defensins
and other small cysteine-rich peptides, 2S albumins,
Exploiting the full potential of disease-resistance genes for agricultural use Rommens and Kishore
123
chitin-binding proteins, lipid-transfer proteins, and
hydrogen-peroxide-generating enzymes [46–48,P3].
systems used by pathogens should enable the design and
development of novel disease-resistance genes.
Many biotechnology companies and universities are
evaluating the performance of AFP-based transgenes
in the field. In fact, hundreds of release permit applications for field trials to test the efficacy of transgenes
for disease control are submitted per year
(http://www.nbiap.vt.edu/cfdocs/fieldtests1.cfm). Field
trials do not only include efficacy tests of the transgenes
but also rigorous tests on agronomic characteristics and
yield of the transgenic plants [49••]. Prior to commercialization, transgenic plants are also assessed for nutritional
composition, and transgenes are evaluated for any food
safety issues. This rigorous process leads to
deprioritization of many transgenes. For example,
expression of the Aspergillus glucose oxidase (AGO) gene
in transgenic potato plants increased disease resistance
in growth chamber experiments [47] but failed to provide commercial levels of disease control in the field. In
addition, AGO gene expression was shown to be correlated with a slightly altered tuber phenotype
(KMM Swords, MS Hakimi, personal communication). A
second hydrogen-peroxide-generating enzyme, the barley oxalate oxidase, is under advanced field evaluation
for control of Sclerotinia in soybean, canola and sunflower
[P4]. Some groups are evaluating the simultaneous
expression of two different AFPs in plants.
Overexpression of an intracellular chitinase and an extracellular β1,3 glucanase resulted in a synergistic effect
providing disease control against Fusarium oxysporum in
tomato [P5]. An abundance of studies on the efficacy of
AFPs in transgenic plants can be expected to be published in the near future. A major issue faced by the AFP
approach is one of identifying AFP proteins that have
fungicidal activity against multiple races of the pathogen
as well as the durability of resistance. In combination
with R-genes, however, these genes may prove extremely useful opening up the possibility of a multi-genic
approach, which could provide efficacious and durable
resistance against these pathogens.
Acknowledgements
Conclusions and future prospects
Disease resistance programs based on R-genes will greatly benefit from the support provided by molecular
breeding and molecular biology. R-genes will be transferred more rapidly into elite germplasm. Better R-genes
will be identified by exploring unconventional sources of
resistance. Resistance will also be enhanced by the engineering of both R-genes and downstream defense
responses. In addition to the progress being made on the
plant side of the equation, an understanding of the genetic make-up of the fungal pathogen and critical genes
involved in the pathogenesis process are expected to
open new horizons in plant and crop protection. An integrated approach, based on the combined knowledge of
the ‘defense’ systems used by the plants and the ‘assault’
The authors thank Kathy Swords for a critical review of the manuscript.
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31. Ellis JG, Lawrence GJ, Luck JE, Dodds PN: Identification of regions
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differences in gene-for-gene specificity. Plant Cell 1999, 11:495-506.
The authors show that specificity differences between R-genes in flax can be
determined by both the LRR and an amino-terminal region with homology to
the Toll/interleukin-1 receptor
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32. Rate DN, Cuenca JV, Bowman GR, Guttman DS, Greenberg JT: The
gain-of-function Arabidopsis acd6 mutant reveals novel regulation
and function of the salicylic acid signaling pathway in controlling cell
death, defenses, and cell growth. Plant Cell 1999, 11:1695-1708.
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33. Hammond-Kosack KE, Tang S, Harrison K, Jones JD: The tomato
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Exploiting the full potential of disease-resistance genes for
agricultural use
Caius M Rommens* and Ganesh M Kishore†
Effective and sustained control of fungal pathogens and
nematodes is an important issue for all agricultural systems.
Global losses caused by pathogens are estimated to be
12% of the potential crop production [1], despite the
continued release of new resistant cultivars and pesticides.
Furthermore, fungi are continually becoming resistant to
existing resistance genes and fungicides, and a few of the
pesticides are being withdrawn from the market for
environmental reasons. In addition to reducing crop yield,
fungal diseases often lower crop quality by producing toxins
that affect humans and human health. Additional methods of
disease control are therefore highly desirable. Breeding
programs based on plant disease-resistance genes are being
optimized by incorporating molecular marker techniques and
biotechnology. These efforts can be expected to result in the
first launches of new disease-resistant crops within the next
five years.
Addresses
Monsanto Company, 700 Chesterfield Parkway, St Louis, MO 63198, USA
*e-mail: [email protected]
† e-mail: [email protected]
Current Opinion in Biotechnology 2000, 11:120–125
0958-1669/00/$ — see front matter
© 2000 Elsevier Science Ltd. All rights reserved.
Abbreviations
AFP
antifungal protein
avr
avirulence
HR
hypersensitive necrotic response
LRR
leucine rich repeat
NBS
nucleotide-binding site
R
resistance
SAR
systemic acquired broad-spectrum resistance
Introduction
The most important class of genes that has been used by
breeders for disease control are the plant resistance (R)
genes: single determinants of an effective and specific
disease resistance that can often be characterized by
localized necrosis at attempted infection sites. Studies
on the utility of hundreds of R-genes for almost a century resulted in the accumulation of a wealth of knowledge
on both the potential and limitations of R-genes.
Although originally believed to provide durable resistance [2], only a few exceptional R-genes proved able to
control pathogen for an extended period of time. The
limited durability of single R-genes for many of the agronomically most important diseases, including wheat
stem rust and rice blast, made it necessary to continue
the discovery and introgression of new R-genes [3]. This
process is time-consuming and laborious, especially if Rgenes are tightly linked to undesirable traits and/or
difficult to score for phenotype. An example of such an
R-gene is the soybean rhg1 gene for control of cyst nematodes, which behaves as a quantitative trait locus (QTL)
and was difficult to separate genetically from a reduced
yield phenotype [4]. R-gene programs for control of certain diseases, such as potato late blight, even had to be
abandoned because R-genes proved too unreliable:
Phytophthora infestans, causal agent of late blight, overcame the resistance provided by all eleven R-genes that
had been introgressed from the wild species Solanum
demissum into cultivated potato for control of this important fungal disease [5].
Over the past decade, many aspects of R-genes have been
molecularly characterized [6•]. In this review, we will
describe how a current understanding of these genes and
the resistance mediated by them makes it possible to
address important issues related to their discovery, transfer
and durability. It will also discuss the development and
implementation of strategies to optimize R-gene-mediated
resistance through genetic engineering.
Efficiency of R-gene discovery and transfer
The introgression of R-genes into elite cultivars via traditional breeding can take up to 15–20 years. This
process is considerably accelerated, however, by using
molecular markers generated via random fragment
length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR)
and single nucleotide polymorphism (SNP) techniques.
These techniques make it possible to screen segregating
populations molecularly rather than for disease phenotypes, which is time-consuming and labor-intensive.
Marker-assisted breeding programs have been estimated
to reduce the time-to-market by 50–70% [7,8]. An example of a multidisciplinary effort to identify and
characterize important traits including disease resistances using modern technologies is the North American
Barley Genome Mapping Project (www.css.orst.edu/barley/nabgmp/nabgmp.htm). Similar efforts are ongoing in
all major crops.
Application of molecular markers has resulted in the isolation of almost twenty R-genes from genetically
well-characterized plant species through map-based
cloning over the past five years [6•]. Molecular analyses
showed that most R-genes isolated to date encode proteins with an amino-terminal nucleotide-binding site
(NBS) and a carboxy-terminal leucine rich repeat (LRR).
R-genes were proven to maintain their activity upon
transfer within — and in some cases even across — plant
species [9,10,11••]. The ability to isolate and transfer
R-genes eliminates the issue of retention of unwanted
and genetically linked germplasm, an important problem
Exploiting the full potential of disease-resistance genes for agricultural use Rommens and Kishore
associated with classical breeding. Genome sequencing
and genetic mapping experiments demonstrated that Rgenes are generally organized in tightly linked clusters
[12••]. Thus, new technologies in the transfer of large
DNA fragments [13] can be used to transfer multiple Rgenes simultaneously. This may enhance the durability
of resistance as it was shown that tightly linked R-genes
could act synergistically [14].
Analysis of expressed sequence tag (EST) libraries
demonstrated that plants such as Arabidopsis and soybean express hundreds of potential R-genes [15]
(AL Balmuth, CM Rommens, unpublished data). Many
such genes have already been mapped to genetically
characterized resistance loci in a variety of plant systems,
including Arabidopsis, potato, soybean, lettuce, maize
and wheat (e.g. see [16,17]). Obviously, the optimized
discovery of R-genes in both domesticated and exotic
germplasm will be of paramount importance in the
future, and these genes can be rapidly transferred into
advanced commercial germplasm based on the molecular
techniques described above.
Durability of R-genes
Exceptional R-genes have proven to provide durable disease control. Genes such as Bs2 in pepper and Xa21 in
rice are important examples that reveal the full potential
of R-genes [18,19]. The durability of Bs2 and Xa21 is a
consequence of their ability to recognize ‘avirulence’
(avr) proteins secreted by most or all races of the bacterial pathogens Xanthomonas campestris and X. oryzae,
respectively. Interestingly, the avr protein recognized by
Bs2 is not only produced by pathovars of X. campestris
that infect pepper but also by pathovars that infect hosts
such as tomato, brassica and citrus [18]. As Bs2 was
recently cloned, its utility may be greatly extended by
transferring it across species boundaries. Expression of
Bs2 in tomato has already been shown to result in resistance against bacterial spot disease caused by
X. campestris pv. vesicatoria [11••]. Other durable R-genes
that have not yet been cloned but may act in a similar
way to Bs2 and Xa21 are the barley Rpg1 gene for control
of stem rust [20] and the Lr34 gene, which protects
wheat against leaf rusts [21].
Can we develop screens to identify broad-spectrum
R-genes such as Bs2 and Xa21 more efficiently? One of
the most promising new screens developed to date is
based on the identification of broad-spectrum R-genes
that recognize ubiquitous proteins secreted by pathogens
and required for their pathogenesis [22••]. The protein
used to validate the efficacy of this screen was the ECP2
protein secreted by the tomato pathogen Cladosporium
fulvum. ECP2 is essential for full pathogenicity and is
produced by all strains of a worldwide collection of C. fulvum. By transiently expressing ECP2 in a variety of
tomato germplasm, plants were identified that responded
with a hypersensitive necrotic response (HR). These
121
plants were genetically analyzed and shown to carry a single dominant gene for ECP2-dependent HR, named
Cf-ECP2. This new plant gene was confirmed to act as a
new R-gene against C. fulvum and is expected to provide
durable disease control.
One of the most interesting R-genes is the barley mlo gene,
which confers resistance to all races of Erysiphe graminis in
barley. Resistance mediated by mlo is more durable than
that provided by most other R-genes because it does not
require activation by specific avr determinants [23].
Defense responses in barley mlo plants are constitutively
potentiated and lead to the rapid formation of subcellular
cell wall appositions, termed papilla, upon infection with
E. graminis. Most fungal penetration attempts are arrested
in these appositions, which were shown to accumulate the
antifungal compound p-coumaroyl-hydroxyagmatine [24•].
The utility of recessive mlo resistance is likely to be
extended by antisense suppression of the dominant Mlo
gene in wheat or any other plant species that is highly susceptible to Erysiphe sp.
Another durable resistance gene that lacks homology with
NBS/LRR genes is the tomato Asc gene, which provides
control of Alternaria alternata f. sp. lycopersici (AAL)
through insensitivity to the AAL toxin [25]. The isolation
of this gene was reported at the International Symposium
on Molecular Plant–Microbe Interactions meeting by
Jacques Hille and co-workers (University of Groningen,
The Netherlands). The Asc gene was shown to share
homology with the longevity gene Lag1. An understanding
of Asc resistance may provide a strategy for detoxification
of fumonisin, a toxin produced by the important corn
pathogen Fusarium moniliforme with a similar mode of
action to the AAL toxin. Fumonisin is a worldwide contaminant in food and feed, and imposes a health risk to both
humans and animals [26].
Even more effective R-genes may be isolated from resistant plant species that are sexually incompatible with
susceptible plants, named ‘non-hosts’. Although this
type of resistance can not be characterized genetically,
indirect evidence suggests that it may in some cases be
controlled by extremely durable R-genes. For example,
the non-host resistance of tobacco against the potato
pathogen Phytophthora infestans is correlated with the
ability of tobacco to respond hypersensitively to an elicitor of this pathogen, indicating the direct involvement
of R-genes [27••]. The product of a tobacco gene that
shares similarities with R-genes was indeed recently
reported to interact with a 10 kDa peptide elicitor produced by P. infestans, named INF1, in the yeast
two-hybrid system (summarized in the meeting report of
the International Symposium on Molecular Plant–
Microbe Interactions meeting [28]). A second factor
required for active non-host responses is a 100 kDa parsley plasma membrane protein that binds to a cell-wall
glycoprotein elicitor of the soybean pathogen
122
Plant biotechnology
Phytophthora sojae. This parsley elicitor receptor is currently being isolated biochemically [29]. The transfer of
non-host genes such as the tobacco and parsley elicitor
receptors to susceptible hosts may have a tremendous
impact on the ability to control Phytophthora sp. and other
aggressive pathogens.
Engineering R-genes and downstream
responses
R-gene disease control programs can be further refined
by optimizing the activity of isolated R-genes before
reintroducing them into the plant. For example, the
activity of the wild-type tomato Pto gene is limited to
certain races of Pseudomonas syringae pv. tomato (Pst) that
contain the avr gene avrPto. Replacement of the weak
endogenous promoter of Pto with the strong promoter of
cauliflower mosaic virus resulted in not only a further
increased resistance to Pst(avrPto) but also a partial control of unrelated pathogens, such as Xanthomonas
campestris and Cladosporium fulvum [30•]. Studies to
probe the function of various R-gene domains by creating recombinant flax R-genes demonstrated that the
LRR region might be involved in R-gene specificity
[31••]. Importantly, exchange of an LRR region resulted
in one case in recognition of a different spectrum of
pathogens than that of the originally used R-genes [31••].
Further studies on the various domains of R-genes may
make it possible to optimize the efficacy and durability
of R-genes.
Efforts to engineer broad-spectrum resistance are not
limited to R-genes but also include approaches around
the plant defense responses elicited by these R-genes:
the rapid and localized HR and the subsequent establishment of the systemic acquired broad-spectrum
resistance (SAR) response. One elegant strategy aims to
alter regulation of the HR in such a way that this
response is induced by both virulent and avirulent
pathogens [P1]. The bottleneck of this approach is the
identification of promoters that respond tightly, rapidly
and in a cell-autonomous manner to infection. This
study may greatly benefit from novel tools in genomics,
which allow the identification of genes (and thus promoters) that only respond to very specific signals. A
variant of the above strategy is to generate a lesion mimic
phenotype via either mutant selection [32] or regulated
expression of genes that trigger the HR [33].
efficacy of either Npr1 or Npr1 homologs in crops.
Preliminary results indicate that overexpression of Npr1
in rice does lead to increased resistance against both
Xanthomonas (reported at the International Symposium
on Molecular Plant–Microbe Interactions meeting by
Pamela Ronald and co-workers, UC Davis) and
Magnaportha (N Srivastava, KMM Swords, personal communication). Overexpression of an Npr1 homolog in
wheat, however, resulted in suppression of pathogenesisrelated genes and enhanced disease susceptibility
(OV Bougri, CM Rommens, unpublished data).
Another signaling gene that has been evaluated for its utility to enhance disease resistance levels is the Myb1 gene,
which is induced by tobacco mosaic virus (TMV) in resistant tobacco plants, and encodes a transcription factor that
binds to a promoter element of the pathogenesis-related
gene PR1a [36]. Modification of Myb1 expression levels in
transgenic tobacco plants was shown to increase resistance
against both a viral (TMV) and a fungal (Rhizoctonia solani)
pathogen [P2].
Two additional highly interesting genes were identified
through mutant screens in Arabidopsis but have not yet
been isolated. These genes, named cpr6 and Ssi1, trigger
not only genes associated with SAR but also genes that
act in a second response pathway activated by jasmonic
acid [37•,38••]. Thus, both cpr6 and Ssi1 genes act as
switches modulating cross-talk between different
defense pathways. The robust resistance controlled by
cpr6 and Ssi1 is unfortunately linked to severe stunting,
and application of this technology for agriculture may
require optimization.
The very recently cloned Arabidopsis Pad4 gene may
prove exceptionally interesting for the development of
broad-spectrum disease resistance [39••]. The interesting aspect of Pad4 is that inactivation of this gene leads
to extreme susceptibility against a wide variety of
pathogens, including Erysiphe orontii, Peronospora parasitica and Pseudomonas syringae [40,41]. Thus,
overexpression of Pad4 in transgenic plants may enhance
disease-resistance signaling. The Pad4 gene has been
hypothesized to act by amplifying weak signals in disease-resistance responses through a positive feedback
cycle with salicylic acid [39••].
Overexpressing defense genes
Another effort is focused on key genes of the SAR
response. One of these key genes is the Npr1 gene,
which encodes a putative transcriptional regulator [34].
Overexpression of Npr1 enhances disease resistance levels against a broad variety of pathogens in Arabidopsis
[35••]. Importantly, this resistance is not associated with
any adverse plant phenotypes, such as stunting or undesired cell death [35••]. These findings make Npr1 an
extremely interesting candidate for agricultural
application. Experiments have been initiated to test the
Many of the genes induced by plant disease-resistance
responses encode proteins with direct antifungal activity
in vitro [42–45]. One of the most important and straightforward strategies in enhancing disease resistance is
based on the identification and expression of such antifungal proteins (AFPs). This is an extension of the
paradigm that has worked extremely well for insect control genes based on insecticidal proteins from Bacillus
thuringienesis. Reported AFP classes include defensins
and other small cysteine-rich peptides, 2S albumins,
Exploiting the full potential of disease-resistance genes for agricultural use Rommens and Kishore
123
chitin-binding proteins, lipid-transfer proteins, and
hydrogen-peroxide-generating enzymes [46–48,P3].
systems used by pathogens should enable the design and
development of novel disease-resistance genes.
Many biotechnology companies and universities are
evaluating the performance of AFP-based transgenes
in the field. In fact, hundreds of release permit applications for field trials to test the efficacy of transgenes
for disease control are submitted per year
(http://www.nbiap.vt.edu/cfdocs/fieldtests1.cfm). Field
trials do not only include efficacy tests of the transgenes
but also rigorous tests on agronomic characteristics and
yield of the transgenic plants [49••]. Prior to commercialization, transgenic plants are also assessed for nutritional
composition, and transgenes are evaluated for any food
safety issues. This rigorous process leads to
deprioritization of many transgenes. For example,
expression of the Aspergillus glucose oxidase (AGO) gene
in transgenic potato plants increased disease resistance
in growth chamber experiments [47] but failed to provide commercial levels of disease control in the field. In
addition, AGO gene expression was shown to be correlated with a slightly altered tuber phenotype
(KMM Swords, MS Hakimi, personal communication). A
second hydrogen-peroxide-generating enzyme, the barley oxalate oxidase, is under advanced field evaluation
for control of Sclerotinia in soybean, canola and sunflower
[P4]. Some groups are evaluating the simultaneous
expression of two different AFPs in plants.
Overexpression of an intracellular chitinase and an extracellular β1,3 glucanase resulted in a synergistic effect
providing disease control against Fusarium oxysporum in
tomato [P5]. An abundance of studies on the efficacy of
AFPs in transgenic plants can be expected to be published in the near future. A major issue faced by the AFP
approach is one of identifying AFP proteins that have
fungicidal activity against multiple races of the pathogen
as well as the durability of resistance. In combination
with R-genes, however, these genes may prove extremely useful opening up the possibility of a multi-genic
approach, which could provide efficacious and durable
resistance against these pathogens.
Acknowledgements
Conclusions and future prospects
Disease resistance programs based on R-genes will greatly benefit from the support provided by molecular
breeding and molecular biology. R-genes will be transferred more rapidly into elite germplasm. Better R-genes
will be identified by exploring unconventional sources of
resistance. Resistance will also be enhanced by the engineering of both R-genes and downstream defense
responses. In addition to the progress being made on the
plant side of the equation, an understanding of the genetic make-up of the fungal pathogen and critical genes
involved in the pathogenesis process are expected to
open new horizons in plant and crop protection. An integrated approach, based on the combined knowledge of
the ‘defense’ systems used by the plants and the ‘assault’
The authors thank Kathy Swords for a critical review of the manuscript.
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