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147
Novel genes for disease-resistance breeding
Leo S Melchers* and Maarten H Stuiver
Plant disease control is entering an exciting period during
which transgenic plants showing improved resistance to
pathogenic viruses, bacteria, fungi and insects are being
developed. This review summarizes the first successful
attempts to engineer fungal resistance in crops, and highlights
two promising approaches. Biotechnology provides the
promise of new integrated disease management strategies that
combine modern fungicides and transgenic crops to provide
effective disease control for modern agriculture.
Addresses
Zeneca MOGEN, PO Box 628, 2300 AP Leiden, The Netherlands;
*e-mail: Leo.Melchers@ageurope.zeneca.com
Current Opinion in Plant Biology 2000, 3:147–152
1369-5266/00/$ — see front matter © 2000 Elsevier Science Ltd.
All rights reserved.
Abbreviations
Avr
avirulence
HR
hypersensitive response
PR
pathogenesis-related
R
resistance gene
transformation and advanced molecular techniques for
plant breeding provides powerful tools for genetically
improving crops so as to enhance their resistance to fungal
diseases. The distinct advantage of transgenic technology
is that it enables the plant breeder to cross species barriers,
allowing genes from non-related plants and other organisms to be introduced into crop plants.
In this review, we discuss the current status of engineered
resistance to fungal pathogens in crops and highlight two
highly promising approaches for generating broad-spectrum fungus-resistant crops [6]. The first strategy relies on
the expression of genes that encode proteins which have
either a direct or indirect inhibitory effect on the growth of
fungi (i.e. the antifungal protein strategy); the second strategy aims to increase fungal resistance in plants by
engineering controlled hypersensitive death of host cells
on the basis of the specific interaction of the product of the
avirulence gene (Avr) and that of the resistance gene (R)
(i.e. Avr/R two-component strategy) [7].
Antifungal proteins
Introduction
Protection against attack by detrimental micro-organisms
and pests is a major challenge for crop production. Fungal
diseases have been one of the main causes of crop losses ever
since mankind started to cultivate plants [1]. At present, the
control of the epidemic spread of fungal diseases mainly
involves three strategies: first, husbandry techniques, such as
crop rotation and avoiding the spread of infested soil and
pathogen-contaminated plant materials; second, breeding of
resistant crop cultivars; and third, the application of agrochemicals. Although conventional plant breeding has made a
significant impact by improving the resistance of many crops
to important diseases, the time-consuming processes of making crosses and back-crosses, and the selection of the desired
resistant progeny make it difficult to react adequately to the
evolution of new virulent fungal races [2,3]. Moreover, these
plant breeding techniques will not provide a solution to
many major fungal diseases because there are simply no natural sources of resistance available to the breeder.
Farmers, therefore, often use fungicides for crop protection in modern agriculture. The use of these compounds is,
however, limited by their high costs and potentially harmful impact on the environment [4,5]. In addition, extended
application can reduce the efficiency of fungicides because
of the evolution of tolerant or resistant pathogens. The
growing concern about the environment, together with a
strong motivation to decrease production costs, encourages
the development of crop cultivars that require less chemicals and/or fewer types of chemical. The advent of plant
Plants have several inducible defense mechanisms that act
to limit pathogen infection, including increased lignification and cell wall cross-linking, the production of small
antibiotic molecules (i.e. phytoalexins), host cell death at
the site of infection (i.e. the hypersensitive response; [8]
and references therein), and the production of reactive
oxygen species [9]. Many plants also develop an increased
resistance to subsequent pathogen infection in uninfected
tissues. This systemic acquired resistance (SAR) can be
effective against viruses, bacteria and fungi, and is accompanied by the expression of a large set of genes termed
pathogenesis-related (PR) genes [10,11].
PR genes probably have an important role in the defense
response of plants against fungal infections. The ‘antifungal protein’ strategy involves the constitutive expression in
transgenic plants of genes encoding proteins that have a
fungitoxic or fungistatic capacity and therefore enhance
resistance to fungal pathogens. Table 1 presents an
overview of the reports of transgenic plants showing
increased resistance obtained using the above approach.
The hydrolytic enzymes chitinase and β-1,3-glucanase,
which are capable of degrading the major cell-wall constituents (i.e. chitin and β-1,3-glucan) of most filamentous
fungi, have been extensively studied in plants. Expression
of a plant or bacterial chitinase gene in transgenic tobacco
was shown to enhance the resistance of plants to Rhizoctonia
solani, an endemic, chitinous, soilborne fungus that infects
numerous plant species [12,13]. Interestingly, similar transgenic tobacco plants showed no enhanced tolerance of
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Plant biotechnology
Table 1
Increased fungal resistance in transgenic plants.
Plant
Transgene(s)
Pathogen
Reference
Tobacco
PR-1a, SAR 8.2
Peronospora tabacina, Phytophthora
parasitica, Pythium
Phytophthora parasitica
Rhizoctonia solani
Rhizoctonia solani
Rhizoctonia solani
Rhizoctonia solani
Rhizoctonia solani
Rhizoctonia solani
Cercospora nicotianae
Alternaria longipes
[18]
[18]
[18]
[12]
[48]
[48]
[13]
[13]
[17]
[23]
Alternaria dauci
Alternaria radicina
Cercospora carotae
Erysiphe heracleï
(a)
(a)
(a)
(a)
Class III chitinase
Chi-I
Bean chitinase (CH5B)
Barley RIP
Serratia marcescens Chi-A
Barley Chi + Glu
Barley Chi + RIP
Rice Chi + alfalfa Glu
Radish Rs-AFP
Carrot
Tobacco Chi-I + Glu-I
Tobacco AP24
Tomato
Tobacco Chi-I + Glu-I
Fusarium oxysporum
[16]
Brassica napus
Bean chitinase
Tomato/tobacco chitinase
Rhizoctonia solani
Cylindrosporium conc.
Sclerotinia sclerotiorum
Phoma lingam
[12]
[29]
[29]
[29]
Potato
AP24
Glux-ox
Phytophthora infestans
Phytophthora infestans
Verticillium dahliae
Verticillium
[19]
[28]
[28]
[22]
Rice
Rice Chi
Rhizoctonia solani
[21]
Wheat
Aly AFP
Fusarium sp.
[22]
Aly AFP
(a) LS Melchers, unpublished data.
Cercospora nicotianae [14], indicating that results obtained in
vitro are difficult to extrapolate to the situation in vivo and
that tolerance to a range of pathogens can require more than
the simple over-expression of a single gene.
Researchers at Zeneca MOGEN were the first to demonstrate that the constitutive co-expression of tobacco
chitinase and β-1,3-glucanase genes in tomato plants confers higher levels of resistance to a fungal pathogen (i.e.
Fusarium oxysporum) than either gene alone, indicating a
synergistic interaction between the two enzymes in vivo
[15,16]. The effectiveness of this approach was further
demonstrated in tobacco, in which the simultaneous
expression of a rice chitinase and an alfalfa glucanase gene
yielded a substantially greater protection against the fungal
pathogen C. nicotianae than either transgene alone [17].
Similar effects were found after co-expression of genes for
class II chitinase, class II glucanase and a type I ribosomeinactivating protein (RIP) from barley in transgenic
tobacco [17]. Certain combinations (i.e. chitinase/glucanase and chitinase/RIP) provided ‘significantly
enhanced protection’ against Rhizoctonia solani. J Ryals’
group at Novartis has made a significant effort to evaluate
these and other PR proteins for their potential to provoke
disease resistance in transgenic tobacco plants (see
Table 1). Transgenic tobacco plants constitutively expressing PR1a, a protein with unknown biochemical function,
showed tolerance to infection by two oomycete pathogens,
Peronospora tabacina and Phytophthora parasitica [18];
although, once more, this resistance did not extend to
other pathogens tested. An interesting observation from
this work was that the apparent disease resistance of a
transgenic line was not correlated with the level of expression of the transgene. This is an observation that has been
made on several occasions in other reported work.
There are several other reports of enhanced tolerance
being conferred by the overexpression of a single gene.
Overexpression of tobacco osmotin, a PR-5 protein, in
potato significantly delayed the appearence of lesions
caused by Phytophthora infestans [19,20]. Interestingly,
recent studies with transgenic monocot crops, including
rice and wheat, have yielded the first promising results
relating to the development of resistance to Rhizoctonia
solani [21] and Fusarium sp. [22], respectively, in these economically most important crops.
In addition to PR proteins, a broad family of small antifungal peptides, termed plant defensins, has been discovered.
Several of these peptides have been isolated from the seeds
of a variety of plants and shown to possess antifungal activity in vitro against a broad spectrum of fungal pathogens.
The constitutive expression of Rs-AFP2 (a small cysteinerich plant defensin from radish) in tobacco conferred a high
level of resistance to the foliar pathogen Alternaria longipes
on plants that produced high levels of Rs-AFP2 [23].
Novel genes for disease-resistance breeding Melchers and Stuiver
149
Figure 1
Schematic drawing of defense responses
activated in a plant–pathogen interaction. A
pathogen secretes elicitor molecules that are
perceived via the plant resistance gene
products. The resulting HR encompasses
many different defense responses, including
induction of cell death, callose deposition at
the site of pathogen entry, and the production
of a number of hormones (e.g. salicyclic acid
[SA], jasmonic acid [JA] and ethylene), which
trigger production of PR-gene products. Some
of the hormones produced may move
systemically, leading to PR protein production
in the neighbouring cells. The boxed
components have all been used in attempts to
engineer disease resistance. Elicitors are used
in the avirulence gene approach (see text).
Resistance genes can be transformed into
plants, thereby increasing the plant’s ability to
recognise pathogenic invaders. Increased
production of active hormones to induce
(subsets of) PR genes is the subject of some
research, as is the over-expression of specific
PR genes (see text). Additionally, the potential
for the engineering of phytoalexin production
and the induction of localized pathogentriggered cell death is being explored.
Pathogen
Elicitors
HR
SA
JA
Resistance
gene
Cell death
Ethylene
PR-genes
Callose
Phytoalexins
Ethylene
SA
PR-genes
Plant cell
Other examples of plant antifungal peptides include thionins from monocots and dicots, hevein (a lectin from Urtica
dioica) and lipid transfer proteins from various plant species
([24] and references therein). The first reports of the efficacy of these peptides in controlling fungal diseases were
published recently. Epple and co-workers [25] demonstrated increased protection of Arabidopsis against Fusarium
oxysporum by the overexpression of an endogenous thionin
(Thi2.1 gene). Interestingly, the induction of the endogenous gene for Thi2.1 after pathogen attack is independent
of PR-gene expression, suggesting that these defenses are
activated through independent pathways. Molina and
Garcia-Olmedo [26] reported that the expression of a barley
non-specific lipid-transfer protein in Arabidopsis and tobacco conferred enhanced resistance to the bacterial pathogen
Pseudomonas syringae. These proteins have also been shown
to demonstrate antifungal effects in vitro.
Finally, there are a number of antifungal proteins that do
not fall into any of the classes described above. For example, the H2O2-producing enzyme oxalate oxidase has been
shown to accumulate in barley that is attacked by powdery
mildew, Erysiphe graminis [27]. Interestingly, Wu et al. [28]
demonstrated that constitutive expression in potato of
another H2O2-generating enzyme, glucose oxidase, provided disease resistance to a range of plant pathogens,
including Erwinia carotovora, Phytophthora infestans and
Verticillium wilt disease.
Most of the promising reports described above are based
solely on observations of the increased fungal resistance of
Current Opinion in Plant Biology
transgenic plants tested in climate-controlled growth chambers or greenhouse facilities. Now, the technical challenge is
to translate such results into a robust effect in the field. A
comprehensive field evaluation of transgenic carrots containing the tobacco class I chitinase and β-1,3-glucanase genes
(LS Melchers, MH Stuiver, unpublished data) has demonstrated that these plants have a high level of resistance to
major carrot pathogens including Alternaria dauci, Alternaria
radicina, Cercospora carotae and Erysiphe heracleï (powdery
mildew). Most of the transgenic carrot lines that were resistant to one pathogen exhibited significant resistance to all
four pathogens. The broad-spectrum fungal resistance in
transgenic carrot plants indicates a proof of the concept of the
‘antifungal gene’ strategy. In another field-testing study,
transgenic canola (Brassica napus) that constitutively
expressed a chimeric chitinase gene showed enhanced resistance to Cylindrosporium concentricum and, to a smaller degree,
Phoma lingam and Sclerotinia sclerotiorum after artificial inoculation with these pathogens [29].
Future studies will be necessary to identify combinations
of defense proteins that might confer effective broad-spectrum protection.
Hypersensitive-response-based strategies
The hypersensitive response (HR) is one of the most powerful mechanisms by which plants resist pathogen attack
(Figure 1). The genetically controlled induction of the HR is
triggered in plant–pathogen interactions only if the plant
contains a disease-resistance protein (R) that recognizes the
corresponding avirulence (Avr) protein from the pathogen. In
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Plant biotechnology
the absence of a functional resistance gene (R) or avirulence
gene product, no recognition occurs and the interaction
between plant and pathogen results in disease. Resistance
genes involved in race-specific interactions often provide full
disease resistance, and are well known from conventional
breeding programs. In recent years, many disease-resistance
genes have been cloned and have been shown to represent
several structurally different classes of gene.
The strength of the hypersensitive reaction makes it
exquisitely designed for combating a broad spectrum of
plant pathogens. The normal use of resistance genes does
not, however, allow such a broad spectrum of activity. The
exclusive specificity of HR-associated disease resistance
for specific pathogen-derived avirulence factors limits their
use to only one pathogen, and sometimes even a limited
number of races or pathotypes of the pathogen. The engineering of broad-spectrum disease resistance relying on
the HR is therefore highly desirable.
The interesting so called ‘Avr/R strategy’ proposed by P de
Wit [7] involves the transfer of an avirulence gene (i.e. the
Cladosporium fulvum avr9 gene) into a plant containing the
corresponding resistance gene (i.e. the tomato Cf9 gene),
and its subsequent expression under the direction of a promoter that is rapidly and locally inducible by a wide range
of fungal pathogens. Pathogen-induced expression of the
avr9 gene will then provoke a resistance reaction manifested by a hypersensitive response. A localized HR will
be induced that prevents the further spread of any invading pathogen that can be inhibited by an HR, followed by
a general defense response. Using this technology we have
shown in collaboration with de Wit and co-workers that
tomato plants can be generated with increased resistance
to a broad-spectrum of diseases: resistance to multiple
fungi with different infection modes, and also to viruses,
was observed (MH Stuiver et al., unpublished data). Along
similar lines, a broad-spectrum responsive HR-induction
system in tobacco plants has been generated [30••].
With the continuing elucidation of the signal transduction
pathways invloved in plant defense responses, more and
more molecular components are becoming available that
can be used as tools to induce the hypersensitive response
either completely or partially [31]. Karrer et al. [32•] have
used a search for tobacco proteins that, when overexpressed, are able to induce necrosis or an HR [32•]; these
are interesting starting points. Further understanding of
the molecular mechanism responsible for the resistance
mediated by the Avr-R genes is critical for establishing
durable resistance in genetically engineered crops. In addition, the elucidation of signal transduction components
functioning downstream of R genes opens avenues for
engineered resistance.
Hypersensitive response signalling
Genetic analysis of HR signalling has revealed a number of
key steps in the signal transduction of the HR. One such
step involves a small number of protein molecules that
function downstream of a very large number of resistance
genes [33]. This convergence is probably the result of
shared signal-transduction cascades leading to the
execution of the processes during the HR. The Arabidopsisderived gene products Ndr1 and Eds1 [34] are good
candidates for the induction of downstream HR defense
responses. Similarly, in the barley–powdery mildew interaction a large number of R genes feed into one central
signalling molecule, Rar1 [35]. The exact function of none
of these proteins is known. Eds1 resembles a lipase, suggesting that lipid signalling may be activated, whereas Ndr1
can be activated by mutations resembling Ca2+-dependent
protein kinase (CDPK)-phosphorylation (MH Stuiver et al.,
unpublished data), suggesting that it functions downstream
of the Ca2+ influx into the cytoplasm that has been
observed in many HR-inducing systems [36].
Besides protein–protein signalling, the generation of second messengers involved in the transduction of the HR
response, such as the above-mentioned Ca2+ ions and
lipids, may serve as a starting point for engineering resistance; so, too, might extracellular nitric oxide [37••] and
reactive oxygen species [31,38,39••], which seem to be
important in mediating at least part of the defense responses, and which, when applied together, might signal
processes downstream of HR induction. The generation of
reactive oxygen species by other means might also induce
systemic acquired resistance [40].
Further downstream in the execution of the HR
response, there is a probable divergence of signals, in
which the induction of cell death is likely to be separated
from other aspects of the induction of the HR, such as
callose deposition, the accumulation of phenolics and the
induction of PR proteins. The hormones salicyclic acid,
jasmonic acid and ethylene are important in the induction
of PR proteins in the HR and also in normal defense
responses triggered in the compatible interaction. These
multiple responses are involved in combating pathogen
infection [41–45]. It is likely that the coordinated or separate activation of these PR-protein induction pathways
[46] leads to a general increased level of resistance. It is
envisaged, however, that the level of control might be
limited in the strength of the resistance and/or the scope
of pathogens inhibited.
Future prospects
The field of plant disease control is undergoing an exciting
period, during which major breakthroughs in elucidating
the molecular basis of resistance to a wide range of plant
pathogens are being made [47]. Improved understanding
of both plant defense mechanisms and pathogen action has
initiated the worldwide development of transgenic strategies for improving the resistance of plants to pathogens. In
contrast with the great success in the production of commercially useful insect- and virus-resistant crops, the
production of fungus-resistant crops with commercially
Novel genes for disease-resistance breeding Melchers and Stuiver
useful levels of resistance has not yet been achieved. The
increasing number of reports of broad-spectrum resistance
to fungi in different transgenic crops, however, indicates
that commercial introductions of fungus-resistant crops can
be expected within the next 4–8 years.
The growing global demand for increased food production
and consumers’ need for high-quality food present considerable challenges to scientists in industry, academia and
government institutions. Fungicides are expected to continue to form the main strategy permitting growers to
achieve high levels of disease control and hence improve
crop yields in the near future. Continuous agrochemical
research has developed a range of modern fungicides with
new modes of action that provide broad-spectrum disease
control in economically important crops. It is now clear that
the introduction of a single transgene is not sufficient to
achieve durable and broad-spectrum disease resistance. A
combination of transgenic strategies will therefore be
needed to reduce the requirement for agrochemicals to
control crop diseases. Future research into disease resistance will focus on the successful integration of transgenic
strategies into breeding programs to develop the durable
resistance of new commercial varieties, in combination
with a selective use of low doses of fungicides. In the
longer term it is envisaged that farmers will achieve optimal disease control in individual crops by balancing the
use of both transgenes and fungicides through integrated
crop management.
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Arabidopsis. Proc Natl Acad Sci USA 1998, 95:10306-10311.
45. Penninckx IA, Thomma BP, Buchala A, Métraux JP, Broekaert WF:
Concomitant activation of jasmonate and ethylene response
pathways is required for induction of a plant defensin gene.
Plant Cell 1998, 10:2103-2113.
35. Lahaye T, Shirasu K, Schulze-Lefert P: Chromosome landing at the
barley Rar1 locus. Mol Gen Genet 1998, 260:92-101.
46. Fraissinet-Tachet L, Baltz R, Chong J, Kaufmann S, Fritig B,
Saindrenan P: Two tobacco genes induced by infection, elicitor
and salicylic acid encode glucosyltransferases acting on
phenylpropanoids and benzoic acid derivatives, including salicylic
acid. FEBS Lett 1998, 437:319-323.
36. Scheel D: Resistance response physiology and signal
transduction. Curr Opin Plant Biol 1998, 4:305-310.
37. Delledonne M, Xia Y, Dixon RA, Lamb C: Nitric oxide functions as a
•• signal in plant disease resistance. Nature 1998, 394:585-588.
A classic paper describing the central role of nitric oxide in plant defence
and the HR using experiments on soybean suspension cells and intact
Arabidopsis leaves.
38. Keller T, Demude HG, Werner D, Doerner P, Dixon RA, Lamb C:
A plant homolog of the neutrophil NADPH oxidase gp91phox
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Dangl J, Jones JD: Plant–microbe interactions. Affairs of the plant:
colonization, intolerance, exploitation and co-operation in
plant–microbe interactions. Curr Opin Plant Biol 1998, 1:285-287.
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barley ribosome inactivating protein leads to increased fungal
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10:305-308.
Novel genes for disease-resistance breeding
Leo S Melchers* and Maarten H Stuiver
Plant disease control is entering an exciting period during
which transgenic plants showing improved resistance to
pathogenic viruses, bacteria, fungi and insects are being
developed. This review summarizes the first successful
attempts to engineer fungal resistance in crops, and highlights
two promising approaches. Biotechnology provides the
promise of new integrated disease management strategies that
combine modern fungicides and transgenic crops to provide
effective disease control for modern agriculture.
Addresses
Zeneca MOGEN, PO Box 628, 2300 AP Leiden, The Netherlands;
*e-mail: Leo.Melchers@ageurope.zeneca.com
Current Opinion in Plant Biology 2000, 3:147–152
1369-5266/00/$ — see front matter © 2000 Elsevier Science Ltd.
All rights reserved.
Abbreviations
Avr
avirulence
HR
hypersensitive response
PR
pathogenesis-related
R
resistance gene
transformation and advanced molecular techniques for
plant breeding provides powerful tools for genetically
improving crops so as to enhance their resistance to fungal
diseases. The distinct advantage of transgenic technology
is that it enables the plant breeder to cross species barriers,
allowing genes from non-related plants and other organisms to be introduced into crop plants.
In this review, we discuss the current status of engineered
resistance to fungal pathogens in crops and highlight two
highly promising approaches for generating broad-spectrum fungus-resistant crops [6]. The first strategy relies on
the expression of genes that encode proteins which have
either a direct or indirect inhibitory effect on the growth of
fungi (i.e. the antifungal protein strategy); the second strategy aims to increase fungal resistance in plants by
engineering controlled hypersensitive death of host cells
on the basis of the specific interaction of the product of the
avirulence gene (Avr) and that of the resistance gene (R)
(i.e. Avr/R two-component strategy) [7].
Antifungal proteins
Introduction
Protection against attack by detrimental micro-organisms
and pests is a major challenge for crop production. Fungal
diseases have been one of the main causes of crop losses ever
since mankind started to cultivate plants [1]. At present, the
control of the epidemic spread of fungal diseases mainly
involves three strategies: first, husbandry techniques, such as
crop rotation and avoiding the spread of infested soil and
pathogen-contaminated plant materials; second, breeding of
resistant crop cultivars; and third, the application of agrochemicals. Although conventional plant breeding has made a
significant impact by improving the resistance of many crops
to important diseases, the time-consuming processes of making crosses and back-crosses, and the selection of the desired
resistant progeny make it difficult to react adequately to the
evolution of new virulent fungal races [2,3]. Moreover, these
plant breeding techniques will not provide a solution to
many major fungal diseases because there are simply no natural sources of resistance available to the breeder.
Farmers, therefore, often use fungicides for crop protection in modern agriculture. The use of these compounds is,
however, limited by their high costs and potentially harmful impact on the environment [4,5]. In addition, extended
application can reduce the efficiency of fungicides because
of the evolution of tolerant or resistant pathogens. The
growing concern about the environment, together with a
strong motivation to decrease production costs, encourages
the development of crop cultivars that require less chemicals and/or fewer types of chemical. The advent of plant
Plants have several inducible defense mechanisms that act
to limit pathogen infection, including increased lignification and cell wall cross-linking, the production of small
antibiotic molecules (i.e. phytoalexins), host cell death at
the site of infection (i.e. the hypersensitive response; [8]
and references therein), and the production of reactive
oxygen species [9]. Many plants also develop an increased
resistance to subsequent pathogen infection in uninfected
tissues. This systemic acquired resistance (SAR) can be
effective against viruses, bacteria and fungi, and is accompanied by the expression of a large set of genes termed
pathogenesis-related (PR) genes [10,11].
PR genes probably have an important role in the defense
response of plants against fungal infections. The ‘antifungal protein’ strategy involves the constitutive expression in
transgenic plants of genes encoding proteins that have a
fungitoxic or fungistatic capacity and therefore enhance
resistance to fungal pathogens. Table 1 presents an
overview of the reports of transgenic plants showing
increased resistance obtained using the above approach.
The hydrolytic enzymes chitinase and β-1,3-glucanase,
which are capable of degrading the major cell-wall constituents (i.e. chitin and β-1,3-glucan) of most filamentous
fungi, have been extensively studied in plants. Expression
of a plant or bacterial chitinase gene in transgenic tobacco
was shown to enhance the resistance of plants to Rhizoctonia
solani, an endemic, chitinous, soilborne fungus that infects
numerous plant species [12,13]. Interestingly, similar transgenic tobacco plants showed no enhanced tolerance of
148
Plant biotechnology
Table 1
Increased fungal resistance in transgenic plants.
Plant
Transgene(s)
Pathogen
Reference
Tobacco
PR-1a, SAR 8.2
Peronospora tabacina, Phytophthora
parasitica, Pythium
Phytophthora parasitica
Rhizoctonia solani
Rhizoctonia solani
Rhizoctonia solani
Rhizoctonia solani
Rhizoctonia solani
Rhizoctonia solani
Cercospora nicotianae
Alternaria longipes
[18]
[18]
[18]
[12]
[48]
[48]
[13]
[13]
[17]
[23]
Alternaria dauci
Alternaria radicina
Cercospora carotae
Erysiphe heracleï
(a)
(a)
(a)
(a)
Class III chitinase
Chi-I
Bean chitinase (CH5B)
Barley RIP
Serratia marcescens Chi-A
Barley Chi + Glu
Barley Chi + RIP
Rice Chi + alfalfa Glu
Radish Rs-AFP
Carrot
Tobacco Chi-I + Glu-I
Tobacco AP24
Tomato
Tobacco Chi-I + Glu-I
Fusarium oxysporum
[16]
Brassica napus
Bean chitinase
Tomato/tobacco chitinase
Rhizoctonia solani
Cylindrosporium conc.
Sclerotinia sclerotiorum
Phoma lingam
[12]
[29]
[29]
[29]
Potato
AP24
Glux-ox
Phytophthora infestans
Phytophthora infestans
Verticillium dahliae
Verticillium
[19]
[28]
[28]
[22]
Rice
Rice Chi
Rhizoctonia solani
[21]
Wheat
Aly AFP
Fusarium sp.
[22]
Aly AFP
(a) LS Melchers, unpublished data.
Cercospora nicotianae [14], indicating that results obtained in
vitro are difficult to extrapolate to the situation in vivo and
that tolerance to a range of pathogens can require more than
the simple over-expression of a single gene.
Researchers at Zeneca MOGEN were the first to demonstrate that the constitutive co-expression of tobacco
chitinase and β-1,3-glucanase genes in tomato plants confers higher levels of resistance to a fungal pathogen (i.e.
Fusarium oxysporum) than either gene alone, indicating a
synergistic interaction between the two enzymes in vivo
[15,16]. The effectiveness of this approach was further
demonstrated in tobacco, in which the simultaneous
expression of a rice chitinase and an alfalfa glucanase gene
yielded a substantially greater protection against the fungal
pathogen C. nicotianae than either transgene alone [17].
Similar effects were found after co-expression of genes for
class II chitinase, class II glucanase and a type I ribosomeinactivating protein (RIP) from barley in transgenic
tobacco [17]. Certain combinations (i.e. chitinase/glucanase and chitinase/RIP) provided ‘significantly
enhanced protection’ against Rhizoctonia solani. J Ryals’
group at Novartis has made a significant effort to evaluate
these and other PR proteins for their potential to provoke
disease resistance in transgenic tobacco plants (see
Table 1). Transgenic tobacco plants constitutively expressing PR1a, a protein with unknown biochemical function,
showed tolerance to infection by two oomycete pathogens,
Peronospora tabacina and Phytophthora parasitica [18];
although, once more, this resistance did not extend to
other pathogens tested. An interesting observation from
this work was that the apparent disease resistance of a
transgenic line was not correlated with the level of expression of the transgene. This is an observation that has been
made on several occasions in other reported work.
There are several other reports of enhanced tolerance
being conferred by the overexpression of a single gene.
Overexpression of tobacco osmotin, a PR-5 protein, in
potato significantly delayed the appearence of lesions
caused by Phytophthora infestans [19,20]. Interestingly,
recent studies with transgenic monocot crops, including
rice and wheat, have yielded the first promising results
relating to the development of resistance to Rhizoctonia
solani [21] and Fusarium sp. [22], respectively, in these economically most important crops.
In addition to PR proteins, a broad family of small antifungal peptides, termed plant defensins, has been discovered.
Several of these peptides have been isolated from the seeds
of a variety of plants and shown to possess antifungal activity in vitro against a broad spectrum of fungal pathogens.
The constitutive expression of Rs-AFP2 (a small cysteinerich plant defensin from radish) in tobacco conferred a high
level of resistance to the foliar pathogen Alternaria longipes
on plants that produced high levels of Rs-AFP2 [23].
Novel genes for disease-resistance breeding Melchers and Stuiver
149
Figure 1
Schematic drawing of defense responses
activated in a plant–pathogen interaction. A
pathogen secretes elicitor molecules that are
perceived via the plant resistance gene
products. The resulting HR encompasses
many different defense responses, including
induction of cell death, callose deposition at
the site of pathogen entry, and the production
of a number of hormones (e.g. salicyclic acid
[SA], jasmonic acid [JA] and ethylene), which
trigger production of PR-gene products. Some
of the hormones produced may move
systemically, leading to PR protein production
in the neighbouring cells. The boxed
components have all been used in attempts to
engineer disease resistance. Elicitors are used
in the avirulence gene approach (see text).
Resistance genes can be transformed into
plants, thereby increasing the plant’s ability to
recognise pathogenic invaders. Increased
production of active hormones to induce
(subsets of) PR genes is the subject of some
research, as is the over-expression of specific
PR genes (see text). Additionally, the potential
for the engineering of phytoalexin production
and the induction of localized pathogentriggered cell death is being explored.
Pathogen
Elicitors
HR
SA
JA
Resistance
gene
Cell death
Ethylene
PR-genes
Callose
Phytoalexins
Ethylene
SA
PR-genes
Plant cell
Other examples of plant antifungal peptides include thionins from monocots and dicots, hevein (a lectin from Urtica
dioica) and lipid transfer proteins from various plant species
([24] and references therein). The first reports of the efficacy of these peptides in controlling fungal diseases were
published recently. Epple and co-workers [25] demonstrated increased protection of Arabidopsis against Fusarium
oxysporum by the overexpression of an endogenous thionin
(Thi2.1 gene). Interestingly, the induction of the endogenous gene for Thi2.1 after pathogen attack is independent
of PR-gene expression, suggesting that these defenses are
activated through independent pathways. Molina and
Garcia-Olmedo [26] reported that the expression of a barley
non-specific lipid-transfer protein in Arabidopsis and tobacco conferred enhanced resistance to the bacterial pathogen
Pseudomonas syringae. These proteins have also been shown
to demonstrate antifungal effects in vitro.
Finally, there are a number of antifungal proteins that do
not fall into any of the classes described above. For example, the H2O2-producing enzyme oxalate oxidase has been
shown to accumulate in barley that is attacked by powdery
mildew, Erysiphe graminis [27]. Interestingly, Wu et al. [28]
demonstrated that constitutive expression in potato of
another H2O2-generating enzyme, glucose oxidase, provided disease resistance to a range of plant pathogens,
including Erwinia carotovora, Phytophthora infestans and
Verticillium wilt disease.
Most of the promising reports described above are based
solely on observations of the increased fungal resistance of
Current Opinion in Plant Biology
transgenic plants tested in climate-controlled growth chambers or greenhouse facilities. Now, the technical challenge is
to translate such results into a robust effect in the field. A
comprehensive field evaluation of transgenic carrots containing the tobacco class I chitinase and β-1,3-glucanase genes
(LS Melchers, MH Stuiver, unpublished data) has demonstrated that these plants have a high level of resistance to
major carrot pathogens including Alternaria dauci, Alternaria
radicina, Cercospora carotae and Erysiphe heracleï (powdery
mildew). Most of the transgenic carrot lines that were resistant to one pathogen exhibited significant resistance to all
four pathogens. The broad-spectrum fungal resistance in
transgenic carrot plants indicates a proof of the concept of the
‘antifungal gene’ strategy. In another field-testing study,
transgenic canola (Brassica napus) that constitutively
expressed a chimeric chitinase gene showed enhanced resistance to Cylindrosporium concentricum and, to a smaller degree,
Phoma lingam and Sclerotinia sclerotiorum after artificial inoculation with these pathogens [29].
Future studies will be necessary to identify combinations
of defense proteins that might confer effective broad-spectrum protection.
Hypersensitive-response-based strategies
The hypersensitive response (HR) is one of the most powerful mechanisms by which plants resist pathogen attack
(Figure 1). The genetically controlled induction of the HR is
triggered in plant–pathogen interactions only if the plant
contains a disease-resistance protein (R) that recognizes the
corresponding avirulence (Avr) protein from the pathogen. In
150
Plant biotechnology
the absence of a functional resistance gene (R) or avirulence
gene product, no recognition occurs and the interaction
between plant and pathogen results in disease. Resistance
genes involved in race-specific interactions often provide full
disease resistance, and are well known from conventional
breeding programs. In recent years, many disease-resistance
genes have been cloned and have been shown to represent
several structurally different classes of gene.
The strength of the hypersensitive reaction makes it
exquisitely designed for combating a broad spectrum of
plant pathogens. The normal use of resistance genes does
not, however, allow such a broad spectrum of activity. The
exclusive specificity of HR-associated disease resistance
for specific pathogen-derived avirulence factors limits their
use to only one pathogen, and sometimes even a limited
number of races or pathotypes of the pathogen. The engineering of broad-spectrum disease resistance relying on
the HR is therefore highly desirable.
The interesting so called ‘Avr/R strategy’ proposed by P de
Wit [7] involves the transfer of an avirulence gene (i.e. the
Cladosporium fulvum avr9 gene) into a plant containing the
corresponding resistance gene (i.e. the tomato Cf9 gene),
and its subsequent expression under the direction of a promoter that is rapidly and locally inducible by a wide range
of fungal pathogens. Pathogen-induced expression of the
avr9 gene will then provoke a resistance reaction manifested by a hypersensitive response. A localized HR will
be induced that prevents the further spread of any invading pathogen that can be inhibited by an HR, followed by
a general defense response. Using this technology we have
shown in collaboration with de Wit and co-workers that
tomato plants can be generated with increased resistance
to a broad-spectrum of diseases: resistance to multiple
fungi with different infection modes, and also to viruses,
was observed (MH Stuiver et al., unpublished data). Along
similar lines, a broad-spectrum responsive HR-induction
system in tobacco plants has been generated [30••].
With the continuing elucidation of the signal transduction
pathways invloved in plant defense responses, more and
more molecular components are becoming available that
can be used as tools to induce the hypersensitive response
either completely or partially [31]. Karrer et al. [32•] have
used a search for tobacco proteins that, when overexpressed, are able to induce necrosis or an HR [32•]; these
are interesting starting points. Further understanding of
the molecular mechanism responsible for the resistance
mediated by the Avr-R genes is critical for establishing
durable resistance in genetically engineered crops. In addition, the elucidation of signal transduction components
functioning downstream of R genes opens avenues for
engineered resistance.
Hypersensitive response signalling
Genetic analysis of HR signalling has revealed a number of
key steps in the signal transduction of the HR. One such
step involves a small number of protein molecules that
function downstream of a very large number of resistance
genes [33]. This convergence is probably the result of
shared signal-transduction cascades leading to the
execution of the processes during the HR. The Arabidopsisderived gene products Ndr1 and Eds1 [34] are good
candidates for the induction of downstream HR defense
responses. Similarly, in the barley–powdery mildew interaction a large number of R genes feed into one central
signalling molecule, Rar1 [35]. The exact function of none
of these proteins is known. Eds1 resembles a lipase, suggesting that lipid signalling may be activated, whereas Ndr1
can be activated by mutations resembling Ca2+-dependent
protein kinase (CDPK)-phosphorylation (MH Stuiver et al.,
unpublished data), suggesting that it functions downstream
of the Ca2+ influx into the cytoplasm that has been
observed in many HR-inducing systems [36].
Besides protein–protein signalling, the generation of second messengers involved in the transduction of the HR
response, such as the above-mentioned Ca2+ ions and
lipids, may serve as a starting point for engineering resistance; so, too, might extracellular nitric oxide [37••] and
reactive oxygen species [31,38,39••], which seem to be
important in mediating at least part of the defense responses, and which, when applied together, might signal
processes downstream of HR induction. The generation of
reactive oxygen species by other means might also induce
systemic acquired resistance [40].
Further downstream in the execution of the HR
response, there is a probable divergence of signals, in
which the induction of cell death is likely to be separated
from other aspects of the induction of the HR, such as
callose deposition, the accumulation of phenolics and the
induction of PR proteins. The hormones salicyclic acid,
jasmonic acid and ethylene are important in the induction
of PR proteins in the HR and also in normal defense
responses triggered in the compatible interaction. These
multiple responses are involved in combating pathogen
infection [41–45]. It is likely that the coordinated or separate activation of these PR-protein induction pathways
[46] leads to a general increased level of resistance. It is
envisaged, however, that the level of control might be
limited in the strength of the resistance and/or the scope
of pathogens inhibited.
Future prospects
The field of plant disease control is undergoing an exciting
period, during which major breakthroughs in elucidating
the molecular basis of resistance to a wide range of plant
pathogens are being made [47]. Improved understanding
of both plant defense mechanisms and pathogen action has
initiated the worldwide development of transgenic strategies for improving the resistance of plants to pathogens. In
contrast with the great success in the production of commercially useful insect- and virus-resistant crops, the
production of fungus-resistant crops with commercially
Novel genes for disease-resistance breeding Melchers and Stuiver
useful levels of resistance has not yet been achieved. The
increasing number of reports of broad-spectrum resistance
to fungi in different transgenic crops, however, indicates
that commercial introductions of fungus-resistant crops can
be expected within the next 4–8 years.
The growing global demand for increased food production
and consumers’ need for high-quality food present considerable challenges to scientists in industry, academia and
government institutions. Fungicides are expected to continue to form the main strategy permitting growers to
achieve high levels of disease control and hence improve
crop yields in the near future. Continuous agrochemical
research has developed a range of modern fungicides with
new modes of action that provide broad-spectrum disease
control in economically important crops. It is now clear that
the introduction of a single transgene is not sufficient to
achieve durable and broad-spectrum disease resistance. A
combination of transgenic strategies will therefore be
needed to reduce the requirement for agrochemicals to
control crop diseases. Future research into disease resistance will focus on the successful integration of transgenic
strategies into breeding programs to develop the durable
resistance of new commercial varieties, in combination
with a selective use of low doses of fungicides. In the
longer term it is envisaged that farmers will achieve optimal disease control in individual crops by balancing the
use of both transgenes and fungicides through integrated
crop management.
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