Download Transgenic approaches to microbial disease resistance in crop

347
Transgenic approaches to microbial disease resistance in crop
plants
John M Salmeron and Bernard Vernooij
Recent progress in the genetic dissection of plant disease
resistance signaling pathways has opened a number of
new avenues towards engineering pathogen resistance in
crops. Genes controlling race-specific and broad-spectrum
resistance responses have been cloned, and novel induced
resistance pathways have been identified in model and crop
systems. Advances continue to be made in identification of
antifungal proteins with effects inhibitory to either pathogen
development or accumulation of associated mycotoxins.
Address
Novartis Agribusiness Biotechnology Research Inc., 3054 Cornwallis
Road, Research Triangle Park, NC 27709, USA
Current Opinion in Plant Biology 1998, 1:347–352
number of these genes has shown that R-gene products share motifs such as leucine-rich repeats (LRRs),
nucleotide binding sites, and kinase domains, consistent
with their roles in signaling pathways and suggesting
common mechanisms for pathogen recognition which
might be exploited for applied goals [1]. Unfortunately, the
instability of most R-genes, along with their highly-specific
activities precludes application for most of them in the
field. Exceptions may include the Hs1-Pro gene from
sugarbeet, which provides an effective source for control
of cyst nematode [2•]. Other R-genes exhibit a broader
spectrum of activity against a given pathogen species.
For example, the Xa21 gene from rice exhibits activity
against 29 distinct races of the bacterial blight pathogen
Xanthomonas oryzae [3].
http://biomednet.com/elecref/1369526600100347
 Current Biology Ltd ISSN 1369-5266
Abbreviations
AFP
antifungal protein
ISR
induced systemic resistance
LRR
leucine-rich repeats
PR
pathogenesis related
R
resistance
SA
salicylic acid
SAR
systemic acquired resistance
Introduction
Diseases caused by bacteria and fungi are currently some
of the major factors limiting crop production worldwide.
In addition to negative effects on yield, diseases can also
impact the post-harvest quality of food. For reasons of
cost, efficacy and environmental concerns, much research
is presently aimed at transgenic expression of genes
that can confer significant levels of disease resistance.
Although recent introductions of plant products for control
of insect pests have been highly successful, transgenic
plants exhibiting resistance to fungal or bacterial diseases
have yet to reach the marketplace. In this review,
we summarize research results with implications for
developing disease resistant transgenic crops. Due to
space limitations, we will focus on control of fungal and
bacterial diseases. Much work in this area centers on
understanding naturally-occuring plant signaling pathways
that control pathogen resistance, and on identifying and
cloning genes encoding antifungal proteins. We highlight
recent developments in both of these areas.
Engineering R-genes for novel resistance phenotypes
may be possible by identifying and modifying R-protein
domains that determine pathogen recognition. Cloning
of the flax M gene controlling rust resistance showed
that mutations leading to loss of R-gene function map
to the 3′ region encoding the LRR [4•]. Differences
between the Cf-9 and Cf-4 gene products from tomato,
controlling resistance to distinct races of leaf mold,
are almost exclusively localized to a region within the
LRR [5•]. Comparison of Cf-9 homologs from resistant
and susceptible plants reveals hypervariability in the
LRR-encoding region [6•]. In combination with future
structural studies, additional comparisons of mutant and
naturally-occuring R-gene alleles may provide insight into
directed tailoring of the LRR for specific pathogen control.
Genes downstream of R-genes
Data from a number of laboratories suggest that R-gene
products act early in signal transduction, perhaps at the
level of initial pathogen perception [7,8]. Genes that
control downstream steps shared by multiple R-gene
pathways would be attractive targets for disease resistance
engineering, and recently a number of these genes have
been identified. The Arabidopsis eds1 (enhanced disease
susceptibility) mutant was isolated through loss of resistance to Peronospora parasitica isolate Noco2 mediated by
the R-gene RPP14 [9]. Further analysis showed that eds1
actually blocks resistance mediated by at least four distinct
Peronospora resistance genes [9]. In contrast, activity of the
Arabidopsis RPM1 gene controlling race-specific resistance
to Pseudomonas pathogens is unaffected by eds1 [9].
Plant disease resistance signaling pathways
R-genes
Much progress has recently been made in understanding
race-specific pathogen resistance controlled by single
dominant resistance (R) genes. The cloning of a large
Other Arabidopsis mutations do affect resistance to Pseudomonas pathogens. While some of these mutations
appear to specifically alter resistance to Pseudomonas [10],
others block resistance to other bacterial phytopathogens
348
Plant–microbe interactions
[10], and some block resistance to both bacterial and
fungal pathogens [11]. One gene controlling resistance to
both bacterial and fungal pathogens, NDR1 (non-specific
disease resistance), has been cloned [12•] and encodes a
putative transmembrane protein. Similarly, mutations in
the PAD4 gene, controlling production of the phytoalexin
camalexin, cause increased susceptibility to bacteria and
fungi [13•]. These genes are very interesting and likely
control steps common to numerous R-gene mediated
signaling pathways.
these genes for engineering of disease resistant crops may
depend upon the ability to separate lesion and disease
resistance phenotypes. Work with some Arabidopsis lsd
mutants suggests that this may be possible [26], and,
most promisingly, recent cloning of some of these genes
opens the door for molecular approaches [22•,24•]. Already,
the necrotic phenotype associated with disease-resistant
barley lines carrying mutations in the Mlo powdery mildew
resistance gene (see below) has been managed through
breeding to allow deployment of these lines in the
field [27].
Systemic acquired resistance
A number of general resistance mechanisms in plants are
inducible by biotic or abiotic agents, and the best studied
of these is systemic acquired resistance (SAR). SAR
is a broad-spectrum resistance, inducible by necrotizing
pathogens or by treatment with chemicals such as salicylic
acid (SA) [14]. SAR leads to induction of a family
of defense genes (pathogenesis related [PR] genes)
thought collectively to confer the observed resistance
to bacterial, fungal and viral pathogens [14]. Genetic
analysis of SAR in Arabidopsis led to the cloning of
the NIM1/NPR1 (noninducible immunity/nonexpressor of
PR) gene, mutations in which abolish SAR induction
[15••,16••]. The NIM1/NPR1 protein shows similarity to
the NF-κB and I-κB factors controlling numerous cellular
responses in mammalian systems, consistent with a key
regulatory role [15••,16••]. Understanding the function of
NIM1/NPR1 and its homologs will be fruitful in elucidating
the mechanism of SAR establishment in Arabidopsis and in
crop plants.
Other Arabidopsis mutants exhibit SAR constitutively.
These so-called cim (constitutive immunity) or cpr (constitutive expressors of PR genes) mutants display high levels
of PR gene expression, and broad-spectrum pathogen
resistance [14,17••]. Although provocative, the pleiotropic
effects often associated with these mutants, such as
reduced size or altered morphology (e.g., [17••]), suggest
that incorporation of cim alleles into crop plants may
result in yield penalties. On the other hand, application
of the SAR-inducing compound benzothiadiazole leads
to protection of wheat against powdery mildew, without
apparent side effects [18]. Constitutive SAR is also
observed in plants expressing certain transgenes. For
example, potatoes expressing the bacterioopsin gene
exhibit systemic necrosis, triggering SAR-like responses
that cause resistance to several pathogens [19]. Similarly,
plants expressing glucose oxidase produce active oxygen
species, which appear to induce SAR as evidenced by
the activation of PR genes in potato [20]. Expression
of an inactive pokeweed antiviral protein induces fungal
disease resistance in tobacco, concomitant with PR protein
expression [21].
Novel induced resistance pathways
An important focus of future research will be the
identification of additional pathways controlling pathogen
resistance. Since a hallmark of SAR is its dependence
upon SA accumulation [15••], testing the function of a
pathway in the presence of the SA-catabolizing enzyme
salicylate hydroxylase can be informative. For example,
the cpr5 mutant of Arabidopsis displays constitutive PR
gene expression and disease resistance, and while bacterial
resistance in the mutant is SA-dependent, resistance
to P. parasitica is unaffected [18]. PR gene expression
and pathogen resistance can be induced in tobacco or
Arabidopsis by root inoculation of Pseudomonas biocontrol
strains in a process known as induced systemic resistance
(ISR), and this process is also SA-independent [28•]. The
cloning of genes such as CPR5 and those controlling ISR
will prove highly interesting.
Other pathways lead to expression of defense genes other
than PR genes. For example, infection of Arabidopsis
with necrotrophs such as Alternaria brassicola leads to
induction of thionin and defensin-like genes such as
PDF1.2, but does not result in PR-1 induction [28•]. As
might be expected, PDF1.2 induction is SA-independent
[29]. Overexpression of the thionin gene in Arabidopsis
leads to partial resistance against Fusarium oxysporum [30•],
indicating that non-SAR pathways may be useful for
disease resistance engineering.
Recently the Mlo gene controlling resistance to powdery
mildew in barley was cloned [31••]. Mutations in Mlo
confer resistance that is not correlated with constitutive
expression of the PR gene PR-1 [32•]. Resistance based on
mlo has been introduced into modern barley cultivars and
has proven durable in the field for more than twenty years
[27]. Interestingly, a mutation designated edr1 (enhanced
disease resistance) has been reported in Arabidopsis that
shares similarities in phenotype to mlo [33•]. The Mlo and
EDR1 genes should provide keys to understanding novel
disease resistance pathways in both monocots and dicots.
Transgenic expression of antifungal proteins
Hydrolytic enzymes
Lesion mimic plant mutants, such as Arabidopsis lsd, acd
and maize Lls, exhibit enhanced PR gene expression, and
often display disease resistance [22•,23,24•,25]. Harnessing
Whereas signaling pathways present novel opportunities
for disease management, transgenic expression of proteins
with antimicrobial activity in vitro has been studied as a
Transgenic approaches to microbial disease resistance in crop plants Salmeron and Vernooij
means to achieve increased disease resistance for many
years. The enzymatically active antimicrobial proteins
include chitinases, glucanases and lysozymes. Chitinases
and glucanases are capable of degrading fungal cell wall
components, and in vitro, some of these enzymes display
strong antifungal activities [34]. Genes for these and other
enzymes have been introduced into transgenic plants,
with varying rates of success. As an illustrative example,
transgenic carrots expressing a particular basic chitinase
from tobacco showed enhanced resistance to three out
of five tested pathogens, but no increased resistance was
detected when the chitinase was derived from petunia or
when any one of three chitinases (including the tobacco
chitinase) was expressed in transgenic cucumber [35].
Thus, it appears that the nature of the recipient plants,
the source of the chitinase gene, and the specific pathogen
tested influence whether or not resistance is achieved [35].
Certainly, the differing levels of antifungal activities
exhibited by chitinases in vitro, and the observation that
some chitinases have lysozyme activity, raises questions
about their specificity and specific activity. By using
a fungally derived chitinase, one might assume to be
working with an enzyme optimized for degrading fungal
cell walls. Consistent with this notion, a Rhizopus chitinase
expressed in tobacco conferred resistance to a Sclerotinia
pathogen. No resistance was found to Botrytis cinerea [36],
however, indicating that this approach cannot be expected
to solve all disease problems.
Chitinases (and other antimicrobial proteins) are induced
in plants upon pathogen infection [37,38] and successful
pathogens of these plants must have evolved ways to
avoid inhibition by these enzymes. It could, therefore,
be unlikely that overexpression of individual endogenous
antimicrobial proteins in plants will impart increased
disease resistance. Indeed, when a number of tobacco
PR genes, including chitinases and glucanases, were
overexpressed in tobacco, only a few were able to
provide some increased level of resistance against tobacco
pathogens, and none provided complete resistance [39].
Another emerging theme is that, although antimicrobial enzymes may provide reduced susceptibility to a
pathogen, they do not result in complete pathogen
control. For instance, transgenic expression in tobacco of a
gene encoding lysozyme, capable of degrading chitin and
bacterial peptidoglycan in vitro, showed the plants initially
to be more resistant to Erysiphe chicoracearum [40]. The
severity of the disease symptoms in the transgenic plants,
however, were equal to those in wild-type, except that
they were delayed by a day. This scenario often appears
in studies of transgenic plants when disease progression
is assayed over time. In the absence of data over such
timecourses, a critical assessment of the efficacy of a given
transgene is difficult.
349
Other proteins with antifungal activity
Genes for numerous antifungal proteins (AFPs) have been
incorporated into transgenic plants. Plant-derived AFPs
include SAR gene products, thionins, and defensins from
seeds [38,41,42]. As with hydrolytic enzymes, the approach
has met with varying degrees of success. Thionins have
been well studied and provide a good case study. Barley
α-thionin expressed in transgenic tobacco was shown
to enhance disease resistance to Pseudomonas syringae
[43]. And, as mentioned previously, overexpression of an
endogenous thionin in Arabidopsis enhanced resistance to
the fungal pathogen Fusarium oxysporum [28•]. Multiple
other attempts to use thionins, however, have failed to give
disease resistance against a number of pathogens [41,43].
AFPs from sources outside the plant kingdom have
also been engineered into plants. These include small
peptides (typically 20–30 amino acids) with antimicrobial
activity derived from various vertebrate and invertebrate
sources [44]. One possible drawback of expression of
such foreign AFPs in plants could be lack of protein
stability, as exemplified by cecropin B. This peptide
is unstable in extracellular fluid, presumably due to
proteolytic degradation [45]. A single amino acid change
increased the half-life of the peptide significantly, and
expression of this mutant peptide in transgenic tobacco
resulted in a decrease in disease symptoms [46•].
In summary, it appears that the exact nature of the
recipient plant and the transgene may influence rates
of success in deployment of antimicrobial proteins. In
general, the data indicate that transgenic plants overexpressing single antimicrobial proteins do not impart
commercially significant enhanced disease resistance.
Synergistic combinations, or activation of entire resistance
pathways, may offer a more successful approach.
Toxins: agents of attack and defense
Phytopathogens often produce toxins during plant infection, and these toxins may act as virulence factors
[47–50,51•,52]. Inactivation of these toxins or their targets in plants has lead to enhanced disease resistance
[49,51•,52]. Promising avenues for disease control in these
systems may include engineering insensitive variants of
toxin targets in the plant, expression of toxin inactivating
enzymes [49,53,54•] or blocking entry of the toxin into the
plant cell [55].
Plants can also produce toxins inhibitory to fungi. For
example, Gaeumannomyces graminis causes take-all disease
in small cereals, and the host range of individual isolates
is determined by avenicin A-1, a toxin present in oat
roots. The host range of G.g. tritici is restricted to wheat
and barley, plants that do not contain avenicin A-1. G.g.
avenae, on the other hand, is capable of infecting oats,
as it contains an avenicin A-1 detoxifying enzyme [56].
Expression of such antifungal compounds in crops may
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Plant–microbe interactions
be one avenue towards fungal control. Today, metabolic
engineering in plants is technically challenging, and few
examples exist (e.g., [57•,58]). In the next few years
we may expect to see additional biochemical pathways
expressed in plants that convert naturally occurring plant
metabolites into antimicrobial compounds.
Conclusions
Rapid progress in understanding the genetic underpinnings of disease resistance in plants has opened a
number of new and exciting opportunities for engineering
pathogen control. The cloning of R-genes and other
signaling pathway components such as NPR1/NIM1 and
Mlo has provided tools for exploring such possibilities in
the short term. Integrating our knowledge of how these
proteins function with the emerging understanding of
other natural defense pathways will lead to an integrated
approach toward engineering of novel and broad-spectrum
defense mechanisms in crops. The combination of these
defenses with the added protection provided by expression of potent antifungal proteins promises the future
delivery to the grower of an effective arsenal to combat the
most important microbial diseases limiting crop production
today.
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A comparison of amino acid sequences of two Cf proteins shows that almost
all amino acid differences lie within the N-terminal portion of the LRR. Most
of the observed differences occur in residues of the LRR predicted to be
exposed to the solvent, consistent with the proposed role of the LRR in
pathogen recognition.
6.
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a large number of the predicted protein products reveals hypervariability
among residues within the LRR that are predicted to be solvent exposed. The
authors also observe a higher than normal rate of nonsynonomous nucleotide
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Note added in proof
The recent publication by Cao et al. [59••] reports that
overexpression of the NPR1/NIM1 gene in Arabidopsis
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effects on plant growth or development.
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The authors describe the molecular cloning of the Arabidopsis NDR1 gene,
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Papers of particular interest, published within the annual period of review,
have been highlighted as:
13.
•
Acknowledgements
We apologize to all investigators whose interesting results could not be
discussed in this review due to space limitations.
• of special interest
•• of outstanding interest
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3.
4.
•
Wang G-L, Song W-Y, Ruan D-L, Sideris S, Ronald PC:
The cloned gene, Xa21, confers resistance to multiple
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of one of the direct repeats of the LRR.
12.
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Glazebrook J, Zook M, Mert F, Kagan I, Rogers EE, Crute IR,
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mutants of Arabidopsis reveal that PAD4 encodes a regulatory
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A genetic dissection of camalexin (phytoalexin) accumulation in Arabidopsis reveals intricacies in the relationship between phytoalexin levels and
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some of the studied genes. At least one regulatory gene controling camalexin
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Cao H, Glazebrook J, Clarke JD, Volko S, Dong X: The
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See annotation for [16••].
16.
••
Ryals J, Weymann K, Lawton K, Friedrich L, Ellis D, Steiner H-Y,
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factor inhibitor I-κB. Plant Cell 1997, 9:425-439.
These two papers [15••, 16••] describe the positional cloning of an Arabidopsis gene required for establishment of SAR by biotic or chemical
agents. The gene is induced by chemical inducers of SAR, and shows homology to the mammalian regulatory proteins NF-κB and I-κB which control
cellular responses to a variety of stimuli including pathogens and other in-
Transgenic approaches to microbial disease resistance in crop plants Salmeron and Vernooij
flammatory agents. The fact that some of these agents also induce pathogen
response genes in plants is provocative.
17.
••
Bowling SA, Clarke JD, Liu Y, Klessig DF, Dong X: The cpr5
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to Peronospora fungus is not dependent on a functional NPR1/NIM1 gene.
This suggests a mechanism for Peronospora resistance in cpr5 plants that
falls outside the classically-defined SAR pathway.
18.
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resistance in wheat. Plant Cell 1996, 8:629-643.
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Similar to Arabidopsis LSD1, the maize Lls1 gene plays a key role in limiting the spread of cell death in leaf tissue. Here, the Lls1 gene is cloned
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suggested as a substrate for the Lls1 protein.
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29.
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Another Arabidopsis gene, THI2.1, is described which is inducible by
necrotrophic pathogens and methyl jasmonate. The authors show that over-
351
expression of this gene, encoding a thionin protein, provides Arabidopsis
with partial resistance against the fungal pathogen Fusarium oxysporum f.
sp. mattholiae.
31.
••
Buschges R, Hollricher K, Panstruga R, Simons G, Wolter
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An elegant demonstration of positional cloning was used by the authors to
clone the Mlo gene from barley. Mutations in Mlo, which lead to constitutive
expression of defense responses and resistance against powdery mildew,
are characterized molecularly. Mlo is predicted to encode a membrane-associated protein with six transmembrane helices.
32.
•
Peterhansel C, Freialdenhoven A, Kurth J, Kolsch R, SchulzeLefert P: Interaction analyses of genes required for resistance
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The paper demonstrates the distinction in genetic control of spontaneous
cell death induced by mlo mutations, and hypersensitive cell death mediated
by race-specific interactions. It also shows that mlo mutations, unlike Arabidopsis cim mutations, do not lead to constitutive defense gene expression
but rather lead to more rapid gene induction in response to pathogen attack.
Finally, the authors show that mlo-based resistance is effective against attack
by a nonhost pathogen in barley.
33.
•
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disease resistance to powdery mildew. Plant Cell 1998, in
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This paper describes the genetic and molecular analysis of an Arabidopsis mutant, edr1, with phenotypic similarities to barley mlo. This mutant is
provocative by its suggestion that additional pathways for disease resistance
may be shared by monocot and dicot plants.
34.
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35.
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This paper reports that overexpression of the NPR1/NIM1 gene in Arabidopsis leads to enhanced resistance against Pseudomonas syringae and
Peronospora parasitica, with no obvious detrimental effects on plant growth
or development.