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are continually becoming resistant to existing resistance genes and fungicides, and a few of the pesticides are being withdrawn from the market for environmental reasons.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 Biotechnology2000, 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 centu-ry resulted in the accumulation of a wealth of knowledge on both the potential and limitations of R-genes. Although originally believed to provide durable resis-tance [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 agro-nomically 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 R -genes are tightly linked to undesirable traits and/or difficult to score for phenotype. An example of such an
R-gene is the soybean rhg1gene for control of cyst nema-todes, 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 cer-tain diseases, such as potato late blight, even had to be abandoned because R-genes proved too unreliable:
Phytophthora infestans, causal agent of late blight, over-came the resistance provided by all eleven R-genes that had been introgressed from the wild species Solanum demissuminto cultivated potato for control of this impor-tant 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 tra-ditional 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 poly-morphic 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 pheno-types, 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 exam-ple of a multidisciplinary effort to identify and characterize important traits including disease resis-tances using modern technologies is the North American Barley Genome Mapping Project (www.css.orst.edu/bar-ley/nabgmp/nabgmp.htm). Similar efforts are ongoing in all major crops.Application of molecular markers has resulted in the iso-lation 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 pro-teins 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
associated with classical breeding. Genome sequencing and genetic mapping experiments demonstrated that R -genes 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 R -genes 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 soy-bean 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 dis-ease control. Genes such as Bs2 in pepper and Xa21in 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 bacter-ial 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 resis-tance 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 Bs2and Xa21are the barley Rpg1gene 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 Xa21more 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. ful-vum. By transiently expressing ECP2 in a variety of tomato germplasm, plants were identified that responded with a hypersensitive necrotic response (HR). These
plants were genetically analyzed and shown to carry a sin-gle 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 mlogene, which confers resistance to all races of Erysiphe graminisin barley. Resistance mediated by mlois 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 mloplants 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 sus-ceptible to Erysiphe sp.
Another durable resistance gene that lacks homology with NBS/LRR genes is the tomato Ascgene, 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 Ascresistance 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 conta-minant in food and feed, and imposes a health risk to both humans and animals [26].
Even more effective R-genes may be isolated from resis-tant 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 elic-itor of this pathogen, indicating the direct involvement of R-genes [27••]. The product of a tobacco gene that
Phytophthora sojae. This parsley elicitor receptor is cur-rently 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 syringaepv. tomato(Pst) that contain the avr gene avrPto. Replacement of the weak endogenous promoter of Ptowith the strong promoter of cauliflower mosaic virus resulted in not only a further increased resistance to Pst(avrPto) but also a partial con-trol of unrelated pathogens, such as Xanthomonas campestris and Cladosporium fulvum [30•]. Studies to
probe the function of various R-gene domains by creat-ing 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 estab-lishment 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 pro-moters) 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].
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 Npr1enhances disease resistance lev-els against a broad variety of pathogens in Arabidopsis
[35••]. Importantly, this resistance is not associated with
any adverse plant phenotypes, such as stunting or unde-sired cell death [35••]. These findings make Npr1 an
extremely interesting candidate for agricultural application. Experiments have been initiated to test the
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 com-munication). Overexpression of an Npr1 homolog in wheat, however, resulted in suppression of pathogenesis-related genes and enhanced disease susceptibility (OV Bougri, CM Rommens, unpublished data).
Another signaling gene that has been evaluated for its util-ity to enhance disease resistance levels is the Myb1gene, which is induced by tobacco mosaic virus (TMV) in resis-tant tobacco plants, and encodes a transcription factor that binds to a promoter element of the pathogenesis-related gene PR1a [36]. Modification of Myb1expression 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 cpr6and 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
cpr6and Ssi1is 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
interest-ing aspect of Pad4is that inactivation of this gene leads to extreme susceptibility against a wide variety of pathogens, including Erysiphe orontii, Peronospora para-sitica and Pseudomonas syringae [40,41]. Thus, overexpression of Pad4in transgenic plants may enhance disease-resistance signaling. The Pad4 gene has been hypothesized to act by amplifying weak signals in dis-ease-resistance responses through a positive feedback cycle with salicylic acid [39••].
Overexpressing defense genes
Many of the genes induced by plant disease-resistance responses encode proteins with direct antifungal activity
chitin-binding proteins, lipid-transfer proteins, and hydrogen-peroxide-generating enzymes [46–48,P3].
Many biotechnology companies and universities are evaluating the performance of AFP-based transgenes in the field. In fact, hundreds of release permit applica-tions 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
commercial-ization, 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 pro-vide commercial levels of disease control in the field. In addition, AGO gene expression was shown to be corre-lated with a slightly altered tuber phenotype (KMM Swords, MS Hakimi, personal communication). A second hydrogen-peroxide-generating enzyme, the bar-ley oxalate oxidase, is under advanced field evaluation for control of Sclerotiniain 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 extra-cellular β1,3 glucanase resulted in a synergistic effect providing disease control against Fusarium oxysporumin tomato [P5]. An abundance of studies on the efficacy of AFPs in transgenic plants can be expected to be pub-lished 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 extreme-ly useful opening up the possibility of a multi-genic approach, which could provide efficacious and durable resistance against these pathogens.
Conclusions and future prospects
Disease resistance programs based on R-genes will great-ly benefit from the support provided by molecular breeding and molecular biology. R-genes will be trans-ferred more rapidly into elite germplasm. Better R-genes will be identified by exploring unconventional sources of resistance. Resistance will also be enhanced by the engi-neering 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 genet-ic make-up of the fungal pathogen and critgenet-ical genes involved in the pathogenesis process are expected to open new horizons in plant and crop protection. An inte-grated approach, based on the combined knowledge of the ‘defense’ systems used by the plants and the ‘assault’
systems used by pathogens should enable the design and development of novel disease-resistance genes.
Acknowledgements
The authors thank Kathy Swords for a critical review of the manuscript.
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