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 Biology1998,1:347–352 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.
Plant disease resistance signaling pathways
R-genesMuch progress has recently been made in understanding race-specific pathogen resistance controlled by single dominant resistance (R) genes. The cloning of a large
number of these genes has shown that R-gene prod-ucts 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 mostR-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].
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-occuringR-gene alleles may provide insight into directed tailoring of the LRR for specific pathogen control.
Genes downstream ofR-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 resis-tance toPeronospora parasiticaisolate Noco2 mediated by the R-gene RPP14[9]. Further analysis showed thateds1
actually blocks resistance mediated by at least four distinct
Peronosporaresistance genes [9]. In contrast, activity of the
Arabidopsis RPM1gene controlling race-specific resistance toPseudomonaspathogens is unaffected byeds1[9].
[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 thePAD4gene, 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.
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 theNIM1/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/NPR1and its homologs will be fruitful in elucidating the mechanism of SAR establishment inArabidopsisand in crop plants.
Other Arabidopsis mutants exhibit SAR constitutively. These so-calledcim(constitutive immunity) orcpr (consti-tutive 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].
Lesion mimic plant mutants, such as Arabidopsis lsd, acd
and maizeLls, exhibit enhanced PR gene expression, and often display disease resistance [22•,23,24•,25]. Harnessing
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 theMlopowdery mildew resistance gene (see below) has been managed through breeding to allow deployment of these lines in the field [27].
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 CPR5and 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, PDF1.2induction is SA-independent [29]. Overexpression of the thionin gene in Arabidopsis
leads to partial resistance againstFusarium oxysporum[30•], indicating that non-SAR pathways may be useful for disease resistance engineering.
Recently the Mlogene 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 genePR-1[32•]. Resistance based on
mlohas been introduced into modern barley cultivars and has proven durable in the field for more than twenty years [27]. Interestingly, a mutation designatededr1 (enhanced disease resistance) has been reported in Arabidopsis that shares similarities in phenotype tomlo[33•]. TheMloand
EDR1genes should provide keys to understanding novel disease resistance pathways in both monocots and dicots.
Transgenic expression of antifungal proteins
Hydrolytic enzymesmeans 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, andin 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 chitinasesin 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, aRhizopuschitinase expressed in tobacco conferred resistance to a Sclerotinia
pathogen. No resistance was found toBotrytis 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 antimicro-bial 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 peptidoglycanin 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.
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 Arabidopsisenhanced 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 over-expressing 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 in-fection, and these toxins may act as virulence factors [47–50,51•,52]. Inactivation of these toxins or their tar-gets 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].
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 under-pinnings 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 expres-sion 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.
Note added in proof
The recent publication by Cao et al. [59••] 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.
Acknowledgements
We apologize to all investigators whose interesting results could not be discussed in this review due to space limitations.
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest
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