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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 350 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. 5. • Thomas CM, Jones DA, Parniske M, Harrison K, Balint-Kurti P, Hatzixanthis K, Jones JDG: Characterization of the tomato Cf4 gene for resistance to Cladosporium fulvum identifies a domain which determines recognitional specificity in Cf-4 and Cf-9. Plant Cell 1997, 9:2209-2224. 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. • Parniske M, Hammond-Kosack KE, Golstein C, Thomas CM, Jones DA, Harrison K, Wulff BBH, Jones JDG: Novel disease resistance specificities result from sequence exchange between tandemly repeated genes at the Cf-4/9 locus of tomato. Cell 1997, 91:821-832. This paper presents a detailed molecular characterization of the Cf gene cluster in cultivars with different recognitional specificities. Comparison of 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 substitutions in this area, suggestive of selection for sequence diversification. 7. Scofield SR, Tobias CM, Rathjen JP, Chang JH, Lavelle DT, Michelmore RW, Staskawicz BJ: Molecular basis for gene-forgene specificity in bacterial speck disease of tomato. Science 1996, 274:2063-2065. 8. Tang X, Frederick RD, Zhou J, Halterman DA, Jia Y, Martin GB: Initiation of plant disease resistance by physical interaction of avrPto and Pto kinase. Science 1996, 274:2060-2063. 9. Parker JE, Holub EB, Frost LN, Falk A, Gunn ND, Daniels MJ: Characterization of eds1, a mutation in Arabidopsis suppressing resistance to Peronospora parasitica specified by several different RPP genes. Plant Cell 1996, 8:2033-2046. 10. Rogers EE, Ausubel FM: Arabidopsis disease susceptibility mutants exhibit enhanced susceptibility to several bacterial pathogens and alterations in PR-1 gene expression. Plant Cell 1997, 9:305-316. 11. Century KS, Holub EB, Staskawicz BJ: NDR1, a locus of Arabidopsis thaliana that is required for disease resistance to both a bacterial and a fungal pathogen. Proc Natl Acad Sci USA 1995, 92:6597-6601. 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. References and recommended reading Century KS, Shapiro AD, Repetti PP, Dahlbeck D, Holub E, Staskawicz BJ: NDR1, a pathogen-induced component required for Arabidopsis disease resistance. Science 1997, 278:19631965. The authors describe the molecular cloning of the Arabidopsis NDR1 gene, perhaps encoding a membrane-associated protein, that plays a role in resistance to both bacterial and fungal pathogens. The broad role played by NDR1 in Arabidopsis disease resistance suggests that it operates downstream of typical R-genes in pathogen response signaling. 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 1. Hammond-Kosack KE, Jones JDG: Plant disease resistance genes. Annu Rev Plant Physiol Plant Mol Biol 1997, 48:575-607. 2. • Cai D, Kleine M, Kifle S, Harloff H-J, Sandal NN, Marcker KA, Klein-Lankhorst RM, Salentijn EMJ, Lange W, Stiekema WJ: Positional cloning of a gene for nematode resistance in sugar beet. Science 1997, 275:832-834. The paper describes the positional cloning, from an important crop, of a resistance gene active against an economically important pest. Translocation lines and Agrobacterium rhizogenes-mediated transformation were two of the tools used to circumvent the lack of sophisticated genetic methodology for sugarbeet. 3. 4. • Wang G-L, Song W-Y, Ruan D-L, Sideris S, Ronald PC: The cloned gene, Xa21, confers resistance to multiple Xanthomonas oryzae pv. oryzae isolates in transgenic plants. Mol Plant–Microbe Interact 1996, 9:850-855. Anderson PA, Lawrence GJ, Morrish BC, Ayliffe MA, Finnegan J, Ellis JG: Inactivation of the flax rust resistance gene M associated with loss of a repeated unit within the leucine-rich repeat coding region. Plant Cell 1997, 9:641-651. Cloning of the M disease resistance gene from flax, and molecular analysis reveal a direct repeat within the LRR region. Mutant alleles with loss of recognitional specificity were found to contain deletions resulting in the loss of one of the direct repeats of the LRR. 12. • Glazebrook J, Zook M, Mert F, Kagan I, Rogers EE, Crute IR, Holub EB, Hammerschmidt R, Ausubel FM: Phytoalexin-deficient mutants of Arabidopsis reveal that PAD4 encodes a regulatory factor and that four PAD genes contribute to downy mildew resistance. Genetics 1997, 146:381-392. A genetic dissection of camalexin (phytoalexin) accumulation in Arabidopsis reveals intricacies in the relationship between phytoalexin levels and pathogen resistance. Whereas camalexin is not required for resistance mediated by some gene-for-gene interactions, resistance to virulent Pseudomonas and avirulent Peronospora pathogens is blocked by mutations in some of the studied genes. At least one regulatory gene controling camalexin production is identified. 14. Ryals JA, Neuenschwander UH, Willits MG, Molina A, Steiner H Y, Hunt MD: Systemic acquired resistance. Plant Cell 1996, 8:1809-1819. 15. •• Cao H, Glazebrook J, Clarke JD, Volko S, Dong X: The Arabidopsis NPR1 gene that control systemic acquired resistance encodes a novel protein containing ankyrin repeats. Cell 1997, 88:57-63. See annotation for [16••]. 16. •• Ryals J, Weymann K, Lawton K, Friedrich L, Ellis D, Steiner H-Y, Johnson J, Delaney TP, Jesse T, Vos P, Uknes S: The Arabidopsis NIM1 protein shows homology to the mammalian transcription 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 mutant of Arabidopsis expressed both NPR1-dependent and NPR1-independent resistance. Plant Cell 1997, 9:1573-1584. The authors describe the identification of the Arabidopsis CPR5 gene. Mutations in cpr5 lead to constitutive expression of defense genes, and resistance to both bacterial and fungal pathogens. Interestingly, the resistance 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. Gorlach J, Volrath S, Knauf-Beiter G, Hengy G, Beckhove U, Kogel K-H, Oostendorp M, Staub T, Ward E, Kessmann H, Ryals J: Benzothiadiazole, a novel class of inducers of systemic acquired resistance, activates gene expression and disease resistance in wheat. Plant Cell 1996, 8:629-643. 19. Abad MS, Hakimi SM, Kaniewski WK, Rommens CMT, Shulaev V, Lam E, Shah DM: Characterization of acquired resistance in lesion-mimic transgenic potato expressing bacterio-opsin. Mol Plant–Microbe Interact 1997, 10:635-645. 20. Wu G, Shortt BJ, Lawrence EB, Leon J, Fitzsimmons KC, Levine EB, Raskin I, Shah DM: Activation of host defense mechanisms by elevated production of H2O2 in transgenic plants. Plant Physiol 1997, 115:427-435. 21. Zoubenko O, Uckun F, Hur Y, Chet I, Tumer N: Plant resistance to fungal infection induced by nontoxic pokeweed antiviral protein mutants. Nat Biotechnol 1997, 15:992-996. 22. • Dietrich RA, Richberg MH, Schmidt R, Dean C, Dangl JL: A novel zinc finger protein is encoded by the Arabidopsis LSD1 gene and functions as a negative regulator of plant cell death. Cell 1997, 88:685-694. The Arabidopsis LSD1 gene functions in suppression of hypersensitivity to pathogens and to agents that induce SAR. In this report, the LSD1 gene is cloned using a positional approach and is found to encode a zinc-finger protein homologous to other proteins with roles in responses to external stimuli. 23. Greenberg JT, Guo A, Klessig DF, Ausubel FM: Programmed cell death in plants: a pathogen-triggered response activated coordinately with multiple defense functions. Cell 1994, 77:551-563. 24. • Gray J, Close PS, Briggs SP, Johal GS: A novel suppressor of cell death in plants encoded by the Lls1 gene of maize. Cell 1997, 89:25-31. 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 and is found to putatively encode a dioxygenase. A phenolic compound is suggested as a substrate for the Lls1 protein. 25. Hu G, Yalpani N, Briggs, SP, Johal GS: A porphyrin pathway impairment is responsible for the phenotype of a dominant disease lesion mimic mutant of maize. Plant Cell 1998, in press. 26. Hunt MD, Delaney TP, Dietrich RA, Weymann KB, Dangl JL, Ryals JA: Salicylate-independent lesion formation in Arabidopsis lsd mutants. Mol Plant–Microbe Interact 1997, 10:531-536. 27. Jorgensen JH: Discovery, characterization and exploitation of Mlo powdery mildew resistance in barley. Euphytica 1992, 63:141-152. 28. • Van Wees SCM, Pieterse CMJ, Trijssenaar A, Van’t Westende YAM, Hartog F, Van Loon LC: Differential induction of systemic resistance in Arabidopsis by biocontrol bacteria. Mol Plant–Microbe Interact 1997, 10:716-724. The paper extends the characterization of induced systemic resistance (ISR), an SA-independent resistance pathway induced by biocontrol Pseudomonads in Arabidopsis and radish. Genetic variation in ISR competency is identified in Arabidopsis, and, by employing mutant biocontrol strains, it is determined that different bacterial factors can trigger ISR in different hosts. 29. 30. • Penninckx IAMA, Eggermont K, Terras FRG, Thomma BPHJ, De Samblanx GW, Buchala A, Metraux J-P, Manners JM, Broekaert WF: Pathogen-induced systemic activation of a plant defensin gene in Arabidopsis follows a salicylic acidindependent pathway. Plant Cell 1996, 8:2309-2323. Epple P, Apel K, Bohlmann H: Overexpression of an endogenous thionin enhances resistance of Arabidopsis against Fusarium oxysporum. Plant Cell 1997, 9:509-520. 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 M, Frijters A, van Daelen R, van der Lee T, Diergaarde P, Groenendijk J et al.: The barley Mlo gene: a novel control element of plant pathogen resistance. Cell 1997, 88:695-705. 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 responses to powdery mildew in barley reveal distinct pathways leading to leaf cell death. Plant Cell 1997, 9:13971409. 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. • Frye CA, Innes RW: An Arabidopsis mutant with enhanced disease resistance to powdery mildew. Plant Cell 1998, in press. 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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Resveratrol levels were not measured in these plants, but preliminary results indicated enhanced disease resistance to rice blast. 58. 59. •• Dixon RA, Lamb CJ, Masoud S, Sewalt VJH, Paiva NL: Metabolic engineering: prospects for crop improvement through the genetic manipulation of phenylpropanoid biosynthesis and defense responses a review. Gene 1996, 179:61-71. Cao H, Li X, Dong X: Generation of broad-spectrum disease resistance by overexpression of an essential regulatory gene in systemic acquired resistance. Proc Natl Acad Sci USA 1998, 95:6531-6536. 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.