Punja GM Resistance Fungal 2001

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216

Past-Presidents contribution / Contribution du prsident sortant

Genetic engineering of plants to enhance

resistance to fungal pathogensa review ofprogress and future prospects

Zamir K. Punja

Abstract: Recent applications of techniques in plant molecular biology and biotechnology to the study of host-pathogen interactions have resulted in the identification and cloning of numerous genes involved in the defense

responses of plants following pathogen infection. These include: genes that express proteins, peptides, or antimicrobial

compounds that are directly toxic to pathogens or that reduce their growth in situ; gene products that directly inhibit

pathogen virulence products or enhance plant structural defense genes, that directly or indirectly activate general plant

defense responses; and resistance genes involved in the hypersensitive response and in the interactions with avirulence

factors. The introduction and expression of these genes, as well as of antimicrobial genes from nonplant sources, in a

range of transgenic plant species have shown that the development of fungal pathogens can be significantly reduced.

The extent of disease reduction varies with the strategy employed as well as with the characteristics of the fungal

pathogen, and disease control has never been complete. Manipulation of salicylic acid, ethylene, and cytokinin levels in

transgenic plants have provided some interesting results with regard to enhanced disease tolerance or susceptibility. The

complex interactions among the expressed gene product, plant species, and fungal pathogen indicate that the response

of transgenic plants cannot be readily predicted. Combinations of defense gene products have shown considerably more

promise in reducing disease than single-transgene introductions. The use of tissue-specific or pathogen-inducible

promoters, and the engineered expression of resistance genes, synthetic antimicrobial peptides, and elicitor molecules

that induce defense responses have the potential to provide commercially useful broad-spectrum disease resistance in

the not-too-distant future. The issues and challenges that will need to be addressed prior to the widespread utilization

of these transgenic plants are highlighted.

Key words: antifungal proteins, antimicrobial peptides, biotechnology, elicitors, hypersensitive response,pathogenesis-related proteins, phytoalexins, resistance genes, transgenic plants.

235Rsum : Les application rcentes au domaine vgtal des techniques de la biologie molculaire et de la

biotechnologie pour ltude des interactions htes-pathognes ont permis didentifier et de cloner plusieurs gnes

impliqus dans les rponses de dfense des plantes par suite dune infection par un agent pathogne. Ceux-ci

comprennent : des gnes qui codent pour des protines, des peptides ou des composs antimicrobiens qui ont une

toxicit directe ou rduisent la croissance in situ des agents pathognes; des produits gniques qui inhibent directement

des produits de virulence des agents pathognes ou stimulent les gnes de dfense structurale des plantes, qui activent

directement ou indirectement les rponses gnrales de dfense des plantes et des gnes de rsistance impliqus dans la

raction dhypersensibilit et dans les interactions avec les facteurs davirulence. Lintroduction et lexpression de ces

gnes, ainsi que celles de gnes antimicrobiens de source non vgtale, dans une gamme despces vgtales

transgniques ont montr que le dveloppement des champignons pathognes peut tre significativement rduit.

Lampleur de la rduction de la maladie dpends de la stratgie utilise aussi bien que des caractristiques du

champignon pathogne; une rpression complte de la maladie na pas encore t atteinte. La manipulation des niveauxdacide salicylique, dthylne et de cytokinines dans des plantes transgniques a fourni quelques rsultats intressants

concernant la tolrance ou la sensibilit accrues la maladie. Les interactions complexes entre les produits gniques

exprims, les espces vgtales et les champignons pathognes laissent voir que la rponse des plantes transgniques ne

peut tre parfaitement prdite. Les combinaisons de produits gniques de dfense ont t beaucoup plus prometteuses

Accepted June 25, 2001.

Z.K. Punja.1 Centre for Environmental Biology, Department of Biological Sciences, Simon Fraser University, Burnaby, BC V581S6, Canada.

1 Corresponding author (e-mail: [email protected]).

Can. J. Plant Pathol. 23: 216-235 (2001)


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Punja: genetic engineering / fungal disease resistance 217

que les introductions dun seul transgne pour lutter contre les maladies. Lemploi de promoteurs spcifiques un tissu ou

inductibles par les agents pathognes et le gnie appliqu lexpression de gnes de rsistance, de peptides

synthtiques antimicrobiens et de molcules inductrices qui stimulent les rponses de dfense ont le potentiel pour

fournir commercialement, dans un futur pas si lointain, un moyen pratique pour rsister un large spectre de maladies. Les

problmes et les dfis qui devront tre surmonts avant darriver une utilisation grande chelle de ces plantes

transgniques sont mis en vidence.

Mots cls : protines antifongiques, peptides antimicrobiens, biotechnologie, liciteurs, raction dhypersensibilit,protines relies la pathogense, phytoalexines, gnes de rsistance, plantes transgniques.

Introductionengineering / fungal disease resistance

One of the challenges facing breeders during the develop-ment of improved crop cultivars for agricultural use is theincorporation of resistance to diseases. Since domesticationof plants for human use began, diseases have caused majoryield losses and have impacted the well-being of humansworldwide (Agrios 1997). The incorporation of disease re-sistance genes into plants has been successfully achievedusing conventional breeding methods, which involve selec-

tion and evaluation of large progeny populations derivedfrom crosses made between resistant and susceptible par-ents and subsequent screening under disease conducive con-ditions. Virtually all agricultural crop cultivars in use todayhave some form of genetic resistance incorporated, gener-ally against a number of diseases. This may involve singleor multiple genes that are characterized as having recessiveor dominant effects (Crute and Pink 1996). Without the in-corporation of these resistance genes, crop productivity andyield would be substantially reduced (Agrios 1997).

With the beginning of the molecular era of plant biologyin the early 1980s, a major area of research has been toidentify, clone, and characterize various genes involved indisease resistance. As a result, many intriguing mecha-

nisms, which plants have evolved to respond to pathogeninfection, have been identified over the past 10 years, andremarkable progress has been made toward elucidating themultitude of genes that are involved in these responses. Theidentification of these genes has made it possible to subse-quently evaluate their specific roles and importance in dis-ease response pathways using transgenic plants developedwith genetic engineering techniques (Fig. 1).

In this paper, I will review advances made in utilizing abroad range of cloned genes (from both plant and nonplantsources) to enhance disease resistance against fungal patho-gens in transgenic plants and address future challenges andprospects. Several other reviews on the subject of geneticengineering for fungal disease resistance provide related in-

formation on this topic (Shah 1997; Swords et al. 1997;Bushnell et al. 1998; Evans and Greenland 1998; Hone1999; Melchers and Stuiver 2000; Rommens and Kishore2000). The approaches that have been taken by researcherscan be grouped into five general categories (see Table 1):(1) The expression of gene products that are directly toxic

to pathogens or that reduce their growth. These includepathogenesis-related proteins (PR proteins) such ashydrolytic enzymes (chitinases, glucanases), antifungalproteins (osmotin- and thaumatin-like), antimicrobialpeptides (thionins, defensins, lectin), ribosome-inactivating proteins (RIP), and phytoalexins.

(2) The expression of gene products Pthatadestroyic or neu-tralize a component of the pathogen arsenal such as

polygalacturonase, oxalic acid, and lipase.(3) The expression of gene products that can potentially

enhance the structural defenses in the plant. These in-clude elevated levels of peroxidase and lignin.

(4) The expression of gene products releasing signals thatcan regulate plant defenses. This includes the produc-tion of specific elicitors, hydrogen peroxide (H2O2),salicylic acid (SA), and ethylene (C2H4).

(5) The expression of resistance gene (R) products in-volved in the hypersensitive response (HR) and in in-teractions with avirulence (Avr) factors.

The selection of genes to genetically engineer into plantsto protect against fungal diseases has been based, in part, onevaluation of the toxicity of the gene product to fungalgrowth or development in vitro, and to the prominence ofthe particular gene(s) in a disease resistance response path-way. Many gene products belong to the group of PR pro-teins (Neuhaus 1999; Van Loon and Van Strien 1999), whileothers are involved in phytoalexin biosynthetic pathwaysand in enhancing plant structural defenses. Although someof these gene products may normally be expressed rela-tively late in the response pathway, e.g. after 48 h, the ratio-

nale for developing transgenic plants was to achieve earlyand high expression (overexpression) of these proteins, usu-ally constitutively throughout most of the plant. In other in-stances, enhanced levels of protein expression werereasoned to provide a greater inhibitory effect on fungal de-velopment than lower naturally occurring or induced levelsin the plant. Other genes were selected in genetic engineer-ing efforts for their ability to induce an array of naturallyoccurring defense mechanisms in the plant. More recently,the cloning of R genes has precipitated interest in utilizingthese genes to provide broad-spectrum disease resistance.Some genetic engineering approaches have been based onnovel approaches of introducing genes from double-stranded RNA entities from viruses found in fungi (Clausen

et al. 2000) and genes of lysozymes cloned from human tis-sues (Nakajima et al. 1997; Takaichi and Oeda 2000) andfrom a range of microbes (Lorito and Scala 1999).

Hydrolytic enzymes

The most widely used approach has been to overexpresschitinases and glucanases, which belong to the group of PRproteins (Neuhaus 1999) and have been shown to exhibitantifungal activity in vitro (Boller 1993; Yun et al. 1997).Since chitins and glucans comprise major components of


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218 Can. J. Plant Pathol. Vol. 23, 2001

Fig. 1. Transgenic plants with enhanced disease resistance have been engineered to express gene products to counterattack fungalvirulence products (from hypha on left), enhanced expression of plant-derived gene products (inside of cell) or gene products from

nonplant sources (outside of cell). The results from these experiments are summarized in Table 1.

the cell wall in many groups of fungi, the overexpression ofthese enzymes in plant cells is postulated to cause thehyphae to lyse and thereby reduce fungal growth (Mauchand Staehelin 1989). The specific roles of these hydrolasesin resistance to disease have been difficult to prove innontransgenic plants, since the enzymes are frequently en-countered in both resistant and susceptible tissues, and theirexpression can also be induced by environmental triggersand plant senescence (Punja and Zhang 1993). However,following expression of different types of chitinases in arange of transgenic plant species, the rate of lesion develop-ment and the overall size and number of lesions were re-

duced upon challenge with many fungal pathogens(Table 1), including those with a broad host range, such asBotrytis cinerea and Rhizoctonia solani. However,chitinaseexpression was ineffective against other pathogens, such asCercospora nicotianae, Colletotrichumlagenarium, andPythium spp., indicating that differences exist in sensitivity

of fungi to chitinase. The characteristics of chitinases fromdifferent sources can vary, e.g. in substrate binding speci-ficity, pH optimum, and localization in the cell, and this canlead to differences in antifungal activity (Sela-Buurlage etal. 1993), highlighting the importance of appropriate selec-

tion of the gene to be used against a targeted pathogen or

group of pathogens. While the results from these effortshave not been spectacular in terms of the level of diseasecontrol, they demonstrate that the rate of disease progressand overall disease severity can be significantly reduced. Afew transgenic crop species expressing chitinases have beenevaluated in field trials and it was demonstrated that diseaseincidence was reduced (Howie et al. 1994; Grison et al.1996; Melchers and Stuiver 2000).

There are fewer examples of the expression of glucanasesin transgenic plants (Table 1) but the results have generally

been similar to that for chitinase expression. The combinedexpression of chitinase and glucanase in transgenic carrot,

tomato, and tobacco was much more effective in preventingdevelopment of disease due to a number of pathogens thaneither one alone (Jongedijk et al. 1995; Van den Elzen et al.1993; Zhu et al. 1994), confirming the synergistic activityof these two enzymes reported from in vitro studies (Sela-Buurlage et al. 1993; Van den Elzen et al. 1993; Melchersand Stuiver 2000). As a general rule, the deployment of ge-netic engineering approaches that involve the expression oftwo or more antifungal gene products in a specific cropshould provide more effective and broad-spectrum diseasecontrol than the single-gene strategy (Lamb et al. 1992;Cornelissen and Melchers 1993; Strittmatter and Wegner


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Table 1. Plant species genetically engineered to enhance resistance to fungal diseases (1991-2001).

Strategia utilizat i specii de plante

modificate Produsul exprsiei genelor Efectul asupra dezvoltrii bolii Referine

Expesia enzimelor hidroliticeAlfalfa (Medicago sativa L.) glucanaza Alfalfa Reducerea dezvoltrii simptomelor datorit

Phytophthora megasperma;

niciun efectAsupra Stemphylium alfalfae

Ginseng american (Panax chitinaza orezului Netestatquinquefolius L.)

Mr (Malus domestica Auth.) endochitinazaTrichoderma harzianumNumrul leziunilor si suprafaa

reduse

datorit Venturia inaequalis

Orz (Hordeum vulgare L.) Trichoderma endo-1, Netestat4--glucanaza

Nap (Brassica napus L.) chitinase bobului de fasole mortalitatea rsadului total i parialredus datorit Rhizoctonia solani

chitinaza tomatei Procentaj sczut al plantelor bolnave

datorit Cylindrosporium

concentricumi Sclerotinia sclerotiorum

Morcov (Daucus carota L.) chitinaza tutunului Reducerea ratei i incidenei finale a boliidatorit Botrytis cinerea,Rhizoctonia solani, i Sclerotiumrolfsii; fr effect asupraThielaviopsisbasicola iAlternaria radicina

Crizantem (Dendranthema chitinaza orezului Dezvoltarea lezrilor redus datoritgrandiflorum) (Ramat.) Kitamura Botrytis cinerea

Castravete (Cucumis sativus L.) chitinaza tutunului Fr effect n dezvoltarea bolii datoriti a petuniei Colletotrichum lagenarium i

Rhizoctonia solanichitinaza orezului Dezvoltarea leziunilor redus datorit

Botrytis cinereaGref (Vitis vinifera L.) chitinaza orezului Dezvoltare redus a Uncinula

necator i leziuni reduse datoritElisinoe ampelina

Trichoderma harzianum Reducerea dezvoltrii Botrytiscinerea

endochitinaza n testele preliminarii

Arahid (Arachis hypogaea) chitinaza tutunului dezvoltare ntrziat a leziunilor isuprafaa leziunii mai mic datoritCercospora arachidicola

Cartof (Solanum tuberosum L.) Trichoderma harzianum Mai puine leziuni ca numr i mrimeendochitinaza datoritAlternaria solani; mortalitate

redus datorit Rhizoctonia solaniOrez (Oryza sativa) chitinaza orezului Instalare ntrziat i severitate redus

a simpomelor datoritMagnaporthe griseachitinaza orezului Mai puine numere de leziuni i mrimi

mai mici datorit Rhizoctoniasolani

Trandafir (Rosa hybrida L.) chitinaza orezului Diametrul leziunii redus datorit punctuluinegru (Diplocarpon rosae)

Cpun (Fragaria ananassa chitinaza orezului Dezvoltare redus a mucegaiuluiDuch.) prfos (Sphaerotheca humuli)

Tutun (Nicotiana benthamiana L.) chitinaza sfeclei de zahr Nu are efect asupra CercosporanicotianaeTutun (Nicotiana sylvestris L.) chitinaza tutunului Nu are efect asupra Cercosporanicotianae

chitinaza tutunului Colonizare redus de ctre Rhizoctonia

solaniTutun (Nicotiana tabacum L.) chitinaza fasolei Mortalitatea sczut a rsadului datorit

Rhizoctonia solani; fr efect asupra

Tutun (N. tabac

chitinaza arahideNetestat


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ud et al. (1996)

Chen and Z.K. Punja

ta nepublicat)

et al. (2000);

ong et al. (1999)

la et al. (1999)

ie et al. (1991)

n et al. (1996)

and Raharjo (1996)

su et al. (1999)

and Raharjo (1996)

et al. (1998)

moto et al. (2000)

rt et al. (2000)

i and Rao (2001)

o et al. (1998)

zawa et al. (1999)

et al. (1995); Datta et

(2000, 2001)hant et al. (1998)

et al. (1997)

en et al. (1993)

aus et al. (1991)

eilig et al. (1993)

ie et al. (1991, 1993)

mann et al. (1996)


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Table 1 (continued).

Strategia utilizat i specii de plante

modificate Expressed gene product Efectul asupra dezvoltrii bolii Reference

Serratia marcescens Incidena bolii redus datorit Howie et al. (1994)chitinaza Rhizoctonia solani pe rsaduri;

fr efect asupra Pythium ultimum

Serratia marcescens Dezvoltarea redus a Rhizoctonia solani Jach et al. (1992)chitinazaRhizopus oligosporus Rata dezvoltrii si msura leziunii pe frunz

Reduse Botrytis cinerea and Sclerotinia sclerotiorum

Streptomyces chitosanaza Netestat El Quakfaoui et al. (Trichoderma harzianum Simptome reduse datorit Alte

Lorito et al. (1998)

endochitinaza alternata, Botrytis cinerea, iRhizoctonia solani

Baculovirus chitinaza Reducerea dezvoltrii leziunilor datorit Shi et al. (2000)

Punctului maro (Alternaria alternata)Glucanaza din soia Dezvoltare redus a Phytophthora Yoshikawa et al. (1993)

parasitica iAlternaria alternata

glucanaza tutunului Reducerea simptomelor bolii datorit Phy- Lusso and Kuc (1996)tophthora parasitica i Peronosporatabacina

Acidothermus Netestatcellulolyticusendoglucanaza

Tomat (Lycopersicon esculentum Tomat slbatic Dezvoltare redus de VerticilliumMill.) (Lycopersicon dahliae rasele 1 i 2

chilense) chitinazaGru (Triticum aestivum L.) Chitinaza orzului Dezvoltare redusa coloniilor de

Blumeria graminis f. sp. triticiChitinaza orzului Dezvoltare redusa coloniilor de

Blumeria graminis and Pucciniarecondita

Expresia Proteinelor PR patogene nruditeNapi (B. napus) Chitinaz din mazre, PR-10.1 Fr efect

asupra Leptosphaeria maculansgen

Gena de rspuns defensiv Dezvoltare i infecie reduscu

defensin Leptosphaeria maculansMorcov (D. carota) TLP din Orez Inciden final i rat redusa bolii

due to Botrytis cinerea iSclerotiniasclerotiorum

Cartof (Solanum commersonii Dun.) Protein asemntoare cu Tolerancrescut la infecie datorit Phy-

Osmotina tophthora infestansCartof (S. tuberosum) Osmotin din tutun Instalare amnat i incidena a bolii

datorit Phytophthora infestansGena PR-10 din mazre Dezvoltare redus de Verticillium

dahliaeGena rspunsului defensiv Fr efect asupra Phytophthorainfestans

STH -2 din cartof

Orez (O. sativa) Gena defensivRir l b dinorez Mai puine leziuni datorit

Magnaporthe griseaTLP din Orez Dezvoltarea leziunilor redus de

Rhizoctonia solaniTutun (N. tabacum) TutunPR-1a Rat i boal final reduse datorit

Peronospora tabacina iPhytoph-

thora parasiticaOsmotin din Tutun Fr efect asupra Phytophthora


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t al. (2000)

eizadeh et al. (1999)

ld et al. (1999)

h et al. (2001)

et al. (1999)

et al. (1999)

Chen and Z.K. Punja

published data)

t al. (1996)

al. (1994)

g et al. (1993)

abel et al. (1993)

frath et al. (2000) Datta et al. (1999)

nder et al. (1993)

al. (1994)


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Strategy used and plant species

engineered Expressed gene product Effect on disease development Reference

Wheat (T. aestivum) Aspergillus giganteus Reduced development of colonies of Oldach et al. (2001)antifungal protein Blumeria graminis and Puccinia

recondita

Rice TLP Delayed development of FusariumgraminearumExpression of antimicrobial proteins, peptides, or compoundsArabidopsis thaliana L. Arabidopsis thionin Reduced development and colonization

by Fusarium oxysporumMistletoe thionin Reduced infection and development of

viscotoxin Plasmodiophora brassicae

Carrot (D. carota) Human lysozyme Enhanced resistance to Erysipheheraclei

and Alternaria dauciGeranium (Pelargonium sp.) Onion antimicrobial Reduced development and sporulation of

protein Botrytis cinereaPotato (S. tuberosum) Alfalfa defensin Enhanced resistance to Verticillium

dahliae

Bacillus Delayed sporulation and reducedamyloliquefaciens sporangia production byPhytophthora

barnase (RNase) infestansSynthetic cationic Reduced development ofFusarium

peptide chimera solani and Phytophthoracactorum

Human lactoferrin Not tested

Rice (O. sativa) Maize RIP No effect on Magnaporthe grisea orRhizoctonia solani

Tobacco (N. tabacum) Amaranthus hevein-typeNo effect on Alternaria longipes orpeptide, Mirabilis Botrytis cinereaknottin-type peptide

Radish defensin Reduced infection and lesion size due toAlternaria longipes

Barley RIP Reduced incidence and severity of

Rhizoctonia solaniMaize RIP Lower damage due to Rhizoctoniasolani

Pokeweed antiviral Lower rate of infection and mortality

protein due to Rhizoctonia solaniSarcotoxin peptide Enhanced seedling survival following

from Sarcophaga inoculation with Rhizoctonia solani,peregrina Pythium aphanidermatum, andPhy-

tophthora nicotianaeStinging nettle (Urtica Not tested

dioica L.) isolectinAntifungal (killing) Not tested

protein from virus

infecting Ustilagomaydis (dsRNA)

Chloroperoxidase from Reduced lesion development by

Pseudomonas Colletotrichum destructivumpyrrocinia

Synthetic antimicrobial Reduced lesion size due to

peptide Colletotrichum destructivumSynthetic magainin- Reduced lesion size and sporulation due

type peptide to Peronospora tabacinaHuman lysozyme Reduced colony size and conidial pro-

duction by Erysiphe

cichoracearumTomato (L. esculentum) Radish defensin Reduced number and size of lesions due

to Alternaria solani

Chen et al. (1999)

Epple et al. (1997)

Holtorf et al. (1998)

Takaichi and Oeda (2000) Bi

et al. (1999)

Gao et al. (2000)

Strittmatter et al. (1995)

Osusky et al. (2000)

Chong and Langridge

(2000)

Kim et al. (1999)

De Bolle et al. (1996)

Terras et al. (1995)

Logemann et al. (1992)

Maddaloni et al. (1997)

Wang et al. (1998);

Zoubenko et al. (1997)

Mitsuhara et al. (2000)

Does et al. (1999)

Park et al. (1996)

Rajasekaran et al. (2000)

Cary et al. (2000)

Li et al. (2001)

Nakajima et al. (1997)

Parashina et al. (2000)


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Wheat (T. aestivum) Barley RIP Slightly reduced development of Bieri et al. (2000)Blumeria graminis

Antifungal (killing) Inhibition ofUstilago maydis and Clausen et al. (2000)

protein from from Tilletia tritici development on seedsvirus infectingUstilago maydis(dsRNA)

Expression of phytoalexins

Alfalfa (M. sativa) Alfalfa isoflavone O- Reduced lesion size due to Phomamethyltransferase medicaginis

Peanut resveratrol Reduced lesion size and sporulation of

synthase Phoma medicaginisBarley (H. vulgare) Grape stilbene Reduced colonization by Botrytiscinerea

(resveratrol) synthase

Rice (O. sativa) Grape stilbene Reduced lesion development due to(resveratrol) synthase Pyricularia oryzae

Tobacco (N. tabacum) Fusarium trichodiene Not testedsynthase

Grape stilbene Reduced colonization by Botrytiscinerea

(resveratrol) synthase

Tomato (L. esculentum) Grape stilbene Reduced lesion development by Phytoph-(resveratrol) synthase thora infestans; no effect on

Alternaria solani orBotrytiscinerea

Wheat (T. aestivum) Grape stilbene Not tested(resveratrol) synthase

Inhibition of pathogen virulence productsCanola (B. napus) Barley oxalate oxidase Not tested

Poplar (Populus euramericana Wheat oxalate oxidase Delayed development ofSeptoriamusiva

Auth.)

Tobacco (N. tabacum) Fusarium Not testedtrichothecene-

degrading enzyme

Wheat oxalate oxidase Not tested

(germin)

Tomato (L. esculentum) Bean polygalacturonase No effect on disease due to Fusariuminhibiting protein oxysporum, Botrytis cinerea, and

Alternaria solaniPear polygalacturonase Reduced rate of development ofBotrytis

inhibiting protein cinereaCollybia velutipes Enhanced resistance to Sclerotinia

oxalate sclerotiorumdecarboxylase

Alteration of structural componentsPotato (S. tuberosum) Cucumber peroxidase No effect on disease due to Fusarium

sambucinum and Phytophthorainfestans

Tomato (L. esculentum) Tobacco anionic No effect on disease due to Fusariumperoxidase oxysporum and Verticilliumdahliae

Wheat (T. aestivum) Wheat germin (no Reduced penetration by Blumeriaoxalate oxidase graminis into epidermal cellsactivity)

Regulation of plant defense responsesA. thaliana Arabidopsis NPR1 Reduced infection and growth of

protein Peronospora parasitica

He and Dixon (2000)

Hipskind and Paiva (2000)

Leckband and Lrz (1998)

Stark-Lorenzen et al.

(1997)

Zook et al. (1996)

Hain et al. (1993)

Thomzik et al. (1997)

Fettig and Hess (1999)

Thompson et al.

(1995)

Liang et al. (2001)

Muhitch et al. (2000)

Berna and Bernier (1997)

Desiderio et al. (1997)

Powell et al. (2000)

Kesarwani et al. (2000)

Ray et al. (1998)

Lagrimini et al. (1993)

Schweizer et al. (1999)

Cao et al. (1998)


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Table 1 (concluded).



Cotton (Gossypium hirsutum L.), Talaromyces flavus Enhanced protection against Rhizoctoniatobacco (N. tabacum) glucose oxidase solani and Verticillium dahliae; no

effect on Fusarium oxysporum

Potato (S. tuberosum) Aspergillus niger Delayed lesion development due to Phy-glucose oxidase tophthora infestans; reduced diseasedevelopment due to Alternaria solaniand Verticillium dahliae

Tobacco catalase Reduced lesion size due toPhytophthora

infestansBacterial salicylate No effect on Phytophthora infestans

hydroxylase

Tobacco (N. tabacum), A. thaliana Bacterial salicylate Enhanced susceptibility to Phytophthorahydroxylase parasitica, Cercosporanicotianae, and

Peronospora parasiticaTobacco (N. tabacum) Bacterial enzymes gen- Enhanced resistance to Oidium

erating salicylic acid lycopersiconArabidopsis ethylene- Enhanced susceptibility to Pythiuminsensitivity gene sylvaticum

Phytophthora cryptogea Reduced infection by Phytophthoraelicitor (-cryptogein) parasitica

Phytophthora cryptogea Enhanced resistance to Phytophthoraelicitor (cryptogein) parasitica, Thielaviopsis basicola,Botry-

tis cinerea, and Erysiphecichoracearum

Expression of combined gene productsCarrot (D. carota) Tobacco chitinase + - Enhanced resistance to Alternariadauci,

1,3-glucanase, Alternaria radicina, Cercospora

osmotin carotae, and Erysiphe heracleiTobacco (N. tabacum) Barley chitinase + - Reduced disease severity due to1,3-glucanase, or Rhizoctonia solanichitinase + RIP

Rice chitinase + alfalfa Reduced rate of lesion development and

glucanase fewer lesions due to Cercosporanicotianae

Tomato (L. esculentum) Tobacco chitinase + - Reduced disease severity due to1,3-glucanase Fusarium oxysporum f. sp.lycopersici

Murray et al. (1999)

Wu et al. (1995, 1997)

Yu et al. (1999)

Yu et al. (1997)

Delaney et al. (1994);

Donofrio and Delaney

(2001)

Verberne et al. (2000)

Knoester et al. (1998)

Tepfer et al. (1998)

Keller et al. (1999)

Melchers and Stuiver

(2000)

Jach et al. (1995)

Zhu et al. (1994)

Jongedijk et al. (1995); Van

den Elzen et al. (1993)

Note: dsRNA, double-stranded RNA; RIP, ribosome-inactivating protein; TLP, thaumatin-like protein.

1993; Jach et al. 1995; Shah 1997; Evans and Greenland1998; Salmeron and Vernooij 1998; Melchers and Stuiver

2000).

Pathogenesis-related proteins

Other PR proteins that exhibit antifungal activity, includ-ing osmotin- and thaumatin-like proteins (TLP), and someuncharacterized PR proteins have been engineered into cropplants (Table 1). Osmotin is a basic 24-kDa protein belong-ing to the PR-5 family whose members have a high degreeof homology to the sweet-tasting protein thaumatin fromThaumatococcus danielli and are produced in plantsunderdifferent stress conditions (Zhu et al. 1995). The PR-5 pro-teins induce fungal cell leakiness, presumably through a

specific interaction with the plasma membrane that resultsin the formation of transmembrane pores (Kitajima and

Sato 1999). Osmotin has been shown to have antifac-

tivity in vitro (Woloshuk et al. 1991; Melchers1993; Liu et al. 1994) and, when testecombination with


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nase and -1,3-glucanase, showed enhanced lyticity (Lorito et al. 1996). When expressed in transgenico, osmotin was shown to delay expression of diseasetoms caused by Phytophthora infestans (Table 1).matin-like proteins are also expressed in plants in re-se to a range of stress conditions and were demon-ed to have antifungal activity in vitro (Malehorn et al.; Koiwa et al. 1997). Expression of TLP in transgenics was reported to delay disease development due to sev-pathogens, including Botrytis, Fusarium, Rhizoctonia,Sclerotinia (Table 1). Combinations of PR-5 protein ex-ion with chitinases or glucanases in transgenic plantsnot been reported and it is anticipated that the level of

se reduction achieved would be significantly higher.

microbial proteins, peptides, and otherpounds

fensins and thionins are low molecular mass (arounda) cysteine-rich peptides (45-54 amino acids in length)


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224

found in monocotyledonous and dicotyledonous plant spe-cies, which were initially derived from seeds and haveantimicrobial activity (Bohlmann 1994; Broekaert et al.1995; Evans and Greenland 1998). Viscotoxin from mistle-toe (Viscum album) is a thionin. It was proposed thatthesepeptides play a role in protecting seeds from infection by

pathogens (Broekaert et al. 1997). Defensins are also foundin insects and mammals, where they play an important rolein curtailing or limiting microbial attack (Rao 1995). Thesepeptides may exert antifungal activity by altering fungalmembrane permeability and (or) inhibiting macromolecule

biosynthesis, and thionins may be toxic to plant and animalcell cultures as well (Broekaert et al. 1997). The over-expression of defensins and thionins in transgenic plants wasdemonstrated to reduce development of several differentpathogens, including Alternaria, Fusarium, andPlasmo-diophora (Table 1), and provided resistance toVerticillium on potato under field conditions (Gao et al.2000).

Chitin-binding peptides (hevein- and knottin-types) are36-40 residues in length and have been recovered from theseeds of some plant species. They contain cysteine residuesand were demonstrated to have antifungal activity in vitro(Broekaert et al. 1997). However, expression ofAmaranthushevein-type peptide and Mirabilis knottin-type peptide intransgenic tobacco did not enhance tolerance to Alternaria

longipes orBotrytis cinerea (De Bolle et al. 1996). Itwaspostulated that the presence of cations, particularly Ca2+,may have inhibited the activity of these peptides in vivo.Modifications to amino acid sequences of peptides may en-

hance the antifungal activity (Evans and Greenland 1998).Ribosome-inactivating proteins are plant enzymes that

have 28 S rRNA N-glycosidase activity, which dependingon their specificity, can inactivate conspecific or foreign ri-

bosomes, thereby shutting down protein synthesis. Themost common cytosolic type I RIP from the endosperm ofcereal grains do not act on plant ribosomes but can affectforeign ribosomes, such as those of fungi (Stirpe et al.1992; Hartley et al. 1996). Expression of barley seed RIPreduced development of Rhizoctonia solani in transgenicto-bacco (Logemann et al. 1992) but had little effect onBlumeria graminis in transgenic wheat (Bieri et al.2000).

In the latter study, the RIP was targeted to the apoplasticspace and may have had less activity against developmentof the intracellular haustoria of the mildew pathogen. It hasbeen demonstrated that combined expression of chitinaseand RIP in transgenic tobacco had a more inhibitory effecton Rhizoctonia solani development than the individualpro-teins (Jach et al. 1995). Therefore, dissolution of the fungalcell wall by hydrolytic enzymes should enhance the efficacyof antifungal proteins and peptides in transgenic plants. Hu-man lysozyme has lytic activity against fungi and bacteria, andwhen expressed in transgenic carrot and tobacco, enhanced re-sistance to several pathogens, including Erysipheand

Alternaria (Nakajima et al. 1997; Takaichi and Oeda 2000).

An antimicrobial protein with homology to lipid transpro-tein was shown to reduce development ofBotrytcinereawhen expressed in transgenic geranium (Bi et al. 1999

Pokeweed (Pytolacca americana) antiviralprotein withtype I RIP activity has been expressed in transgenictobaccoand shown to reduce development ofRhizoctonsolani(Wang et al. 1998). Because of some toxicity to pla

cells,


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Can. J. Plant Pathol. Vol. 23, 2001

oxic mutant proteins were derived and their expression ingenic plants led to the activation of defense-related signalling

ways and PR-protein induction (e.g., chitinase and glucanase),h in turn enhanced plant resistance to infection byoctonia solani (Zoubenko et al. 1997). The induction ofse pathways in transgenic plants using other strategies willscussed later.ntimicrobial peptides have been synthesized in the labo-

y to produce smaller (10-20 amino acids in length)cules that have enhanced potency against fungi (Cary et al.). In addition, a synthetic cationic peptide chi-

(cecropin-melittin) with broad-spectrum antimicrobiality has been produced (Osusky et al. 2000). When ex-ed in transgenic potato and tobacco, these synthetic pep-have provided enhanced resistance against a number of

al pathogens, including Colletotrichum, Fusarium, andtophthora (Table 1). These peptides may demonstrate

activity against fungal hyphae, inhibit cell wall forma-and (or) enhance membrane leakage. The ability to cre-

synthetic recombinant and combinatorial variants ofdes that can be rapidly screened in the laboratory couldde additional opportunities to engineer resistance to a

e of pathogens simultaneously. Enhancement of the spe-activities of antifungal enzymes or the creation of variantsbroad activity using directed molecular evolution (DNA

fling) has also been proposed as a method to enhance theacy of transgenic plants in the future (Lassner androok 2001).

toalexins

ese are low molecular mass secondary metabolites pro-d in a broad range of plant species, which were demon-ed to have antimicrobial activity and are induced byogen infection and elicitors (Hammerschmidt 1999;er and Kokubun 2001). Phytoalexins are synthesized

gh complex biochemical pathways (Dixon et al. 1996),as the shikimic acid pathway, and genetic manipula-

of these pathways to suppress or enhance phytoalexinuction has been difficult to achieve. Similar to theolytic enzymes, it has not been easy to conclusivelyonstrate the role played by phytoalexins in enhancingance to disease in many host-pathogen interactions. Ant of Arabidopsis deficient in the production of thee-type phytoalexin camalexin was shown to be more

eptible to infection by Alternaria brassicicola but nototrytis cinerea (Thomma et al. 1999b). Using transgenics, it has been possible to also show that the over-

ession of genes encoding certain phytoalexins, such ass-resveratrol and medicarpin, resulted in delayed devel-

nt of disease and symptom production by a number ofogens on several plant species (Table 1). These studiesencouraging in light of the difficulties of engineeringcomplex biochemical pathways leading to phytoalexinmulation in plants (Dixon et al. 1996).

bition of pathogen virulence products

e plant cell wall acts as a barrier to penetration by fungalgens and numerous strategies have evolved among plantgens to overcome this (Walton 1994). These include se-


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Punja: genetic engineering / fungal disease resistance

cretion of a range of plant cell wall degrading enzymes(depolymerases) and the production of toxins such as oxalicacid by fungal pathogens. Several strategies to engineer resis-tance against fungal infection have targeted the inactivation ofthese pathogen virulence products. Polygalacturonase-inhibiting proteins (PGIP) are glycoproteins present in the cellwall of many plants and that can inhibit the activity of fungal

endopolygalacturonases (Powell et al. 1994; Desiderio et al.1997). The expression of PGIP in transgenic plants led tocontrasting results: in transgenic tomato expressing a beanPGIP, resistance to Fusarium, Botrytis, orAlternariawasnot enhanced (Desiderio et al. 1997) while in transgenic to-mato expressing a pear PGIP, colonization of leaves andfruits by Botrytis was reduced (Powell et al. 2000). In the

former study, it was shown that PGIPs from bean differedin specificity to fungal polygalacturonase in vitro, and thePGIP-1 that had been selected for transformation was notinhibitory (Desiderio et al. 1997). Thus, appropriate in vitroscreening of PGIPs would be required prior to undertaking

transformation experiments. As with the PR proteins andantifungal compounds, disease development was reduced byPGIPs but not totally prevented in the transgenic plants.

Another developed strategy that could have potential toreduce pathogen infection is immunomodulation, the ex-pression of genes encoding antibodies or antibody frag-ments in plants (plantibodies) that could bind to pathogenvirulence products (De Jaeger et al. 2000; Schillberg et al.2001). The antibodies can be expressed inter- or extra-cellularly and can bind to and inactivate enzymes, toxins, orother pathogen factors involved in disease development.Currently, there are no published reports on the expressionof antifungal antibodies in transgenic plants that have led toa reduction in disease. However, it has been demonstrated

that antilipase antibodies inhibited infection of tomato byBotrytis cinerea, when mixed with spore inoculum, by

pre-venting fungal penetration through the cuticle (Commnil etal. 1998). Similarly, infection by Colletotrichumgloeo-sporioides on various fruits was inhibited using

polyclonal antibodies that bound to fungal pectate lyase(Wattad et al. 1997). Genetic engineering of antibodyexpression in plants is extremely challenging technically andthe applications to fungal disease control (immunization)have yet to be deter-mined, although success against virus diseases has been re-ported (De Jaeger et al. 2000).

Production of phytotoxic metabolites, such as mycotoxinsand oxalic acid, by fungal pathogens has been shown to fa-cilitate infection of host tissues following cell death. Degra-dation of these compounds by enzymes expressed intransgenic plants could provide an opportunity to enhanceresistance to disease. Expression of a trichothecene-degrading enzyme from Fusarium sporotrichioides intrans-genic tobacco reduced plant tissue damage and enhancedseedling emergence in the presence of the trichothecene(Muhitch et al. 2000). The effect on pathogen developmentwas not tested. Germin-like oxalate oxidases are stableglycoproteins first discovered in cereals, which are presentduring seed germination and are induced in response to fun-gal infection and abiotic stress (Dumas et al. 1995; Zhang etal. 1995; Berna and Bernier 1997). Their activity on the

substrate oxalic acid results in the production oandH2O2; the latter can induce defense responses iplant


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225

enhance strengthening of cell walls (Brisson et al.; Mehdy 1994). The expression of barley oxalatese in oilseed rape enhanced tolerance to the phytotoxicts of oxalic acid, although the effect on the targetogen Sclerotinia sclerotiorum was not evaluatedmpson et al. 1995). Expression of oxalate oxidase ingenic hybrid poplar enhanced resistance to Septoria, oxalate decarboxylase expression enhanced resistance

mato to Sclerotinia sclerotiorum (Table 1). These re-indicate that the inactivation of specific pathogen vir-ce factors, such as toxins, by gene products expressedansgenic plants has the potential to reduce developmentecific fungal pathogens.

ration of structural components

gnification of plant cells around sites of infection or le-has been reported to be a defense response of plants

can potentially slow down pathogen spread (NicholsonHammerschmidt 1992). The enzyme peroxidase is re-d for the final polymerization of phenolic derivativeslignin and may also be involved in suberization or

nd healing. A decrease in polyphenolic compounds,as lignin, in potato tubers by redirection of tryptophan

ansgenic plants through expression of tryptophan decar-lase rendered tissues more susceptible to Phytophthorastans (Yao et al. 1995), illustrating the role of phenolicounds in defense. Reduction of phenylpropanoid me-ism through inhibition of phenylalanine ammonia-

activity in transgenic tobacco also rendered tissuessusceptible to Cercospora nicotianae (Maher et al.

). Overexpression of a cucumber peroxidase gene ingenic potato, however, did not increase resistance ofes to infection by Fusarium or Phytophthora, andn levels were not significantly affected, in spite of ele-d peroxidase expression (Ray et al. 1998). It was sug-

d that peroxidase levels may not have been the limitingfor lignification or that the native peroxidase activityhave been cosuppressed. Overexpression of a tobacco

nic peroxidase gene in tomato did enhance lignin levelsresistance to fungal pathogens was not enhancedimini et al. 1993). Lignin levels were also significantly

er following expression of the H2O2-generating enzymeose oxidase in transgenic potato (Wu et al. 1997) andxpression of the hormone indoleacetic acid (IAA) ingenic tobacco (Sitbon et al. 1999). In the former case,ance to several fungal pathogens was enhanced (Ta-1). Peroxidase overexpression in plants can, however,negative effects on plant growth and development

rimini et al. 1997), and the results to date indicate that

approach appears to hold less promise for enhancingse resistance.

reduction in large callose deposits surroundingoria ofPeronospora parasitica infecting Arabidopsis

ana was indirectly achieved in transgenic plants not ac-ulating SA by expression of the enzyme salicylate hy-ylase (Donofrio and Delaney 2001). These plants alsoreduced expression of the PR-1 gene and exhibited sig-antly enhanced susceptibility to the pathogen, suggest-hat callose deposition during normal defense responses

e plant was influenced by the reduced levels of SA.


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Activation of plant defense responses

One activator of host defense responses are elicitor mole-cules from an invading pathogen. These can trigger a net-work of signalling pathways that coordinate the defenseresponses of the plant, including HR, PR protein, and

phytoalexin production (Heath 2000; McDowell and Dangl

2000; Shirasu and Schulze-Lefert 2000). A gene encodingthe elicitor cryptogein (a small basic protein, 98 amino ac-ids in length) from the pathogen Phytophthoracryptogeawas cloned and expressed in transgenic tobacco under con-trol of a pathogen-inducible promoter (Keller et al. 1999).Challenge inoculation with a range of fungi induced the HRas well as several defense genes, and growth of the patho-gens was concomitantly restricted (Table 1). Resistance tothe pathogens was not complete, possibly because of thetime needed for production of the transgenic elicitor follow-ing initial infection (Keller et al. 1999). Another elicitor,INF1, was shown to act as an Avr factor in the tobacco -Phytophthora infestans interaction and triggered the

onset of the HR (Kamoun et al. 1998). Expression of the geneen-coding the AVR9 peptide elicitor from Cladosporiumfulvum in transgenic tomatoes containing the Cf-9 genere-sulted in a necrotic defense response (Hammond-Kosack etal. 1994; Hone et al. 1995). The development of lesions re-sembling the HR induced through expression of a bacterial

proton pump gene (bacterio-opsin) fromHalobacteriumhalobium activated multiple defense systems in transgenic

tobacco plants (Mittler et al. 1995) in the absence of patho-gen challenge. In transgenic potato, expression of bacterio-

opsin enhanced resistance to some pathogens but had no ef-fect on others (Abad et al. 1997), while in poplar, there wasno effect on disease development (Mohamed et al. 2001).Antisense inhibition of catalase, a H2O2-degrading enzyme,resulted in development of necrotic lesions and PR-proteinaccumulation (Takahashi et al. 1997). While these and otherreports indicate that induction of the HR and necrosis, withthe resulting activation of general defense pathways, couldpotentially result in broad-spectrum disease resistance (Bent1996; Hone 1999; Melchers and Stuiver 2000), the use ofsuch an approach would require tight regulation of the ex-

pressed phenotype, in addition to ensuring that no deleteri-ous side effects, such as abnormal or suppressed growth,occurred on the transgenic plants. If successful, the activa-

tion of general defense responses in these transgenic plantswould provide protection against viral and bacterial patho-gens in addition to fungi.

Another activator of defense responses that has been en-gineered in transgenic plants is H2O2 generated through ex-pression of genes encoding for glucose oxidase (Table 1).H2O2 has been shown to directly inhibit pathogen growth(Wu et al. 1995) and to induce PR proteins, SA, and ethyl-ene (Wu et al. 1997; Chamnongpol et al. 1998), as well as

phytoalexins (Mehdy 1994). It is produced during the earlyoxidative burst in plant cell response to infection (Bakerand Orlandi 1995) and can trigger the HR (Levine et al.1994; Tenhaken et al. 1995), strengthen cell walls (Brissonet al. 1994), and enhance lignin formation (Wu et al. 1997).Expression of elevated levels of H2O2 in transgenic cotton,tobacco, and potato reduced disease development due to a

number of different fungi, including RhizoctVerti-


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m, Phytophthora, and Alternaria (Table 1); high levelshowever, be phytotoxic (Murray et al. 1999). In one

y, necrotic lesions from the HR enhanced infection byecrotrophic pathogen Botrytis cinerea (Govrin and Le-2000). Therefore, the widespread induction of cellh in a transgenic plant to induce disease resistance has to beoached with caution.

her activators of plant defense responses include sig-

ng molecules such as SA, ethylene, and jasmonic acidg et al. 1997; Dong 1998; Reymond and Farmer 1998;psey et al. 1999). The roles of SA as a signal moleculehe activation of plant defense responses to pathogen in-on and as an inducer of systemic acquired resistanceR) have been extensively studied (Ryals et al. 1996;ter et al. 1997; Dempsey et al. 1999; Mtraux 2001).g transgenic plants, evidence for the role of SA in de-

response activation has been obtained. Plants express-the SA-metabolizing enzyme salicylate hydroxylase, arial protein that converts SA to the inactive formhol, did not accumulate high levels of SA and had en-ed susceptibility to pathogen infection (Gaffney et al.; Delaney et al. 1994; Donofrio and Delaney 2001) or

unaltered susceptibility (Yu et al. 1997). A mutant ofbidopsis nonresponsive to induction of SAR showed en-ed susceptibility to fungal infection (Delaney et al.; Donofrio and Delaney 2001). The overexpression ofn transgenic tobacco was recently shown to enhancerotein production and provide resistance to fungal

ogens (Verberne et al. 2000). Expression of tobaccoase, an enzyme with SA-binding activity, in transgenico enhanced defense gene expression leading to SARnhanced tolerance to Phytophthora infestans (Yu et al.). Overexpression of the NPR1 gene, which regulatesSA-mediated signal leading to SAR induction, in trans-

Arabidopsis increased the level of PR proteins duringtion and enhanced resistance to Peronospora parasitica

et al. 1998). It was postulated that synergistic interac-between PR proteins and products of other down-

m defense-related genes provided the enhancedance. In addition, NPR1 was only activated upon in-

on or by induction of SAR, avoiding potential side ef-on plant growth from constitutive expression. These

es demonstrate that manipulation of SA levels in trans-c plants has the potential to lead to enhanced diseaseance by inducing PR-protein expression and other de-gene products.

hylene and jasmonic acid appear to be signals used innse of plants to necrotrophic pathogen attack (in con-to biotrophic infection) and that work independently of,possibly antagonistic to, SA-mediated responses (Dong

; Thomma et al. 1999a; McDowell and Dangl 2000; Lee et001). Mutant or transformed plantsesponsive to either jasmonate or ethylene were found tomore susceptible to infection by root- and foliar-ting fungi (Knoester et al. 1998; Staswick et al. 1998;yan et al. 1998; Hoffmann et al. 1999; Thomma et al.a), confirming a role for these signals in certain host-

ogen interactions. In contrast, ethylene-insensitive mu-may exhibit reduced disease symptoms, as described

usarium oxysporum on tomato (Lund et al. 1998). Ge-engineering efforts to alter ethylene or jasmonate pro-


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duction in plants may, however, result in unpredictableeffects on disease response, depending on the pathogen, aswell as induce potential side effects in view of the multipleroles played by these signal molecules in plants (ODonnellet al. 1996; Weiler 1997; Wilkinson et al. 1997).

Ethylene production and extracellular PR-protein expres-sion were found to be induced by expression of cytokinins

in transgenic tomato cells (Bettini et al. 1998). The engi-neering of hormone biosynthetic gene expression in trans-genic plants has been accomplished (Hedden and Phillips2000). Whether or not reduced or elevated levels of hor-mones, such as auxins, cytokinins, gibberellins, andjasmonate, can lead to the development of transgenicplants with enhanced disease resistance remains to beseen, given their broad range of physiological effects onplant development. Interestingly, inhibition of IAA pro-duction by antisense transformation of the nitrilase 1 genein Arabidopsis reduced levels of IAA and developmentofroot galls due to Plasmodiophora brassicae(Neuhaus et

al. 2000). In contrast, overexpression of IAA in tobacco en-hanced ethylene production and peroxidase activity and in-creased lignin content although the response to disease wasnot tested (Sitbon et al. 1999). Altered auxin or cytokininexpression has the potential to also affect mycorrhizal colo-nization of plant roots (Barker and Tagu 2000).

Resistance genes

Resistance gene products may serve as receptors forpathogen Avr factors or recognize the Avr factor indirectlythrough a coreceptor (Staskawicz et al. 1995). This gene-for-gene interaction triggers one or more signal transductionpathways that in turn activate defense responses in the plant

to prevent pathogen growth (Hammond-Kosack and Jones1996; De Wit 1997). These defense responses include thedevelopment of the HR, expression of PR proteins, and ac-cumulation of SA and can lead to the development of SAR(Ryals et al. 1996; Dempsey et al. 1999; Kombrink andSchmelzer 2001). Ethylene and jasmonic acid may also beinvolved in signalling the defense responses in the gene-for-gene interaction (Deikman 1997; Dong 1998). Efforts toclone an array of R genes involved in fungal disease resis-tance have met with some success (Bent 1996; Crute andPink 1996; Baker et al. 1997; De Wit 1997; Hammond-Kosack and Jones 1997; Ellis and Jones 1998;). The R-gene

products that have been cloned from tomato, tobacco, rice,flax, Arabidopsis, and several other plant species sharedoneor more similar motifs: a serine or threonine kinase domain,a nucleotide binding site, a leucine zipper, or a leucine-richrepeat region, all of which may contribute to recognitionspecificity (Shirasu and Schulze-Lefert 2000; Takken andJoosten 2000). The Hm1 R gene cloned from maize is anexception, as it encodes for a NADPH-dependent reductasethat inactivates the potent toxin produced by race 1 strainsof Cochliobolus carbonum (Johal and Briggs 1992).Many R genes belong to tightly linked multigene families, e.g.Cf-4 to Cf-9 encoding resistance to Cladosporiumfulvum mold oftomato (Thomas et al. 1997).

There are several examples of the expression of R genesin transgenic plants. The overexpression of the HRT gene,which controls the HR to turnip crinkle virus in Arabidopsis,

227

did not confer enhanced resistance to Peronosporatabacina (Cooley et al. 2000). The authors proposed thatmultiple factors may be involved in determining theresistance re-sponse, or that the resistance may be HR-independent. Ex-

pression of the Cf-9 gene, which confers resistance intomato to races of Cladosporium fulvum, in transgenic

to-bacco and potato gave rise to the HR when challenged withAVR9 peptide (Hammond-Kosack et al. 1998), indicatingthat the Cf-9 gene product was produced. However, for thedisease resistance mediated by R gene - Avr factor to befully expressed, several additional loci may be required(Hammond-Kosack and Jones 1996; Baker et al. 1997), inaddition to elevated levels of SA (Delaney et al. 1994). Re-sults to date suggest that the expression of cloned R genesin heterologous transgenic plants is unlikely by itself to en-hance tolerance to fungal pathogens because of the com-plexity of the interacting signalling pathways. Acombination of several interacting genes, similar to that forthe antifungal proteins, will likely be required. An enhanced

understanding of R-gene structure and function could, how-ever, make it possible to modify functional domains in thefuture to tailor R genes for use in providing broad-spectrumresistance to diseases in transgenic plants (Bent 1996;Dempsey et al. 1998). Other potential approaches to the use ofR genes for engineering disease resistance in plants arediscussed by Rommens and Kishore (2000).

Scientific challenges

Besides identifying and cloning potentially useful genesto engineer into plants, the development of transgenic plantswith enhanced fungal disease resistance faces additionalchallenges. Depending on the plant species, transformation

frequencies can be as low as 1-10%, and out of hundreds ofconfirmed transgenic lines, only a few may have appropri-ate transgene expression levels. Recent advances in planttransformation should provide new opportunities to over-come some of these difficulties (Gelvin 1998; Hansen andWright 1999; Newell 2000). The positive relationship ofhigh levels of PR proteins and antifungal compounds withenhanced disease resistance in plants has been documentedin many but not all cases. However, as indicated previously,there are a number of examples where transgene productsexpressed at high levels induced plant cell damage or hadother undesirable effects. These include the engineered ex-

pression of thionins, RIP, peroxidase, H2O2, elicitor mole-cules, and growth regulators. In most instances, constitutive

promoters have been used to achieve high expression levelsthroughout most tissues of the plant. In crops affected bypathogens that colonize more than one type of organ, e.g.roots and leaves, this is advantageous. In instances whereonly specific tissues need to be protected, e.g. leaves, fruit,or seed, or where the antifungal compounds need to be ex-

pressed at certain targeted sites in the cell, specific promot-ers would need to be identified (Bushnell et al. 1998;Dahleen et al. 2001). Wound-inducible and pathogen-inducible promoters, which have advantages for engineeringspecific disease resistance against fungal pathogens by ex-

pressing antifungal compounds only at sites of infection orwounds, have also been described (Roby et al. 1990;Strittmatter et al. 1995; Keller et al. 1999). Targeting of the


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engineered protein to the apoplastic space or to the vacuolehas been achieved in numerous previous studies and mayenhance the antifungal activity, depending on the mode ofinfection of the pathogen. Future research will require thefine tuning of engineered gene expression and establish-ment of the optimal expression levels and target site in thecell needed to prevent pathogen infection.

Commercial development

Compared to the demonstrable scientific and economicsuccess in engineering crop plants for resistance to herbi-cides, insect pests, and virus diseases (Shah et al. 1995), en-gineering of plants with enhanced disease resistance haslagged behind. Despite close to 100 published scientific re-ports (Table 1), of which about 30% are on tobacco used asa model system, only a few of the transgenic crops havebeen field-tested, and wide-scale deployment may not yetbe realized for another 5-10 years. Development of trans-genic plants with enhanced disease resistance is also beingactively pursued in the private sector, and recent unpub-

lished developments may not be included here. It remains tobe demonstrated under field conditions to which level dis-ease resistance is achieved and whether it is against a rangeof phytopathogens or only specific diseases. It is notewor-thy that many of the successfully controlled pathogens inlaboratory and greenhouse evaluations are those with a widehost range, such as Rhizoctonia solani and Botrytiscinerea,for which there are few available sources of resistancethrough conventional breeding in most crops. This is partic-ularly true also for seedling-infecting pathogens, for whichthere are few examples of genetic resistance in the host.Therefore, genetic engineering of novel disease resistancetraits in crop plants has the potential to provide control of

devastating pathogens with reduced fungicide applications.Expression of an antifungal trait throughout the growingseason, from seed to harvest, under prolonged disease-conducive conditions, can also provide significant advan-tages for disease management using this technology.

Issues to be addressed

The current challenges and issues surrounding the accep-tance of genetically modified foods will need to be ad-dressed before crop plants engineered for increasedresistance to fungal pathogens are successfully brought tomarket (Hilder and Boulter 1999; Barton and Dracup 2000;Kaeppler 2000). These issues include: potential spread ofthe trait to closely related weedy species and impact ontheir ecology; possibility of nontarget effects on other dis-eases, pests, or beneficial microorganisms; potential healtheffects of the overexpression of antimicrobial proteins infoods; and possibility of promoting new pathogen strainswith resistance to or that are able to overcome the novel en-gineered trait.

The spread of novel engineered genes from crop plants toweedy relatives has been demonstrated in some plant spe-cies through movement of pollen (Hails 2000; Wilkinson etal. 2000). Could a weedy sexually compatible relative of acrop species benefit from the potential introduction and ex-pression of a disease-resistant phenotype? Weeds are known


to harbour inoculum of a wide range of fungal pathogens(Agrios 1997) and these pathogens may maintain a balanceover weed growth. The potentially accelerated growth of aweedy species via introgression of the disease-resistanttransgene, however, may be balanced by the reduction inprimary inoculum that can be generated from weedy hostsadjacent to commercial crop fields, thereby reducing dis-

ease pressure. The extent to which the fitness of a weedyspecies may be enhanced by introgression of a disease-resistant phenotype has yet to be evaluated.

Nontarget effects on other diseases, pests, or beneficialmicroorganisms will have to be monitored in crop plants en-gineered to express antifungal or antimicrobial compounds.While unpredicted beneficial effects against other relatedfungal pathogens may be a positive aspect, an assessment ofthe effects on unrelated fungi, viruses, or bacteria may needto be conducted. It is unwieldy for researchers involved inthe development of genetically engineered plants to screenagainst a multitude of diseases or pathogens common tothat crop, an approach that may be taken by plant breedersduring development of a new cultivar. The results in Table 1

demonstrate the specificity of the evaluation approach usedfor transgenic plants, which is conducted mostly underaxenic or controlled environment conditions, and which in-frequently includes more than one pathogen for challengeinoculation. A report of enhanced resistance to tobacco mo-saic virus and potato virus X in a -1,3-glucanase-deficienttobacco mutant (Iglesias and Meins 2000) suggests that

plants overexpressing this enzyme to enhance fungal resis-tance should be tested for potentially enhanced susceptibil-ity to viruses. Antisense transformation of tobacco to

produce -1,3-glucanase-deficient plants showed that theseplants had increased deposition of callose in response to in-fection and had fewer lesions due to tobacco mosaic virusand tobacco necrosis virus (Beffa et al. 1996). How would

the overexpression of antimicrobial compounds in the rootsof genetically engineered plants alter their compatibilitywith mycorrhizae or beneficial endophytic fungi, or withvarious rhizosphere-colonizing microbes that could inhibitthe development of soilborne pathogens? Evaluations ofthese potential effects have been conducted in a few studies(Vierheilig et al. 1993, 1995; Lottmann et al. 2000; Lukowet al. 2000; Lottmann and Berg 2001). No side effects havebeen found so far, except for one study in which cultivar-specific alterations in rhizosphere bacteria were found in atransgenic canola line (Siciliano and Germida 1999). Jointcollaborations between molecular biologists and plant pa-thologists should foster the appropriate evaluations of thesetransgenic plants.

The potential of antimicrobial compounds to act as aller-gens or toxins when consumed by humans (Franck-Oberaspach and Keller 1997) would require that the guide-lines established by the appropriate regulatory agencies befollowed for the countries where the crops are grown andmarketed (Kaeppler 2000).

The possibility of selecting pathogen strains with resis-tance to the engineered trait may be increased with thewidespread deployment of transgenic crops expressing spe-cific antimicrobial compounds or that have broad-spectrumdisease resistance. Fungal pathogens have demonstrated thecapability for rapid change in genetic structure in the face


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of selection forces, such as highly specific fungicides, ma-jor disease resistance genes, and environmental factors. Theselection imposed by antimicrobial proteins, for example,could force the evolution of adaptive strategies in the patho-gen to defend against the inhibitory compounds. Such a co-evolution has been proposed for chitinases (Bishop et al.2000), in which adaptive functional modifications of the en-

zyme active site have occurred. Similarly, changes in sensi-tivity of pathogens to antimicrobial proteins overexpressedin transgenic plants could be selected. The use of combinedgenes that target different sites could reduce the selection

pressure imposed on the pathogen. Genetically engineeredplants with successfully enhanced disease resistance shouldnot be viewed as a panacea and continual monitoring forunexpected events will be necessary.

Future prospects

The tremendous scientific progress made since 1991 ingenetic engineering of plants for enhanced resistance tofungal pathogens as described in this paper is an indication

of the high level of interest in the scientific community onthis subject. As the technology evolves toward the use oftissue-specific or pathogen-inducible promoters, the expres-sion of engineered traits that are effective against a broadrange of pathogens, and the utilization of synthetically de-rived peptides and of R genes, the impact on disease man-agement will be enhanced. Evaluation of these transgenicplants for response to disease will need to be extended tofield trials and appropriate agronomic data collected to en-sure that this technology can be successfully implementedin farmers fields to augment on-going disease managementpractices. Transgenic plants with enhanced disease resis-tance can become a valuable component of a disease man-agement program in the future.

Acknowledgements

I thank The Canadian Phytopathological Society for theopportunity to prepare this paper as the Past-presidentscontribution, W.P. Chen for bringing to my attention rele-vant research articles, M. Nguyen for typing the manuscript,and the Natural Sciences and Engineering Research Councilof Canada for their continued financial support.

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