University of Groningen

Molecular genetic characterisation of the Asc locus of tomato conferring resistance to thefungal pathogen Alternaria alternata f. sp. lycopersiciBiezen, E.A. van der; Overduin, B.; Kneppers, T.J.A.; Mesbah, L.A.; Nijkamp, H.J.J.; Hille, J.

Published in:Euphytica

DOI:10.1007/BF00022521

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Citation for published version (APA):Biezen, E. A. V. D., Overduin, B., Kneppers, T. J. A., Mesbah, L. A., Nijkamp, H. J. J., & Hille, J. (1994).Molecular genetic characterisation of the Asc locus of tomato conferring resistance to the fungal pathogenAlternaria alternata f. sp. lycopersici. Euphytica, 79(3). https://doi.org/10.1007/BF00022521

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https://doi.org/10.1007/BF00022521https://research.rug.nl/en/publications/molecular-genetic-characterisation-of-the-asc-locus-of-tomato-conferring-resistance-to-the-fungal-pathogen-alternaria-alternata-f-sp-lycopersici(628af7fa-05e8-4509-bf9f-56c067611142).htmlhttps://doi.org/10.1007/BF00022521

Euphytica 79: 205-217,1994 .© 1994 Kluwer Academic Publishers . Printed in the Netherlands .

Molecular genetic characterisation of the Asc locus of tomato conferringresistance to the fungal pathogen Alternaria alternata f. sp. lycopersici

E.A. van der Biezen, B . Overduin, T.J.A. Kneppers, L.A. Mesbah, H.J.J. Nijkamp & J . HilleDepartment of Genetics, Institute for Molecular Biological Sciences, BioCentrum Amsterdam, Vrije Universiteit,De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands

Key words: AAL-toxins, Alternaria alternata, Asc locus, Lycopersicon esculentum, positional cloning, transposontagging

Abstract

The Alternaria stem canker disease of tomato is caused by the fungal pathogen Alternaria alternata f. sp . lycopersiciand its host-selective AAL-toxins . Resistance to the pathogen and insensitivity to the toxins are conferred by theAsc locus on chromosome 3L . Sensitivity to AAL-toxins is a relative character ; the toxins inhibit developmentof all tested tomato tissues but susceptible cultivars are much more sensitive than resistant cultivars . In additionto tomato, some other plant and animal species are sensitive to the toxins as well. The likely mode of action ofAAL-toxins is interference with sphingolipid biosynthesis by specific inhibition of ceramide synthase activity . Tomolecularly isolate Asc, transposon tagging and positional cloning strategies are applied . As a first step, transposoninsertions and restriction fragment length polymorphism (RFLP) markers are identified in proximity of the Asclocus. Subsequently, the transposons are used to inactivate Asc by insertion mutagenesis, and the RFLP markers areused to identify yeast artificial chromosomes (YACs) with tomato DNA inserts . Once an Asc-insertion mutant and/ora YAC encompassing Asc has been obtained, physical isolation and characterisation of Asc will be conceivable .Elucidation of the molecular role ofAsc will illuminate the specificity of host recognition by Alternaria alternataf. sp . lycopersici .

Abbreviations: AAL-toxin - Alternaria alternata lycopersici-toxin ; A . a. lycopersici - Alternaria alternata f . sp .lycopersici ; Asc - Alternaria stem canker ; HST - host-selective toxin

205

Introduction

Understanding the molecular specificity of host recog-nition by plant pathogens offers an attractive chal-lenge for phytopathologists. Although plants are con-tinuously exposed to potential parasites, only a fewinfect their hosts . The molecular mechanisms under-lying the specificity of these successful host-pathogeninteractions remain to be solved . However, the basisof host recognition is generally unpredictable becausethe pathogens have multiple effects on the host cells,and several host strategies are employed to preventpathogen infection . Additionally, the dominant orrecessive nature of inheritance of a resistance locushas little bearing on the function of the gene products

involved . Therefore, the physical isolation and char-acterisation of resistance and susceptibility genes willgreatly contribute to an understanding of the specificityof host recognition .

Present host plants and fungal pathogens are sup-posedly the result of co-evolution : through parasiticadaptation, necrotrophic fungi developed to plantpathogens . The lowly adapted facultative saprophyteskill host cells by toxins and obtain the nutrients fromdead cells, while the highly adapted obligate biotroph-ic parasites achieve nutrients from living host cells(Heath, 1987). It is anticipated that for these differenttypes of pathogens, plants developed different defencestrategies (De Wit, 1992 ; Walton & Panaccione, 1993) .The biotrophic pathogens illustrate gene-for-gene rela-

206

6w

PLANT GENOTYPEplant rcsisluncc + target

W

WFo TOx+w05 TOX"

R •1' rT

V

PLANT ( : KNOTY I'IStarget

1' t

Fig . 1 . Plant-fungal HST interaction models . In both cases thetarget for the HST is encoded by the T locus . Plants with an activedefence mechanism (left) have a resistance locus R that acts upon theHST rendering it nonactive and, consequently, have an incompatibleinteraction (I) with the fungus . Absence of the resistance locus (r)results in the inability to induce a resistance response and, hence,in a compatible interaction (C) . Plants that do not have a defencemechanism at their disposal (right) can resist fungal infection (I) ifthey also lack the target locus (t) . Plants that do contain the T locusare susceptible to fungal infection (C) (modified from Heath, 1993) .Abbreviations: T - target present, t - target absent, R - resistancepresent, r - resistance absent, I - incompatible interaction, C -compatible interaction.

tionships by releasing elicitors which are specifical-ly recognised by the hosts that subsequently activateresistance responses . Host susceptibility in these cas-es results from the inability to respond with a resis-tance reaction and is inherited as a recessive trait (e .g .tomato-Cladosporiumfulvum, Van den Ackerveken etal., 1992) .

Different plant defence strategies operate againstsaprophytic pathogens . Some strains of the saprophyticfungi Cochliobolus (Helminthosporium) and Alternar-ia alternata secrete metabolic compounds that areselectively toxic to their corresponding hosts (mono-cots and dicots, respectively) . These host-selective tox-ins (HSTs) display the same host specificity as thepathogen itself and are the primary pathogenicity fac-tors (Otani & Kohmoto, 1992 ; Walton & Panaccione,1993). Research on the effects of the HSTs from thesepathogens resulted in accumulating support that sus-ceptibility can, apart from the inability to induce aresistance response, also be a consequence of the lackof a resistance mechanism (Fig . 1, right) . This situ-ation is illustrated by two host-Cochliobolus interac-tions; 1) C. heterostrophus secretes T -toxin causingsouthern corn leaf blight in cms-T lines of maize (Zeamays). Susceptibility is maternally inherited becausethe biochemical target for T -toxin, the URF13 protein,is mitochondrially encoded. Some maize lines do nothave a resistance mechanism but simply lack the gene

coding for URF13, and therefore, are resistant to fun-gal infection (Levings III, 1991 ; Levings III & Siedow,1992). 2) C. victoriae produces victorin (HV toxin)that causes victoria blight in oats (Avena sativa) . Theproduct of the Vb gene is assumed to be involved information of the target of victorin . Lines containingVb are susceptible to pathogen infection, and thereforesusceptibility is inherited as a dominant trait (Wolpert& Macko, 1991 ; Akimitsu et al ., 1993) . Host suscep-tibility, in these cases, is a genotype specific propertyand relies on the presence of susceptibility genes (urf13or Vb) which encode targets for the HSTs (T toxin orvictorin) . The resistant hosts lack the targets or havetargets with low affinity to HSTs and thus, active resis-tance mechanisms are absent .

Conversely, hosts that do harbour functional resis-tance mechanisms respond with active strategies, e .g.HST detoxification (Fig. 1, left) . This possibility isexemplified by C. carbonum that produces HC-toxinand causes leaf spot in susceptible maize lines . Thedominantly inherited HMI gene encodes an enzymethat chemically modifies HC-toxin . The target for HC-toxin is not known yet (Meeley et al ., 1992 ; Johal &Briggs, 1992) .

The fungal pathogen Alternaria alternata f. sp .lycopersici secretes host-selective AAL-toxins that areinvolved in the molecular recognition of susceptiblehosts . Only tomato (Lycopersicon esculentum) geno-types harbouring the Alternaria stem canker (Asc)locus are insensitive to the toxins and, hence, resis-tant to fungal infection. This paper deals with theprogress in understanding the molecular specificity ofhost recognition by the pathogen that is determined bythe two alleles of the Asc locus .

Alternaria stem canker disease of tomato

The Asc locus

The Alternaria stem canker disease of tomato and thecausal fungus, Alternaria alternata f . sp . lycopersici,were first reported in 1975 in California, USA (Groganet al ., 1975) . The typical symptoms are slowly enlarg-ing dark-brown-to-black cankers on the stems near thesoil line or aboveground . Topmost leaflets developnecrotic areas between the veins or, in later stages,become completely necrotic (Fig . 2). Associated withthe disease are the AAL-toxins, produced by the fun-gus, which can be isolated from diseased plants andelicit identical symptoms to those that develop on nat-

207

Fig . 2 . Interaction between tomato Asc genotypes and Alternaria alternata f. sp. lycopersici and its host-selective AAL-toxins . (A) Necroticlesions on a leaflet from a susceptible cultivar (asc/asc) infected with fungal spores ; (B, C) Alternaria stem cankers of infected susceptible plants(asc/asc) at the seedling and the mature stage, respectively ; (D) Fungal cultures growing on liquid (left) and solid (right) media ; (E) Computerprediction of the three-dimensional structure of AAL-toxins (white = hydrogen, grey = carbon, red = oxygen, blue = nitrogen) ; (F) Necrosis onleaflets of the three Asc genotypes in bioassays using 0 .2 MM AAL-toxins (see text for description) ; (G) Inhibited development of seedlings ofthe three Asc genotypes germinated in the presence of 0.6 pM and 3 µM AAL-toxins ; controls contain water only .

208

TOMATO CHROMOSOME 3

o- TG479

21- TG585

Fig. 3. Genetic map of the long arm of chromosome 3 containing theAsc locus, some RFLP markers and the morphological markers bls(baby lea syndrome) and sf (solanifolia) . Distances are expressed incM, the grey rectangle denotes the centromere . SIJ1515T and ET90are T-DNA insertions carrying an Ac and a Ds element, respectively .

urally infected plants (Gilchrist & Grogan, 1976 ; Siler& Gilchrist, 1983). In 1977 the disease was detected inJapan in susceptible tomatoes and shown to be causedby the same fungal species (Kohmoto et al ., 1982). Thepathogen was also identified in other parts of the USA(Abbas & Vesonder, 1993 ; Abbas et al ., 1993b), and inSouthern Europe (Smith et al ., 1988) . It is not knownwhether the occurrence in the different geographicalareas resulted from independent development of newpathotypes or introduction of the pathotype into anoth-er region .

Following the identification of susceptible andresistant tomato lines, the segregation of resistancewas investigated . One single locus with two allelessegregated in a Mendelian fashion and was designat-ed Asc for Alternaria stem canker (Gilchrist & Gro-gan, 1976). Heterozygotes (Asclasc) show intermedi-

ate phenotypes in AAL-toxin sensitivity assays, but canfully resist fungal infection . Therefore, theAsc locus isdominant for resistance to infection by the pathogen butsemi-dominant for insensitivity to AAL-toxins . Near-isogenic Asc lines (NILs) were made by continuousselfing (F9) of heterozygotes and have proven to bevery useful in characterising the sensitivity to AAL-toxins (Clouse & Gilchrist, 1987) . Genetic mappingwith morphological markers revealed Asc to be flankedby solanifolia (sf) and baby lea syndrome (bls) on thelong arm of chromosome 3 (Witsenboer et al ., 1989 ;Van der Biezen et al ., 1994b). BecauseAsc has not beenintrogressed from wild relatives and there are virtual-ly no DNA polymorphisms among tomato cultivars,the resistant wild tomato L. pennellii was crossed witha susceptible line. The interspecific progenies segre-gated for resistance and permitted restriction fragmentlength polymorphism (RFLP) analysis. Chromosome3 specific RFLP markers (Tanksley et al ., 1992) placedAsc on the middle of chromosome 3L (Fig . 3) (Van derBiezen et al ., 1994a) .

Alternaria and its AAL-toxins

Alternaria alternata (Fries) Keissler (A. tenuis Nees)is a widespread saprophytic filamentous fungus thatis commonly found in the soil and on agriculturalproducts after harvest (Tiejin & Ceponis, 1982) . Thespecies is composed of many strains (formae speciales)that are classified according to morphological similari-ties at the asexual stage (Simmons, 1967) . Seven strainsare known that, in addition to a saprophytic existence,also infect and colonise specific plant species and,therefore, are referred to as pathotypes of A. alternata(Otani & Kohmoto, 1992) . These facultative sapro-phytes are distinguished by their host range and pro-duce different HSTs which, in most cases, consist ofmultiple closely related molecules (Table 1) . Frequent-ly, A . alternata isolates spontaneously lose their abilityto produce toxins . The toxin-less mutants simultane-ously fail to infect their host plants and become indis-tinguishable from strains that are exclusively sapro-phytic. Two tomato diseases are known that are causedby A . alternata : 1) blackmold, a ripe tomato fruit rotcaused by a saprophytic form (Grogan et al ., 1975 ;Pearson & Hall, 1975), and 2) Alternaria stem canker,caused by A. alternata f . sp . lycopersici that affectstomato stems, leaves and green fruits (Grogan et al .,1975) .

A. alternata f. sp . lycopersici has a limited hostrange. Following inoculation of 265 tomato cultivars

Table 1 . The Alternaria alternata pathotypes with their host-selective toxins and thedisease of the susceptible hosts .

Fig . 4. Chemical structure of the AAL-toxins . The 17-carbon back-bone of all AAL-toxins is the 1-amino-dimethyl-heptadecapentol(upper panel) . The AAL-toxin analogues TA, TB, Tc, TD, and TEdiffer with respect to the attached molecules at C-I (R I ), C-4 (R2)and C-5 (R3) (lower panel). Only the structures of the AAL-toxinisomers-I are shown . Isomers-2 have opposite groups at the C-13and C-14 positions, i .e . hydrogen at C-13 and propane-tricarboxylicacid at C-14 .

with a spore suspension, 25% were susceptible topathogen infection (Grogan et al ., 1975) . Infection

209

assays of divergent families showed all tested plantspecies to be resistant to the fungus and insensitiveto the AAL-toxins (Gilchrist & Grogan, 1976) . There-fore, tomato was initially thought to be the only speciessusceptible to A. a. lycopersici infection. However,examination of additional species showed that Lycop-ersicon cheesmanii, a wild tomato relative from theGalapagos Islands, was susceptible to fungal infection(Van der Biezen et al ., 1994a). The AAL-toxins possesstypical host-selective characteristics: in all cases testeda positive correlation was found between susceptibilityto fungal infection and sensitivity to AAL-toxins .

The chemical structure of AAL-toxins was elu-cidated by mass spectroscopy and nuclear mag-netic resonance (NMR) spectrometry. AAL-toxinsare 1-amino-dimethyl-heptadecapentols esterified topropane-tricarboxylic acid with an average molecu-lar weight of 521 Da (Fig . 2). The toxins consistof at least five related molecules : TA, TB, Tc, TD,and TE, and each toxin itself consists of two iso-mers (Fig. 4) (Bottini & Gilchrist, 1981 ; Bottini etal., 1981 ; Clouse et al., 1985 ; Caldas et al., 1994) .The TA and TB analogues have similar specific activ-ity which is 30-400 times higher than that of the otherforms (Caldas et al ., 1994) . Therefore, only TA and TBtoxins have been used in experiments described hereand are referred to as AAL-toxins . Concentrations ofAAL-toxins are determined by a quantitative chemicalassay using high performance liquid chromatography(HPLC) (Siler & Gilchrist, 1982) . Determination of thethree-dimensional structure and synthetic productionof AAL-toxins are in progress (Oikawa et al ., 1994) .The fungus maintains its capacity to produce toxinswhen cultured on various artificial media. Differentamounts of toxins are produced, however, among dif-ferent culture substrates and different fungal isolates

Pathotype Host-selective toxins Disease

Apple AM-toxin I, II, III Alternaria blotchJapanese pear AK-toxin I, II Altemaria black spotRough Lemon ACRL-toxin I Alternaria brown spotStrawberry AF-toxin I, II, III Altemaria black spotTangerine ACT toxin A, B Altemaria brown spotTobacco AT toxin Altemaria brown spotTomato AAL-toxin TA, TB, Tc, TD, TE Alternaria stem canker

TOXIN R 1 R2 R3

TA H OH OH

TB H OH H

TC H H H

TD C (=0) CH3 OH H

TE C (=0) CH3 H H

2 10

Table 2 . Physiological effects of AAL-toxins on tomato Asc genotypes .

(Siler & Gilchrist, 1983 ; Gilchrist et al ., 1992 ; Abbas& Vesonder, 1993 ; Shepard et al ., 1993) .

Sensitivity to AAL-toxins

The AAL-toxins have been invaluable for the charac-terisation of the tomato-A . a. lycopersici interaction .The responses to AAL-toxins were studied of varioustissues from resistant (Asc/Asc), susceptible (asc/asc)and heterozygous (Asc/asc) genotypes (Fig. 2). Thephysiological effects of the toxins include 1) develop-ment of necrotic lesions on leaves and fruits ; 2) inhi-bition of in vitro development of calli, pollen, rootsand shoots ; and 3) reduced viability of protoplasts andsuspension cells (Table 2). It has been demonstratedthat AAL-toxins inhibit plant cell development at var-ious levels of differentiation, that sensitivity to AAL-toxins is present at vegetative and generative tissues,and that Asc is expressed at the level of an individu-al cell . The concentrations of AAL-toxins necessaryto cause symptom development vary for the differenttissues, indicating differences in effectiveness of thetoxins. All tissues showed similar genotype-specificdifferential responses with the resistant tissues beingless sensitive than the susceptible tissues . For tissuesfrom plants that are heterozygous for Asc, intermedi-ate responses to AAL-toxins were demonstrated . Therelative insensitivity of these heterozygotes (Asc/asc)can be overcome by application of a high concentra-tion of AAL-toxins. Accordingly, the toxin concentra-tion employed in the bioassays determines the sensi-tivity responses to AAL-toxins and, hence, the mode

Physiological effect

browning, growth inhibitiongrowth inhibition, reduced viabilitynecrotic lesionsshoot induction inhibition/necrotic lesionsswollen, vesiculatedswollen, leached matrix, reduction of cristaereduced viabilitynecrotic lesionsgermination and tube growth inhibitiongrowth inhibition

I - = no effect; +, ++ and +++= relative differences of effect ; nd = not determined2 (1) Bino et al., 1988 ; (2) Fuson & Pratt, 1988 ; (3) Gilchrist et al., 1992 ; (4) Kodama et al., 1991 ; (5) Moussatos et al., 1993b; (6) Parket al., 1981 ; (7) Van der Biezen et al., 1994a ; (8) Witsenboer et al., 1988 ; (9) Witsenboer et al., 1989 ; (10) Witsenboer et al., 1992

of inheritance of insensitivity . Insensitivity to AAL-toxins inherits as a (semi)dominant trait when low toxinconcentrations are used, when high concentrations areapplied insensitivity inherits in a recessive fashion .

For sake of sensitivity and simplicity, leaflet bioas-says are most often applied for the determination ofsensitivity to AAL-toxins (Fig. 2) . After incubationof detached leaflets on filter papers saturated with adilution of AAL-toxins in sealed petridishes, the lev-el of necrosis is assessed (Clouse & Gilchrist, 1987) .Because susceptibility to fungal infection has until nowalways been correlated with sensitivity to AAL-toxins,leaflet bioassays are also performed for plant genotyp-ing. The percentage of necrosis is directly proportionalto the concentration of AAL-toxins, and the durationof exposure to AAL-toxins . Therefore, the leaves areexposed to specific concentrations (usually 0 .2 µM)and during defined periods (usually 72 h) . In addition,the leaves need to be exposed to light for a minimumof 12 h to allow necrosis to develop (Witsenboer et al .,1992 ; Moussatos et al., 1993b). Finally, aging playsa role, indicating a developmental regulation of Ascsensitivity . The youngest leaflets on a leaf are about 3times more sensitive then the older following leaflets(Moussatos et al., 1993b) .

In the physiological complexity of AAL-toxin-treated tomato tissues, it is difficult to distinguish caus-es from effects. Moreover, the conclusions are limit-ed of experiments that did not include time-responseand AAL-toxin dosage-response courses . The physi-ological studies have shown the consequences of theAAL-toxins rather than their mode of action . To deter-

Asc/Asc Asc/asc asc/asc reference 2

+ 1 nd ++ (4)(8)(9)+ nd ++ (2)(4)(8)

nd + (3)+ nd ++ (8)(9)

nd + (6)nd + (6)

+ ++ +++ (5)(10)+ ++ +++ (5)(7)(10)+ nd ++ (1)+ ++ +++ (7)(8)(9)

Tissues/cells/organelles [AAL](1M)

Calli 0.1-1Cell suspensions 3Fruits 0 .02Leaf discs 0 .02/1Leaf endoplasmatic reticula 20Leaf mitochondria 20Leaf protoplasts 10Leaves 1Pollen 60Roots 0.2

mine the cause of toxicity to sensitive tissue, and toexplain the mechanism of insensitivity to the toxinsand resistance to the fungus, the biochemical target ofthe AAL-toxins needs to be revealed .

Targets ofAAL-toxins

Before necrotic symptoms can be detected on detachedleaflets of a susceptible line, AAL-toxins at low con-centration cause a specific decline in the sucrose uptakecapacity, and an accumulation of the ethylene precursor1-aminocyclopropane-l-carboxylic acid (ACC), fol-lowed by an increase in the production of ethylene(Moussatos et al ., 1993a, 1994). Using exogenous sup-plied ACC and inhibitors of endogenous ACC, a cor-relation between ethylene production and AAL-toxin-induced necrosis was demonstrated . In addition, it wasobserved that the pyrimidine precursor dihydro-oroticacid (DHO) reduced the AAL-toxin-induced necrosison tomato leaflets, possibly by suppression of ACCsynthesis. From these results it was hypothesised thatthe action ofAsc is linked to sucrose transport, ethylenebiosynthesis, pyrimidine metabolism and cell death(Moussatos et al ., 1994). The suggestion that AAL-toxins disruptpyrimidine metabolism by inhibiting theactivity of aspartate carbamoyltransferase (ACTase)(Gilchrist, 1983) could not be confirmed experimental-ly (Fuson & Pratt, 1988 ; Kodama et al ., 1991 ; Abbaset al ., 1992) . In addition, it was demonstrated that Ascdoes not encode ACTase (Overduin et al ., 1993) .

By analysing the contents of 18 amino acids and5 related compounds in leaves from infected plantsand in AAL-toxin-treated leaves, a specific accumula-tion of ethanolamine (EA) and phosphoethanolamine(PEA) was observed (Kawaguchi et al ., 1991) . SinceEA and PEA are the primary and secondary inter-mediate metabolites of the phospholipid biosynthe-sis pathway in higher plants, the possible interfer-ence of AAL-toxins with this pathway was furtherinvestigated. Following application of 14C-EA pre-cursor to susceptible AAL-toxin-treated leaf discs,strong inhibition of the incorporation of EA into phos-phatidylethanolamine (PtdEA) was observed (Orolazaet al ., 1992) . Therefore, enzymes of the phospholipidpathway were suggested as potential biochemical tar-gets for AAL-toxins .

The involvement of AAL-toxins in sphingolipidbiosynthesis was found following experiments withfumonisins (reviewed in Abbas et al ., 1993a; Norred,1993 ; Riley et al ., 1993 ; Merrill et al ., 1993) . Fumon-isins are a group of toxins that share structural homolo-

21 1

gy with AAL-toxins and were first identified in culturesof the fungus Fusarium moniliforme (Sheldon) and,later, also in A . a. lycopersici cultures (Bezuidenhout etal., 1988; Gelderblom et al ., 1988; Chen et al ., 1992) . Fmoniliforme and, hence, the fumonisins are a commoncontaminant of maize grain (Zea mays) throughout theworld. Fumonisins are not acutely toxic to maize itself,but are carcinogenic in laboratory rats and cause iden-tical symptoms to domestic animals as various diseasesattributed to F moniliforme-contaminated feed. Asso-ciated with human consumption of infected grains isa higher incidence of esophageal cancer . Results ofphysiological and biochemical experiments indicatethat AAL-toxins and fumonisins have a similar modeof action . Both toxins have the same spectrum of phy-totoxicity : they elicit similar genotype-specific symp-toms in tomato (Gilchrist et al., 1992; Mirocha et al .,1992) and have identical effects on jimsonweed (Datu-ra stramonium), black nightshade (Solanum nigrum)(Abbas et al ., 1992, 1993b), and duckweed (Lemnapausicostata and L. minor) (Vesonder et al ., 1992a ;1992b ; Tanaka et al ., 1993). In addition, fumonisinsand AAL-toxins showed to be toxic to rat liver and dogkidney cells (Shier et al ., 1991 ; Mirocha et al., 1992 ;Vesonder et al ., 1993) .

The suggested mechanism of action of AAL-toxinsand fumonisins was derived from the observation thatthese compounds bear structural similarity to sphin-ganine, a primary constituent of sphingolipids (Fig .5) . It was observed in rat hepatocytes that the toxinsinhibit the incorporation of 14C-serine into sphingo-sine and that the level of 14C-sphinganine increased .Subsequently, it was shown that the fumonisins andthe AAL-toxins disrupt de novo sphingolipid biosyn-thesis by inhibiting ceramide synthase (sphinganineN-acyltransferase) (Wang et al., 1991 ; Merrill et al .,1993) . From their structural resemblance to sphin-ganine it was suggested that fumonisins and AAL-toxins are recognised by ceramide synthase as sub-strate analogues. As a result of the inhibition of thisenzyme, the ceramide biosynthesis decreases, leadingto the accumulation of sphinganine and a depletionof complex sphingolipids in several mammals, higherplants and yeast (Pichia ciferri) (Kaneshiro et al ., 1992,1993 ; Merrill et al., 1993; Riley et al ., 1993). Sphin-golipids have numerous functions in controlling cellbehaviour, including the regulation of cell receptorsand cell growth, differentiation, cell-cell communica-tions, stabilisation of membranes, sorting of lipids andproteins, and interactions with cytoskeletal elements(Hannun & Bell, 1989; Shier, 1992) . Evidently, AAL-

2 12

0

coo -SCoA +

H~.CH20H

Palmitoyl-CoA

Serene NH3

Sphinganine

CO,+ CoASH

0CH2H

3-Ketosphinganine

NH3N ADPHI

OHCH-OH

NH3Fany acyl-CoAI

OHCH20H

NH

OHCH20H

NTH

Ceramide

0

Fig. 5. Sphingolipid biosynthesis pathway and site of action ofAAL-toxins and fumonisins (after Merrill et al ., 1993) . AAL-toxinsinhibit ceramide synthase (sphinganine N-acyltransferase), leadingto accumulation of sphinganine and reduced synthesis of complexsphingolipids .

toxins would have a wide spectrum of effects at a widerange of tissues which is validated by the observedphysiological responses .

The genotype-specific differential sensitivity ofresistant and susceptible tomato lines that is regulatedby the different alleles of theAsc locus remains unclear .It can be postulated that host resistance or susceptibil-ity is due to chemical modification of the AAL-toxins.In explaining resistance, the dominant Asc allele mightbe involved in the detoxification of AAL-toxins . Fol-lowing modification, the toxins are not recognised byceramide synthase as substrate analogues and, hence,have lost their toxicity . Conversely, to explain suscepti-bility, the recessive asc allele might code for ceramidesynthase that upon inactivation by the AAL-toxinsleads to cell death. Another option is that the AAL-toxins themselves are harmless but are modified to tox-ic metabolites by an asc-encoded enzyme. In the latter

Sphinganine

Dihydroceramide

0

1Sphingosine

two cases, resistance would be based on the inability torecognise or convert AAL-toxins . Alternatively, boththe Asc alleles could encode ceramide synthase withdifferential affinity to AAL-toxins ; the dominant allele(conferring resistance) having less affinity to AAL-toxins than the recessive allele (conferring susceptibil-ity) . This suggestion also explains the relative actionofAsc (Van der Biezen et al ., 1993) . These hypothesesmay be tested by incubation of 14C-AAL-toxins withextracts from resistant and susceptible tomato leavesand subsequent determination of the structural conse-quences (Meeley et al., 1992 ; Alberts et al ., 1993) .As an alternative to these biochemical approaches, thefunction of Asc could also be clarified by following amolecular genetic approach .

Molecular genetic characterisation of the Asclocus

Transposon tagging of the Asc locus

For the isolation of genes that are only known by theirphenotype several procedures are applicable, includ-ing transposon tagging and positional cloning. Bothstrategies have successfully been used in tomato for theisolation of genes involved in resistance to pathogens(Martin et al ., 1993 ; Jones et al ., 1994) . For effectivetransposon mutagenesis several criteria have to be met(Van der Biezen et al ., 1994c) . First, inactivation of thetarget gene should result in a recognisable phenotype .With respect to the Asc locus, however, the outcome ofinsertional mutagenesis cannot be predicted . Consid-ering the hypotheses mentioned in the last paragraphexplaining the mode of resistance and susceptibility,either one or both Asc alleles could be active . Inactiva-tion of the Asc or asc allele would consequently leadto loss of resistance or loss of susceptibility, respec-tively. Therefore, two independent transposon taggingexperiments were conducted, each aiming at the inacti-vation of a different Asc allele . The second parameter intransposon tagging involves the observed small genet-ic distance of transposition . The efficiency of trans-poson mutagenesis can therefore be increased about100-fold by starting the experiment with a transpo-son close to the gene-of-interest . In collaboration withseveral laboratories, numerous transposon-containingT-DNA insertions were mapped on the tomato genome .The two T DNAs that mapped closest to Asc were cho-sen for transposon tagging of this locus : SLJ1515T

Inactivation of the resistance allele

I ` DI

Ac line

Inactivation of the susceptibility allele

transactivation of Ds:

Ds line

sAc line

asc wex

x

tester

F!

x

,,,T{{{,,,r{{{asc asc

AciAsc f fast

outcrossing and selfing :

F/

tester

i

i

(sAd.)asc

Fl

(sAc/sAc) - -

(MAd-)act aseOs

OC, and 2

asc //

me

Dr-4cFig. 6. Transposon tagging directed to different alleles of the Asclocus. Upper panel: Inactivation of resistance . Following crossingof the resistant (Asc/Asc) Ac line to the susceptible tester (asc/asc),heterozygous (Asc/asc) progenies are tested for lost resistance (i.e .susceptibility) as a result of insertional inactivation of the domi-nant Asc allele . Lower panel: Inactivation of susceptibility . First,the closely linked Ds transposon is transactivated by the stabilisedAc element (sAc). Subsequently, the F1 is selfed and outcrossed .The OCl and F2 progenies are screened for lost susceptibility (i .e .resistance) as a result of inactivation of the recessive asc allele .

(Thomas et al ., 1994) and ET90 (K. Theres, personalcommunication ; Knapp et al ., 1994) (Fig . 3) .

SLJ1515T contains an autonomous Ac element andwas located 21 cM distal of Asc. Because this Ac ele-ment had been introduced in an A . a . lycopersici resis-tant line (Asc/Asc), it was used for inactivation of thedominant Asc allele (Overduin et al ., in preparation)(Fig. 6). The Ac line was crossed to a susceptible cul-tivar (asc/asc) and heterozygous progenies (Asc/asc)were screened for loss of resistance as a result of trans-poson insertion into the dominant Asc allele. Out ofapproximately 20,000 progenies, one plant was identi-fied that was susceptible to A. a. lycopersici infectionand contained the original T -DNA and several trans-posed Ac elements . However, association of a specif-ic Ac insertion with susceptibility to fungal infectioncould not yet be demonstrated . Resolving the origin ofthe susceptibility of this plant is hampered by the factthat its progeny segregates for resistance . This remark-able mutant is currently being further characterised .

To test the hypothesis that the recessive asc allele isrequired for susceptibility to A. a . lycopersici infection,

213

a random chemical mutagenesis was conducted (Vander Biezen et al., in preparation) . Seeds of a suscepti-ble line (asc/asc) were treated with ethyl methanesul-fonate (EMS), and after selfing, the M2 progenies werescreened for insensitivity to AAL-toxins . In additionto numerous plants with mutant morphological phe-notypes, plants showing insensitivity to AAL-toxinswere recovered with high frequency . No differencesin the necrotic response to AAL-toxins were observedin leaves from these mutagenised and regular resistantlines (Asc/Asc and Asc/asc) . The mutants were resis-tant to fungal infection following inoculation with aspore suspension . Allelic tests and genetic mapping of9 independently derived resistant mutants demonstrat-ed that the recessive asc allele was mutated to dominantalleles . Therefore, it is concluded that the recessive ascallele confers susceptibility to A . a. lycopersici . ThisEMS mutagenesis approach to obtain resistance to A. a .lycopersici resembles the somaclonal variation-basedtechniques employed for inducing resistance. Resis-tance against various pathogens in diverse crops wasobtained by tissue culture and in vitro selection tech-niques, e .g . resistance against Fusarium oxysporum f.sp . lycopersici in tomato (Shahin and Spivey, 1986 ;reviewed in Van den Bulk, 1991) . With respect to oth-er A. alternata pathotypes, resistance and insensitivityto AK- and AM-toxins have been induced by y-rayirradiation of Japanese pear and apple (Tabira et al .,1993), and to AT toxins by in vitro selection of tobac-co protoplasts (Thanutong et al ., 1983) .

The results of the EMS mutagenesis ofasc indicatethat resistant plants can potentially also be obtainedby transposon mutagenesis . However, the nature ofan EMS-induced mutation differs from a transposon-induced mutation . A basepair alteration (EMS) in theasc gene leading to insensitivity to AAL-toxins couldresult in a gene product that is still functional but hasless affinity to AAL-toxins . In general, transposoninsertions in genes cause loss-of-function mutations .If AAL-toxins cause cell death by direct interventionwith the asc gene product, then transposon insertionsin this gene are expected to be lethal . However, thehigh frequency by which resistant plants were obtainedindicates that EMS caused non-lethal loss-of-functionmutations in asc .

For insertional inactivation of the asc allele, theET90 tomato transformant was used . The T-DNAof this susceptible (asc/asc) line contains a nonau-tonomous Ds transposon and was located 10 cM fromthe Asc locus (Fig . 3). For transactivation of Ds, astabilised Ac line was used in the same genetic back-

2 1 4

ground . Following selfing and outcrossing of the F1 to asusceptible tester stock, progenies are screened for lossof sensitivity to AAL-toxins by seedling assays (Fig .6) . Molecular analysis showed that Ds is somaticallyactive and that transposed Ds elements are germinallytransmitted with a frequency of 7% . Presently, 110,000progenies have been germinated on AAL-toxins, how-ever, no insensitive plants have been found . The recov-ery of such insertion mutant depends on the conditionsthat transposon inactivation of the susceptibility allele,using the Ds element at the ET90 position, resultsin a viable mutant that is insensitive to AAL-toxins .Estimations that include the frequency and distanceof transposition predict a mutation rate in the rangeof 10-5 (Van der Biezen et al ., 1994c). Based on themutation frequency and population size it is calculatedthat a 67% probability of finding an insertion mutanthas been reached .

Positional cloning of the Asc locus

To construct a high resolution map of the Asc locus,chromosome 3L specific RFLP markers (Tanksley etal., 1992) were hybridised with DNA of F2 plantsderived from an interspecific cross between L. pennelliiand L. esculentum (Van der Biezen et al ., 1994a). Twoclosely linked markers, TG134 (0 .7 cM) and TG442(1 .4 cM), were identified at both sides of the Asc locus(Fig. 3). The DNA sequence of the RFLP markerswas used to design oligomers which were employedin polymerase chain reactions (PCR) to screen a yeastartificial chromosome (YAC) library . For each RFLPmarker (TG 134 and TG442) a corresponding YAC wasisolated : Y-134 (630 kb) and Y-442 (700 kb), respec-tively. Presently, the YAC end-probes are being iso-lated to permit each insert to be placed in the correctorientation relative to Asc. It remains to be determinedwhether Y 134 and/or Y-442 contain Asc or that addi-tional YACs need to be isolated for the construction ofa contig .

To determine the position of the end-probes rel-ative to Asc, two sets of plants harbouring chromo-somal recombinations at either side of the Asc locusare required. The resistant wild tomato L. pennellii(Asc/Asc) was crossed to a susceptible chromosome 3tester line (asc/asc) containing the markers baby leasyndrome (bls) and solanifolia (sf) (Fig. 3) (Van derBiezen et al ., 1994a). To increase the efficiency ofrecombinant selection, first F2 plants are selected forrecombinations between these phenotypical markers .Following visual selection, plants are scored for Asc

by leaflet bioassays with AAL-toxins . Only plants thatare sensitive to AAL-toxins (asc/asc) and that eithercarry a recombination between asc and bls or betweenasc and sf are selected for subsequent DNA analysis .The former plants (asc, Bls) are molecularly analysedfor recombinations between asc and TG442, the lattergroup (asc, Sf) for recombinations between asc andTG134. Since the genetic distance between Asc andboth morphological markers is approximately 20 cM,the efficiency of recombinant selection is about 5 timeshigher than without preselection . For the detection ofrecombinations between L. pennellii and L. esculen-tum chromosomes, Southern hybridisation (RFLPs)and PCR techniques (polymorphisms in product size orrestriction patterns) are applied on pooled plant selec-tions (Churchill et al ., 1993) .

Concluding remarks

The fungus A. a. lycopersici secretes host-selectiveAAL-toxins that are the primary molecular determi-nants of pathogenicity for the Alternaria stem cankerdisease in tomato . Most likely, the toxins are recog-nised by the enzyme ceramide synthase which leads toinhibition of the sphingolipid synthesis and results incell death . The ability to produce AAL-toxins enablesthe saprophyte to destroy host cells and to obtain nutri-ents and, hence, allows the pathogen to proliferate andfurther infect susceptible tissues . Resistance and sus-ceptibility to fungal infection are conceivably the resultof the concomitant insensitivity and sensitivity to theAAL-toxins, respectively. Sensitivity to AAL-toxinsin tomato is regulated by the Asc locus which, conse-quently, encodes a host recognition factor . Likewise,the interaction between the fungal pathogen and tomatocomes down to a direct or indirect interaction betweenthe product(s) of the Asc locus and the AAL-toxins .The molecular basis of the specificity of host recogni-tion is determined by the alleles of the Asc locus .

It is expected that the physical isolation and char-acterisation of Asc will elucidate its function in theinteraction between A . a. lycopersici and tomato . Tothat end, approaches are applied that are based on dif-ferent characteristics . Product-based strategies dependon the identification of the biochemical target(s) ofAAL-toxins. However, ceramide synthase is a veryunstable membrane-bound enzyme and therefore dif-ficult to purify. Moreover, this enzyme might not beencoded by Asc. A phenotype-based approach suchas transposon tagging relies on the ability to detect

the phenotype (susceptibility or resistance) followinginactivation of an Asc allele by the transposon . Fortransposon mutagenesis of Asc or asc, it is crucialthat the functional allele is inactivated resulting in anon-lethal and recognisable insertion mutant . Finally,position-based strategies are straightforward and arepotentially not dependent on the function or biochemi-cal characteristics of the Asc product(s) . Following theidentification of a YAC or cosmid vector harbouringAsc, complementation of resistance or susceptibilitycan be achieved by transforming susceptible or resis-tant lines, respectively .

Harmful effects ofA. a . lycopersici to tomato cropsremained limited as yet. No (intentional) selection forresistant plants carryingAsc has been carried out duringdevelopment of commercial tomato varieties . Howev-er, some of the present lines are susceptible (asc/asc)to fungal infection, indicating that the aggressivenessand/or the occurrence of the pathogen is restricted .Consequently, the question arises as to whether resis-tance toA . a. lycopersici infection, conferred by theAsclocus, is a result of adaptation to coexistence with thepathogen . Is the dominant Asc allele derived from therecessive asc allele? Since the EMS-induced mutationsat the recessive asc allele result in dominant alleles thismight be the case .

The compatible interaction between tomato and A .alternata could, alternatively, also be the result ofadaptation at the fungal side. A mutation in a geneinvolved in a biosynthetic pathway of A . alternatacould result in accumulation of metabolic compounds .Possibly, increased concentrations of certain interme-diates could be fortuitously toxic to some plants . Theobservation that different quantities of toxins are pro-duced among different culture media and different fun-gal isolates supports this hypothesis . If the sensitivetissues are destroyed by the toxin-producing fungus,a new nutritional source is established . According-ly, such mutation would change a normally exclu-sive saprophyte to a facultative saprophyte or plantpathogen .

Acknowledgements

David Gilchrist, Jonathan Jones, Maarten Koorn-neef, Charles Rick, Klaus Theres and John Yoder areacknowledged for their generous gifts of tomato lines,Steven Tanksley for RFLP markers, and Pieter Vos forselecting YAC clones . Hanneke Witsenboer is thankedfor critically reading the manuscript. This work was

215

financially supported in part by the BRIDGE programof the European Community (BIOT 900192) .

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