Characterization of Phytophthora infestans genes regulated during the interaction with potato

MOLECULAR PLANT PATHOLOGY

(2002)

3

(6 ) 473ndash485

copy 2002 BLACKWELL SC IENCE LTD

473

Blackwell Science Ltd

Characterization of

Phytophthora infestans

genes regulated during the interaction with potato

KAT INKA BEYER

1

SON IA J IMEacuteNEZ J IMEacuteNEZ

1

THOMAS A RANDALL

2

S TEPHEN LAM

2

ANDRES B INDER

3

THOMAS BOLLER

1

AND MARGARET A COLL INGE

1

1

Friedrich Miescher Institute PO Box 2543 CH-4002 Basel Switzerland

2

Syngenta Biotechnology Inc Research Triangle Park 3054 Cornwallis Road Research Triangle Park NC 27709 USA

3

Syngenta Crop Protection AG Werk Stein Schaffhauserstrasse CH-4332 Stein Switzerland

SUMMARY

Suppression Subtractive Hybridization (SSH) was used to searchfor genes of


that are induced during theinfection of potato To avoid having to distinguish the genes ofthe pathogen from the plant genes involved in defence responsesand to isolate the genes involved in the early stages of interac-tion mycelium of

P infestans

was induced by contact with thehost plant and then separated from the plant tissue A differentialcDNA library was generated by SSH that compared such inducedmycelium with mycelium incubated in water The expression ofabout 100 cDNA fragments from this differential cDNA librarywas analysed by hybridization of the arrayed PCR products withmRNA from control and induced mycelium Twenty per cent ofthem showed increased transcript levels in mycelium within thefirst 24 h after exposure to a potato leaf For six of these cDNAclones the elevated expression in response to the potato leafcould be proven by RNA gel blot analysis Five of these cDNAclones have predicted amino acid sequence homologies toentries in the databases including an amino acid transporter asucrose transporter a spliceosome-associated factor an ABCtransporter and a cell division control protein We showed thatthe genes corresponding to these six cDNA clones are differen-tially regulated during their life Reliable gene expression analysisof

Phytophthora

in infected leaf tissue is not possible until

c

48 hpost-infection but for two of the genes we identified inductionduring

in planta

growth was detectable by RNA gel blot analysisTherefore the SSH library that we have created provides a basisfor the identification of

P infestans

genes that are up-regulatedduring the interaction with the plant which could be important

for pathogenicity

INTRODUCTION

The oomycete


causes the devastatinglate blight disease of potato and tomato Historically it isnotorious as the causal agent of the Irish potato famine in 1845(Ristaino

et al

2001) that led to the death of over a millionpeople and the emigration of many more (Duncan 1999) Today

P infestans

remains a destructive and economically importantpathogen Throughout the world the costs of fighting thispathogen and the losses in yield due to late blight are enormous(Duncan 1999) Since

P infestans

was first described as thecause of late blight disease intense research was devoted toan understanding of its genetics and physiology (eg Erwin

et al

1983 Ingram and Williams 1991) as well as its interactionwith potato (Freytag

et al

1994 Kamoun

et al

1999b) Howeverknowledge of the molecular processes involved in pathogenicityis still limited A better understanding of the molecular mecha-nisms underlying this plantndashmicrobe interaction could lead to thedevelopment of new strategies to reduce crop losses and therebybenefit agricultural practices

One approach to identifying important factors in the establish-ment of an infection lies in defining the transcriptional changesoccurring therein Genes differentially expressed during contactwith the plant would be of special interest since such genesmight contribute to pathogenicity or virulence Componentsessential for pathogenesis could be involved in different phasesof plant colonization eg attachment germination appresso-rium formation invasive growth nutrient uptake and sporula-tion In addition these factors could also play a role in protectionagainst plant defence responses

Several previous studies focusing on potato genes regulatedduring colonization by

P infestans

identified a broad range ofinduced genes (eg Avrova

et al

1999 Beyer

et al

2001 Collingeand Boller 2001 Zhu

et al

1995) From

P infestans

howeveronly a few genes could be identified that were differentiallyexpressed during infection of its host (Goumlrnhardt

et al

2000Kamoun

et al

1997 Pieterse

et al

1991 Pieterse

et al

19921993ab 1994)

Correspondence

Department of Plant Biology University of Zuumlrich Zollikerstrasse 107CH-8008 Zuumlrich Switzerland E-mail collingebotinstunizhch

MPP_143fm Page 473 Tuesday October 22 2002 852 PM

474

K BEYER

et al


(2002)

3

(6 ) 473ndash485 copy 2002 BLACKWELL SC IENCE LTD

In this study we performed a PCR-based differential screenusing Suppression Subtractive Hybridization (SSH) to identify the

P infestans

genes involved in the infection of potato Previouslywe reported the successful application of this method to create adifferential library from infected leaf tissue and the identificationof several potato genes highly induced in response to

P infestans

(Beyer

et al

2001) The clear under-representation of

Phytoph-thora

genes in this library made it inefficient for isolatingregulated genes of the pathogen and thus a more pathogenorientated experimental set-up had to be sought Goumlrnhardt

et al

(2000) generated an SSH library from

in vitro

germinatedcysts of

P infestans

in order to identify genes important for theinitial step of the infection process It is however not yet knownif the genes identified in this approach also play a role in the laterstages of the potatondash

Phytophthora

interactionIn the screen described here the differentiation of

in planta

-induced genes of

P infestans

from the high background ofpathogen-activated plant genes was circumvented by exposingmycelium to potato leaves and then separating it from the planttissue Several

Phytophthora

genes were identified that areinduced in response to potato including genes with predictedhomologies to two nutrient transporters an ATP binding cassette-(ABC) transporter a protein involved in cell division a spliceo-some associated factor as well as an unknown protein Thus thescreening system described enables the analysis of genes from ahemibiotrophic pathogen that might play a role in the interactionwith its plant host

EXPERIMENTAL PROCEDURES

Growth of potato plants and

P infestans


strain 88069 was kindly provided byFrancine Govers (University of Wageningen Netherlands) It wasgrown on rye agar (Caten and Jinks 1968) at 18

deg

C in the darkVirulence was maintained by infecting leaves and re-isolatingevery 3ndash4 months Sporangia zoospores and cysts were obtainedas described by van West

et al

(1998) Germinated cysts wereobtained by incubation of cysts in water at 18

deg

C in the dark for4ndash6 h To obtain mycelium zoospores were germinated andgrown in liquid rye medium at 18

deg

C in the dark for 2ndash6 daysIsolation and purity of the different developmental stages wasconfirmed microscopically

Axenic potato plants cultivar Bintje were kindly provided byPatrick Schweizer (University of Fribourg Switzerland) The plantswere grown and maintained as described in Beyer

et al

(2001)

Media preparation

Defined medium was prepared according to Xu

et al

(1982) Forsolution A 20 g glucose 132 g (NH

4

)

2

SO

4

132 g CaCl

2

times

H

2

O

05 g MgSO

4

times

7H

2

O 07 g KH

2

PO

4

03 g K

2

HPO

4

001 gFe(SO4)

3

were mixed with 500 mL H

2

O and the pH was adjustedto 46 For solution B 50 mg each of MnSO

4

times

H

2

O

ZnSO

4

andthiamine were dissolved in 100 mL H

2

O For solution C 232 gfumaric acid was dissolved in 300 mL H

2

O and the pH wasadjusted to 46 Solutions A and C were mixed together with 1 mLof solution B and the pH was adjusted to 46 For the N-starvationmedium the defined medium was prepared without (NH

4

)

2

SO

4

while the C-starvation medium was prepared with only 2 gglucose

Infection of potato

Leaves were excised from 6-week-old plants placed with theabaxial side up on moist filter paper in Petri dishes and inoculatedwith

P infestans

zoospores Droplets (about 10

micro

L) containingapproximately 1000 zoospores were spotted on the abaxial sideof the leaf To maintain the high humidity required for infectionthe Petri dishes were closed with Parafilm They were placed inthe dark at 18

deg

C for 16 h and then transferred to a growthchamber (16ndash20

deg

C)

Differential screen

Mycelium was grown in liquid rye medium at 18

deg

C in the darkfor 6 days The mycelium was washed with water and smallportions were placed into a vessel containing 50 mL water(control) or on the abaxial side of a potato leaf floating in 50 mLwater (tester) The mycelium was incubated for 4 8 or 24 h andthe samples were pooled to create one control and one testersample The tester samples were harvested by removing themycelium from the potato leaf using forceps and the controlsample was handled in a similar manner The mycelium was driedquickly on Whatman paper and frozen in liquid nitrogen Poly(A)RNA was isolated from the mycelium using a Poly(A) Pure Kit(Ambion Witney Oxon) Suppression Subtractive Hybridization(SSH) was performed as described in Beyer

et al

(2001)

Dot blot assay

The cDNA inserts of the clones from the SSH library were ampli-fied by PCR using primers N1 and N2R (Clontech PCR-Select Kit)that are specific for the adaptors ligated on to the cDNA frag-ments during the SSH procedure Then NaOH and EDTA pH 8 wereadded to each PCR reaction to a final concentration of 04

M

NaOH and 10 m

M

EDTA and the PCR products were denatured at95

deg

C for 10 min A 30

micro

L aliquot of the PCR products was thenloaded in duplicate on two Zeta-Probe Blotting Membranes(Bio-Rad) using a dot blot manifold The slots of the dot blotmanifold were each pre-washed with 500

micro

L H

2

O then the PCRsamples were applied and each slot was rinsed again with 04

M


P infestans

genes regulated during infection

475



(2002)

3

(6 ) 473ndash485

NaOH The membranes were then quickly rinsed in 2

times

SSC UVcrosslinked and hybridized with the radioactively labelledcDNA probes For this poly(A) RNA was prepared from a pool ofmycelium incubated in water for 4 8 and 24 h and myceliumco-incubated with a potato leaf for the same time periods Tocreate radioactively labelled cDNA probes first strand cDNAsynthesis was carried out in the presence of

α

-

32

P dCTP asdescribed in Beyer

et al

(2001) The blots were hybridized andwashed at 65

deg

C as described by Church and Gilbert (1984)

DNA sequence analysis

Sequencing was performed using Dye Terminators from PEApplied Biosystems using a Perkin-Elmer GeneAmp PCR systemthermocycler and analysed using an ABI PRISM 377 DNAsequencer Sequence data were analysed using the GCG package(Wisconsin Package ver 101 Genetics Computer Group Madi-son WI) Similarity searches were made with B

LAST

programs atthe National Center for Biotechnology Information lthttpwwwncbinlmnihgovgt

RNA and DNA gel blot analysis

A series of duplicate RNA blots was made with total RNAextracted from different stages of

P infestans

and necroticparts of infected potato leaves RNA of mycelium sporangiazoospores and cysts was extracted using the Trizol reagent (LifeTechnologies) RNA of germinated cysts was isolated using theRNAwiz Reagent (Ambion) and RNA of infected leaves wasextracted as described by Chomczynski and Sacchi (1987) TotalRNA samples (10ndash20

micro

g) were separated on formaldehyde-agarose gels and transferred to positively charged nylonmembrane (Zeta-probe Bio-Rad) (Sambrook

et al

1989) DNAprobes were labelled using the Prime It II random primedlabelling kit (Stratagene) and hybridization and washing werecarried out for RNA gel blots at 65

deg

C and for DNA gel blots at50

deg

C as described in Church and Gilbert (1984) For low-leveltranscripts RNA gel blots were hybridized at 42

deg

C using theUltraHyb Buffer (Ambion) for greater sensitivity The resultingradioactive signals were compared and quantified using aPhosphor imager (Storm 860 Molecular Dynamics)

RESULTS

Creation of a subtracted library

We screened for genes that are induced in

P infestans

during theinteraction with potato using the PCR-based method SuppressionSubtractive Hybridization (SSH) (Diatchenko

et al

1996) Tospecifically identify

P infestans

genes involved in the interactionwith potato we used mycelium incubated in water as the control

sample and mycelium that had been stimulated by the presenceof a potato leaf as the tester Importantly this creates a subtractedlibrary that contains only sequences of

P infestans

This approachis similar to the one used by Munoz and Bailey (1998) and enablesthe mechanical separation of the two interacting organismsbefore the applying the SSH procedure To test the suitability ofthis

in vitro

system we analysed the incubated leaves micro-scopically after staining for fungal infection structures (Wilsonand Coffey 1980) In leaves that had been exposed to themycelium for 3 days colonization of the leaf by

P infestans

couldbe detected and infection structures were indistinguishablefrom those seen after infection of leaves by zoospores (Fig 1A)Furthermore the recognition of the potato leaf as a potentialhost must take place very quickly since the mycelium becomesfirmly attached to the leaf after only 30 min of incubation andhas to be removed from the leaf using forceps To determine if theregulation of genes occurs upon co-incubation with a potato leafsimilar to that seen in infection of leaves with zoospores theexpression of the known

in planta

induced

Phytophthora

gene

ipiO

(Pieterse

et al

1993a 1994) was analysed and a clearup-regulation was detected (Fig 1B) Thus our

in vitro

infectionconditions closely mirror the infection process caused byinoculation with zoospores and we used this system to generatea differential library using the lsquoinduced myceliumrsquo ( IM) as the testerand the water-incubated mycelium as control

Differential screening of the library clones

A dot blot assay was performed to determine what proportion ofthe clones in the SSH library represent transcripts that accumu-late in the presence of a potato leaf Ninety-six PCR-amplifiedcDNA inserts of about 200ndash800 bp were blotted on to two replicamembranes using a dot blot manifold Two genes that had beenidentified in a previous SSH screen and shown to be constitutivelyexpressed in

in vitro

grown mycelium and

in planta

(data notshown) were loaded as controls on the membranes The DNAblots were hybridized with the cDNA populations derived fromthe original control and tester samples that had been used togenerate the SSH library Level of induction for each clone wascalculated by dividing the signal on the blot hybridized withtester cDNA by the signal obtained on the blot hybridizedwith control cDNA The two control genes as well as 80 of thetested clones showed induction values in the range of 1ndash3-foldTwenty clones corresponding to 20 of clones from theSSH library showed an induction of at least fourfold (datanot shown)

In addition to the dot blot assay about 600 random cloneswere sequenced and subsequently B

LAST

X searches were per-formed against G

EN

B

ANK

data Hits were considered significant ifthe expectation value was less than 1e-5 Genes were groupedinto different categories using the same functional classification


476

K BEYER

et al


(2002)

3


as was employed for

S cerevisiae

in MIPS (Munich informationcentre for protein sequences lthttpwwwmipsgsfdegt data notshown) Seventy per cent of the sequences could not be classifiedsince they did not have significant hits in the databases or showhomologies to proteins of unknown function The sequences thatcould be assigned fell mainly into the functional classes of basiccellular processes such as metabolism energy protein synthesisand fate and transport

Transcriptional characterization of selected clones

Expression in mycelium induced by the plant

Twenty-five cDNA clones from the differential library werechosen for further analysis in RNA gel blot experiments Firstthe 20 cDNA fragments that showed increased transcript levels

in the dot blot experiment were chosen Two of these IM-002Aand IM-003C exhibit significant homology to an amino acidtransporter and a sucrose transporter (accession nos AF159856and AL445063) respectively Furthermore they show homologyto similar transporters of the rust fungus

Uromyces fabae

thatare up-regulated during the infection of pea (Hahn

et al

1997Voegele

et al

2001) Since transporters are conceptually of clearimportance for the interaction with the plant we took advantageof our sequence collection from the subtracted library andsearched for additional clones showing homologies to differentkinds of transporters Five such clones were found and includedin our RNA gel blot analysis They have homologies to a phos-phate transporter two different ABC transporters a tSNARE anda putative vesicular transport protein in yeast (SRO9) The expres-sion of the 25 selected cDNA clones was analysed on RNA gel

Fig 1 (A) Infected potato leaves stained for infection structures (1) and (3) potato leaf infected by spraying the leaf with a spore suspension of P infestans (1) 2 days after infection (3) 3 days after infection (2) and (4) potato leaf infected by co-incubation with P infestans mycelium in water for 3 days a = infecting spore b = appressorium c = appressorium or swollen hypha d = infecting hypha e = intercellular spreading hypha Magnifications (1) and (2) 320times (3) 100times (4) 200times (B) Expression of ipiO in mycelium treated with water (Con) and incubated with a potato leaf (Ind) Top panel RNA gel blot Bottom panel ethidium bromide stained gel before transfer


P infestans


477



(2002)

3

(6 ) 473ndash485

blots containing either total or poly(A) RNA from pooled samplesof mycelium collected at different time points after incubation inwater only or with a potato leaf Of the 25 clones that wereanalysed on RNA gel blots 15 were detectable and 10 of theseshowed higher transcript levels in the induced mycelium (Table 1and Fig 2)

The differential expression in response to the potato leaf vsthe water control observed in the pooled samples was thenanalysed at different time points of incubation namely after 4 h8 h and 24 h The five cDNAs showing at least twofold inductionwere selected for this analysis These clones have homologies toa sucrose transporter (IM-003C) a spliceosome-associated factor(IM-008B) an ABC transporter (IM-01F1) a cell division control(CDC) protein (cdc6) (IM-06D11) and one clone without any clearhomology in the database (IM-004 A) In addition we includedIM-002A which has a lower level of induction but shows signif-icant homology to a known

in planta

induced gene of

Uromycesfabae

(Hahn

et al

1997) For all of the six selected clones theanalysis was performed at least twice in independent experimentsand the differential expression was confirmed for all of them(Fig 3) In these time-course experiments IM-004A showed theweakest induction which is in contrast to the level of inductiondetected on the pooled RNA gel blots Some of the genes IM-003C IM-008B and IM-06D11 showed a decrease in differentialexpression after 24 h (data not shown) This could be due to ageneral decrease in fitness caused by the long incubation inwater Before beginning the SSH screen when testing the expres-sion of different genes we noted that actin transcript levelsdecreased after 24 h Therefore we suggest that the inducedmycelium system only be used for the identification of earlyinduced genes

Expression at different stages of the life cycle

Expression of the genes represented by the six selected cDNAclones at different stages of the life cycle was analysed on RNAgel blots of total RNA from sporangia zoospores cysts germi-nated cysts mycelium grown in liquid rye medium and infectedplant material (Fig 4) Ten

micro

g of RNA were loaded on the gelexcept for germinated cysts from which less RNA of lower qualitywas loaded because of consistent problems in RNA isolation fromthis stage The RNA of infected plant tissue was a mixture of RNAfrom potato leaves 3 4 and 5 days after infection To assay therelative amount of

P infestans

RNA loaded the blot was subse-quently hybridized with a fragment of the actin gene (actA) of

Pinfestans

a gene that is expressed at similar levels in all devel-opmental stages tested (Unkles

et al

1991) The strength of theactin signal in Fig 4 demonstrates the lower amount of RNAloaded for the germinated cysts as well as the dilution with plantRNA in the sample from infected potato leaves

Three of the analysed genes IM-002A (homologous to anamino acid transporter) IM-003C (homologous to a sugar trans-porter) and IM-008B (homologous to a spliceosome-associatedfactor) show a similar expression pattern They were expressedmainly in sporangia in mycelium and in the infected plant tissueIM-002A was also expressed in zoospores and cysts but IM-003Cand IM-008B were not Relative to the expression of actin IM-008B also showed a relatively high expression in germinatedcysts IM-004A (no clear homology in the database) showed asimilar expression pattern to that of actin IM-01F1 (homologousto ABC transporter) was expressed in all stages but was clearly up-regulated in mycelium and in the infected plant material IM-06D11(homologous to cdc6) was mainly expressed in zoospores andcysts the main infectious agents but also in mycelium and the

Table 1 Genes of P infestans induced upon co-incubation with a potato leaf

Clonename Homology

Protein match identityprobability

Length offragment

Foldinductiondagger

IM-002A Amino acid transporter Mus musculus AF159856 355e-11 500 bp 15IM-003CDagger D-Xylose-proton symporter Thermoplasma acidophilum AL445063 487e-5 300 bp 31IM-004 A No clear hits 370 bpsect 26IM-007F No clear hits 360 bp 16IM-008B Spliceosome-associated protein Schizosaccharomyces pombe AL023589 301e-12 690 bpsect 25IM-008DDagger Retinoblastoma protein Notophthalamus viridescens Y09226 343e-06 740 bp 14IM-01F1Dagger ATP-binding cassette 2 subfamily 2 member 2 Mus musculus X75927 562e-59 700 bpsect 26IM-06D11 Cell division control protein 6 Xenopus laevis U66558 302e-7 480 bp 64IM-06E10 Phosphate transporter Pho91 Saccharomyces cerevisiae Z71628 338e-24 590 bp 15IM-09E10 Similarity to SRO9 Schizosaccharomyces pombe AL355653 382e-9 530 bp 15

Length is indicated in base pairsdaggerFold induction calculated by normalizing the expression level in the control against the expression level in the induced sampleDaggerFound twice among the analysed clonessectLonger cDNA fragments have been isolated additionally and are available at the database


478 K BEYER et al

MOLECULAR PLANT PATHOLOGY (2002) 3 (6 ) 473ndash485 copy 2002 BLACKWELL SC IENCE LTD

infected plant tissue This cDNA clone detected three hybridizingbands of different sizes in all the P infestans in vitro stages but onlythe largest band of 36 kb was detected in the infected plant tissue

To analyse the in planta expression in more detail transcriptlevels were determined 1 2 3 4 and 5 days after infection RNAfrom mycelium grown in liquid rye medium and incubated for 8 hin water was used as a comparison Expression of all the analysedgenes was first detectable 3 days after infection (Fig 5) and acontinuous increase in transcript level was observed which wascomparable to the expression of actin The relative amount ofactin mRNA which specifically hybridizes to P infestans actinwas taken as a measure of the P infestans biomass in theinfected leaf Accordingly the level of expression was determinedby normalizing the signals for each gene against those for actinThis level of expression was then compared to the expression inin vitro grown mycelium (data not shown) Importantly twogenes IM-01F1 and IM-06D11 showed a higher expressionin planta than in vitro which can also be seen in Fig 5 IM-01F1shows highest level of expression 5 days after infection twofoldhigher than in the in vitro mycelium IM-06D11 hybridizes only toa fragment of 3600 bp in RNA from infected leaves whereas it

hybridizes more strongly to smaller fragments in RNA from thein vitro grown mycelium It is the only clone with a clearly inducedexpression at the first detectable time point after infection Threedays after infection its expression was fourfold greater thanin vitro and then subsequently its transcript level slightlydecreased This is consistent with the result obtained in theexpression analysis of different stages (Fig 4) where it wasmainly expressed in zoospores and cysts the main infectiousagents Therefore this gene might be important at early timepoints during the infection process It is possible that theexpression of the other genes is up-regulated before they aredetectable in infected leaves or perhaps later in the infectionprocess since their signals still seemed to be increasing 5 daysafter infection

Expression analysis in response to different mediaIt has been reported that various genes of fungal pathogensexpressed during infection are also regulated by nutrient starva-tion which indicates that nutrient deprivation may be one of theconditions that biotrophic pathogens encounter during in plantagrowth (reviewed in Oliver and Osbourn 1995) To determine if

Fig 2 Differential expression of selected library clones in response to co-incubation with a potato leaf Bottom panel ethidium bromide-stained agarose gel before transfer Top panel autoradiograph with signals after hybridization with radioactive probe Mycelium was incubated in water (Con) or in water together with a potato leaf (Ind) for 2 4 8 and 24 h The RNA was isolated and equal amounts from each time-point were pooled to create one control sample and one tester sample (A) Fifteen microg of total RNA was loaded in each lane (B) Two and a half microg of poly(A)RNA were loaded in each lane


P infestans genes regulated during infection 479

copy 2002 BLACKWELL SC IENCE LTD MOLECULAR PLANT PATHOLOGY (2002) 3 (6 ) 473ndash485

the genes that were identified in our screen are regulated inresponse to nutrient starvation their expression was analysedin mycelium incubated in different media Northern blots wereprepared with 15 microg of total RNA from mycelium grown for6 days in rye medium followed by incubation for 8 h in water indefined medium (Xu et al 1982) in defined medium withouteither nitrogen or carbon source or in rye medium (Fig 6) Threeof the analysed genesmdashIM-003C IM-004A and IM-008Bmdash

showed similar expression in all the different media In contrasttranscript levels of IM-002A increased slightly in response to theN-starvation medium and strongly in response to the C-starvationmedium as well as to the rye medium Moreover IM-01F1 andIM-06D11 were both induced in response to the completedefined medium and they showed a slight induction in responseto the N-starvation medium and a strong induction to the C-starvation medium compared to their expression in water and

Fig 3 Changes in transcript level over time of the differential clones in response to co-incubation with a potato leaf Bottom panel ethidium bromide-stained agarose gel before transfer Top panel autoradiograph with hybridization signals after radioactive detection Mycelium was incubated in water (Con) or in water together with a potato leaf (Ind) for the indicated times Fifteen microg of total RNA was loaded in each lane


480 K BEYER et al


rye medium It is possible that their expression is induced inresponse to a nitrogen source

Database accession numbers

The new cDNA sequences reported here have been deposited inthe EMBL Nucleotide Sequence Database with the accessionnumbers AJ487842 to AJ487851

DISCUSSION

The destructiveness of late blight disease makes it very importantto investigate the causal organism in more detail The infectionprocess involves specific morphological changes and manymetabolic processes which require the regulation of a widevariety of genes

Genes regulated during infection have been described for anumber of phytopathogenic fungi and oomycetes For examplefrom Uromyces fabae a total of 31 in planta induced genes (PIGs)were isolated from a haustorium specific library (Hahn andMendgen 1997) Two proteins of Cladosporium fulvum that aresecreted into the intercellular space of infected tomato leaves(Wubben et al 1994) as well as the race specific elicitors AVR4and AVR9 were shown to be transcriptionally induced during

plant infection (Laugeacute and de Wit 1998) From Magnaporthegrisea a putative hydrophobin gene MPG1 that is induced duringinfectious growth was identified by differential cDNA cloning(Talbot et al 1993) and several differentially expressed geneswere isolated by screening cDNA libraries of infected rice plants(Kim et al 2001 Rauyaree et al 2001) In Phytophthora capsicia cutinase-encoding gene is induced in the interaction withpepper (Munoz and Bailey 1998) From P infestans nine in plantainduced genes have previously been isolated (Pieterse et al1993a) In addition several genes have been reported to beexpressed during specific stages of the life cycle which areimportant for infection (Goumlrnhardt et al 2000 Judelson andMichelmore 1990) MPG1 of M grisea (Talbot et al 1993) wasshown to be a determinant of pathogenicity which demonstratesthe success of differential screening approaches

Here we describe an efficient system for isolating P infestansgenes that may play a role in the interaction with potato Thisin vitro experimental system allows the physical separation of theinduced mycelium from the plant tissue after the initiation ofinfection It has the advantage that genes of the pathogen do nothave to be discriminated from the host genes a process that isvery difficult in the analysis of infected plant tissue due to theoverwhelming representation of plant sequences (Beyer et al2001)

Fig 4 Transcript levels of selected clones at different stages of the P infestans life cycle Bottom panel ethidium bromide-stained agarose gel before transfer Top panel autoradiograph with signals after hybridization with radioactive probe Ten microg of total RNA were loaded in lanes A B C E and F and 5 microg total RNA was loaded in lane D (A) sporangia (B) zoospores (C) cysts (D) germinated cysts (E) mycelium and (F) infected potato leaves




About 600 clones from the subtracted library were sequencedand grouped into different classes according to their predictedsequence homologies This classification suggested that growthand the maintenance of metabolism are important processes inmycelial growth stage A similar grouping was described for ESTsof a mycelial library of P infestans (Kamoun et al 1999a) and forother host-pathogens Most of the expressed genes identified inMagnaporthe grisea during the colonization of rice were groupedaccording to geneprotein expression and metabolism (Kim et al2001) Furthermore the most abundant P sojae-derived ESTs ofan interaction library are involved in intermediary metabolismindicating that rapid growth and invasion of the host tissue makea massive demand on central metabolic processes (Qutob et al2000)

One hundred of the clones from the subtracted library werescreened by dot blot analysis Twenty-five clones were selectedfor further analysis on RNA gel blots comparing mycelium

incubated with a potato leaf with mycelium incubated only inwater Ten of the cDNAs that showed induction on the dot blot alsoshowed induction in this assay (Figs 2 and 3) The increases intranscript level observed in these experiment were moderate andlevel of induction ranged from 15- to 3-fold This may be becausein this system only a few hyphae of the induced mycelium havedirect contact with the plant surface and therefore only a part ofthe lsquoinducedrsquo mycelium is really induced Another reason couldbe that we searched for expression changes triggered by thepresence of the host plant in the mycelium of a hemibiotrophicpathogen that does not normally exist in an independent mycelialform in the field (Hohl 1991) This developmental stage probablydoes not undergo major changes in terms of adaptation to newconditions and therefore even small changes in gene expressioncould have an important impact on in planta growth Indeed itwas shown in other systems that genes showing a low level ofinduction following a certain stimulus can be very important

Fig 5 Transcript levels during in planta growth and in in vitro grown mycelium Bottom panel ethidium bromide-stained agarose gel before transfer Top panel autoradiograph with hybridization signals after radioactive detection Fifteen microg of total RNA was loaded in each lane (AndashE) Infected plant tissue at (A) 1 (B) 2 (C) 3 (D) 4 and (E) 5 days after infection (F) mycelium incubated in H2O for 8 h


482 K BEYER et al


for the corresponding adaptation In yeast for example a geneknock-out study revealed that a heat shock protein exhibitinga twofold induction following sorbic acid treatment conferredresistance to this treatment (de Nobel et al 2001)

Six genes whose transcript levels were at least twofold higherin lsquoinduced myceliumrsquo were identified in this small-scale screen ofthe subtracted library These were an amino acid transportera sucrose transporter an ABC transporter a spliceosome associ-ated protein a cdc6 homologue and one with no homology toentries in the databases The changes in transcript levels werealready detectable after 4 h of exposure to a potato leafTranscript levels of these genes were also regulated in differentdevelopmental stages and by nutrient conditions

The fact that we identified two genes similar to those inducedin planta in another plant-pathogen indicates a convergentevolution of gene expression changes in distinct plant patho-gens One of these clones IM-003C exhibits homology to asugar transporter (D-Xylose-proton symporter Thermoplasmaacidophilum AL445063) and also shows significant homology toa hexose transporter of Uromyces fabae that is exclusivelyexpressed in haustoria while colonizing pea and is needed for

sugar uptake (Voegele et al 2001) The other clone IM-002Ashows homology to an amino acid transporter of Mus musculus(AF159856) and is also homologous to an amino acid transporterthat is mainly expressed in haustoria of Uromyces (Hahn et al1997) In Uromyces these two genes are expressed exclusively inhaustoria and infected plants but their expression has not beenanalysed in teliospores or in in vitro mycelium since Uromyces isan obligate biotroph The two transporters that we isolated areexpressed in mycelium in planta and in sporangia There was noclear induction in planta compared to expression in in vitrogrown mycelium However it has been observed that unliketypical haustoria of many higher fungi those of P infestans oftenlook like side branches or infection hyphae and it was thereforesuggested that the nutrient uptake of P infestans may not berestricted to haustoria but include intercellular intracellular andtranscellular hyphae (reviewed in Hohl 1991) Thus these trans-porters may be important during in planta growth for nutrientuptake

Since it is very likely that transporters are needed during infec-tion for the uptake of nutrients and metabolites in the colonizedhost tissue we selected other clones from the subtracted library

Fig 6 Changes in transcript level in response to different media Bottom panel ethidium bromide-stained agarose gel before transfer Top panel autoradiograph with hybridization signals after radioactive detection Fifteen microg of total RNA was loaded in each lane Six-day-old mycelium was incubated for 8 h in different media (A) H2O (B) defined medium (C) N-starvation medium (D) C-starvation medium (E) Rye medium




with homologies to transporters Indeed one of them IM-01F1which exhibits homology to an ABC transporter showed a clearinduction ABC transporters are highly conserved traffic ATPasesthat occur ubiquitously and transport a wide spectrum ofcompounds over various membranes (Senior et al 1995) It hasbeen postulated that the function of these transporters in plantpathogens could be the secretion of endogenous pathogenicityfactors (eg toxins) or exogenous plant defence compounds Indeedseveral studies have shown that ABC transporters are involved inthe secretion of antibiotics toxins plant defence compounds andfungicides (reviewed in Del Sorbo et al 2000) However all ofthese fungal ABC transporters belong to the class of multidrugor pleiotropic drug resistance (MDR or PDR) type transporterswhereas the ABC transporter that we identified in P infestansdisplays a rather weak homology to this subgroup A greaterhomology of IM-01F1 is found with a human ATP-bindingcassette transporter (X75927) called ABCA2 This transporter wasshown to be involved in drug resistance in the brain (Dean et al2001) but interestingly it was also shown to be transcriptionallyup-regulated by sterols (Kaminski et al 2001) IM-01F1 is clearlyinduced in response to a potato leaf in the in vitro system and itis also clearly in planta-induced (Fig 5) During the life cycle it ishighly expressed only in mycelium and infected plants indicatingthat it plays a role during colonization of the host Like otherin planta-induced genes it is transcriptionally activated understarvation conditions defined medium and carbon starvationBased on its homology it is possible that IM-01F1 is involved insterol uptake from the host which is thought to be necessarysince Phytophthora species are unable to produce their ownsterols (Hendrix 1970) It is also possible that it plays a role inthe secretion of exogenous plant toxins and fungicides since thehomolog in humans is also involved in drug resistance Theprecise function of IM-01F1 remains to be elucidated howeverit represents a novel type of ABC transporter that may play a rolein the interaction with potato

Another interesting gene identified in the subtracted libraryrepresented by clone IM-06D11 shows significant homology tocdc6 from Xenopus laevis (U66558) a protein that is crucial tothe control of DNA replication during the cell cycle (Colemanet al 1996 and references therein) P infestans has to undergomorphological changes to infect the host plant and it has toadapt to the new environmental conditions as well as to growrapidly to overcome the defence responses of the host Thereforecell division control could play a crucial role in the infectionprocess The gene that we identified is induced in response to thepotato leaf and it is also clearly induced in planta at early timepoints of infection During the life cycle it shows a high up-regulation in the zoospore and cyst stages Therefore it could beimportant for the establishment of infection and colonizationLike IM-01F1 it shows an induction in defined medium as wellas in response to carbon starvation Interestingly IM-06D11

hybridizes to three bands on RNA gel blots and the largest bandpredominates in the in planta growing mycelium This could bedue to cross-hybridization to a potato gene induced by theinfection with P infestans but this is rather unlikely since nohybridization occurred with potato genomic DNA The possibilityof cross-hybridization to a homologous gene product of Pinfestans can be also excluded since on the genomic DNA gelblot analysis IM-06D11 only recognizes one gene The differentRNA bands most likely correspond to different splicing productsthat may play different roles during the life cycle

Interestingly we also identified a clone IM-008B that showshomology to a spliceosome-associated factor (Schizosaccharo-myces pombe AL023589) which is possibly involved in theprovision of specific proteins necessary for the diverse adapta-tions during colonization of the plant Importantly it is mainlyexpressed in the stages necessary for colonization of the hostsuch as germinated cysts mycelium and in planta growth andalso in sporangia suggesting an involvement during infectionand colonization

Of the six genes that were shown to be induced in the inducedmycelium system used to create the subtracted library two alsoshowed a clear in planta induction in infected leaf tissuecompared with in vitro grown mycelium (Figs 4 and 5) The othersmight be specifically induced by first contact with plantcompounds or they might be induced later or more likely earlier inthe course of infection when their expression in infected leaveswas still undetectable This highlights the value of the inducedmycelium system First it allowed the production of a differentiallibrary containing only P infestans sequences Second changesin expression patterns within hours of exposure to the host (ornon-host) can be assayed time points that cannot be accuratelyanalysed in infected leaf tissue because of the small amount ofP infestans biomass

In our screen we have identified a number of genes that areregulated following co-incubation of mycelium with the host leafSome of them are mainly expressed in mycelium and in plantaindicating a role during colonization Future work will revealwhat role they play in pathogenicity or virulence

ACKNOWLEDGEMENTS

We are grateful to Herbert Angliker and Peter Muumlller for the DNAsequencing and oligonucleotide synthesis services We thankRoman Ulm for critically reading the manuscript KB is fundedby the Syngenta Research Foundation within the framework ofthe Syngenta Phytophthora Consortium

REFERENCES

Avrova AO Stewart HE De Jong W Heilbronn J Lyon GD andBirch PRJ (1999) A cysteine protease gene is expressed early in resistant


484 K BEYER et al


potato interactions with Phytophthora infestans Mol Plant-MicrobeInteract 12 1114ndash1119

Bernard K Auphan N Granjeaud S Victorero G Schmitt-Verhulst A-MJordan BR and Nguyen C (1996) Multiplex messenger assay simultaneousquantitative measurement of expression of many genes in the context ofT cell activation Nucl Acids Res 24 1435ndash1442

Beyer K Binder A Boller T and Collinge MA (2001) Identificationof potato genes induced during colonization by Phytophthora infestansMol Plant Path 2 125ndash134

Caten CE and Jinks JL (1968) Spontaneous variability of singleisolates of Phytophthora infestans I Cultural variation Can J Bot 46329ndash347

Chomczynski P and Sacchi N (1987) Single step method of RNAisolation by acid guanidinium thiocyanate-phenol-chloroform extractionAnal Biochem 162 156ndash159

Church GM and Gilbert W (1984) Genomic sequencing Proc NatlAcad Sci USA 81 1991ndash1995

Coleman TR Carpenter PB and Dunphy WG (1996) The XenopusCdc6 protein is essential for the initiation of a single round of DNAreplication in cell-free extracts Cell 87 53ndash63

Collinge M and Boller T (2001) Differential induction of two potatogenes Stprx2 and StNAC in response to infection by Phytophthorainfestans and to wounding Plant Mol Biol 46 521ndash529

Dean M Hamon Y and Chimini G (2001) The human ATP-bindingcassette (ABC) transporter superfamily J Lipid Res 42 1007ndash1017

Del Sorbo G Schoonbeek H and De Waard MA (2000) Fungaltransporters involved in efflux of natural toxic compounds and fungicidesFungal Genet Biol 30 1ndash15

Diatchenko L Lau YF Campbell AP Chenchik A Moqadam FHuang B Lukyanov S Lukyanov K Gurskaya N Sverdlov EDSiebert PD (1996) Suppression subtractive hybridization a method forgenerating differentially regulated or tissue-specific cDNA probes andlibraries Proc Natl Acad Sci USA 93 6025ndash6030

Duncan J (1999) Phytophthoramdashan abiding threat to our crops MicrobiolToday 26 114ndash116

Erwin DC Bartnicki-Garcia S and Tsao PH eds (1983) Phytophthoraits Biology Taxonomy Ecology and Pathology St Paul MN TheAmerican Phytopathological Society

Freytag S Arabatzis N Hahlbrock K and Schmelzer E (1994)Reversible cytoplasmic rearrangements precede wall appositionhypersensitive cell death and defense-related gene activation in potatoPhytophthora infestans interactions Planta 194 123ndash135

Goumlrnhardt B Rouhara I and Schmelzer E (2000) Cyst germinationproteins of the potato pathogen Phytophthora infestans share homologywith human mucins Mol Plant-Microbe Interact 13 32ndash42

Hahn M and Mendgen K (1997) Characterization of in planta-inducedrust genes isolated from a haustorium-specific cDNA library Mol Plant-Microbe Interact 10 427ndash437

Hahn M Neef U Struck C Goettfert M and Mendgen K (1997)A putative amino acid transporter is specifically expressed in haustoriaof the rust fungus Uromyces fabae Mol Plant-Microbe Interact 10438ndash445

Hendrix JW (1970) Sterols in growth and reproduction of fungi AnnuRev Phytopathol 8 111ndash130

Hohl HR (1991) Nutrition In Phytophthora Infestans the Cause of LateBlight on Potato Advances in Plant Pathology 7 (Ingram DS andWillams PH eds) London UK Academic Press pp 53ndash83

Ingram DS and Williams PH eds (1991) Phytophthora Infestans the

Cause of Late Blight of Potato Advances in Plant Pathology 7 LondonUK Academic Press

Judelson HS and Michelmore RW (1990) Highly abundant andstage-specific mRNAs in the obligate pathogen Bremia lactucae MolPlant-Microbe Interact 3 225ndash232

Kaminski WE Piehler A Pullman K Porsch-Ozcurumez MDuong C Bared GM Buchler C and Schmitz G (2001) Completecoding sequence promoter region and genomic structure of the humanABCA2 gene and evidence for sterol-dependent regulation in macro-phages Biochem Biophys Res Commun 281 249ndash258

Kamoun S Hraber P Sobral B Nuss D and Govers F (1999a)Initial assessment of gene diversity for the oomycete pathogenPhytophthora infestans based on expressed sequences Fungal GenBiol 28 94ndash106

Kamoun S Huitema E and Vleeshouwers VGAA (1999b) Resistanceto oomycetes a general role for the hypersensitive response TrendsPlant Sci 4 196ndash200

Kamoun S van West P de Jong AJ de Groot KE VleeshouwersVGAA and Govers F (1997) A gene encoding a protein elicitor ofPhytophthora infestans is donwn-regulated during infection of potatoMol Plant-Microbe Interact 10 13ndash20

Kim S Ahn I and Lee Y (2001) Analysis of genes expressed duringricendashMagnaporthe grisea interactions Mol Plant-Microbe Interact 141340ndash1346

Laugeacute R and de Wit PJGM (1998) Fungal avirulence genes structureand possible functions Fungal Gen Biol 24 285ndash297

Munoz CI and Bailey AM (1998) A cutinase-encoding gene fromPhytophthora capsici isolated by differential-display RT-PCR Curr Genet33 225ndash230

de Nobel H Lawrie L Brul S Klis F Davis M Alloush H andCoote P (2001) Parallel and comparative analysis of the proteome andtranscriptome of sorbic acid-stressed Saccharomyces cerevisiae Yeast18 1413ndash1428

Oliver R and Osbourn A (1995) Molecular dissection of fungalphytopathogenicity Microbiology 141 1ndash9

Pieterse CMJ Derksen A-MCE Folders J and Govers F (1994)Expression of the Phytophthora infestans ipiB and ipiO genes in plantaand in vitro Mol Gen Genet 244 269ndash277

Pieterse CMJ Riach MBR Bleker T Van Den Berg-Velthuis GCMand Govers F (1993a) Isolation of putative pathogenicity genes ofthe potato late blight fungus Phytophthora infestans by differentialhybridization of a genomic library Phys Mol Plant Path 43 69ndash79

Pieterse CMJ Risseeuw EP and Davidse LC (1991) An in plantainduced gene of Phytophthora infestans codes for ubiquitin Plant MolBiol 17 799ndash811

Pieterse CMJ Verbakel HM Spaans JH Davidse LC andGovers F (1993b) Increased expression of the calmodulin gene of thelate blight fungus Phytophthora infestans during pathogenesis onpotato Mol Plant-Microbe Interact 6 164ndash172

Qutob D Hraber PT Sobral BWS and Gijzen M (2000) Comparativeanalysis of expressed sequences in Phytophthora sojae Plant Physiol123 243ndash253

Rauyaree P Choi W Fang E Blackmon B and Dean RA (2001)Genes expressed during early stages of rice infection with the rice blastfungus Magnaporthe grisea Mol Plant Pathol 2 347ndash354

Ristaino JB Groves CT and Parra GR (2001) PCR amplification ofthe Irish potato famine pathogen from historic specimens Nature 411695ndash697




Sambrook J Fritsch EF and Maniatis T (1989) Molecular Cloning aLaboratory Manual Cold Spring Harbour NY Cold Spring HarbourLaboratory Press

Senior AE Alshawi MK and Urbatsch IL (1995) The catalytic cycleof P-glycoprotein FEBS Lett 377 285ndash289

Talbot NJ Ebbole DJ and Hamer JE (1993) Identification andcharacterization of MPG1 a gene involved in pathogenicity from therice blast fungus Magnaporthe grisea Plant Cell 5 1575ndash1590

Unkles SE Moon RP Hawkins AR Duncan JM and Kinghorn JR(1991) Actin in the oomycetous fungus Phytophthora infestans is theproduct of several genes Gene 100 105ndash112

Voegele RT Struck C Hahn M and Mengden K (2001) Therole of haustoria in sugar supply during infection of broad bean bythe rust fungus Uromyces fabae Proc Natl Acad Sci USA 988133ndash8138

van West P de Jong AJ Judelson HS Emons AMC and Govers F(1998) The ipiO gene of Phytophthora infestans is highly expressed ininvading hyphae during infection Fungal Gen Biol 23 126ndash138

Wilson UE and Coffey MD (1980) Cytological evaluation of general resist-ance to Phytophthora infestans in potato foliage Ann Bot 45 81ndash90

Wubben JP Joosten MHAJ and De Wit PJGM (1994) Expressionand localization of two in planta induced extracellular proteins of thefungal tomato pathogen Cladosporium fulvum Mol Plant-MicrobeInteract 7 516ndash524

Xu D Huang H and Wang C (1982) Polyacrylamide gel disc electro-phoresis of proteins from species of Phytophthora Acta Mycol Sin 140ndash47

Zhu B Chen THH and Li PH (1995) Expression of three osmotin-likeprotein genes in response to osmotic and fungal infection in potatoPlant Mol Biol 28 17ndash26


474

K BEYER

et al


(2002)

3


In this study we performed a PCR-based differential screenusing Suppression Subtractive Hybridization (SSH) to identify the

P infestans

genes involved in the infection of potato Previouslywe reported the successful application of this method to create adifferential library from infected leaf tissue and the identificationof several potato genes highly induced in response to

P infestans

(Beyer

et al

2001) The clear under-representation of

Phytoph-thora

genes in this library made it inefficient for isolatingregulated genes of the pathogen and thus a more pathogenorientated experimental set-up had to be sought Goumlrnhardt

et al

(2000) generated an SSH library from

in vitro

germinatedcysts of

P infestans

in order to identify genes important for theinitial step of the infection process It is however not yet knownif the genes identified in this approach also play a role in the laterstages of the potatondash

Phytophthora

interactionIn the screen described here the differentiation of

in planta

-induced genes of

P infestans

from the high background ofpathogen-activated plant genes was circumvented by exposingmycelium to potato leaves and then separating it from the planttissue Several

Phytophthora

genes were identified that areinduced in response to potato including genes with predictedhomologies to two nutrient transporters an ATP binding cassette-(ABC) transporter a protein involved in cell division a spliceo-some associated factor as well as an unknown protein Thus thescreening system described enables the analysis of genes from ahemibiotrophic pathogen that might play a role in the interactionwith its plant host

EXPERIMENTAL PROCEDURES

Growth of potato plants and

P infestans


strain 88069 was kindly provided byFrancine Govers (University of Wageningen Netherlands) It wasgrown on rye agar (Caten and Jinks 1968) at 18

deg

C in the darkVirulence was maintained by infecting leaves and re-isolatingevery 3ndash4 months Sporangia zoospores and cysts were obtainedas described by van West

et al

(1998) Germinated cysts wereobtained by incubation of cysts in water at 18

deg

C in the dark for4ndash6 h To obtain mycelium zoospores were germinated andgrown in liquid rye medium at 18

deg

C in the dark for 2ndash6 daysIsolation and purity of the different developmental stages wasconfirmed microscopically

Axenic potato plants cultivar Bintje were kindly provided byPatrick Schweizer (University of Fribourg Switzerland) The plantswere grown and maintained as described in Beyer

et al

(2001)

Media preparation

Defined medium was prepared according to Xu

et al

(1982) Forsolution A 20 g glucose 132 g (NH

4

)

2

SO

4

132 g CaCl

2

times

H

2

O

05 g MgSO

4

times

7H

2

O 07 g KH

2

PO

4

03 g K

2

HPO

4

001 gFe(SO4)

3

were mixed with 500 mL H

2

O and the pH was adjustedto 46 For solution B 50 mg each of MnSO

4

times

H

2

O

ZnSO

4

andthiamine were dissolved in 100 mL H

2

O For solution C 232 gfumaric acid was dissolved in 300 mL H

2

O and the pH wasadjusted to 46 Solutions A and C were mixed together with 1 mLof solution B and the pH was adjusted to 46 For the N-starvationmedium the defined medium was prepared without (NH

4

)

2

SO

4

while the C-starvation medium was prepared with only 2 gglucose

Infection of potato

Leaves were excised from 6-week-old plants placed with theabaxial side up on moist filter paper in Petri dishes and inoculatedwith

P infestans

zoospores Droplets (about 10

micro

L) containingapproximately 1000 zoospores were spotted on the abaxial sideof the leaf To maintain the high humidity required for infectionthe Petri dishes were closed with Parafilm They were placed inthe dark at 18

deg

C for 16 h and then transferred to a growthchamber (16ndash20

deg

C)

Differential screen

Mycelium was grown in liquid rye medium at 18

deg

C in the darkfor 6 days The mycelium was washed with water and smallportions were placed into a vessel containing 50 mL water(control) or on the abaxial side of a potato leaf floating in 50 mLwater (tester) The mycelium was incubated for 4 8 or 24 h andthe samples were pooled to create one control and one testersample The tester samples were harvested by removing themycelium from the potato leaf using forceps and the controlsample was handled in a similar manner The mycelium was driedquickly on Whatman paper and frozen in liquid nitrogen Poly(A)RNA was isolated from the mycelium using a Poly(A) Pure Kit(Ambion Witney Oxon) Suppression Subtractive Hybridization(SSH) was performed as described in Beyer

et al

(2001)

Dot blot assay

The cDNA inserts of the clones from the SSH library were ampli-fied by PCR using primers N1 and N2R (Clontech PCR-Select Kit)that are specific for the adaptors ligated on to the cDNA frag-ments during the SSH procedure Then NaOH and EDTA pH 8 wereadded to each PCR reaction to a final concentration of 04

M

NaOH and 10 m

M

EDTA and the PCR products were denatured at95

deg

C for 10 min A 30

micro

L aliquot of the PCR products was thenloaded in duplicate on two Zeta-Probe Blotting Membranes(Bio-Rad) using a dot blot manifold The slots of the dot blotmanifold were each pre-washed with 500

micro

L H

2

O then the PCRsamples were applied and each slot was rinsed again with 04

M


P infestans


475



(2002)

3

(6 ) 473ndash485


times


α

-

32


et al


deg




LAST




P infestans


micro


et al


deg


deg


deg


RESULTS



P infestans


et al


P infestans



P infestans


in vitro


P infestans


in planta

induced

Phytophthora

gene

ipiO

(Pieterse

et al


in vitro




in vitro

grown mycelium and

in planta



LAST


EN

B

ANK



476

K BEYER

et al


(2002)

3


as was employed for

S cerevisiae






Uromyces fabae


et al

1997Voegele

et al




P infestans


477



(2002)

3

(6 ) 473ndash485



in planta

induced gene of

Uromycesfabae

(Hahn

et al




micro


P infestans


Pinfestans


et al




Clonename Homology


Length offragment

Foldinductiondagger




478 K BEYER et al














480 K BEYER et al





DISCUSSION














482 K BEYER et al

















ACKNOWLEDGEMENTS


REFERENCES



484 K BEYER et al






















































P infestans


475



(2002)

3

(6 ) 473ndash485


times


α

-

32


et al


deg




LAST




P infestans


micro


et al


deg


deg


deg


RESULTS



P infestans


et al


P infestans



P infestans


in vitro


P infestans


in planta

induced

Phytophthora

gene

ipiO

(Pieterse

et al


in vitro




in vitro

grown mycelium and

in planta



LAST


EN

B

ANK



476

K BEYER

et al


(2002)

3


as was employed for

S cerevisiae






Uromyces fabae


et al

1997Voegele

et al




P infestans


477



(2002)

3

(6 ) 473ndash485



in planta

induced gene of

Uromycesfabae

(Hahn

et al




micro


P infestans


Pinfestans


et al




Clonename Homology


Length offragment

Foldinductiondagger




478 K BEYER et al














480 K BEYER et al





DISCUSSION














482 K BEYER et al

















ACKNOWLEDGEMENTS


REFERENCES



484 K BEYER et al






















































476

K BEYER

et al


(2002)

3


as was employed for

S cerevisiae






Uromyces fabae


et al

1997Voegele

et al




P infestans


477



(2002)

3

(6 ) 473ndash485



in planta

induced gene of

Uromycesfabae

(Hahn

et al




micro


P infestans


Pinfestans


et al




Clonename Homology


Length offragment

Foldinductiondagger




478 K BEYER et al














480 K BEYER et al





DISCUSSION














482 K BEYER et al

















ACKNOWLEDGEMENTS


REFERENCES



484 K BEYER et al






















































P infestans


477



(2002)

3

(6 ) 473ndash485



in planta

induced gene of

Uromycesfabae

(Hahn

et al




micro


P infestans


Pinfestans


et al




Clonename Homology


Length offragment

Foldinductiondagger




478 K BEYER et al














480 K BEYER et al





DISCUSSION














482 K BEYER et al

















ACKNOWLEDGEMENTS


REFERENCES



484 K BEYER et al






















































478 K BEYER et al














480 K BEYER et al





DISCUSSION














482 K BEYER et al

















ACKNOWLEDGEMENTS


REFERENCES



484 K BEYER et al




























































480 K BEYER et al





DISCUSSION














482 K BEYER et al

















ACKNOWLEDGEMENTS


REFERENCES



484 K BEYER et al






















































480 K BEYER et al





DISCUSSION














482 K BEYER et al

















ACKNOWLEDGEMENTS


REFERENCES



484 K BEYER et al





























































482 K BEYER et al

















ACKNOWLEDGEMENTS


REFERENCES



484 K BEYER et al






















































482 K BEYER et al

















ACKNOWLEDGEMENTS


REFERENCES



484 K BEYER et al






























































ACKNOWLEDGEMENTS


REFERENCES



484 K BEYER et al






















































484 K BEYER et al



































































Documents

Characterization of Phytophthora infestans genes regulated during the interaction with potato