Probing the contractile vacuole as Achilles' heel of the biotrophic grapevine pathogen Plasmopara

15 Pages·2017·3.75 MB·English

by  Viktoria Tröster

Protoplasma(2017)254:1887–1901
DOI10.1007/s00709-017-1123-y
ORIGINALARTICLE
’
Probing the contractile vacuole as Achilles heel of the biotrophic
Plasmopara viticola
grapevine pathogen
ViktoriaTröster1&TabeaSetzer1&ThomasHirth1&AnnaPecina1&
AndreasKortekamp2&PeterNick1
Received:3March2017/Accepted:10May2017/Publishedonline:26May2017
#Springer-VerlagWien2017
Abstract ThecausativeagentofGrapevineDownyMildew, activitydepends onthe activityofNADPH oxidase.The ef-
the oomycete Plasmopara viticola, poses a serious threat to fectoftheextractcanbephenocopiedbyitsmajorcompound,
viticulture.Inthecurrentwork,thecontractilevacuoleofthe glycyrrhizin,suggestingamodeofactionforthisbiologically
zoosporeisanalysedaspotentialtargetfornovelplantprotec- safealternativetocopperproducts.
tion strategies. Using a combination of electron microscopy,
spinningdiscconfocalmicroscopy,andvideodifferentialin- Keywords Contractilevacuole .DownyMildew.
terferencecontrastmicroscopy,wehavefollowedthegenesis Glycyrrhizin .Plasmoparaviticola .RGDpeptides
and dynamics of this vacuole required during the search for
thestomata,whenthenon-walledzoosporeisexposedtohy-
potonic conditions. This subcellular description was com- Introduction
bined with a pharmacological study, where the functionality
of the contractile vacuole was blocked by manipulation of Plasmopara viticola is the causative agent of Grapevine
actin, by Na, Cu, and Al ions or by inhibition of the Downy Mildew, a severe disease in viticulture with tremen-
NADPH oxidase. We further observe that RGD peptides dous economic impact worldwide. This parasitic protist be-
(mimickingbindingsitesforintegrinsattheextracellularma- longstotheclassofOomycetes,organisms,whichoriginally
trix)caninhibitthefunctionofthecontractilevacuoleaswell. had been considered as fungi. As already pointed out in the
Finally, we show that an extract from Chinese liquorice famoustextbookbyPeterSitte(KleinigandSitte1986),phy-
(Glycyrrhiza uralensis) proposed as biocontrol for Downy topathologicalresearchduringthesecondhalfofthelastcen-
Mildews can efficiently induce zoospore burst and that this turyhasunequivocallydemonstratedthattheOomycetesare
not fungi (for a historical overview, see Kutschera and
Hossfeld2012).Meanwhile,theseareclassifiedintothephy-
ThisstudyisdedicatedtothememoryofPeterSitte,Albert-Ludwigs-
lumofbiflagellateheterokontalgae,theStramenopila(Beakes
UniversityofFreiburg,whopassedawayin2016.
etal.2012).Distinctfeaturesare,forinstance,theirdeviating
HandlingEditor:UliKutschera ploidy(oomycetesarediplonts),andacellwallcomposedof
Electronicsupplementarymaterial Theonlineversionofthisarticle cellulose rather than chitin. The Oomycetes pass through a
(doi:10.1007/s00709-017-1123-y)containssupplementarymaterial, motilestageoftheirlifecycle,wheretheyharbourtwodiffer-
whichisavailabletoauthorizedusers.
entlyshapedflagella.NativetoNorthAmerica,P.viticolahas
been accidentally introduced into Europe in the nineteenth
* PeterNick
century on grapevine root stocks used as biological control
[email protected]
against the pathogenic insect Phylloxera (Gessler et al.
2011).SincetheEuropeanGrape,Vitisvinifera,isanaïvehost
1 MolecularCellBiology,BotanicalInstituteKarlsruheInstituteof
forP.viticola,theconsequencesweredevastating:thepatho-
Technology(KIT),Fritz-Haber-Weg4,Bld.30.43,
76131Karlsruhe,Germany genrapidlyconqueredallEuropeanvineyards,andisnow,in
2 InstituteofPlantProtectionStateEducationandResearchCenter additiontoPowderyMildew,onemajorreasonfortheintense
(DLR)Rheinpfalz,Breitenweg71,67435Neustadt,Germany useofchemicalplantprotection(basedonfungicidesthatare
1888 V.Trösteretal.
also used against true fungi) characteristic for conventional (Schaubschlägeretal.1994),andthelossesinyieldandqual-
viticulture. itybytheresultingdryberriesaresubstantial.
Acentralfactorofthisefficientspreadisthehighratesof Control of Downy Mildew in the highly susceptible
fastasexual reproduction. After hibernation inold foliage or EuropeangrapevinespeciesV.viniferaisdifficult,sincemost
soil,therobustlyarmouredoosporesinitiateprimaryinfection ofthelifecycleproceedsinsidethehost.Traditionally,differ-
duringspringrainfalls,andproduceagerminationpegtermi- ent copper compounds have been used, but since copper is
nating ina primary sporangium(Müller and Sleumer1934). toxictomanyorganismsandnotonlyaffectsbiologicaldiver-
From here, rapid cycles of asexual propagation proceed on sityinthesoilbutalsoposesaseriousthreattothequalityof
healthy grapevine organs, especially during humid and mild ground water (reviewed inVan Zwieten etal. 2004), copper
summer periods, resulting in epidemic spread all over the might be banned from conventional viticulture in the near
vineyardwithinfewdays.Numeroussporangiaareproduced future. However, copper preparations are still admitted for
andthentransportedbywindandraindrops.Athighhumid- organicviticulture(upto4kgha−1inGermany,andevenup
ity, the sporangia release five to eight motile zoospores to6kgha−1inFrance)withtheargumentthatherenoother
(Riemann et al. 2002) that swim towards the stomata of the alternatives for conventional fungicides are available.
hostandstartnewinfectioncycles.Obviously,thesuccessof Nevertheless,thispracticeisseenprogressivelyasproblemat-
the infection cycle depends on the efficiency by which the icandacompletebanofcopperproductshasbeendiscussed
motile zoospore can sense and target the stomata (Kiefer since several years. Conventional fungicides against Downy
etal.2002).Zoosporesaccumulateatthestomatawithinsev- Mildew includethe strobilurins(Bartlett etal. 2002) and as-
eral minutes and only in rare cases attach elsewhere. This sociatedcompoundsbelongingtothegroupofQoIfungicides.
stomatal targeting is impaired, when stomatal closure is in- Thesecompoundsaredoomedtofailure,sincealreadywithin
duced by abscisic acid. Also, the subsequent steps of early 10yearsofusage,P.viticolastrainsthathaveacquiredresis-
pathogen development, such as germ-tube morphogenesis, tance against these compounds have spread all over Europe
seem to be coordinated by unknown signals from the host. (Chen et al. 2007), and there are even cases of multidrug
Thesesignalsseemtobespecificforthehostspecies,because resistance against all relevant groups of fungicides (Giraud
zoospores of Plasmopara halstedii (the causative agent for et al. 2013). To increase the efficiency of fungicide applica-
SunflowerDownyMildew)didnottargettostomataongrape- tions and to confine further spread of fungicide resistance,
vineleavesasefficientlyasP.viticola(Kortekamp2003). interactivepredictionsystems,basedonepidemiological,me-
Oncethebiflagellatezoosporeshavesuccessfullylocateda teorological, and biological data, have been developed and
stoma, they attach to the guard cells by means of adhesive successfullyimplemented(Bleyeretal.2011).
mucus, shed their flagella, and encyst there. During Asalternativetofungicides,newvarietieshavebeengen-
encystation,zoosporesundergomassivechangesinstructure: erated by introgression of resistance loci from North
Theyroundup,rapidlygenerateacellulosiccellwall,ejectthe American or Siberian grapes (Eibach et al. 2007). However,
two flagella, and polarise their cytoskeleton (Riemann et al. theinitialsuccessofthisstrategyisprogressivelyendangered
2002). After several hours, cysts germinate and produce a bythe evolution ofnewstrains ofP. viticolathatare ableto
germination peg which reaches into the substomatal cavity. overcome these resistances and as a result can infect even
There, a substomatal vesicle is built from which a primary theseresistantcultivars(Rouxeletal.2013;Gómez-Zeledón
hypha emerges that penetrates into the mesophyll cells and etal.2013).
formshaustoriatoacquirenutrientsfromthehostplant,with- TheselimitationsinchemicalorgeneticcontrolofDowny
outbreachingthehostmembrane.Thehyphaedevelopfurther Mildew demonstrate that alternative targets are warranted.
intomyceliacolonisingtheintercellularspaceofoneintercos- During most of its life cycle, the pathogen is well protected
talfield.Atthisstage,theinfectioncanbeseenwiththenaked againstfungicides,eitherbecauseitisnotaccessible(hidden
eye as yellow Boil spots^ on top of the leaves. After a few insidethehostleaf)orbecauseitisprotectedbycellwalls,as
days, the mycelium develops sporangiophores that protrude in the case for sporangiophores, sporangia, and cysts. There
through the stomata and form tree-like branched structures, exists only a very short phase in the life cycle, where the
whichcarrylemon-shapedsporangia.Thissporulationisvis- pathogenisreallyvulnerable:betweenhatchingfromthespo-
ibleaswhitishpowderonthebottomsideoftheleavesinspir- rangium and the encystation at the stoma. This short phase
ingthenameofthedisease(DownyMildew).Duringhumid representssomething like the BAchilles’ Heel^ ofthe patho-
conditions and night temperatures ranging between 16 and genandmightbeusedastargetforchemicalplantprotection.
23 °C, sporangia detach and are spread by wind and rain to Zoosporesprovideatleasttwotargetsthatareinteresting:they
start a new infection cycle (Müller and Sleumer, 1934). need to locate the stomata, and they need to maintain their
Zoosporescaninfectallgreenpartsofvinesincludingberries integrityduringthismobilephase.
and inflorescences. Although Downy Mildew seems to be Stomataltargetingofmobilezoosporesmightcontributeto
non-toxic for humans, it can cause allergic reactions differences of susceptibility between different grapevines as
Achilles’heelofabiotrophicgrapevinepathogen 1889
concluded from a comparative infection study (Jürges et al. membrane,termedtheplasmalemmalreticulum(reviewedin
2009) on grapevine species from North America, Asia and Pickard2008),sharesmorphologicalandmolecularsimilari-
Europe.WhereasEuropeanspeciesaresuccessfulcolonised, ties with adhesion sites of mammalian cells and has been
species from North American and one species from Siberia implicated with mechanosensory ion channels. Whether
can arrestthe colonisation ofP. viticola briefly after the for- Oomycetes are endowed with a plasmalemmal reticulum is
mationofa germtube. Inseveral Asiatic species,a third re- not known. However, the observation of fungal RGD-
sponse pattern was observed, where stomatal targeting was mediatedadhesion(reviewedinHostetter2000)andspecific
impaired. As a consequence,a small and aberrantmycelium effects of RGD peptides in Achlya and Saprolegnia
was produced on the surface of the leaf that failed to infect (Chitcholtan and Garrill 2005; Kaminskyj and Heath 1995)
successfully(Jürgesetal.2009).Thefactthatmistargetingof indicatesthatfunctionalintegrinanaloguesalsospantheplas-
zoospores correlates with a failure of colonisation suggests mamembraneinOomycetesandmightintegratetheextracel-
thatspecific(host)signalscontroltheinteractionbetweenhost lularmatrixwithintracellularactin.
andpathogen.Chemotaxisofzoosporeshasalsobeenreport- The actin cytoskeleton of P. viticola is re-organised de-
ed for other Oomycetes, for instance, for the guiding of pending on stage, forming granulose plaque-like structures
Phytophthora cinnamomi to the root (Allen and Newhook, along with filaments in encysted and germinated spores, as
1973). wellaslongitudinalstrandsingermtubes.Oomyceteactinis
Thesecondtargetislinkedwiththefactthatzoosporeslack stronglypolarisednotonlyatthesiteofgermtubeemergence
cellwalls.Theyareonlyprotectedbyacellmembrane,mak- (P.cinnamomi,HydeandHardham1993)butalsowithinthe
ingthemasfragileasprotoplasts.Duringatimeintervalofup germtubeitself(P.viticola,Riemannetal.2002).
to30min,zoosporesareswimmingunprotectedovertheleaf Aparticipationofactinfilamentsandanaloguesorhomo-
surface, and have to cope with a constant influx of water. logues of a plasmalemmal reticulum for the function of the
Duringthisperiod, theyare probablyattractedbyplant sub- contractile vacuole is likely, but the structural detection of
stancesreleasedfromthestomata.Onlywhentheyattachand these components in vivo is far from trivial, since the zoo-
encyst, they reacquire a cell wall that safeguards the cell sporesaremovingvividlyandareverysmall.However,itis
against osmotic fluctuations. To avoid bursting, zoospores possible to probe the functionality of the contractile vacuole
are endowed with contractile vacuoles, collecting water and by quantifying zoospore burst in response to compounds
expulsingitinregularlycycles.Contractilevacuolesarepiv- supposedly interfering with the plasmalemmal reticulum
otalfortheregulationofcellvolumeandosmoticpotentialin (RGD peptides) or apoplastic oxidative burst (Na, Cu, and
freshwater protists (Patterson 1980). The contractile vacuole Al ions) as important signal for osmosensing (reviewed in
represents an interesting potential target for chemical plant Ismail et al. 2014). These functional data of the contractile
protection, because this structure is only found in the vacuole were combined with kinetic microscopy at high-
Oomycete pathogen, but not in its plant host, such that it resolution by both differential interference contrast, as well
shouldbepossibletofindcompoundsthatspecificallyinhibit asafterfluorescentvisualisationofactinandendomembrane
thecontractilevacuolewhileleavingthehostcellsuntouched. system. Using these approaches, the developmental se-
Nevertheless, knowledge about volume control of plant quence of the contractile vacuole was followed from its
cellsmightcontributetounderstandalsothephysiologyand genesisduringcellularisationofthesyncytialsporangiumtill
regulationofthecontractilevacuole.Infact,plantprotoplasts its disintegration upon encystment. To get insight into the
as well, although lacking a contractile vacuole, are able to membrane dynamics during cellularisation, different stages
adjust cellvolumetoa certain extentinresponsetoosmotic of sporangium germination till the hatching of zoospores
challenge byreleasing orrecycling membranematerialfrom wereinvestigatedbytransmission electron microscopylead-
internalstores(Liuetal.2013).Thisvolumecontrolislinked ingtoamodelformodeofactionforthetraditional,copper-
with a dynamic remodelling of submembraneous actin that, basedcontrolofDownyMildewand,thus,alsosuggeststhe
thus, must physically interact with the cell membrane and contractile vacuole as promising target for alternative, eco-
integrate mechanic load acting upon the membrane logically safer, ways of chemical control of this pathogen.
(reviewed in Nick 2011). Although plants lack canonical We explore one ofthese alternatives: extracts fromliquorice
integrinsthatconveythisfunctioninmammaliancells,there (Glycyrrhiza spec.) that have been reported to be effective
seem to exist functional analogues. For instance, specific againstdifferentOomycetepathogens(Schusteretal.2010).
heptapeptides (YGRGDSP, usually referred to as RGD pep- We can show that extracts from Glycyrrhiza uralensis, a
tides)thatcan titrate the interaction ofintegrins withthe ex- plant used in Traditional Chinese Medicine, as well as the
tracellularmatrix(reviewedinRuoslahti1996)arealsoeffec- respective active compound, glycyrrhizin, efficiently induce
tive in plant cells (Canut et al. 1998; Zaban et al. 2013). In zoosporeburstandthatthiseffectdependsontheactivityof
additiontothisfunctionalanalogy,therearestructuralanalo- NADPH oxidase, a membrane-based enzyme complex re-
gies as well. A meshwork of adhesion sites at the plasma sponsible for apoplastic oxidative burst.
1890 V.Trösteretal.
Materialandmethods addedtoareactiontubewith1mlofazoosporesuspension,
released from around 5 × 104 sporangia ml−1 and from a
Pathogen material All observations were made with 10-μgml−1stockinDMSOresultinginafinalconcentration
sporangia and zoospores of the plant pathogenic Oomycete of10ngml−1forthedyeand0.1%forthesolvent(DMSO).
P. viticola (Berk. & Curtis) Berl. & De Toni. To improve Suspensionanddyeweremixedgentlybyinvertingthetube
standardisation, strains derived from single sporangia were and incubated for 30 min, before sporangia and zoospores
used (Gómez-Zeledón etal. 2013).These strains, 1191_B11 were observed by spinning disc confocal microscopy under
and1191_B15,hadbeenbothcollectedfromthehostcultivar excitationwith488nm(detailsaregivenbelow).Duetothe
LembergerinLauffen,Germanyandwerekindlyprovidedby incubationtime,thesporangiawerefullyturgescentandsome
the lab of Prof. Dr. Otmar Spring at Hohenheim University. ofthemevenexhibitedactivecontractilevacuoles.Compared
Thestrainswherepropagatedonexcisedleavesofthehighly tothe protocolofKoningetal. (1993) using yeast cells, the
susceptibleviniferacultivarMüller-Thurgau.Forinoculation, concentration of DiOC was reduced by a factor of 1000,
6
a fully expanded Müller-Thurgau leaf was placed with the which was still perfectly sufficient to yield a good signal,
lowersurfaceonanaqueoussuspensionofmaturesporangia butshouldminimisepotentialsideeffects ofthedye onvia-
(∼104sporangiaml−1)inaPetridishof14cmindiameterand bilityandphysiologyofthecells.
incubatedovernightinthedarkat16°C(PercivalI-30BLL,
Percival Scientific, USA). The leaf was then removed from Visualisation of endosomes Endosomes were visualised by
the suspension at the following day and transferred with the thepolystyryldyeFM®4-64(MolecularProbes,Invitrogen)
inoculatedabaxialsideturnedupwardsontomoisttissueina atafinalconcentrationof1μMaddedinthesamemannertoa
fresh Petri dish further incubating with a cycle of 14 h light sporangialsuspensionfroma1-mMstocksolution.Afterin-
(TL-DSuper8018W/840,Phillips,25μmolm−2s−1accord- cubationfor30min,sporangiaandzoosporeswereobserved
ing to Williams et al. 2007) and 10 h of night at a constant by spinning disc confocal microscopy under excitation with
temperature of16 °C. Under these conditions, the sporangia 561nm(detailsaregivenbelow).Duetotheincubationtime,
emerged after 4 days. Sporangia were harvested from dried the sporangia were fully turgescent and some of them even
leaves using a custom-built vacuum cleaner. For all experi- exhibitedactivecontractilevacuoles.
ments,onlyfreshlyharvestedsporangiawereemployed.
Video microscopy of contractive vacuoles Sporangia were
Visualisation of actin filaments The spatial organisation of collectedfrominfectedgrapevineleaveswithheavysporula-
actininP.viticolasporangia,phalloidinconjugatedtoAlexa tion by excising leaf discs of around 1 cm2 that were trans-
Fluor488(MolecularProbes,Invitrogen),wasfollowedafter ferredinto600μlofdistilledwaterandinvertedafewtimesto
mild fixation according to the protocol of Riemann et al. rinse off the sporangia. The donor leaf piece was then re-
(2002) with minor modifications. Five hundred microlitres moved and the sporangia suspension incubated for 1–2 h in
of an aqueous zoospore suspension was fixed for 10 min in the dark at 16 °C in a phytotrone (I-30 BLL, Percival
the same volume of a freshly prepared 2-fold fixation stock Scientific,USA),andthereleaseofzoosporeswasmonitored
yieldingafinalconcentrationof1.85%w/vparaformaldehyde by checking aliquots microscopically. When the release was
in microfilament buffer (final concentration 100 mM complete, 80 μl of zoospore suspension was transferred to
K HPO , 100 mM KH PO , pH 7.3, 100 mM KCl, 0.25% custom-made slides. These slides were wrapped twice with
2 4 2 4
Triton X-100). The sporangia were briefly spun down and adhesive tape (tesa, Offenburg, Germany) in perpendicular
washed twice with 1 ml of distilled water by carefully direction to produce a central pool of 15 mm × 15 mm size
invertingthetube.Aftercollectingthesporangiabyafurther and 1 mm depth which was important to prevent zoospore
centrifugationstep,thesupernatantwasremovedtoaresidual burst through the pressure exerted by the coverslip during
volumeof100μl,andthefluorescentphalloidinwasaddedto observation. For video microscopy, the AxioImager.Z.1 mi-
a final concentration of 66 nM from a methanolic stock of croscope (Carl Zeiss, Jena, Germany) was used with a 63×
6.6 μM. After staining for 10 min, the sporangia were ob- LCI− NeofluarImmCorr differential interference contrast
servedbyspinningdiscconfocalmicroscopy(detailsaregiv- (DIC) objective (NA 1.3) and the time-lapse mode of the
en below) using an excitation at 488 nm line of an Argon- camera (Axio-Cam MRm, Carl Zeiss, Jena, Germany) at a
Kryptonlaser. frequency of 4 Hz. Videos were analysed by the
AxioVisionLE64software(CarlZeiss,Jena,Germany).
VisualisationofEREndoplasmicreticulum(ER)andnuclear
envelope in sporangia and zoospores of P. viticola were Spinning disc confocal microscopy Sporangia and zoo-
stained in vivo by 3,3′-dihexyloxa carbocyanine iodide sporesstained by different fluorochromes toprobe for actin,
(DiOC , Molecular Probes, Invitrogen) according toKoning ER,andendosomes(seeabove)wereviewedbyspinningdisc
6
etal.(1993)withminormodifications.Thedyewasdirectly confocalmicroscopy(Zeiss,Jena,Germany)usinga63×/1.44
Achilles’heelofabiotrophicgrapevinepathogen 1891
DICoilobjective(Zeiss,Jena,Germany)andaspinning-disc investigated along with the non-specific, charged peptide
device (YOKOGAWA CSU-X1 5000). Confocal z-stacks polyaminesulphate(Fluka,Buchs,Switzerland).Theconcen-
were collected with an AxioObserver.Z1 (Zeiss, Jena, tration of these peptides was varied, but never exceeded
Germany). For Alexa Fluor 488 phalloidin and DiOC , the 50 ng ml−1. In some experiments, a commercial
6
488nm,andforFM®4-64,the561nmlineofanAr-Krlaser phytotherapeutical extract used for Traditional Chinese
were used. Emission was at 518 and 670 nm, respectively. Medicine from G. uralensis (Nr. 118A, Glycyrrhizae radix,
Images were analysed with the Zeiss software (ZEN, blue Gan Cao, PhÿtoComm®, Kehl, Germany) was used. The
edition,Zeiss,Jena,Germany). concentrationoftheactivecompoundglycyrrhizininthisex-
tractwasestimatedtorangebetween8and20mgml−1based
TransmissionelectronmicroscopyofsporangiaTofollow on the typical values found in these roots (Chen and Sheu
ultrastructural changes of sporangia maturation, freshly har- 1993, Yokozawa et al. 2000). Since the precise content of
vestedsporangiawereincubatedinwaterfor1hinthedark glycyrrhizininthis extractwasnotknown,wealsousedthe
andcollectedbyabriefcentrifugationstep.Thesedimentwas pure compound glycyrrhizin in some experiments (Roth,
resuspendedin1.5mlof0.1Mcacodylatebuffer,pH7.2,and Karlsruhe, Germany) at 20 or 50 ng ml−1. The effect of
then fixed in 2.5% glutaraldehyde according to Heumann glycyrrhizinwas comparedtothe effectofeither250nMof
(1992) by a microwave-assisted fixation protocol, followed the actin polymerisation inhibitor Latrunculin B (Sigma,
by secondary fixation in OsO . After dehydration in an in- Deisenhofen,Germany), 250 nM ofthe membranerigidifier
4
creasing ethanol series (30, 50, 70, 80, 90, 100% ethanol), dimethylsulfoxide(DMSO,Roth,Karlsruhe,Germany),or2
specimenswereembeddedintoEponandsectionedbyultra- to 200 nM of diphenyleneiodonium (DPI, Sigma,
microtomy (Ultracut R, Leica, Bensheim, Germany) with a Deisenhofen,Germany),aspecificinhibitorofNADPHoxi-
diamond knife (DiATOME, Hatfield, PA). Ultra-thin slides dases,respectively.
wereplacedonpioloform-coatedgridsandkeptdryinagrid
box. After staining with 10% methanolic uranyl acetate and
lead citrate, samples were viewed by transmission electron Results
microscopyat80kV(Zeiss912).
Contractilevacuoleactivityprecedeszoosporeindividua-
Quantification of zoospore responses to different com- tion The mobile phase of the life cycle in P. viticola isvery
poundsMaturesporangiaweresuspendedtoaconcentration short(lessthan2h)andfollowsadevelopmentalsequenceof
of1.5×104sporangiaml−1indistilledwater,andzoospores sporangialswelling;zoosporecellularisation;hatching;amo-
wereallowedtohatchbyincubationofthesuspensioninthe bilephase,wherezoosporesswimtowardsthe stomata;and,
dark at 16 °C for 1–2 h till the release of zoospores was asfinalpoint,attachmentandencystation(Fig.1).Duringthis
complete, which was monitored by checking aliquots of the timeinterval,thecytoplasmexperiencesseveralsharpchang-
suspension by bright-field microscopy. The respective com- esinwaterpotential.Togetinsightintothecellulardetailsof
pound was then diluted directly into the suspension to the thetransitionsduringsporangialswellingandzoosporeindi-
corresponding final concentration, and the suspension was viduation,wefollowedchangesofultrastructurebytransmis-
then transferred into a haemocytometer (Fuchs-Rosenthal) sion electron microscopy after chemical fixation (Fig. 2).
andfollowedbydifferentialinterferencecontrastmicroscopy. Whereasthedrysporangiashowsacrinkledraisin-likeshape
Imagesoftheobservationfieldwererecordedatregulartime (Fig.2a),theyimmediatelyswellintolemon-shapedballoons
intervalsoverthefollowing90min,andtheincidenceofzoo- (Fig. 2c) upon contact with water, indicating that the water
sporeburstwasscoredandrelatedtothevalueobservedatthe potentialofthecytoplasminthedrysporangiummustbevery
sametimepointinacontrolexperimentconductedinparallel negative.Alreadypriortoswelling,thecytoplasmisinclose
with the same suspension, but without addition of this com- physicalcontactwiththesporangialwall(Fig.2b,spw),such
pound.Inthiscontrol,nozoosporeburstwasobservedupto thattheincreaseinvolumewillbuildupaconsiderableturgor
20 min, and also subsequently, only a low number ofbursts withinafewseconds.Thepartitioningofthesyncytialcyto-
wereseen.Theimpactoflowconcentrations(upto40μM)of plasmintoindividualzoosporespriortohatching(Fig.1)re-
sodium chloride, copper sulphate, and aluminium chloride quires that the cytoplasm has to detach from the sporangial
was tested, as well. To address a potential influence of the wall(Fig.2e,g),suchthattheturgorcomponentofthewater
extracellular matrix, the specific heptapeptide YGRGDSP potential will return to zero, and this situation will persist
(RGD;Panatecs,Tübingen,Germany,purity>75%),mimick- throughout the entire mobile phase. Only when zoospores
ingtheadhesivemotivesofanimalfibronectinandvitronectin encyst and again generate their own individual cell wall
recognised by animal integrins (shown to interact with un- (Fig. 1), the negative water potential of the cytoplasm will
known plant analogues), and the inactive invert YGDGRSP beagaincompensatedbyturgorpressure.Thesesharpchang-
(DGR; Panatecs, Tübingen, Germany, purity >75%) were es of water potential are accompanied by dramatic
1892 V.Trösteretal.
Fig.1 Schematicrepresentation
ofearlydevelopmentbetween
swellingofsporangiaand
encystationofzoospores.The
timepointofresuspendingdry
sporangiainwaterisdefinedas
0min.Timepointsareaverage
valuesforahost-freecondition
derivedfromKieferetal.(2002)
remodellingofthecytoplasm(Fig.2).Priortoswelling,when smallearlydense-bodyvesicles(Fig.2b,edbv)areobserved.
thesporangialwallisstillwrinkled(Fig.2b,spw),numerous Their grey colour may indicate lipids, which have been
Fig. 2 Ultrastructural details of sporangial swelling and zoospore maturatingsporangium.fSecretionofelectron-densematerialinamature
individuation followed by transmission electron microscopy. Dry sporangiumwithpartiallycompletedseparationofzoospores.edbvearly
sporangia prior to swelling (a, b) in comparison with fullyturgescent dense-bodyvesicle,prpmprospectiveplasmamembrane,exvexocytotic
sporangia (c, d) after aniline blue staining (a, c) and TEM (b, d) is vacuole.Notethatthezoosporehasalreadydetachedfromthesporangial
compared to fully swollen sporangia; nu nucleus, edbv early dense- wall (spw). g, h putative basal bodies (bb) in mature sporangia with
bodyvesicles,mtmitochondrionnotethestillwrinkledsporangialwall almostcompletedindividuationofzoospores;notethecontiguousplasma
(spw)inc.eEmergenceofnumerousmaturedense-bodyvesicles(mdbv) membrane(pm)inh
progressively replacing the early dense-body vesicles (edbv) in a
Achilles’heelofabiotrophicgrapevinepathogen 1893
proposedasenergysourceforthesyncytium(Gayetal.1971). 20–30s)couldbefollowedrepeatedly,showingthattheactiv-
Theseearlydense-bodyvesiclesarealsoseeninfullyturges- ity of contractile vacuoles starts prior to cellularisation of the
centsporangia,buthaveincreasedinsizeandinsteadappear syncytium.Whenthisactivitywasinvestigatedathightempo-
lighterincolour(Fig. 2d,edbv).Additionalorganelles, such ral resolution (4 frames per second), further details became
as individual nuclei (Fig. 2b, nu) or mitochondria (Fig. 2d, observable: The bifurcated state persisted for the first 6 s
mt),canbeencounteredaswell.Atlaterstages,thematurating (Fig.3c(1-3)),startingwithtwovacuolechambersofcompa-
sporangium contains numerous mature dense-body vesicles rablesize(Fig.3c(1)),followedbyshrinkageofonechamber
(Fig.2e,f,mdbv),progressively emergingand replacing the (Fig.3c(2)),andfusionofbothparts(Fig.3c(3))toalarger
greyearlydense-bodyvesicles.Hereby,transitionalstagescan singlevacuole(Fig.3c(4)).Thissinglevacuolewasseenover
be encountered, where a smaller and denser structure that is thenext6s(Fig.3c(4–6)),butunderwentphasesofexpansion
clearlydelineatedbyamembraneisseeninsidealargerand and shrinkage (Fig. 3c (4, 6)), when it appeared rough and
white vesicle that is, by itself, lined by a membrane. With granular and dynamic, whereas the fully turgescent vacuole
progressive maturation of the sporangium, a clear gap be- appeared translucent lined by a membrane that was clearly
tween sporangial wall and cell membrane develops (Fig. 2f, visible in the differential interference contrast (Fig. 3c (5)).
g),wheregranularmaterialseemstobedepositedbyexocy- Then,thenextcyclestartedwiththebifurcatedinitialsituation
toticvesicles(Fig.2f,exv).Moreover,longersheetslinedby (Fig. 3c (7)). Cellularisation proceeds by formation of mem-
twomembranesinparallelcanbefoundthatprobablycorre- branesina star-likemanner incentrifugaldirection(Fig.3d).
spondtoprospectiveplasmamembranesbetweenfuturezoo- Thecytoplasmispartitionedsuchthatonecontractilevacuole
spores in different stages of individuation (Fig. 2f, prpm; isassignedtoeachprospectivezoospore.
Fig.2h,pm).Occasionally,pairsofovalstructureswithelec-
tron dense parallel stripes are encountered in some sections Contractile vacuole activity is linked with dynamic
(Fig.2g,h,bb),probablyrepresentingthebasalbodiesofthe endomembraneremodellingThe structure of actin in tur-
twoflagella. gescent syncytial sporangia was visualised by fluorescent
Sporangia are syncytial cells, containing several nuclei in phalloidin after mild chemical fixation (Fig. 4a). When
thecytoplasm.Weobservedthatcontractilevacuoleswereal- subsequent sections from confocal z-stacks were follow-
readyactiveinthesyncytialcytoplasmpriortocellularisation ed, the mature sporangium was found to contain star-like,
into single zoospores (Fig. 3a). A contraction cycle lasted brightlyfluorescent,actinplaques,fromwhichconnecting
around 30 s and comprised phases, where the vacuole was actin cables emanated, consistent with previous observa-
bifurcatedfollowedbyphases,wherethetwochambersfused tions(Riemannetal.2002).Whereasthefixationrequired
toonelargervacuole(Fig.3a,b).Thissequence(lastingaround to label actin by phalloidin did not allow to observe
Fig.3 Activityofcontractile
vacuolepriortocellularisationof
thesyncytium.a,bMature
sporangiumwithbifurcated
contractilevacuoleatthe
beginning(a)andend(b)ofa
recordedtime-lapseseriesover
40s.cIndividualframesfroma
time-lapseseriesrecordedata
frequencyof4framespersecond
inthezoom-inindicatedbythe
whitesquareinaandb,timein-
tervalbetweentheshownframes
is2s.Whitearrowheadsindicate
thepositionofthecontractile
vacuole.dTwosporangiaatin-
cipientcellularisation,theensuing
cellmembranesareindicatedby
whitearrows;notethecontractile
vacuoleintheuppersporangium
(whitearrowhead)
1894 V.Trösteretal.
Fig.4 Cytologyoftheturgescent
sporangium.aActinvisualisedby
fluorescentphalloidinafter
chemicalfixation.Subsequent
sectionsfromaconfocalz-stack.
bERvisualisedbyDioC6
invivo.Subsequentframesfroma
confocalsection.TheDioC6
signalisshownalongwiththe
correspondingimageby
differentialinterferencecontrast
(DIC).Arrowsindicatearelease
siteofcontractilevacuoles;
asterisksindicateastar-likeER
structuresubtendingthecontrac-
tilevacuole.nunucleus
potential dynamic changes nor the interrelation between Afterindividuation,theindividualzoosporeswerealready
theactinplaqueswithcontractilevacuoles,itwaspossible motilewithinasporangiumandwereswimmingactivelyeven
to follow the endoplasmic reticulum (ER) in vivo using priortohatching(Fig.5a;thetimeintervalbetweentheframes
the fluorescent dye DiOC (Fig. 4b, supplemental movie 1–8 was 250 ms). Upon staining with the membrane imper-
6
S2).Thisallowstoseedetailsofthecontractilevacuolein meable endocytosis tracker FM4-64, the cell interior was
the prehatching sporangium that would be hard to pick up stronglystainedwithinafewminutesindicatingahighinten-
in the rapidly moving zoospores. When the fluorescent sity ofendocytotic uptake (Fig. 5b). This was alsoobserved
DiOC signal in individual sections from a confocal z- forthemature,butstillsyncytialsporangium.Alreadyinthe
6
stack was followed over time in comparison with the cor- prehatching state, the individual zoospores showed active
responding image collected by differential interference contractilevacuoles.Inthefreezoospores,thecontractilevac-
contrast (DIC), two prominent arrays were observed dur- uolewasobservedinproximityoftheflagellarroots(Fig.5c).
ingthephase,whenthecontractilevacuolewasroughand To characterisefunctionaland cytological aspects ofcon-
granular (Fig. 4 (b1, b2)): A star-like ER structure (aster- tractilevacuolesofP.viticola,singlezoosporeswerefollowed
isks in Fig. 4 (b1, b2)) consisting of converging, multiple, on their way by video microscopy and time series were re-
ER cisternae separated the chambers of the contractile corded by differential interference contrast (shown
vacuole, and disappeared upon fusion of the chambers. representativelyinFig.6a;SupplementalMovieS2).Theen-
Rosette-like structures composed of a central small cavity tirecyclelastedaround3s(meanvalue3.28s,standarderror
surrounded by several smaller or larger cavities were seen of the mean of 0.24 s,n = 42) and, thus, was strongly (by
around the site of release (arrows in Fig. 4 (b1)) and around 10-fold) accelerated over the situation in the mature
would fuse with one ray of the ER star during the same sporangium. The contractile vacuole was established during
phase (arrows in Fig. 4 (b1, b2)). During the subsequent thefirst∼20%ofthecycle,remainedstabletill~90%ofthe
phase of the contractile cycle, when the vacuole appeared cycle,andwasthenrapidlyreleasedduringthelast~10%of
smooth and translucent, the ER did not exhibit a pattern the cycle. During the granular phase of the cycle, numerous
that was obviously linked with the contractile vacuole. It vesicles could be seen to merge with the vacuole, but were
should be mentioned that the fluorescent signal appeared replaced by radial channels in the translucent phase. These
to be especially strong at the poles of the sporangium. channelsweredynamicaswell,andpersistedonlyashorttime
Achilles’heelofabiotrophicgrapevinepathogen 1895
Fig.5 Cytologyofzoospores.aZoosporesarereleasedfromturgorprior Mobilezoosporerecordedbydifferentialinterferencecontrast.fflfront
tohatching.bIntensivestainingofsyncytialsporangium(uppercell)and flagellum,rflrearflagellum,cvcontractilevacuole
hatchingzoospore(lowercell)bytheendocytosistrackerFM®4-64.c
afteremergence.Assumingasphericalshapeforbothvacuole toresultinzoosporeburst.Theincidenceofzoosporeburstin
andcell,themeanvolumeflowpersecondwasestimatedto response to different compounds can therefore be used as
be 3.8% (±0.30%) of total volume, which means that in readoutfortheeffectofthesecompoundsonthefunctionality
around 25 cycles (around 75 s), one entire cell volume of ofthecontractilevacuole.Undercontrolconditions,nozoo-
watermustbesecretedtopreventthezoosporefrombursting. spore burst was observedup to20 min;from 30min, a low
number (200 cases in a total population of around 120,000)
Contractile vacuole activity can be blocked by Cu2+, by couldbeobservedat30min,andthisvalueincreasedto400at
Al3+, and by RGD peptides Due to the massive secretion 90 min. The variability in this number between biological
ofwaternecessarytopreserveintegrityofamotilezoospore, replicas was 21%. Addition of 10 μM of sodium chloride
evenminorperturbationofthecontractilevacuoleisexpected increased the incidence of zoospore burst by a factor of 4
Fig.6 Functionalandcytologicaldetailsofthecontractilevacuoleina thecycle.bMagnificationofthecontractilevacuolefromthetimeseries
free zoospore. a Time series over one contraction cycle recorded by showninatohighlightcytologicaldetails.Numbersasina.Whitearrows
differential interference contrast. Theentire cycle lasted 3 s; numbers indicate vesicular structures that fuse during the early phase of the
indicatetherelativetimeoftherespectiveframeinpercentoftheentire contractile cycle, white arrowheads indicate channels of changing
cycle (100% corresponding to one completed cycle). The contractile width that appear and disappear during the stable phase of the
vacuoleisestablishedduringthefirst~20%ofthecycle,remainsstable contractilecycle
till~90%ofthecycle,andisthenrapidlyreleasedduringthelast~10%of
1896 V.Trösteretal.
Fig. 7 Chemical manipulation of zoospore burst. Metal ion (a, b), foraconcentrationadjustedto10μM;incaseofECM-interactingpep-
peptides interacting with the extracellular matrix (c, d), compounds tides(c),theconcentrationwasadjustedto20ngml−1.Dose-response
actinonactin,membranefluidity,andgenerationofsuperoxide(e)have curvesformetalions(b)weremeasuredat30minafteradditionofthe
beentested,aswellasanextractofGlycyrrhizauralensis,aplantusedin ions,incaseofECM-interactingpeptides(d),at90minafteradditionof
Traditional Chinese Medicine (f). The incidence of zoospore burst as the peptides. Identicalconcentrationsofsporangia (15,000 ml−1) were
readout for impaired function of the contractile vacuole is shown in used,andeachexperimentwasaccompaniedbyanuntreatedcontrolas
relativevaluesoveracontrolwithouttreatment.Timecourses(a,c)and internalstandard.Valuesgivetheincidenceofburstingzoosporesrelative
dose-responsecurvesforafixedtimeoftreatment(b,d)areshown.As tothisinternalstandardatthefinaltimepointoftheexperiment(30minin
metalions(a,b),aluminium(Al),copper(Cu),andsodium(Na)were caseofmetalions,90minincaseofECM-interactingpeptides).Values
tested,as peptidesinteractingwiththeextracellularmatrix(ECM),the representmeansandstandarderrorsfromtwoindependentexperimental
heptapeptideYGRGDSP(RGD)mimickingtheadhesivemotivesofan- series;undercontrolconditions,nozoosporeburstwasobservedupto
imalfibronectinandvitronectinrecognisedbyanimalintegrins(shownto 20min,alownumber(200ml−1ofsporangialsuspension)wasobserved
interact with unknown plant analogues), and the inactive invert at30min,andthisvalueincreasedto400ml−1at90min.Thevariability
YGDGRSP(DGR)weretestedalongwiththenon-specific,chargedpep- inthisnumberbetweenbiologicalreplicaswas21%
tidepolyaminesulphate.Timecoursesformetalions(a)wererecorded
(Fig. 7a). In contrast, addition of the same concentration of (DGR), and the non-specific, charged peptide polyamine sul-
either copper sulphate or aluminium chloride drastically en- phate.Time-coursesofzoosporeburstwererecordedforthese
hancedtheincidenceofzoosporebursttomorethantwoor- peptides(Fig.7c)usingapeptideconcentrationof20ngml−1
dersofmagnitudewithin30mincomparedtothecontrol.A (Fig.7c).Afteralagphaseof20min,zoosporesburstprogres-
dose-responsecurverecordedat30minaftertheonsetofthe sivelyreachingaplateauof10-foldoverthecontrolwithalag
treatment(Fig.7b)showedthattheeffectwassaturatedfrom phase of 40 min (polyamine sulphate) or 80 min (RGD). In
20μMincaseofaluminiumions,whereasincaseofcopper, contrast,theDGRpeptideswerefoundtobemostlyineffective.
similar values were reached for 40 μM. For sodium, from Dose-responsecurvesassessedat90minafter additionofthe
20μM,aplateauataround25-foldincreasecomparedtothe peptides (Fig. 7d) showed a strong increase of burst with in-
plateauwasobserved. creasingconcentrationsfortheRGDpeptide,resultinginalmost
Inanimalcells,theheptapeptideYGRGDSP(abbreviatedas 20-foldhighervaluescomparedtocontrol,whereastherewas
RGD) is found in the extracellular matrix proteins fibronectin little effect of DGR even at the highest concentration
and vitronectin, and is recognised by animal integrins. The (50ngml−1).Polyaminesulphatelevelledoffataplateauthat
bindingconfersmutualadhesionbetweenanimalcellsortheir wasaroundhalfthevalueobservedfortheRGDpeptide.These
interaction with the extracellular matrix. To test whether ana- observationsindicatethatRGDcanimpairthefunctionofthe
loguesofintegrinsarerelevantforthefunctionofthecontractile contractilevacuole.Thisinhibitionisspecific,sinceonlyapart
vacuole,theinfluenceofRGDpeptideswastestedalongwith ofthiseffectcanbephenocopiedbytheunspecific,positively
thebiologicallyinactive,butequallychargedinvertYGDGRSP chargedpolyaminesulphate.