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ThisarticleispublishedinPlantPhysiologyOnline,PlantPhysiologyPreviewSection,whichpublishesmanuscriptsacceptedfor publicationaftertheyhavebeeneditedandtheauthorshavecorrectedproofs,butbeforethefinal,completeissueispublished online.Earlypostingofarticlesreducesnormaltimetopublicationbyseveralweeks. Differential Antifungal and Calcium Channel-Blocking 1[w] Activity among Structurally Related Plant Defensins Robert G. Spelbrink, Nejmi Dilmac, Aron Allen, Thomas J. Smith, Dilip M. Shah*, and Gregory H. Hockerman The Donald Danforth Plant Science Center, St. Louis, Missouri 63132 (R.G.S., A.A., T.J.S., D.M.S.); and Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana 47907 (N.D., G.H.H.) PlantdefensinsareafamilyofsmallCys-richantifungalproteinsthatplayimportantrolesinplantdefenseagainstinvading fungi.Structuresofseveralplant defensinssharea Cys-stabilizeda/b-motif. Structural determinants inplantdefensins that govern their antifungal activity and the mechanisms by which they inhibit fungal growth remain unclear. Alfalfa (Medicago sativa) seed defensin, MsDef1, strongly inhibits the growth of Fusarium graminearum in vitro, and its antifungal activity is markedly reduced in the presence of Ca21. By contrast, MtDef2 from Medicago truncatula, which shares 65% amino acid sequence identity with MsDef1, lacks antifungal activity against F. graminearum. Characterization of the in vitro antifungal activity of the chimeras containing portions of the MsDef1 and MtDef2 proteins shows that the major determinants of antifungal activity residein the carboxy-terminal region (amino acids 31–45) of MsDef1. We furtherdefine the active site by demonstratingthattheArgatposition38ofMsDef1iscriticalforitsantifungalactivity.Furthermore,wehavefoundforthe firsttime,toourknowledge,thatMsDef1blocksthemammalianL-typeCa21channelinamannerakintoavirallyencoded and structurally unrelated antifungal toxin KP4 from Ustilago maydis, whereas structurally similar MtDef2 and the radish (Raphanussativus)seeddefensinRs-AFP2failtoblocktheL-typeCa21channel.Fromtheseresults,wespeculatethatthetwo unrelatedantifungalproteins,KP4andMsDef1,haveevolutionarilyconvergeduponthesamemoleculartarget,whereasthe twostructurally related antifungal plantdefensins, MtDef2 andRs-AFP2,have divergedtoattack different targetsinfungi. Plant defensins are small (45–54 amino acids) Cys- a broad spectrum of filamentous fungi. The morpho- rich proteins implicated in the first-line host defense genic antifungal defensins reduce hyphal elongation against fungal pathogens (Thomma et al., 2002). The and induce hyperbranching, whereas nonmorpho- tertiary structures of these proteins are quite similar genic defensins reduce hyphal elongation without and share a common Cys-stabilized a/b-motif com- causing any morphological distortions (Broekaert posed of three antiparallel b-strands and one a-helix. etal.,1995;Thommaetal.,2003).Despitesomeprogress This motif is also found in insect defensins and scor- made in the past few years, the structure-activity pion neurotoxins (Fontecilla-Camps, 1989; Bontems relationshipsandmodesofactionformostoftheplant et al., 1991; Kobayashi et al., 1991). Despite their defensinsremainunknown.Mutationalanalysisofthe structuralsimilarity,plantdefensinsarehighlyvaried radish(Raphanussativus)Rs-AFP2hasrevealedthatthe in their primary amino acid sequences, with only amino acid residues important for antifungal activity eight structure-stabilizing Cys residues in common are clustered into two adjacent sites. The first site is (Thomma et al., 2002). The variation in the primary aroundthetypeVIb-turnconnectingb-strands2and3, sequences may account for the different biological andthe second site isformed by residues onthe loop activities reported for plant defensins, including anti- connecting b-strand 1 and the a-helix and the contig- fungalactivity(Terrasetal.,1995),antibacterialactivity uous residues on the a-helix and b-strand 3 (De (Segura et al., 1998), proteinase activity (Wijaya et al., Samblanx et al., 1997). Unlike the mammalian and 2000), and a-amylase inhibitory activity (Bloch and insect defensins, antifungal plant defensins induce Richardson,1991). membrane permeabilization through specific interac- Some plant defensins exhibit potent antifungal tion with high-affinity binding sites on fungal cells activity in vitro at micromolar concentrations against (Thevissen et al., 1997, 2000) but do not form ion- permeableporesinartificiallipidbilayers,nordothey change their electrical properties (Thevissen et al., 1996). When fungal hyphae are treated with Rs-AFP2 1ThisworkwassupportedbytheNationalInstitutesofHealth or the Dahlia merckii defensin Dm-AMP1, there is (grantno.GM–10704toT.J.S.). arapidinfluxofCa21,effluxofK1,andalkalinization *Corresponding author; e-mail [email protected]; fax of the growth medium (Thevissen et al., 1996). How 314–587–1581. [w]TheonlineversionofthisarticlecontainsWeb-onlydata. this interaction of defensin proteins with fungal Article,publicationdate,andcitationinformationcanbefoundat hyphae generates plasma membrane ion fluxes www.plantphysiol.org/cgi/doi/10.1104/pp.104.040873. leading to fungal growth inhibition remains unclear. Plant Physiology, August 2004, Vol. 135, pp. 1–13, www.plantphysiol.org (cid:1) 2004 American Society of Plant Biologists 1 of 13 Downloaded from on April 16, 2019 - Published by www.plantphysiol.org Copyright © 2004 American Society of Plant Biologists. All rights reserved. Spelbrink et al. The characterization of defensin-resistant mutants of RESULTS unicellularandfilamentousfungihasimplicatedarole Alfalfa Defensins, MsDef1 and MtDef2, Differ in Their forfungalsphingolipidsandglucosylceramidesinthe Antifungal Activity mechanism of growth inhibition by these defensins (Thevissenetal.,2000,2004;Ferketetal.,2003;Thomma The cloning and sequence analysis of the cDNA et al., 2003). More recently, it has been shown that encoding the alfalfa seed defensin MsDef1 has been Rs-AFP2 interacts with fungal glucosylceramides in reported previously (Gao et al., 2000). A cDNA clone afirststepleadingtofungalgrowtharrest(Thevissen encoding MtDef2 was cloned and sequenced as de- etal.,2004). scribed in ‘‘Materials and Methods.’’ The amino acid Itislikelythatnotallplantdefensinsactbythesame sequences of MsDef1 and MtDef2 are 65% identical modeofaction.Forexample,patchclampexperiments and carry a net positive charge of 13 and 21, re- demonstratedthatplantdefensinsisolatedfrommaize spectively (Fig. 1).The in vitro antifungal activitiesof (Zea mays) inhibit sodium currents in a rat tumor cell thesetwoproteinsagainstafungalpathogen,F.grami- line.However,theirinvitroantifungalactivitywasnot nearum, were determined and compared with the reported (Kushmerick et al., 1998). Some scorpion antifungal activities of a previously characterized neurotoxins are structurally related to defensins and radish seed defensin, Rs-AFP2 (Terras et al., 1995), areknown to block potassium channels (Garcia et al., and the virally encoded killer toxin KP4 from U. 2001). Based on surface topology similarities with maydis (Young, 1987; Park et al., 1994; Fig. 2; Table I). potassium channel blockers, a similar mode of action MsDef1, Rs-AFP2, and KP4 exhibited strong dose- has been proposed for a pea (Pisum sativum) seed dependent antifungal activity against this fungus, defensin Psd1 (Almeida et al., 2002). whereasMtDef2didnot.Althoughlesspotent,MsDef1 We have previously reported the isolation and andKP4inducedmorepronouncedhyperbranchingof characterization of the broad-spectrum antifungal de- thefungalhyphaethanRs-AFP2(Fig.2B).Itshouldbe fensin (MsDef1) from alfalfa (Medicago sativa) seed notedthatthemostdramaticeffectofthesedefensinsis previouslyreferredtoasAlfAFP(Gaoetal.,2000).This an altered growth pattern or hyperbranching that is protein was found to inhibit the hyphal elongation of accompaniedbyinhibitionofhyphalelongation. the fungal pathogen Fusarium graminearum in a dose- dependentmanner,causingahyperbranchingpheno- C-Terminal Region (Residues 31–45) of MsDef1 Is type. We demonstrate here that the carboxy-terminal Important for Its Antifungal Activity region(residues31–45)ofMsDef1containsmajordeter- minants for antifungal activity with the N-terminal InordertoidentifytheregionsofMsDef1molecules region (residues 1–15) contributing to the antifun- that are important for antifungal activity and for in- gal activity in a relatively minor way. In addition, we duction of hyperbranching phenotype in the fungus, showthatArgatposition38iscriticalfortheantifun- chimeric defensins consisting of portions of MsDef1 gal activity of MsDef1. This residue lies in a position and MtDef2 proteins were tested for their antifungal homologous to the active site of the known Ca21 activity and ability to induce hyperbranching in F. channel blocker KP4 and the Na1 channel blocker graminearum. MsDef1 and MtDef2 were divided into scorpion toxin AaHII. Furthermore, we show for the threeregionsofsimilarlength,baseduponthesecond- first time, to our knowledge, that a plant defensin, ary structural elements predicted by sequence align- MsDef1, selectively blocks the mammalian L-type ments with plant defensins whose three-dimensional Ca21 channel in a manner similar to that of KP4, (3D)structureshavebeendetermined(Fantetal.,1998; astructurallyunique,virallyencodedkillertoxinfrom Almeidaetal.,2002;Layetal.,2003).Chimericproteins theP4strainofthecornsmutfungus,Ustilagomaydis. correspondingtoallsixpossiblecombinations(Fig.3), However, two other structurally similar defensins, termedDef1-2C1throughDef1-2C6,wereobtainedby MtDef2 and Rs-AFP2, do not block the L-type Ca21 expressingthesyntheticgenesencodingtheseproteins channel.Theblockagewasfoundtobeverystrong(up in Pichia pastoris. All six chimeric proteins were to 90% blockage) and highly specific for the L-type screenedforantifungalactivityagainstF.graminearum channel. Finally, we report functional homology be- andNeurosporacrassa,alongwithMsDef1andMtDef2 tween MsDef1 and a known Ca21 channel blocker, proteins. Antifungal activity was assessed both by KP4, suggestinga common mode of actionin fungi. measuring hyphal growth inhibition after 16 h of Figure1. SequencecomparisonofRs-AFP2and Medicago defensins. Significant differences in residues between the two Medicago defensins areshowninboldface.Thechargeofeachpro- teinisshowninparentheses.Symbolsrepresent approximate positions of the predicted a-helix (spiral)andb-sheets(arrows). 2 of 13 Plant Physiol. Vol. 135, 2004 Downloaded from on April 16, 2019 - Published by www.plantphysiol.org Copyright © 2004 American Society of Plant Biologists. All rights reserved. Alfalfa Defensin Blocks a Mammalian Calcium Channel Figure2. ComparisonbetweentheeffectsofKP4,MsDef1,Rs-AFP2,andMtDef2onF.graminearum.A,MsDef1,KP4,and Rs-AFP2allexhibitadose-dependentinhibitionofhyphae.Assayswereconductedinlowionicstrengthsyntheticfungalmedia inoculatedwithfungalspores.Micrographspreparedaftera16-hincubation.B,HyperbranchingeffectonFusarium.Invitro assayswerepreparedinthesamemannerasdescribedinA,with25mgmL21ofantifungalprotein.Theaveragenumberof hyphalbudspergermlinewasdeterminedbycounting50germlingsafter9hofincubation.Thereisamarkeddifferenceinthe numberofbudsamongtheproteinstested,withMsDef1andKP4inducingthemostbuds,Rs-AFP2causingalessextremeeffect, andMtDef2causingnohyperbranching. Plant Physiol. Vol. 135, 2004 3 of 13 Downloaded from on April 16, 2019 - Published by www.plantphysiol.org Copyright © 2004 American Society of Plant Biologists. All rights reserved. Spelbrink et al. Figure3. AntifungalactivityofthechimericDef1/Def2defensinsonF.graminearum.MsDef1andMtDef2weredividedinto threeregionsofsimilarlength,andchimericproteinscorrespondingtoallsixpossiblecombinations,termedDef1-2C1through Def1-2C6,wereobtainedbyexpressingthesyntheticgenesencodingtheseproteinsusingaP.pastorisexpressionsystem.Allsix chimeric proteinswere screenedforantifungal activityagainstF. graminearumand N. crassa (data not shown), along with MsDef1andMtDef2proteins.Antifungalactivitywasassessedbymeasuringbothhyphalgrowthinhibitionafter16hofexposure totheproteinsanddeterminingthedegreeofhyperbranchingafter9hofexposuretotheproteins(TableII). exposuretotheproteinsandbydeterminingthedegree MsDef1. We selected Arg-38 as a candidate for muta- ofhyperbranchingafter9hofexposuretotheproteins. genesisforthefollowingreasons:first,theresiduelies As shown in Figure 3 and Table II, the C-terminal inthemotifofMsDef1mostsimilartotheactivesiteof sequence (residues 31–45) of MsDef1 contains the theionchannelblockerKP4andscorpiontoxinAaHII major determinants of the in vitro antifungal activity (Fontecilla-Camps,1989;Guetal.,1995).Bothproteins sinceDef1-2C2containingthissequencewasnearlyas have been shown to have a basic residue at the base active as MsDef1, whereas Def1-2C3 containing of the b2-b3 loop that is critical for its antifungal the corresponding sequence of MtDef2 was inactive activity—Lys-42 for KP4 and Lys-58 for AaHII againstF.graminearum.TheN-terminalsequence(res- idues 1–15) of MsDef1, however, does contribute somewhattotheantifungalactivity oftheCterminus TableI. IC valuesforpeptides sinceDef1-2C4ismorepotentthaneitherDef1-2C2or 50 Antifungal activity of defensins. The growth inhibition and hyper- Def1-2C5. As shown in Figure 3, both the N-terminal branchinginducedbydefensinswerequantified.Theconcentrationof (residues 1–15) and C-terminal (residues 31–45) se- defensins required to inhibit 50% of the overall growth (IC ) was 50 quences of MsDef1 are required to observe the same determined spectrophotometrically and confirmed visually. Fungal degree of hyperbranching as that observed for the sporesweregrowninsyntheticmediasupplementedwith2-foldserial wild-type MsDef1. The middle region of the defensin dilutions of defensins. The absorbance at 595 nm was measured (residues 16–30) apparently does not contain signifi- after48h. cantdeterminantsofantifungalactivity.Similarresults IC IC 50 50 were obtained when the chimeric defensins were AntifungalProtein F.graminearum N.crassa tested on a more sensitive test fungus, N. crassa. mgmL21 mgmL21 It should be noted that, at concentrations higher than 12 m g mL21, all proteins, including Def1-2C1, MsDef1 6–12 1–3 MtDef2 .100 25–50 Def1-2C3, Def1-2C6, and MtDef2, showed modest Rs-AFP2 1–3 ,1 antifungal activity against N.crassa. KP4 .100 50–100 Def1-2C1 .100 25–50 Arg-38 Is Important for Antifungal Activity of MsDef1 Def1-2C2 6–12 1–3 Def1-2C3 .100 25–50 In an effort to further define the region of MsDef1 Def1-2C4 6–12 1–3 conferring antifungal activity, we performed site- Def1-2C5 6–12 1–3 directed mutagenesis on MsDef1 and expressed the Def1-2C6 .100 25–50 variantproteininP.pastoris.Theantifungalactivityof Def2-Q39R .100 1–3 Def1-R38Q .100 25–50 the purified protein was compared with that of 4 of 13 Plant Physiol. Vol. 135, 2004 Downloaded from on April 16, 2019 - Published by www.plantphysiol.org Copyright © 2004 American Society of Plant Biologists. All rights reserved. Alfalfa Defensin Blocks a Mammalian Calcium Channel TableII. Quantitationofdefensin-inducedhyperbranchinginF.graminearum Thedegreeofhyperbranchingwasdeterminedbycountingthenumberofhyphalbuds8hafterincubationwithdefensin.Numbersinthetableare averagesofatleast50sporespertreatment. Protein NoPeptides MsDef1 MtDef2 Rs-AFP2 KP4 C1 C2 C3 C4 C5 C6 Ave 2.0 4.6 2.0 3.0 3.6 2.0 3.1 2.1 3.8 4.1 2.1 StD 0.0 1.3 0.1 1.0 0.9 0.1 1.0 0.2 1.0 1.2 0.2 (Fontecilla-Camps, 1989; Gu et al., 1994; Gage et al., fungus is poorly recognized by Def2-Q39R and that 2001). Second, Arg-38 lies in a homologous region additional residues in proximity to Arg-38 may be showntoinfluence theantifungalactivity ofRs-AFP2 important for full recognition. (De Samblanx et al., 1997). Third, we noticed that Arg-38 is replaced by Gln in MtDef2. We suspected that the difference in charge at this position might Antifungal Activity of MsDef1, Like That of KP4, account for a large difference in the antifungal activ- Is Strongly Abrogated by Exogenous Ca21 ities of these two proteins (Table I). Therefore, we made a single amino acid substitution to both the It has been shown previously that the antifungal MsDef1protein(R38Q)andtheMtDef2protein(Q39R) activity of KP4 is specifically abrogated by exoge- and compared their antifungal activities against N. nously added Ca21 (Gu et al., 1995). Similarly, the crassa and F. graminearum. We found that, against N. antifungal activity of some plant defensins is signifi- crassa hyphae, the Def2-Q39R was vastly morepotent cantlyreducedwhenthecationicstrengthofthefungal thanthewild-typeMtDef2protein(Fig.4;TableI)and growth medium is increased (Terras et al., 1992, almost identical to that of MsDef1. Conversely, the 1993). Therefore, we tested the effects of Ca21 on the Def1-R38Q variant showed a dramatic decrease in in vitro antifungal activity of MsDef1 and Rs-AFP2 antifungalactivitycomparedtothewild-typeMsDef1 and compared them with that of KP4. As shown in protein and had similar antifungal activity as wild- Figure 5, a relatively low concentration of Ca21 type MtDef2. Therefore, the exchange of a single abrogates KP4 and MsDef1 activity against F. grami- amino acid between MsDef1 and MtDef2 was able to nearumbutdoesnotaffecttheactivityofRs-AFP2.As switch the antifungal activity of the two defensins. littleas0.5mMCa21reducedtheantifungalactivityof Similar results were obtained against F. graminearum, KP4 and MsDef1, with total abrogation occurring at withtheexceptionthattheDef2-Q39Rmutantwasnot 2mMCa21.TheantifungalactivityofKP4waspartially appreciably more potent than the wild-type defensin. reducedby2mMMg21,butmorethan5mMMg21was It is likely that the molecular target of MsDef1 in this required for complete abrogation. With MsDef1, no Figure4. Arg-38ofMsDef1isimportantforantifungalactivity.Sporesofbothfungiwereallowedtogerminateandgrowin syntheticfungalmediasupplementedwith12mgmL21oftheindicateddefensinforN.crassaand25mgmL21defensinsfor F.graminearum.Growthinhibitionandhyperbranchingweredeterminedasdescribedin‘‘MaterialsandMethods.’’ Plant Physiol. Vol. 135, 2004 5 of 13 Downloaded from on April 16, 2019 - Published by www.plantphysiol.org Copyright © 2004 American Society of Plant Biologists. All rights reserved. Spelbrink et al. MsDef1, but Not MtDef2 and Rs-AFP2, Blocks the Mammalian L-Type Ca21Channel IthasbeendemonstratedpreviouslythatKP4inhib- itsCa21uptakeinfungalcells(Gageetal.,2001).Rather unexpectedly,KP4wasfoundtospecificallyblockthe L-type voltage-gated Ca21 channels in a weakly volt- age-dependent fashion (Gu et al., 1995; Gage et al., 2002). Since the antifungal activity of MsDef1 closely parallelsthatofKP4,wetestedMsDef1foritsabilityto blockaCa21channelinmammaliancells.Theactivity ofMsDef1onBa21currentsconductedbythreedistinct voltage-gated Ca21 channels, Ca 2.1 (De Weille et al., v 1991), Ca 1.2 (Snutch et al., 1991), and Ca 2.3 (Soong v v et al., 1993) from rat brain coexpressed with auxiliary subunitsb (Pragnelletal.,1991)anda d(Ellisetal., 1b 2 1988) in tsA-201 cells, was assayed as described pre- viously(Gageetal.,2002).AsshowninFigure6A,10 mM MsDef1 blocks approximately 90% of the Ca21 current through the Ca 1.2 (L-type) channel, with the v maximuminhibitionoccurringafterexposingthecells tothedefensinforapproximately13min.Theblockby 2 mM MsDef1developedslowlyover several minutes, such that 0.56% 6 0.03% of current remained at equilibrium (n 5 3; Fig. 6B). A lower concentration wasusedinordertoascertainwhetherMsDef1affected thevoltagedependencyofthecurrent.Figure6Cshows the current-voltage relationship of Ca 1.2 in the pres- v ence or absence of 2 mM MsDef1. Cells expressing Ca 1.2, as described above, wereheld at 260 mVand v depolarizedtotheindicatedvoltagefor100msbefore (blackcircles)orafter(whitecircles)equilibrationin2 mM MsDef1. While MsDef1 decreases peak current, it doesnotappreciablyshiftthecurrent-voltagerelation- shipofCa 1.2(Fig.6C).MsDef1didnotblockeitherthe Figure5. AbrogationoftheantifungalactivityofMsDef1andKP4by v Ca21. Shown here is an example of one of the tested metals and Cav2.1ortheCav2.3channel(Fig.6,DandE).Surpris- concentrations;50mgmL21KP4and5mgmL21MsDef1andRs-AFP2 ingly, Rs-AFP2 failed to block any of the three Ca21 wereusedinthisexperiment. channels despite sharing a 3D structure similar to MsDef1andhavingpotentactivityagainstF.graminea- rum (Fig. 6, F and G). As expected, MtDef2, which is closelyrelatedtoMsDef1butlacksinvitroantifungal reduction in antifungal activity was observed until mMosDreefth1,anm4ormeMthMang2215wmaMs aNdad1edo.rF2o5r bmoMthKK1P4isanred- Lac-ttyivpiteyCaag2a1inchstaFn.ngerla(mFiinge.a6rHum).,alsofailedtoblockthe quired to reduce their antifungal activity and 50 mM is needed for complete abrogation. In terms of ionic 45Ca21 Uptake in N. crassa Hyphae strength, 3-fold more Mg21 and 10-fold more K1 or Na1arerequiredtocausethesamedegreeofreduction There have been several previous studies that at- of antifungal activity of MsDef1 as Ca21. Therefore, tempted to measure Ca21 flux across fungal mem- the in vitro antifungal activity of MsDef1, like that of branes in response to defensins and KP4 (Thevissen KP4, is particularly sensitive to exogenously added etal.,1996,1999;DeSamblanxetal.,1997;Gageetal., Ca21. Because the abrogation of MsDef1 and KP4 2001). We tested the ability of MsDef1, Rs-AFP2, and antifungal activity is metal specific and not simply known Ca21channel blockers to affect 45Ca flux in N. due to ionic effects, it is likely that Ca21 is involved crassahyphae.Briefly,N.crassasporeswereallowedto in the mode of action of these proteins. The relative germinate in synthetic fungal media for 8 h. The insensitivity of Rs-AFP2 to cations may be due to the cultures were then supplemented with 1 mCi mL21 fact that it binds to a different target than MsDef1 of 45CaCl . After a 20-min incubation, samples were 2 and KP4 or that it binds to the same target with collected and filtered onto Whatman filter paper, significantly higher affinity, thereby making it harder washed extensively with unlabeled CaCl , and 2 to be displaced by Ca21. As shown below, it seems counted in a liquid scintillation counter. We found morelikelythattheformerpossibilityistrue. that25mgmL21ofbothMsDef1andRs-AFP2 caused 6 of 13 Plant Physiol. Vol. 135, 2004 Downloaded from on April 16, 2019 - Published by www.plantphysiol.org Copyright © 2004 American Society of Plant Biologists. All rights reserved. Alfalfa Defensin Blocks a Mammalian Calcium Channel Figure6. MsDef1,butnotRs-AFP2orMtDef2,selectivelyblocksL-typeCa21channels.A,TimedependencyoftheMsDef1 blockofCav1.2channels.Asshownhere,10mMMsDef1blocksapproximately90%oftheCa21current,withthemaximum inhibitionoccurringafterexposingthecellstothedefensinforapproximately13min.B,Blockby2mMMsDef1developed slowly, over several minutes, such that 0.56% 6 0.03% of current remained at equilibrium (n 5 3). C, Current-voltage relationshipofCav1.2inthepresenceandabsenceof2mMMsDef1.CellsexpressingCav1.2,asdescribedabove,wereheldat 260mVanddepolarizedtotheindicatedvoltagefor100msbefore(blackcircles)orafter(whitecircles)equilibrationin2mM MsDef1.WhileMsDef1decreasespeakcurrent,itdoesnotappreciablyshiftthecurrent-voltagerelationshipofCa1.2.DandE, v MsDef1wasappliedtothenon-L-typechannelsCa2.1andCa 2.3,respectively,asinB,exceptthattheholdingpotentialwas v v 280mVforCa 2.1and2100mVforCa 2.3.NoinhibitionofeitherCa2.1orCa 2.3byMsDef1wasdetected.F,Whole-cell v v v v voltageclampoftsA-201cellsexpressingCa1.2channels,asinB,before(control)andseveralminutesaftertheapplicationof v 10mMRs-AFP2.G,ThefractionofcontrolcurrentremainingafterseveralminutesofperfusionwithRs-AFP2intsA-201cells Plant Physiol. Vol. 135, 2004 7 of 13 Downloaded from on April 16, 2019 - Published by www.plantphysiol.org Copyright © 2004 American Society of Plant Biologists. All rights reserved. Spelbrink et al. an influx of Ca21 (Table III). This result is consistent TableIII. 45Ca21uptakeexperimentsinN.crassa withwhatwasreportedpreviouslyforRs-AFP2(The- Sporeswereallowedtogerminatefor8hinsyntheticfungalmedia vissen et al., 1996). However, we found that four before the additionof 1 mCi mL21 of 45CaCl. MsDef1 and Rs-AFP2 knownCa21channelblockers—cadmium,lanthanum, wereusedataconcentrationof25mgmL21;250mgmL21KP4were gadolinium,andKP4—alsocauseddramaticinfluxof testedalongwith1mMofcadmium,lanthanum,andgadolinium.After Ca21. Therefore, while this experimental approach is 20min,thehyphaewerecollectedandvacuumfilteredontoWhatman conceptually simple, it is apparently not adequate 5 filterpaper, washedwith10mLof10mM CaCl2,andcountedfor for measuring the activity of Ca21 channel blockers. 45Ca.Therelativeinfluxwascomputedbytakingtheaveragecpmand Indeed, this potential problem has been noted in dividingbythecpmofthecontrol(untreated). a previous publication(Corzo andSanders, 1992). Treatment RelativeInflux No.Experiments MsDef1 4.462.2 6 Rs-AFP2 14.865.2 3 DISCUSSION KP4 1.860.1 3 Plantdefensins possesspotent andbroad-spectrum Cd21 3.161.5 3 growth-inhibitoryactivityagainst fungi.Our findings La31 153.5635.2 4 Gd31 102.7638.8 5 shed light on the structure-activity relationships and modesofactionofdefensinsfromalfalfa.Ourstudies on the MsDef1 and MtDef2 chimeric defensins have shownthattheC-terminalaminoacidresidues31to45 MsDef1, we selected the Arg-38 for mutagenesis. As in MsDef1 are important for antifungal activity. The shown in Figure 4, this one residue was sufficient to comparison of this C-terminal region in these two reverse the antifungal activity of the mutant proteins defensins indicates the presence of four positively when compared to the wild-type proteins. These re- chargedaminoacids(LysorArg)inMsDef1butnone sults further demonstrate functional similarity be- inMtDef2.Ouranalysisofthedefensinchimerasalso tweenMsDef1andtheCa21channelblockerKP4. indicates that the N-terminal region (residues 1–15) Theresultspresentedherefurtherdemonstratethat contributes to the antifungal activity of MsDef1. This thereisagreatdealofmechanisticsimilaritybetween regionisremarkablyconservedinbothproteins,with MsDef1 and the structurally different antifungal pro- onlytwoaminoaciddifferencesatpositions5and9.It teinKP4(Fig.7).Thedatasuggestthat,althoughanti- is thus likely that the presence of Asn-5 and Lys-9 in fungal potency of these proteins differs significantly, MsDef1contributestotheoverallantifungalactivityof their mechanism of action is similar. We show for the MsDef1. Our analysis of the defensin chimeras indi- first time, to our knowledge, that a plant defensin catesthattheloopconnectingb-strand2andb-strand blocks the L-type Ca21 channel in mammalian cells. 3, as well as b-strand 3, are the important secondary Like KP4 (Gu et al., 1995; Gage et al., 2002), MsDef1 structure elements for the antifungal activity of is a potent inhibitor of the Ca 1.2 (L-type) channel v MsDef1. but has little or no effect on the Ca 2.3 or Ca 2.1 v v ThestructuralhomologyofMsDef1withtheknown Ca21 channels. Interestingly, MsDef1 blocks up to voltage-gated Ca21 channel blocker KP4 and the Na1 90% of the L-type Ca21 channel activity (Fig. 6A), channelblockerscorpiontoxinAaHIIprovidedaguide whereas KP4 blocks only 60% of the L-type Ca21 forextendingmutagenesisstudiesand,moreprecisely, channel activity (Gu et al., 1995; Gage et al., 2002). In definingtheactivesite.Allthreeoftheseproteinshave addition,MsDef1takesapproximately13mintoreach commonstructuralfeatures,suggestingtheactivesite equilibrium and is very reminiscent of the time- lies near the b2-b3 loop. Like KP4 and scorpion toxin dependent effects of calciseptine (Teramoto et al., AaHII, this loop is extremely basic and stabilized by 1996).LikeKP4,thisblockdoesnotseemtochangethe a disulfide bond with the C terminus. In the scorpion voltage dependency of the channel appreciably (Fig. toxin, modification of Lys-58 at the base of the loop 6C). These observations, along with the abrogation of inactivatesthetoxin(Fontecilla-Camps,1989).KP4also antifungalactivitybyCa21,suggestthatMsDef1binds has a Lys at the base of this loop (K42). When this totheextracellularsideoftheCa 1.2poreregionmuch v residue was changed to a Gln, KP4 exhibited a 90% like the blockage of K1 channels by charybdotoxin decrease in antifungal activity (Gage et al., 2001). (MacKinnon and Miller, 1989) or the blockage of Na1 MsDef1, like KP4 and scorpion toxin, has a basic channelsbytetrodotoxin(Terlauetal.,1991). residueatthispositionoftheprotein(Arg-38).Further- In order to test whether other morphogenic defen- more, MtDef2, which has limited antifungal activity, sins share this property with MsDef1, Rs-AFP2 was has Gln at this position. Because of the homology to chosen since its 3D structure is similar to that of expressingCa1.2,Ca2.1,orCa2.3channels.Ba21currentsinthesechannelswereelicitedusingtheprotocoldescribedinB v v v andD.ThedefensinRs-AFP2didnotinhibitcurrentconductedbyanyofthesevoltage-gatedCa21channels(valuesaremeans6 SE;n53).H,L-typeCav1.2channelsarenotblockedbyMtDef2.Thecellwasheldat260mMandpulsedto110mVbefore (control)or5minafterinitiationofperfusionwith10mMMtDef2. 8 of 13 Plant Physiol. Vol. 135, 2004 Downloaded from on April 16, 2019 - Published by www.plantphysiol.org Copyright © 2004 American Society of Plant Biologists. All rights reserved. Alfalfa Defensin Blocks a Mammalian Calcium Channel Figure7. Three-dimensionalstructuresoftheplantdefensinRs-AFP1(1AYJ;Fantetal.,1998),thescorpiontoxinAaHII(1PTX; Houssetetal.,1994),andthefungaltoxinKP4(1KPT;Guetal.,1995).Inallthreefigures,theCyssidechainsarerepresentedin ballandstick,whiletheArgandLysresiduesareshownasbluestickmodels.InthediagramofRs-AFP1,thecolorsofthe secondarystructuralelementscorrespondtotheregionsselectedforMsDef1hybridanalysis.Orange,purple,andgreenindicate theN-terminal(residues1–15),middle(residues16–30),andC-terminal(residues31–45)portions,respectively.Forscorpion toxinandKP4,theribbondiagramsarecoloredinagradientfromredtopurpleastheproteinistracedfromtheNtotheC termini. MsDef1 but differs substantially in its primary amino Ca21 generated by tip-localized Ca21 channels (Tsien acid sequence (Fant et al., 1998). Rs-AFP2 is a more andTsien,1990).Indeed,disruptionofthistipgradient potentgrowthinhibitorofF.graminearumandN.crassa has been shown to cause hyperbranching in growing than MsDef1, but it induces less pronounced hyper- hyphaemuchlikethatobservedwithKP4andMsDef1 branching in these fungi. Rs-AFP2 failed to block any (Jacksonand Heath,1993). ofthethreeCa21channels(Fig.6,FandG),indicating IfMsDef1causeshyphalgrowthdefectsbyblocking different modes of action for these two structurally the uptake of Ca21, then N. crassa growth should also related defensins (Lay et al., 2003). The fact that be inhibited by EGTA and lanthanum, a known Ca21 MsDef1, like KP4, specifically blocks the L-type Ca21 channelblocker.WhenN.crassasporesweregrownin channelandthatitsantifungalactivityisabrogatedby synthetic media supplemented with EGTA or lantha- Ca21 makes it likely that MsDef1 targets fungal Ca21 num, fungal growth was inhibited and hyperbranch- channels.Althoughtheremaybeanalternativemode ing was induced in a manner resembling that of of action, the data presented here are consistent with MsDef1 (see Supplemental Figs. 1 and 2, available at the notion that MsDef1 blocks a specific fungal Ca21 www.plantphysiol.org).ConcentrationsofEGTAover channel.WhileMsDef1hasnotbeendirectlyshownto 100 mM greatly inhibited the growth of N. crassa, and block a fungal Ca21 channel, it should be noted that concentrations over 500 mM inhibited the growth of F. there is no known example of an ion channel blocker graminearum.GrowthinhibitionofU.maydisbyEGTA that has an unrelated function in its active biological was previously demonstrated to be similar to that system. Furthermore, it should be noted that disrup- caused by KP4 (Gage et al., 2002). This finding is tion of fungal Ca21 gradients is known to cause consistent with the notion that defensins may be hyperbranching (Jackson and Heath, 1993) and that inhibiting normal hyphal growth by disrupting Ca21 theefficacyof theseproteins onL-type Ca21channels transport. correlates wellwith theirhyperbranchingeffects. It is possible that the antifungal effects of MsDef1 It is reasonable to suggest that MsDef1 acts via result from the disruption of Ca21 gradients required disruption of a Ca21 gradient, since Ca21 is a ubiqui- for filamentous growth and budding rather than in- tous signaling molecule that plays important roles in hibition of nutritional uptake of Ca21 (Jackson and the life cycle of fungi. In fungi, Ca21 is involved in, Heath, 1993). It has been reported that mutations in but not limited to, bud formation (Davis, 1995), either adenylyl cyclase (Gold et al., 1994) or cAMP- hyphal elongation (Jackson and Heath, 1993), and dependent protein kinase (Durrenberger et al., 1998) cAMPregulation(Iidaetal.,1990).ThisCa21gradient can affect the control of filamentous growth versus is maintained through a series of Ca21 channels, budding in fungi. The addition of the secondary antiporters,andpumps(CunninghamandFink,1994; messenger, cAMP, to the growth media can revert Tsien et al., 1995). Hyphal tip growth is a highly themutantphenotypetoawild-typephenotype.Inthe dynamicandcomplexprocessthatinvolvescontrolof case of KP4, exogenously added cAMP was found to localized synthesis and expansion of the growing tip. rescue the growth inhibition induced by this Ca21 Thisprocessiscontrolledbyagradientinthecytosolic channel blocker (Gage et al., 2001). Because of the Plant Physiol. Vol. 135, 2004 9 of 13 Downloaded from on April 16, 2019 - Published by www.plantphysiol.org Copyright © 2004 American Society of Plant Biologists. All rights reserved. Spelbrink et al. functional similarity of MsDef1 with KP4, we deter- tions,datageneratedfrom45Ca21uptakeexperiments mined if abrogation of MsDef1-induced antifungal mustbeinterpretedwithextremecautionandmaynot activityoccurredwithcAMPtreatment.Asinthecase necessarily reflect actual Ca21 uptake (Corzo and with KP4, cAMP reduced the antifungal activity of Sanders, 1992). Due to the complexity of Ca21 regula- MsDef1inadose-dependentmannerforbothN.crassa tion in fungal hyphae, it is difficult to assess the Ca21 and F. graminearum. Spores of both fungi were germi- channel-blocking activity of defensins with this sim- nated in the presence of inhibitory concentrations of plistic technique. With the availability of the Ca21 MsDef1(2mgmL21forN.crassaand25mgmL21forF. channel gene sequences in N. crassa, it may now be graminearum). cAMP at concentrations above 10 mM possible to test MsDef1 for its ability to block these wasabletoreducetheantifungaleffectsofMsDef1in channels individually using the same approach we a dose-dependent manner (see Supplemental Fig. 3). report for the mammalian channels. This result suggests a link between growth inhibition and fungal Ca21 signaling (Gage et al., 2001). In N. crassa, there are three distinct Ca21 signal MATERIALS AND METHODS transduction pathways based on the unique Ca21 signatures associated with mechanical perturbation, Cloning and Sequence Analysis of Medicago hypo-osmotic shock, and high external Ca21. KP4 Def1 and Def2 Genes inhibits the intracellular Ca21 responses to hypo-os- Thecloningandnucleotidesequenceofafull-lengthcDNAcloneencoding moticshockandhighexternalCa21butnottomechan- MsDef1hasbeenpublishedpreviously(Gaoetal.,2000).AsearchofThe Institute of Genomic Research M. truncatula Gene Index (MtGI) using the ical perturbation (Nelson et al., 2004). In addition, MsDef1sequenceasaqueryyieldedonesingletonexpressedsequencetag physiological evidence suggests the presence of clone(TC50237)of483bpwith75%identityatthenucleotidesequencelevel. two stretch-activated and two intracellular inositol- ThiscDNAcloneencodingMtDef2wasgenerouslyprovidedbyTheSamuel 1,4,5-triphosphate-activatedCa21channelsinN.crassa RobertsNobleFoundation(Ardmore,OK)andsequenced(J.N.Hanks,A.K. Snyder, M.A. Graham, R.K. Shah, L.A. Blaylock, M.J. Harrison, and D.M. (Levinaetal., 1995;Silverman-GavrilaandLew,2001, Shah,unpublisheddata). 2002). ThesequenceforRs-AFP2waspublishedpreviously(Terrasetal.,1992). The complete sequencing of the N. crassa genome We obtained a synthetic gene encoding the mature Rs-AFP2 protein from hasrevealedthepresenceofthreeCa21channelgenes Integrated DNATechnologies (Coralville, IA) and cloned it into the Pichia (Galaganetal.,2003).ThreenewCa21channelproteins pastorisexpressionvectorasdescribedbelow. have been identified in Neurospora (Borkovich et al., 2004). These three proteins have close homologs to Expression of Defensins in Pichia pastoris the three Ca21 channel proteins in Saccharomyces AlldefensinproteinswereexpressedintheyeastPichiapastoris.ThepPIC9 cerevisiae—Mid1p, Cch1p, and Yvc1p (Fischer et al., vector(Invitrogen,Carlsbad,CA)allowsthemethanol-inducibleexpressionof 1997;PaidhungatandGarrett,1997;Mulleretal.,2001). the recombinant protein in P. pastoris and its secretion using the a-factor The sequence of CCH1 is similar to the a -subunit of secretionsignalofSaccharomycescerevisiae.TheDNAsequencescodingforthe animalvoltage-gatedCa21channels.Yvc1p1isavacuo- maturedefensinsequenceofMsDef1,MtDef2,andRs-AFP2wereclonedin framewiththeinitiationcodonofthesignalsequenceattheXhoIrestriction lar voltage-gated Ca21 channel (Palmer et al., 2001). siteofpPIC9.Thesequenceencodingthelastfouraminoacidsofthea-factor MID1doesnothaveanysequencesimilaritytoknown signalproteinsequencewerenotincludedintheexpressionconstruct.The ion channels but has been reported to be a stretch- plasmids were transformed in Escherichia coli DH5a. The resulting vector activatedcationchannel(Kanzakietal.,1999). containedthecodingregionforthematuredefensinsequencefusedinframe InordertotestMsDef1directlyforitsCa21channel- withthea-factorsignalsequencedownstreamoftheP.pastorisalcoholoxidase promoter.ThevectorwasthenlinearizedbydigestionwithSalIandintegrated blocking activity in N. crassa, it will be necessary to intoP.pastorisstrainGS115(Invitrogen)byelectroporation.His1transform- conduct electrophysiological studies with specific ants were selected by plating on minimal dextrose plates. Clones were Ca21 channels in the same manner as reported for cultured in buffered minimal glycerol media and induced with methanol. the mammalian Ca21 channels. Currently, it has been ThepresenceofeachdefensininthegrowthmediumwasconfirmedbyELISA (Gaoetal.,2000). difficult to directly measure defensin-induced Ca21 flux in a fungal system. Previous studies have looked at 45Ca in N. crassa hyphae treated with Rs-AFP2 and Design of Defensin Chimeras reported a rapid influx (Thevissen et al., 1996; De Def1-2C5,Def1-2C6,Def1-R38Q,andDef2-Q39Rexpressionvectorswere Samblanx et al., 1997). In an effort to determine preparedbysite-directedmutagenesisoftheMsDef1andMtDef2expression if the treatment of the fungus with MsDef1 leads vectors. The mutagenesis was done by PCR using the QuickChange site- to significant changes in Ca21 flux across the plasma directed mutagenesis kit purchased from Stratagene (La Jolla, CA). This membrane, we performed a 45Ca21 uptake assay techniquechangedtheAsn-5residueandLys-9ofMsDef1toHisandThr, respectively,creatingMsDef1-2C5.Similarly,theHis-5andThr-9residuesof using N. crassa hyphae. All defensins tested caused theMtDef2constructweremutatedtoAsnandLystocreateDef1-2C6.The a strong influx of Ca21 similar to that previously synthetic genes for Def1-2C1, Def1-2C2, Def1-2C3, and Def1-2C4 were reported for Rs-AFP2 (Table III; Thevissen et al., obtainedfromMCLAB(SanFrancisco)andclonedintopPIC9inamanner identicaltothatemployedforMsDef1,MtDef2,andRs-AFP2. 1996).Whilethisresultappearstosuggestthatdefen- sins act as Ca21channel activators, hyphae treated with the known Ca21 channel blockers—cadmium, Expression and Purification of Defensins gadolinium, lanthanum, and KP4—also caused Pichiaculturesweregrownovernightinbufferedminimalglycerolmedia astronginfluxofCa21.Asnoted inpreviouspublica- andtheninducedwithmethanolevery24h,accordingtothemanufacturer’s 10 of 13 Plant Physiol. Vol. 135, 2004 Downloaded from on April 16, 2019 - Published by www.plantphysiol.org Copyright © 2004 American Society of Plant Biologists. All rights reserved.

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Aug 6, 2004 Alfalfa Defensin Blocks a Mammalian Calcium Channel .. Snyder, M.A. Graham, R.K. Shah, L.A. Blaylock, M.J. Harrison, and D.M.. Shah . chloride. Growth was measured at the indicated times by determining optical density at We are grateful to Dr. Roger Beachy for his encouragement and
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