Differential Antifungal and Calcium Channel-Blocking Activity among Structurally Related Plant ...

13 Pages·2004·0.55 MB·English

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.
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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).
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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.
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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
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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.’’
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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
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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
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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
shed light on the structure-activity relationships and Gd31 102.7638.8 5
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
Figure6. (Continued.)
expressingCa1.2,Ca2.1,orCa2.3channels.Ba21currentsinthesechannelswereelicitedusingtheprotocoldescribedinB
v v v
andD.ThedefensinRs-AFP2didnotinhibitcurrentconductedbyanyofthesevoltage-gatedCa21channels(valuesaremeans6
SE;n53).H,L-typeCav1.2channelsarenotblockedbyMtDef2.Thecellwasheldat260mMandpulsedto110mVbefore
(control)or5minafterinitiationofperfusionwith10mMMtDef2.
2062 Plant Physiol. Vol. 135, 2004
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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 2063
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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
Cloning and Sequence Analysis of Medicago
signatures associated with mechanical perturbation,
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
ical perturbation (Nelson et al., 2004). In addition, Institute of Genomic Research M. truncatula Gene Index (MtGI) using the
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.
(Levinaetal., 1995;Silverman-GavrilaandLew,2001, Snyder, M.A. Graham, R.K. Shah, L.A. Blaylock, M.J. Harrison, and D.M.
2002). Shah,unpublisheddata).
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
1
animalvoltage-gatedCa21channels.Yvc1pisavacuo- maturedefensinsequenceofMsDef1,MtDef2,andRs-AFP2wereclonedin
lar voltage-gated Ca21 channel (Palmer et al., 2001). framewiththeinitiationcodonofthesignalsequenceattheXhoIrestriction
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
difficult to directly measure defensin-induced Ca21 (Gaoetal.,2000).
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,creatingDef1-2C5.Similarly,theHis-5andThr-9residuesofthe
using N. crassa hyphae. All defensins tested caused
MtDef2 construct were mutated to Asn and Lys to create Def1-2C6. 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
1996).Whilethisresultappearstosuggestthatdefen- identicaltothatemployedforMsDef1,MtDef2,andRs-AFP2.
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
2064 Plant Physiol. Vol. 135, 2004
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Copyright © 2004 American Society of Plant Biologists. All rights reserved.