Differential Antifungal and Calcium Channel-Blocking Activity

Differential Antifungal and Calcium Channel-BlockingActivity among Structurally Related Plant Defensins1[w]

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.); andMedicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana 47907(N.D., G.H.H.)

Plant defensins are a family of small Cys-rich antifungal proteins that play important roles in plant defense against invadingfungi. Structures of several plant defensins share a Cys-stabilized a/b-motif. Structural determinants in plant defensins thatgovern their antifungal activity and the mechanisms by which they inhibit fungal growth remain unclear. Alfalfa (Medicagosativa) seed defensin, MsDef1, strongly inhibits the growth of Fusarium graminearum in vitro, and its antifungal activity ismarkedly reduced in the presence of Ca21. By contrast, MtDef2 from Medicago truncatula, which shares 65% amino acidsequence identity with MsDef1, lacks antifungal activity against F. graminearum. Characterization of the in vitro antifungalactivity of the chimeras containing portions of the MsDef1 and MtDef2 proteins shows that the major determinants ofantifungal activity reside in the carboxy-terminal region (amino acids 31–45) of MsDef1. We further define the active site bydemonstrating that the Arg at position 38 of MsDef1 is critical for its antifungal activity. Furthermore, we have found for thefirst time, to our knowledge, that MsDef1 blocks the mammalian L-type Ca21 channel in a manner akin to a virally encodedand structurally unrelated antifungal toxin KP4 from Ustilago maydis, whereas structurally similar MtDef2 and the radish(Raphanus sativus) seed defensin Rs-AFP2 fail to block the L-type Ca21 channel. From these results, we speculate that the twounrelated antifungal proteins, KP4 and MsDef1, have evolutionarily converged upon the same molecular target, whereas thetwo structurally related antifungal plant defensins, MtDef2 and Rs-AFP2, have diverged to attack different targets in fungi.

Plant defensins are small (45–54 amino acids) Cys-rich proteins implicated in the first-line host defenseagainst fungal pathogens (Thomma et al., 2002). Thetertiary structures of these proteins are quite similarand share a common Cys-stabilized a/b-motif com-posed of three antiparallel b-strands and one a-helix.This motif is also found in insect defensins and scor-pion neurotoxins (Fontecilla-Camps, 1989; Bontemset al., 1991; Kobayashi et al., 1991). Despite theirstructural similarity, plant defensins are highly variedin their primary amino acid sequences, with onlyeight structure-stabilizing Cys residues in common(Thomma et al., 2002). The variation in the primarysequences may account for the different biologicalactivities reported for plant defensins, including anti-fungal activity (Terras et al., 1995), antibacterial activity(Segura et al., 1998), proteinase activity (Wijaya et al.,2000), and a-amylase inhibitory activity (Bloch andRichardson, 1991).

Some plant defensins exhibit potent antifungalactivity in vitro at micromolar concentrations against

a broad spectrum of filamentous fungi. The morpho-genic antifungal defensins reduce hyphal elongationand induce hyperbranching, whereas nonmorpho-genic defensins reduce hyphal elongation withoutcausing any morphological distortions (Broekaertet al., 1995; Thomma et al., 2003). Despite some progressmade in the past few years, the structure-activityrelationships and modes of action for most of the plantdefensins remain unknown. Mutational analysis of theradish (Raphanus sativus) Rs-AFP2 has revealed that theamino acid residues important for antifungal activityare clustered into two adjacent sites. The first site isaround the type VIb-turn connectingb-strands 2 and 3,and the second site is formed by residues on the loopconnecting b-strand 1 and the a-helix and the contig-uous residues on the a-helix and b-strand 3 (DeSamblanx et al., 1997). Unlike the mammalian andinsect defensins, antifungal plant defensins inducemembrane permeabilization through specific interac-tion with high-affinity binding sites on fungal cells(Thevissen et al., 1997, 2000) but do not form ion-permeable pores in artificial lipid bilayers, nor do theychange their electrical properties (Thevissen et al.,1996). When fungal hyphae are treated with Rs-AFP2or the Dahlia merckii defensin Dm-AMP1, there isa rapid influx of Ca21, efflux of K1, and alkalinizationof the growth medium (Thevissen et al., 1996). Howthis interaction of defensin proteins with fungalhyphae generates plasma membrane ion fluxesleading to fungal growth inhibition remains unclear.

1 This work was supported by the National Institutes of Health(grant no. GM–10704 to T.J.S.).

* Corresponding author; e-mail [email protected]; fax314–587–1581.

[w] The online version of this article contains Web-only data.Article, publication date, and citation information can be found at

www.plantphysiol.org/cgi/doi/10.1104/pp.104.040873.

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The characterization of defensin-resistant mutants ofunicellular and filamentous fungi has implicated a rolefor fungal sphingolipids and glucosylceramides in themechanism of growth inhibition by these defensins(Thevissen et al., 2000, 2004; Ferket et al., 2003; Thommaet al., 2003). More recently, it has been shown thatRs-AFP2 interacts with fungal glucosylceramides ina first step leading to fungal growth arrest (Thevissenet al., 2004).

It is likely that not all plant defensins act by the samemode of action. For example, patch clamp experimentsdemonstrated that plant defensins isolated from maize(Zea mays) inhibit sodium currents in a rat tumor cellline. However, their in vitro antifungal activity was notreported (Kushmerick et al., 1998). Some scorpionneurotoxins are structurally related to defensins andare known to block potassium channels (Garcia et al.,2001). Based on surface topology similarities withpotassium channel blockers, a similar mode of actionhas been proposed for a pea (Pisum sativum) seeddefensin Psd1 (Almeida et al., 2002).

We have previously reported the isolation andcharacterization of the broad-spectrum antifungal de-fensin (MsDef1) from alfalfa (Medicago sativa) seedpreviously referred to as AlfAFP (Gao et al., 2000). Thisprotein was found to inhibit the hyphal elongation ofthe fungal pathogen Fusarium graminearum in a dose-dependent manner, causing a hyperbranching pheno-type. We demonstrate here that the carboxy-terminalregion (residues 31–45) of MsDef1 contains major deter-minants for antifungal activity with the N-terminalregion (residues 1–15) contributing to the antifun-gal activity in a relatively minor way. In addition, weshow that Arg at position 38 is critical for the antifun-gal activity of MsDef1. This residue lies in a positionhomologous to the active site of the known Ca21

channel blocker KP4 and the Na1 channel blockerscorpion toxin AaHII. Furthermore, we show for thefirst time, to our knowledge, that a plant defensin,MsDef1, selectively blocks the mammalian L-typeCa21 channel in a manner similar to that of KP4,a structurally unique, virally encoded killer toxin fromthe P4 strain of the corn smut fungus, Ustilago maydis.However, two other structurally similar defensins,MtDef2 and Rs-AFP2, do not block the L-type Ca21

channel. The blockage was found to be very strong (upto 90% blockage) and highly specific for the L-typechannel. Finally, we report functional homology be-tween MsDef1 and a known Ca21 channel blocker,KP4, suggesting a common mode of action in fungi.

RESULTS

Alfalfa Defensins, MsDef1 and MtDef2, Differ in TheirAntifungal Activity

The cloning and sequence analysis of the cDNAencoding the alfalfa seed defensin MsDef1 has beenreported previously (Gao et al., 2000). A cDNA cloneencoding MtDef2 was cloned and sequenced as de-scribed in ‘‘Materials and Methods.’’ The amino acidsequences of MsDef1 and MtDef2 are 65% identicaland carry a net positive charge of 13 and 21, re-spectively (Fig. 1). The in vitro antifungal activities ofthese two proteins against a fungal pathogen, F. grami-nearum, were determined and compared with theantifungal activities of a previously characterizedradish seed defensin, Rs-AFP2 (Terras et al., 1995),and the virally encoded killer toxin KP4 from U.maydis (Young, 1987; Park et al., 1994; Fig. 2; Table I).MsDef1, Rs-AFP2, and KP4 exhibited strong dose-dependent antifungal activity against this fungus,whereas MtDef2 did not. Although less potent, MsDef1and KP4 induced more pronounced hyperbranching ofthe fungal hyphae than Rs-AFP2 (Fig. 2B). It should benoted that the most dramatic effect of these defensins isan altered growth pattern or hyperbranching that isaccompanied by inhibition of hyphal elongation.

C-Terminal Region (Residues 31–45) of MsDef1 IsImportant for Its Antifungal Activity

In order to identify the regions of MsDef1 moleculesthat are important for antifungal activity and for in-duction of hyperbranching phenotype in the fungus,chimeric defensins consisting of portions of MsDef1and MtDef2 proteins were tested for their antifungalactivity and ability to induce hyperbranching in F.graminearum. MsDef1 and MtDef2 were divided intothree regions of similar length, based upon the second-ary structural elements predicted by sequence align-ments with plant defensins whose three-dimensional(3D) structures have been determined (Fant et al., 1998;Almeida et al., 2002; Lay et al., 2003). Chimeric proteinscorresponding to all six possible combinations (Fig. 3),termed Def1-2C1 through Def1-2C6, were obtained byexpressing the synthetic genes encoding these proteinsin Pichia pastoris. All six chimeric proteins werescreened for antifungal activity against F. graminearumand Neurospora crassa, along with MsDef1 and MtDef2proteins. Antifungal activity was assessed both bymeasuring hyphal growth inhibition after 16 h of

Figure 1. Sequence comparison of Rs-AFP2 andMedicago defensins. Significant differences inresidues between the two Medicago defensinsare shown in boldface. The charge of each pro-tein is shown in parentheses. Symbols representapproximate positions of the predicted a-helix(spiral) and b-sheets (arrows).

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Figure 2. Comparison between the effects of KP4, MsDef1, Rs-AFP2, and MtDef2 on F. graminearum. A, MsDef1, KP4, andRs-AFP2 all exhibit a dose-dependent inhibition of hyphae. Assays were conducted in low ionic strength synthetic fungal mediainoculated with fungal spores. Micrographs prepared after a 16-h incubation. B, Hyperbranching effect on Fusarium. In vitroassays were prepared in the same manner as described in A, with 25 mg mL21 of antifungal protein. The average number ofhyphal buds per germline was determined by counting 50 germlings after 9 h of incubation. There is a marked difference in thenumber of buds among the proteins tested, with MsDef1 and KP4 inducing the most buds, Rs-AFP2 causing a less extreme effect,and MtDef2 causing no hyperbranching.

Alfalfa Defensin Blocks a Mammalian Calcium Channel

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exposure to the proteins and by determining the degreeof hyperbranching after 9 h of exposure to the proteins.

As shown in Figure 3 and Table II, the C-terminalsequence (residues 31–45) of MsDef1 contains themajor determinants of the in vitro antifungal activitysince Def1-2C2 containing this sequence was nearly asactive as MsDef1, whereas Def1-2C3 containingthe corresponding sequence of MtDef2 was inactiveagainst F. graminearum. The N-terminal sequence (res-idues 1–15) of MsDef1, however, does contributesomewhat to the antifungal activity of the C terminussince Def1-2C4 is more potent than either Def1-2C2 orDef1-2C5. As shown in Figure 3, both the N-terminal(residues 1–15) and C-terminal (residues 31–45) se-quences of MsDef1 are required to observe the samedegree of hyperbranching as that observed for thewild-type MsDef1. The middle region of the defensin(residues 16–30) apparently does not contain signifi-cant determinants of antifungal activity. Similar resultswere obtained when the chimeric defensins weretested on a more sensitive test fungus, N. crassa.It should be noted that, at concentrations higherthan 12 mg mL21, all proteins, including Def1-2C1,Def1-2C3, Def1-2C6, and MtDef2, showed modestantifungal activity against N. crassa.

Arg-38 Is Important for Antifungal Activity of MsDef1

In an effort to further define the region of MsDef1conferring antifungal activity, we performed site-directed mutagenesis on MsDef1 and expressed thevariant protein in P. pastoris. The antifungal activity ofthe purified protein was compared with that of

MsDef1. We selected Arg-38 as a candidate for muta-genesis for the following reasons: first, the residue liesin the motif of MsDef1 most similar to the active site ofthe ion channel blocker KP4 and scorpion toxin AaHII(Fontecilla-Camps, 1989; Gu et al., 1995). Both proteinshave been shown to have a basic residue at the baseof the b2-b3 loop that is critical for its antifungalactivity—Lys-42 for KP4 and Lys-58 for AaHII

Figure 3. Antifungal activity of the chimeric Def1/Def2 defensins on F. graminearum. MsDef1 and MtDef2 were divided intothree regions of similar length, and chimeric proteins corresponding to all six possible combinations, termed Def1-2C1 throughDef1-2C6, were obtained by expressing the synthetic genes encoding these proteins using a P. pastoris expression system. All sixchimeric proteins were screened for antifungal activity against F. graminearum and N. crassa (data not shown), along withMsDef1 andMtDef2 proteins. Antifungal activity was assessed bymeasuring both hyphal growth inhibition after 16 h of exposureto the proteins and determining the degree of hyperbranching after 9 h of exposure to the proteins (Table II).

Table I. IC50 values for peptides

Antifungal activity of defensins. The growth inhibition and hyper-branching induced by defensins were quantified. The concentration ofdefensins required to inhibit 50% of the overall growth (IC50) wasdetermined spectrophotometrically and confirmed visually. Fungalspores were grown in synthetic media supplemented with 2-fold serialdilutions of defensins. The absorbance at 595 nm was measuredafter 48 h.

Antifungal ProteinIC50 IC50

F. graminearum N. crassa

mg mL21 mg mL21

MsDef1 6–12 1–3MtDef2 .100 25–50Rs-AFP2 1–3 ,1KP4 .100 50–100Def1-2C1 .100 25–50Def1-2C2 6–12 1–3Def1-2C3 .100 25–50Def1-2C4 6–12 1–3Def1-2C5 6–12 1–3Def1-2C6 .100 25–50Def2-Q39R .100 1–3Def1-R38Q .100 25–50

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(Fontecilla-Camps, 1989; Gu et al., 1994; Gage et al.,2001). Second, Arg-38 lies in a homologous regionshown to influence the antifungal activity of Rs-AFP2(De Samblanx et al., 1997). Third, we noticed thatArg-38 is replaced by Gln in MtDef2. We suspectedthat the difference in charge at this position mightaccount for a large difference in the antifungal activ-ities of these two proteins (Table I). Therefore, wemade a single amino acid substitution to both theMsDef1 protein (R38Q) and the MtDef2 protein (Q39R)and compared their antifungal activities against N.crassa and F. graminearum. We found that, against N.crassa hyphae, the Def2-Q39R was vastly more potentthan the wild-type MtDef2 protein (Fig. 4; Table I) andalmost identical to that of MsDef1. Conversely, theDef1-R38Q variant showed a dramatic decrease inantifungal activity compared to the wild-type MsDef1protein and had similar antifungal activity as wild-type MtDef2. Therefore, the exchange of a singleamino acid between MsDef1 and MtDef2 was able toswitch the antifungal activity of the two defensins.Similar results were obtained against F. graminearum,with the exception that the Def2-Q39R mutant was notappreciably more potent than the wild-type defensin.It is likely that the molecular target of MsDef1 in this

fungus is poorly recognized by Def2-Q39R and thatadditional residues in proximity to Arg-38 may beimportant for full recognition.

Antifungal Activity of MsDef1, Like That of KP4,

Is Strongly Abrogated by Exogenous Ca21

It has been shown previously that the antifungalactivity of KP4 is specifically abrogated by exoge-nously added Ca21 (Gu et al., 1995). Similarly, theantifungal activity of some plant defensins is signifi-cantly reduced when the cationic strength of the fungalgrowth medium is increased (Terras et al., 1992,1993). Therefore, we tested the effects of Ca21 on thein vitro antifungal activity of MsDef1 and Rs-AFP2and compared them with that of KP4. As shown inFigure 5, a relatively low concentration of Ca21

abrogates KP4 and MsDef1 activity against F. grami-nearum but does not affect the activity of Rs-AFP2. Aslittle as 0.5 mM Ca21 reduced the antifungal activity ofKP4 and MsDef1, with total abrogation occurring at2 mM Ca21. The antifungal activity of KP4 was partiallyreduced by 2 mM Mg21, but more than 5 mM Mg21 wasrequired for complete abrogation. With MsDef1, no

Table II. Quantitation of defensin-induced hyperbranching in F. graminearum

The degree of hyperbranching was determined by counting the number of hyphal buds 8 h after incubation with defensin. Numbers in the table areaverages of at least 50 spores per treatment.

Protein No Peptides 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 2.1StD 0.0 1.3 0.1 1.0 0.9 0.1 1.0 0.2 1.0 1.2 0.2

Figure 4. Arg-38 of MsDef1 is important for antifungal activity. Spores of both fungi were allowed to germinate and grow insynthetic fungal media supplemented with 12 mg mL21 of the indicated defensin for N. crassa and 25 mg mL21 defensins forF. graminearum. Growth inhibition and hyperbranching were determined as described in ‘‘Materials and Methods.’’





reduction in antifungal activity was observed untilmore than 4 mM Mg21 was added. For both KP4 andMsDef1, more than 25 mM Na1 or 25 mM K1 is re-quired to reduce their antifungal activity and 50 mM

is needed for complete abrogation. In terms of ionicstrength, 3-fold more Mg21 and 10-fold more K1 orNa1 are required to cause the same degree of reductionof antifungal activity of MsDef1 as Ca21. Therefore,the in vitro antifungal activity of MsDef1, like that ofKP4, is particularly sensitive to exogenously addedCa21. Because the abrogation of MsDef1 and KP4antifungal activity is metal specific and not simplydue to ionic effects, it is likely that Ca21 is involvedin the mode of action of these proteins. The relativeinsensitivity of Rs-AFP2 to cations may be due to thefact that it binds to a different target than MsDef1and KP4 or that it binds to the same target withsignificantly higher affinity, thereby making it harderto be displaced by Ca21. As shown below, it seemsmore likely that the former possibility is true.

MsDef1, but Not MtDef2 and Rs-AFP2, Blocks the

Mammalian L-Type Ca21 Channel

It has been demonstrated previously that KP4 inhib-its Ca21 uptake in fungal cells (Gage et al., 2001). Ratherunexpectedly, KP4 was found to specifically block theL-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 closelyparallels that of KP4, we tested MsDef1 for its ability toblock a Ca21 channel in mammalian cells. The activityof MsDef1 on Ba21 currents conducted by three distinctvoltage-gated Ca21 channels, Cav2.1 (De Weille et al.,1991), Cav1.2 (Snutch et al., 1991), and Cav2.3 (Soonget al., 1993) from rat brain coexpressed with auxiliarysubunits b1b (Pragnell et al., 1991) and a2d (Ellis et al.,1988) in tsA-201 cells, was assayed as described pre-viously (Gage et al., 2002). As shown in Figure 6A, 10mM MsDef1 blocks approximately 90% of the Ca21

current through the Cav1.2 (L-type) channel, with themaximum inhibition occurring after exposing the cellsto the defensin for approximately 13 min. The block by2 mM MsDef1 developed slowly over several minutes,such that 0.56% 6 0.03% of current remained atequilibrium (n 5 3; Fig. 6B). A lower concentrationwas used in order to ascertain whether MsDef1 affectedthe voltage dependency of the current. Figure 6C showsthe current-voltage relationship of Cav1.2 in the pres-ence or absence of 2 mM MsDef1. Cells expressingCav1.2, as described above, were held at 260 mV anddepolarized to the indicated voltage for 100 ms before(black circles) or after (white circles) equilibration in 2mM MsDef1. While MsDef1 decreases peak current, itdoes not appreciably shift the current-voltage relation-ship of Cav1.2 (Fig. 6C). MsDef1 did not block either theCav2.1 or the Cav2.3 channel (Fig. 6, D and E). Surpris-ingly, Rs-AFP2 failed to block any of the three Ca21

channels despite sharing a 3D structure similar toMsDef1 and having potent activity against F. graminea-rum (Fig. 6, F and G). As expected, MtDef2, which isclosely related to MsDef1 but lacks in vitro antifungalactivity against F. graminearum, also failed to block theL-type Ca21 channel (Fig. 6H).

45Ca21 Uptake in N. crassa Hyphae

There have been several previous studies that at-tempted to measure Ca21 flux across fungal mem-branes in response to defensins and KP4 (Thevissenet al., 1996, 1999; De Samblanx et al., 1997; Gage et al.,2001). We tested the ability of MsDef1, Rs-AFP2, andknown Ca21channel blockers to affect 45Ca flux in N.crassa hyphae. Briefly, N. crassa spores were allowed togerminate in synthetic fungal media for 8 h. Thecultures were then supplemented with 1 mCi mL21

of 45CaCl2. After a 20-min incubation, samples werecollected and filtered onto Whatman filter paper,washed extensively with unlabeled CaCl2, andcounted in a liquid scintillation counter. We foundthat 25 mg mL21 of both MsDef1 and Rs-AFP2 caused

Figure 5. Abrogation of the antifungal activity of MsDef1 and KP4 byCa21. Shown here is an example of one of the tested metals andconcentrations; 50 mg mL21 KP4 and 5 mg mL21 MsDef1 and Rs-AFP2were used in this experiment.

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Figure 6. MsDef1, but not Rs-AFP2 or MtDef2, selectively blocks L-type Ca21 channels. A, Time dependency of the MsDef1block of Cav1.2 channels. As shown here, 10 mM MsDef1 blocks approximately 90% of the Ca21 current, with the maximuminhibition occurring after exposing the cells to the defensin for approximately 13 min. B, Block by 2 mM MsDef1 developedslowly, over several minutes, such that 0.56% 6 0.03% of current remained at equilibrium (n 5 3). C, Current-voltagerelationship of Cav1.2 in the presence and absence of 2 mM MsDef1. Cells expressing Cav1.2, as described above, were held at260 mV and depolarized to the indicated voltage for 100 ms before (black circles) or after (white circles) equilibration in 2 mM

MsDef1. While MsDef1 decreases peak current, it does not appreciably shift the current-voltage relationship of Cav1.2. D and E,MsDef1 was applied to the non-L-type channels Cav2.1 and Cav 2.3, respectively, as in B, except that the holding potential was280 mV for Cav 2.1 and2100 mV for Cav 2.3. No inhibition of either Cav2.1 or Cav 2.3 by MsDef1 was detected. F, Whole-cellvoltage clamp of tsA-201 cells expressing Cav1.2 channels, as in B, before (control) and several minutes after the application of10 mM Rs-AFP2. G, The fraction of control current remaining after several minutes of perfusion with Rs-AFP2 in tsA-201 cells





an influx of Ca21 (Table III). This result is consistentwith what was reported previously for Rs-AFP2 (The-vissen et al., 1996). However, we found that fourknown Ca21 channel blockers—cadmium, lanthanum,gadolinium, and KP4—also caused dramatic influx ofCa21. Therefore, while this experimental approach isconceptually simple, it is apparently not adequatefor measuring the activity of Ca21 channel blockers.Indeed, this potential problem has been noted ina previous publication (Corzo and Sanders, 1992).

DISCUSSION

Plant defensins possess potent and broad-spectrumgrowth-inhibitory activity against fungi. Our findingsshed light on the structure-activity relationships andmodes of action of defensins from alfalfa. Our studieson the MsDef1 and MtDef2 chimeric defensins haveshown that the C-terminal amino acid residues 31 to 45in MsDef1 are important for antifungal activity. Thecomparison of this C-terminal region in these twodefensins indicates the presence of four positivelycharged amino acids (Lys or Arg) in MsDef1 but nonein MtDef2. Our analysis of the defensin chimeras alsoindicates that the N-terminal region (residues 1–15)contributes to the antifungal activity of MsDef1. Thisregion is remarkably conserved in both proteins, withonly two amino acid differences at positions 5 and 9. Itis thus likely that the presence of Asn-5 and Lys-9 inMsDef1 contributes to the overall antifungal activity ofMsDef1. Our analysis of the defensin chimeras indi-cates that the loop connecting b-strand 2 and b-strand3, as well as b-strand 3, are the important secondarystructure elements for the antifungal activity ofMsDef1.

The structural homology of MsDef1 with the knownvoltage-gated Ca21 channel blocker KP4 and the Na1

channel blocker scorpion toxin AaHII provided a guidefor extending mutagenesis studies and, more precisely,defining the active site. All three of these proteins havecommon structural features, suggesting the active sitelies near the b2-b3 loop. Like KP4 and scorpion toxinAaHII, this loop is extremely basic and stabilized bya disulfide bond with the C terminus. In the scorpiontoxin, modification of Lys-58 at the base of the loopinactivates the toxin (Fontecilla-Camps, 1989). KP4 alsohas a Lys at the base of this loop (K42). When thisresidue was changed to a Gln, KP4 exhibited a 90%decrease in antifungal activity (Gage et al., 2001).MsDef1, like KP4 and scorpion toxin, has a basicresidue at this position of the protein (Arg-38). Further-more, MtDef2, which has limited antifungal activity,has Gln at this position. Because of the homology to

MsDef1, we selected the Arg-38 for mutagenesis. Asshown in Figure 4, this one residue was sufficient toreverse the antifungal activity of the mutant proteinswhen compared to the wild-type proteins. These re-sults further demonstrate functional similarity be-tween MsDef1 and the Ca21 channel blocker KP4.

The results presented here further demonstrate thatthere is a great deal of mechanistic similarity betweenMsDef1 and the structurally different antifungal pro-tein KP4 (Fig. 7). The data suggest that, although anti-fungal potency of these proteins differs significantly,their mechanism of action is similar. We show for thefirst time, to our knowledge, that a plant defensinblocks the L-type Ca21 channel in mammalian cells.Like KP4 (Gu et al., 1995; Gage et al., 2002), MsDef1is a potent inhibitor of the Cav1.2 (L-type) channelbut has little or no effect on the Cav2.3 or Cav2.1Ca21 channels. Interestingly, MsDef1 blocks up to90% of the L-type Ca21 channel activity (Fig. 6A),whereas KP4 blocks only 60% of the L-type Ca21

channel activity (Gu et al., 1995; Gage et al., 2002). Inaddition, MsDef1 takes approximately 13 min to reachequilibrium and is very reminiscent of the time-dependent effects of calciseptine (Teramoto et al.,1996). Like KP4, this block does not seem to change thevoltage dependency of the channel appreciably (Fig.6C). These observations, along with the abrogation ofantifungal activity by Ca21, suggest that MsDef1 bindsto the extracellular side of the Cav1.2 pore region muchlike the blockage of K1 channels by charybdotoxin(MacKinnon and Miller, 1989) or the blockage of Na1

channels by tetrodotoxin (Terlau et al., 1991).In order to test whether other morphogenic defen-

sins share this property with MsDef1, Rs-AFP2 waschosen since its 3D structure is similar to that of

Figure 6. (Continued.)expressing Cav1.2, Cav2.1, or Cav2.3 channels. Ba21 currents in these channels were elicited using the protocol described in Band D. The defensin Rs-AFP2 did not inhibit current conducted by any of these voltage-gated Ca21 channels (values are means6SE; n 5 3). H, L-type Cav1.2 channels are not blocked by MtDef2. The cell was held at 260 mM and pulsed to 110 mV before(control) or 5 min after initiation of perfusion with 10 mM MtDef2.

Table III. 45Ca21 uptake experiments in N. crassa

Spores were allowed to germinate for 8 h in synthetic fungal mediabefore the addition of 1 mCi mL21 of 45CaCl2. MsDef1 and Rs-AFP2were used at a concentration of 25 mg mL21; 50 mg mL21 KP4 weretested along with 1 mM of cadmium, lanthanum, and gadolinium. After20 min, the hyphae were collected and vacuum filtered onto Whatman5 filter paper, washed with 10 mL of 10 mM CaCl2, and counted for45Ca. The relative influx was computed by taking the average cpm anddividing by the cpm of the control (untreated).

Treatment Relative Influx No. Experiments

MsDef1 4.4 6 2.2 6Rs-AFP2 14.8 6 5.2 3

KP4 1.8 6 0.1 3Cd21 3.1 6 1.5 3La31 153.5 6 35.2 4Gd31 102.7 6 38.8 5

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MsDef1 but differs substantially in its primary aminoacid sequence (Fant et al., 1998). Rs-AFP2 is a morepotent growth inhibitor of F. graminearum and N. crassathan MsDef1, but it induces less pronounced hyper-branching in these fungi. Rs-AFP2 failed to block anyof the three Ca21 channels (Fig. 6, F and G), indicatingdifferent modes of action for these two structurallyrelated defensins (Lay et al., 2003). The fact thatMsDef1, like KP4, specifically blocks the L-type Ca21

channel and that its antifungal activity is abrogated byCa21 makes it likely that MsDef1 targets fungal Ca21

channels. Although there may be an alternative modeof action, the data presented here are consistent withthe notion that MsDef1 blocks a specific fungal Ca21

channel. While MsDef1 has not been directly shown toblock a fungal Ca21 channel, it should be noted thatthere is no known example of an ion channel blockerthat has an unrelated function in its active biologicalsystem. Furthermore, it should be noted that disrup-tion of fungal Ca21 gradients is known to causehyperbranching (Jackson and Heath, 1993) and thatthe efficacy of these proteins on L-type Ca21 channelscorrelates well with their hyperbranching effects.

It is reasonable to suggest that MsDef1 acts viadisruption of a Ca21 gradient, since Ca21 is a ubiqui-tous signaling molecule that plays important roles inthe life cycle of fungi. In fungi, Ca21 is involved in,but not limited to, bud formation (Davis, 1995),hyphal elongation (Jackson and Heath, 1993), andcAMP regulation (Iida et al., 1990). This Ca21 gradientis maintained through a series of Ca21 channels,antiporters, and pumps (Cunningham and Fink, 1994;Tsien et al., 1995). Hyphal tip growth is a highlydynamic and complex process that involves control oflocalized synthesis and expansion of the growing tip.This process is controlled by a gradient in the cytosolic

Ca21 generated by tip-localized Ca21 channels (Tsienand Tsien, 1990). Indeed, disruption of this tip gradienthas been shown to cause hyperbranching in growinghyphae much like that observed with KP4 and MsDef1(Jackson and Heath, 1993).

If MsDef1 causes hyphal growth defects by blockingthe uptake of Ca21, then N. crassa growth should alsobe inhibited by EGTA and lanthanum, a known Ca21

channel blocker. When N. crassa spores were grown insynthetic media supplemented with EGTA or lantha-num, fungal growth was inhibited and hyperbranch-ing was induced in a manner resembling that ofMsDef1 (see Supplemental Figs. 1 and 2, available atwww.plantphysiol.org). Concentrations of EGTA over100 mM greatly inhibited the growth of N. crassa, andconcentrations over 500 mM inhibited the growth of F.graminearum. Growth inhibition of U. maydis by EGTAwas previously demonstrated to be similar to thatcaused by KP4 (Gage et al., 2002). This finding isconsistent with the notion that defensins may beinhibiting normal hyphal growth by disrupting Ca21

transport.It is possible that the antifungal effects of MsDef1

result from the disruption of Ca21 gradients requiredfor filamentous growth and budding rather than in-hibition of nutritional uptake of Ca21 (Jackson andHeath, 1993). It has been reported that mutations ineither adenylyl cyclase (Gold et al., 1994) or cAMP-dependent protein kinase (Durrenberger et al., 1998)can affect the control of filamentous growth versusbudding in fungi. The addition of the secondarymessenger, cAMP, to the growth media can revertthe mutant phenotype to a wild-type phenotype. In thecase of KP4, exogenously added cAMP was found torescue the growth inhibition induced by this Ca21

channel blocker (Gage et al., 2001). Because of the

Figure 7. Three-dimensional structures of the plant defensin Rs-AFP1 (1AYJ; Fant et al., 1998), the scorpion toxin AaHII (1PTX;Housset et al., 1994), and the fungal toxin KP4 (1KPT; Gu et al., 1995). In all three figures, the Cys side chains are represented inball and stick, while the Arg and Lys residues are shown as blue stick models. In the diagram of Rs-AFP1, the colors of thesecondary structural elements correspond to the regions selected for MsDef1 hybrid analysis. Orange, purple, and green indicatethe N-terminal (residues 1–15), middle (residues 16–30), and C-terminal (residues 31–45) portions, respectively. For scorpiontoxin and KP4, the ribbon diagrams are colored in a gradient from red to purple as the protein is traced from the N to the Ctermini.





functional similarity of MsDef1 with KP4, we deter-mined if abrogation of MsDef1-induced antifungalactivity occurred with cAMP treatment. As in the casewith KP4, cAMP reduced the antifungal activity ofMsDef1 in a dose-dependent manner for both N. crassaand F. graminearum. Spores of both fungi were germi-nated in the presence of inhibitory concentrations ofMsDef1 (2 mg mL21 for N. crassa and 25 mg mL21 for F.graminearum). cAMP at concentrations above 10 mM

was able to reduce the antifungal effects of MsDef1 ina dose-dependent manner (see Supplemental Fig. 3).This result suggests a link between growth inhibitionand fungal Ca21 signaling (Gage et al., 2001).

In N. crassa, there are three distinct Ca21 signaltransduction pathways based on the unique Ca21

signatures associated with mechanical perturbation,hypo-osmotic shock, and high external Ca21. KP4inhibits the intracellular Ca21 responses to hypo-os-motic shock and high external Ca21 but not to mechan-ical perturbation (Nelson et al., 2004). In addition,physiological evidence suggests the presence oftwo stretch-activated and two intracellular inositol-1,4,5-triphosphate-activated Ca21 channels in N. crassa(Levina et al., 1995; Silverman-Gavrila and Lew, 2001,2002).

The complete sequencing of the N. crassa genomehas revealed the presence of three Ca21 channel genes(Galagan et al., 2003). Three new Ca21 channel proteinshave been identified in Neurospora (Borkovich et al.,2004). These three proteins have close homologs tothe three Ca21 channel proteins in Saccharomycescerevisiae—Mid1p, Cch1p, and Yvc1p (Fischer et al.,1997; Paidhungat and Garrett, 1997; Muller et al., 2001).The sequence of CCH1 is similar to the a1-subunit ofanimal voltage-gated Ca21 channels. Yvc1p is a vacuo-lar voltage-gated Ca21 channel (Palmer et al., 2001).MID1 does not have any sequence similarity to knownion channels but has been reported to be a stretch-activated cation channel (Kanzaki et al., 1999).

In order to test MsDef1 directly for its Ca21 channel-blocking activity in N. crassa, it will be necessary toconduct electrophysiological studies with specificCa21 channels in the same manner as reported forthe mammalian Ca21 channels. Currently, it has beendifficult to directly measure defensin-induced Ca21

flux in a fungal system. Previous studies have lookedat 45Ca in N. crassa hyphae treated with Rs-AFP2 andreported a rapid influx (Thevissen et al., 1996; DeSamblanx et al., 1997). In an effort to determineif the treatment of the fungus with MsDef1 leadsto significant changes in Ca21 flux across the plasmamembrane, we performed a 45Ca21 uptake assayusing N. crassa hyphae. All defensins tested causeda strong influx of Ca21 similar to that previouslyreported for Rs-AFP2 (Table III; Thevissen et al.,1996). While this result appears to suggest that defen-sins act as Ca21channel activators, hyphae treatedwith the known Ca21 channel blockers—cadmium,gadolinium, lanthanum, and KP4—also causeda strong influx of Ca21. As noted in previous publica-

tions, data generated from 45Ca21 uptake experimentsmust be interpreted with extreme caution and may notnecessarily reflect actual Ca21 uptake (Corzo andSanders, 1992). Due to the complexity of Ca21 regula-tion in fungal hyphae, it is difficult to assess the Ca21

channel-blocking activity of defensins with this sim-plistic technique. With the availability of the Ca21

channel gene sequences in N. crassa, it may now bepossible to test MsDef1 for its ability to block thesechannels individually using the same approach wereport for the mammalian channels.

MATERIALS AND METHODS

Cloning and Sequence Analysis of MedicagoDef1 and Def2 Genes

The cloning and nucleotide sequence of a full-length cDNA clone encoding

MsDef1 has been published previously (Gao et al., 2000). A search of The

Institute of Genomic Research M. truncatula Gene Index (MtGI) using the

MsDef1 sequence as a query yielded one singleton expressed sequence tag

clone (TC50237) of 483 bp with 75% identity at the nucleotide sequence level.

This cDNA clone encoding MtDef2 was generously provided by The Samuel

Roberts Noble Foundation (Ardmore, OK) and sequenced (J.N. Hanks, A.K.

Snyder, M.A. Graham, R.K. Shah, L.A. Blaylock, M.J. Harrison, and D.M.

Shah, unpublished data).

The sequence for Rs-AFP2 was published previously (Terras et al., 1992).

We obtained a synthetic gene encoding the mature Rs-AFP2 protein from

Integrated DNA Technologies (Coralville, IA) and cloned it into the Pichia

pastoris expression vector as described below.

Expression of Defensins in Pichia pastoris

All defensin proteins were expressed in the yeast Pichia pastoris. The pPIC9

vector (Invitrogen, Carlsbad, CA) allows the methanol-inducible expression of

the recombinant protein in P. pastoris and its secretion using the a-factor

secretion signal of Saccharomyces cerevisiae. The DNA sequences coding for the

mature defensin sequence of MsDef1, MtDef2, and Rs-AFP2 were cloned in

frame with the initiation codon of the signal sequence at the XhoI restriction

site of pPIC9. The sequence encoding the last four amino acids of the a-factor

signal protein sequence were not included in the expression construct. The

plasmids were transformed in Escherichia coli DH5a. The resulting vector

contained the coding region for the mature defensin sequence fused in frame

with the a-factor signal sequence downstream of the P. pastoris alcohol oxidase

promoter. The vector was then linearized by digestion with SalI and integrated

into P. pastoris strain GS115 (Invitrogen) by electroporation. His1 transform-

ants were selected by plating on minimal dextrose plates. Clones were

cultured in buffered minimal glycerol media and induced with methanol.

The presence of each defensin in the growth medium was confirmed by ELISA

(Gao et al., 2000).

Design of Defensin Chimeras

Def1-2C5, Def1-2C6, Def1-R38Q, and Def2-Q39R expression vectors were

prepared by site-directed mutagenesis of the MsDef1 and MtDef2 expression

vectors. The mutagenesis was done by PCR using the QuickChange site-

directed mutagenesis kit purchased from Stratagene (La Jolla, CA). This

technique changed the Asn-5 residue and Lys-9 of MsDef1 to His and Thr,

respectively, creating Def1-2C5. Similarly, the His-5 and Thr-9 residues of the

MtDef2 construct were mutated to Asn and Lys to create Def1-2C6. The

synthetic genes for Def1-2C1, Def1-2C2, Def1-2C3, and Def1-2C4 were

obtained from MCLAB (San Francisco) and cloned into pPIC9 in a manner

identical to that employed for MsDef1, MtDef2, and Rs-AFP2.

Expression and Purification of Defensins

Pichia cultures were grown overnight in buffered minimal glycerol media

and then induced with methanol every 24 h, according to the manufacturer’s

Spelbrink et al.




directions (Invitrogen). The cultures were grown for 7 d at 29�C, and cells were

removed by centrifugation at 2,000g for 15 min.

Defensins were purified from the growth medium by first dialyzing

against 25 mM sodium acetate, pH 4.5, and then passing the dialyzate through

CM-Sephadex C-25 cation-exchange resin (Amersham Biosciences, Piscat-

away, NJ) equilibrated with 25 mM sodium acetate, pH 4.5. Resin was

extensively washed with binding buffer (25 mM sodium acetate, pH 4.5),

and the bound protein was then eluted in 1 M NaCl, 50 mM Tris, pH 7.6.

Fractions containing the protein were manually collected and analyzed by

SDS-PAGE for the presence of the defensin. Fractions containing the defensin

protein were concentrated using a Minitan II ultrafiltration system (Millipore,

Bedford, MA) with a 3-kD cutoff membrane and dialyzed against 10 mM Tris,

pH 7.6. Purity was assessed using Homogenous 20 SDS gels on a Phastgel

system (Amersham Biosciences). The identity of the defensin was confirmed

by MALDI-MS in the positive ion mode using the matrices a-cyano-4-hydroxy

cinnamic acid (reflector mode) and sinapinic acid (linear mode). All defensins

were found to be pure and have the predicted mass-to-charge ratio (data not

shown).

The protein concentration for all expressed defensins was determined by

bicinchoninic acid protein concentration assay using the test tube protocol

provided by the manufacturer (Pierce, Rockford, IL).

Purification of KP4

KP4 was purified as reported previously (Gu et al., 1994, 1995). Briefly, the

toxin was isolated from the supernatant of the KP4 toxin expressing strains of

Ustilago maydis strain P4 using ion-exchange chromatography similar to that

described above.

Antifungal Assays

The antifungal activity of MsDef1 and MtDef2 and chimeric defensins was

measured in an in vitro assay using 96-well microtiter plates. Fifty microliters

of each protein dilution were added to each well of the microtiter plate

containing 50 mL of spore suspension prepared in 23 synthetic low-salt

fungal medium at a concentration of 40,000 spores mL21 (Liang et al., 2001).

Fusarium graminearum and Neurospora crassa spores were harvested from

potato dextrose (Difco, Sparks, MD) and Vogel’s medium N (Vogel, 1964) agar

plates, respectively, by washing the plate with water. The fungal cultures were

incubated at 24�C for 24 h. The IC50 values were determined by microscopic

analysis of hyphal growth after a 16-h incubation at room temperature and

microspectrophotometrically after 48 h (Broekaert et al., 1990). Values were

obtained from data collected in at least three independent experiments. It

should be noted that accurate measurements of growth inhibition with N.

crassa are difficult because of the aerial hyphae that develop. IC50 values do not

always indicate the degree of antifungal activity. Extreme hyperbranching

can cause denser growth and thereby higher absorbance in this assay. The

photographs were taken using an Olympus CK40 inverted microscope at

2003 magnification using Kodak T-max 400 (Rochester, NY) film after 16 h of

growth, unless otherwise noted. The degree of hyperbranching of F. grami-

nearum hyphae was quantified by counting the number of hyphal buds visible

on the germlings after 9 h of incubation with defensin proteins. More than 50

spores from each treatment were counted per experiment. The hyperbranch-

ing assay was repeated at least twice.

To test the growth-inhibitory effects of EGTA and lanthanum, spores were

used to inoculate 100 mL of synthetic fungal medium in a 96-well microplate at

a concentration of 2,000 spores per well (Broekaert et al., 1990). The cultures

were supplemented with various concentrations of EGTA or lanthanum

chloride. Growth was measured at the indicated times by determining optical

density at 595 nm in a spectrophometer (Spectra Max; Molecular Devices,

Sunnyvale, CA). Optical density readings were taken after 15, 37, and 48 h of

growth at 25�C. The antifungal assay for cAMP abrogation was done similarly

to that described above. For N. crassa, 2 mg mL21 of MsDef1 were added to the

culture in addition to the indicated concentrations of cAMP.

Effect of Defensins on Mammalian Ca21 Channels

The activity of MsDef1 and Rs-AFP2 on Ba21 currents passing through

three distinct Ca21 channels was tested by an assay described previously

(Gage et al., 2002). Briefly, whole-cell voltage clamp recording of Ba21 currents

in tsA-201 cells expressing the L-type channel Cav1.2 along with the b1b and

a2d subunits was made. The cell was held at 260 mV, and current was elicited

using 100-ms depolarizations to 110 mV at a frequency of 0.05 Hz. After

a steady baseline of current was established, defensin protein was applied

to cells via perfusion in the extracellular bath. The bath solution consisted of

150 mM Tris, 4 mM MgCl2, and 10 mM BaCl2, pH 7.3. The pipette solution

consisted of 130 mM N-methyl, d-glucamine, 60 mM HEPES, 10 mM EGTA,

2 mM MgATP, and 1 mM MgCl2, pH 7.3. Currents were recorded using an

Axopatch 200B amplifier (Axon Instruments, Union City, CA). Defensins were

applied in the extracellular bath at the indicated concentrations using an RSC

160 perfusion system (Bio-Logic, Claix, France). Data acquisition and analysis

were performed using Clampex and ClampFit software (Axon, Raleigh,

NC). All current recordings were leak subtracted using a standard P/N proce-

dure and filtered at 1 kHz.

45Ca21 Uptake in N. crassa Hyphae

In order to measure the flux of 45Ca21 across fungal hyphae, N. crassa

spores were used to inoculate cultures of synthetic fungal media at a concen-

tration of 3 3 105 spores mL21 in 50-mL Falcon tubes. The spores were

allowed to germinate by incubating the cultures at 28�C for 8 h in a shaking

incubator (200 rpm); 250-mL aliquots of the culture were prepared and added

to 1.5-mL microcentrifuge tubes. The cultures were then supplemented with

1 mCi mL21 of 45CaCl2 and 25 mg mL21 of MsDef1, Rs-AFP2, KP4, or 1 mM

gadolinium chloride, lanthanum chloride, or cadmium chloride. After a

20-min incubation at room temperature, the hyphae were collected and

vacuum filtered onto Whatman 5 filter paper. Each filter was washed with

10 mL of 10 mM CaCl2. The filter paper was then counted for 45Ca in a Beckman

LS 6500 liquid scintillation counter (Beckman Instruments, Fullerton, CA).

Sequence data from this article have been deposited with the EMBL/

GenBank data libraries under accession number MtDef2 AY313169.

ACKNOWLEDGMENTS

We are grateful to Dr. Roger Beachy for his encouragement and financial

support of this work. We thank Jennifer Hanks and Oluyemi Aladejebi for

their technical assistance, and Dr. Julia Gross for her help with the mass

spectrometry analysis of defensins. The program MolViewX (http://www.

danforthcenter.org/smith/molview.htm) was used to create Figure 7.

Received February 12, 2004; returned for revision May 12, 2004; accepted May

13, 2004.

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Differential Antifungal and Calcium Channel-Blocking Activity