p e p t i d e s 2 9 ( 2 0 0 8 ) 2 0 9 0 – 2 1 0 0

Isolation, characterization and cloning of a cDNA encoding anew antifungal defensin from Phaseolus vulgaris L. seeds

Patrıcia D. Games a,1, Izabela S. dos Santos a,1, Erica O. Mello a, Mariangela S.S. Diz a,Andre O. Carvalho a, Goncalo A. de Souza-Filho b, Maura Da Cunha c,Ilka M. Vasconcelos d, Beatriz dos S. Ferreira b, Valdirene M. Gomes a,*aUniversidade Estadual do Norte Fluminense, Laboratorio de Fisiologia e Bioquımica de Microrganismos, Campos dos Goytacazes,

28013-602 RJ, BrazilbUniversidade Estadual do Norte Fluminense, Laboratorio de Biotecnologia, Campos dos Goytacazes, 28013-602 RJ, BrazilcUniversidade Estadual do Norte Fluminense, Laboratorio de Biologia Celular e Tecidual, Campos dos Goytacazes, 28013-602 RJ, BrazildUniversidade Federal do Ceara, Departamento de Bioquımica, Laboratorio de Toxinas Vegetais, Fortaleza, 60451-970 CE, Brazil

a r t i c l e i n f o

Article history:

Received 1 July 2008

Received in revised form

12 August 2008

Accepted 12 August 2008

Published on line 22 August 2008

Keywords:

Antimicrobial peptides

Plant defensin

Pathogenic yeasts

Phytopathogenic fungi

Molecular cloning

Phylogenetic analysis

a b s t r a c t

The PvD1 defensin was purified from Phaseolus vulgaris (cv. Perola) seeds, basically as

described by Terras et al. [Terras FRG, Schoofs HME, De Bolle MFC, Van Leuven F, Ress

SB, Vanderleyden J, Cammue BPA, Broekaer TWF. Analysis of two novel classes of plant

antifungal proteins from radish (Raphanus sativus L.) seeds. J Biol Chem 1992;267(22):15301–

9], with some modifications. A DEAE-Sepharose, equilibrated with 20 mM Tris–HCl, pH 8.0,

was initially utilized for the separation of peptides after ammonium sulfate fractionation.

The basic fraction (the non-retained peak) obtained showed the presence of one unique

band in SDS–Tricine gel electrophoresis with a molecular mass of approximately 6 kDa. The

purification of this peptide was confirmed after a reverse-phase chromatography in a C2/C18

column by HPLC, where once again only one peak was observed and denominated H1. H1

was submitted to N-terminal sequencing and the comparative analysis in databanks

revealed high similarity with sequences of different defensins isolated from other plants

species. The N-terminal sequence of the mature defensin isolated was used to produce a

degenerated primer. This primer allowed the amplification of the defensin cDNA by RT-PCR

from mRNA of P. vulgaris seeds. The sequence analysis of the cloned cDNA, named PVD1,

demonstrated 314 bp encoding a polypeptide of 47 amino acids. The deduced peptide

presented high similarity with plant defensins of Vigna unguiculata (93%), Cicer arietinum

(95%) and Pachyrhizus erosus (87%). PvD1 inhibited the growth of the yeasts, Candida albicans,

Candida parapsilosis, Candida tropicalis, Candida guilliermondii, Kluyveromyces marxiannus and

Saccharomyces cerevisiae. PvD1 also presented an inhibitory activity against the growth of

phytopathogenic fungi including Fusarium oxysporum, Fusarium solani, Fusarium lateritium and

Rizoctonia solani.

# 2008 Elsevier Inc. All rights reserved.

avai lable at www.sc iencedi rec t .com

journal homepage: www.e lsev ier .com/ locate /pept ides

* Corresponding author at: Laboratorio de Fisiologia e Bioquımica de Microrganismos do Centro de Biociencias e Biotecnologia daUniversidade Estadual do Norte Fluminense, Campos dos Goytacazes, RJ, Brazil. Tel.: +55 22 27261689; fax: +55 22 27261520.

E-mail address: valmg@uenf.br (V.M. Gomes).1 These authors contributed equally to this work.

0196-9781/$ – see front matter # 2008 Elsevier Inc. All rights reserved.doi:10.1016/j.peptides.2008.08.008

p e p t i d e s 2 9 ( 2 0 0 8 ) 2 0 9 0 – 2 1 0 0 2091

1. Introduction

Plant defensins are antimicrobial peptides recognized as part

of the armamentarium of plant innate immune system [10].

They are small and basic peptides of 45–54 amino acid residues

comprised in a three-dimensional structure formed by three

anti-parallel b-strands and one a-helix which is stabilized by

four disulfide bonds [13,33]. The four disulfide bonds form a

cysteine-stabilized a-helix b-strand motif, common to these

peptides [2,33]. Plant defensins, like insect and mammal

defensins, possess antimicrobial activity. In plant defensins,

this activity is directed mainly against fungi [19,26], including

agronomic important plant fungal pathogens belonging to the

genera Fusarium, Alternaria and Verticillium, besides some

bacteria are also inhibited. Terras et al. [27] demonstrated

that these peptides play an important role in the protection of

radish seeds during germination. Furthermore, transgenic

expression of defensin demonstrated that they enhance

resistance against fungal plant pathogens [16], not only in

greenhouse experiments, but also under field conditions [14].

Two defensins peptides had their receptors identified on the

plasma membrane of fungi. They bind to sphingolipids

(glucosylceramide and mannosyldiinositolphosphoryl-cera-

mide) of fungal membranes, causing them an insurmountable

injury [28,30,31]. During the inhibition process, an interaction

between the peptide and the sphingolipid occurs at a first

moment, and then the peptide became inserted into the

plasma membrane, causing its permeabilization [28,30,31].

However, it was not known whether the arresting of spawned

growth is due just to membrane permeabilization or whether

the defensins interact with an intracellular target [31].

Recently, the surmised defensin intracellular target was

reported as being the cyclin F for the Pisum sativum defensin

1 (Psd1) [18]. Psd1 interacts with the cyclin F and is localized in

the nuclei of fungal hyphae, in vivo. This interaction and its

localization were related to cause interference in the cell cycle,

as confirmed by the model system of interkinetic nuclear

migration in the mouse retinal neuroblast [18].

The common bean (PhaseolusvulgarisL.) originated in Central

and South America [34] and their seeds are of great importance

because they represent the main legume grain used as human

foodinthe world.Brazil is thelargestproducerofcommonbean,

and its culture is socially important because it is farmed as a

subsistence culture and is the main income for small farmers.

The aim of this study was to isolate and characterize a new

antifungal defensin from P. vulgaris seeds, as well as to obtain

the cDNA encoding this defensin. In addition, the antimicrobial

activity was investigated under the growth of different

phytopathogenic fungi and bake and pathogenic yeasts.

2. Materials and methods

2.1. Plant material

Seeds of P. vulgaris L. (cv. Perola) were provided by the

Laboratorio de Melhoramento Genetico Vegetal, Centro de

Ciencias e Tecnologias Agropecuarias, Universidade Estadual

do Norte Fluminense, Campos dos Goytacazes, Rio de Janeiro,

Brazil.

2.2. Fungi

The yeasts, Candida parapsilosis (CE002), Candida guilliermondii

(CE013), Candida tropicalis (CE017), Candida albicans (CE022),

Kluyveromyces marxiannus (CE025) and Saccharomyces cerevisiae

(1038) were obtained from the Departamento de Biologia,

Universidade Federal do Ceara, Fortaleza, Ceara, Brazil. Yeasts

were maintained on Sabouraud agar (1% peptone, 2% glucose

and 1.7% agar–agar).

The phytopathogenic fungi Fusarium oxysporum, Fusarium

solani, Fusarium laterithium and Rizoctonia solani were obtained

from the Laboratorio de Fisiologia e Bioquımica de Micro-

rganismos, Universidade Estadual do Norte Fluminense,

Campos dos Goytacazes, Rio de Janeiro, Brazil. Fungi also

were maintained on Sabouraud agar (1% peptone, 2% glucose

and 1.7% agar–agar).

2.3. Bacterial strain

The chemically competent cells of Escherichia coli bacterial

strain DH5-a (genotype: F� F80lacZDM15 D(lacZYA-argF)U169

recA1 endA1 hsdR17(rk�, mk

+) phoA supE44 thi-1 gyrA96 relA1 l�)

was acquired from Invitrogen.

2.4. Purification of the P. vulgaris defensin

The purification of the defensin from P. vulgaris (cv. Perola)

seeds, was performed basically as described by Terras et al.

[25] with some modifications. Proteins from thirty grams of

seed flour were extracted for 2 h with 150 mL of extraction

buffer (10 mM Na2HPO4, 15 mM NaH2PO4, 100 mM KCl, 1.5%

EDTA), pH 5.4, at 4 8C. The precipitate obtained with 70%

ammonium sulfate saturation was solubilized in distilled

water and heated at 80 8C for 15 min. The resulting suspen-

sion was clarified by centrifugation and the supernatant was

extensively dialyzed against distilled water. The dialyzed

proteic extract (F-0/70) was recovered by freeze drying and

submitted to chromatographic methods. An anion-exchange

DEAE-Sepharose column was employed for further separa-

tion of peptides from the F-0/70. This column (3 cm � 7 cm)

was equilibrated and initially eluted with 20 mM Tris–HCl, pH

8.0. Elution of the bound fraction was carried out using 1 M

NaCl in the equilibration buffer. All chromatographic steps

were performed at the flow rate of 50 mL h�1. The basic

fraction (the non-retained peak) derivated from the DEAE-

Sepharose was recovered and diluted in a solution containing

0.1% (v/v) TFA and 2% (v/v) acetonitrile and injected onto an

HPLC C2/C18 ST 4.6/100 reverse-phase column (GE Health-

care). The chromatography was performed at a flow rate of

0.5 mL min�1 with 100% solvent A (0.1% TFA and 2%

acetonitrile) for 10 min, 0–100% solvent B (80% acetonitrile

containing 0.1% TFA) over 30 min, 100% solvent B over 5 min

and finally 100% solvent A for the remaining time. Proteins

were monitored by on-line measurement of the absorbance at

220 nm.

2.5. Gel electrophoresis

SDS–Tricine gel electrophoresis was performed according to

the method of Schagger and Von Jagow [22].

Fig. 2 – SDS–Tricine gel electrophoresis of proteins fractions

obtained during the purification of the P. vulgaris defensin.

F, F-0/70 fraction; D1, non-retained fraction from anion-

exchange DEAE-Sepharose chromatography; D2, retained

p e p t i d e s 2 9 ( 2 0 0 8 ) 2 0 9 0 – 2 1 0 02092

2.6. Amino acid sequence analysis

The peak obtained from HPLC was freeze dried and

submitted to amino acid sequence analysis. N-terminal

amino acid sequence of the peptide was determined by

Edman degradation carried out in a Shimadzu PSQ-23A

protein sequencer (Shimadzu, Kyoto, Japan). PTH-amino

acids were detected at 269 nm after separation on a reverse-

phase C18 column (0.46 cm � 25 cm) under isocratic condi-

tions, according to the manufacturer’s instructions.

Searches for sequence similarity were performed with the

BLASTp program [3].

2.7. RNA extraction from P. vulgaris seeds andreverse transcription

Total RNA from P. vulgaris seeds were extracted in buffer

contained 0.2 M Tris–HCl pH 7.5, 0.1 M LiCl, 5 mM EDTA and 1%

SDS followed by LiCl–phenol extraction method [20]. Poly-A+

mRNA was purified using the PolyATract1 mRNA Isolation

Systems IV (Promega). A reverse transcription reaction

consisting of 10 mL was prepared by mixing 4 mg mRNA,

1 � first strand buffer (Invitrogen), 80 pmol oligo dT18 primer,

5 mM DTT, 0.5 mM dNTP, 20 units RNaseOUT and 100 units

Fig. 1 – (A) Anion-exchange DEAE-Sepharose

chromatography. Column was previously equilibrated

with 20 mM Tris–HCl, pH 8.0. Elution was carried out

using 1 M NaCl in the equilibration buffer, at 50 mL hS1.

Arrows indicate the initial and final elution with

NaCl; (B) reverse-phase HPLC chromatography.

D1 fraction was applied to a C2/C18 ST 4.6/100

reverse-phase column and run in a Shimadzu

apparatus. Elution was carried out, as described in item

2.4. The dark line represents the protein elution profile at

220 nm and the oblique line represents the acetonitrile

gradient.

fraction from anion-exchange DEAE-Sepharose

chromatography eluted with 1 M NaCl; PvD1, H1 from D1

peak eluted from the reverse-phase column; M, molecular

mass markers (kDa).

SuperScript III (Invitrogen). The reaction was incubated for

80 min at 50 8C prior to PCR.

2.8. PCR amplification of cDNA encoding a P. vulgarisdefensin

The PCR degenerated primer, 50-AA(AG)ACITG(CT)GA(A-

G)AA(CT)CTIGC-30 (MWG-Biotech, USA), was designed based

on the first seven amino acids (KTCENLA) of the amino

terminal sequence of the mature P. vulgaris defensin purified

in HPLC. Oligo dT18 primer was used in conjunction with

template specific primer. PCR reaction mixture contained

1 � Taq buffer (GE Healthcare), dNTP 0.2 mM, 10 pmol oligo

dT18, 20 pmol of degenerated primer, 0.8 units Taq DNA

polymerase I (Fermentas) in a final volume of 20 mL. PCR

was carried out in a Mastercycler gradient 22331 (Eppendorf).

The reaction was initially warmed at 95 8C for 5 min and

subjected to a 40 cycle program as followed: 95 8C for 45 s;

temperature gradient (41.1, 42.2, 43.1, 44.4, 45.3, 46.4, 47.4, 48.3

and 49.4 8C) for 45 s; 72 8C for 2 min; last PCR step included

extension at 72 8C for 5 min. Further amplifications were

carried out at the same conditions but at 44.4 8C instead of the

temperature gradient.

2.9. Cloning and DNA sequencing of the P. vulgarisdefensin cDNA

The cDNA amplified in the RT-PCR was cloned into the

pTZ57R/T vector (InsTAcloneTM PCR Cloning Kit, Fermentas),

according to the instruction manual. The resulting DNA

Fig. 3 – Comparison of the N-terminal amino acid sequences of the PvD1 purified from P. vulgaris seeds and other

homologous peptides. For the amino acids sequenced, I% indicates the percentage of identical residues and P% indicates

the percentage of positive residues (representing amino acids with the same biochemical characteristics such as charge and

hydrophobicity) are shown in gray. Sequences from the following proteins were presented: PvD1 from P. vulgaris; defensin

from Vigna radiata (gi18146788); defensin from V. unguiculata [7]; defensin from V. unguiculata (gi112671); defensin from

Tephrosia platycarpa (gi62549225); defensin from Pisum sativum (gi20139322).

p e p t i d e s 2 9 ( 2 0 0 8 ) 2 0 9 0 – 2 1 0 0 2093

construction was named pTZ57R/T-PVD1. The nucleotide

sequence of the cDNA insert was determined for both strands

at ABI 3110 Genetic Analyzer (Applied Biosystems) using a

reaction mixture prepared with Big Dye1 Terminator v3.1 Kit

(Applied Biosystems) and the sequencing primers M13 forward

(�20) (50-GTAAAACGACGGCCAG-30) and M13 reverse (50-

CAGGAAACAGCTATGAC-30) according to the instruction

manual. The deduction of the amino acids sequence and

sequence alignment were carried out using the programs

Expasy-Translate tool (http://ca.expasy.org/tools/dna.html)

and BLAST (http://www.ncbi.nlm.nih.org/BLAST) [3], respec-

tively. The deduction of pI and molecular mass was done using

the Expasy-Compute pI/Mw tool (http://www.expasy.org/

tools/pi_tool.html) [4,5,15].

2.10. Phylogenetic analysis

For the phylogenetic analysis, sequences of 34 defensins

(mature proteins, without the signal peptide) from different

plant species were obtained from SWISSPROT (http://ca.

expasy.org/sprot) and aligned with CLUSTAL W2 (http://

www.ebi.ac.uk/Tools/clustalw2/index.html) using the default

settings [17]. The graphic display was done with the PHYLIP

Drawtree program (http://bioweb.pasteur.fr/seqanal/phylo-

geny/phylip-fr.html).

Fig. 4 – Nucleotide sequence of the fragment amplified by RT-P

seeds defensin and cloned into the pTZ57R/T vector. The cDNA

the PVD1 codify to a peptide of 47 amino acid as shown below th

amino terminal sequence used for the primer design is shown

2.11. Preparation of fungal cells and effect of the P.vulgaris defensin on phytopathogenic fungal and yeastgrowth

For the preparation of yeast cell cultures, an inoculum was

transferred to Petri dishes containing Sabouraud agar and

allowed to grow at 28 8C for 2 days. After this period, cells were

transferred to 10 mL of fresh Sabouraud broth and quantified

in a Neubauer chamber for further calculation of appropriate

dilutions. For the preparation of spores of phytopathogenic

fungi, fungal cultures were transferred to Petri dishes

containing Sabouraud agar and allowed to grow at 28 8C for

10 days; after this period, 10 mL of Sabouraud broth was added

to the dishes and these were gently agitated with the help of a

Drigalski loop for spore liberation. Spores were quantified in a

Neubauer chamber for appropriate dilutions.

To monitor the effect of the defensin on the growth of

fungal cells and spores, these were incubated at 28 8C in

microplates at 200 mL of final volume (10,000 spores/cells in

1 mL of Sabouraud broth) in the presence or absence (general

control) of the D1 fraction (25, 50 and 100 mg mL�1). Optical

readings at 660 nm were taken at zero time and at every 6 h for

the following 36 h for the yeasts and 60 h for the phytopatho-

genic fungi. Readings were taken against a blank containing

only the culture medium. All the experiments were run in

CR using the primer designed from the mature P. vulgaris

presents 314 bp and the deduced amino acid sequence of

e nucleotide sequence. The stop codon (TAA) is in bold. The

in light gray.

p e p t i d e s 2 9 ( 2 0 0 8 ) 2 0 9 0 – 2 1 0 02094

triplicate and the reading averages, the standard errors and

coefficients of variation were calculated.

2.12. Scanning electron microscopy

For scanning electron microscopy, yeast cells grown for 36 h

in Sabouraud broth in the presence or absence of the D1

fraction (100 mg mL�1) were fixed for 30 min at room tem-

perature in a solution containing 2.5% glutaraldehyde, 4.0%

formaldehyde in 0.05 M cacodylate buffer, pH 7.0. Subse-

quently, the materials were rinsed three times 0.1 M

cacodylate buffer, pH 7.0; post-fixed for 30 min at room

temperature with 2.0% osmium tetroxide diluted in 0.1 M

cacodylate buffer, pH 7.0; and rinsed again with this same

buffer. After this procedure, the yeast cells were dehydrated

in alcohol, critical point dried in CO2, covered with 20 nm gold

and observed in a DSEM 962 Zeiss scanning electron

microscope. The yeast cells grown without the addition of

peptides were also determined.

Fig. 5 – Comparison of the complete amino acid sequences of va

the clone pTZ57R/T-DEF. I% indicates the percentage of identica

residues (representing amino acids with the same biochemical

indicate the degree of homology of the defensins with the deduc

to optimize alignment and means a space introduced into an al

sequence relative to another; the consensus sequence is shown

possess an aromatic amino acid; the numbers above the seque

numbers and numbering is based on the sequence of the defen

the sequences indicate the position of the amino acids in the s

were deduced from cDNA clones with omission of the putative s

persica, Arabidopsis thaliana, Helianthus annus, Solanum tuberosu

Wasabia japonica; the lines underneath the sequences indicate

strands and double lines a-helixes, according to Fant et al. [13].

accession number or article reference are shown as follows: PV

deduced peptide from the clone pCR2.1-DEF from V. unguiculata

SPE10 (gi50659050), Pachyrhizus erosus defensin; Psd1 (gi201393

defensin 2; C. annuum defensin (gi40794499); P. persica defensin

protein from A. thaliana; H. annuus defensin (gi8099184); S. tube

G. biloba defensin (gi5149374331); T. aestivum defensin (gi223243

(gi1173437), S. bicolor inhibitor of a-amylase 3; H. vulgare Gamma

Ah-AMP1 (gi1049478), Aesculus hippocastanum antifungal protein

protein 1; Dm-AMP1 (gi1049480), Dahlia merckii antimicrobial pro

W. japonica defensin (gi11691894); Hs-AFP1 (gi1049482), Heucher

3. Results

3.1. Purification of the P. vulgaris defensin

After extraction, the F-0/70 was initially fractionated by anion-

exchange chromatography, a DEAE-Sepharose. Two protein

peaks, named D1 and D2 were eluted with equilibrium buffer

and 1 M NaCl, respectively (Fig. 1A). The non-adsorbed basic

fraction (D1) from the ion-exchanger step was further

fractionated by reverse-phase chromatography in the C2/

C18 ST 4.6/100 column. The D1 fraction was separated in one

unique peak, named H1 (Fig. 1B).

The analysis of the protein profile of the F-0/70 fraction (F)

and D2 fraction (adsorbed acid fraction) demonstrated the

presence of several proteins and peptides in SDS–Tricine gel

electrophoresis under reducing conditions (Fig. 2). However,

the D1 fraction was composed of one unique band with a

molecular mass of approximately 6 kDa (Fig. 2). Corroborating

with this data, H1, as determined by SDS–Tricine gel

rious plant defensins with the PVD1 protein deduced from

l residues and P% indicates the percentage of positive

characteristics such as charge and hydrophobicity), both

ed PVD1 peptide from common bean; (S) gaps are included

ignment to compensate for insertions and deletions in one

below, (*) means that in these positions the peptides

nce indicate the extension of the peptides in amino acid

sin Rs-AFP1 from Raphanus sativus; the numbers flanking

equence of the mature peptides. The following sequences

ignal peptides: Vigna unguiculata, Capsicum annuum, Prunus

m, Picea glauca, Gingko giloba, Triticum aestivum, R. sativus,

secondary structure elements, single lines represent b-

The name of the species, abbreviations and the data bank

D1, deduced peptide from the clone pTZ57R/T-DEF; VUDEF,

[8]; Psas10 (gi112671), clone PSAS10 from V. unguiculata;

22), Pisum sativum defensin 1; Psd2 (gi20139323), P. sativum

(gi28624546); PDF2.3 (gi15226878), plant defensin-fusion

rosum defensin (gi129350); P. glauca defensin (gi40362748);

63); SIa2 (gi134486), S. bicolor inhibitor of a-amylase 2; SIa3

hordothioni (gi135793); Beta vulgaris defensin (gi1839272);

1; Ct-AMP1 (gi1049479), Clitoria ternatea antimicrobial

tein 1; Rs-AFP1 (gi2914400), R. sativus antifungal protein 1;

a sanguinea antifungal protein 1.

Fig. 6 – Unrooted phylogenetic tree for known and putative

plant defensins. Protein accession number or article

reference is preceded by the name of the peptide and/or

species in which the peptide was identified: PVD1, deduced

peptide obtained in this work; Vu, deduced peptide from the

clone pCR2.1-DEF from Vigna unguiculata [8]; Pe, SPE10,

Pachyrhizus erosus defensin (gi50659050); Ca, Cicer arietinum

defensin (Q6XW14); Ps1, Psd1, Pisum sativum defensin 1

(gi20139322); Ps2, Psd2, P. sativum defensin 2 (gi20139323);

Ah, Ah-AMP1, Aesculus hippocastanum antifungal protein 1

(gi1049478); Ct, Ct-AMP1, Clitoria ternatea antimicrobial

protein 1 (gi1049479); Dm, Dm-AMP1, Dahlia merckii

antimicrobial protein 1 (gi1049480); At1, AFP1, cysteine-rich

antifungal protein 1 precursor from Arabidopsis thaliana

(P30224); At2, AFP3, probable cysteine-rich antifungal

protein At2g26020 precursor from A. thaliana (O80994); Wj,

Wasabia japonica defensin (gi11691894); Hs, Hs-AFP1,

Heuchera sanguinea antifungal protein 1 (gi1049482); BV,

Beta vulgaris defensin (gi1839272); Sb1, SIa2, S. bicolor

inhibitor of a- amylase 2 (gi134486); Sb2, SIa3, S. bicolor

inhibitor of a-amylase 3 (gi1173437); Hv, Gamma

hordothioni from H. vulgare (gi135793); Pa, Picea abies

defensin (gi:1360108); Gb, Gingko biloba defensin

(gi5149374331); Ta, Triticum aestivum defensin (gi22324363);

Tm, Tm-AMP, T. monococcum defensin (P84964); Tk, Tk-AMP,

T. kiharae defensin (P84965); Gm, SRP-SOYBN, 8.4 kDa

sulfur-rich protein precursor (Protein SE60) from Glycine max

(Q07502); St, Solanum tuberosum defensin (gi129350); Os,

Oryza sativa defensin (Q7F8K7); Ca, Capsicum annuum

defensin (gi40794499); Sc, S. chacoense Gamma-thionin/

defensin (gi170773916); At3, PDF2.3, plant defensin-fusion

protein from A. thaliana (gi15226878); Ha, SD2, Helianthus

annuus defensin (gi8099184); Pp, Prunus persica defensin

(gi28624546); Na, Nicotiana attenuata defensin (gi42374733).

p e p t i d e s 2 9 ( 2 0 0 8 ) 2 0 9 0 – 2 1 0 0 2095

electrophoresis, was also composed of only one peptide with

molecular mass of 6 kDa. Thus, this peptide was named PvD1

(Fig. 2). The partial N-terminal amino acid sequence of PvD1

showed high similarity, between 100% and 85%, to defensin

isolated from seeds of Vigna radiata (gi18146788), Vigna

unguiculata [7], Vigna unguiculata (gi112671), Tephrosia platycarpa

(gi62549225) and P. sativum (gi20139322) (Fig. 3).

3.2. Cloning and sequence analysis of the P. vulgarisdefensin cDNA

RT-PCR amplification of mRNA isolated from common bean

seeds resulted in a fragment that was cloned into the pTZ57R/

T vector and sequenced. Nucleotide sequence analysis

revealed that cDNA consists of 314 bp and in the cDNA

sequence lacks the AUG initiation codon, which is consistent

with the fact that this cDNA was amplified using a primer

specific for a mature protein, i.e. without the signal peptide

(Fig. 4). The cDNA encodes a predicted peptide of 47 amino

acids which presents theoretical pI and molecular weight of

8.2 and 5448.11 Da, respectively (Fig. 4).

Furthermore, the alignment of the predicted peptide PVD1

with the protein database confirmed the partial alignment

result of the previously purified peptide showing again

similarity with plant defensins from V. unguiculata (93%), Cicer

arietinum (95%) and Pachyrhizus erosus (87%), among others

(Fig. 5). It may be clearly observed from the sequence

comparison that eight strictly conserved Cys residues of plant

defensins, that play an important role in peptide stabilization,

are present in PvD1. Other conserved residues, such as an

aromatic residue Tyr11 and Gly13 were also found in the

sequence (Fig. 5).

To investigate the evolutionary relationships among PvD1

and other plant defensin, an unrooted phylogenetic tree was

constructed based on the deduced amino acid sequences of

predicted PVD1 and members of the defensin family from

other plant species (Fig. 6). It may be observed that the

phylogenetic tree is grouped, in general, in a family-specific

manner. The result highlights that P. vulgaris defensin is

clustered with four other Fabaceae species such as V.

unguiculata, P. erosus, C. arietinum and P. sativum, indicating

the closer relationship within the defensins of this family

(Fig. 6, PVD1 underlined). Defensins from Arabidopsis thaliana

and Wasabia japonica, members of the Brassicaceae family,

have a closer relationship than others. The tree also

demonstrates that defensins from the Poacea especies,

Sorghum bicolor and Hordeum vulgare, form a cluster, as do

species from the Solanaceae family, Capsicum annuum and

Solanum chacoense. Furthermore, Ginkgo biloba was clustered

with Picea abies, another gymnosperm specie (Fig. 6).

3.3. Effect of the P. vulgaris defensin on fungi growth

Initially, we tested the effect of PvD1 on growth of some

phytopathogenic fungi, which included F. solani, F. laterithium,

F. oxysporum and R. solani. Fig. 7 shows the patterns of fungal

growth in the presence of PvD1 and in the general control. An

inhibitory effect on the growth of all fungi tested was

observed. Inhibition was detected particularly in the presence

of 100 mg mL�1 of PvD1, as compared with the growth in the

Fig. 7 – The effect of PvD1 on the growth of phytopathogenic fungi. The absorbance at 660 nm was taken as a measure of

Fusarium solani (A), F. laterithium (B), Rizoctonia solani (C) and F. oxysporum (D) growth. (&) Control; (*) 25 mg mLS1; (~)

50 mg mLS1; (^) 100 mg mLS1 of PvD1. Experiments were run in triplicate and the standard errors (coefficients of variation

were less than 20%) were omitted for clarity.

p e p t i d e s 2 9 ( 2 0 0 8 ) 2 0 9 0 – 2 1 0 02096

control and in the other concentrations used 25 and 50 mg mL�1.

In this study, we also tested the PvD1 effect on the growth of

some pathogenic yeasts and the bake yeast S. cerevisiae. The

growth of C. albicans, C. parapsilosis, C. guilliermondii, and K.

marxiannus was inhibited in the presence of PvD1 at all

concentrations used. The IC50 value for C. albicans and C.

guilliermondii can be observed at the concentrations of

<50 mg mL�1 (Fig. 8A and D, respectively). An accentuated

reduction in the growth of theS. cerevisiaeyeast was observed at

the concentration of 100 mg mL�1 (Fig. 8F). It was not observed a

significant inhibition of the growth of the C. tropicalis yeast.

We further analyzed, through scanning electron micro-

scopy, possible alterations caused by the PvD1 in C. albicans

(Fig. 9A and B), C. parapsilosis (Fig. 9C and D), C. tropicalis (Fig. 9E

and F) and S. cerevisiae (Fig. 9G, H and I) morphology. Normal

growth development was observed for control cells (Fig. 9A, C,

E and G), nevertheless cultures of S. cerevisiae and C. albicans

treated with PvD1 exhibited notable alterations in bud

formation, with difficultly in liberating buds and cell agglom-

eration, respectively (Fig. 9B, H and I).

4. Discussion

During recent years, an increasing number of cysteine-rich

antimicrobial peptides have been isolated from plants and,

particularly, from seeds [6]. More recently, Carvalho et al. [7,8]

purified and cloned a plant defensin from cowpea seeds that

presented the ability to inhibit the growth of phytopathogens in

vitro. In this work, we showthepurificationof a defensin fromP.

vulgaris seeds through one unique chromatography step.

Initially, a rich peptide fraction was obtained through the

ammonium sulfate fractionation. Then, this fraction was

separated in two other fractions, D1 and D2, by a chromato-

graphic step on a DEAE-Sepharose (Fig. 1A). The D1 fraction

represents the minor protein concentration. SDS–Tricine gel

electrophoresis under reducing conditions revealed that this

fraction was composed of a single protein band with molecular

mass of approximately 6 kDa (Fig. 2). The purification of the

peptide was confirmed after a reverse-phase chromatography,

where only one peak was observed, denominated H1 (Fig. 1B).

H1 was also composed of one unique peptide with molecular

mass of 6 kDa as determined by SDS–Tricine gel electrophoresis

(Fig. 2). This peptide was further namedPvD1. The values for the

peptides lie in the range of the molecular masses found by

several authors for defensins isolated from other plants [6].

Alignment of partial N-terminal amino acids sequence with the

protein database show that the peptide PvD1 really belongs to

the defensin family, displaying similarity to other defensins

isolated from different plants species (Fig. 3).

Among the primary structure of plant defensins, eight Cys

residues are conserved in all sequences [9,11]. The cloned P.

Fig. 8 – The effect of PvD1 on the growth of the yeasts C. albicans (A), C. parapsilosis (B), K. marxiannus (C), C. guilliermondii (D),

C. tropicalis (E) and S. cerevisiae (F). (&) Control; (*) 25 mg mLS1; (~) 50 mg mLS1; (*) 100 mg mLS1 of PvD1. Experiments were

performed in triplicate and the standard errors (coefficients of variation were less than 20%) were omitted for clarity.

p e p t i d e s 2 9 ( 2 0 0 8 ) 2 0 9 0 – 2 1 0 0 2097

vulgaris seed defensin protein of 47 amino acids presents all of

these residues at similar positions (Fig. 5). By using the

Computer pI/Mw Tool (http://www.expasy.org), the calculated

pI and molecular weight of the deduced protein were inferred

to be 8.2 and 5448.11 Da, respectively. Both characteristics are

shared among plant defensin members [25,33]. These residues

are engaged to each other, forming four disulfide bridges that

are important for the stabilization of the protein global fold.

Two of these bridges, formed between the Cys21 and Cys25 of

the a-helix region with the Cys45 and Cys47 of the last b-sheet,

respectively, encompass the structural array, denominated

‘‘cysteine stabilized ab motif’’, commonly found in peptides

that present biological activity [2,11,13]. In addition to the eight

conserved Cys, other residues were also found to be conserved

in the sequence alignments of plant defensins. These include

the aromatic residue Tyr11 Gly13, Glu29 and Gly34 (Rs-AFP1

numbering). The molecular cloning of PvD1 cDNA enabled us

to know the sequence of the whole peptide. The P. vulgaris

predicted protein PVD1 has the eight conserved Cys residues

and the aromatic residues Tyr11 and Gly13 (Fig. 5).

Using the ClustalW-algorithm a multiple alignment of over

34 defensin sequences from distinct plant species was

performed and a molecular phylogeny tree was constructed

with the PHYLIP Drawtree program. Our results show that, in

general, the phylogenetic tree is grouped in a family-specific

manner (Fig. 6). This phenomenon has previously also been

observed byShenetal. [23].PvD1sharesa commonevolutionary

origin with the defensin from the Fabaceae species, based on its

conserved structural and sequence characteristics, such as

amino acid homologies and conserved motifs (Fig. 6).

Most of the work on cysteine-rich antimicrobial peptides is

based on the assumption that they are involved in plant

Fig. 9 – Scanning electron microscopy of yeast cells in the presence of PvD1. (A, C, E and G) control, absence of defensin; (B, D,

F, H, I) presence of defensin (100 mg mLS1). C. albicans (A and B), C. parapsilosis (C and D), C. tropicalis (E and F), and S.

cerevisiae (G, H and I). Arrows show difficulty in liberating buds. Bars = A–I, 5 mm.

p e p t i d e s 2 9 ( 2 0 0 8 ) 2 0 9 0 – 2 1 0 02098

defense mechanisms against phytopathogenic fungi [6,9]. The

antimicrobial activity of defensins was first reported for two

isoforms isolated from radish seeds and has now been

extended for defensins from different species [7,18,25]. Based

on the antimicrobial effects observed on fungi, at least two

groups can be distinguished; the ‘‘morphogenic’’ defensins

that cause reduced hyphal elongation with a concomitant

increase in hyphal branching, and the ‘‘nonmorphogenic’’

defensins that only slow down hyphal elongation without

inducing marked morphological distortions [33].

We investigate the effect of PvD1 on growth of some

phytopathogenic fungi, F. solani, F. laterithium, F. oxysporum and

R. solani. Inhibition was detected when these fungi was

allowed to growth in the presence of 100 mg mL�1 of PvD1

p e p t i d e s 2 9 ( 2 0 0 8 ) 2 0 9 0 – 2 1 0 0 2099

(Fig. 7). Osborn et al. [19] related that plant defensins isolated

from different seeds cause morphological alterations that are

often very distinct in some, but not all, tested fungi.

Thevissen et al. [32] reviewed the effect of defensins on the

growth of some pathogenic yeasts of the Candida genus. The

plant defensins Hs-AFP1 and Rs-AFP2 and the insect defensin

heliomicin were all able to inhibit the growth of C. albicans and

C. krusei but not of C. glabrata. We also scrutinized the effect of

PvD1 on the growth of the yeasts C. albicans, C. parapsilosis, C.

guilliermondii, C. tropicalis, K. marxiannus and S. cerevisiae. In the

presence of PvD1, the growth of the yeasts C. albicans, C.

parapsilosis, C. guilliermondii andK.marxiannuswere inhibited at

all concentrations of PvD1 tested (Fig. 7A, B, D and C,

respectively). Nevertheless, the growth of the bake yeast S.

cerevisiae was inhibited only at the concentration of

100 mg mL�1 (Fig. 8F) and it was not observed inhibition of

the growth of the C. tropicalis yeast (Fig. 8E).

The saccharomycetales accomplish cell division by bud

formation, thus the mother cell forms a bud and when the new

cell is completely formed the process is finished by the

separation of the daughter cell from the mother cell. Our

photomicrography revealed that cultures of C. albicans and S.

cerevisiae treated with PvD1 exhibited notable cell agglomera-

tion (Fig. 9B) and alterations in bud formation, as well as

difficulty in liberating buds (Fig. 9H and I), respectively. Other

antimicrobial peptides obtained from plants were shown to

cause morphological alterations on yeast cells such as these

described in this work. A peptide with homology to 2S albumin

from Passiflora edulis seeds had provoked aberrations in cell

shape, cell wall and bud formation on the yeast S. cerevisiae [1].

Fraction enriched with antimicrobial peptides from C. annuum

seeds had caused morphological alterations on diverse yeast

species and some of the alterations were reported to be on the

cell wall, on bud formation and also to induce yeast-

pseudhyphae transition [12,21].

For the plant defensins Dm-AMP1 and Rs-AMP1 had already

identified the receptors to which they bind on the plasma

membrane of yeast cells. Dm-AMP1 bind to the mannosyldii-

nositolphoshorylceramide and Rs-AMP1 bind to glucosylcer-

amide causing membrane permeabilization [29,31]. The

membrane damage are commonly described as part of the

action mechanism involved in growth inhibition but other

mechanism seems to be also associated with the growth

inhibition. In fact an intracellular target had been described for

the Psd1 which bind to cyclin F and thus impair progression of

cell cycle [18]. In addition, the demonstration that Rs-AMP1

and Rs-AMP2 had no effect on mammal cells [25] point out to a

possible use of these antimicrobial peptides as new ther-

apeutic compounds to treat not only invasive fungal infection

in humans but also some kind of human cancer that has as

cause the uncontrolled cyclin expression [24,35].

Acknowledgements

This study forms part of the PhD degree thesis of ISS, carried

out at the Universidade Estadual do Norte Fluminense. We

also thanks to the financial support to Andre O. Carvalho at the

Universidade Estadual do Norte Fluminense through a fellow-

ship to FAPERJ (E-26-150-015/2006). We also acknowledge the

financial support of the Brazilian agencies CNPq, CAPES,

FAPERJ and TECNORTE/FENORTE.

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