Planta (2011) 234:183–193
DOI 10.1007/s00425-011-1392-1
O R I G I N A L A R T I CL E
Laticifer proteins play a defensive role against hemibiotrophic
and necrotrophic phytopathogens
Diego P. Souza · Cleverson D. T. Freitas ·
Danielle A. Pereira · Fábio C. Nogueira ·
Fredy D. A. Silva · Carlos E. Salas · Márcio V. Ramos
Received: 7 February 2011 / Accepted: 22 February 2011 / Published online: 11 March 2011
© Springer-Verlag 2011
Abstract Proteins from latex of Calotropis procera
(CpLP), Plumeria rubra (PrLP), Carica candamarcensis
(P1G10) and Euphorbia tirucalli (EtLP) were tested for
antifungal activity against phytopathogens. CpLP and
P1G10 inhibited each fungi analyzed. PrLP and EtLP did
not exert inhibition. CpLP and P1G10 exhibited preferen-
tial inhibitory activity towards R. solani (IC50 = 20.7 and
25.3 g/ml, respectively). The inhibitory activity was lost
after heat treatment or proteolysis, providing evidence for
the involvement of proteins in the inhibitory eVect. Treat-
ment of CpLP or P1G10 with Dithiothreitol improved both,
the endogenous proteolytic activity and the antifungal prop-
erties. Conversely, pre-treatment of CpLP or P1G10 with
D. P. Souza · D. A. Pereira · F. D. A. Silva · M. V. Ramos (&)
Departamento de Bioquímica e Biologia,
Molecular da Universidade Federal do Ceará, Campus do Pici,
Cx. Postal 6033, Fortaleza, Ceará CEP 60451-970, Brazil
e-mail: vramos@ufc.br
C. D. T. Freitas (&)
Departamento de Biologia, Universidade Federal do Piauí,
Campus Floriano, BR 343, Km 3, 5,
Bairro Melladão CEP 64800-000, Brazil
e-mail: cleversondiniz@hotmail.com
F. C. Nogueira
Departamento de Bioquímica, Instituto de Química da
Universidade Federal do Rio de Janeiro,
Rede Proteômica do Rio de Janeiro, Rio de Janeiro, Brazil
C. E. Salas
Departamento de Bioquímica e Imunologia,
Instituto de Ciências Biológicas, Escola de Enfermagem,
Faculdade de Medicina, UFMG,
Belo Horizonte 31270-901, Brazil
iodoacetamide drastically reduced endogenous proteolytic
activities and partially abrogated antifungal activity. Simi-
lar results were observed when spores were challenged to
germinate in the presence of laticifer proteins. The puriWed
cysteine proteinase CMS2MS2 from Carica candamarcen-
sis latex or papain (E.C. 3.4.22.2), a cysteine proteinase
from latex of Carica papaya L., but not trypsin (EC
3.4.21.4) or chymotrypsin (EC 3.4.21.1), two serine prote-
ases, replicated the results obtained with CpLP or P1G10,
thus restricting the antifungal property to latex plant cys-
teine proteinases. CpLP, CMS2MS2 and papain induced
production of reactive oxygen species in spores of F.
solani, suggesting that inhibition could be linked to oxida-
tive stress. Proteome analysis of CpLP by 2-D electropho-
resis and MALDI-TOF-TOF conWrmed the existence of
various pathogenic-related proteins such as chitinases, per-
oxidases and osmotins. The results support that laticifer
proteins are part of plant defense repertoire against phyto-
pathogenic fungi.
Keywords Aspergilus niger · Fusarium solani ·
MALDI-TOF-TOF · Pathogenesis-related proteins ·
Proteomic
Abbreviations
BSA Bovine serum albumin
CMS2MS2 The cysteine proteinase puriWed of the latex
of Carica candamarcensis
DAB 3,3⬘-Diaminobenzidine
DTT Dithiothreitol
IAA Iodoacetamide
IC50 The protein concentration reducing growth to
50% of control values
LP Laticifer proteins
ROS Reactive oxygen species
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Introduction
Canal networks containing latex are widely disseminated in
the plant kingdom. These Xuids are chemically complex
and contain a wide array of secondary metabolites exhibit-
ing intense metabolism (Kekwick 2001). Despite their ori-
gin, latex is the cytoplasm of specialized cells growing
intrusively into organized tissues and organs, forming an
interconnected network that allows latex exudation imme-
diately after tissue damage (Hagel et al. 2008). Cardeno-
lides, Xavonoids and terpenes are among the toxic
compounds found in laticifers of Antiaris toxicaria, Calot-
ropis procera and Asclepias curassavica, respectively
(Giordani et al. 2000; Salunke et al. 2005; Gan et al. 2009).
Pathogenesis-related (PR) proteins are commonly found
in latex and their presence linked with a defensive role of
this Xuid in plants. Proteases (Freitas et al. 2007), -amy-
lase inhibitor (Farias et al. 2007), chitinases (Taira et al.
2005), proteinase inhibitors (Azarkan et al. 2006), -1,3-
glucanase (Churngchow et al. 1995), osmotin (Freitas et al.
2011) and oxidative enzymes (Mura et al. 2008) are
PR-proteins already described in latex of diVerent plants.
Their role in plant defense has been specially investigated
by using insect models. Proteins extracted from latex of
C. procera were detrimental to some Lepidoptera and
Coleoptera insects when incorporated into artiWcial diets
(Ramos et al. 2007, 2010). In addition, laboratory observa-
tions suggest that latex can contribute to repel insects.
The studies of latex activity against phytopathogens are
still incipient. Literature about hevein, the most celebrated
latex protein from Hevea brasiliensis (the rubber tree) com-
prises the most consistent information about antifungal
activity of latex (Parijs et al. 1991). Interestingly, hevein-
like proteins were further reported in other plants but rarely
reported in other laticifer Xuid (Wasano et al. 2009). It is
therefore relevant to investigate the presence of antifungal
proteins in other laticifer Xuids. Here, latices from four
plants were collected and their soluble proteins recovered
and tested for antifungal activity on phytopathogenic fungi.
Attempts were made to identify the proteins eliciting anti-
fungal activity and their possible mechanism of action.
Materials and methods
Chemicals
Papain (E.C. 3.4.22.2) from Carica papaya latex, trypsin
(E.C. 3.4.21.4) and chymotrypsin (E.C. 3.4.21.1) from
bovine pancreas, protease (pronase) from Streptomyces
griseus (E.C. 3.4.24.31), Bovine serum albumin (BSA),
azocasein and 3,3⬘-diaminobenzidine (DAB) were pur-
chased from Sigma, Brazil. Acrylamide, bis-acrylamide,
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Planta (2011) 234:183–193
3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulf-
onate (CHAPS), dithiothreitol (DTT), sodium dodecyl
sulfate (SDS), iodoacetamide (IAA) and molecular size
markers were from GE Healthcare, Brazil. Other chemicals
were of analytical grade.
Plant material
Healthy plants of C. procera (Ait.) R.Br., Plumeria rubra
L. (both, Apocynaceae) and Euphorbia tirucalli L.
(Euphorbiaceae) growing in the vicinity of Fortaleza, state
of Ceará, Brazil, were used as source of fresh latex. Each
plant material was identiWed and the vouchers N. 32663 (C.
procera), N. 15018 (P. rubra) and N. 38702 (E. tirucalli)
were deposited at the Prisco Bezerra Herbarium of the Uni-
versidade Federal do Ceará, Brazil. Unripe fruits of Carica
candamarcensis L. (Caricaceae) plants, aged between 2 and
4 years, were the source of latex used in this study (Silva
et al. 2003). A voucher specimen of the plant was deposited
at the herbarium of the Universidad de La Serena, Chile,
with # 15063.
Latex processing
The latices of C. procera, P. rubra and E. tirucalli were
collected by cutting the ending branches and collecting the
exudates into distilled water at 1:2 (v/v) dilution. The mixes
were gently agitated during collection to overcome the ten-
dency of latex spontaneous coagulation-like process. The
suspensions were centrifuged at 5,000£g for 10 min at
4°C. The resulting rubber-like precipitates were discarded
and the supernatants were exhaustively dialyzed against
distilled water using membranes of 8,000 Da cut-oV at 8°C
for 60 h with three daily changes of water. The dialysates
were then centrifuged as described before and the clean,
rubber-free supernatants containing the soluble protein
were freeze-dried and the dried white-oV powder stored at
¡20°C until use. These protein fractions were designated
laticifer proteins (LP) and named according to the source
as: CpLP, PrLP and EtLP, corresponding to C. procera,
P. rubra and E. tirucalli, respectively. Laticifer proteins
(P1G10) of Carica candamarcensis Hook (Vasconcella
cundinamarcensis) were prepared as reported by Teixeira
et al. (2008). Protein content in the samples was measured
according to the Bradford’s method (1976) with bovine
serum albumin as the protein standard.
Treatment of protein fractions
Heat-inactivation of laticifer proteins from CpLP and
P1G10 was done by dissolving 100 mg of the sample in
50 ml of water and immersing in boiling water for 30 min
followed by immersion in ice. The insoluble material was
Planta (2011) 234:183–193 185

Table 1 Some hosts and diseases caused by fungi used in this study
Fungi Main hosts Disease caused Injured tissue/organ References

Hemibiotrophic
Colletotrichum Carica papaya Papaya anthracnose Fruit Cia et al. (2007)
gloeosporioides
Fusarium oxysporum More than 100 species Vascular wilt Root Agrios (2005)
of plants and root rot
Fusarium solani Glycine max, Root crown rot, Tap roots and Fu and Chang (1999);
Phaseolus vulgaris dieback and wilt hypocotyls Iqbal et al. (2005);
Abeysinghe (2007)
Necrotrophic
Rhizoctonia solani Oryza sativa, Sheath blight Hypocotyls Datta et al. (1999);
Phaseolus vulgaris and roots Abeysinghe (2007)
Neurospora sp. Anacardium accidentale Green mold Fruits Andrade et al. (1990)
Aspergillus niger Vitis vinifera, Allium cepa, Black mold Fruits Samson et al. (2001)
Arachis hypogaea

removed by centrifugation at 25°C and 10,000£g for Preparation of spore suspension

10 min and the clear supernatant was freeze-dried and des-
ignated Heated-LP.
Laticifer proteins of CpLP and P1G10 [100 mg in 50 ml
of 50 mM Tris–HCl buVer (pH 7.5)] were digested at 37°C
with pronase from Streptomyces griseus, during 24 h with
three enzyme additions at 0-, 12- and 18-h intervals. The
pronase/sample ratio was 1:50 (w/w). Following digestion
the samples were dialyzed against distilled water, freeze
dried and named pronase-LP. Pronase was chosen because
it is a non-selective mixture of proteolytic enzymes suitable
for extensive digestion of LP. Both treatments were per-
formed as an attempt to obtain samples free of protein
activity and thus provide evidence for the involvement of
laticifer protein in antifungal activity.
Microorganisms
Fungal isolates utilized were: Fusarium oxysporum, Fusar-
ium solani, Colletotrichum glorosporioides, Neurospora
sp, Rhizoctonia solani and Aspergilus niger. The fungi
were obtained from a local collection maintained in the
Laboratory of Microbiology at the Departamento de Biolo-
gia, Universidade Federal do Ceará, Brazil, where they
were properly identiWed by a taxonomist. The fungi were
maintained on Sabouraud Dextrose Agar (0.5% Enzymatic
Digest of Casein, 0.5% Enzymatic Digest of Animal Tis-
sue, 4% dextrose and 1.5% agar–agar, Wnal pH 5.6) at
27°C, 12 h light/dark cycle and 70% relative humidity. All
fungi assayed had their identity conWrmed after DNA puri-
Wcation, PCR ampliWcation and DNA sequencing of ITS
speciWc regions (results not shown). These fungi were
selected on the basis of their relevance for agricultural
losses of important crops within the region, as summarized
in Table 1.
The spore suspensions were obtained from their respective
2-week-old cultures in sterile distilled water yielding a
homogenous spore suspension. Conidia were quantiWed on
a Neubauer chamber from an appropriate dilution (2 £ 105
spore/ml) by using a BX60 Olympus Light Microscope.
Growth inhibitory assays
A quantitative assay to assess inhibition of fungal growth
was performed following the protocol developed by
Broekaert et al. (1990). To evaluate the eVect of CpLP,
PrLP, EtLP and P1G10 on fungal growth, 10 l of spore
suspension (2 £ 105/ml) were incubated in 96-well Xat-bot-
tom microplates (NuncTM) with 90 l Yeast Peptone Dex-
trose broth (5% m/v) at 27°C, 70% relative humidity. After
16 h, 100 l of each LP solution was dissolved in 50 mM
sodium acetate buVer (pH 5.0), cleared by centrifugation at
10,000£g for 10 min at 4°C and Wltered through a 0.22 m
membrane (Millipore). The fungal growth was monitored
from 0 to 48 h by turbidimetry at 620 nm in an automated
microplate reader (Biotrak II Plate Reader, Amersham
Biosciences). The amount of protein solution (triplicate)
that reduced growth by 50% relative to the control after
48 h incubation was deWned as IC50 and expressed as g
protein/ml.
Germination inhibitory assay
To evaluate the eVect of laticifer proteins on spore germina-
tion, 10 l of the spore suspension (2 £ 105/ml in water)
was incubated with 10 l LP solutions (1 mg/ml in 50 mM
acetate buVer, pH 5.0) on sterile reticulated plastic plates.
The plates were maintained at 27°C and 70% relative

123
186
humidity. After 24 h, plates were visualized with a light
Olympus Microscope (BX60). Microphotography of each
preparation was obtained and the rate of spore germination
visually assessed. Each experiment was done twice, with
two replicates per treatment.
Detection of H2O2
Production of reactive oxygen species (ROS) such as H2O2
by spores treated with LP was estimated by adding 3,3⬘-
diaminobenzidine (DAB), as described by Thordal-
Christensen et al. (1997). DAB forms a dark brownish pre-
cipitate in the presence of peroxidases and H2O2 causing a
visible cytochemical stain. Spores of F. solani seeded at
Wnal concentration of 2 £ 105 spores/ml were incubated in
sodium acetate buVer pH 5.0 or Tris–HCl buVer pH 7.5
(controls) or buVer plus papain, trypsin, chymotrypsin,
CMS2MS2, BSA or CpLP at 1 mg/ml or 0.5 mg/ml in the
presence of 0.1 mg/ml DAB for 2 h at 27°C and 70% rela-
tive humidity. Formation of dark brownish precipitate was
detected with a BX60 Olympus Light Microscope.
Proteolytic assays
Proteolytic activity of C. procera, P. rubra and E. tirucalli
was already characterized and described previously (Freitas
et al. 2007, 2010). Here, azocasein was used as a non-spe-
ciWc substrate to estimate proteolysis in the assays. The
color developed in all enzymatic reactions was measured
by absorbance at 420 nm (Xavier-Filho et al. 1989). One
unit of activity was deWned as the amount of enzyme capa-
ble of increasing absorbance by 0.01 at 420 nm.
As a Wrst attempt to investigate the participation of cys-
teine proteinases in the antifungal activity, CpLP and
P1G10 (2 mg/ml) were dissolved in 50 mM sodium acetate
(pH 5.0) containing 10 mM Iodoacetamide (IAA, a speciWc
cysteine proteinase inhibitor). The mixtures were incubated
at 25°C for 30 min followed by dialysis in distilled water to
eliminate the free IAA and thus, freeze-dried. The samples
were used to measure the residual cysteine proteinase activ-
ity as described above with azocasein substrate and assayed
for inhibition of fugal growth. In addition, papain and
CMS2MS2, two puriWed cysteine proteinases of latex ori-
gin, were tested and compared with two puriWed serine pro-
teinases (trypsin and chymotrypsin).
Two-dimensional polyacrylamide gel electrophoresis
(2D-PAGE)
Eleven centimeter Immobiline DryStrips (pH 3–10 and pH
4–7) were rehydrated overnight with solution (7 M urea/
2 M thiourea/1% CHAPS/2% IpG-buVer pH 3–10 and pH
4–7/1% DTT/bromophenol blue) containing LP from
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Planta (2011) 234:183–193
C. procera (300 g). Runs were performed in an Ettan™
IPGPhor II™ system (GE-Healthcare). Electrical condi-
tions were as described by the supplier. Electrophoresis in
the second dimension was performed in a vertical system
with uniform 12.5% separating gel (14 £ 14 cm) as
described in Freitas et al. (2007).
In-gel tripsin digestion, mass spectrometry database search
and protein identiWcation
Selected spots in 2D-gels were excised and processed for
mass spectrometric analysis (Hellman et al. 1995). Matrix-
assisted laser desorption ionization-time of Xight-tandem
mass spectrometry (MALDITOF-TOF–MS/MS) acquisi-
tion was performed in an ABI 4700 Proteomics Analyzer
(Applied Biosystems) using 3,5-dimethoxy-4-hydroxycin-
namic acid as matrix. Data collected were further analyzed
by GPS Explorer TM software (Applied Biosystems). The
acquired mass spectral data were searched against the
NCBI database using the MASCOT (Matrix Science Ltd.,
London, UK) search engine.
Statistical analysis
Results of Fig. 1 are reported as mean § SEM for tripli-
cates. Statistical signiWcance (p < 0.05) was assessed by
analysis of variance followed by Tukey’s test performed in
Prisma Software.
Results and discussion
Laticifer proteins inhibit in vitro growth
of phytopathogenic fungi
CpLP and P1G10 exhibited important inhibitory activity
against all the fungi analyzed (Table 2). With CpLP, the
lowest IC50 was 20.7 g/ml on R. solani, and the largest
IC50 1,368 g/ml for Aspergillus niger, a 66-fold diVerence
between these IC50. The remaining IC50 were dispersed
between these values, somewhat closer to the IC50 for
R. solani. Furthermore, the largest IC50 found was 5.8-fold
lower than the protein concentration in latex of C. procera
(8 mg/ml). P1G10 was highly toxic towards each of the
studied fungi, as well. The IC50 values ranged 25–137 g/
ml and similarly, IC50 determined with P1G10 LP was
below the physiological protein concentration found in
latex from C. candamarcensis. Laticifer proteins from
P. rubra and E. tirucalli did not aVect fungal growth, even
at the maximal assayed dose (10 mg/ml). The concentration
of soluble proteins in whole latex of P. rubra was estimated
to be 0.32 mg/ml; thus, the largest assayed dose for
P. rubra signiWcantly exceeded the estimated amount on its
Planta (2011) 234:183–193 187

Fig. 1 EVect of activator (DTT) C. gloeosporioides F. solani

and inhibitor (IAA) of cysteine
Control CpLP Control CpLP
proteinase on antifungal activity
P1 G10 Papain P1 G10 Papain
of laticifer proteins from Calot- 100 # 100
#
ropis procera (CpLP), Carica

Fungal growth (%)

Fungal growth (%)

candamarcensis (P1G10) and 80 80 *
papain from Carica papaya. *# *#
*#
60 * 60
Percent values correspond to the *#
antifungal activity of each *#
40
40
sample determined after 48 h of *# *# *#
assay. Samples were dissolved 20 * 20 * *#
*#
in sodium acetate buVer (pH 5.0) * *
at 1 mg/ml (w/v). *p < 0.05 indi- 0 0
Nativa DDT IAA Nativa DDT IAA
cates statistical diVerence com-
pared with control and #p < 0.05
compared with CpLP (n = 3, F. oxysporium R. solani
ANOVA, Tukey’s test)
Control CpLP Control CpLP
Papain P1 G10 Papain
100 #
*
100

Fungal growth (%)

Fungal growth (%)

*
80 * 80
*#

60 60 *#
*

40 *
40
* *
20 *#
20 * *
*#
* *
0 0
Nativa DDT IAA Nativa DDT IAA

Neurospora sp
.. A. niger
Control CpLP Control CpLP
P1 G10 Papain P1G10 Papain
#
100 *# 100 *#
*#
Fungal growth (%)

*
Fungal growth (%)

80 80 *#
*# *
*
60 *# * 60
*#
40 *# 40
*#
*# *#
20 * 20
*
0 0
Nativa DDT IAA Nativa DDT IAA

latex. E. tirucalli did not show inhibitory activity and the
protein content in its latex was negligible.
The involvement of proteins in antifungal activity was
also studied in heat-denatured or proteolytically digested
laticifer fractions of CpLP and C. candamarcensis P1G10.
Both, heat and protein digestion of LP impaired the inhibi-
tory eVect on vegetative growth of every fungi studied (data
not shown). Antifungal proteins have been reported previ-
ously in other latices. Parijs et al. (1991) showed that the
hevein, a puriWed protein from Hevea brasiliensis latex,
exhibited antifungal activity upon eight phytopathogens
and three chitinases from Ficus microcarpa latex showed
antifungal activity on Trichoderma viride (Taira et al.
2005). More recently, an osmotin from Calotropis procera
latex was shown to be active against F. solani, Neurospora
sp. and C. gloeosporioides (Freitas et al. 2011). The defen-
sive role of a thaumatin-like protein was also stressed by
Looze et al. (2009) that showed no antifungal activity of
puriWed thaumatin from Carica papaya latex to Candida
albicans and Saccharomyces cerevisiae that are not phyto-
pathogens.
Cysteine proteinase activity correlates with inhibition
of fungal growth
While latex of E. tirucalli had no detectable proteolytic
activity, latex of P. rubra exhibited moderate activity. Con-
versely, CpLP and P1G10 LP were highly active and their
activities were drastically reduced upon IAA treatment
(Table 3). The proteolytic activity of P1G10 is about two-
fold higher than C. procera, suggesting a higher content in
cysteine proteinases. These results were in agreement with

123
188 Planta (2011) 234:183–193

Table 2 Antifungal activity of laticifer proteins of C. procera F. solani, F. oxysporum and R. solani. CMS2MS2 inhibited
(CpLP), Ca. candamarcensis (P1G10), P. rubra (PrLP) and E. tirucalli both fungi with IC50 34.0, 37.6 and 41.8 g/ml, respec-
(EtLP)
tively. These values are close to those determined for
Fungi Antifungal activity (IC50 g proteina/mL) papain. Papain antifungal action was reversed by IAA sug-
CpLP P1G10 PrLP EtLP
gesting that its proteolytic activity fully accounts for the
antifungal action. However, the partial fungal growth rever-
Colletotrichum 455.0 § 9.3 137.0 § 4.3 NI NI sion in IAA-treated LP fractions (CpLP, P1G10) suggests
gloeosporioides that in addition to proteinases, additional non-proteolytic
Fusarium oxysporum 29.4 § 2.1 – NI NI latex proteins might display antifungal activity. This is par-
Fusarium solani 134.5 § 8.1 56.1 § 6.5 NI NI ticularly true in CpLP where an antifungal protein osmotin
Rhizoctonia solani 20.7 § 1.6 25.3 § 2.4 NI NI was recently described (Freitas et al. 2011).
Neurospora sp. 549.9 § 14.3 40.2 § 3.2 NI NI In another series of experiments, three of the proteolytic
Aspergillus niger 1368.0 § 11.2 104.0 § 3.4 NI NI fractions with antifungal activity (CpLP, P1G10 and papain)
Results are expressed as mean § SD of three independent experi- were treated with DTT to thereafter assess their eVect on fun-
ments. IC50 was estimated after 48 h gal growth. The rationale is that cysteine proteinases are acti-
NI no-inhibition at the maximal dose tested (10 mg/ml) vated by reducing agents (DTT, mercaptoethanol, cysteine)
a
Soluble proteins determined by Bradford (1976) that reduce the cysteinyl residue located in the active site,
essential for proteolytic activity. Consequently, after DTT
Table 3 Proteolytic activities of laticifer proteins studied activation, inhibition of fungal growth was enhanced to a
Protein samples Proteolytic Proteolytic activity larger extent compared with the respective untreated LP frac-
and puriWed activity (AU/g in presence of IAA tions (Fig. 1), suggesting that proteolytic fractions are par-
proteases proteina) (AU/g protein) tially active before DTT addition. The proteolytic activation
of LP proteins was conWrmed by dosage of proteolytic activ-
CpLP 2.71 § 0.09 0
ity with azocasein in the presence of DTT (6-fold higher than
P1G10 4.54 § 0.15 0
native LP, data not shown). The inhibition of fungal growth
PrLP 0.75 § 0.02 0.27 § 0.01
was also compared with two non-plant proteolytic enzymes.
EtLP Nd –
Trypsin and chymotrypsin did not exert inhibitory activity at
Papain 1.54 § 0.09 0
all (data not shown). It is important to cite that the speciWc
Trypsin 6.02 § 0.21 –
activity of trypsin used in these assays was fourfold higher
Chymotrypsin 2.56 § 0.54 –
than papain (6.02 vs. 1.54 AU/g; Table 3). Any work that
Values are mean § SD of triplicate points describes the antifungal activity of cysteine proteinases from
Nd not detected, IAA iodoacetamide latex has not been reported in literature so far. However, cys-
a
Soluble proteins determined by Bradford (1976) teine proteinases from diVerent plants have been associated
with disease resistance (Avrova et al. 1999; van der Hoorn
prior reports for these latices (Freitas et al. 2007; Teixeira and Jones 2002; Kruger et al. 2002). These results provide
et al. 2008). Proteolytically active and IAA-inhibited CpLP preliminary evidence that cysteine proteinases in latices par-
and P1G10 were investigated for their antifungal activity. ticipate defensively against phytopathogens.
As observed in Fig. 1, each of these latex samples or papain In this study, we further investigated the role of CpLP
strongly inhibited fungal growth. Overall, the inhibitory and CMS2MS2 on spore germination. We compared the
eVect of CpLP was more eYcient than P1G10 or papain, eVect of native LP, heat-treated LP, pronase-LP, IAA-LP,
except in A. niger where P1G10 was more active than CMS2MS2, papain, trypsin, chymotrypsin and BSA on
CpLP, while papain was barely active. The explanation for spore germination. Of these samples, native LP, CMS2MS2
the diVerences in eYciency by these lattices could be and papain inhibited spore germination of each of studied
related to an adaptive response by fungi or to a lack of prior fungi, while BSA (used as protein control), trypsin and
exposure to fungi by these plant species. The inhibitory chymotrypsin showed no activity (Fig. 2). In addition,
eVect on fungal growth was partially suppressed when IAA-CpLP retained the ability to inhibit Neurospora sp,
CpLP proteinases were inhibited with IAA. A similar similarly to what was observed in Fig. 1. It is likely that in
inhibitory trend was observed on IAA-treated P1G10. addition to cysteine proteinases other laticifer proteins of
These observations suggest that cysteine-like proteases in C. procera participate in this antifungal response. In sup-
both latices are involved in fungal growth inhibition. In an port of this hypothesis, identiWcation of additional defense
attempt to better correlate cysteine proteinases with proteins in CpLP is reported in the following. Finally, the
antifungal activity, a puriWed cysteine proteinase of Ca. proteins of E. tirucalli and P. rubra failed to induce
candamarcensis (CMS2MS2) was challenged to inhibit changes in spore germination (data not shown).

123
Planta (2011) 234:183–193 189

CpLP

Control Nativa Heated Pronase IAA BSA Papain

F. solani

Neurospora sp.

C. gloeosporioides

Control H2O2 CMS2MS2 Papain Trypsin Chymotrypsin

F. solani

F. oxysporum

R. solani

Fig. 2 Inhibition of spore germination of laticifer proteins and pro- Assays were performed at 27°C for 24 h and 70% RH. Inhibition of
teinases. Control: Sodium acetate buVer (pH 5.0); Nativa, heated, pron- germination was observed under light microscopy on a BX60 Olympus
ase and IAA-CpLP (1 mg/ml, w/v); BSA (1 mg/ml); Trypsin, microscopy
chymotrypsin, CMS2MS2 and papain (0.5 mg/ml). Bars 40 m.

IdentiWcation of PR-proteins in latex of C. procera
The CpLP proteins were resolved in 2-DE using two
ampholyte systems during the Wrst eletrophoretic separa-
tion. The Wrst 2-DE proWle using a pH range 3–10 allows
identiWcation of a moderate number of proteins (60–80);
however, many of the most basic proteins were poorly
resolved under this condition (Fig. 3). The lack of eYcient
ampholyte systems creating pH gradients above 10 ham-
pers the chances for eYcient separation of these basic pro-
teins. A similar pattern was obtained in 2-DE gels of
P1G10 proteins, where predominated a discrete group of
proteins localized in the basic region of the gel (not shown).
By focusing on the most acidic proteins we used ampholyte
system pH 4–7, that aVorded a cleaner proWle suitable for
isolation of spots for MS analysis. Overall, 20 spots from

123
190 Planta (2011) 234:183–193

Fig. 3 Two-dimensional poly-

acrylamide gel electrophoresis
(12.5%) of laticifer proteins
(300 g) from Calotropis pro-
cera. Protein spots, visualized
by 0.1% PhastGel Blue R-250,
were excised of the gels, digest
by trypsin and analyzed by
MALDI-TOF-TOF

2-DE gels (pH 3–10) and 14 from 2-DE gels (pH 4–7) were
excised and treated for MS/MS MALDI-TOF-TOF analy-
sis. A total of nine diVerent sequences were determined and
17 candidate proteins were identiWed with aid of MASCOT
and NCBI databases. The list of identiWed proteins is
shown in Table 4. Eight spots (21–28) that appear in pI tan-
dem (pH 4–7) with apparent mass around 65 KDa were
identiWed as having the same amino acid sequence and clas-
siWed as a glycoside hydrolases. This protein is shown to be
increased in latex of plants attacked by Monarch butterXy
(Pereira et al. 2010). The corresponding peptide sequence
(ELFEEMLSR) of isoforms found here did not match any
amino acid sequence of plant proteins deposited in dat-
abases. Instead, these isoforms are similar to glycoside
hydrolases of bacterial origin. Nine pathogenesis-related
proteins were identiWed; including a lipid-associated pro-
tein, a peroxidase, a proteinase inhibitor and isoforms of
cysteine proteinases (2), osmotins (2) and chitinases (2).
The presence of chitinases, osmotin, proteases, protease
inhibitor and peroxidase was previously described in CpLP
(Freitas et al. 2007, 2011; Ramos et al. 2007, 2010). The
presence of these PR-proteins in C. procera latex conWrmed
the results that additional proteins along with cysteine pro-
teinases are involved in antifungal activity. One of the pio-
neer studies of latex proteins on 2-DE gels reported latex
allergens characterized by electrophoresis and microse-
quencing (Posch et al. 1997). Recently, Wang et al. (2010)
reported a suitable method based on 2-DE electrophoresis
compatible with mass spectrometry for separation and anal-
ysis of latex proteins from Hevea latex. Apart of these stud-
ies, literature is still deWcient in latex proteomic analysis.
As a Wrst attempt in this direction, we successfully identi-
Wed defensive proteins on latex of Calotropis procera,
despite the very low resolution of basic proteins in gels.

Table 4 List of identiWed proteins from Calotropis procera latex by MALDI-TOF-TOF

Spot Teoric/experimental Protein Parental Sequences of all ID (NCBI) Protein description
no score ions identiWed peptides
Molecular mass (kDa) pI

2 38.5/43.2 8.4/5.5 108 1602.86 MGQLNVLTGSKGEIR gi|55057256 Peroxidase (Picea abies)

13 51.2/25.4 6.7/7.4 105 1659.71 NSWGSDWGEDGYMR gi|85000505 Cysteine protease precursor
TacP (Theileria annulata
strain Ankara)
14 25.6/24.9 8.1/7.9 137 1421.60 APGGCNNPCTVFK gi|161375756 Osmotin (Piper colubrinum)
1788.72 DDPTSTFTCPGGTNYR
15 25.6/24.5 8.1/8.1 137 1421.60 APGGCNNPCTVFK gi|161375756 Osmotin (Piper colubrinum)
1788.72 DDPTSTFTCPGGTNYR
19–20 20.4/14.6 4.9/7.4 80 978.49 GNLDIFSGR gi|15236014 Lipid-associated family protein
(Arabidopsis thaliana)
21–28 68.2/64.9 5.3/4.6 68 1153.55 ELFEEMLSR gi|148556511 Glycoside hydrolase 15-related
(Sphingomonas wittichii RW1)
32 34.8/27.3 8.6/5.4 87 1948.02 VPGYGVITNIINGGVECGK gi|222139388 Class I chitinase (Pyrus pyrifolia)
33 22.1/27.0 9.0/5.5 60 1036.60 YYILPVIR gi|75264047 Hypothetical protein (Miraculin)
[Arabidopsis thaliana]
34 39.8/29.0 6.6/7.7 65 1199.62 VPPSVDWR gi|75295919 Cysteine proteinase (Glycine max)
38 34.8/26.2 8.6/5.4 87 1948.02 VPGYGVITNIINGGVECGK gi|222139388 Class I chitinase (Pyrus pyrifolia)

123
Planta (2011) 234:183–193
Fig. 4 Detection of H2O2 in Control
spores of Fusarium solani
induced by latex cysteine
proteinases. Control: Sodium
acetate buVer (pH 5.0); BSA
(1 mg/ml, w/v); Trypsin,
CMS2MS2 and papain
(0.5 mg/ml); CpLP and
CpLP-IAA (1 mg/ml). Bars
10 m. Uptake of DAB was
conWrmed by the dark staining
(reddish-brown) reaction in the
spores, indicated by arrows CpLP
Antifungal activity of laticifer proteins induces
oxidative stress
Oxidative stress related to antifungal activity by CpLP or
puriWed proteases (CMS2MS2 and papain) was investi-
gated by estimating hydrogen peroxide production in
spores of F. solani. As seen in Fig. 4, spores exposed to
BSA (protein control) or trypsin did not produce any dark
brownish precipitate, indicating low levels of H2O2 in
spores. However, spores of F. solani incubated with CpLP,
papain or CMS2MS2 exhibited dark staining indicative of
peroxide production. Treatment of CpLP with IAA fully
eliminated the oxidative damage of spores. Reactive oxy-
gen species (ROS) are known mediators of intracellular sig-
naling cascades. Over production of ROS may, however,
lead to oxidative stress, loss of cell function and ultimately
cell death by apoptosis or necrosis (Nordberg and Arner
2001). Here, we have shown that cysteine proteinases from
latex induced the generation of H2O2 in fungal spores of F.
solani. These Wnding is similar to the results previously
reported for an aspartic proteinase from Solanum tubero-
sum (Mendieta et al. 2006).
Conclusion
Many species of laticifer plants are reported to contain
large amounts of cysteine proteinases within latex.
Involvement of plant cysteine proteinases on insecticidal
activity is consistently reported (Lopes et al. 2007; Konno
et al. 2004; van der Hoorn and Jones 2004). However, very
limited studies describe the involvement of laticifer cys-
teine proteinases in protection against phytopathogens. The
present study reports the growth inhibition of diVerent
191
BSA Trypsin H 2 O2
CpLP+IAA CMS2MS2 Papain
phytopathogenic fungi by latex proteolytic fractions.
Several lines of evidence link the presence of cysteine pro-
teinases with this inhibition. Accordingly, two puriWed cys-
teine proteinase of latex origin from C. papaya (papain)
and Carica candamarcensis (CMS2MS2) were also active.
It is therefore concluded that cysteine proteinases from
latices of C. procera and C. candamarcensis are directly
associated with antifungal activity. On the other hand, the
antifungal activity of C. procera on Neurospora sp. was
not clearly associated with cysteine proteinases, because
IAA-LP was able to inhibit fungal growth and spore germi-
nation like native-LP suggesting the existence of additional
mechanisms controlling fungal growth. This hypothesis is
supported by the occurrence of other known defensive pro-
teins indentiWed in latex. These results conWrm the involve-
ment of cysteine proteinases in latex as a mechanism of
plant defense against phytopathogenic fungi.
Acknowledgments Biochemical, functional and applied studies of
latex proteins were supported by grants from FUNCAP, CNPq,
CAPES, RENORBIO and IFS (M.V.R.). Authors are in debit with
Prof. José Tadeu Abreu de Oliveira which provided fungi sources for
assays.
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