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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 iodoacetamide drastically reduced endogenous proteolytic
(CpLP), Plumeria rubra (PrLP), Carica candamarcensis activities and partially abrogated antifungal activity. Simi-
(P1G10) and Euphorbia tirucalli (EtLP) were tested for lar results were observed when spores were challenged to
antifungal activity against phytopathogens. CpLP and germinate in the presence of laticifer proteins. The puriWed
P1G10 inhibited each fungi analyzed. PrLP and EtLP did cysteine proteinase CMS2MS2 from Carica candamarcen-
not exert inhibition. CpLP and P1G10 exhibited preferen- sis latex or papain (E.C. 3.4.22.2), a cysteine proteinase
tial inhibitory activity towards R. solani (IC50 = 20.7 and from latex of Carica papaya L., but not trypsin (EC
25.3 g/ml, respectively). The inhibitory activity was lost 3.4.21.4) or chymotrypsin (EC 3.4.21.1), two serine prote-
after heat treatment or proteolysis, providing evidence for ases, replicated the results obtained with CpLP or P1G10,
the involvement of proteins in the inhibitory eVect. Treat- thus restricting the antifungal property to latex plant cys-
ment of CpLP or P1G10 with Dithiothreitol improved both, teine proteinases. CpLP, CMS2MS2 and papain induced
the endogenous proteolytic activity and the antifungal prop- production of reactive oxygen species in spores of F.
erties. Conversely, pre-treatment of CpLP or P1G10 with 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
D. P. Souza · D. A. Pereira · F. D. A. Silva · M. V. Ramos (&) proteins are part of plant defense repertoire against phyto-
Departamento de Bioquímica e Biologia, pathogenic fungi.
Molecular da Universidade Federal do Ceará, Campus do Pici,
Cx. Postal 6033, Fortaleza, Ceará CEP 60451-970, Brazil
e-mail: vramos@ufc.br
Keywords Aspergilus niger · Fusarium solani ·
MALDI-TOF-TOF · Pathogenesis-related proteins ·
C. D. T. Freitas (&) Proteomic
Departamento de Biologia, Universidade Federal do Piauí,
Campus Floriano, BR 343, Km 3, 5,
Abbreviations
Bairro Melladão CEP 64800-000, Brazil
e-mail: cleversondiniz@hotmail.com BSA Bovine serum albumin
CMS2MS2 The cysteine proteinase puriWed of the latex
F. C. Nogueira of Carica candamarcensis
Departamento de Bioquímica, Instituto de Química da
DAB 3,3⬘-Diaminobenzidine
Universidade Federal do Rio de Janeiro,
Rede Proteômica do Rio de Janeiro, Rio de Janeiro, Brazil DTT Dithiothreitol
IAA Iodoacetamide
C. E. Salas IC50 The protein concentration reducing growth to
Departamento de Bioquímica e Imunologia,
Instituto de Ciências Biológicas, Escola de Enfermagem,
50% of control values
Faculdade de Medicina, UFMG, LP Laticifer proteins
Belo Horizonte 31270-901, Brazil ROS Reactive oxygen species

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184 Planta (2011) 234:183–193

Introduction 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulf-
onate (CHAPS), dithiothreitol (DTT), sodium dodecyl
Canal networks containing latex are widely disseminated in sulfate (SDS), iodoacetamide (IAA) and molecular size
the plant kingdom. These Xuids are chemically complex markers were from GE Healthcare, Brazil. Other chemicals
and contain a wide array of secondary metabolites exhibit- were of analytical grade.
ing intense metabolism (Kekwick 2001). Despite their ori-
gin, latex is the cytoplasm of specialized cells growing Plant material
intrusively into organized tissues and organs, forming an
interconnected network that allows latex exudation imme- Healthy plants of C. procera (Ait.) R.Br., Plumeria rubra
diately after tissue damage (Hagel et al. 2008). Cardeno- L. (both, Apocynaceae) and Euphorbia tirucalli L.
lides, Xavonoids and terpenes are among the toxic (Euphorbiaceae) growing in the vicinity of Fortaleza, state
compounds found in laticifers of Antiaris toxicaria, Calot- of Ceará, Brazil, were used as source of fresh latex. Each
ropis procera and Asclepias curassavica, respectively plant material was identiWed and the vouchers N. 32663 (C.
(Giordani et al. 2000; Salunke et al. 2005; Gan et al. 2009). procera), N. 15018 (P. rubra) and N. 38702 (E. tirucalli)
Pathogenesis-related (PR) proteins are commonly found were deposited at the Prisco Bezerra Herbarium of the Uni-
in latex and their presence linked with a defensive role of versidade Federal do Ceará, Brazil. Unripe fruits of Carica
this Xuid in plants. Proteases (Freitas et al. 2007), -amy- candamarcensis L. (Caricaceae) plants, aged between 2 and
lase inhibitor (Farias et al. 2007), chitinases (Taira et al. 4 years, were the source of latex used in this study (Silva
2005), proteinase inhibitors (Azarkan et al. 2006), -1,3- et al. 2003). A voucher specimen of the plant was deposited
glucanase (Churngchow et al. 1995), osmotin (Freitas et al. at the herbarium of the Universidad de La Serena, Chile,
2011) and oxidative enzymes (Mura et al. 2008) are with # 15063.
PR-proteins already described in latex of diVerent plants.
Their role in plant defense has been specially investigated Latex processing
by using insect models. Proteins extracted from latex of
C. procera were detrimental to some Lepidoptera and The latices of C. procera, P. rubra and E. tirucalli were
Coleoptera insects when incorporated into artiWcial diets collected by cutting the ending branches and collecting the
(Ramos et al. 2007, 2010). In addition, laboratory observa- exudates into distilled water at 1:2 (v/v) dilution. The mixes
tions suggest that latex can contribute to repel insects. were gently agitated during collection to overcome the ten-
The studies of latex activity against phytopathogens are dency of latex spontaneous coagulation-like process. The
still incipient. Literature about hevein, the most celebrated suspensions were centrifuged at 5,000£g for 10 min at
latex protein from Hevea brasiliensis (the rubber tree) com- 4°C. The resulting rubber-like precipitates were discarded
prises the most consistent information about antifungal and the supernatants were exhaustively dialyzed against
activity of latex (Parijs et al. 1991). Interestingly, hevein- distilled water using membranes of 8,000 Da cut-oV at 8°C
like proteins were further reported in other plants but rarely for 60 h with three daily changes of water. The dialysates
reported in other laticifer Xuid (Wasano et al. 2009). It is were then centrifuged as described before and the clean,
therefore relevant to investigate the presence of antifungal rubber-free supernatants containing the soluble protein
proteins in other laticifer Xuids. Here, latices from four were freeze-dried and the dried white-oV powder stored at
plants were collected and their soluble proteins recovered ¡20°C until use. These protein fractions were designated
and tested for antifungal activity on phytopathogenic fungi. laticifer proteins (LP) and named according to the source
Attempts were made to identify the proteins eliciting anti- as: CpLP, PrLP and EtLP, corresponding to C. procera,
fungal activity and their possible mechanism of action. P. rubra and E. tirucalli, respectively. Laticifer proteins
(P1G10) of Carica candamarcensis Hook (Vasconcella
cundinamarcensis) were prepared as reported by Teixeira
Materials and methods et al. (2008). Protein content in the samples was measured
according to the Bradford’s method (1976) with bovine
Chemicals serum albumin as the protein standard.

Papain (E.C. 3.4.22.2) from Carica papaya latex, trypsin Treatment of protein fractions
(E.C. 3.4.21.4) and chymotrypsin (E.C. 3.4.21.1) from
bovine pancreas, protease (pronase) from Streptomyces Heat-inactivation of laticifer proteins from CpLP and
griseus (E.C. 3.4.24.31), Bovine serum albumin (BSA), P1G10 was done by dissolving 100 mg of the sample in
azocasein and 3,3⬘-diaminobenzidine (DAB) were pur- 50 ml of water and immersing in boiling water for 30 min
chased from Sigma, Brazil. Acrylamide, bis-acrylamide, followed by immersion in ice. The insoluble material was

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

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186 Planta (2011) 234:183–193

humidity. After 24 h, plates were visualized with a light C. procera (300 g). Runs were performed in an Ettan™
Olympus Microscope (BX60). Microphotography of each IPGPhor II™ system (GE-Healthcare). Electrical condi-
preparation was obtained and the rate of spore germination tions were as described by the supplier. Electrophoresis in
visually assessed. Each experiment was done twice, with the second dimension was performed in a vertical system
two replicates per treatment. with uniform 12.5% separating gel (14 £ 14 cm) as
described in Freitas et al. (2007).
Detection of H2O2
In-gel tripsin digestion, mass spectrometry database search
Production of reactive oxygen species (ROS) such as H2O2 and protein identiWcation
by spores treated with LP was estimated by adding 3,3⬘-
diaminobenzidine (DAB), as described by Thordal- Selected spots in 2D-gels were excised and processed for
Christensen et al. (1997). DAB forms a dark brownish pre- mass spectrometric analysis (Hellman et al. 1995). Matrix-
cipitate in the presence of peroxidases and H2O2 causing a assisted laser desorption ionization-time of Xight-tandem
visible cytochemical stain. Spores of F. solani seeded at mass spectrometry (MALDITOF-TOF–MS/MS) acquisi-
Wnal concentration of 2 £ 105 spores/ml were incubated in tion was performed in an ABI 4700 Proteomics Analyzer
sodium acetate buVer pH 5.0 or Tris–HCl buVer pH 7.5 (Applied Biosystems) using 3,5-dimethoxy-4-hydroxycin-
(controls) or buVer plus papain, trypsin, chymotrypsin, namic acid as matrix. Data collected were further analyzed
CMS2MS2, BSA or CpLP at 1 mg/ml or 0.5 mg/ml in the by GPS Explorer TM software (Applied Biosystems). The
presence of 0.1 mg/ml DAB for 2 h at 27°C and 70% rela- acquired mass spectral data were searched against the
tive humidity. Formation of dark brownish precipitate was NCBI database using the MASCOT (Matrix Science Ltd.,
detected with a BX60 Olympus Light Microscope. London, UK) search engine.

Proteolytic assays Statistical analysis

Proteolytic activity of C. procera, P. rubra and E. tirucalli Results of Fig. 1 are reported as mean § SEM for tripli-
was already characterized and described previously (Freitas cates. Statistical signiWcance (p < 0.05) was assessed by
et al. 2007, 2010). Here, azocasein was used as a non-spe- analysis of variance followed by Tukey’s test performed in
ciWc substrate to estimate proteolysis in the assays. The Prisma Software.
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- Results and discussion
ble of increasing absorbance by 0.01 at 420 nm.
As a Wrst attempt to investigate the participation of cys- Laticifer proteins inhibit in vitro growth
teine proteinases in the antifungal activity, CpLP and of phytopathogenic fungi
P1G10 (2 mg/ml) were dissolved in 50 mM sodium acetate
(pH 5.0) containing 10 mM Iodoacetamide (IAA, a speciWc CpLP and P1G10 exhibited important inhibitory activity
cysteine proteinase inhibitor). The mixtures were incubated against all the fungi analyzed (Table 2). With CpLP, the
at 25°C for 30 min followed by dialysis in distilled water to lowest IC50 was 20.7 g/ml on R. solani, and the largest
eliminate the free IAA and thus, freeze-dried. The samples IC50 1,368 g/ml for Aspergillus niger, a 66-fold diVerence
were used to measure the residual cysteine proteinase activ- between these IC50. The remaining IC50 were dispersed
ity as described above with azocasein substrate and assayed between these values, somewhat closer to the IC50 for
for inhibition of fugal growth. In addition, papain and R. solani. Furthermore, the largest IC50 found was 5.8-fold
CMS2MS2, two puriWed cysteine proteinases of latex ori- lower than the protein concentration in latex of C. procera
gin, were tested and compared with two puriWed serine pro- (8 mg/ml). P1G10 was highly toxic towards each of the
teinases (trypsin and chymotrypsin). studied fungi, as well. The IC50 values ranged 25–137 g/
ml and similarly, IC50 determined with P1G10 LP was
Two-dimensional polyacrylamide gel electrophoresis below the physiological protein concentration found in
(2D-PAGE) latex from C. candamarcensis. Laticifer proteins from
P. rubra and E. tirucalli did not aVect fungal growth, even
Eleven centimeter Immobiline DryStrips (pH 3–10 and pH at the maximal assayed dose (10 mg/ml). The concentration
4–7) were rehydrated overnight with solution (7 M urea/ of soluble proteins in whole latex of P. rubra was estimated
2 M thiourea/1% CHAPS/2% IpG-buVer pH 3–10 and pH to be 0.32 mg/ml; thus, the largest assayed dose for
4–7/1% DTT/bromophenol blue) containing LP from P. rubra signiWcantly exceeded the estimated amount on its

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

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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).

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

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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 cysteine proteinases (2), osmotins (2) and chitinases (2).
excised and treated for MS/MS MALDI-TOF-TOF analy- The presence of chitinases, osmotin, proteases, protease
sis. A total of nine diVerent sequences were determined and inhibitor and peroxidase was previously described in CpLP
17 candidate proteins were identiWed with aid of MASCOT (Freitas et al. 2007, 2011; Ramos et al. 2007, 2010). The
and NCBI databases. The list of identiWed proteins is presence of these PR-proteins in C. procera latex conWrmed
shown in Table 4. Eight spots (21–28) that appear in pI tan- the results that additional proteins along with cysteine pro-
dem (pH 4–7) with apparent mass around 65 KDa were teinases are involved in antifungal activity. One of the pio-
identiWed as having the same amino acid sequence and clas- neer studies of latex proteins on 2-DE gels reported latex
siWed as a glycoside hydrolases. This protein is shown to be allergens characterized by electrophoresis and microse-
increased in latex of plants attacked by Monarch butterXy quencing (Posch et al. 1997). Recently, Wang et al. (2010)
(Pereira et al. 2010). The corresponding peptide sequence reported a suitable method based on 2-DE electrophoresis
(ELFEEMLSR) of isoforms found here did not match any compatible with mass spectrometry for separation and anal-
amino acid sequence of plant proteins deposited in dat- ysis of latex proteins from Hevea latex. Apart of these stud-
abases. Instead, these isoforms are similar to glycoside ies, literature is still deWcient in latex proteomic analysis.
hydrolases of bacterial origin. Nine pathogenesis-related As a Wrst attempt in this direction, we successfully identi-
proteins were identiWed; including a lipid-associated pro- Wed defensive proteins on latex of Calotropis procera,
tein, a peroxidase, a proteinase inhibitor and isoforms of 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) 234:183–193 191

Fig. 4 Detection of H2O2 in Control BSA Trypsin H 2 O2


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 CpLP+IAA CMS2MS2 Papain

Antifungal activity of laticifer proteins induces phytopathogenic fungi by latex proteolytic fractions.
oxidative stress Several lines of evidence link the presence of cysteine pro-
teinases with this inhibition. Accordingly, two puriWed cys-
Oxidative stress related to antifungal activity by CpLP or teine proteinase of latex origin from C. papaya (papain)
puriWed proteases (CMS2MS2 and papain) was investi- and Carica candamarcensis (CMS2MS2) were also active.
gated by estimating hydrogen peroxide production in It is therefore concluded that cysteine proteinases from
spores of F. solani. As seen in Fig. 4, spores exposed to latices of C. procera and C. candamarcensis are directly
BSA (protein control) or trypsin did not produce any dark associated with antifungal activity. On the other hand, the
brownish precipitate, indicating low levels of H2O2 in antifungal activity of C. procera on Neurospora sp. was
spores. However, spores of F. solani incubated with CpLP, not clearly associated with cysteine proteinases, because
papain or CMS2MS2 exhibited dark staining indicative of IAA-LP was able to inhibit fungal growth and spore germi-
peroxide production. Treatment of CpLP with IAA fully nation like native-LP suggesting the existence of additional
eliminated the oxidative damage of spores. Reactive oxy- mechanisms controlling fungal growth. This hypothesis is
gen species (ROS) are known mediators of intracellular sig- supported by the occurrence of other known defensive pro-
naling cascades. Over production of ROS may, however, teins indentiWed in latex. These results conWrm the involve-
lead to oxidative stress, loss of cell function and ultimately ment of cysteine proteinases in latex as a mechanism of
cell death by apoptosis or necrosis (Nordberg and Arner plant defense against phytopathogenic fungi.
2001). Here, we have shown that cysteine proteinases from
latex induced the generation of H2O2 in fungal spores of F. Acknowledgments Biochemical, functional and applied studies of
latex proteins were supported by grants from FUNCAP, CNPq,
solani. These Wnding is similar to the results previously
CAPES, RENORBIO and IFS (M.V.R.). Authors are in debit with
reported for an aspartic proteinase from Solanum tubero- Prof. José Tadeu Abreu de Oliveira which provided fungi sources for
sum (Mendieta et al. 2006). assays.

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