Osmotin from Calotropis procera latex: New insights into structure and antifungal properties

Biochimica et Biophysica Acta 1808 (2011) 2501–2507

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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a m e m

Osmotin from Calotropis procera latex: New insights into structure and antifungal properties Cleverson Diniz Teixeira de Freitas a,⁎, José Luiz de Souza Lopes b, Leila Maria Beltramini b, Raquel Sombra Basílio de Oliveira c, José Tadeu Abreu Oliveira c, Márcio Viana Ramos c,⁎ a b c

Departamento de Biologia, Universidade Federal do Piauí, Campus Amilcar Ferreira Sobral, BR 343, Km 3,5, Bairro Melladão, CEP 64800-000, Floriano-Piauí, Brazil Universidade de São Paulo, Instituto de Física de São Carlos, Grupo de Biofísica Molecular “Sérgio Mascarenhas”, São Paulo, Brazil Departamento de Bioquímica e Biologia Molecular da Universidade Federal do Ceará, Campus do Pici, Cx. Postal 6033, CEP 60451-970, Fortaleza-Ceará, Brazil

a r t i c l e

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Article history: Received 20 April 2011 Received in revised form 29 June 2011 Accepted 12 July 2011 Available online 23 July 2011 Keywords: Calcein Fusarium solani Laticifer protein Propidium iodide Thaumatin-like protein

a b s t r a c t This study aimed at investigating the structural properties and mechanisms of the antifungal action of CpOsm, a purified osmotin from Calotropis procera latex. Fluorescence and CD assays revealed that the CpOsm structure is highly stable, regardless of pH levels. Accordingly, CpOsm inhibited the spore germination of Fusarium solani in all pH ranges tested. The content of the secondary structure of CpOsm was estimated as follows: α-helix (20%), β-sheet (33%), turned (19%) and unordered (28%), RMSD 1%. CpOsm was stable at up to 75 °C, and thermal denaturation (Tm) was calculated to be 77.8 °C. This osmotin interacted with the negatively charged large unilamellar vesicles (LUVs) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-1glycerol (POPG), inducing vesicle permeabilization by the leakage of calcein. CpOsm induced the membrane permeabilization of spores and hyphae from Fusarium solani, allowing for propidium iodide uptake. These results show that CpOsm is a stable protein, and its antifungal activity involves membrane permeabilization, as property reported earlier for other osmotins and thaumatin-like proteins. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Phytopathogens, such as fungi and bacteria, are widely distributed in nature, and their infectious processes result in major losses of various economically important crops [1,2]. Although plants are constantly exposed to numerous pathogens, disease rarely leads to plant death [3]. This seems to be due to various defense mechanisms that plants have evolved, including the de novo synthesis of proteins that play crucial roles during the invasions and infections of different pathogens [4]. Proteins exhibiting distinct activities and displaying antimicrobial properties have been purified and characterized from different plants. Most of them are recognized as pathogenesis-related proteins (PR-proteins) and include chitinases [5], defensins [6], proteinase inhibitors [7], proteinases [8] and thaumatin-like proteins [9], among others. Osmotins have been implicated in osmotic adjustment or cryoprotection in plants [10,11]. However, some osmotins have been shown to participate in plant defenses against fungal infections [12,13]. These proteins belong to the PR-5 family and share high primary, secondary and tertiary structural similarities to thaumatin [14]. Osmotins and thaumatin-like proteins have been purified and characterized from different plant tissues, including laticifer fluids [15]. These proteins have molecular masses ranging from 20 up to 30 kDa and can be acidic,

neutral or basic [16]. Another important feature of osmotins is their high concentrations of cysteine residues, which are involved in various disulfide bonding [14]. Recently, an antifungal protein was purified from the latex of Calotropis procera [17]. The protein, named CpOsm, exhibited two isoforms with masses of 22,340 and 22,536 Da and 8.9 and 9.1 pI, respectively. CpOsm drastically reduced spore germination and inhibited the mycelial growth of the phytopathogens Fusarium solani, Neurospora sp. and Colletotrichum gloeosporioides. In this work, we report on new structural features of CpOsm and describe aspects of its antifungal mechanism. 2. Experimental 2.1. Materials 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-1-glycerol (POPG), 1,2- dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2distearoyl-sn-glycero-3-phosphoetanolamine (DSPE) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Calcein and propidium iodide, used as fluorescence probes, were purchased from Sigma (Brazil). Others chemicals were of analytical grade. 2.2. Latex and laticifer proteins

⁎ Corresponding authors. Tel.: +55 85 3366 9403; fax: +55 85 3366 9789. E-mail addresses: [email protected] (C.D.T. de Freitas), [email protected] (M.V. Ramos). 0005-2736/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamem.2011.07.014

The latex of Calotropis procera was collected from the aerial parts of the wild plants in tubes containing distilled water to give a dilution

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rate of 1:2 (v/v), and the protein fraction of the latex was obtained as previously described [18]. Native plants were located in the vicinity of Fortaleza-Ceará, Brazil. The plant voucher (sample specimen N.32663) was deposited at the Prisco Bezerra Herbarium of the Universidade Federal do Ceará, Brazil, where the botanical material was identified.

with average diameters of 100 nm were prepared in 25 mM Tris–HCl (pH 7.0) at a final concentration of 1 mM by extrusion through a polycarbonate filter 11 times. Fluorescence and CD spectra of CpOsm incubated with each LUV were recorded under the conditions described above.

2.3. Purification of CpOsm through ion exchange chromatography

2.7. Release of entrapped fluorescent marker from large unilamellar vesicles (LUVs)

CpOsm was purified strictly as previously described [17]. Briefly, laticifer proteins prepared in 50 mM Na-acetate buffer, pH 5.0 (5 mg/ml) were fractionated by ion exchange chromatography on a CM Sepharose fast flow column (Amersham Biosciences, Brazil). After dialysis and lyophilization, the fraction that had been eluted with 0.2 M NaCl (PII-CM) was subjected to an additional round of ion exchange chromatography on a Resource-S column (Amersham Biosciences, Brazil), previously equilibrated with 25 mM Na-phosphate buffer (pH 6.0) and assisted by a fast protein liquid chromatography (FPLC) system. The peak that eluted with 0.1 M NaCl (P2-R) represented CpOsm. The homogeneity of CpOsm was confirmed by polyacrylamide gel electrophoresis in the presence of SDS as described by Laemmli [19]. For the mass spectrometry analysis, CpOsm was dissolved in 1 ml 0.1% TFA, and an aliquot of 0.5 μl (0.5 μg) was mixed with 0.5 μl of sinapinic acid matrix and spotted onto a MALDI plate. A MALDI-TOF analysis in linear mode was performed with an ABI 4700 Proteomics Analyzer. 2.4. Steady-state fluorescence measurements The CpOsm fluorescence emission spectra were obtained from an ISS K2 spectrofluorimeter (ISS Fluorescence, Analytical and Biomedical Instruments, Illinois, USA), using a 1 cm path length rectangular quartz cuvette at 25 °C. CpOsm (5 μM) was excited at 280 and 295 nm, and the emission spectra were recorded from 280 to 450 nm. Reference spectra (buffers) were recorded and subtracted after each measurement. The structural stability of CpOsm at different pH levels was evaluated by incubating the protein for 30 min in each buffer; 25 mM glycine-HCl (pH 3.0), 25 mM Na-acetate (pH 5.0) and 25 mM Tris-HCl (pH 7.0 and 9.0).

The membrane-damaging activity was determined by measuring the release of the dye calcein from POPG LUVs [21]. POPG were dissolved in a 4:1 chloroform/methanol (v/v) mixture and dried with a N2 stream. Buffer (10 mM Tris–HCl pH 7.4, 150 mM NaCl, 0.1 mM EDTA and 35 mM calcein) was added to the film of lipids, and, after hydration, the suspension was shaken vigorously and freeze–thawed in liquid nitrogen for 10 cycles. LUV mixtures were submitted to chromatography on Sephadex G-50 columns (2 × 30 cm) to separate the vesicles from the free calcein. Vesicle suspensions were estimated by measuring the lipid phosphorus content [22], and a final phospholipid concentration of 100 μM was used for the calcein release assay. The kinetics of the membrane damage induced by CpOsm was monitored for 700 s by evaluating the increases in fluorescence emissions at 514 nm after excitation at 492 nm. The signals were expressed as percentages of total calcein released after the addition of 0.1% Triton X-100. Each experiment was performed twice, consisting of two replicates per treatment.

2.8. Preparation of spore suspension Fusarium solani was obtained from the local collection of the Microbiology Laboratory at the Department of Biology of the Federal University of Ceara and maintained on Sabouraud Dextrose Agar at 27 °C, with a 12 h light/dark cycle and 70% relative humidity. Homogenous spore suspensions were obtained from 2 to 3 week-old cultures in sterile distilled water and conidia were quantified with a Neubauer chamber to obtain an appropriate dilution of 2×106 spores/ml, using a BX60 Olympus Light Microscope.

2.5. Far-UV circular dichroism (CD) and estimation of the secondary structural content 2.9. Inhibition of spore germination assay CpOsm (10 μM) CD spectra were recorded with a JASCO J-715 spectropolarimeter (Jasco Instruments, Tokyo, Japan) at 190 to 250 nm as an average of 16 scans, using a 0.1 cm path length rectangular quartz cuvette at 25 °C. The CpOsm structural stability was investigated at different pH values as described above. A thermal denaturation analysis of CpOsm was performed after heating the protein gradually at pH 7.0 (in 5 °C steps) from 25 to 95 °C, using a circular TC-100 water bath (Jasco). To stress the involvement of disulfide bonds on CpOsm structural conformation CD spectrum was performed after the protein incubation (pH 7.0) for 30 min at 25 °C with 10 mM DTT (Dithiothreitol). The estimation of secondary structure elements was performed by CD spectrum deconvolution, using the CDPro package [20].

The possible inhibitory effect of purified CpOsm on spore germination was evaluated by mixing 10 μl of a spore suspension (2× 106/ml in water) with 10 μM of the CpOsm samples prepared in different buffers [25 mM glycine-HCl (pH 3.0), 25 mM Na-acetate (pH 5.0) and 25 mM Tris–HCl (pH 7.0 and 9.0)]. The plates were closed to avoid evaporation or contamination and were maintained for 24 h at 27 °C with a 12 h light/dark cycle and 70% relative humidity. Fungal growth was observed, using a BX60 Olympus Light Microscope. Spores were considered germinated if any hyphal structures were present. Each experiment was performed twice and consisted of two replicates per treatment.

2.10. Evaluation of membrane damage after CpOsm treatment 2.6. Vesicle preparation Large unilamellar vesicles (LUVs) of 1-palmitoyl-2-oleoyl-snglycero-3-phospho-rac-1-glycerol (POPG, negatively charged LUV), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC, zwitterionic LUV) and 1,2-distearoyl-sn-glycero-3-phosphoetanolamine (DSPE, zwitterionic LUV) were prepared by dissolving each phospholipid in a 4:1 chloroform/methanol (v/v) mixture and slowly evaporating the solvent under a N2 stream to yield a dry lipid film, that was run through a SpeedVac system for 2 h. Large unilamellar vesicles (LUVs)

Spores and hyphae (obtained after 24 h of germination) of Fusarium solani were incubated at 25 °C for 30 min in 25 mM Tris–HCl, pH 7.0 (controls) or CpOsm (10 μM). Propidium iodide was added at a final concentration of 1 mM, and uptake was observed after 30 min of incubation at 25 °C. Fluorescence was measured using a fluorescence measurement system (Olympus System Microscopy) at an excitation wavelength of 480 nm and an emission wavelength of 580 nm. Each experiment was performed twice, consisting of two replicates per treatment.

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3. Results and discussion

preparation was used for all further experiments. The relative abundance of each protein isoform into latex is difficult to measure and properly estimate. However, taking in account that both protein forms were co-purified by equal step by step, it is expected that their proportion were maintained throughout their purification. The relative abundance of each isoform was previously accessed by twodimensional electrophoresis [17]. The isoform with pI 8.9 represented about 35% and the isoform with pI 9.1 about 65%. The fact that a unique forty identical amino acid residues at N-terminus has been determined; almost identical tryptic peptide fingerprint and unique CD and Fluorescence spectra have been found for this protein sample support the hypothesis of two protein isoforms which strongly suggest a single protein in terms of activity and function. However, blast search osmotin genes show that osmotins are commonly coded by a family of genes. Thus under this view, it is also possible that the two osmotins described in this work are products of two distinct genes rather than products of post-translational events. This point remains to be determined.

3.1. CpOsm mass spectrum

3.2. Structure–activity relationships

The purity of CpOsm was confirmed by MALDI-MS/TOF as shown in Fig. 1. Both protein isoforms appeared as mono-charged peaks of 22,340.47 and 22,536.81 Da and a single double charged peak, corresponding to one-half of the mass (11189.78 Da). This preparation exhibited an identical electrophoresis pattern to that reported recently in a study evaluating osmotin purification [17]. This osmotin

After excitation at 280 and 295 nm, the fluorescence spectra of CpOsm showed λmax values of 337 and 338 nm, respectively (Fig. 2a–b). As observed in the figure, dissolved CpOsm at different pH conditions gave rise to identical spectra in terms of their maximum emissions, revealing the structural stability of the protein. Differences were seen in peak intensities, corresponding to exposures to different pH conditions.

Fig. 1. Mass spectrometry analysis of osmotin from Calotropis procera latex (CpOsm). MALDI-MS/TOF was used to determine the degree of purity and molecular mass of CpOsm. Sample was mixed with sinapinic acid (matrix) dissolved in 0.1% trifluoroacetic acid before analysis.

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Fig. 2. Effect of pH on conformation and antifungal activity of CpOsm. CpOsm (5 μM) was incubated at 25 °C for 30 min in different buffers and excited at 280 (a) or (b) 295 nm, and fluorescence spectra were determined. (c) Far-UV circular dichroism spectra of CpOsm (10 μM). (d) Antifungal activity of CpOsm (10 μM) on Fusarium solani spore germination. Bars: 30 μm. After 24 h, CpOsm completely inhibited spore germination while a dense web of hyphae is seen in controls.

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These alterations can be attributed to the protonation statuses of the side-chain residues or collisional quenching. Fluorescence spectroscopy is one of the most sensitive methods for studying changes in the conformations of proteins in solution, so the results observed with CpOsm suggest that the protein structure is maintained [23]. These results are in agreement with the behaviors of other osmotins and thaumatin-like proteins excited at 280 nm. The thaumatin-like protein from Carica papaya latex presented with a λmax fluorescence value of

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Wavelength (nm) Fig. 4. Effect of DTT on conformation of CpOsm. Far-UV circular dichroism spectra of native CpOsm at pH 7.0 (CpOsm), CpOsm in presence of 10 mM DTT (CpOsm + DTT) and CpOsm incubated with 10 mM DTT and dialyzed against Tris-buffer pH 7.0 (CpOsm Dialysed). Inset: Evidence of lost of the 232 nm peaks after DTT incubation.

342 nm at pH levels ranging from 2.0 to 10.0 [15]. Zeamatin, the osmotin from the corn of Zea mays, and thaumatin, a sweet-tasting protein purified from the fruit of Thaumatoccus daniellii, showed their maximum fluorescence emissions at 333 and 337 nm, respectively, when assayed at pH levels of 5.5, 7.5 and 9.5 [24]. CpOsm appeared to be in its native conformation, because its fluorescence peak was at 337–338 nm. When a protein is in its denatured state, it exposes its hydrophobic amino acids to the aqueous environment, characterized by a fluorescence peak between 355 and 360 nm, which is related to the maximum fluorescence of tryptophan in solution [23,25]. The CD spectrum of CpOsm showed a weakly positive band at 232 nm with a minimum band in the region of 212 nm and a positive maximum at 195 nm. This profile was seen independent of pH conditions (Fig. 2c). These bands are features commonly observed in the spectra of proteins with disulfide bonds and elevated concentrations of β-sheet segments. These results agree with previous structural information available regarding other osmotins and thaumatin-like proteins, such as those from C. papaya latex recorded at pH 5.2 and Z. mays and T. daniellii at pH 5.5 and 7.5 [15,24]. It is therefore expected that the CpOsm structure is established by disulfide bonds, although a unique cysteine residue (Cys-9) has been discovered in the first 40 amino acid residues of its N-terminal [17]. The secondary structure content of CpOsm (pH 7.0) was estimated, using the CDSSTR and CONTIN programs, and the following results were obtained: 20% α-helix, 33% β-sheet, 19% turned and 28% unordered, with 1% RMSD. These results are in agreement with those observed in osmotin or thaumatin-like proteins from Nicotiana tabacum, Zea mays and Lycopersicon esculentum, which are proteins with known crystallographic structures [26–28].

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Wavelength (nm) Fig. 5. Analysis of the interaction of CpOsm with different LUVs measured by fluorescence and circular dichroism. Fluorescence emission spectra of CpOsm (5 μM) excited at 280 (a) or 295 nm (b) and far-UV circular dichroism spectra (c) of CpOsm (10 μM) in presence of LUVs. POPG: 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-1-glycerol; DMPC: 1,2 dimyristoyl-sn-glycero-3-phosphocholine; DSPE: 1,2-distearoyl-sn-glycero-3-phosphoetanolamine.

Studies evaluating the thermal stability of CpOsm have provided additional information regarding the protein structure. As observed in Fig. 3a, CpOsm remained stable at temperatures up to 75 °C. The temperature of denaturation (Tm) was calculated to be 77.8 °C. It was calculated by the loss in ellipticity of the band at 205 nm (Fig. 3b). In another approach, after heating CpOsm from 25 up to 95 °C, in increments of 5 °C at 10 min intervals, the sample was reincubated at 25 °C for 20 min. A new CD spectrum that was

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recorded under similar conditions was comparable to that of native CpOsm (Fig. 3c). This data suggested that CpOsm partially reordered its structure. The thaumatin-like protein from the emperor banana (Musa basjoo) has been described as a highly stable protein [9]. This protein inhibited the hyphae growth of Myscosphaerella arachidicola after thermal treatment (70 °C) and was still active at pH 12.0. Similarly, the thaumatin-like protein from Castanopsis chinensis was active on M. arachidicola fungi after being heated to

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Fig. 7. Membrane permeabilization induced by CpOsm (10 μM in pH 7.0) in spores and hyphae of Fusarium solani. After 30 min incubation at 25 °C, fluorescence was detected, using fluorescence microscopy of propidium iodide uptake. Bars: 10 μm (spores) and 40 μm (hyphae).

60 °C [29]. CpOsm seems to exhibit considerable resistance to temperature changes, thus retaining its biological activity. A weakly positive band around 232 nm found in the CpOsm CD spectra (Fig. 2c) was also observed in the CD spectra of the thaumatinlike proteins from Carica papaya latex, zeamatin, and thaumatin [15,24]. As cited before, this spectrum shape is frequently associated with the presence of disulfide bonds in proteins [30]. Disulfide bonds are known to be stabilizers of protein structures. For instance, the thaumatin-like proteins from Carica papaya latex, zeamatin, and thaumatin have eight disulfide bridges [15,26]. The internal patterns of disulfide bonds seem to be very conserved structural characteristics of osmotins and thaumatin-like proteins, and it is therefore expected that they contribute to the high structural stabilities of these proteins [14]. The CD spectrum profile of CpOsm incubated with 10 mM DTT changed from the CpOsm native CD spectrum and decreased the intensity of the band at 232 nm (Fig. 4) probably due to disruption of the disulfide bonds. After the removal of the reducing agent by dialysis, the obtained CD spectrum was very similar to the CpOsm native spectrum, but the intensity of the band at 232 nm was not reestablished. The antifungal activity of CpOsm is dependent on its threedimensional structure, which is stabilized by disulfide bonds, shown by its loss of antifungal activity against F. solani, Neurospora sp. and Colletotrichum gloeosporioides when it was incubated with dithiothreitol (DTT) [17]. The suppressive effect of CpOsm on spore germination of phytopathogens has been demonstrated previously at pH 5.0 [17]. Here, F. solani was used as a model to determine the inhibitory activity of CpOsm at varying pH conditions. The inhibitory activity of CpOsm was similar at all pH ranges tested (3.0, 5.0, 7.0 and 9.0) (Fig. 2d). These results confirmed the high structural stability of CpOsm deduced from the fluorescence and CD data. The mechanism underlying the inhibitory activity of CpOsm on fungi spores was further investigated by studying its interaction with negatively charged large unilamellar vesicles (LUVs) using spectroscopic tools and by examining membrane damage of fungi spores exposed to CpOsm in presence of Propidium iodide. 3.3. CpOsm interacts with negatively charged LUVs and causes membrane permeabilization Osmotin and thaumatin-like proteins have been described as membrane-binding proteins. These interactions have been shown to cause membrane leakage in microorganisms and artificial membranes and have been generally considered to be the bases

of their toxicities [31]. To confirm the presence of CpOsm activity, the fluorescence and CD spectra of CpOsm were obtained after mixing the protein with large unilamellar vesicles (LUVs). The fluorescence emission spectra of CpOsm showed excitation at 280 and 295 nm and was measured either in 25 mM Tris–HCl, pH 7.0 or in the presence of LUVs as shown in Fig. 5a–b. As observed, the addition of the DMPC, DSPE and POPG LUVs did not change the shape of the CpOsm fluorescence spectra, despite the reductions in signal intensities. Thus, no structural changes to the protein were revealed. On the other hand, the CpOsm CD spectrum was considerably altered in the presence of POPG LUVs (Fig. 5c). Negatively charged phospholipids largely predominate in POPG vesicles, while DMPC and DSPE are zwitterionic vesicles. The CD spectrum of CpOsm mixed with POPG suggests the effective interactions of CpOsm with these vesicles. CpOsm was shown to be a basic protein with isoforms above pI 8.9 [17]. In agreement with this, at pH 7.0, CpOsm is positively charged, and its interaction with negative POPG LUVs is favored. Furthermore, CpOsm was shown to induce membrane permeabilization in POPG LUVs as demonstrated by the leakage of calcein from POPG vesicles in a dose-dependent manner (Fig. 6). CpOsm was able to cause 21% permeability in these vesicles at doses as low as 1 μM, equivalent to 22 μg/ml of protein. This concentration is almost 10-fold lower than estimates from the naturally occurring latex of Calotropis procera (216 μg/ml). At the highest dose tested (10 μM), CpOsm caused 59% calcein leakage. These results are compatible with those of data from the literature, since osmotins and other basic proteins, such as cytochrome C or polycations (poly-L-lysine), have been reported to induce leakage of intracellular contents [32]. The experiments with the POPG LUVs indicated that membrane disruption involves a step of electrostatic interaction between the CpOsm and negative phospholipid head groups on the membrane surface. Although the negative phospholipids are not the major component in the eukaryotic membrane, they can be organized in rafts-like domains in the membrane that can also lead to this interaction. In the Fusarium solani membrane, such electrostatic interaction is probably suggested to occur with the negatively charged phospholipid of phosphatidylinositol [33]. Several antifungal proteins with wide varieties of three-dimensional structures have been reported to act on the plasma membrane. However, they all have at least two characteristics in common: net positive charges under physiological conditions that promote interactions with the negatively charged surfaces of the plasma membranes of microorganisms and a neutral or hydrophobic structural region that allows for the formation of pores [34–36].

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3.4. CpOsm induces membrane permeabilization in vivo In the presence of CpOsm, the uptake of the fluorogenic dye propidium iodide was observed in both the hyphae and the spores of F. solani (Fig. 7). Propidium iodide only penetrates cells that have damaged plasma membranes, and the fluorescence observed is a consequence of DNA-dye binding. As shown in Fig. 7, no fluorescent spores or hyphae were observed in the absence of CpOsm (control), while intense fluorescence was seen in the presence of 10 μM CpOsm. These results indicate that CpOsm induced membrane permeabilization in the spores and hyphae of F. solani. As previously reported, osmotin purified from NaCl-adapted tobacco cells caused cellular disruption and leakage of the intracellular contents of Saccharomyces cerevisiae when tested at 4 μM [31]. Additionally, they showed that tobacco osmotin induced apoptosis by the accumulation of reactive oxygen species in S. cerevisiae cells. CpOsm had no observable effects on rabbit erythrocytes or human peripheral mononuclear blood cells, suggesting a certain degree of specificity (data not shown). The osmotin purified from the latex of Calotropis procera is a constitutive laticifer protein that is involved in phytopathogen defense indicating the defensive role of latex in plants. Acknowledgements Biochemical, functional and applied studies of the latex from Calotropis procera have been supported by grants from FUNCAP, CNPq (Universal, RENORBIO and Brazil/India cooperation) and IFS (M.V.R.). References [1] G.N. Agrios, Plant Pathology, fourth ed. Academic Press, San Diego, 1997. [2] R.B. Ferreira, S. Monteiro, R. Freitas, C.N. Santos, Z. Chen, L.M. Batista, J. Duarte, A. Borges, A.R. Teixeira, The role of plant defense proteins in fungal pathogenesis, Mol. Plant Pathol. 8 (2007) 677–700. [3] P. Tiffin, D. Moeller, Molecular evolution of plant immune system genes, Trends Genet. 22 (2006) 662–670. [4] L.C. van Loon, M. Rep, C.M.J. Pieterse, Significance of inducible defense-related proteins in infected plants, Annu. Rev. Phytopathol. 44 (2006) 135–162. [5] A.K. Patel, V.K. Singh, R.P. Yadav, A.J. Moir, M.V. Jagannadham, ICChI, a glycosylated chitinase from the latex of Ipomoea carnea, Phytochemistry 70 (2009) 1210–1216. [6] J.H. Wong, T.B. Ng, Sesquin, a potent defensin-like antimicrobial peptide from ground beans with inhibitory activities toward tumor cells and HIV-1 reverse transcriptase, Peptides 26 (2005) 1120–1126. [7] J.L.S. Lopes, N.F. Valadares, D.I. Moraes, J.C. Rosa, H.S.S. Araújo, L.M. Beltramini, Physico-chemical and antifungal properties of protease inhibitors from Acacia plumosa, Phytochemistry 70 (2009) 871–879. [8] D.P. Souza, C.D.T. Freitas, D.A. Pereira, F.C. Nogueira, F.D.A. Silva, C.E. Salas, M.V. Ramos, Laticifer proteins play a defensive role against hemibiotrophic and necrotrophic phytophatogens, Planta 234 (2011) 183–193. [9] V.S.M. Ho, J.H. Wong, T.B. Ng, A thaumatin-like antifungal protein from the emperor banana, Peptides 28 (2007) 760–766. [10] S.S. Newton, J.G. Duman, An osmotin-like cryoprotective protein from the bittersweet nightshade Solanum dulcamara, Plant Mol. Biol. 44 (2000) 581–589. [11] M. Onishi, H. Tachi, T. Kojima, M. Shiraiwa, H. Takahara, Molecular cloning and characterization of a novel salt-inducible gene encoding an acidic isoform of PR-5 protein in soybean (Glycine max [L.] Merr), Plant Physiol. Biochem. 44 (2006) 574–580. [12] M.V. Rajam, N. Chandola, P.S. Goud, D. Singh, V. Kashyap, M.L. Choudhary, D. Sihachakr, Thaumatin gene confers resistance to fungal pathogens as well as

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