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Conidiogenic effects of mannose-binding lectins isolated from cotyledons of red kidney bean (Phaseolus vulgaris) on Alternaria alternata
Phytochemistry 72 (2011) 94–99
Contents lists available at ScienceDirect
Phytochemistry journal homepage: www.elsevier.com/locate/phytochem
Conidiogenic effects of mannose-binding lectins isolated from cotyledons of red kidney bean (Phaseolus vulgaris) on Alternaria alternata Hossein Alizadeh a,b, David W.M. Leung a,⇑, Anthony L.J. Cole a a b
School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch 8140, New Zealand Department of Plant Protection, Aboureyhan Pardis, University of Tehran, Tehran, Iran
a r t i c l e
i n f o
Article history: Received 3 August 2010 Received in revised form 1 November 2010 Available online 25 November 2010 Keywords: Brown spot Conidiation Lectins PvFRIL Sporulation
a b s t r a c t Effect of proteinaceous extracts from red kidney bean cotyledons on mycelium of Alternaria alternata growing on potato dextrose agar (PDA) plates was investigated. Unexpectedly, conidia formation was induced in response to applied crude extracts. A PDA disc method was developed to quantify conidia formed. A purified fraction retaining conidiation inducing effect (CIE) was obtained following several protein purification procedures including the last step of eluting bound proteins from an Affi-gel blue gel column. Based on MALDI (matrix assisted laser desorption/ionization) mass spectrometric analysis, a previously identified mannose-binding lectin (MBL) called PvFRIL (Phaseolus vulgaris fetal liver tyrosine kinase 3-receptor interacting lectin) was present in this conidiation inducing fraction. The PvFRIL was subsequently purified using a single step mannose–agarose affinity column chromatography. When the lectin was applied exogenously to A. alternata, increased conidiation resulted. The conidia produced in response to the MBL were similar to those induced by other methods and their germ tubes were longer after 12 h growth than those induced under white light. To our knowledge this is the first report of exogenous application of a PvFRIL or another purified protein from a plant inducing conidia formation in a fungus. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Leguminous seeds are a good source of bioactive proteins (Reynoso-Comacho et al., 2006; Sridhar and Bhat, 2007; Leung and Alizadeh, 2009). These include different lectins that can recognize and reversibly bind to specific sugars including mannose, galactose and N-acetylglucosamine of glycolipids, glycoproteins and other glycoconjugates on surfaces of cells (Pusztai et al., 1993; Lei and Chang, 2009). While the physiological roles of plant lectins are still not clear (Van Damme et al., 2008), their effects on other organisms have been widely investigated (Vasconcelos and Oliveira, 2004; Van Damme et al., 2008). In general, plant lectins may be antiviral, antibacterial, antifungal, anti-insect and even poisonous to higher animals including humans (Cowan, 1999; Ng, 2004; Kaur et al., 2006; Singh et al., 2006; Keyaerts et al., 2007; Sitohy et al., 2007). In contrast to these frequently-reported activities of plant lectins associated with biological defence in nature, a mannose-binding lectin (MBL) called PvFRIL isolated from Phaseolus vulgaris (red kidney bean seed) has been shown to be able to preserve the viability of human progenitor cells and prevent their differentiation
⇑ Corresponding author. Tel.: +64 3 364 2650; fax: +64 3 364 2590. E-mail address: [email protected] (D.W.M. Leung). 0031-9422/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2010.11.002
and proliferation (Moore et al., 2000). Furthermore, the FRIL family proteins including the PvFRIL of red kidney bean and other legumes such as hyacinth bean (Dolichos lablab) could be of additional medical importance for treating inflammation-related diseases (Dinarello and Moore, 2006). Some filamentous fungi are important agents of disease and others can be used as biocontrol agents, biotransformation catalysts or in food and pharmaceutical industries (Alexopoulos et al., 1996). For instance, different strains of Alternaria alternata have been used as biological control agents against insects and Plasmopara viticola, a pathogen causing downy mildew on grapevine (Hatzipapas et al., 2002; Musetti et al., 2006). Moreover, A. alternata f.sp. sphenocleae has been reported as a useful biological control agent for gooseweed (Sphenoclea zeylanica) (Masangkay et al., 2000). Spore production is also useful for identification and for experimentation but sometimes conidiation is a problem. A. alternata, an important pathogen from citrus, is an example of a fungus for which spore production has been problematic (Carvalho et al., 2008). Therefore, it is of great interest to find new practical means to induce fungal conidiation in vitro. Conidiation in fungi may be triggered by a variety of environmental factors including nutrient deprivation, osmotic stress, moisture stress, specific volatile organic chemicals, ozone and near ultra violet light (NUV) (Pascual et al., 1998; Masangkay et al., 2000; Yoshida and shirata, 2000; Chovanec et al., 2001;
H. Alizadeh et al. / Phytochemistry 72 (2011) 94–99
Mills et al., 2004; Carvalho et al., 2008; Nemcovic et al., 2008; Antony-Babu and Singleton, 2009). To date, mulberry leaf extracts containing biotin have been shown to induce conidiation in a fungus (Yoshida and Shirata, 2000). However, it is generally not known if purified plant proteins have conidiation inducing activity. Here we report investigations of application of protein extracts of red kidney bean cotyledons exhibiting bioactivity effectively on A. alternata. It was concluded that red kidney bean MBLs, particularly PvFRIL, possessed the ability to induce conidiation in an A. alternata isolate.
2. Results 2.1. CIE of MBLs isolated from red kidney bean cotyledons In preliminary investigations, we observed, besides a growth inhibition crescent, darkening of the periphery or growing region of mycelium of A. alternata in the path of diffusing red kidney bean cotyledonary extract from a paper disc placed on the surface of a potato dextrose agar (PDA) plate (Fig. 1A). This darkening of mycelium did not occur in the absence of the extract but it was similar to that in the older part (the central part) of the mycelium which had the ability to form conidia. Closer inspection of mycelium removed from the vicinity of the interface between the mycelium and a paper disc with crude red kidney bean extract under a light microscope (Fig. 1A) showed the observed darkening was due to the induction of conidia at the mycelial periphery (Fig. 1B). Further investigations are the focus of the present report. The conventional PDA plate method of culturing fungal mycelium was useful for visual detection of conidiation (Fig. 1A) but it was not suitable for the purpose of quantitative determination of the number of conidia formed in response to application of a plant extract or protein solution. A variant of this method involving cutting out discs of growing mycelium from a PDA plate before transferring to a fresh Petri plate containing sterile paper strips impregnated with a test solution was also not useful to investigate induction of conidiation as wounding itself appeared to increase conidiation of A. alternata. Therefore, we used a small PDA disc (1.5 cm in diameter) which was essentially the mini version of the conventional PDA plate method to culture fungal mycelium to enable quantitative recovery and determination of conidia formed in the present study (Fig. 1B). The crude cotyledonary extracts induced conidia formation in a concentration-dependent manner (Fig. 2). To identify the bean proteins possessing this previously unknown bioactive property, crude extracts of bean cotyledons were purified through several protein purification steps. In the final purification step, bound proteins were eluted from an Affi-gel blue column. This purified fraction still retained CIE on A. alternata and was resolved into three protein bands with apparent molecular masses of 31, 21 and 14 kDa after SDS–PAGE and staining with Coomassie brilliant blue (Fig. 3). When the respective bands were analyzed using MALDI TOF/TOF, the 31 kDa band was identified as PHA-L (score 821) and the 14 and 21 kDa ones were identified as MBLs which were also known as the FRIL of P. vulgaris or PvFRIL (scores 579 and 713, respectively and the mass spectra data shown in Fig. 4). Another commercially purified lectin from red kidney bean, PHA-L did not increase conidiation of A. alternata (result not shown). A well characterized and commercially prepared MBL from Jack bean (Canavalia ensiformis), the concanavalin A type VI, used at five times the concentration of MBL from red kidney bean also had no CIE (result not shown). Another FRIL from hyacinth bean (kindly supplied by Dr. F. Apone, Arterra Bioscience, Italy) used at a similar concentration as PvFRIL had no CIE on A. alternata (unpublished results).
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Subsequently, MBLs eluted from mannose–agarose column chromatography of crude extracts of red kidney bean cotyledons were confirmed to have, in a dose-dependent manner, CIE on A. alternata compared to the control without addition of any bean protein (Figs. 1A and 2). Interestingly, the isolated MBLs did not cause growth inhibition in the mycelia periphery (Fig. 1A).
2.2. Effect of MBLs on conidia germination The germination ability of the conidia produced in response to white light control, or to addition of crude bean cotyledon extract, or to isolated bean MBLs was not significantly different (Fig. 5). However, germ tubes were significantly longer from conidia induced by addition of 20 lg of MBLs isolated from red kidney bean cotyledons than those induced under white light control (Fig. 6). Addition of crude extract containing 20 lg soluble protein resulted in shorter germ tubes than the control (Fig. 6).
3. Discussion Previous investigations about inducing factors of conidiation have been mainly limited to food sources, media, and light wavelengths (Alexopoulos et al., 1996). Proteins in the crude cotyledonary extracts were implicated in a preliminary experiment to be responsible for the observed CIE because overnight incubation with papain effectively abolished this effect (Alizadeh et al., unpublished results). In the present study, it was revealed that a previously identified MBL, the PvFRIL (Moore et al., 2000) from red kidney bean seed, could also increase conidiation of a fungus. This is the first report on the effect of a purified protein of another organism on conidiation in a fungus. The PHA of red kidney bean, FRIL of hyacinth bean and concanavalin A of Jack bean did not induce conidiation of A. alternata, suggesting specificity of PvFRIL to increase conidiation of A. alternata. Further studies are required to determine the range of fungi that the MBLs from red kidney bean cotyledons can induce conidiation. It is possible that there might be proteins from a variety of sources that have CIE on A. alternata and other fungi including those that might be recalcitrant to other conidiation inducing factors. Exogenous application of a conidiation inducing protein is a new tool for induction of conidiation. With this it might be possible to aid taxonomy of cryptic fungi. It could also be possible to produce conidia on a large scale for fungal species that have medical importance, or potentials as biological control agents of agricultural pests or weeds. The conidia formed in response to PvFRIL treatment did not appear to differ from those induced under other conditions tested as far as conidia germination was concerned. However, the length of germ tubes from MBL-induced conidia was significantly longer than those induced under other conditions. In the treatment with crude extracts of red kidney bean cotyledons, there might be an antifungal protein present together with MBL and the former could have inhibited elongation of germ tubes. In comparison with white light, the result suggested that conidia produced in cultures grown in the presence of added MBL were more vigorous as far as growth of germ tubes was concerned. The physiological basis for this difference is intriguing and worthy of further investigations. The mechanism whereby applied PvFRIL can increase conidiation remains to be determined. It will also be of interest to determine whether this new approach to experimentally induce conidia formation involves similar or distinct underpinning cellular and molecular mechanisms compared to other conidia induction treatments.
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Fig. 1. (A) Effects on conidiation of crude extract or mannose-binding lectins, each with 20 lg of soluble proteins, prepared from red kidney bean cotyledons and applied to paper discs compared with buffer (10 mM Tris–HCl); (B) a PDA disc method to study conidiation of Alternaria alternata. Crude extract of red kidney bean cotyledons or buffer only as control applied to a paper strip was placed closed to the growing mycelium on the same agar disc. Following incubation in the dark at 26 °C overnight, mycelium in the vicinity of the paper strip was removed and viewed under a light microscope (shown in insets; magnification = 100).
Transition from vegetative growth to conidiation of many fungi is often accompanied by an increase in production of secondary metabolites such as melanin which was likely to be at least partially responsible for the darkening appearance of the mycelium (Calvo et al., 2002). As shown in this report, however, it might be possible to uncover additional conidiation factors, including proteins, that have been previously unrecognized in plant extracts.
4. Experimental 4.1. Protein purification and isolation of MBLs In an initial experiment, crude extracts were purified in a sequence of steps using ammonium sulfate (Sigma) fractionation, DEAE-cellulose (Sigma) column chromatography and Affi-gel blue gel (Bio-Rad, Hercules, USA) column chromatography as previously
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using 0.2 M D-mannose (Sigma). The eluted lectins were freeze-dried, re-dissolved in 10 mM Tris buffer (pH 7.3) and dialyzed against the same buffer at 4 °C overnight. After this, they were evaluated for CIE. 4.2. Evaluation of crude extracts and lectins on conidiation of A. alternata
Fig. 2. Effect of crude extract (A – 20, B – 10, and C – 5 lg of soluble proteins) and mannose-binding lectins (D – 20, E – 10, F – 5 lg, isolated from red kidney bean cotyledons) and G, buffer on conidiation of Alternaria alternata. Means ± S.E. (standard error) labeled with the same letter do not differ significantly according to Duncan’s MRT (P < 0.05).
A potato dextrose agar (PDA) disc method was developed in our lab to determine effect of lectins on conidiation. Discs of PDA (1.5 cm in diameter) were cut from a solidified PDA medium using a sterile test tube and transferred to a sterile Petri plate (four discs in each Petri plate). Three-day-old mycelium of A. alternata grown from an inoculum on a sterile PDA plate was used to generate inocula for conidiation experiments. A piece of growing mycelium (1 mm2) was removed from the periphery of the 3-day-old mycelium and placed on a PDA disc. The fungus was cultured on one side of PDA discs for 2 days at 26 °C. Then sterile paper strips (3 10 mm) were also placed one each on PDA discs but one mm away from growing zone of the fungal mycelium. To each paper strip, 20 ll containing 5, 10 or 20 lg of lectins eluted from the mannose–agarose column, or crude extract containing 5, 10 or 20 lg of soluble protein prepared from red kidney bean cotyledons or 10 mM Tris buffer (pH 7.3) as control, was applied. Also in one experiment, commercially prepared PHA-L (Cat. No. L2769-2MG) and concanavalin (Cat. No. L7647-10 mg) purchased from Sigma were applied. The Petri plates containing the PDA discs were wrapped with aluminium foil and transferred to a dark room at 26 °C. After 24 h of incubation, each of the PDA discs was transferred to five ml of a washing solution containing 0.025% Tween 20 and 0.8 M NaCl and then sonicated to isolate conidia (Nemcovic et al., 2008), which were counted using a hemacytometer. Following conidia induction, mycelia materials in the vicinity of the applied test solution were picked up with the aid of a piece of cellophane tape which was viewed under a light microscope to confirm the presence or absence of conidia and their external appearance. 4.3. Conidium germination assay
Fig. 3. Red kidney bean cotyledon proteins (12 lg) from Affi-gel blue gel column chromatography were fractionated using SDS–PAGE with molecular mass standards.
described (Alizadeh et al., 2010). Mannose–agarose column (Sigma, St. Louis, USA) was subsequently used to purify MBLs from red kidney bean cotyledons according to Moore et al. (2000) for confirmation of the initial finding suggesting that MBLs might have CIE. Briefly, cotyledons of red kidney bean (100 g) were ground in 50 mM Tris/HCl buffer (400 ml) pH 8.0 containing 1 mM each of CaCl2 and MgCl2 and centrifuged at 10,000g for 10 min at 4 °C. The pH of the supernatant was adjusted to pH 4.0 with HCl and after another centrifugation the supernatant (referred to as crude extract) was readjusted to pH 8.0. The extract was stirred continuously with mannose–agarose overnight at 4 °C before this slurry was applied to a column. The mannose–agarose beads were washed with TBS (Tris buffered saline comprised of 10 mM Tris and 150 mM NaCl at pH 7.5) and the bound MBLs were eluted
Conidia obtained from different experimental treatments were used immediately for germination assay. They were suspended in sterile distilled water and then spread on glass microscope slides coated with a thin layer of PDA. The slides were placed in sterile Petri plates which were incubated at 26 °C for 12 h. There were four slides (one in a Petri plate) for each treatment. The experiment was carried out two times with similar results. The number of germinated conidia (those capable of producing a germ tube) were counted and the length of germ tubes of 25 randomly selected germinated conidia on a glass slide was measured under a light microscope. Conidia obtained from the fungus grown under white light were used as control. Germ tube length measurements were carried out in the following order, one slide at a time: the conidia grown from cultures treated with MBLs, crude extract, from white light control and then this order was repeated again until all the slides were examined. The slides were kept at 4 °C until germ tube lengths were measured. 4.4. Protein content Soluble proteins in crude extracts or fractions eluted from the mannose–agarose column were quantified using the principle of quantitative binding of proteins with the Coomassie brilliant blue dye (Bradford, 1976). Bovine serum albumin was used as a standard.
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Fig. 4. MALDI MS/MS mass spectra of the two polypeptides with apparent molecular masses of approximately 14 and 21 kDa as shown in Fig. 3.
Fig. 5. Germination of conidia formed in response to crude extract or mannosebinding lectins (20 lg) isolated from red kidney bean cotyledons or conidia from cultures grown under white light. Means ± S.E. labeled with the same letter do not differ significantly according to Duncan’s MRT (P < 0.05).
Fig. 6. Length of germ tube (lm) of conidia produced in response to crude extract or mannose-binding lectins (20 lg) isolated from red kidney bean cotyledons. Means ± S.E. labeled with the same letter do not differ significantly according to Duncan’s MRT (P < 0.05).
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4.5. Electrophoresis and molecular mass determination of MBLs The proteins eluted from the mannose–agarose column or the Affi-gel blue gel column were separated using SDS–PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) with a 12% resolving gel and a 4% stacking gel (Laemmli, 1970). SDS–PAGE gels were stained with the Coomassie brilliant blue dye. Pre-stained low range molecular mass protein markers (Bio-Rad, Catalog 161-0303) were also included in each gel. 4.6. Protein identification based on MALDI mass spectrometric analysis Individual bands were cut from a gel after SDS–PAGE and sent to the Center for Protein Research, Department of Biochemistry, Otago University (Dunedin, New Zealand) for MALDI tandem Time-of-Flight analysis (MALDI TOF/TOF, Applied Biosystemes, MA). The MS/MS data generated were searched against the UniProt/SWISS-PROT amino acid sequence database using the Mascot search engine (http://www.matrixscience.com). 4.7. Statistical analysis Data were subjected to analysis of variance. Mean values in each treatment (with at least three replications) were compared using Duncan’s Multiple Range test at 5% level of significance (Clewer and Scarisbrick, 2001). References Alexopoulos, C.J., Mims, C.W., Blackwell, M., 1996. Introductory Mycology, fourth ed. Wiley & Sons, New York. 869p. Alizadeh, H., Leung, D.W.M., Cole, A.L.J., 2010. Concurrent occurrence of a-amylase inhibitor and stimulator in red kidney bean seed: physiological implications. Biol. Plant. 54, 195–197. Antony-Babu, S., Singleton, I., 2009. Effect of ozone on spore germination, spore production and biomass production in two Aspergillus species. Antonie Van Leeuwenhoek 96, 413–422. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Calvo, A.M., Wilson, R.A., Bok, J.W., Keller, N.P., 2002. Relationship between secondary metabolism and fungal development. Microbiol. Mol. Biol. Rev. 66, 447–459. Carvalho, D.C., Alves, E., Batista, T.R.S., Camargos, R.B., Lopes, E.A.G.L., 2008. Comparison of methodologies for conidia production by Alternaria alternata from citrus. Braz. J. Microbiol. 39, 792–798. Chovanec, P., Hudecova, D., Varecka, L., 2001. Vegetative growth, aging and lightinduced conidiation of Trichoderma viride cultivated on different carbon sources. Folia Micrbiol. 46, 417–422. Clewer, A.G., Scarisbrick, D.H., 2001. Practical Statistics and Experimental Design for Plant and Crop Science. John Wiley & Sons LTD, Chichester, UK. Cowan, M.M., 1999. Plant products as antimicrobial agents. Clin. Microbiol. Rev. 12, 564–582.
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Dinarello, C.A., Moore, J.G., 2006. Use of FRIL proteins for reducing the production of pro-inflammatory cytokines. WIPO patent No. WO/2006/110577. Hatzipapas, P., Kalosaka, K., Dara, A., Christias, C., 2002. Spore germination and appressorium formation in the entomopathogenic Alternaria alternata. Mycol. Res. 106, 1349–1359. Kaur, M., Singh, K., Jai Rup, P., Kamboj, S.S., Saxena, A.K., Sharma, M., Bhagat, M., Sood, S.K., Singh, J., 2006. A tuber lectin from Arisaema jacquemontii Blume with anti-insect and anti-proliferative properties. J. Biochem. Mol. Biol. 39, 432–440. Keyaerts, E., Vijgen, L., Pannecouque, C., Van Damme, E., Peumanc, W., Egberink, H., Balzarini, J., Jan Ranst, M., 2007. Plant lectins are potent inhibitors of coronaviruses by interfering with two targets in the viral replication cycle. Antiviral Res. 75, 179–187. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Lei, H.Y., Chang, C.P., 2009. Lectin of concanavalin A as an anti-hepatoma therapeutic agent. J. Biomed. Sci. 16, 1–12. Leung, D.W.M., Alizadeh, H., 2009. Lessons from evolution of a-amylase inhibitor gene in common bean (Phaseolus vulgaris) for the public concern about genetic engineering of crop plants. Int. J. Sci. Soc. 1, 189–194. Masangkay, R.F., Paulitz, T.C., Hallet, S.G., Watson, A.K., 2000. Characterization of Alternaria alternata f.sp. sphenocleae. Biocont. Sci. Technol. 10, 385–397. Mills, A.A.S., Platt, H.W., Hurta, R.A.R., 2004. Effect of salt compounds on mycelial growth, sporulation and spore germination of various potato pathogens. Postharvest Biol. Technol. 34, 341–350. Moore, J.G., Fuchs, C.A., Hata, Y.S., Hicklin, D.J., Colucci, G., Chrispeels, M.J., Feldman, M., 2000. A new lectin in red kidney beans called PvFRIL stimulates proliferation of NIH 3T3 cells expressing the Flt3 receptor. Biochim. et Biophys. Acta 1475, 216–224. Musetti, R., Vecchione, A., Strinhger, L., Borselli, S., Zulini, L., Marzani, C., Ambrosio, M.D., Sanita di Toppi, L., Pertot, I., 2006. Inhibition of sporulation and ultrastructural alterations of grapevine downy mildew by the endophytic fungus Alternaria alternata. Disease Cont. Pest Manag. 96, 689–698. Nemcovic, M., Jakubikova, L., Viden, I., Farkas, V., 2008. Induction of conidiation by endogenous volatile compounds in Trichoderma spp.. FEMS Micobiol. Lett. 284, 231–236. Ng, T.B., 2004. Antifungal proteins and peptides of leguminous and non-leguminous origins. Peptides 25, 1215–1222. Pascual, S., Melgarejo, P., Magan, N., 1998. Induction of submerged conidiation of the biocontrol agent Penicillium oxalicum. Appl. Microbiol. Biotechnol. 48, 389– 392. Pusztai, A., Ewen, S.W., Grant, G., Brown, D.S., Stewart, J.C., Peumans, W.J., Van Damme, E.J., Bardocz, S., 1993. Antinutritive effects of wheat-germ agglutinin and other N-acetylglucosamine-specific lectins. Br. J. Nutrit. 70, 313–321. Reynoso-Comacho, R., Ramos-Gomez, M., Loarca-Pina, G., 2006. Bioactive components in common beans (Phaseolus vulgaris). In: Guevara-Gonzalez, R.G., Torres-Pacheco, I. (Eds.), Advances in Agricultural and Food Biotechnology. Research Signpost, Kerala, India, pp. 217–236. Singh, K., Kaur, M., Rup, P.J., Singh, J., 2006. Exploration for anti-insect properties of lectin from seeds of soybean (Glycine max) using Bactrocera cucurbitae as a model. Phytoparasitica 34, 463–473. Sitohy, M., Doheim, M., Badr, H., 2007. Isolation and characterization of a lectin with antifungal activity from Egyptian Pisum sativum seeds. Food Chem. 104, 971– 979. Sridhar, K.R., Bhat, R., 2007. Agrobotanical, nutritional and bioactive potential of unconventional legume–Mucuna. Livestock Res. Rural Develop. 19, 126. Van Damme, E.J.M., Lannoo, N., Peumans, W.J., 2008. Plant lectins. Adv. Bot. Res. 48, 109–209. Vasconcelos, I.M., Oliveira, J.T.A., 2004. Antinutritional properties of plant lectins. Toxicon 44, 385–403. Yoshida, S., Shirata, A., 2000. Biotin induces sporulation of mulberry anthracnose fungus, Colletotrichum dematium. J. General Plant Pathol. 66, 117–122.
Contents lists available at ScienceDirect
Phytochemistry journal homepage: www.elsevier.com/locate/phytochem
Conidiogenic effects of mannose-binding lectins isolated from cotyledons of red kidney bean (Phaseolus vulgaris) on Alternaria alternata Hossein Alizadeh a,b, David W.M. Leung a,⇑, Anthony L.J. Cole a a b
School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch 8140, New Zealand Department of Plant Protection, Aboureyhan Pardis, University of Tehran, Tehran, Iran
a r t i c l e
i n f o
Article history: Received 3 August 2010 Received in revised form 1 November 2010 Available online 25 November 2010 Keywords: Brown spot Conidiation Lectins PvFRIL Sporulation
a b s t r a c t Effect of proteinaceous extracts from red kidney bean cotyledons on mycelium of Alternaria alternata growing on potato dextrose agar (PDA) plates was investigated. Unexpectedly, conidia formation was induced in response to applied crude extracts. A PDA disc method was developed to quantify conidia formed. A purified fraction retaining conidiation inducing effect (CIE) was obtained following several protein purification procedures including the last step of eluting bound proteins from an Affi-gel blue gel column. Based on MALDI (matrix assisted laser desorption/ionization) mass spectrometric analysis, a previously identified mannose-binding lectin (MBL) called PvFRIL (Phaseolus vulgaris fetal liver tyrosine kinase 3-receptor interacting lectin) was present in this conidiation inducing fraction. The PvFRIL was subsequently purified using a single step mannose–agarose affinity column chromatography. When the lectin was applied exogenously to A. alternata, increased conidiation resulted. The conidia produced in response to the MBL were similar to those induced by other methods and their germ tubes were longer after 12 h growth than those induced under white light. To our knowledge this is the first report of exogenous application of a PvFRIL or another purified protein from a plant inducing conidia formation in a fungus. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Leguminous seeds are a good source of bioactive proteins (Reynoso-Comacho et al., 2006; Sridhar and Bhat, 2007; Leung and Alizadeh, 2009). These include different lectins that can recognize and reversibly bind to specific sugars including mannose, galactose and N-acetylglucosamine of glycolipids, glycoproteins and other glycoconjugates on surfaces of cells (Pusztai et al., 1993; Lei and Chang, 2009). While the physiological roles of plant lectins are still not clear (Van Damme et al., 2008), their effects on other organisms have been widely investigated (Vasconcelos and Oliveira, 2004; Van Damme et al., 2008). In general, plant lectins may be antiviral, antibacterial, antifungal, anti-insect and even poisonous to higher animals including humans (Cowan, 1999; Ng, 2004; Kaur et al., 2006; Singh et al., 2006; Keyaerts et al., 2007; Sitohy et al., 2007). In contrast to these frequently-reported activities of plant lectins associated with biological defence in nature, a mannose-binding lectin (MBL) called PvFRIL isolated from Phaseolus vulgaris (red kidney bean seed) has been shown to be able to preserve the viability of human progenitor cells and prevent their differentiation
⇑ Corresponding author. Tel.: +64 3 364 2650; fax: +64 3 364 2590. E-mail address: [email protected] (D.W.M. Leung). 0031-9422/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2010.11.002
and proliferation (Moore et al., 2000). Furthermore, the FRIL family proteins including the PvFRIL of red kidney bean and other legumes such as hyacinth bean (Dolichos lablab) could be of additional medical importance for treating inflammation-related diseases (Dinarello and Moore, 2006). Some filamentous fungi are important agents of disease and others can be used as biocontrol agents, biotransformation catalysts or in food and pharmaceutical industries (Alexopoulos et al., 1996). For instance, different strains of Alternaria alternata have been used as biological control agents against insects and Plasmopara viticola, a pathogen causing downy mildew on grapevine (Hatzipapas et al., 2002; Musetti et al., 2006). Moreover, A. alternata f.sp. sphenocleae has been reported as a useful biological control agent for gooseweed (Sphenoclea zeylanica) (Masangkay et al., 2000). Spore production is also useful for identification and for experimentation but sometimes conidiation is a problem. A. alternata, an important pathogen from citrus, is an example of a fungus for which spore production has been problematic (Carvalho et al., 2008). Therefore, it is of great interest to find new practical means to induce fungal conidiation in vitro. Conidiation in fungi may be triggered by a variety of environmental factors including nutrient deprivation, osmotic stress, moisture stress, specific volatile organic chemicals, ozone and near ultra violet light (NUV) (Pascual et al., 1998; Masangkay et al., 2000; Yoshida and shirata, 2000; Chovanec et al., 2001;
H. Alizadeh et al. / Phytochemistry 72 (2011) 94–99
Mills et al., 2004; Carvalho et al., 2008; Nemcovic et al., 2008; Antony-Babu and Singleton, 2009). To date, mulberry leaf extracts containing biotin have been shown to induce conidiation in a fungus (Yoshida and Shirata, 2000). However, it is generally not known if purified plant proteins have conidiation inducing activity. Here we report investigations of application of protein extracts of red kidney bean cotyledons exhibiting bioactivity effectively on A. alternata. It was concluded that red kidney bean MBLs, particularly PvFRIL, possessed the ability to induce conidiation in an A. alternata isolate.
2. Results 2.1. CIE of MBLs isolated from red kidney bean cotyledons In preliminary investigations, we observed, besides a growth inhibition crescent, darkening of the periphery or growing region of mycelium of A. alternata in the path of diffusing red kidney bean cotyledonary extract from a paper disc placed on the surface of a potato dextrose agar (PDA) plate (Fig. 1A). This darkening of mycelium did not occur in the absence of the extract but it was similar to that in the older part (the central part) of the mycelium which had the ability to form conidia. Closer inspection of mycelium removed from the vicinity of the interface between the mycelium and a paper disc with crude red kidney bean extract under a light microscope (Fig. 1A) showed the observed darkening was due to the induction of conidia at the mycelial periphery (Fig. 1B). Further investigations are the focus of the present report. The conventional PDA plate method of culturing fungal mycelium was useful for visual detection of conidiation (Fig. 1A) but it was not suitable for the purpose of quantitative determination of the number of conidia formed in response to application of a plant extract or protein solution. A variant of this method involving cutting out discs of growing mycelium from a PDA plate before transferring to a fresh Petri plate containing sterile paper strips impregnated with a test solution was also not useful to investigate induction of conidiation as wounding itself appeared to increase conidiation of A. alternata. Therefore, we used a small PDA disc (1.5 cm in diameter) which was essentially the mini version of the conventional PDA plate method to culture fungal mycelium to enable quantitative recovery and determination of conidia formed in the present study (Fig. 1B). The crude cotyledonary extracts induced conidia formation in a concentration-dependent manner (Fig. 2). To identify the bean proteins possessing this previously unknown bioactive property, crude extracts of bean cotyledons were purified through several protein purification steps. In the final purification step, bound proteins were eluted from an Affi-gel blue column. This purified fraction still retained CIE on A. alternata and was resolved into three protein bands with apparent molecular masses of 31, 21 and 14 kDa after SDS–PAGE and staining with Coomassie brilliant blue (Fig. 3). When the respective bands were analyzed using MALDI TOF/TOF, the 31 kDa band was identified as PHA-L (score 821) and the 14 and 21 kDa ones were identified as MBLs which were also known as the FRIL of P. vulgaris or PvFRIL (scores 579 and 713, respectively and the mass spectra data shown in Fig. 4). Another commercially purified lectin from red kidney bean, PHA-L did not increase conidiation of A. alternata (result not shown). A well characterized and commercially prepared MBL from Jack bean (Canavalia ensiformis), the concanavalin A type VI, used at five times the concentration of MBL from red kidney bean also had no CIE (result not shown). Another FRIL from hyacinth bean (kindly supplied by Dr. F. Apone, Arterra Bioscience, Italy) used at a similar concentration as PvFRIL had no CIE on A. alternata (unpublished results).
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Subsequently, MBLs eluted from mannose–agarose column chromatography of crude extracts of red kidney bean cotyledons were confirmed to have, in a dose-dependent manner, CIE on A. alternata compared to the control without addition of any bean protein (Figs. 1A and 2). Interestingly, the isolated MBLs did not cause growth inhibition in the mycelia periphery (Fig. 1A).
2.2. Effect of MBLs on conidia germination The germination ability of the conidia produced in response to white light control, or to addition of crude bean cotyledon extract, or to isolated bean MBLs was not significantly different (Fig. 5). However, germ tubes were significantly longer from conidia induced by addition of 20 lg of MBLs isolated from red kidney bean cotyledons than those induced under white light control (Fig. 6). Addition of crude extract containing 20 lg soluble protein resulted in shorter germ tubes than the control (Fig. 6).
3. Discussion Previous investigations about inducing factors of conidiation have been mainly limited to food sources, media, and light wavelengths (Alexopoulos et al., 1996). Proteins in the crude cotyledonary extracts were implicated in a preliminary experiment to be responsible for the observed CIE because overnight incubation with papain effectively abolished this effect (Alizadeh et al., unpublished results). In the present study, it was revealed that a previously identified MBL, the PvFRIL (Moore et al., 2000) from red kidney bean seed, could also increase conidiation of a fungus. This is the first report on the effect of a purified protein of another organism on conidiation in a fungus. The PHA of red kidney bean, FRIL of hyacinth bean and concanavalin A of Jack bean did not induce conidiation of A. alternata, suggesting specificity of PvFRIL to increase conidiation of A. alternata. Further studies are required to determine the range of fungi that the MBLs from red kidney bean cotyledons can induce conidiation. It is possible that there might be proteins from a variety of sources that have CIE on A. alternata and other fungi including those that might be recalcitrant to other conidiation inducing factors. Exogenous application of a conidiation inducing protein is a new tool for induction of conidiation. With this it might be possible to aid taxonomy of cryptic fungi. It could also be possible to produce conidia on a large scale for fungal species that have medical importance, or potentials as biological control agents of agricultural pests or weeds. The conidia formed in response to PvFRIL treatment did not appear to differ from those induced under other conditions tested as far as conidia germination was concerned. However, the length of germ tubes from MBL-induced conidia was significantly longer than those induced under other conditions. In the treatment with crude extracts of red kidney bean cotyledons, there might be an antifungal protein present together with MBL and the former could have inhibited elongation of germ tubes. In comparison with white light, the result suggested that conidia produced in cultures grown in the presence of added MBL were more vigorous as far as growth of germ tubes was concerned. The physiological basis for this difference is intriguing and worthy of further investigations. The mechanism whereby applied PvFRIL can increase conidiation remains to be determined. It will also be of interest to determine whether this new approach to experimentally induce conidia formation involves similar or distinct underpinning cellular and molecular mechanisms compared to other conidia induction treatments.
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Fig. 1. (A) Effects on conidiation of crude extract or mannose-binding lectins, each with 20 lg of soluble proteins, prepared from red kidney bean cotyledons and applied to paper discs compared with buffer (10 mM Tris–HCl); (B) a PDA disc method to study conidiation of Alternaria alternata. Crude extract of red kidney bean cotyledons or buffer only as control applied to a paper strip was placed closed to the growing mycelium on the same agar disc. Following incubation in the dark at 26 °C overnight, mycelium in the vicinity of the paper strip was removed and viewed under a light microscope (shown in insets; magnification = 100).
Transition from vegetative growth to conidiation of many fungi is often accompanied by an increase in production of secondary metabolites such as melanin which was likely to be at least partially responsible for the darkening appearance of the mycelium (Calvo et al., 2002). As shown in this report, however, it might be possible to uncover additional conidiation factors, including proteins, that have been previously unrecognized in plant extracts.
4. Experimental 4.1. Protein purification and isolation of MBLs In an initial experiment, crude extracts were purified in a sequence of steps using ammonium sulfate (Sigma) fractionation, DEAE-cellulose (Sigma) column chromatography and Affi-gel blue gel (Bio-Rad, Hercules, USA) column chromatography as previously
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using 0.2 M D-mannose (Sigma). The eluted lectins were freeze-dried, re-dissolved in 10 mM Tris buffer (pH 7.3) and dialyzed against the same buffer at 4 °C overnight. After this, they were evaluated for CIE. 4.2. Evaluation of crude extracts and lectins on conidiation of A. alternata
Fig. 2. Effect of crude extract (A – 20, B – 10, and C – 5 lg of soluble proteins) and mannose-binding lectins (D – 20, E – 10, F – 5 lg, isolated from red kidney bean cotyledons) and G, buffer on conidiation of Alternaria alternata. Means ± S.E. (standard error) labeled with the same letter do not differ significantly according to Duncan’s MRT (P < 0.05).
A potato dextrose agar (PDA) disc method was developed in our lab to determine effect of lectins on conidiation. Discs of PDA (1.5 cm in diameter) were cut from a solidified PDA medium using a sterile test tube and transferred to a sterile Petri plate (four discs in each Petri plate). Three-day-old mycelium of A. alternata grown from an inoculum on a sterile PDA plate was used to generate inocula for conidiation experiments. A piece of growing mycelium (1 mm2) was removed from the periphery of the 3-day-old mycelium and placed on a PDA disc. The fungus was cultured on one side of PDA discs for 2 days at 26 °C. Then sterile paper strips (3 10 mm) were also placed one each on PDA discs but one mm away from growing zone of the fungal mycelium. To each paper strip, 20 ll containing 5, 10 or 20 lg of lectins eluted from the mannose–agarose column, or crude extract containing 5, 10 or 20 lg of soluble protein prepared from red kidney bean cotyledons or 10 mM Tris buffer (pH 7.3) as control, was applied. Also in one experiment, commercially prepared PHA-L (Cat. No. L2769-2MG) and concanavalin (Cat. No. L7647-10 mg) purchased from Sigma were applied. The Petri plates containing the PDA discs were wrapped with aluminium foil and transferred to a dark room at 26 °C. After 24 h of incubation, each of the PDA discs was transferred to five ml of a washing solution containing 0.025% Tween 20 and 0.8 M NaCl and then sonicated to isolate conidia (Nemcovic et al., 2008), which were counted using a hemacytometer. Following conidia induction, mycelia materials in the vicinity of the applied test solution were picked up with the aid of a piece of cellophane tape which was viewed under a light microscope to confirm the presence or absence of conidia and their external appearance. 4.3. Conidium germination assay
Fig. 3. Red kidney bean cotyledon proteins (12 lg) from Affi-gel blue gel column chromatography were fractionated using SDS–PAGE with molecular mass standards.
described (Alizadeh et al., 2010). Mannose–agarose column (Sigma, St. Louis, USA) was subsequently used to purify MBLs from red kidney bean cotyledons according to Moore et al. (2000) for confirmation of the initial finding suggesting that MBLs might have CIE. Briefly, cotyledons of red kidney bean (100 g) were ground in 50 mM Tris/HCl buffer (400 ml) pH 8.0 containing 1 mM each of CaCl2 and MgCl2 and centrifuged at 10,000g for 10 min at 4 °C. The pH of the supernatant was adjusted to pH 4.0 with HCl and after another centrifugation the supernatant (referred to as crude extract) was readjusted to pH 8.0. The extract was stirred continuously with mannose–agarose overnight at 4 °C before this slurry was applied to a column. The mannose–agarose beads were washed with TBS (Tris buffered saline comprised of 10 mM Tris and 150 mM NaCl at pH 7.5) and the bound MBLs were eluted
Conidia obtained from different experimental treatments were used immediately for germination assay. They were suspended in sterile distilled water and then spread on glass microscope slides coated with a thin layer of PDA. The slides were placed in sterile Petri plates which were incubated at 26 °C for 12 h. There were four slides (one in a Petri plate) for each treatment. The experiment was carried out two times with similar results. The number of germinated conidia (those capable of producing a germ tube) were counted and the length of germ tubes of 25 randomly selected germinated conidia on a glass slide was measured under a light microscope. Conidia obtained from the fungus grown under white light were used as control. Germ tube length measurements were carried out in the following order, one slide at a time: the conidia grown from cultures treated with MBLs, crude extract, from white light control and then this order was repeated again until all the slides were examined. The slides were kept at 4 °C until germ tube lengths were measured. 4.4. Protein content Soluble proteins in crude extracts or fractions eluted from the mannose–agarose column were quantified using the principle of quantitative binding of proteins with the Coomassie brilliant blue dye (Bradford, 1976). Bovine serum albumin was used as a standard.
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Fig. 4. MALDI MS/MS mass spectra of the two polypeptides with apparent molecular masses of approximately 14 and 21 kDa as shown in Fig. 3.
Fig. 5. Germination of conidia formed in response to crude extract or mannosebinding lectins (20 lg) isolated from red kidney bean cotyledons or conidia from cultures grown under white light. Means ± S.E. labeled with the same letter do not differ significantly according to Duncan’s MRT (P < 0.05).
Fig. 6. Length of germ tube (lm) of conidia produced in response to crude extract or mannose-binding lectins (20 lg) isolated from red kidney bean cotyledons. Means ± S.E. labeled with the same letter do not differ significantly according to Duncan’s MRT (P < 0.05).
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4.5. Electrophoresis and molecular mass determination of MBLs The proteins eluted from the mannose–agarose column or the Affi-gel blue gel column were separated using SDS–PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) with a 12% resolving gel and a 4% stacking gel (Laemmli, 1970). SDS–PAGE gels were stained with the Coomassie brilliant blue dye. Pre-stained low range molecular mass protein markers (Bio-Rad, Catalog 161-0303) were also included in each gel. 4.6. Protein identification based on MALDI mass spectrometric analysis Individual bands were cut from a gel after SDS–PAGE and sent to the Center for Protein Research, Department of Biochemistry, Otago University (Dunedin, New Zealand) for MALDI tandem Time-of-Flight analysis (MALDI TOF/TOF, Applied Biosystemes, MA). The MS/MS data generated were searched against the UniProt/SWISS-PROT amino acid sequence database using the Mascot search engine (http://www.matrixscience.com). 4.7. Statistical analysis Data were subjected to analysis of variance. Mean values in each treatment (with at least three replications) were compared using Duncan’s Multiple Range test at 5% level of significance (Clewer and Scarisbrick, 2001). References Alexopoulos, C.J., Mims, C.W., Blackwell, M., 1996. Introductory Mycology, fourth ed. Wiley & Sons, New York. 869p. Alizadeh, H., Leung, D.W.M., Cole, A.L.J., 2010. Concurrent occurrence of a-amylase inhibitor and stimulator in red kidney bean seed: physiological implications. Biol. Plant. 54, 195–197. Antony-Babu, S., Singleton, I., 2009. Effect of ozone on spore germination, spore production and biomass production in two Aspergillus species. Antonie Van Leeuwenhoek 96, 413–422. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Calvo, A.M., Wilson, R.A., Bok, J.W., Keller, N.P., 2002. Relationship between secondary metabolism and fungal development. Microbiol. Mol. Biol. Rev. 66, 447–459. Carvalho, D.C., Alves, E., Batista, T.R.S., Camargos, R.B., Lopes, E.A.G.L., 2008. Comparison of methodologies for conidia production by Alternaria alternata from citrus. Braz. J. Microbiol. 39, 792–798. Chovanec, P., Hudecova, D., Varecka, L., 2001. Vegetative growth, aging and lightinduced conidiation of Trichoderma viride cultivated on different carbon sources. Folia Micrbiol. 46, 417–422. Clewer, A.G., Scarisbrick, D.H., 2001. Practical Statistics and Experimental Design for Plant and Crop Science. John Wiley & Sons LTD, Chichester, UK. Cowan, M.M., 1999. Plant products as antimicrobial agents. Clin. Microbiol. Rev. 12, 564–582.
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