Plant Biochemistry

4.60 Plant Biochemistry: Antifungal Proteins Protecting Plants from Fungal Pathogens JH Wong and TB Ng, The Chinese University of Hong Kong, Hong Kong, China © 2011 Elsevier B.V. All rights reserved.

4.60.1 4.60.2 4.60.2.1 4.60.2.1.1 4.60.2.1.2 4.60.2.2 4.60.2.2.1 4.60.2.2.2 4.60.2.2.3 4.60.2.2.4 4.60.2.3 4.60.2.3.1 4.60.2.3.2 4.60.2.4 4.60.2.4.1 4.60.2.4.2 4.60.2.5 4.60.2.5.1 4.60.2.5.2 4.60.2.5.3 4.60.2.6 4.60.2.7 4.60.2.8 4.60.2.8.1 4.60.2.9 4.60.2.9.1 4.60.2.9.2 4.60.2.9.3 4.60.2.10 4.60.2.10.1 4.60.2.10.2 4.60.2.10.3 4.60.2.10.4 4.60.2.10.5 4.60.2.11 4.60.2.11.1 4.60.2.11.2 4.60.2.11.3 4.60.2.11.4 4.60.2.12 4.60.2.12.1 4.60.2.12.2 4.60.2.13 4.60.2.13.1 4.60.2.14 4.60.2.15 4.60.3 4.60.4 References

Introduction Leguminous Antifungal Proteins Peanut (Arachis hypogaea) Two class II chitinase genes and expression studies in transgenic tobacco plants Hypogin, an antifungal peptide with sequence similarity to peanut allergen Chickpea (Cicer arietinum) Thaumatin-like proteins Cyclophilin-like protein Cicerin and arietin, novel peptides Cicerarin, a novel antifungal peptide from the green chickpea Rice Bean (Delandia umbellata) Chitinase-like protein Antifungal peptide Field Bean (Dolichos lablab or Lablab purpuneus) Chitinase-like antifungal protein Inhibition of growth of Aspergillus flavus and fungal α-amylases by a lectin-like protein Soybean (Glycine max) Polygalacturonase-inhibiting protein New basic peroxidase cDNA from soybean hypocotyls infected with Phytophthora sojae f.sp. glycines Glysojanin from black soybean (G. soja) Defensin-like antifungal peptide Leucaena leucocephala chitinases Red Bean Peptides from red bean and pinto bean Mung Bean (Phaseolus mungo) Mung bean defensin Cyclophilin-like antifungal protein A nonspecific lipid transfer protein Phaseolus vulgaris The gene encoding EPGIP TLP from French bean legumes A hemagglutinin from red kidney bean Peroxidase from French bean Vulgin, antifungal polypeptide with mitogenic activity from pinto bean Pea (Pisum sativum) Defensins Miraculin-like protein from sugar snap P. sativum var. macrocarpon Beta-1,3-glucanase gene in pea (P. sativum) Pisumin, a novel antifungal protein from sugar snap pea P. sativum var macrocarpon Broad Beans (Vicia faba) Bowman–Birk-type trypsin–chymotrypsin inhibitors Fabin, a novel calcyon-like and glucanase-like protein with mitogenic, antifungal and translation-inhibitory activities Cow Pea (Vigna unguiculata) Cyclophilin-like protein Legume Vicilins (7S Storage Globulins) Inhibitory Effects of Antifungal Proteins on HIV-1 Reverse Transcriptase, Protease, and Integrase Nonleguminous Antifungal Proteins Conclusion and Future Perspectives

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Glossary chitinase Enzyme catalyzing the hydrolysis of chitin in fungal cell wall. cyclophilin-like proteins Proteins with sequence homology to cyclophilins. Cyclophilins catalyze cis–trans isomerization of amide bonds in peptides and proteins and may be involved in protein folding, signal transduction, trafficking, assembly, and cell-cycle regulation. Cyclophilins may be stress-related proteins. glucanases Proteins that hydrolyze glucans, a predominant component of fungal cell wall.

miraculin A sweet plant protein. polygalacturonase-inhibiting proteins Proteins that inhibit fungal polygalacturonases. thaumatin-like proteins (TLPs) Proteins with substantial sequence homology to the sweet protein thaumatin. TLPs have no sweet taste but possess antifungal activity, whereas thaumatin lacks antifungal activity. trypsin–chymotrypsin inhibitors Proteins that inhibit gut trypsin and chymotrypsin in phytophagous insects.

4.60.1 Introduction The seeds of plants, especially those of leguminous plants, are rich in proteins. Proteins such as lectins (Wong JH and Ng TB (2005)), [48] arcelins [12], protease inhibitors [71], α-amylase inhibitors [Ishimoto and Chrispeels, 1999 [20], [33], and ribosomeinactivating proteins [25] have been isolated from seeds. All of the aforementioned proteins are defense proteins, alternatively called pathogenesis-related proteins and antipathogenic proteins because they protect plants from predators such as insects and pathogenic microbes such as fungi, bacteria, and viruses. Some of these proteins are present in plant tissues at relatively high concentrations and play a role as storage proteins. In addition to the aforementioned proteins, plants also produce antifungal proteins. The objective of this article is to summarize recent literature on plant (leguminous and nonleguminous) antifungal proteins. As fungal invasion can produce devastating damage on crops resulting in huge economic losses, research on antifungal proteins has captured the attention of many researchers. Hopefully, transgenic plants expressing antifungal proteins will acquire resistance to pathogenic fungi. This article encompassed antifungal proteins of both leguminous and nonleguminous origins. These proteins can be easily isolated from seed extracts by using affinity chromatography, cation and anion exchange chromatography, and gel filtration.

4.60.2 Leguminous Antifungal Proteins Most of the leguminous antifungal proteins isolated in the author’s laboratory are adsorbed on Affi-gel blue gel, CMcellulose and Mono S, and unadsorbed on Diethylaminoethyl (DEAE)-cellulose. A typical isolation procedure consists of ion-exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion-exchange chromatogra­ phy on CM-cellulose or fast protein liquid chromatography (FPLC) on Mono S, followed by FPLC-gel filtration on Superdex 75. The molecular mass of the antifungal protein/peptide is estimated by gel filtration on a Superdex 75 or Superdex Peptide column, and by sodium dodecyl sulfate–polyacrylamide gel electrophoresis or tricine gel electrophoresis. Homogeneity of the antifungal protein/peptide is evidenced by a single electrophoretic band and a single amino acid detected in each round of the sequencing reaction.

4.60.2.1 4.60.2.1.1

Peanut (Arachis hypogaea) Two class II chitinase genes and expression studies in transgenic tobacco plants

Cloning of two different genes encoding class II chitinases from peanut (Arachis hypogaea L. cv. NC4), A.h.Chi2;1 and A.h.Chi2;2, has been accomplished. In peanut cell suspension cultures, the level of A.h.Chi2;2 messenger RNA (mRNA) was heightened after treatment with ethylene or salicylate and in the presence of Botrytis cinerea conidia. The gene A.h.Chi2;1 was only expressed following treatment with fungal spores. Transgenic tobacco plants harboring the complete peanut A.h.Chi2;1 gene demonstrated a similar expression pattern in leaves as observed in cell cultures [22].

4.60.2.1.2

Hypogin, an antifungal peptide with sequence similarity to peanut allergen

A protein designated as hypogin, with a marked suppressive action on mycelial growth in the fungi Mycosphaerella arachidicola, Fusarium oxysporum, and Coprinus comatus, was purified from seeds of the peanut A. hypogaea. The protein inhibited human immunodeficiency virus (HIV) reverse transcriptase and enzymes associated with HIV infection including α-glucosidase and β-glucosidase. The proliferative response of mouse splenocytes was attenuated after exposure to the protein. Hypogin exhibited a molecular mass of 7.2 kDa in tricine gel electrophoresis and gel filtration on Superdex 75 and an N-terminal sequence homologous to peanut allergen Ara H1. The purification protocol encompassed affinity chromatography on Affi-gel blue gel and ion-exchange chromatography on CM-Sepharose. The protein was adsorbed on both chromatographic media [51].

Plant Biochemistry: Antifungal Proteins Protecting Plants from Fungal Pathogens

4.60.2.2 4.60.2.2.1

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Chickpea (Cicer arietinum) Thaumatin-like proteins

A pathogenesis-related protein induced by infection with Ascochyta rabiei was isolated from intercellular washing fluid of chickpea (Cicer arietinum L.) leaves. The amino-terminal sequence of the protein was typical of a thaumatin-like protein (TLP). The isoelectric point was 6.5 and the molecular mass was 16 kDa; thus, chickpea PR-5a is a small TLP. PR-5a lacked antifungal activity toward A. rabiei. Screening of a chickpea complementary DNA (cDNA) library resulted in isolation of a cDNA clone (p5a-241) for this protein. A second cDNA clone (ELR112) encoding a TLP was isolated using differential hybridization of cDNA libraries obtained from elicited and water-treated cell suspension cultures of chickpea. The deduced protein (PR-5b) had a molecular mass of 22 kDa. PR-5b was localized in the vacuole owing to the presence of a respective N-terminal signal peptide and a carboxy-terminal extension. Southern blot analyses revealed that ELR112 and p5a-241 represented single-copy genes. During fungal infection of chickpea plants, expression of both genes proceeded much faster in an A. rabiei-resistant cultivar than in a susceptible one [17].

4.60.2.2.2

Cyclophilin-like protein

An 18-kDa protein designated chickpea cyclophilin-like antifungal protein (CLAP) was isolated from seeds of the chickpea (C. arietinum). It exhibited an N-terminal sequence analogous to cyclophilins. The protein was isolated with a protocol comprising affinity chromatography on Affi-gel blue gel and ion-exchange chromatography on CM-Sepharose. In addition to a suppressive effect on the growth of fungi, including Rhizoctonia solani, M. arachidicola, and B. cinerea, the protein was capable of inhibiting HIV type 1(HIV-1) reverse transcriptase. Chickpea CLAP did not demonstrate lectin and ribonuclease activities but it weakly suppressed translation in a rabbit reticulocyte lysate system. The protein augmented [methyl-3H]-thymidine incorporation by murine spleno­ cytes [54]. Marivet et al. [31] reported bean cyclophilin gene expression under stressful conditions.

4.60.2.2.3

Cicerin and arietin, novel peptides

Two antifungal peptides with novel N-terminal sequences, designated as cicerin and arietin, respectively, were isolated from seeds of the chickpea (C. arietinum). Both peptides were adsorbed on Affi-gel blue gel and CM-Sepharose and demonstrated a molecular mass of 8.2 and 5.6 kDa, respectively. Arietin was more strongly adsorbed on CM-Sepharose than cicerin and more potently inhibited translation-inhibiting activity in a rabbit reticulocyte lysate system and mycelial growth in M. arachidicola, F. oxysporum and B. cinerea. Both were void of mitogenic and anti-HIV-1 reverse transcriptase activities [68].

4.60.2.2.4

Cicerarin, a novel antifungal peptide from the green chickpea

An 8-kDa peptide designated as cicerarin, with a novel N-terminal amino acid sequence, was isolated from seeds of the green chickpea C. arietinum cv ‘Green Chickpea’. Cicerarin was isolated with a procedure that entailed ion-exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, and gel filtration by FPLC on Superdex 75. Cicerarin was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel in 10-mM Tris–HCl buffer (pH 7.3). Cicerarin manifested antifungal activity against B. cinerea, M. arachidicola, and Physalospora piricola, which was retained after exposure to 100 °C for 15 min [9].

4.60.2.3 4.60.2.3.1

Rice Bean (Delandia umbellata) Chitinase-like protein

A 28-kDa antifungal protein with a chitinase-like N-terminal sequence, designated as delandin, was isolated from the rice bean. It was adsorbed on both Affi-Gel blue gel and SP-Toyopearl. It impeded mycelial growth in M. arachidicola, B. cinerea, F. oxysporum, R. solani, and Colletotrichum gossypii, and inhibited the activity of HIV-1 reverse transcriptase. The protein slightly inhibited translation in rabbit reticulocyte lysate. It evoked a mitogenic response from mouse splenocytes [55].

4.60.2.3.2

Antifungal peptide

A 5-kDa peptide, demonstrating striking sequence resemblance to the cowpea 10-kDa protein precursor and garden pea diseaseresistance response protein, was isolated from rice bean seeds. The defensin-like peptide was adsorbed on CM-Sepharose and Affi­ gel blue gel. It impeded mycelial growth in the fungi B. cinerea, F. oxysporum, R. solani, and M. arachidicola. It enhanced incorporation of [methyl-3H] thymidine into mouse splenocytes, inhibited the activity of HIV-1 reverse transcriptase, and attenuated in vitro protein synthesis by rabbit reticulocyte lysate [56].

4.60.2.4 4.60.2.4.1

Field Bean (Dolichos lablab or Lablab purpuneus) Chitinase-like antifungal protein

An antifungal protein, exhibiting a molecular mass of 28 kDa and an N-terminal sequence resembling chitinases, has been isolated from the seeds of the field bean Dolichos lablab. The purification procedure involved extraction with aqueous buffer, affinity chromatography on Affi-gel blue gel, and ion-exchange chromatography on CM-Sepharose. The protein, designated as dolichin, elicited antifungal activity against the fungi F. oxysporum, R. solani and C. comatus. Dolichin was inhibitory to HIV reverse transcriptase and α- and β-glucosidases, which are glycohydrolases implicated in HIV infection. It exhibited very low cell-free translation-inhibitory activity [64].

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4.60.2.4.2

Inhibition of growth of Aspergillus flavus and fungal α-amylases by a lectin-like protein

Aspergillus flavus is a fungus pathogenic to maize causing an important ear rot disease when plants are exposed to drought and heat stress. The alpha-amylase of A. flavus promoted aflatoxin production in the endosperm of infected maize kernels. A 36-kDa alphaamylase inhibitor from Lablab purpureus (AILP) inhibited several fungal alpha-amylases but was devoid of any effect on animal and plant α-amylases. It inhibited conidial germination and hyphal growth of A. flavus. AILP was similar in amino acid sequence to lectin members of a lectin–arcelin-α-amylase inhibitor family described in common bean. AILP exhibited hemagglutinating activity on papain-treated human and rabbit erythrocytes and it represents a novel variant in the lectin–arcelin-α-amylase inhibitor family of proteins with lectin-like and α-amylase inhibitory activities [13].

4.60.2.5 4.60.2.5.1

Soybean (Glycine max) Polygalacturonase-inhibiting protein

A polygalacturonase (PG)-inhibiting protein (PGIP) was purified from germinating soybean (Glycine max (L.) Merr.). There were at least three components with similar molecular masses (37–40 kDa) but distinct N-terminal sequences. The nucleotide sequence comprised 942 bp with a single open reading frame that encoded a polypeptide of 313 residues with a predicted molecular mass of 33984 Da and an isoelectric point of 8.21. Wounding of soybean hypocotyls strongly induced the expression of the PGIP gene. The PGIP manifested different activities toward endopolygalacturonases (EPGs) from Sclerotinia sclerotiorum and A. niger. Soybean PGIP is possibly involved in plant defense against fungal pathogens [14].

4.60.2.5.2

New basic peroxidase cDNA from soybean hypocotyls infected with Phytophthora sojae f.sp. glycines

The GMIPER1 gene encoding a putative pathogen-induced peroxidase may play a crucial role in inducing resistance in soybean to Phytophthora sojae f.sp. glycines and in response to various external stresses [70].

4.60.2.5.3

Glysojanin from black soybean (G. soja)

A monomeric 25-kDa protein, with N-terminal sequence similarity to a segment of chitin synthase, was purified from the seeds of the black soybean G. soja. The protein, designated as glysojanin displayed strong antifungal activity against the fungi F. oxysporum and M. arachidicola. It inhibited HIV-1 reverse transcriptase with an IC50 of 47 μM, [methyl-3H] thymidine incorporation by mouse splenocytes with an IC50 of 175 μM, and translation in the rabbit reticulocyte lysate with an IC50 of 20 μM. Glysojanin was isolated with a procedure that included ion-exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ionexchange chromatography by FPLC on Mono S, and gel filtration by FPLC on Superdex 75 [36].

4.60.2.6

Defensin-like antifungal peptide

From the seeds of the Yunnan bean, a 6.5-kDa defensin-like antifungal peptide was isolated by affinity chromatography on Affi-gel blue gel, FPLC–ion-exchange chromatography on Mono S, and FPLC–gel filtration on Superdex 75. The antifungal peptide was adsorbed on Affi-gel blue gel at pH 7.8 and Mono S at pH 4.5. The peptide exerted antifungal activity with an IC50 of 2 μM for the fungus F. oxysporum and 10 μM for M. arachidicola. It exhibited a weaker mitogenic activity toward murine splenocytes than Concanavalin A. It demonstrated antiproliferative activity on a murine leukemia (L1210), a hepatoma (HepG2), and a murine leukemia (M1) cell line, and inhibited HIV-1 reverse transcriptase with an IC50 of 200 μM [47].

4.60.2.7

Leucaena leucocephala chitinases

Chitinase cDNAs from Leucaena leucocephala seedlings were cloned by polymerase chain reaction (PCR) amplification with degenerate primers based on conserved class I chitinase sequences and cDNA library screening. Two closely related chitinase cDNAs were sequenced and inferred to encode precursor proteins of 323 (KB1) and 326 (KB2) amino acids. Expression of the KB2 chitinase from a pET32a plasmid in Origami (DE3) Escherichia coli yielded high chitinase activity in the cell lysate. The recombinant thioredoxin fusion protein was purified and cleaved to form a 32-kDa chitinase. The recombinant chitinase hydrolyzed colloidal chitin with endochitinase-type activity, and suppressed growth in 13 of the 14 fungal strains tested [21].

4.60.2.8 4.60.2.8.1

Red Bean Peptides from red bean and pinto bean

Peptides with a molecular mass of 5 kDa were isolated from seeds of the pinto bean and red bean, respectively. The peptides manifested an N-terminal sequence with striking similarity to those of cowpea 10-kDa protein precursor and garden pea diseaseresistance response protein. The defensin-like bean peptides displayed potent antifungal activity toward a variety of fungal species including B. cinerea, M. arachidicola, and F. oxysporum. The proteins also exhibited mitogenic activity toward mouse splenocytes and an inhibitory action on HIV-1 reverse transcriptase [52]. An antifungal peptide was purified from the red bean with a protocol entailing affinity chromatography on Affi-gel blue gel and ion-exchange chromatography on CM-Sepharose. The 8-kDa protein, designated angularin, was adsorbed on both chromatographic media. Angularin exhibited antifungal activity against various fungal species including M. arachidicola and B. cinerea. It inhibited

Plant Biochemistry: Antifungal Proteins Protecting Plants from Fungal Pathogens

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mycelial growth in B. cinerea with an IC50 of 14.3 μM; F. oxysporum and R. solani were unaffected. Angularin displayed inhibitory activity on translation in the rabbit reticulocyte lysate system (IC50 = 8.0 μM) but had no effect on proliferation of splenocytes. The activity of HIV-1 reverse transcriptase was inhibited in the presence of angularin [57].

4.60.2.9

Mung Bean (Phaseolus mungo)

4.60.2.9.1

Mung bean defensin

Mungbean defensin VrCRP defensin cDNA was expressed in Pichia pastoris and the recombinant defensin (rVrD1) was purified. The recombinant VrD1 suppressed mycelial growth in fungi including F. oxysporum, Pyricularia oryza, R. solani, and Trichophyton rubrum and development of bruchid larvae. It also inhibited in vitro protein synthesis. These biological activities resembled those of the bacterially expressed defensin. Functional expression of VrD1 in P. pastoris is a highly feasible system for ascertaining the structure– function relationship of VrD1 by employing mutagenesis [7]. A cDNA encoding a small cysteine-rich protein designated VrCRP was isolated from a bruchid-resistant mung bean. VrCRP encodes a protein of 73 amino acids containing a 27 amino acid signal peptide and eight cysteines. VrCRP is a member of the plant defensin family. Artificial seeds containing 0.2% (w/w) of the purified VrCRP-TSP were lethal to larvae of the bruchid Callosobruchus chinensis. VrCRP is apparently the first reported plant defensin exhibiting in vitro insecticidal activity against C. chinensis [8].

4.60.2.9.2

Cyclophilin-like antifungal protein

A protein designated as mungin, isolated from mung bean (Phaseolus mungo) seeds, hindered mycelial growth in the fungi R. solani, C. comatus, M. arachidicola, B. cinerea, and F. oxysporum. The 18-kDa protein also displayed an N-terminal sequence similar to cyclophilins. It exerted an inhibitory action against alpha- and beta-glucosidases and suppressed [methyl-3H]thymidine incorporation by mouse splenocytes [50].

4.60.2.9.3

A nonspecific lipid transfer protein

A nonspecific lipid transfer peptide was purified from mung bean seeds. The procedure involved aqueous extraction, ion-exchange chromatography on CM-Sephadex and high-performance liquid chromatography (HPLC) on POROS-HS-20. The peptide displayed a molecular mass of 9.03 kDa in mass spectrometry. It exerted antifungal action toward F. solani, F. oxysporum, Pythium aphanider­ matum, and Sclerotium rolfsii, and antibacterial action against Staphylococcus aureus but not against Salmonella typhimurium. The lipid binding of this peptide resembled that of previously described lipid transfer protein extracted from seeds of wheat and maize, indicating that it possessed lipid transfer activity [46]. It was crystallized at 297 K using ammonium sulfate as a precipitant by means of the hanging-drop vapor-diffusion method. Native X-ray diffraction data were collected to a resolution of 2.4 Å. The crystals were rhombohedral, belonging to space group P2(1)2(1)2(1), with unit-cell parameters a = 38.671, b = 51.785, and c = 55.925 Å. Assuming the presence of one molecule in the crystallographic asymmetric unit resulted in a Matthews coefficient (V(M)) of approximately 3.0 Å(3) Da(−1), corresponding to a solvent content of about 58% [46].

4.60.2.10 4.60.2.10.1

Phaseolus vulgaris The gene encoding EPGIP

PGIP, a cell-wall protein isolated from bean (P. vulgaris L.) hypocotyls, inhibits fungal EPGs and plays an important role in plant resistance to phytopathogenic fungi. The nucleotide and deduced amino acid sequences of the PGIP gene showed no significant similarity with any known databank sequence [42]. The levels of PGIP and its mRNA rose in P. vulgaris hypocotyls in response to wounding or treatment with salicylic acid. In bean hypocotyls infected with C. lindemuthianum, the level of PGIP was raised in cells enveloping the infection site. Synthesis of PGIP constitutes a defense mechanism elicited by signal molecules that induce plant defense genes [4]. Transgenic tomato plants with dissimilar expression levels of PGIP-1 were infected with the pathogenic fungi F. oxysporum f. sp. lycopersici, B. cinerea, and Alternaria solani. No increase in resistance was detected. The PGIP-1 gene was also transiently expressed in Nicotiana benthamiana with potato virus X (PVX) as a vector. PGIP-1 isolated from transgenic tomatoes and PGIP-1 in crude protein extracts of PVX-infected N. benthamiana displayed a specificity different from that of PGIP purified from P. vulgaris. PGIP-1 failed to interact with a PG from F. moniliforme as revealed by surface-plasmon resonance analysis, while the bulk bean PGIP interacted with and inhibited this enzyme. PGIP-1 expressed in tomato and N. benthamiana had only limited capability to inhibit crude PG preparations from F. oxysporum f. sp. lycopersici, B. cinerea, and A. solani. Differential affinity chromatography was utilized to resolve PGIP proteins in P. vulgaris extracts. A PGIP-A with specificity akin to that of PGIP-1 was separated from a PGIP-B capable of interacting with PGs from both A. niger and F. moniliforme. Thus, PGIPs with different specificities are expressed in P. vulgaris and that the high-level expression of one member (PGIP-1) of the PGIP gene family in transgenic plants is inadequate for conferring general, enhanced resistance to fungi [10]. Pressey [37] found that the amount of PGIP was 14 times higher in bean pods than in etiolated hypocotyls. Two PGIP isoforms were isolated from bean pods by chromatography on S-Sepharose, DEAE-Sephadex A-50, Sephadex G-75, and Mono Q column. They differed slightly in pI value and N-terminal amino acid sequence. Two members of the PGIP gene family (PGIP-1 and PGIP-2) of P. vulgaris L. were expressed separately in N. benthamiana and the ligand specificity of their products was analyzed by surface plasmon resonance (SPR). PGIP-1 did not interact with PG from

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F. moniliforme but interacted with PG from A. niger. PGIP-2 interacted with both PGs. Eight amino acid variations distinguish the two proteins: five of them lie within the beta-sheet/beta-turn structure and two of them are adjacent to this region. By site-directed mutagenesis, each of the variant amino acids of PGIP-2 was replaced with the corresponding amino acid of PGIP-1. The mutated PGIP-2s were expressed individually in N. benthamiana, purified and subjected to SPR analysis. Each single mutation brought about a reduction in affinity for PG from F. moniliforme; residue Q253 made a substantial contribution, and its substitution with a lysine resulted in a drastic decline in the binding energy of the complex. Conversely, amino acid K253 of PGIP-1 was mutated into the corresponding amino acid of PGIP-2, a glutamine. With this single mutation, PGIP-1 acquired the ability to interact with F. moniliforme PG [27]. A leucine-rich repeat (LRR) plant protein involved in resistance to pathogens, a PGIP-1 from P. vulgaris, has been crystallized and preliminary X-ray characterization has been performed. The protein contains 10 repeats of a short (24 amino-acid) LRR motif. Single crystals of the protein were grown from vapor–diffusion experiments using PEG 2K monomethylether as precipitant; these crystals diffract to at least 2.3 Å resolution. The space group is P2(1), with two molecules of PGIP-1 in the asymmetric unit; the crystals contain approximately 38% solvent [28]. PGIPs interact with EPGs secreted by phytopathogenic fungi, inhibit their enzymatic activity, and favor the accumulation of oligogalacturonides, which activate plant defense responses. PGIPs are members of the LRR protein family that in plants play crucial roles in development, defense against pathogens, and recognition of beneficial microbes. The crystal structure at 1.7-Å resolution of a PGIP from P. vulgaris has been reported. There are two beta-sheets instead of the single one originally predicted by modeling studies. The structure also reveals a negatively charged surface on the LRR concave face, likely involved in binding PGs. The structural information on PGIP provides a basis for designing more efficient inhibitors for plant protection [11]. The secondary structure of the PGIP from P. vulgaris, a LRR protein present in the cell wall of many plants, has been elucidated. Far-UV CD and infrared spectroscopy coupled to constrained secondary structure prediction methods revealed the existence of 12 α- and 12 β-segments, thus enabling a schematic representation of three domains of the protein, that is, the central LRR region and the two cysteine-rich flanking domains. Peptides from endoproteinase-degraded PGIP were analyzed by mass spectrometry, and four disulfide bonds were identified. Mass spectrometric analysis in conjunction with glycosidase treatments revealed two N-linked oligosaccharides located on Asn 64 and Asn 141. The main structure was similar to the typical complex plant N-glycan consisting of a core pentasaccharide β-1, 2-xylosylated, carrying an α-1, 3-fucose linked to the innermost N-acetylglucosamine and an outer arm N-acetylglucosamine residue [32]. The interaction between fungal EPGs and PGIPs found in plant cell walls is characterized by high affinity, reversibility, and a 1:1 stoichiometry that brings about a reduction of the catalytic rate of a particular EPG by up to 99.7%. EPG/PGIP interactions have prompted many researchers to suspect the involvement of these proteins in the production of specific signals (oligosaccharins) during plant pathogenesis for certain EPG/PGIP combinations; the specific activity of EPG is increased beyond that characteristic of the enzyme alone. A detailed analysis of the product of the interaction of native P. vulgaris PGIP-2 with five EPGs from A. niger, namely PGI, PGII, PGA, PGB, and PGC, in the presence of homogalacturonan has been presented. It has been demonstrated that for PGA and PGC, the interaction with PGIP-2 may result either in inhibition or activation in a manner that is pH dependent. These data suggest the need for a reevaluation of the conventional description applied to PGIPs; suggestions include PG-binding protein and PG-modulating protein [23]. The cell wall acts as the first line of defense during pathogen invasion. PGPGs are a class of cell wall-modifying enzymes with precise temporal and organ-specific expression. PG inhibitors have been found in bean pods [49]. A 350-bp fragment with high homology to PGs was identified by differential display analysis of soybean cyst nematode (SCN) race 3-resistant PI 437654 and susceptible cultivar Essex. The fragment was strongly expressed in Essex, 2 days after inoculation. Complete coding sequences of two PG cDNAs, PG1 and PG2, were isolated by 3′ and 5′ rapid amplification of cDNA ends PCR (RACE PCR). PI 437654 and Essex had identical PG1 and PG2 sequences. A transversion from A to C created a PstI restriction site in the PG2 cDNA that was used to distinguish the two PG cDNAs by cleaved amplified polymorphic sequence analysis. A cDNA encoding a PGIP that is 89% identical to the P. vulgaris PGIP was isolated from soybean roots by reverse transcription (RT)–PCR. Steady-state levels of PG and PGIP were investigated by RNA gel blot analysis in roots 1–5 days after infection and, hypocotyls and leaves. Differences in the constitutive levels of PG mRNAs were observed in roots of different soybean genotypes. Steady-state levels of PG mRNAs were enhanced during compatible interactions with SCN and reduced in incompatible interactions and in mechanically wounded roots. Enhanced PGIP transcription was observed in response to mechanical wounding in both PI 437654 and Essex, but only in compatible interactions with SCN, suggesting uncoupling of PGIP functions in developmental and stress cues. Constitutive expression in incompatible interactions shows PGIP is not a factor in SCN resistance. Thus, the upregulation of endogenous PG transcription in soybean roots early after SCN infection could facilitate successful parasitism by SCN [30]; PGIPs have also been reported in other species (e.g., tomato) [41].

4.60.2.10.2

TLP from French bean legumes

A 20-kDa protein with an N-terminal sequence analogous to those of TLPs and thaumatins was isolated from the legume of the French bean P. vulgaris cv Kentucky wonder using a simple procedure involving affinity and ion-exchange chromatography. The protein was adsorbed on both CM-Sepharose and Affi-gel blue gel. It exerted antifungal activity against F. oxysporum, Pleurotus ostreatus, and C. comatus but not against R. solani [63].

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4.60.2.10.3

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A hemagglutinin from red kidney bean

A homodimeric 67-kDa hemagglutinin adsorbed on Affi-gel blue gel and CM-Sepharose was isolated from red kidney beans. The hemagglutinating activity of this lectin was inhibited by glycoproteins, but not by simple sugars. It inhibited HIV-1 reverse transcriptase and α-glucosidase. The N-terminal sequence of the lectin displayed some variations from previously reported lectins from P. vulgaris, but showed some resemblance to chitinases. It thwarted mycelial growth of the fungal species F. osysporum, C. comatus, and R. solani [69].

4.60.2.10.4

Peroxidase from French bean

A novel 37-kDa antifungal protein with its N-terminal sequence showing resemblance to the C-terminal sequences of peroxidases was isolated from French bean legumes. It was adsorbed on Affi-gel blue gel and CM-Sepharose. The protein displayed peroxidase activity with a Km of 58 μM and a Vmax of 3.36 U nmol−1. Optimal peroxidase activity was observed at 22 °C and pH 4. It exerted antifungal activity against a variety of fungal species including C. comatus, M. arachidicola, F. oxysporum, and B. cinerea. It inhibited the activities of α- and β-glucosidases but was without any inhibitory effect on HIV-1 reverse transcriptase [58].

4.60.2.10.5

Vulgin, antifungal polypeptide with mitogenic activity from pinto bean

An antifungal protein with some N-terminal resemblance to chitinase was purified from an extract of pinto beans. The polypeptide, designated as vulgin, exerted antifungal activity toward M. arachidicola, C. comatus, F. oxysporum, and B. cinerea. Vulgin inhibited translation in a rabbit reticulocyte lysate system with an IC50 of 4.3 μM and HIV-1 reverse transcriptase activity with an IC50 of 58 μM. Vulgin increased in vitro incorporation of [methyl-3H] thymidine into mouse splenocytes [61]. From the seeds of the pinto bean (P. vulgaris cv. ‘Pinto’), a chitinase and a novel antifungal protein, both with the ability of enhancing nitrite production by murine peritoneal macrophages, were isolated. The antifungal proteins, designated as phaseins A and B, demonstrated molecular masses of 28 and 32 kDa, respectively. Phaseins A and B were adsorbed on Affi-gel blue gel and CMSepharose, and were eluted as adjacent peaks from CM-Sepharose. Phasein A exhibited potent antifungal activity toward F. oxysporum and P. piricola. Phasein B was more potent than phasein A toward P. piricola but less potent than phasein A toward F. oxysporum and R. solani. Both antifungal proteins inhibited the activity of HIV-1 reverse transcriptase and translation in a rabbit reticulocyte lysate system, with phasein B having a higher potency. Nitric oxide production by mouse macrophages was consider­ ably elevated in the presence of both phaseins A and B, with the effect of phasein A being more pronounced. The bioactivities of phaseins were in general potent compared with those of other antifungal proteins [59].

4.60.2.11 4.60.2.11.1

Pea (Pisum sativum) Defensins

Two small cysteine-abundant polypeptides (Psd1 and Psd2) with antifungal activity against A. niger have been isolated from seeds of the pea (Pisum sativum) by ammonium sulfate fractionation followed by gel filtration on Sephadex G-75 and reverse-phase HPLC. They were localized primarily in vascular bundles and epidermal tissues of pea pods and exhibited potent antifungal activity toward several fungi, displaying IC50 values ranging from 0.04 to 22 μg ml−1. This inhibitory activity was attenuated when A. niger growth medium was supplemented with cations such as Ca2+, Mg2+, Na+, and K+ ions. Although the primary sequence of both Psd1 and Psd2 is homologous with other plant defensins, they cannot easily be assigned to any established group [2]. Almeida et al. [3] reported the cDNA cloning, expression in P. pastoris, purification, and characterization of recombinant P. sativum defensin 1 (rPsd1). It is a novel Cys-rich protein with four disulfide bridges and potent antifungal activity. The recombinant rPsd1 was purified to homogeneity by cation-exchange chromatography, followed by reversed-phase HPLC, and subjected to automated amino acid sequencing, which revealed four additional amino acids (EAEA) at the N-terminal region. Circular dichroism, intrinsic fluorescence, and nuclear magnetic resonance spectroscopy analysis disclosed that the recombinant protein possessed an analogous folding and a correct disulfide-bonding pattern when compared to native Psd1. Nevertheless, the rPsd1 presented a more species-specific antifungal activity. The importance of the N- and C-termini for Psd1 activity was revealed. P. sativum defensin 1 (Psd1) is a 46-amino-acid-residue plant defensin possessing a globular fold with a triple-stranded antiparallel β-sheet and an α-helix (from residue Asn17 to Leu27). It has a ‘cysteine-stabilized alpha/beta motif’ and identical three-dimensional topology in the backbone with other defensins and neurotoxins. Comparison of the electrostatic surface potential among proteins with high three-dimensional topology provided insights into the mode of action of Psd1. The surface topologies between proteins that present antifungal activity or sodium-channel-inhibiting activity are different. The surface topology has several common characteristics with potassium channel inhibitors, suggesting that Psd1 probably possesses this activity. Other common features with potassium channel inhibitors include the presence of a lysine residue crucial for inhibitory activity [1].

4.60.2.11.2

Miraculin-like protein from sugar snap P. sativum var. macrocarpon

A 38-kDa antifungal protein designated as sativin was isolated from the legumes of the sugar snap (also known as honey pea) P. sativum var. macrocarpon. The procedure comprised extraction, affinity chromatography on Affi-gel blue gel, and ion-exchange chromatography on CM-Sepharose. It possessed an N-terminal amino acid sequence similar to those of miraculin (a sweet protein) and pisavin (a ribosome-inactivating protein from P. sativum var. arvense Poir manifesting similarity to miraculin). Unlike pisavin,

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Intergration of Biotechnologies

however, sativin inhibited translation in a rabbit reticulocyte lysate system with a very low potency (IC50 = 14 μM). Sativin demonstrated antifungal activity against F. oxysporum, C. comatus and P. ostreatus but not against R. solani [65].

4.60.2.11.3

Beta-1,3-glucanase gene in pea (P. sativum)

As part of a search for seed coat-specific expressed genes in P. sativum cv. ‘Finale’ by PCR-based methods, a cDNA encoding a beta1,3-glucanase, designated PsGNS2 was identified and isolated. The deduced peptide sequence of PsGNS2 is similar to a subfamily of beta-1,3-glucanases, characterized by a long amino acid extension at the C-terminal end compared to the other beta-1,3­ glucanases. PsGNS2 is expressed in young flowers and in the seed coat and is weakly expressed in vegetative tissues (roots and stems) during seedling development. It is not inducible by environmental stress or in response to fungal infection. In developing pea flowers, the transcript is detectable in all four whorls. In the seed coat, the expression is temporally and spatially regulated. High abundance of the transcript became visible in the seed coat when the embryo reached the late heart stage and remained until the mid-seed-filling stage. In situ hybridization data demonstrated that the expression of PsGNS2 is restricted to a strip of the inner parenchyma tissue of the seed coat, which is involved in temporary starch accumulation and embryo nutrition. Also, this tissue showed less callose deposits than the other ones. The 5′ genomic region of PsGNS2 was isolated and promoter activity studies in transgenic Medicago truncatula showed a seed-specific expression. Highest activity of the promoter was found in the seed coat and in the endosperm part of the seed [5].

4.60.2.11.4

Pisumin, a novel antifungal protein from sugar snap pea P. sativum var macrocarpon

A 31-kDa antifungal protein with a novel N-terminal sequence was isolated from the legumes of the sugar snap pea P. sativum var. macrocarpon. The protein, designated as pisumin, displayed antifungal activity against C. comatus and P. ostreatus and much weaker activity against F. oxysporum and R. solani. Pisumin inhibited cell-free translation in a rabbit reticulocyte lysate system with an IC50 of 6 μM. Pisumin was similar to other leguminous antifungal proteins in that it was adsorbed on Affi-gel blue gel and CM-Sepharose [62]. The expression in P. pastoris, purification and characterization of the recombinant P. sativum defensin 1(rPsd1), a pea defensin, which presents four disulfide bridges and high antifungal activity, have been accomplished. The recombinant protein was purified to homogeneity by gel filtration, followed by reversed-phase HPLC. Mass spectrometry of native and recombinant Psd1 disclosed that the protein expressed heterologously was posttranslationally processed to the same mature protein as the native one. Circular dichroism and nuclear magnetic resonance spectroscopy analysis revealed that the recombinant protein had the same folding when compared to native Psd1. In addition, the rPsd1 was fully active against A. niger, when compared with native Psd1[6].

4.60.2.12 4.60.2.12.1

Broad Beans (Vicia faba) Bowman–Birk-type trypsin–chymotrypsin inhibitors

An isolation procedure comprising affinity chromatography on Affi-gel blue gel, ion-exchange chromatography on SP-Toyopearl, and FPLC on Mono S was employed to isolate a 7.5-kDa peptide from broad beans that manifested antifungal activity toward M. arachidicola, F. oxysporum, and B. cinerea. N-terminal sequence analysis revealed the identity of the antifungal peptide to be a trypsin– chymotrypsin inhibitor. The trypsin–chymotrypsin inhibitor inhibited chymotrypsin activity and HIV-1 reverse transcriptase activity, but stimulated proliferation of murine splenocytes [67]. A new trypsin–chymotrypsin inhibitor, with an N-terminal sequence showing some differences from the previously reported trypsin–chymotrypsin inhibitor, was isolated from the broad bean Vicia faba. The inhibitor was a 13-kDa peptide adsorbed on Affi­ gel blue gel and CM-Sepharose. It demonstrated antifungal activity toward M. arachidicola and P. piricola. In addition, the trypsin– chymotrypsin inhibitor elicited a mitogenic response from mouse splenocytes and inhibited the activity of HIV-1 reverse tran­ scriptase [60].

4.60.2.12.2

Fabin, a novel calcyon-like and glucanase-like protein with mitogenic, antifungal and translation-inhibitory activities

A 34-kDa protein, termed fabin, with an N-terminal sequence exhibiting similarities to N-terminal sequences of human calcyon and barley endo-1,4-glucanase, and to C-terminal sequences of human translation initiation factor 4 gamma and yeast super-killer viralicidic (virus killing) activity, was isolated from the broad bean V. faba. Antifungal activity of the protein was observed against several fungal species including R. solani, B. cinerea, F. oxysporum, and M. arachidicola. Fabin inhibited HIV-1 reverse transcriptase with an IC50 of 34 μM and translation in a rabbit reticulocyte lysate with an IC50 of 2.4 μM. At a concentration of about 1.5 μM, fabin elicited a ninefold increase in the mitogenic response of murine splenocytes [34].

4.60.2.13 4.60.2.13.1

Cow Pea (Vigna unguiculata) Cyclophilin-like protein

A protein designated as unguilin was isolated from seeds of cow pea (Vigna unguiculata). It exhibited a molecular mass of 18 kDa and an N-terminal sequence similar to that of cyclophilins and the CLAP from mung beans, and was adsorbed on Affi-gel blue gel and CM-Sepharose. Unguilin exerted an antifungal effect toward fungi including C. comatus, M. arachidicola, and B. cinerea. In addition, unguilin inhibited HIV-1 reverse transcriptase and the glycohydrolases α- and β-glucosidases, which are involved in HIV

Plant Biochemistry: Antifungal Proteins Protecting Plants from Fungal Pathogens

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infection. Unguilin inhibited [methyl-3H]-thymidine uptake by mouse splenocytes and it weakly inhibited translation in a rabbit reticulocyte lysate system [53]. Two proteins with antifungal and antiviral potency exist in cowpea seeds. The proteins, designated α- and β-antifungal proteins in accordance with their order of elution from the CM-Sepharose column, inhibited HIV reverse transcriptase and one of the glycohydrolases associated with HIV infection, α-glucosidase, but β-glucuronidase was not affected. α-Antifungal protein was more potent in retarding mycelial growth in most of the fungi tested. β-Antifungal protein was more active in only one instance. Both antifungal proteins had weak cell-free translation-inhibitory activity. The proteins were adsorbed on Affi-gel blue gel and CMSepharose but could be separated from one another during chromatography on the latter medium by means of a linear NaCl concentration gradient. These alpha- and beta-antifungal proteins exhibited different molecular masses, 28 and 12 kDa, respec­ tively. α-Antifungal protein was characterized by an N-terminal sequence showing similarity to sequences of chitinases. β-Antifungal protein exhibited a novel N-terminal sequence [66].

4.60.2.14

Legume Vicilins (7S Storage Globulins)

Vicilin (7S storage proteins), isolated from different leguminous seeds, inhibited yeast growth and glucose-stimulated acidification of the medium by yeast cells. The degree of growth inhibition varied according to the origin of vicilins: more than 90% for vicilins from cowpea (V. unguiculata, cultivar ‘Pitiuba’) and 65% for vicilins from V. radiata. Vicilins from cowpea seeds inhibited glucosestimulated acidification of the medium by Saccharomyces cerevisae. It was suggested that vicilins bind to chitin-containing structures of yeast cells and that such association could result in inhibition of H+ pumping, cell growth, and spore formation. A final consequence of the yeast growth inhibition by vicilins is spore formation [16].

4.60.2.15

Inhibitory Effects of Antifungal Proteins on HIV-1 Reverse Transcriptase, Protease, and Integrase

Antifungal proteins from seeds of leguminous plants including French bean, cowpea, field bean, mung bean, peanut, and red kidney bean were assayed for the ability to inhibit HIV-1 reverse transcriptase, protease, and integrase, enzymes essential to the life cycle of HIV-1. It was noted that cowpea β-antifungal protein was potent in inhibiting HIV-1 protease and HIV-1 integrase. Cowpea α-antifungal protein was potent in inhibiting HIV-1 reverse transcriptase and HIV-1 integrase. Peanut antifungal protein exhibited a high inhibitory activity against HIV-1 integrase and an intermediate potency in inhibiting HIV-1 reverse transcriptase and HIV-I protease. French bean TLP expressed low HIV-I protease inhibitory activity and red kidney bean lectin inhibited HIV-I integrase by only a very small extent. Antifungal proteins from the field bean and mung bean had an intermediate potency in inhibitory HIV-1 protease and integrase. However, mung bean antifungal protein was not capable of inhibiting HIV-1 reverse transcriptase. The results indicate that nearly all leguminous antifungal proteins examined were able to inhibit HIV-1 reverse transcriptase, protease and integrase to some extent [35]. The N-terminal sequences of some antifungal proteins are presented in Table 1. A diversity of structures can be seen.

4.60.3 Nonleguminous Antifungal Proteins A 15-kDa antifungal protein from Panax notoginseng (sanchi ginseng) roots, with an N-terminal sequence similar to those of chitinases, demonstrated potent antifungal activity against F. oxysporum [24]. The chive (Allium tuberosum) chitinase-like protein, showing striking N-terminal sequence similarity to chitinases from leek (A. porrum) and garlic (A. sativum), exhibited inhibitory activity against a variety of fungi such as R. solani, F. oxysporum, M. arachidicola, and B. cinerea. Its antifungal activity was stable over a wide range of pH (1.6–12.3) and at temperatures up to 60 °C for 5 min. It had mitogenic activity toward mouse splenocytes and antiproliferative activity on breast cancer cells (Lam et al., 2000 [26]). Two 30-kDa chitinase-like antifungal proteins were isolated from ripe emperor bananas (Musa basjoo). The protein more strongly adsorbed on Mono S showed a higher antifungal potency toward F. oxysporum than the less strongly adsorbed protein. There was, however, no activity against M. arachidicola [18]. From Amaranthus hypochondriacus seeds, a 3184-Da chitin-binding protein with a single Cys-/Gly-rich chitin-binding domain was isolated. It was thermostable and protease resistant. It inhibited a variety of fungal species including A. alternata, A. candidus, Table 1

N-terminal sequences of some antifungal proteins

Rice bean defensin-like protein Broad bean antifungal protein (fabin) Green chickpea antifungal protein (cicerarin) Chickpea antifungal protein (arietin) Chickpea antifungal protein (cicerin) Pinto bean antifungal protein (phasein A) Pinto bean antifungal protein (phasein B)

RTHENLANTYKGPPITTG GDPGDQNGKA VKSTGRADDDLAVKTKYLPP GVGYKVVVTTTAAADDDDVV ARCENFADSYRQPPISSSQT CDVGSVISASLFEQ GARKDDHAKLVFLLKD

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Intergration of Biotechnologies

A schraceus, C albicans, F. solani, Geotrichum candidum, Penicillium chrysogenum, and Trichoderma species (Rivillas-Acevedo and Soriano-Garcia, 2007 [39]) Antifungal peptides from radish (Raphanus sativus) seeds belong to the plant defensin family. Linear synthetic 19-mer peptides display activity analogous to the native antifungal proteins. Replacement of cysteines in the 19-mer peptides with α-aminobutyric acid resulted in an augmented antifungal activity. Analogous cyclic 19-mer peptides also demonstrate high antifungal activity (Schaaper et al., 2001 [40]). A 30-kDa deoxyribonuclease from asparagus (Asparagus officinalis) seeds manifested antifungal activity against B. cinerea, but not against F. oxysporum, M. arachidicola, and R. solani. Its optimal pH for DNase activity toward herring sperm DNA is pH 7.5. It inhibits cell-free translation with an IC50 of 20 μM but did not attenuate HIV-1 reverse transcriptase activity [43]. Ginkbilobin, a 13-kDa antifungal protein from Ginkgo biloba seeds, displayed an N-terminal sequence with resemblance to white spruce embryo-abundant protein. It hindered mycelial growth in B. cinerea, M. arachidicola, F. oxysporum, C. comatus, and R. solani with an IC50 of 0.25, 6.5, 3.6, 3.4, and 8.7 μM, respectively. It also exerted moderate antibacterial activity against S. aureus, Pseudomonas aeruginosa, and E. coli. It manifested antimitogenic activity on mouse splenocytes and inhibitory activity toward HIV­ 1 reverse transcriptase (Wang and Ng, 2000b [44]). The lipid transfer protein from seeds of the motherwort (Leonurus japonicus Houtt), a Chinese medicinal herb, inhibited B. maydis, A. niger, F. oxysporum, P. digitatum, and S. cerevisiae with an IC50 value of 5.5, 6.1, 9.3, 40, and 76 μM, respectively. It also inhibited A. brassicae, R. cerealis, and the bacterium Bacillus subtilis (Yang et al., 2006 [49]). Lin et al. [29] isolated a 9412-Da lipid transfer protein from Brassica campestris seeds. It retarded mycelial growth in F. oxysporum and M. arachidicola with an IC50 of 8.3 and 4.5 μM, respectively. It demonstrateed dose-dependent binding to lyso-α-lauroyl phosphatidylcholine, indicating its lipid transfer activity. Like its counterpart from the mung bean, it exhibited pH stability, thermostability, and protease stability. However, unlike mung bean lipid transfer protein, it manifested HIV-1 reverse transcriptase inhibitory activity and antiproliferative activity toward tumor cells. A 20.5-kDa Kunitz-type trypsin inhibitor from Pseudostellaria heterophylla roots, a Chinese medicinal herb, inhibited mycelial growth in F. oxysporum (Wang and Ng, 2006 [45]). A 18-kDa trypsin inhibitor from the Chinese medicinal herb, malaytea scurfpea (Psoralea corylifolia), exerted an inhibitory action on A. brassicae, A. niger, F. oxysporum, and R. solani (Yang et al., 2006) [15]. A 28-kDa trypsin–chymotrypsin inhbitor from Withania somnnifera root tubers inhibited spore germination and hyphal growth in A. flavus, F. oxysporum, and F. verticilloides. It also inhibited C. michiganensis subsp, michiganensis (Girish et al., 2006 [15]). Chilli pepper (Capsicum annuum) seeds contained a proteinase inhibitor with an inhibitory action toward yeasts (Ribeiro et al., 2007 [38]). The ripe fruits of the emperor banana (M. basjoo cv. ‘Emperor Banana’) produce a 20-kDa TLP that retards mycelial growth in F. oxysporum and M. arachidicola. It lacked mitogenic activity toward splenocytes and only slightly inhibited HIV-1 reverse transcriptase [19].

4.60.4 Conclusion and Future Perspectives It can be seen from the foregoing account that a wide range of antifungal proteins with different amino acid sequences and molecular masses have been detected or isolated from various parts, in particular the seeds, of plants. They can be categorized into different groups including chitinases, glucanses, TLPs, protease inhibitors, peroxidases, cyclophilin-like proteins, miraculin-like proteins, lipid transfer proteins, lectins, and hemagglutinins. Hopefully, more plant antifungal proteins will be isolated in the future and used for the benefit of mankind.

References [1] Almeida MS, Cabral KM, Kurtenbach E, et al. (2002) Solution structure of Pisum sativum defensin 1 by high resolution NMR: Plant defensins, identical backbone with different mechanisms of action. Journal of Molecular Biology 315: 749–757. [2] Almeida MS, Cabral KM, Zingali RB, and Kurtenbach E (2000) Characterization of two novel defense peptides from pea (Pisum sativum) seeds. Archives of Biochemistry and Biophysics 378: 278–286. [3] Almeida MS, Cabral KS, de Medeiros LN, et al. (2001) cDNA cloning and heterologous expression of functional cysteine-rich antifungal protein Psd1 in the yeast Pichia pastoris. Archives of Biochemistry and Biophysics 395: 199–207. [4] Bergmann CW, Ito Y, Singer D, et al. (1994) Polygalacturonase-inhibiting protein accumulates in Phaseolus vulgaris L. in response to wounding, elicitors and fungal infection. Plant Journal 5: 625–634. [5] Buchner P, Rochat C, Wuilleme S, and Boutin JP (2002) Characterization of a tissue-specific and developmentally regulated beta-1,3-glucanase gene in pea (Pisum sativum). Plant Molecular Biology 49: 171–186. [6] Cabral KM, Almeida MS, Valente AP, et al. (2003) Production of the active antifungal Pisum sativum defensin 1 (Psd1) in Pichia pastoris: overcoming the inefficiency of the STE13 protease. Protein Expression and Purification 31: 115–122. [7] Chen JJ, Chen GH, Hsu HC, et al. (2004) Cloning and functional expression of a mungbean defensin VrD1 in Pichia pastoris. Journal of Agricultural and Food

Chemistry 52:2256–2261.

[8] Chen KC, Lin CY, Kuan CC, et al. (2002) A novel defensin encoded by a mungbean cDNA exhibits insecticidal activity against bruchid. Journal of Agricultural and Food Chemistry 50: 7258–7263. [9] Chu KT, Liu KH, and Ng TB (2003) Cicerarin, a novel antifungal peptide from the green chickpea. Peptides 24: 659–663. [10] Desiderio A, Aracri B, Leckie F, et al. (1997) Polygalacturonase-inhibiting proteins (PGIPs) with different specificities are expressed in Phaseolus vulgaris. Molecular Plant – Microbe Interactions 10: 852–860.

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[11] Di Matteo A, Federici L, Mattei B, et al. (2003) The crystal structure of polygalacturonase-inhibiting protein (PGIP), a leucine-rich repeat protein involved in plant defense. Proceedings of the National Academy of Sciences of the United States of America 100: 10124–10128. [12] Fabre C, Causse H, Mourey L, et al. (1998) Characterization and sugar properties of arcelin-1, and insecticidal lectin-like protein isolated from kidney bean (Phaseolus vulgaris L. cv. RAZ-2) seeds. Biochemical Journal 329: 551–560. [13] Fakhoury AM and Woloshuk CP (2001) Inhibition of growth of Aspergillus flavus and fungal alpha-amylases by a lectin-like protein from Lablab purpureus. Molecular Plant – Microbe Interactions 14: 955–961. [14] Favaron F, D’Ovidio R, Porceddu E, and Alghisi P (1994) Purification and molecular characterization of a soybean polygalacturonase-inhibiting protein. Planta 195: 80–87. [15] Girish KS, Machiah KD, Ushanandhini S, et al. (2006) Antimicrobial properties of a non-toxic glycoprotein (WSG) from Withania somnifera (Ashwagandha). Journal of Basic Microbiology 46: 365–374. [16] Gomes VM, Okorokov LA, Rose TL, et al. (1998) Legume vicilins (7S storage globulins) inhibit yeast growth and glucose stimulated acidification of the medium by yeast cells. Biochimica et Biophysica Acta 1379: 207–216. [17] Hanselle T, Ichinoseb Y, and Barz W (2001) Biochemical and molecular biological studies on infection (Ascochyta rabiei)-induced thaumatin-like proteins from chickpea plants (Cicer arietinum L.). Zeitschrift fur Naturforschung C-A Journal of Biosciences 56: 1095–1107. [18] Ho VS and Ng TB (2007) Chitinase-like proteins with antifungal activity from emperor banana fruits. Protein and Peptide Letters 14: 828–831 [19] Ho VS, Wong JH, and Ng TB (2007) A thaumatin-like antifungal protein from the emperor banana. Peptides 28: 760–766. [20] Ishimoto M and Chrispeels MJ (1996) Protective mechanism of the Mexican bean weevil against high levels of alpha-amylase inhibitor in the common bean. Plant Physiology 111: 393–401. [21] Kaomek M, Mizuno K, Fujimura T, et al. (2003) Cloning, expression, and characterization of an antifungal chitinase from Leucaena leucocephala de Wit. Bioscience, Biotechnology, and Biochemistry 67: 667–676. [22] Kellmann JW, Kleinow T, Engelhardt K, et al. (1996) Characterization of two class II chitinase genes from peanut and expression studies in transgenic tobacco plants. Plant Molecular Biology 30: 351–358. [23] Kemp G, Stanton L, Bergmann CW, et al. (2004) Polygalacturonase-inhibiting proteins can function as activators of polygalacturonase. Molecular Plant–Microbe Interactions 17:888–894. [24] Lam SK and Ng TB (2001) Isolation of a small chitinase-like antifungal protein from Panax notoginseng (sanchi ginseng) roots. International Journal of Biochemistry and Cell Biology 33: 287–292. [25] Lam SSL, Wang HX, and Ng TB (1998) Purification and novel ribosome inactivating proteins, alpha- and beta-pisavins, from seeds of the garden pea Pisum sativum. Biochemical and Biophysical Research Communications 253: 135–142. [26] Lam YW, Wang HX, and Ng TB(2000) A robust cysteine-deficient chitinase-like antifungal protein from inner shoots of the edible chive Allium tuberosum. Biochemical and Biophysical Research Communications 279: 74–80. [27] Leckie F, Mattei B, Capodicasa C, et al. (1999) The specificity of olygalacturonase-inhibiting protein (PGIP): A single amino acid substitution in the solvent-exposed beta-strand/ beta-turn region of the leucine-rich repeats (LRRs) confers a new recognition capability. EMBO Journal 18: 2352–2363. [28] Leech A, Mattei B, Federici L, et al. (2000) Preliminary X-ray crystallographic analysis of a plant defence protein, the polygalacturonase-inhibiting protein from Phaseolus vulgaris. Acta Crystallographica D Biologica Crystallographica 56: 98–100. [29] Lin P, Xia L, and Ng TB (2007) First isolation of an antifungal lipid transfer peptide from seeds of a Brassica species. Peptides 28: 1514–1519. [30] Mahalingam R, Wang G, and Knap HT (1999) Polygalacturonase and polygalacturonase inhibitor protein: Gene isolation and transcription in Glycine max–Heterodera glycines interactions., Molecular Plant–Microbe Interactions 12: 490–498. [31] Marivet J, Margis-Pinheiro M, Frendo G, and Burkard P (1994). Bean cyclophilin gene expression during plant development and stress conditions. Plant Molecular Biology 26:1181–1189. [32] Mattei B, Bernalda MS, Federici L, et al. (2001) Secondary structure and post-translational modifications of the leucine-rich repeat protein PGIP (polygalacturonase-inhibiting protein) from Phaseolus vulgaris. Biochemistry 40: 569–576. [33] Morton RL, Schroeder HE, Bateman KS, et al. (2000) Bean alpha-amylase inhibitor 1 in transgenic peas (Pisum sativum) provides complete protection from pea weevil (Bruchus pisorum) under field conditions. Proceedings of the National Academy of Sciences of the United States of America 97: 3820–3825. [34] Ng TB and Ye XY (2003) Fabin, a novel caleyon-like and glucanase-like protein with mitogenic, antifungal and translation-activities from broad beans. Biological Chemistry 384:811–s815. [35] Ng TB, Au TK, Lam TL, et al. (2002) Inhibitory effects of antifungal proteins on human immunodeficiency virus type 1 reverse transcriptase, protease and integrase. Life Sciences 70: 927–935. [36] Ngai PHK and Ng TB (2003) Purification of glysojanin, an antifungal protein, from the black soybean Glycine soja. Biochemistry and Cell Biology 81: 387–394. [37] Pressey R (1996) Polygalacturonase inhibitors in bean pods. Phytochemistry 42: 1267–1270. [38] Ribeiro SF, Carvalho AO, Da Cunha M, et al. (2007) Isolation and characterization of novel peptides from chilli pepper seeds: aAntimicrobial activities against pathogenics yeasts. Toxicon 50: 600–611. [39] Rivillas-Acevado LA and Soriano-Garcia M (2007) Isolation and biochemical characterization of an antifungal peptide from Amaranthus hypochondriacus seeds. Journal of Agricultural and Food Chemistry 55: 10156–10161. [40] Schaaper WM, Posthuma GA, Plasman HH, et al. (2001) Synthetic peptides derived from the beta2-beta3 loop of Raphanus sativus antifungal protein 2 that mimic the active site. Journal of Peptide Research 57: 409–418. [41] Stotz HU, Contos JJ, Powell AL, et al. (1994) Structure and expression of an inhibitor of fungal polygalacturonases from tomato. Plant Molecular Biology 25: 607–617. [42] Toubart P, Desiderio A, Salvi G, et al. (1992) Cloning and characterization of the gene encoding the endopolygalacturonase-inhibiting protein (PGIP) of Phaseolus vulgaris L. Plant Journal 2: 367–373. [43] Wang HX and Ng TB (2001) Isolation of a novel deoxyribonuclease with antifungal activity from Asparagus officinalis seeds. Biochemical and Biophysical Research Communications 289: 120–124. [44] Wang HX and Ng TB (2000) Ginkbilobin a novel antifungal protein from Ginkgo biloba seeds with sequence similarity to embryo abundant protein. Biochemical and Biophysical Research Communications 279: 407–411. [45] Wang HX and Ng TB (2006) Concurrent isolation of a Kunitz-type trypsin inhibitor with antifungal activity and a novel lectin from Pseudostellaria heterophylla roots. Biochemical and Biophysical Research Communications 342: 349–353. [46] Wang SY, Wu JH, Ng TB, et al. (2004) A non-specific lipid transfer protein with antifungal and antibacterial activities from the mung bean. Peptides 25: 1235–1242. [47] Wong JH and Ng TB (2003) Gymnin, a potent defensin-like antifungal peptide from the Yunnan bean (Gymnocladus chinensis Baill). Peptides 24: 963–968. [48] Wong JH and Ng TB (2005) Isolation and characterization of a glucose/mannose/rhammose-specific lectin from the knife bean Canavalia gladiata. Archives of Biochemistry and Biophysics 439: 91–98. [49] Yang X, Li J, Li X, et al. (2006) Isolation and characterization of a novel thermostable non-specific lipid transfer protein from motherwort (Leonurus japonicus Houtt) seeds. Peptides 27: 3122–3128. [50] Ye XY and Ng TB (2000) Mungin, a novel cyclophilin-like antifungal protein from the mung bean. Biochemical and Biophysical Research Communications 273: 1111–1115. [51] Ye XY and Ng TB (2001a) Hypogin, a novel antifungal peptide from peanuts with sequence similarity to peanut allergan. Journal of Peptide Research 57: 330–336. [52] Ye XY and Ng TB (2001b) Peptides from pinto bean and red bean with sequence homology to cowpea 10-kDa protein precursor exhibit antifungal, mitogenic, and HIV-1 reverse transcriptase-inhibitory activities. Biochemical and Biophysical Research Communications, 285: 424–429.

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Intergration of Biotechnologies

[53] Ye XY and Ng TB (2001c) Isolation of unguilin, a cyclophilin-like protein with anti-mitogenic, antiviral, and antifungal activities, from black-eyed pea. Journal of Protein Chemistry 20: 353–359. [54] Ye XY and Ng TB (2002a) Isolation of a new cyclophilin-like protein from chickpeas with mitogenic, antifungal and anti-HIV-1 reverse transcriptase activities. Life Sciences 70: 1129–1138. [55] Ye XY and Ng TB (2002b) Delandin, a chitinase-like protein with antifungal, HIV-1 reverse transcriptase inhibitory and mitogenic activities from the rice bean Delandia umbellata. Protein Expression and Purification 24: 524–529. [56] Ye XY and Ng TB (2002c) A new antifungal peptide from rice beans. Journal of Peptide Research 60: 81–87. [57] Ye XY and Ng TB (2002d) Purification of angularin, a novel antifungal peptide from adzuki beans. Journal of Peptide Science 8: 101–106. [58] Ye XY and Ng TB (2002e) Isolation of a novel peroxidase from French bean legumes and first demonstration of antifungal activity of a non-milk peroxidase. Life Sciences 71: 1667–1680. [59] Ye XY and Ng TB (2002f) A new antifungal protein and a chitinase with prominent macrophage-stimulating activity from seeds of Phaseolus vulgaris cv. pinto. Biochemical and Biophysical Research Communications 290: 813–819. [60] Ye XY and Ng TB (2002g) A new peptidic protease inhibitor from Vicia faba seeds exhibits antifungal, HIV-1 reverse transcriptase inhibiting and mitogenic activities. Journal of Peptide Science 8: 656–662. [61] Ye XY and Ng TB (2003a) Isolation of vulgin, a new antifungal polypeptide with mitogenic activity from the pinto bean. Journal of Peptide Science 9: 114–119. [62] Ye XY and Ng TB (2003b) Isolation of pisumin, a novel antifungal protein from legumes of the sugar snap pea Pisum sativum var. macrocarpon. Comparative Biochemistry and Physiology Part C Toxicology and Pharmacology 134: 235–240. [63] Ye XY, Wang HX, and Ng TB (1999) First chromatographic isolation of an antifungal thaumatin-like protein from French bean legumes and demonstration of its antifungal activity. Biochemical and Biophysical Research Communications 263: 130–134. [64] Ye XY, Wang HX, and Ng TB (2000a) Dolichin, a new chitinase-like antifungal protein isolated from field beans (Dolichos lablab). Biochemical and Biophysical Research Communications 269: 155–159. [65] Ye XY, Wang HX, and Ng TB (2000b) Sativin, a novel antifungal miraculin-like protein isolated from legumes of the sugar snap Pisum sativum var. macrocarpon. Life Sciences 67: 775–781. [66] Ye XY, Wang HX, and Ng TB (2000c) Structurally dissimilar proteins with antiviral and antifungal potency from cowpea (Vigna unguiculata) seeds. Life Sciences 67: 3199–3207. [67] Ye XY, Ng TB, and Rao PF (2001) A Bowman-Birk-type trypsin-chymotrypsin inhibitor from broad beans. Biochemical and Biophysical Research Communications 289: 91–96. [68] Ye XY, Ng TB, and Rao PF (2002) Cicerin and arietin, novel chickpea peptides with different antifungal potencies. Peptides 23: 817–822. [69] Ye XY, Ng TB, Tsang PW, and Wang J (2001) Isolation of a homodimeric lectin with antifungal and antiviral activities from red kidney bean (Phaseolus vulgaris) seeds. Journal of Protein Chemistry 20: 367–375. [70] Yi SY and Hwang BK (1998) Molecular cloning and characterization of a new basic peroxidase cDNA from soybean hypocotyls infected with Phytophthora sojae f.sp. glycines. Molecular Cell 8: 556–564. [71] Zhang JH, Wang CZ, and Qin JD (2000) The interactions between soybean trypsin inhibitor and delta-endotoxin of Bacillus thuringiensis in Helicoverpa armigera larva. Journal of Invertebrate Pathology 75: 259–266.