Partitioning recombinant chitinase from Nicotiana benthamiana by an aqueous two-phase system based on polyethylene glycol and phosphate salts.

The objectives of this study were to purify 42 kDa chitinase derived from Trichoderma asperellum SH16 produced in Nicotiana benthamiana by a polyethylene glycol (PEG)/salt aqueous two-phase system (ATPS). The specific activities of the crude chitinase and the partially purified chitinase from N. benthamiana were about 251 unit/mg and 386 unit/mg, respectively. The study found the 300 g/L PEG 6000 + 200 g/L potassium phosphate (PP) and 300 g/L PEG 6000 + 150 g/L sodium phosphate (SP) systems had the highest partitioning efficiency for each salt in primary extraction. However, among the two types of salt, PP displayed higher efficiency than SP, with a partitioning coefficient K of 4.85 vs. 3.89, a volume ratio V of 2.94 vs. 2.68, and a partitioning yield Y of approximately 95 % vs. 83 %. After back extraction, the enzymatic activity of purified chitinase was up to 834 unit/mg (PP) and 492 unit/mg (SP). The purification factors reached 3.32 (PP) and 1.96 (SP), with recovery yields of about 59 % and 61 %, respectively. SDS-PAGE and zymogram analysis showed that the recombinant chitinase was significantly purified by using ATPS. The purified enzyme exhibited high chitinolytic activity, with the hydrolysis zone's diameter being around 2.5 cm-3 cm. It also dramatically reduced the growth of Sclerotium rolfsii; the colony diameter after treatment with 60 unit of enzyme for 104 spores was only about 1 cm, compared to 3.5 cm in the control. The antifungal effect of chitinase suggests that this enzyme has great potential for applications in agricultural production as well as postharvest fruit and vegetable preservation.

The potential of plant and fungal proteins in the control of gastrointestinal nematodes from animals.

Gastrointestinal nematode infection is an important cause of high economic losses in livestock production. Nematode control based on a synthetic chemical approach is considered unsustainable due to the increasing incidence of anthelmintic resistance. Control alternatives such as the use of natural products are therefore becoming relevant from an environmental and economic point of view. Proteins are macromolecules with various properties that can be obtained from a wide range of organisms, including plants and fungi. Proteins belonging to different classes have shown great potential for the control of nematodes. The action of proteins can occur at specific stages of the nematode life cycle, depending on the composition of the external layers of the nematode body and the active site of the protein. Advances in biotechnology have resulted in the emergence of numerous protein and peptide therapeutics; however, few have been discussed with a focus on the control of animal nematodes. Here, we discuss the use of exogenous proteins and peptides in the control of gastrointestinal.

The potential of plant and fungal proteins in the control of gastrointestinal nematodes from animals / Potencial de proteínas de plantas e fungos no controle de nematoides gastrintestinais de animais

Abstract Gastrointestinal nematode infection is an important cause of high economic losses in livestock production. Nematode control based on a synthetic chemical approach is considered unsustainable due to the increasing incidence of anthelmintic resistance. Control alternatives such as the use of natural products are therefore becoming relevant from an environmental and economic point of view. Proteins are macromolecules with various properties that can be obtained from a wide range of organisms, including plants and fungi. Proteins belonging to different classes have shown great potential for the control of nematodes. The action of proteins can occur at specific stages of the nematode life cycle, depending on the composition of the external layers of the nematode body and the active site of the protein. Advances in biotechnology have resulted in the emergence of numerous protein and peptide therapeutics; however, few have been discussed with a focus on the control of animal nematodes. Here, we discuss the use of exogenous proteins and peptides in the control of gastrointestinal.

Resumo A infecção por nematoides gastrintestinais é uma importante causa de grandes perdas econômicas na pecuária. O controle de nematoides com compostos químicos sintéticos é considerado insustentável devido ao aumento da resistência anti-helmíntica. Alternativas de controle, como o uso de produtos naturais, estão se tornando relevantes do ponto de vista ambiental e econômico. As proteínas são macromoléculas com várias propriedades que podem ser obtidas de uma ampla gama de organismos, incluindo plantas e fungos. Proteínas pertencentes a diferentes classes têm mostrado grande potencial para o controle de nematoides. A ação das proteínas pode ocorrer em estágios específicos do ciclo de vida do nematoide, dependendo da composição das camadas externas do parasito e do sítio ativo da proteína. Avanços na biotecnologia resultaram no surgimento de numerosas terapias de proteínas e peptídeos; no entanto, pouco foi discutido com foco no controle de nematoides parasitos de animais. Na presente revisão foi discutido o uso de proteínas exógenas e peptídeos no controle de nematoides gastrintestinais, os mecanismos sugeridos de ação, e os desafios e perspectivas para o uso dessas biomoléculas como uma classe de anti-helmínticos.

Glycan-dependent chikungunya viral infection divulged by antiviral activity of NAG specific chi-like lectin.

Highly pathogenic alphaviruses display complex glycans on their surface. These glycans play a crucial role in viral pathogenesis by facilitating glycan-host interaction during viral entry which can be targeted. Various studies have reported antiviral activity of lectins that bind to the glycans present on the surface of enveloped viruses. This study evaluates the antiviral potential of a chitinase (chi)-like lectin from Tamarind (TCLL) having specificity for N-acetylglucosamine (NAG). Thus, TCLL might bind to N-glycan rich surface of alphavirus and inhibit the entry of virus into the host cells. The direct treatment of TCLL with virus reduced the virus infection. Remarkably, the addition of NAG to TCLL abolished antiviral activity confirming that NAG binding property of TCLL is accountable for its antiviral activity. Further, an ELISA assay confirmed the binding of TCLL to alphaviruses. Taken together, this study will prove to be beneficial in developing lectin therapeutics targeting alphavirus glycan.

Improved antifungal activity of barley derived chitinase I gene that overexpress a 32 kDa recombinant chitinase in Escherichia coli host

Abstract Agricultural crops suffer many diseases, including fungal and bacterial infections, causing significant yield losses. The identification and characterisation of pathogenesis-related protein genes, such as chitinases, can lead to reduction in pathogen growth, thereby increasing tolerance against fungal pathogens. In the present study, the chitinase I gene was isolated from the genomic DNA of Barley (Hordeum vulgare L.) cultivar, Haider-93. The isolated DNA was used as template for the amplification of the ∼935 bp full-length chitinase I gene. Based on the sequence of the amplified gene fragment, class I barley chitinase shares 93% amino acid sequence homology with class II wheat chitinase. Interestingly, barley class I chitinase and class II chitinase do not share sequence homology. Furthermore, the amplified fragment was expressed in Escherichia coli Rosetta strain under the control of T7 promoter in pET 30a vector. Recombinant chitinase protein of 35 kDa exhibited highest expression at 0.5 mM concentration of IPTG. Expressed recombinant protein of 35 kDa was purified to homogeneity with affinity chromatography. Following purification, a Western blot assay for recombinant chitinase protein measuring 35 kDa was developed with His-tag specific antibodies. The purified recombinant chitinase protein was demonstrated to inhibit significantly the important phytopathogenic fungi Alternaria solani, Fusarium spp, Rhizoctonia solani and Verticillium dahliae compared to the control at concentrations of 80 µg and 200 µg.

Antifungal activity and patterns of N-acetyl-chitooligosaccharide degradation via chitinase produced from Serratia marcescens PRNK-1.

Serratia marcescens PRNK-1, which has strong chitinolytic activity, was isolated from cockroaches (Periplaneta americana L.). The chitinase from S. marcescens PRNK-1 was characterized after incubation in a 0.5% colloidal chitin medium at 30 °C for 3 days. The molecular weights of three bands after staining for chitinase activity were approximately 34, 41, and 48 kDa on an SDS-PAGE gel. S. marcescens PRNK-1 strain strongly inhibited hyphal growth of Rhizoctonia solani and Fusarium oxysporum. Thin-layer chromatography (TLC) and high performance liquid chromatograph (HPLC) analyses were conducted to investigate the degradation patterns of N-acetyl-chitooligosaccharides by PRNK-1 chitinase. The N-acetyl-chitooligosaccharides: N-acetyl-chitin dimer (GlcNAc)2, N-acetyl-chitin trimer (GlcNAc)3, and N-acetyl-chitin tetramer (GlcNAc)4 were degraded to (GlcNAc)1-3 on a TLC plate. In an additional experiment, (GlcNAc)6 was degraded to (GlcNAc)1-4 on a TLC plate. The optimal temperature for chitinase activity of the PRNK-1 was 50 °C, producing 32.8 units/mL. As seen via TLC, the highest degradation of (GlcNAc)4 by PRNK-1 chitinase occurred with 50 °C incubation. The optimal pH for chitinase activity of PRNK-1 was pH 5.5, producing 24.6 units/mL. As seen via TLC, the highest degradation of (GlcNAc)4 by PRNK-1 chitinase occurred at pH 5.0-6.0. These results indicate that chitinase produced from S. marcescens PRNK-1 strain showed strong antifungal activity and potential of production of N-acetyl-chitooligosaccharides.

Cytotoxicity against tumor cell lines and anti-inflammatory properties of chitinases from Calotropis procera latex.

The role of chitinases from the latex of medicinal shrub Calotropis procera on viability of tumor cell lines and inflammation was investigated. Soluble latex proteins were fractionated in a CM Sepharose Fast-Flow Column and the major peak (LPp1) subjected to ion exchange chromatography using a Mono-Q column coupled to an FPLC system. In a first series of experiments, immortalized macrophages were cultured with LPp1 for 24 h. Then, cytotoxicity of chitinase isoforms (LPp1-P1 to P6) was evaluated against HCT-116 (colon carcinoma), OVCAR-8 (ovarian carcinoma), and SF-295 (glioblastoma) tumor cell lines in 96-well plates. Cytotoxic chitinases had its anti-inflammatory potential assessed through the mouse peritonitis model. We have shown that LPp1 was not toxic to macrophages at dosages lower than 125 µg/mL but induced high messenger RNA expression of IL-6, IL1-ß, TNF-α, and iNOs. On the other hand, chitinase isoform LPp1-P4 retained all LPp1 cytotoxic activities against the tumor cell lines with IC50 ranging from 1.2 to 2.9 µg/mL. The intravenous administration of LPp1-P4 to mouse impaired neutrophil infiltration into the peritoneal cavity induced by carrageenan. Although the contents of pro-inflammatory cytokines IL-6, TNF-α, and IL1-ß were high in the bloodstreams, such effect was reverted by administration of iNOs inhibitors NG-nitro-L-arginine methyl ester and aminoguanidine. We conclude that chitinase isoform LPp1-P4 was highly cytotoxic to tumor cell lines and capable to reduce inflammation by an iNOs-derived NO mechanism.

Defense-related Proteins from Chelidonium majus L. as Important Components of its Latex.

The aim of this review is to cover most recent research on plant pathogenesis- and defenserelated proteins from latex-bearing medicinal plant Chelidonium majus (Papaveraceae) in the context of its importance for latex activity, function, pharmacological activities, and antiviral medicinal use. These results are compared with other latex-bearing plant species and recent research on proteins and chemical compounds contained in their latex. This is the first review, which clearly summarizes pathogenesisrelated (PR) protein families in latex-bearing plants pointing into their possible functions. The possible antiviral function of the latex by naming the abundant proteins present therein is also emphasized. Finally latex-borne defense system is hypothesized to constitute a novel type of preformed immediate defense response against viral, but also non-viral pathogens, and herbivores.

Purification and biochemical characterization of novel acidic chitinase from Paenicibacillus barengoltzii.

The novel chitinase (PbChi67) from the marine bacterium Paenicibacillus barengoltzii CAU904 was purified and biochemically characterized. PbChi67 was purified to apparent homogeneity with 10.2 fold purification and 8.0% recovery yield. The molecular mass of the enzyme was 67.0kDa by SDS-PAGE and 67.9kDa by gel filtration, respectively. PbChi67 was most active at pH 3.5 and was stable within pH 3.0-9.0. The optimal temperature of PbChi67 was 60°C and it was stable up to 55°C with a thermal denaturing half-life of 43min at 65°C. The enzyme exhibited strict substrate specificity towards colloidal chitin and glycol chitin but showed no or trace activities towards other tested substrates. The Km and Vmax values of PbChi67 for colloidal chitin and glycol chitin were 3.35mg/mL and 17.1µmol/min/mg, and 2.66mg/mL and 15.0µmol/min/mg, respectively. PbChi67 hydrolyzed colloidal chitin to yield N-acetyl chitooligosaccharides (COSs) with degree of polymerization (DP) of 2-4 at the initial hydrolysis stage, indicating that it is an endo-type chitinase. These properties make the enzyme as a good candidate for recycling of chitin materials.

The Ifchit1 chitinase gene acts as a critical virulence factor in the insect pathogenic fungus Isaria fumosorosea.

The filamentous fungus, Isaria fumosorosea, is a promising insect biological control agent. Chitinases have been implicated in targeting insect cuticle structures, with biotechnological potential in insect and fungal control. The I. fumosorosea chitinase gene, Ifchit1, was isolated and determined to encode a polypeptide of 423 amino acids (46 kDa, pI = 6.53), present as a single copy in the I. fumosorosea genome. A split marker transformation system was developed and used to construct an Ifchit1 gene knockout. The ΔIfchit1 strain displayed minor alterations in mycelial growth on diverse media at 26 °C compared to the wild type and complemented (ΔIfchit1::Ifchit1) strains; however, colony morphology was affected, and the mutant strain had a temperature sensitive phenotype (32 °C). Although sporulation was delayed for the mutant, overall conidial production was almost twice than that of wild type. Biochemical assays indicated decreased chitinase activity during growth in Czapek-Dox liquid media for the ΔIfchit1 strain. Insect bioassays using diamondback moth, Plutella xylostella, larvae revealed decreased infectivity, i.e., increased LC 50 (threefold to fourfold) and a significantly delayed time to death, LT 50 from 3 to 6 days, for the ΔIfchit1 strain compared to the wild type and complemented strains. These data indicate an important role for the Ifchit1 chitinase as a virulence factor in I. fumosorosea.

Gene cloning, expression and characterization of an exo-chitinase with high ß-glucanase activity from Aeromonas veronii B565.

Objective: We aimed to express and characterize biochemical properties of Chi92, a chitinase from Aeromonas veronii B565, and study its potential application as aquafeed supplement. Methods: The chitinase gene chi92 was cloned from A. veronii strain B565 and expressed in Pichia pastoris GS115. The recombinant chitinase (Chi92) was purified and characterized. Chi92 was supplemented in diets containing P. pastoris powder and fed to zebrafish for 14 days. By comparing with the control group, effect of Chi92 supplementation on growth, feed utilization, microvilli morphology, and disease resistance was investigated. Results: The complete gene sequence encoded a polypeptide with 864 amino acids. Chi92 exhibited optimal activity at pH 6.0 and 40℃, and was resistant to proteases and not substantially inhibited by metal ions. Chi92 had high chitinase activity (69.4 U/mL). The specific activity was 809.2 U/mg and 235.6 U/mg on colloidal chitin and ß-1,3-1,4-glucan, respectively. Thin-layer chromatography and electrospray ionization-coupled mass spectrometry revealed that N-diacetylglucosamine was the dominant product of Chi92 when colloidal chitin was used as substrate. Moreover, Chi92 showed advantages over other chitinases for degradation of yeast cell wall. Supplementation of Chi92 in diet containing yeast product significantly improved the intestine microvilli length and density of zebrafish after two weeks of feeding. Marginally improved growth performance, feed utilization, as well as disease resistance were also observed in the Chi92 supplement group. Conclusion: The pH stability, resistance against metal ions/chemical reagents/proteases, and high yeast cell wall degradation activity of Chi92 suggest its potential use as feed additive enzyme for warm water aquaculture.

Purification, characterization, and gene cloning of a chitinase from Stenotrophomonas maltophilia N4.

The Stenotrophomonas maltophilia synthesises high-activity chitinase in response to chitin or chitosan induction. The enzyme was purified 8.5 fold and subjected to characterisation. The optimum hydrolysis conditions for this enzyme when using colloidal chitin as substrate were pH 5.6 and temperature of 45 °C. The enzyme demonstrated high thermal stability at 45 °C within 2 h. The studied chitinase exhibited high activity towards colloidal chitin, glycol chitin and chitosan, while it did not hydrolyse glycosidic bonds in carboxymethylcellulose. The enzyme exhibited the highest activity, equalling 90 U/ml, towards Nitrophenyl ß-D-N,N',N"-triacetylchitotriose and activity of 37 U/ml towards 4-Nitrophenyl N,N'-diacetyl-ß-D-chitobioside. The K(m) value in the presence of the two former substrates was:1.2 and 3.9 mM, respectively, which classifies the studied enzyme as an endochitinase. Cysteine and 2-mercaptoethanol stimulated to a small degree the activity of the chitinase which may indicate the involvement of cysteine residues in the catalysis mechanism. The full length of the nucleotide sequence of this chitinase gene is 2106 bp, which amounts to 702 amino acids.

Bacillus subtilis chitinase identified by matrix-assisted laser desorption/ionization time-of flight/time of flight mass spectrometry has insecticidal activity against Spodoptera litura Fab.

An extracellular chitinase was identified and purified (CS1 and CS2) from Bacillus subtilis. The 16S rRNA sequencing was submitted in GenBank (accession numbers KC336487 and KC412256). The purified crude enzymes were identified through matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF MS) analysis. The peptide sequences were matched with chitinase sequences. The peak m/z with 1297. 592 and 3094.570 mascot search resulted sequence was blasted with NCBI protein sequences and confirmed that it is a chitinase enzyme. The effects of chitinase on gut enzymes lactate dehydrogenase, acid phosphatase, alkaline phosphatase and adenosine triphosphatase of the tobacco cutworm Spodoptera litura larvae were investigated. At all concentrations tested, chitinase decreased the activities of these gut enzymes relative to the control. When chitinase treated leaves were fed to larvae in bioassays, gut tissue and gut enzymes were affected. The histological study clearly shows the chitinase treated larval gut, peritrophic membrane and epithelial cells were affected significantly. Chitinase isolated from B. subtilis has effectively reduced the gut enzyme activity and growth of S. litura. The chitin based bioformulation may serve as an effective biocide against the polyphagous pest like S. litura.

Purification of Chitinase enzymes from Bacillus subtilis bacteria TV-125, investigation of kinetic properties and antifungal activity against Fusarium culmorum.

BACKGROUND: Chitin is the main structural component of cell walls of fungi, exoskeletons of insects and other arthropods and shells of crustaceans. Chitinase enzyme is capable of degrading chitin, and this enzyme can be used as a biological fungicide against phytopathogenic fungi, as well as an insecticide against insect pests. METHODS: In this study, 158 isolates, which were derived from bacteria cultures isolated from leaves and root rhizospheres of certain plants in Turkey, were selected after confirming that they are not phytopathogenic based on the hypersensitivity test performed on tobacco; and antifungal activity test was performed against Fusarium culmorum, which is a pathogenic fungi that cause decomposition of roots of vegetables. Accordingly, chitinase enzyme activity assay was performed on 31 isolates that have an antifungal activity, and among them the isolate of Bacillus subtilis TV-125 was selected, which has demonstrated the highest activity. RESULTS: Chitinase enzyme was purified by using ammonium sulphate and DEAE-sephadex ion exchange chromatography. Ammonium sulphate precipitation of chitinase enzyme from Bacillus subtilis TV-125 isolate was performed at maximum range of 0-20%, and 28.4-fold purification was obtained with a 13.4% of yield. Optimum activity of the purified enzyme was observed at pH 4.0 and at 50°C of temperature. In addition, it was identified that Bacillus subtilis TV-125A isolate retains 42% of its activity at 80°C temperature. CONCLUSION: In the last phase of the study, chitinase enzyme purified from Bacillus subtilis TV-125A was tested on four fungal agents, although all the results were positive, it was particularly effective on F. culmorum according to the findings.

Heterologous expression and functional characterization of a novel chitinase from the chitinolytic bacterium Chitiniphilus shinanonensis.

Chitiniphilus shinanonensis strain SAY3(T) is a chitinolytic bacterium isolated from moat water of Ueda Castle in Nagano Prefecture, Japan. Fifteen genes encoding putative chitinolytic enzymes (chiA-chiO) have been isolated from this bacterium. Five of these constitute a single operon (chiCDEFG). The open reading frames of chiC, chiD, chiE, and chiG show sequence similarity to family 18 chitinases, while chiF encodes a polypeptide with two chitin-binding domains but no catalytic domain. Each of the five genes was successfully expressed in Escherichia coli, and the resulting recombinant proteins were characterized. Four of the recombinant proteins (ChiC, ChiD, ChiE, and ChiG) exhibited endo-type chitinase activity toward chitinous substrates, while ChiF showed no chitinolytic activity. In contrast to most endo-type chitinases, which mainly produce a dimer of N-acetyl-D-glucosamine (GlcNAc) as final product, ChiG completely split the GlcNAc dimer into GlcNAc monomers, indicating that it is a novel chitinase.

Optimization of nutrition factors on chitinase production from a newly isolated Chitiolyticbacter meiyuanensis SYBC-H1

The present study reports statistical medial optimization for chitinase production by a novel bacterial strain isolated from soil recently, which the name Chitinolyticbacter meiyuanensis SYBC-H1 is proposed. A sequential statistical methodology comprising of Plackett-Burman and response surface methodology (RSM) was applied to enhance the fermentative production of chitinase, in which inulin was firstly used as an effective carbon source. As a result, maximum chitinase activity of 5.17 U/mL was obtained in the optimized medium, which was 15.5-fold higher than that in the basal medium. The triplicate verification experiments were performed under the optimized nutrients levels which indicated that it well agreed with the predicted value.

Generation and characterization of anti-chitinase monoclonal antibodies.

Class IV chitinase, an allergenic protein of Vitis vinifera (grape), was purified by anion exchange chromatography and used for immunization of Balb/c mice. Monoclonal antibodies (MAbs) were raised by hybridoma technology using Sp2/0 myeloma cells. Finally after three limiting dilutions, six stable clones were generated. Antibody isotyping showed that IgG(2a), IgG(2b), and IgM were produced by one, two, and three of the clones, respectively. All of the MAbs had kappa light chain. The affinities were in the range of 3 × 10(8) to 1.2 × 10(9) M(-1). The MAbs were specific for grape chitinase as confirmed by Western blotting. In conclusion, we successfully produced several MAbs against grape class IV chitinase, which could be used for assessment of this allergen in different grape cultivars.

Production of chitooligosaccharides and their potential applications in medicine.

Chitooligosaccharides (CHOS) are homo- or heterooligomers of N-acetylglucosamine and D-glucosamine. CHOS can be produced using chitin or chitosan as a starting material, using enzymatic conversions, chemical methods or combinations thereof. Production of well-defined CHOS-mixtures, or even pure CHOS, is of great interest since these oligosaccharides are thought to have several interesting bioactivities. Understanding the mechanisms underlying these bioactivities is of major importance. However, so far in-depth knowledge on the mode-of-action of CHOS is scarce, one major reason being that most published studies are done with badly characterized heterogeneous mixtures of CHOS. Production of CHOS that are well-defined in terms of length, degree of N-acetylation, and sequence is not straightforward. Here we provide an overview of techniques that may be used to produce and characterize reasonably well-defined CHOS fractions. We also present possible medical applications of CHOS, including tumor growth inhibition and inhibition of T(H)2-induced inflammation in asthma, as well as use as a bone-strengthener in osteoporosis, a vector for gene delivery, an antibacterial agent, an antifungal agent, an anti-malaria agent, or a hemostatic agent in wound-dressings. By using well-defined CHOS-mixtures it will become possible to obtain a better understanding of the mechanisms underlying these bioactivities.

Purification and characterization of a viral chitinase active against plant pathogens and herbivores from transgenic tobacco.

The Autographa californica nucleopolyhedrovirus chitinase A (AcMNPV ChiA) is a chitinolytic enzyme with fungicidal and insecticidal properties. Its expression in transgenic plants enhances resistance against pests and fungal pathogens. We exploited tobacco for the production of a biologically active recombinant AcMNPV ChiA (rChiA), as such species is an alternative to traditional biological systems for large-scale enzyme production. The protein was purified from leaves using ammonium sulfate precipitation followed by anion exchange and gel-filtration chromatography. Transgenic plants produced an estimated 14 mg kg(-1) fresh leaf weight, which represents 0.2% of total soluble proteins. The yield of the purification was about 14% (2 mg kg(-1) fresh leaf weight). The comparison between the biochemical and kinetic properties of the rChiA with those of a commercial Serratia marcescens chitinase A indicated that the rChiA was thermostable and more resistant at basic pH, two positive features for agricultural and industrial applications. Finally, we showed that the purified rChiA enhanced the permeability of the peritrophic membrane of larvae of two Lepidoptera (Bombyx mori and Heliothis virescens) and inhibited spore germination and growth of the phytopatogenic fungus Alternaria alternata. The data indicated that tobacco represents a suitable platform for the production of rChiA, an enzyme with interesting features for future applications as "eco-friendly" control agent in agriculture.

Glucanases and chitinases as causal agents in the protection of Acacia extrafloral nectar from infestation by phytopathogens.

Nectars are rich in primary metabolites and attract mutualistic animals, which serve as pollinators or as an indirect defense against herbivores. Their chemical composition makes nectars prone to microbial infestation. As protective strategy, floral nectar of ornamental tobacco (Nicotiana langsdorffii x Nicotiana sanderae) contains "nectarins," proteins producing reactive oxygen species such as hydrogen peroxide. By contrast, pathogenesis-related (PR) proteins were detected in Acacia extrafloral nectar (EFN), which is secreted in the context of defensive ant-plant mutualisms. We investigated whether these PR proteins protect EFN from phytopathogens. Five sympatric species (Acacia cornigera, A. hindsii, A. collinsii, A. farnesiana, and Prosopis juliflora) were compared that differ in their ant-plant mutualism. EFN of myrmecophytes, which are obligate ant-plants that secrete EFN constitutively to nourish specialized ant inhabitants, significantly inhibited the growth of four out of six tested phytopathogenic microorganisms. By contrast, EFN of nonmyrmecophytes, which is secreted only transiently in response to herbivory, did not exhibit a detectable inhibitory activity. Combining two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis with nanoflow liquid chromatography-tandem mass spectrometry analysis confirmed that PR proteins represented over 90% of all proteins in myrmecophyte EFN. The inhibition of microbial growth was exerted by the protein fraction, but not the small metabolites of this EFN, and disappeared when nectar was heated. In-gel assays demonstrated the activity of acidic and basic chitinases in all EFNs, whereas glucanases were detected only in EFN of myrmecophytes. Our results demonstrate that PR proteins causally underlie the protection of Acacia EFN from microorganisms and that acidic and basic glucanases likely represent the most important prerequisite in this defensive function.