Chitin-binding Lectin Research Articles

Evaluation of non-clinical safety of Wsmol, a chitin binding lectin of Moringa oleifera seeds in adult zebrafish (Danio rerio)

Previous studies have shown several biotechnological and lectin biomedical activities provided from Moringa oleifera seeds. The Wsmol (water soluble chitin binding lectin) has affinity for n-acetyl-D-glucosamine-glycans present in various cell receptors and it is able to realize many biological activities. This study was conducted to evaluate the nonclinical safety of Wsmol in adult Zebrafish (Danio rerio). The action of Wsmol (1.0 mg / mL; 20 μL; ip) and Vehicle (0.9 % NaCl, 20 μL, ip) on the locomotor activity of adult zebrafish (ZFa; n = 6 / group) was evaluated in the open field test. A naïve group was included. After 24 h of treatments, mortality rate was analyzed and the concentration able to kill 50 % (LC50) of the zebrafish was calculated. Wsmol, active in the physiological environment of Zebrafish did not modify the locomotor activity nor did it prove to be toxic to the animals, suggesting that its lectin activity, although can promote pharmacological activities when recognizing glycans of several ion channels and membrane protein receptors, can not be associated with toxicity or locomotor damage.

Spectroscopic and thermodynamic characterization of the interaction between sugar-stabilised silver nanoparticles and wheat germ agglutinin (WGA), a chitin binding lectin

Nanomaterials have lately been investigated in agriculture as eco-friendly and effective antifungal agents. Many nanomaterials, notably metal nanoparticles, have strong antifungal properties. Among metal nanoparticles, Ag nanoparticles have received the most attention as antifungal agents. Many plant lectins have been identified as antifungal agents. Conjugating AgNPs with antifungal lectins is thus expected to improve Ag nanoparticle antifungal efficacy. Understanding the molecular interactions and physical features of lectin-sugar-stabilised nanoparticle conjugates is critical for future applications. WGA has traditionally been used as an anti-tumor and antifungal agent. To investigate the prospect of developing an effective biocompatible antifungal system with applications in medicine and agriculture, fluorescence spectroscopy was used to investigate the interaction between sugar-stabilised silver nanoparticles and WGA. During the association, protein intrinsic fluorescence emission is suppressed by about ∼15 % at saturation, with no significant shift in fluorescence emission maxima. Binding tests reveal a strong bond. Stern-Volmer analysis of the quenching data indicates that the interaction happens via a static quenching process that induces complex formation. The study of hemagglutination activity and interaction experiments in the presence of particular sugar shows that the lectin's sugar-binding site is separate from the nanoparticle-binding site, and cell recognition is conserved in the lectin-nanoparticle complex. The Van't Hoff plot thermodynamic parameters suggest that the contact is hydrophobic. The fact that ΔGo is negative shows that the binding is a spontaneous process. CD spectroscopy experiments reveal that the lectin's secondary structure is not affected while binding to the nanoparticle. Our findings suggest that a stable WGA-silver nanoparticle combination may emerge for a variety of applications.

Effects of saline extract and lectin from Punica granatum seed sarcotesta on Aedes aegypti larvae

• Salin extract contained lectin, ellagic acid, α and β punicalagins, ellagic acid derivatives, and cinnamic acid derivatives. • The extract was able to induce mortality in A. aegypti larvae. • The lectin PgTeL did not show larvicidal activity. • The extract altered midgut morphology and inhibited amylase and trypsin activity in the larval gut. Currently, one of the main strategies to reduce the incidence of the arboviruses such as Zika, dengue, and chikungunya is through the population control of their vector, Aedes aegypti . Lectins are carbohydrate-binding proteins with several biological properties, including insecticidal activity against A. aegypti. Punica granatum sarcotesta contains a chitin-binding lectin (PgTeL), which prompted our evaluation of its toxicity against A. aegypti . Here, we analyzed the effects of saline extract from P. granatum sarcotesta and PgTeL on the survival of A. aegypti larvae. Saline extract of P. granatum was evaluated for the presence of secondary metabolites; PgTeL was further isolated and purified from this extract. Larvicidal assay was performed using P. granatum saline extract (0.17–1.20 mg/mL of protein) and isolated PgTeL (0.1–1.0 mg/mL). Histological analysis was performed to evaluate the effects of the extract on the larvae midgut morphophysiology. Additionally, the activity of digestive enzymes in midgut extracts of the larvae was determined. P. granatum extract, containing protein (17.1 mg/mL), ellagic acid (2.1 mg/mL), α-punicalagin (2.2 mg/mL), β-punicalagin (2.3 mg/mL), ellagic acid derivatives (2.2 mg/mL), and cinnamic acid derivatives (3.9 mg/mL), caused larvae mortality (LC 50/48 h : 0.67 mg/mL of protein) and altered the midgut morphology; midgut cells were affected, showing protrusions in the apical region and a thickening of the peritrophic matrix. P. granatum extract inhibited amylase and trypsin activities in the larval gut. However, PgTeL did not display larvicidal activity. In conclusion, the sarcotesta saline extract from pomegranate shows larvicidal activity against A. aegypti, and the mode of action may involve disruption of the digestive processes of the larvae. These findings may represent a first step towards the availability of a new tool to control A. aegypti .

Acute toxicity and genotoxicity assessment of PgTeL, a lectin from pomegranate sarcotesta, in mice

• PgTeL did not show hemolytic and cytotoxic effects in vitro . • It also did not cause toxicity when administered intraperitoneally in mice. • No damage to genetic material was observed. • PgTeL induced an increase in HDL levels and a decrease in TG and LDL levels, suggesting a hypolidemic effect. • The results open several possibilities for the application of PgTeL in vivo . PgTeL is a chitin-binding lectin from Punica granatum sarcotesta and shows antimicrobial activity against a wide range of microorganisms of medical interest. PgTeL did not show cytotoxicity in human peripheral blood mononuclear cells; however, there have been no studies to date on PgTeL toxicity in vivo . The present study evaluated PgTeL for cytotoxicity in murine splenocyte cells, in vitro hemolytic activity, and in vivo acute toxicity and genotoxicity in mice. In the acute toxicity assay with PgTeL at 100 mg/kg (i.p.), behavioral, biochemical, hematological, hemostatic, and histopathological parameters were evaluated. Comet and micronucleus tests were performed to assess the genotoxicity. After 24 h of incubation, there was only a slight decrease in splenocyte viability in treatments with PgTeL at 200 μg/mL (13.2%) and 100 μg/mL (9.9%) when compared to the control. No damage to the membranes of erythrocytes (0% hemolysis) was observed when they were incubated with all concentrations (3.9–1000 µg/mL) of PgTeL. There were no behavioral, hematological, hemostatic, or histopathological abnormalities in the in vivo acute toxicity assay. PgTeL caused an increase in HDL levels and decreased levels of triglycerides, LDL, and VLDL. The results provide several possibilities for the application of PgTeL in vivo , such as in formulations with antimicrobial potential.

Antibacterial, antifungal and in vivo anticancer activities of chitin-binding lectins from Tomato (Solanum lycopersicum) fruits

A mixture of chitin-binding lectins from Tomato ( Solanum lycopersicum ) fruits, designated as Tomato chitin-binding lectins (TCLs), was isolated through affinity chromatography using an acetylated chitin column. Molecular weights of TCLs were determined to be 30 to 115 KDa which possessed mild toxicity with an LC 50 value of 521 µg/ml examined by the brine shrimp nauplii toxicity assay. Strong antibacterial activity of TCLs was found against Escherichia coli , Staphylococcus aureus and Shigella boydii at a concentration of 500 µg/ml by using disc diffusion method. Minimum inhibitory concentrations (MIC) of TCLs against Staphylococcus aureus and Escherichia coli were found to be 200 µg/ml and 140 µg/ml, respectively whereas minimum bactericidal concentrations (MBC) against the same bacterial species were 840 and 600 µg/ml, respectively. TCLs also exerted antibiofilm activity (53.32% at 250 μg/ml) against Escherichia coli . Strong antifungal activity of TCLs against Aspergillus niger was found at 600 µg/ml whereas the lectin mixture agglutinated A. niger spores at 200 µg/ml. TCLs exhibited 19.63% and 59.91% anti-proliferative activity against Ehrlich ascites carcinoma (EAC) cells in vivo in Swiss albino mice when intraperitonealy injected at doses 1.0 mg/kg/day and 2.0 mg/kg/day, respectively for five consecutive days. Morphological changes of apoptosis in EAC cells under fluorescence microscope and alteration of the expression of apoptosis-related genes (Fas, Caspase 8 and Caspase 3) had also been observed. MTT assay showed 27.61%, 38.74% and 49.23% of in vitro anticancer activity of the tomato lectins at concentrations of 37.5, 75 and 150 µg/ml, respectively.

Structure and Function of Plant Chitin-binding Lectins and Tomato Lectin

Structure and Function of Plant Chitin-binding Lectins and Tomato Lectin

Structure and Function of Plant Chitin-binding Lectins and Tomato Lectin

キチンに特異的に結合する構造上異なる複数のレクチンドメインが植物に分布している。その一つヘベインドメインはCBM18としても知られ、約40残基のアミノ酸配列の中に保存された8個のシステインとグリシン残基を含む。このドメインを構成要素とするタンパク質の遺伝子は単独であるいは他のドメインと組み合わさって機能している。トマトレクチンはヘベインドメインと植物細胞壁糖タンパク質エクステンシンに類似のドメインを構成単位とするキメラレクチンである。キチンのみならずポリラクトサミンに結合する性質から、糖鎖染色や糖鎖の分画に利用されてきた。本稿では植物におけるキチン結合性レクチンについてまとめ、トマトレクチンの植物における役割を考察する。

Myracrodruon urundeuva leaf lectin damages exochorionic cells and binds to the serosal cuticle of Aedes aegypti eggs.

In recent years, lectins have been identified as alternative agents against Aedes aegypti during the aquatic phases of its life cycle. For example, chitin-binding lectin from Myracrodruon urundeuva leaf (MuLL) can function as a larvicide. In this study, we investigated whether MuLL can also act as an ovicide against this insect. Aedes aegypti eggs were incubated with MuLL for 72h to determine the concentration at which the hatching rate reduces by 50% (EC50). The effects of MuLL on the egg surface structure were evaluated using scanning electron microscopy (SEM), and the possible interaction of MuLL with the internal structures of eggs and embryos was investigated using MuLL-fluorescein isothiocyanate (FITC) conjugate. MuLL acted as an ovicidal agent with an EC50 of 0.88mg/mL. The SEM analysis revealed that eggs treated with MuLL for 24 and 48h no longer had tubercles and did not show a well-defined exochorionic network. In addition, deformation and degeneration of the surface were observed after 72h. Fluorescence microscopy showed that MuLL penetrated the eggs 48h after incubation and was detected in the upper portion of the embryo's gut. After 72h, MuLL was observed in the serosal cuticle and digestive tract. In conclusion, MuLL can function as an ovicidal agent against A. aegypti through damage to the surface and internal structures of the eggs.

Comparative structural and functional analysis of STL and SLL, chitin-binding lectins from Solanum spp.

Therapeutically important chitin-binding lectins have been already reported in the literature for Solanum tuberosum and Solanum lycopersicum, but their structural data are unavailable. Therefore, we have done comparative structural and functional analysis of chitin-binding lectins from S. tuberosum (STL) and S. lycopersicum (SLL). From the sequence analysis, it has been observed that there is high percentage of proline residues in STL and SLL, 21% and 30% respectively. We utilized the hybrid homology modeling-ab initio approaches to predict the 3D structures of STL and SLL, which are used for in silico interaction studies with N,N’-Diacetylchitobiose, Triacetylchitotriose and Tetra-N-acetylglucosamine. The best STL-glycan and SLL-glycan complexes were subjected to Molecular dynamics simulation to understand and compare the structural stability and the binding patterns of glycans toward STL and SLL. We observed that the structural stability of the STL and SLL had been improved significantly due to the binding of glycans. Together with the results of global, essential dynamics and MM-PBSA analysis, indicated that N,N’-Diacetylchitobiose has more binding affinity towards STL, whereas Triacetylchitotriose has more binding affinity with SLL. This comprehensive and comparative structural and functional analysis provides critical insights about the structures and their binding sites, binding orientation, and binding affinity of chitin oligomers towards the structures of STL and SLL. These findings can be used to design further experimental studies to explore the potential therapeutic properties of STL and SLL. Communicated by Ramaswamy H. Sarma

Structural and functional characterization of chitin binding lectin from Datura stramonium: insights from phylogenetic analysis, protein structure prediction, molecular docking and molecular dynamics simulation

Chitin binding lectin, found in seeds of Datura stramonium (DSL), is an important glycan binding protein that has great therapeutic properties. The objective of the study is to understand the evolutionary significance, structural and functional characterization of chitin binding lectin from D. stramonium, thus will facilitate to explore in deeper structural insights about the protein and its interactions with substrates. In this study, initially the sequence analysis was performed for chitin binding lectin to understand the sequential properties followed by using similarity search, multiple sequence alignment and phylogenetic analysis to identify the closely related protein sequences of DSL. After this, we utilized hybrid homology modeling-ab initio approaches to predict the 3D model of DSL, which is subsequently used for interaction studies with four ligands namely N,N′-Diacetylchitobiose, Triacetylchitotriose and Chitin tetramer, which are all oligomers of chitin. Docking analysis was also performed for N-Acetyllactosamine, which is reported as a potent inhibitor of haemagglutination by Datura lectin. Interestingly we observed two binding sites of substrate. The active site residues in predicted binding site are Glu272, Arg62 and Thr246. Moreover, the best four DSL-ligand complexes along with unbounded form of DSL were subjected to MD simulation to understand the structural stability, integrity and compactness. Together the results of docking and MD simulation, the chitotriose oriented in center of the DSL showing more binding affinity towards binding pocket of DSL. This comprehensive analysis of DSL provides key insights about the structure, active site, binding affinity and mode of binding of the substrates. Communicated by Ramaswamy H. Sarma

Succinylated Wheat Germ Agglutinin Colocalizes with the Toxoplasma gondii Cyst Wall Glycoprotein CST1.

The glycosylated mucin domain of the Toxoplasma gondii cyst wall glycoprotein CST1 is heavily stained by Dolichos biflorus agglutinin, a lectin that binds to N-acetylgalactosamine. The cyst wall is also heavily stained by the chitin binding lectin succinylated wheat germ agglutinin (s-WGA), which selectively binds to N-acetylglucosamine-decorated structures. Here, we tracked the localization of N-acetylglucosamine-decorated structures that bind to s-WGA in immature and mature in vitro cysts. s-WGA localization was observed at the cyst periphery 6 h after the differentiation of the tachyzoite-stage parasitophorous vacuole. By day 1 and at all later times after differentiation, s-WGA was localized in a continuous staining pattern at the cyst wall. Coinciding with the maturation of the cyst matrix by day 3 of cyst development, s-WGA also localized in a continuous matrix pattern inside the cyst. s-WGA localized in both the outer and inner layer regions of the cyst wall and in a continuous matrix pattern inside mature 7- and 10-day-old cysts. In addition, s-WGA colocalized in the cyst wall with CST1, suggesting that N-acetylglucosamine- and N-acetylgalactosamine-decorated molecules colocalized in the cyst wall. In contrast to CST1, GRA4, and GRA6, the relative accumulation of the molecules that bind s-WGA in the cyst wall was not dependent on the expression of GRA2. Our results suggest that GRA2-dependent and GRA2-independent mechanisms regulate the trafficking and accumulation of glycosylated molecules that colocalize in the cyst wall.IMPORTANCE Chronic Toxoplasma gondii infection is maintained in the central nervous system by thick-walled cysts. If host immunity wanes, cysts recrudesce and cause severe and often lethal toxoplasmic encephalitis. Currently, there are no therapies to eliminate cysts, and little biological information is available regarding cyst structure(s). Here, we investigated cyst wall molecules recognized by succinylated wheat germ agglutinin (s-WGA), a lectin that specifically binds to N-acetylglucosamine-decorated structures. N-Acetylglucosamine regulates cell signaling and plays structural roles at the cell surface in many organisms. The cyst wall and cyst matrix were heavily stained by s-WGA in mature cysts and were differentially stained during cyst development. The relative accumulation of molecules that bind to s-WGA in the cyst wall was not dependent on the expression of GRA2. Our findings suggest that glycosylated cyst wall molecules gain access to the cyst wall via GRA2-dependent and GRA2-independent mechanisms and colocalize in the cyst wall.

Bacterial expression, purification and biophysical characterization of wheat germ agglutinin and its four hevein-like domains.

Wheat germ agglutinin (WGA), a chitin binding lectin, has attracted increasing interest because of its unique characteristics such as conformational stability, binding specificity and transcytosis capacity. To pave the way for the study of the molecular basis of WGA's structural stability and binding capacity, as well as to facilitate its use in biomedical and biotechnological developments, we produced recombinant WGA and its 4 isolated hevein-like domains in a bacterial system. All the proteins were expressed as fusion constructs linked to a thioredoxin domain, which was enzymatically or chemically released. The structural and ligand-binding properties of recombinant WGA were similar to the wild lectin. The 4 isolated domains folded and were ligand-binding competent, indicating that each domain constitutes an independent folding unity. The biophysical characterization of the recombinant domains sheds new light on the intricate folding and binding behavior of this emblematic lectin.

A rapid and effective method for purification of a heat-resistant lectin from potato (Solanum tuberosum) tubers.

The potato lectin has been identified to consist of two chitin-binding modules, each containing twin hevein domains. Based on the thermotolerance of the hevein polypeptide, a simple, rapid, and effective protocol for the small-scale purification of the potato lectin has been developed in this study. The method involves only one anion exchange chromatographic step beyond the ammonium sulfate precipitation and the heating treatment. With this method, the potato lectin, a glycoprotein with molecular mass of approximately 60kDa was found and purified to homogeneity with 9513.3 u/mg of specific hemagglutination (HA) activity in 76.8% yield. The homogeneity was confirmed by SDS-PAGE electrophoresis and reverse-phase HPLC analysis. The purified lectin was identified using MS-based peptide sequencing (MALDI-TOF/TOF) and showed a 100% Confidence Interval as being homologous to hevein domains in potato lectin. The periodic acid-Schiff staining and ferric-orcinol assay for pentose, as well as its HA activity inhibition by chitosan oligomers further confirmed the purified lectin as a potato chitin-binding lectin. It is noteworthy that the purified potato lectin exhibited heat resistance, by which, together with a short time precipitation by ammonium sulfate, more than 96% of the total proteins in the crude extract were removed. The lectin therefore was easily resolved from the other remining proteins on a DEAE-methyl polyacrylate column.

Functional characterization of chitin-binding lectin from Solanum integrifolium containing anti-fungal and insecticidal activities

BackgroundAlong with the rapid development of glycomic tools, the study of lectin–carbohydrate interactions has expanded, opening the way for applications in the fields of analytic, diagnostic, and drug delivery. Chitin-binding lectins (CBLs) play roles in immune defense against chitin-containing pathogens. CBLs from species of the Solanaceae family, such as tomato, potato and jimsonweed, display different binding specificities to sugar chains containing poly-N-acetyllactosamine.ResultsIn this report, CBLs from Solanum integrifolium were isolated by ion exchange chromatography. The fractions showed hemagglutination activity (HA). The recombinant CBL in the 293F cell culture supernatant was able to inhibit the growth of Rhizoctonia solani and Colletotrichum gloeosporioide. Furthermore, the carbohydrate-binding property of CBLs was confirmed with the inhibition of HA. Binding of CBL to Spodoptera frugiperda (sf21) insect cells can partly be inhibited by N-Acetylglucosamine (GlcNAc), which is related to decrease mitochondrial membrane potential of sf21 cells.ConclusionsThe results showed that CBL exhibited antifungal properties and inhibited insect cell growth, which is directly correlated to the lectin-carbohydrate interaction. Further identification and characterization of CBLs will help to broaden their scope of application in plant defense and in biomedical applications.

PgTeL, the lectin found in Punica granatum juice, is an antifungal agent against Candida albicans and Candida krusei

The pomegranate (Punica granatum) sarcotesta contains a chitin-binding lectin (PgTeL) with antibacterial activity against human pathogenic species. In this work, the structural stability of PgTeL was evaluated by fluorimetric analysis and the lectin was evaluated for cytotoxicity to human peripheral blood mononuclear cells (PBMCs) and antifungal activity against Candida albicans and Candida krusei. PgTeL folding was impaired when lectin was incubated at pH≥6.0. On the other hand, the lectin did not undergo unfolding even when heated at 100°C. PgTeL (1, 10, and 100μg/mL) was not cytotoxic to PBMCs. Antifungal activity was detected for C. albicans (MIC: 25μg/mL; MFC: 50μg/mL) and C. krusei (MIC and MFC of 12.5μg/mL). Treatment of yeast cells with PgTeL resulted in decrease of intracellular ATP content even at sub-inhibitory concentrations (½MIC and ¼MIC) and induced lipid peroxidation. In addition, PgTeL damaged the integrity of fungal cell wall of both species, with more pronounced effects in C. krusei. The lectin showed significant antibiofilm activity on C. albicans at sub-inhibitory concentrations (0.195 and 0.39μg/mL). In conclusion, PgTeL is an anti-Candida agent whose action mechanism involves oxidative stress, energetic collapse, damage to the cell wall and rupture of yeast cells.

Sugar-Binding Profiles of Chitin-Binding Lectins from the Hevein Family: A Comprehensive Study.

Chitin-binding lectins form the hevein family in plants, which are defined by the presence of single or multiple structurally conserved GlcNAc (N-acetylglucosamine)-binding domains. Although they have been used as probes for chito-oligosaccharides, their detailed specificities remain to be investigated. In this study, we analyzed six chitin-binding lectins, DSA, LEL, PWM, STL, UDA, and WGA, by quantitative frontal affinity chromatography. Some novel features were evident: WGA showed almost comparable affinity for pyridylaminated chitotriose and chitotetraose, while LEL and UDA showed much weaker affinity, and DSA, PWM, and STL had no substantial affinity for the former. WGA showed selective affinity for hybrid-type N-glycans harboring a bisecting GlcNAc residue. UDA showed extensive binding to high-mannose type N-glycans, with affinity increasing with the number of Man residues. DSA showed the highest affinity for highly branched N-glycans consisting of type II LacNAc (N-acetyllactosamine). Further, multivalent features of these lectins were investigated by using glycoconjugate and lectin microarrays. The lectins showed substantial binding to immobilized LacNAc as well as chito-oligosaccharides, although the extents to which they bound varied among them. WGA showed strong binding to heavily sialylated glycoproteins. The above observations will help interpret lectin-glycoprotein interactions in histochemical studies and glyco-biomarker investigations.

A chitin-binding lectin from Moringa oleifera seeds (WSMoL) impairs the digestive physiology of the Mediterranean flour larvae, Anagasta kuehniella

Biotechnological techniques allow the investigation of alternatives to outdated chemical insecticides for crop protection; some investigations have focused on the identification of molecules tailored from nature for this purpose. We, herein, describe the negative effects of water-soluble lectin from Moringa oleifera seeds (WSMoL) on Anagasta kuehniella development. The chitin-binding lectin, WSMoL, impaired the larval weight gain by 50% and affected the activity of the pest's major digestive enzymes. The commitment of the digestive process became evident after controlled digestion studies, where the capacity of protein digestion was compromised by >90%. Upon acute exposure, the lectin was not resistant to digestion; however, chronic ingestion of WSMoL was able to reverse this feature. Thus, we show that resistance to digestion may not be a prerequisite for a lectin's ability to exert negative effects on larval physiology. The mechanism of action of WSMoL involves binding to chitin with possible disruption to the peritrophic membrane, causing disorder between the endo- and ectoperitrophic spaces. Additionally, results suggest that WSMoL may trigger apoptosis in gut cells, leading to the lower enzymatic activity observed in WSMoL-fed larvae. Although assays employing an artificial diet did not demonstrate effects of WSMoL on A. kuehniella mortality, this lectin may hold potential for exerting insecticide effects on other pest insects, as well for use in other experimental approaches, such as WSMoL-expressing plants. Moreover, the use of WSMoL with other biotechnological tools, such as ‘pyramid’ crops, may represent a strategy for delaying the evolution of pest resistance to transgenic crops, since its multiple site targets could act in synergism with other insecticide compounds.

Structural Analysis of an Avr4 Effector Ortholog Offers Insight into Chitin Binding and Recognition by the Cf-4 Receptor.

Chitin is a key component of fungal cell walls and a potent inducer of innate immune responses. Consequently, fungi may secrete chitin-binding lectins, such as the Cf-Avr4 effector protein from the tomato pathogen Cladosporium fulvum, to shield chitin from host-derived chitinases during infection. Homologs of Cf-Avr4 are found throughout Dothideomycetes, and despite their modest primary sequence identity, many are perceived by the cognate tomato immune receptor Cf-4. Here, we determined the x-ray crystal structure of Pf-Avr4 from the tomato pathogen Pseudocercospora fuligena, thus providing a three-dimensional model of an Avr4 effector protein. In addition, we explored structural, biochemical, and functional aspects of Pf-Avr4 and Cf-Avr4 to further define the biology of core effector proteins and outline a conceptual framework for their pleiotropic recognition by single immune receptors. We show that Cf-Avr4 and Pf-Avr4 share functional specificity in binding (GlcNAc)6 and in providing protection against plant- and microbial-derived chitinases, suggesting a broader role beyond deregulation of host immunity. Furthermore, structure-guided site-directed mutagenesis indicated that residues in Pf-Avr4 important for binding chitin do not directly influence recognition by Cf-4 and further suggested that the property of recognition is structurally separated or does not fully overlap with the virulence function of the effector.

High-throughput transcriptome analysis of barley (Hordeum vulgare) exposed to excessive boron

Boron (B) is an essential micronutrient for optimum plant growth. However, above certain threshold B is toxic and causes yield loss in agricultural lands. While a number of studies were conducted to understand B tolerance mechanism, a transcriptome-wide approach for B tolerant barley is performed here for the first time. A high-throughput RNA-Seq (cDNA) sequencing technology (Illumina) was used with barley (Hordeum vulgare), yielding 208 million clean reads. In total, 256,874 unigenes were generated and assigned to known peptide databases: Gene Ontology (GO) (99,043), Swiss-Prot (38,266), Clusters of Orthologous Groups (COG) (26,250), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) (36,860), as determined by BLASTx search. According to the digital gene expression (DGE) analyses, 16% and 17% of the transcripts were found to be differentially regulated in root and leaf tissues, respectively. Most of them were involved in cell wall, stress response, membrane, protein kinase and transporter mechanisms. Some of the genes detected as highly expressed in root tissue are phospholipases, predicted divalent heavy-metal cation transporters, formin-like proteins and calmodulin/Ca2+-binding proteins. In addition, chitin-binding lectin precursor, ubiquitin carboxyl-terminal hydrolase, and serine/threonine-protein kinase AFC2 genes were indicated to be highly regulated in leaf tissue upon excess B treatment. Some pathways, such as the Ca2+–calmodulin system, are activated in response to B toxicity. The differential regulation of 10 transcripts was confirmed by qRT-PCR, revealing the tissue-specific responses against B toxicity and their putative function in B-tolerance mechanisms.

English

Cytotoxicity of tuber lectins from two potato cultivars was assessed and their anti-tumor potential against experimentally induced Ehrlich ascites carcinoma in Swiss albino mice was evaluated. Twenty (20) kDa chitin-binding lectins from Solanum tuberosum tubers, STL-S and STL-D were purified through ion-exchange and affinity chromatographic methods, hemagglutinating activity and blood group specificity of the lectins were checked whereas the cytotoxicity was determined using brine shrimp (Artemia salina L.) nauplii lethality assay. The lectins showed no specificity to animal and human erythrocytes. LC50 values for STL-S and STL-D were found to be 75 and 90 μg/ml, respectively with a dose-dependent intermediary toxic effect. After inducing ascites by intraperitoneal propagation, the Swiss albino mice were treated by administering the lectins at a dose of 1.38 mg/kg/day for five consecutive days. STL-S and STL-D showed 79.84 and 83.04% of growth inhibition of EAC cells, respectively. Additionally, hemoglobin and RBC levels became considerably increased with a drop off in the WBC levels in the treated mice group indicating moderate anticancer activities exhibited by the potato lectins.   Key words: Chitin-binding lectins, antitumor activity, LC50, cell growth inhibition.