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Background: Chitinases are plant defense-related proteins with a high biotechnological potential to be applied in agriculture. Objectives: This study aimed to purify a chitinase from the latex of Ficus benjamina. Methods: An antifungal class I chitinase, named FbLx-Chi-1, was purified from the latex of Ficus benjamina after precipitation with 30-60% ammonium sulfate and affinity chromatography on a chitin column and antifungal potential assay against phytopathogenic fungi important to agriculture. Results: FbLx-Chi-1 has 30 kDa molecular mass, as estimated by SDS-PAGE and the optimal pH and temperature for full chitinolytic activity were 5.5 and 60 ºC, respectively. FbLx-Chi-1 is a high pH-, ion-tolerant and thermostable protein. Importantly, FbLx-Chi-1 hindered the growth of the phytopathogenic fungi Colletotrichum gloeosporioides, Fusarium pallidoroseum, and Fusarium oxysporum. The action mode of FbLx-Chi-1 to hamper F. pallidoroseum growth seems to be correlated with alterations in the morphology of the hyphal cell wall, increased plasma membrane permeability, and overproduction of reactive oxygen species. Conclusion: These findings highlight the biotechnological potential of FbLx-Chi-1 to control important phytopathogenic fungi in agriculture. In addition, FbLx-Chi-1 could be further explored to be used in industrial processes such as the large-scale environmentally friendly enzymatic hydrolysis of chitin to produce its monomer N-acetyl-β-D-glucosamine, which is employed for bioethanol production, in cosmetics, in medicine, and for other multiple applications.

The textile industry is essential, but it is also responsible for causing environmental problems, particularly the discharge of dyes. In this context, this study aimed to immobilize a previously purified peroxidase, called MoPOX, on glutaraldehyde-activated chitosan beads in order to improve the potential for textile dye decolorization. The chitosan beads were activated with 8% glutaraldehyde for 1 h, and the immobilization was performed at 30 °C, pH 5.2 for 4 h. Scanning Electron Microscopy (SEM) analyses were used to observe the differences in the chitosan beads after immobilization. The optimum temperature dropped from 70 °C to 30 °C after immobilization, but immobilized MoPOX demonstrated excellent heat stability. The optimum pH remained 5.2, while the apparent kinetic constant value (Km) of immobilized MoPOX (14.67 mM) was lower in comparison to free MoPOX (46.8 mM). The immobilized enzyme showed improved activity after long storage times, and it could retain 40 % of its original activity even after 5 cycles. The potential for decolorization of different textile dyes was considerably enhanced after immobilization, reaching more than 80 %. Also, MoPOX showed no toxicity towards Artemia salina. Overall, the findings point to the promising potential of using immobilized MoPOX as a biocatalyst in a variety of biotechnological applications.

BACKGROUND Disease vector insects are barriers for human development. The use of synthetic chemicals to control these vectors has caused damage to the environment and contributed to the arising of resistant insect populations. This has led to an increased search for plant-derived molecules with insecticidal activity or that show synergistic effects with known insecticidal substances, such as protease inhibitors. Thus, we aimed to evaluate the effect of Enterolobium contortisiliquum trypsin inhibitor (EcTI) on Aedes aegypti development as well as its effect on insecticidal activity of Bacillus thuringiensis toxins. RESULTS EcTI showed an apparent molecular mass about of 20 kDa in SDS-PAGE and was able to inhibit in vitro the activity of trypsin and proteases from midgut of Ae. aegypti larvae. EcTI was not able to cause acute toxicity on mosquito larvae even at 1000 μg mL-1 , however it promoted a delay in larval development after prolonged exposure. The zymogram results for EcTI-treated larvae (from 50 to 200 μg mL-1 ) showed an increase of midgut proteases activity as a larvae defense mechanism, however no changes in the enzyme profile was observed. These same concentrations were able to enhance up to three fold the insecticidal activity of B. thuringiensis toxins without causing toxicity to Artemia sp. nauplii, a non-target organism. CONCLUSIONS The results offer a novel approach by combining EcTI and B. thuringiensis toxins for combating Ae. aegypti larvae. © 2020 Society of Chemical Industry.

Cowpea miRNAs and Argonaute genes showed differential expression patterns in response to CPSMV challenge Several biotic stresses affect cowpea production and yield. CPSMV stands out for causing severe negative impacts on cowpea. Plants have two main induced immune systems. In the basal system (PTI, PAMP-triggered immunity), plants recognize and respond to conserved molecular patterns associated with pathogens (PAMPs). The second type (ETI, Effector-triggered immunity) is induced after plant recognition of specific factors from pathogens. RNA silencing is another important defense mechanism in plants. Our research group has been using biochemical and proteomic approaches to learn which proteins and pathways are involved and could explain why some cowpea genotypes are resistant whereas others are susceptible to CPSMV. This current study was conducted to determine the role of cowpea miRNA in the interaction between a resistant cowpea genotype (BRS-Marataoã) and CPSMV. Previously identified and deposited plant microRNA sequences were used to find out all possible microRNAs in the cowpea genome. This search detected 617 mature microRNAs, which were distributed in 89 microRNA families. Next, 4 out of these 617 miRNAs and their possible target genes that encode the proteins Kat-p80, DEAD-Box, GST, and SPB9, all involved in the defense response of cowpea to CPSMV, had their expression compared between cowpea leaves uninoculated and inoculated with CPSMV. Additionally, the differential expression of genes that encode the Argonaute (AGO) proteins 1, 2, 4, 6, and 10 is reported. In summary, the studied miRNAs and AGO 2 and AGO4 associated genes showed differential expression patterns in response to CPSMV challenge, which indicate their role in cowpea defense.

Dermatophytes belonging to Trichophyton ssp. are important anthropophilic and zoophilic pathogens, which developed resistance to griseofulvin, the common antifungal drug used to treat dermatophytosis. In this context, Moringa oleifera seed proteins have been described as antifungal agents with potential applications. Thus, this work aimed to evaluate the antidermatophytic in vitro, focusing on mechanisms, and in vivo potential of Mo-CBP4, purified from M. oleifera seeds. Mo-CBP4was purified after protein extraction with 50 mM Tris-HCl buffer, pH 8.0, and chromatography on chitin and CM Sepharose™ columns and antidermatophytic potential of Mo-CBP4 evaluated in vitro and in vivo. In vitro, Mo-CBP4 reduced in 50% the germination of microconidia of Trichophyton mentagrophytes at 45 μM; but did not show inhibition of mycelial growth. Mo-CBP4 (45 μM) presents the inhibitory activity even when incubated with N-acetyl- d -glucosamine (NAG). Analysis of the mechanisms of Mo-CBP4 revealed an increase in membrane permeability, ROS overproduction and damage to cell wall leading to microconidia death. Furthermore, using in vivo models, Mo-CBP4 (5, 10 and 20 mg g−1) reduced the severity and time of dermatophytosis. Altogether, these findings indicate that Mo-CBP4 has great potential for the development of novel antifungal drugs for the clinical treatment of dermatophytosis.

Plant proteins are emerging as an alternative to conventional treatments against candidiasis. The aim of this study was to better understand the mechanism of action of Mo-CBP2 against Candida spp, evaluating redox system activity, lipid peroxidation, DNA degradation, cytochrome c release, medium acidification, and membrane interaction. Anti-candida activity of Mo-CBP2 decreased in the presence of ergosterol, which was not observed with antioxidant agents. C. albicans treated with Mo-CBP2 also had catalase and peroxidase activities inhibited, while superoxide dismutase was increased. Mo-CBP2 increased the lipid peroxidation, but it did not alter the ergosterol profile in live cells. External medium acidification was strongly inhibited, and cytochrome c release and DNA degradation were detected. Mo-CBP2 interacts with cell membrane constituents, changes redox system enzymes in C. albicans and causes lipid peroxidation by ROS overproduction. DNA degradation and cytochrome c release suggest apoptotic or DNAse activity. Lipid peroxidation and H+-ATPases inhibition may induce the process of apoptosis. Finally, Mo-CBP2 did not have a cytotoxic effect in mammalian Vero cells. This study highlights the biotechnological potential of Mo-CBP2 as a promising molecule with low toxicity and potent activity. Further studies should be performed to better understand its mode of action and toxicity.

Mo-CBP3 is a chitin-binding 2S albumin from Moringa oleifera. This seed storage protein is resistant to thermal denaturation and shows biological activities that might be of practical use, such as antifungal properties against Candida sp., a pathogen that causes candidiasis, and against Fusarium solani, a soil fungus that can cause diseases in plants and humans. Previous work has demonstrated that Mo-CBP3 is a mixture of isoforms encoded by members of a small multigene family. Mature Mo-CBP3 is a small protein (∼14 kDa), constituted by a small chain of approximately 4 kDa and a large chain of 8 kDa, which are held together by disulfide bridges. However, a more comprehensive picture on the spectrum of Mo-CBP3 isoforms which are found in mature seeds, is still lacking. In this work, genomic DNA fragments were obtained from M. oleifera leaves, cloned and completely sequenced, thus revealing new genes encoding Mo-CBP3. Moreover, mass spectrometry analysis showed that the mature protein is a complex mixture of isoforms with a remarkable number of molecular mass variants. Using computational predictions and calculations, most (∼86%) of the experimentally determined masses were assigned to amino acid sequences deduced from DNA fragments. The results suggested that the complex mixture of Mo-CBP3 isoforms originates from proteins encoded by closely related genes, whose products undergo different combinations of distinct post-translational modifications, including cleavage at the N- and C-terminal ends of both subunits, cyclization of N-terminal Gln, as well as Pro hydroxylation, Ser/Thr phosphorylation, and Met oxidation.

In nature, plants are simultaneously challenged by biotic and abiotic stresses. However, little is known about the effects of these combined stresses for most crops. This work aimed to evaluate the responsed of the virus-resistant cowpea genotype BRS-Marataoa to the exposure of salt stress combined with CPSMV infection. Cowpea plants were exposed to 200 mM NaCl either simultaneously (SV plant group) or 24 h prior to the CPSMV infection [S(24 h)V plant group]. Physiological, biochemical, and proteomic analyses at 2 and 6 days post salt stress (DPS) revealed that cowpea significantly reprogrammed its cellular metabolism. Indeed, plant size, photosynthetic parameters (net photosynthesis, transpiration rate, stomatal conductance, and internal CO2 partial pressure) and chlorophyll and carotenoid contents were reduced in S(24 h)V compared to SV. Moreover, accumulation of viral particles at 6 DPS in S(24 h)V was observed indicating that the salt stress imposed prior to virus infection favors viral particle proliferation. Proteomic analysis showed differential contents of 403 and 330 proteins at 2 DPS and 6 DPS, respectively, out of 733 differentially abundant proteins between the two plant groups. The altered leaf proteins are involved in energy and metabolism, photosynthesis, stress response, and oxidative burst. Biological significance This is an original study in which a virus-resistant cowpea genotype (BRS-Marataoa) was (i) exposed simultaneously to 200 mM NaCl and inoculation with CPSMV (SV plant group) or (ii) exposed to 200 mM NaCl stress 24 h prior to inoculation with CPSMV [S(24 h)V plant group]. The purpose was to shed light on how this CPSMV resistant cowpea responded to the combined stresses. Numerous key proteins and associated pathways were altered in the cowpea plants challenged with both stresses, but unexpectedly, the salt stress imposed 24 h prior to CPSMV inoculation allowed viral proliferation, turning the cowpea genotype from resistant to susceptible.

The efficiency of current antimicrobial drugs is noticeably decreasing and thus the development of new treatments is necessary. Natural and synthetic antimicrobial peptides (AMPs) have attracted great attention as promising candidates. Inspired on Mo-CBP3, an antimicrobial protein from Moringa oleifera seeds, we designed and synthesized three AMPs named Mo-CBP3-PepI, Mo-CBP3-PepII, and Mo-CBP3-PepIII. All these three peptides inhibited the growth of Candida species and pathogenic bacteria, penetrate into microbial cells, but none is hemolytic or toxic to human cells. Mo-CBP3-PepIII, particularly, showed the strongest antimicrobial activity against Staphylococcus aureus and Candida species, important human pathogens. Additionally, Mo-CBP3-PepIII did not exhibit hemolytic or toxic activity to mammalian cells, but increased Staphylococcus aureus plasma membrane permeabilization. In Candida parapsilosis, Mo-CBP3-PepIII induced pore formation in the plasma membrane and overproduction of reactive oxygen species. Bioinformatics analysis suggested that Mo-CBP3-PepIII is resistant to pepsin digestion and other proteolytic enzymes present in the intestinal environment, which opens the possibility of oral delivery in future treatments. Together, these results suggest that Mo-CBP3-PepIII has great potential as an antimicrobial agent against the bacterium S. aureus and the fungi C. parapsilosis.

Correlations between the transcriptional responses of genes that encode superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and peroxiredoxin (Prx) enzymes and Colletotrichum gloeosporioides development in cowpea leaves were assessed. Each of these genes is involved in the redox metabolism and hydrogen peroxide balance. Although electron microscopy revealed that conidia adhered to and germinated on the leaf cuticle, the inoculated cowpea leaves did not show any characteristic anthracnose symptoms. The adhered and germinated conidia showed irregular surfaces and did not develop further. This was apparently due to increased leaf H2O2 levels in response to inoculation with C. gloeosporioides. During the early stages post inoculation, cowpea leaves elevated the H2O2 content and modulated the defense gene expression, as well as associated pathways. During the later stages, the increased expression of the CuZnSODI and CuZnSODII genes suggested an active superoxide dismutation to further elevate H2O2 levels, which indicated that higher H2O2 content may function as a toxic agent that kills the fungus. The second increase in H2O2 production above the threshold level was correlated with the expression of the APXI, CATI, CATII, PrxIIBCD, and PrxIIE genes, which resulted in a coordinated pattern to establish an appropriate balance between H2O2 generation and scavenging. Therefore, appropriate H2O2 content in cowpea leaves inhibited C. gloeosporioides development and maintained intracellular redox homeostasis to avoid uncontrolled programmed cell death and necrosis in cowpea leaves.

Chitin-binding proteins behave as storage and antifungal proteins in the seeds of Moringa oleifera. Moringa oleifera is a tropical multipurpose tree. Its seed constituents possess coagulant, bactericidal, fungicidal, and insecticidal properties. Some of these properties are attributed to a group of polypeptides denominated M. oleifera chitin-binding proteins (in short, Mo-CBPs). Within this group, Mo-CBP2, Mo-CBP3, and Mo-CBP4 were previously purified to homogeneity. They showed high amino acid similarity with the 2S albumin storage proteins. These proteins also presented antimicrobial activity against human pathogenic yeast and phytopathogenic fungi. In the present study, the localization and expression of genes that encode Mo-CBPs and the biosynthesis and degradation of the corresponding proteins during morphogenesis and maturation of M. oleifera seeds at 15, 30, 60, and 90 days after anthesis (DAA) and germination, respectively, were assessed. The Mo-CBP transcripts and corresponding proteins were not detected at 15 and 30 days after anthesis (DAA). However, they accumulated at the latter stages of seed maturation (60 and 90 DAA), reaching the maximum level at 60 DAA. The degradation kinetics of Mo-CBPs during seed germination by in situ immunolocalization revealed a reduction in the protein content 48 h after sowing (HAS). Moreover, Mo-CBPs isolated from seeds at 60 and 90 DAA prevented the spore germination of Fusarium spp. Taken together, these results suggest that Mo-CBPs play a dual role as storage and defense proteins in the seeds of M. oleifera.

Staphylococcus aureus is a multidrug-resistant bacterium responsible for several cases of hospital-acquired infections, which constitute a global public health problem. The introduction of new healthcare strategies and/or the discovery of molecules capable of inhibiting the growth or killing S. aureus would have a huge impact on the treatment of S. aureus-mediated diseases. Herein, a Bowman–Birk protease inhibitor (LzaBBI), with strong in vitro antibacterial activity against S. aureus, was purified to homogeneity from Luetzelburgia auriculata seeds. LzaBBI in its native form is a 14.3 kDa protein and has a pI of 4.54, and its NH2-terminal sequence has high identity with other Bowman–Birk inhibitors. LzaBBI showed a mixed-type inhibitory activity against both trypsin and chymotrypsin, respectively, and it remained stable after both boiling at 98 °C for 120 min and incubation at various pHs. Scanning electron microscopy revealed that LzaBBI disrupted the S. aureus membrane integrity, leading to bacterial de...

Various plant species have long been used in traditional medicine worldwide to treat diabetes. Among the plant-based compounds with hypoglycemic properties, studies on insulin-like proteins isolated from leaves, fruits and seeds are rarely reported in the relevant literature. Our research group has been investigating the presence of insulin-like proteins in Moringa oleifera, a plant species native to India, and we have obtained a leaf protein isolate and semi-purified derived fractions, as well as a seed coat protein fraction (Mo-SC), with hypoglycemic activity in chemically induced diabetic mice that have increased tolerance to orally administered glucose. Equally importantly, Mo-SC possesses insulin-like antigenic epitopes. In this context, the present review aims to highlight that prospection of insulin-like proteins in plants is of the utmost importance both for finding new drugs for the treatment of diabetes and for shedding light on the mechanisms involved in diabetes.

Proteins extracted from Capsicum annuum L. fruits were initially subjected to reversed-phase chromatography on HPLC, resulting in eight peptide-rich fractions. All the fractions obtained were tested for their ability to inhibit porcine trypsin and amylase from both human saliva and from larval insect in vitro. All fractions were also tested for their ability to inhibit growth of the phytopathogenic fungi. Several fractions inhibited the activity of human salivary amylase and larval insect amylase, especially fraction Fa5. No fraction tested was found to inhibit trypsin activity, being Fa2 fraction an exception. Interestingly fraction Fa5 also displayed high antimicrobial activity against the species of the Fusarium genus. Fraction Fa5 was found to have two major protein bands of 17 and 6.5 kDa, and these were sequenced by mass spectrometry. Two peptides were obtained from the 6.5-kDa band, which showed similarity to antimicrobial peptides. Fraction Fa5 was also tested for its ability to permeabilize membranes and induce ROS. Fraction Fa5 was able to permeabilize the membranes of all the fungi tested. Fungi belonging to the genus Fusarium also showed an increase in the endogenous production of ROS when treated with this fraction. Antimicrobial peptides were also identified in the fruits from other Capsicum species.

A cowpea class I chitinase (VuChiI) was expressed in the methylotrophic yeast P. pastoris. The recombinant protein was secreted into the culture medium and purified by affinity chromatography on a chitin matrix. The purified chitinase migrated on SDS-polyacrylamide gel electrophoresis as two closely-related bands with apparent molecular masses of 34 and 37 kDa. The identity of these bands as VuChiI was demonstrated by mass spectrometry analysis of tryptic peptides and N-terminal amino acid sequencing. The recombinant chitinase was able to hydrolyze colloidal chitin but did not exhibit enzymatic activity toward synthetic substrates. The highest hydrolytic activity of the cowpea chitinase toward colloidal chitin was observed at pH 5.0. Furthermore, most VuChiI activity (approximately 92%) was retained after heating to 50 °C for 30 min, whereas treatment with 5 mM Cu2+ caused a reduction of 67% in the enzyme's chitinolytic activity. The recombinant protein had antifungal activity as revealed by its ability to inhibit the spore germination and mycelial growth of Penicillium herquei. The three-dimensional structure of VuChiI was resolved at a resolution of 1.55 A by molecular replacement. The refined model had 245 amino acid residues and 381 water molecules, and the final R-factor and Rfree values were 14.78 and 17.22%, respectively. The catalytic domain of VuChiI adopts an α-helix-rich fold, stabilized by 3 disulfide bridges and possessing a wide catalytic cleft. Analysis of the crystallographic model and molecular docking calculations using chito-oligosaccharides provided evidences about the VuChiI residues involved in sugar binding and catalysis, and a possible mechanism of antifungal action is suggested.

Jaburetox (JBTX) is an insecticidal and antifungal peptide derived from jack bean (Canavalia ensiformis) urease that has been considered a candidate for developing genetically modified crops. This study aimed to perform the risk assessment of the peptide JBTX following the general recommendations of the two-tiered, weight-of-evidence approach proposed by International Life Sciences Institute. The urease of C. ensiformis (JBU) and its isoform JBURE IIb (the JBTX parental protein) were assessed. The history of safe use revealed no hazard reports for the studied proteins. The available information shows that JBTX possesses selective activity against insects and fungi. JBTX and JBU primary amino acids sequences showed no relevant similarity to toxic, antinutritional or allergenic proteins. Additionally, JBTX and JBU were susceptible to in vitro digestibility, and JBU was also susceptible to heat treatment. The results did not identify potential risks of adverse effects and reactions associated to JBTX. However, further allergen (e.g. serum IgE binding test) and toxicity (e.g. rodent toxicity tests) experimentation can be done to gather additional safety information on JBTX, and to meet regulatory inquiries for commercial approval of transgenic cultivars expressing this peptide.

Candida albicans has emerged as a major public health problem in recent decades. The most important contributing factor is the rapid increase in resistance to conventional drugs worldwide. Synthetic antimicrobial peptides (SAMPs) have attracted substantial attention as alternatives and/or adjuvants in therapeutic treatments due to their strong activity at low concentrations without apparent toxicity. Here, two SAMPs, named Mo-CBP3 -PepI (CPAIQRCC) and Mo-CBP3 -PepII (NIQPPCRCC), are described, bioinspired by Mo-CBP3 , which is an antifungal chitin-binding protein from Moringa oleifera seeds. Furthermore, the mechanism of anticandidal activity was evaluated as well as their synergistic effects with nystatin. Both peptides induced the production of reactive oxygen species (ROS), cell wall degradation, and large pores in the C. albicans cell membrane. In addition, the peptides exhibited high potential as adjuvants because of their synergistic effects, by increasing almost 50-fold the anticandidal activity of the conventional antifungal drug nystatin. These peptides have excellent potential as new drugs and/or adjuvants to conventional drugs for treatment of clinical infections caused by C. albicans.

Soybean toxin (SBTX) is a protein isolated from soybean seeds and composed of two polypeptide subunits (17 and 27 kDa). SBTX has in vitro activity against phytopathogenic fungi such as Cercospora sojina, Aspergillus niger, and Penicillium herguei, and yeasts like Candida albicans, C. parapsilosis, Kluyveromyces marxiannus, and Pichia membranifaciens. The present study aimed to analyze in vitro whether SBTX causes any side effects on non-target bacterial and mammalian cells that could impede its potential use as a novel antifungal agent. SBTX at 100 mu g/mL and 200 mu g/mL did not hinder the growth of the bacteria Salmonella enterica (subspecies enterica serovar choleraesuis), Bacillus subtilis (subspecies spizizenii) and Staphylococcus aureus. Moreover, SBTX at concentrations up to 500 mu g/mL did not significantly affect the viability of erythrocytes, neutrophils, and human intestinal Caco-2 cells. To study whether SBTX could induce relevant alterations in gene expression, in vitro DNA microarray experiments were conducted in which differentiated Caco-2 cells were exposed for 24 h to 100 mu g/mL or 200 mu g/mL SBTX. SBTX up-regulated genes involved in cell cycle and immune response pathways, but downregulated genes that play a role in cholesterol biosynthesis and platelet degranulation pathways. Thus, although SBTX did not affect bacteria, nor induced cytotoxity in mammalian cells, it affected some biological pathways in the human Caco-2 cell line that warrants further investigation.

SBTX has defensive role against C. kikuchii, and therefore, its constituent genes SBTX17 and SBTX27 are promising candidates to engineer pathogen resistant plants. Soybean (Glycine max [L.] Merr.) is economically the most important legume crop in the world. Its productivity is strongly affected by fungal diseases, which reduce soybean production and seed quality and cause losses of billions of dollars worldwide. SBTX is a protein that apparently takes part in the defensive chemical arsenal of soybean against pathogens. This current study provides data that reinforce this hypothesis. Indeed, SBTX inhibited in vitro the mycelial growth of Cercospora kikuchii, it is constitutively located in the epidermal region of the soybean seed cotyledons, and it is exuded from mature imbibed seeds. Moreover, RT-qPCR analysis of the SBTX associated genes, SBTX17 and SBTX27, which encode for the 17 and 27 kDa polypeptide chains, showed that both genes are expressed in all studied plant tissues during the soybean development, with the highest levels found in the mature seeds and unifoliate leaves. In addition, to assess a local response of the soybean secondary leaves from 35-day-old plants, they were inoculated with C. kikuchii and treated with salicylic acid. It was verified using RT-qPCR that SBTX17 and SBTX27 genes overexpressed in leaves compared to controls. These findings strongly suggest that SBTX has defensive roles against C. kikuchii. Therefore, SBTX17 and SBTX27 genes are promising candidates to engineer pathogen resistant plants.

Disease vector insects are barriers for human development. The use of synthetic chemicals to control these vectors has caused damage to the environment and contributed to the arising of resistant insect populations. This has led to an increased search for plant-derived molecules with insecticidal activity or that show synergistic effects with known insecticidal substances, such as protease inhibitors. Thus, we aimed to evaluate the effect of Enterolobium contortisiliquum trypsin inhibitor (EcTI) on Aedes aegypti development as well as its effect on insecticidal activity of Bacillus thuringiensis toxins.EcTI showed an apparent molecular mass about of 20 kDa in SDS-PAGE and was able to inhibit in vitro the activity of trypsin and proteases from midgut of Ae. aegypti larvae. EcTI was not able to cause acute toxicity on mosquito larvae even at 1000 μg mLThe results offer a novel approach by combining EcTI and B. thuringiensis toxins for combating Ae. aegypti larvae. © 2020 Society of Chemical Industry.

Plant seeds can exudate active molecules with inhibitory effects against several soil pathogens, including nematodes. This study aimed to characterize and evaluate the nematicidal properties against Meloidogyne incognita of exuded proteins from Moringa oleifera seeds. M. oleifera seeds were soaked in distilled water, and exudates were harvested and analyzed for the presence of defense proteins and anthelmintic activity. Enzymatic assays revealed the existence of PR-proteins such as β-1,3-glucanases (0.18 ± 0.003 nkatal mg−1 of protein), chitinases (0.22 ± 0.004 nkatal mg−1 of protein), proteases (261.30 ± 6.405 AU mg−1 of protein min−1), serine (190.30 ± 5.574 IA mg−1 of protein) and cysteine (231.70 ± 0.923 IA mg−1 of protein), protease inhibitors. The exuded proteins presented ovicidal activity and caused 100% mortality of second-stage juveniles (J2s). Scanning electron microscopy (SEM) revealed deleterious effects on M. incognita eggs, such as invaginations, cracks, scratched surface, and loss of internal content. These findings confirm the presence of anthelmintic proteins in M. oleifera seed exudate, possibly involved in plant defense during seed germination. Besides this, the exuded proteins exhibited strong biotechnological potential for use in the biocontrol of M. incognita infections, which are responsible for millions of dollars in staple crop losses every year.

Antimicrobial peptides (AMPs) are important constituents of the innate immunity system of all living organisms. They participate in the first line of defense against invading pathogens such as viruses, bacteria, and fungi. In view of the increasing difficulties to treat infectious diseases due to the emergence of antibiotic-resistant bacterial strains, AMPs have great potential to control infectious diseases in humans and animals. In this study, two small peptides, RcAlb-PepI and RcAlb-PepII, were designed based on the primary structure of Rc-2S-Alb, a 2S albumin from the seed cake of Ricinus communis, and their antimicrobial activity assessed. RcAlb-PepII strongly inhibited the growth of Klebsiella pneumoniae and Candida parapsilosis, and induced morphological alterations in their cell surface. C. parapsilosis exposed to RcAlb-PepII presented higher cell membrane permeabilization and elevated content of reactive oxygen species. RcAlb-PepII also degraded and reduced the biofilm formation in C. parapsilosis and in K. pneumonia cells. Experimentally, RcAlb-PepII was not hemolytic and had low toxicity to mammalian cells. These are advantageous characteristics, which suggest that RcAlb-PepII is safe and apparently effective for its intended use and has great potential for the future development of an antimicrobial agent with the ability to kill or inhibit K. pneumoniae and C. parapsilosis cells.

Cassia leiandra is an Amazonian plant species that is used popularly for the treatment of mycoses. Recently, a protease inhibitor, named ClTI, with insecticidal activity against Aedes aegypti, was purified from the mature seeds of C. leiandra. In this work, we show that ClTI has antifungal activity against Candida species and describe its mode of action towards Candida albicans. This study is relevant because the nosocomial infections caused by Candida species are a global public health problem that, together with the growing resistance to current drugs, has increased the urgency of the search for new antifungal compounds. ClTI inhibited the growth of Candida albicans, Candida tropicalis, Candida parapsilosis, and Candida krusei. However, ClTI was more potent against C. albicans. The candidicidal mode of action of ClTI on C. albicans involves enhanced cell permeabilization, alteration of the plasma membrane proton-pumping ATPase function (H+ -ATPase), induction of oxidative stress, and DNA damage. ClTI also exhibited antibiofilm activity and non-cytotoxicity to mammalian cells. These results indicate that ClTI is a promising candidate for the future development of a new, natural, and safe agent for the treatment of infections caused by C. albicans.

During the last few years, a growing number of antimicrobial peptides have been isolated from plants and particularly from seeds. Recent results from our laboratory have shown the purification of a new trypsin inhibitor, named CaTI, from chilli pepper (Capsicum annuum L.) seeds. This study aims to evaluate the antifungal activity and mechanism of action of CaTI on phytopathogenic fungi and detect the presence of protease inhibitors in other species of this genus.; Results: Our results show that CaTI can inhibit the growth of the phytopathogenic fungi Colletotrichum gloeosporioides and C. lindemuthianum. CaTI can also permeabilize the membrane of all tested fungi. When testing the inhibitor on its ability to induce reactive oxygen species, an induction of reactive oxygen species (ROS) and nitric oxide (NO) particularly in Fusarium species was observed. Using CaTI coupled to fluorescein isothiocyanate (FITC), it was possible to determine the presence of the inhibitor inside the hyphae of the Fusarium oxysporum fungus. The search for protease inhibitors in other Capsicum species revealed their presence in all tested species.; Conclusion: This paper shows the antifungal activity of protease inhibitors such as CaTI against phytopathogenic fungi. Antimicrobial peptides, among which the trypsin protease inhibitor family stands out, are present in different species of the genus Capsicum and are part of the chemical arsenal that plants use to defend themselves against pathogens. © 2017 Society of Chemical Industry.; © 2017 Society of Chemical Industry.

The seed treatment of a CPSMV-susceptible cowpea genotype with the mutagenic agent EMS generated mutagenized resistant plantlets that respond to the virus challenge by activating biochemical and physiological defense mechanisms. Cowpea is an important crop that makes major nutritional contributions particularly to the diet of the poor population worldwide. However, its production is low, because cowpea is naturally exposed to several abiotic and biotic stresses, including viral agents. Cowpea severe mosaic virus (CPSMV) drastically affects cowpea grain production. This study was conducted to compare photosynthetic and biochemical parameters of a CPSMV-susceptible cowpea (CE-31 genotype) and its derived ethyl methanesulfonate-mutagenized resistant plantlets, both challenged with CPSMV, to shed light on the mechanisms of virus resistance. CPSMV inoculation was done in the fully expanded secondary leaves, 15 days after planting. At 7 days post-inoculation, in vivo photosynthetic parameters were measured and leaves collected for biochemical analysis. CPSMV-inoculated mutagenized-resistant cowpea plantlets (MCPI) maintained higher photosynthesis index, chlorophyll, and carotenoid contents in relation to the susceptible (CE-31) CPSMV-inoculated cowpea (CPI). Visually, the MCPI leaves did not exhibit any viral symptoms neither the presence of the virus as examined by RT-PCR. In addition, MCPI showed higher SOD, GPOX, chitinase, and phenylalanine ammonia lyase activities, H2O2, phenolic contents, and cell wall lignifications, but lower CAT and APX activities in comparison to CPI. All together, these photosynthetic and biochemical changes might have contributed for the CPSMS resistance of MCPI. Contrarily, CPI plantlets showed CPSMV accumulation, severe disease symptoms, reduction in the photosynthesis-related parameters, chlorophyll, carotenoid, phenolic compound, and H2O2 contents, in addition to increased β-1,3-glucanase, and catalase activities that might have favored viral infection.

Chitinases (EC 3.2.1.14) found in the latex of Calotropis procera (Ait) R. Br. were studied. The proteins were homogeneously obtained after two ion exchange chromatography steps. Most proteins were identified individually in 15 spots on 2-D gel electrophoresis with isoelectric points ranging from 4.6 to 6.0 and molecular masses extending from 27 to 30 kDa. Additionally, 66 kDa proteins were identified as chitinases in SDS-PAGE. Their identities were further confirmed by mass spectrometry (MS) analysis of the tryptic digests of each spot and MS analysis of the non-digested proteins. Positive reaction for Schiff's reagent suggested the proteins are glycosylated. The chitinases exhibited high catalytic activity toward to colloidal chitin at pH 5.0, and this activity underwent decay in the presence of increasing amounts of reducing agent dithiothreitol. Spore germination and hyphae growth of two phytopathogenic fungi were inhibited only marginally by the chitinases but were affected differently. This suggested a complex relationship might exist between the specificity of the proteins toward the fungal species. The chitinases showed potent insecticidal activity against the Bruchidae Callosobruchus maculatus, drastically reducing survival, larval weight and adult emergence. It is concluded that closely related chitinases are present in the latex of C. procera, and the first experimental evidence suggests these proteins are involved more efficiently in defence strategies against insects rather than fungi.

BACKGROUND: Nowadays, the Aedes aegypti mosquito represents a serious public health issue in view of the large outbreaks of the arboviral diseases zika, dengue, chikungunya and yellow fever. This holometabolous insect has midgut digestive enzymes that are trypsin‐ and chymotrypsin‐like proteins. Protease inhibitors are able to bind to proteolytic enzymes and promote a blockage in digestion and nutrition, leading to death. Thus, we investigated the effect of trypsin inhibitor of Leucaena leucocephala (LTI) seeds on egg hatching, larval development and digestive midgut proteases. RESULTS: LTI was obtained by trichloroacetic acid precipitation followed by a single chromatography step on anhydrous trypsin sepharose. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate showed a single protein band with a molecular mass close to 20 kDa. After exposure of Ae. aegypti eggs to LTI (0.3 mg mL⁻¹), egg hatching was reduced (50%). LTI did not show acute toxicity on newly hatched larvae incubated under the same conditions, but after 10 days of exposure a high mortality rate (86%) was observed and the surviving larvae had a 25% delay in development. LTI was able to inhibit in vitro the midgut enzymatic activity (70%), and when larvae were incubated with LTI solution we observed an inhibition of 56%. CONCLUSIONS: LTI is a promising new tool to control critical points of Ae. aegypti development. © 2016 Society of Chemical Industry

Mo-CBP

Infections caused by Candida tropicalis have increased significantly worldwide in parallel with resistance to antifungal drugs. To overcome resistance novel drugs have to be discovered. The objective of this work was to purify and characterize a cysteine protease inhibitor from the seeds of the Amazon rainforest tree Cassia leiandra and test its inhibitory effect against C. tropicalis growth. The inhibitor, named ClCPI, was purified after ion exchange and affinity chromatography followed by ultrafiltration. ClCPI is composed of a single polypeptide chain and is not a glycoprotein. The molecular mass determined by SDS-PAGE in the absence or presence of β-mercaptoethanol and ESI-MS were 16.63 kDa and 18.362 kDa, respectively. ClCPI was stable in the pH range of 7.0–9.0 and thermostable up to 60 °C for 20 min. ClCPI inhibited cysteine proteases, but not trypsin, chymotrypsin neither alpha-amylase. Inhibition of papain was uncompetitive with a Ki of 4.1 × 10−7 M and IC50 of 8.5 × 10−7 M. ClCPI at 2.6 × 10−6 M reduced 50% C. tropicalis growth. ClCPI induced damages and morphological alterations in C. tropicalis cell surface, which led to death. These results suggest that ClCPI have great potential for the development of an antifungal drug against C. tropicalis.

The efficiency of current antimicrobial drugs is noticeably decreasing and thus the development of new treatments is necessary. Natural and synthetic antimicrobial peptides (AMPs) have attracted great attention as promising candidates. Inspired on Mo-CBP

This study aimed to detect and characterize antimicrobial proteins, especially antimicrobial peptides (AMPs) from leaves and roots of Capsicum annuum and to evaluate their inhibitory activities against different phytopathogenic fungi and the bacterium Xanthomonas euvesicatoria. Two methodologies were used for the extraction of peptides from leaves and roots of C. annuum: acid and ethanolic extraction. Extracts were subjected to reversed-phase chromatography on HPLC. The extraction and purification procedures were analysed by uni- and bi-dimensional electrophoresis in tricine gels. Our results show that alcoholic and acid extracts from both tissues can inhibit the growth of the phytopathogenics fungi C. lindemuthianum and C. gloeosporioides. The acid extracts from both tissues are active against X. euvesicatoria and only leaf extracts displayed specific inhibitory activity towards trypsin and α-amylase activity. The data compiled here aim to contribute to establish the multiplicity of potential uses of plant AMPs for the control of pests and pathogens of agricultural relevance.

Quantitative PCR (qPCR) is a robust technology for comparing the expression profiles of target genes, however the data produced need normalization with appropriate reference genes. The purpose of this study was to evaluate the expression stability of eight candidate reference genes (EF1A 1a1, EF1A 2a, EF1A 2b, EF1B, ACT11, UKN 1, ACT and SKIP 16) for qPCR assays in soybean leaves under fungus (Cercospora kikuchii) infection and treatments with salicylic and jasmonic acids. Four programs, GeNorm, NormFinder, BestKeeper, and RefFinder were used to evaluate the expression stability. The leaves were treated according the following conditions: 1) infected with Cercospora kikuchii (CK); (2) treated with salicylic acid (SA); (3) treated with SA and infected with C. kikuchii; (4) treated with jasmonic acid (JA); and (5) treated with JA and infected with C. kikuchii. For all studied conditions, GeNorm analyses revealed that combinations of six genes were always needed for gene expression normalization. Three EF1A genes (EF1A 2a, EF1A 1a1 and EF1A 2b) were the most stable in all tested conditions and then, they were always included in gene combinations. The other three genes varied according the different conditions. In analyses with other programs, at least two EF1A genes were often ranked among the three best stable genes. The expression of a PR3 (class I chitinase) gene was used to validate the reference genes across the total samples. Our results provide a shortlist of reference genes to normalize qPCR assays in soybean under CK infection and treatments with SA and JA.

The latex from Thevetia peruviana is rich in plant defense proteins, including a 120 kDa cysteine peptidase with structural characteristics similar to germin-like proteins. More than 20,000 plant species produce latex, including Apocynaceae, Sapotaceae, Papaveraceae and Euphorbiaceae. To better understand the physiological role played by latex fluids, a proteomic analysis of Thevetia peruviana (Pers.) Schum latex was performed using two-dimensional gel electrophoresis and mass spectrometry. A total of 33 proteins (86 %) were identified, including storage proteins, a peptidase inhibitor, cysteine peptidases, peroxidases and osmotins. An unusual cysteine peptidase, termed peruvianin-I, was purified from the latex by a single chromatographic step involving gel filtration. The enzyme (glycoprotein) was inhibited by E-64 and iodoacetamide and exhibited high specific activity towards azocasein (K m 17.6 µM), with an optimal pH and temperature of 5.0–6.0 and 25–37 °C, respectively. Gel filtration chromatography, two-dimensional gel electrophoresis, and mass spectrometry revealed that peruvianin-I possesses 120 kDa, pI 4.0, and six subunits (20 kDa). A unique N-terminal amino acid sequence was obtained to oligomer and monomers of peruvianin-I (1ADPGPLQDFCLADLNSPLFINGYPCRNPALAISDDF36). High-resolution images from atomic force microscopy showed the homohexameric structure of peruvianin-I may be organized as a trimer of dimers that form a central channel similar to germin-like proteins. Peruvianin-I exhibited no oxalate oxidase and superoxide dismutase activity or antifungal effects. Peruvianin-I represents the first germin-like protein (GLP) with cysteine peptidase activity, an activity unknown in the GLP family so far.

Antimicrobial peptides (AMPs) are produced by a range of organisms as a first line of defense against invaders or competitors. Owing to their broad antimicrobial activity, AMPs have attracted attention as a potential source of chemotherapeutic drugs. The increasing prevalence of infections caused by Candida species as opportunistic pathogens in immunocompromised patients requires new drugs. Lecythis pisonis is a Lecythydaceae tree that grows in Brazil. The AMPs produced by this tree have not been described previously. We describe the isolation of 12 fractions enriched in peptides from L. pisonis seeds. Of the 12 fractions, at 10 μg/ml, the F4 fraction had the strongest growth inhibitory effect (53.7%) in Candida albicans, in addition to a loss of viability of 94.9%. The F4 fraction was separated into seven sub-fractions by reversed-phase chromatography. The F4.7' fraction had the strongest activity at 10 μg/ml, inhibiting C. albicans growth by 38.5% and a 69.3% loss of viability. The peptide in F4.7' was sequenced and was found to be similar to plant defensins. For this reason, the peptide was named L. pisonis defensin 1 (Lp-Def1). The mechanism of action that is responsible for C. albicans inhibition by Lp-Def1 includes a slight increase of reactive oxygen species induction and a significant loss of mitochondrial function. The results described here support the future development of plant defensins, specifically Lp-Def1, as new therapeutic substances against fungi, especially C. albicans.

A novel trypsin inhibitor, named RcTI, was purified from castor bean cake (Ricinus communis L.) by heat treatment followed by chromatography on anhydrotrypsin-Sepharose 4B and Resource Q. RcTI is a 14 kDa competitive inhibitor with pI 5.2 and a dissociation constant (Ki) of 1.9 × 10−5 mM. The amino-terminal sequence showed similarity with a 2S sulfur-rich seed storage protein (83%) and napin-like protein (48%). RcTI was stable over a broad pH range and is exceptionally resistant to heating as it retained high inhibitory activity toward trypsin after incubation at 100 °C for 2 h. RcTI (13 μg) inhibited the spore germination of the phytopatogenic fungus Colletotrichum gloeosporioides and promoted 91% inhibition of the proteases from the midgut of Aedes aegypti larvae. The results of the present study indicate that RcTI has biotechnological potential as an alternative agent to combat the important phytopathogen C. gloeosporioides and the larvae of A. aegypti.

The biochemical and nutritional attributes of two soybean (Glycine max (L.) Merr.) cultivars, one susceptible (Seridó) and the other resistant (Seridó-RCH) to stem canker, were examined to assess whether the resistance to pathogens was related to levels of antinutritional and/or defense proteins in the plant and subsequently affected the nutritional quality. Lectin, urease, trypsin inhibitor, peroxidase and chitinase activities were higher in the resistant cultivar. Growing rats were fed with isocaloric and isoproteic diets prepared with defatted raw soybean meals. Those on the Seridó-RCH diet showed the worst performance in terms of protein quality indicators. Based on regression analysis, lectin, trypsin inhibitor, peroxidase and chitinase appear to be involved in the resistance trait but also in the poorer nutritional quality of Seridó-RCH. Thus, the development of cultivars for disease resistance may lead to higher concentrations of antinutritional compounds, affecting the quality of soybean seeds. Further research that includes the assessment of more cultivars/genotypes is needed.

Toxoplasma gondii is a parasite of great medical and veterinary importance that has worldwide distribution and causes toxoplasmosis. There are few treatments available for toxoplasmosis and the search for plant extracts and compounds with anti-Toxoplasma activity is of utmost importance for the discovery of new active drugs. The objective of this study was to investigate the action of a protein extract and a protease inhibitor enriched fraction from J. curcas seed cake on developing tachyzoites of T. gondii-infected Vero cells. The protein extract (JcCE) was obtained after solubilization of the J. curcas seed cake with 100 mM sodium borate buffer, pH 10, centrifugation and dialysis of the resulting supernatant with the extracting buffer. JcCE was used for the in vitro assays of anti-Toxoplasma activity at 0.01, 0.1, 0.5, 1.5, 3.0 and 5.0 mg/ml concentration for 24 h. The results showed that JcCE reduced the percentage of infection and the number of intracellular parasites, but had no effect on the morphology of Vero cells up to 3.0 mg/mL. The cysteine protease inhibitor enriched fraction, which was obtained after chromatography of JcCE on Sephadex G-75 and presented a unique protein band following SDS–PAGE, reduced both the number of T. gondii infected cells and intracellular parasites. These results suggest that both JcCE and the cysteine protease inhibitor enriched fraction interfere with the intracellular growth of T. gondii.

BACKGROUND Arboviroses such as dengue, Zika and chikungunya represent a serious public health issue as a consequence of the absence of approved vaccines or specific antiviral drugs against the arboviruses that cause them. One way to prevent these diseases is by combating the vector mosquito, Aedes aegypti (Diptera), which has serine proteases in the midgut. Protease inhibitors are molecules that can block enzyme activity, impairing digestion and nutrition, which can lead to death. Thus, we purified and characterized a novel chymotrypsin-trypsin inhibitor (LsCTI) from Lonchocarpus sericeus seeds and investigated its effect upon Ae. aegypti egg hatching, larval development and digestive proteases. RESULTS LsCTI showed a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the molecular mass determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was 8870.45 Da. Kinetics analyses revealed a noncompetitive type of inhibition and low inhibition constant (Ki ) for chymotrypsin (8.24 x 10-8 m). The thermal resistance was remarkable, even at 100 °C for 180 min. The inhibitor concentration required for 50-percent enzyme inhibition (IC50 ) of LsCTI was 4.7 x 10-7 m for Ae. aegypti midgut larval enzymes. LsCTI did not affect egg hatchability at 0.3 mg mL-1 , but caused a high larval mortality rate (77%) and delayed development (37%). CONCLUSIONS LsCTI is a novel protease inhibitor with remarkable biochemical characteristics and is a potential tool to control Ae. aegypti development. © 2017 Society of Chemical Industry.

Infection with Cowpea severe mosaic virus (CPSMV) represents one of the main limitations for cowpea (Vigna unguiculata L. Walp.) productivity due to the severity of the disease symptoms, frequency of incidence, and difficulties in dissemination control. This study aimed to identify the proteins and metabolic pathways associated with the susceptibility and resistance of cowpea plants to CPSMV. Therefore, we treated the seeds of a naturally susceptible cowpea genotype (CE-31) with the mutagenic agent ethyl methane sulfonate (EMS) and compared the secondary leaf proteomic profile of the mutagenized resistant plants inoculated with CPSMV (MCPI plant group) to those of the naturally susceptible cowpea genotype CE-31 inoculated (CPI) and noninoculated (CPU) with CPSMV. MCPI responded to CPSMV by accumulating proteins involved in the oxidative burst, increasing H2O2 generation, promoting leaf cell death (LCD), increasing the synthesis of defense proteins, and decreasing host factors important for the establishment of CPSMV infection. In contrast, CPI accumulated several host factors that favor CPSMV infection and did not accumulate H2O2 or present LCD, which allowed CPSMV replication and systemic dissemination. Based on these results, we propose that the differential abundance of defense proteins and proteins involved in the oxidative burst, LCD, and the decrease in cowpea protein factors required for CPSMV replication are associated with the resistance trait acquired by the MCPI plant group.

A trypsin inhibitor from Cassia leiandra seeds, named ClTI, was purified, characterized, and its insecticidal activity against Aedes aegypti evaluated. ClTI was purified by DEAE-Cellulose and trypsin-Sepharose 4B chromatography, with a 15.5-fold purification and 2.4% yield. ClTI is composed of a 19,484 Da polypeptide chain as revealed by mass spectrometry, it is not a glycoprotein, its amino acid sequence is similar to other Kunitz-type inhibitors, and it comprises 35% β-sheets, 14% β-turns, and 50% disordered secondary structures. ClTI is an uncompetitive inhibitor of bovine trypsin (IC50 of 33.81 × 10−8 M, Ki of 6.25 × 10−8 M) stable over a broad range of pHs (2.2–10.0) and temperatures (30–70 °C), but dithiothreitol led to a partial loss of the inhibitory activity. ClTI, at 4.65 × 10−6 M, reduced in 50% the activity of the Ae. aegypti midgut proteases. ClTI also promoted acute toxicity on the 3rd instar larvae of Ae. aegypti, with an LC50 of 2.28 × 10−2 M. Moreover, it caused a 24-h delay of the larvae development and 44% mortality after ten days of exposure. Altogether, these results suggest that ClTI has potential as a natural compound to control Ae. aegypti, a vector of several infection diseases.

An antifungal class III peroxidase was purified from Marsdenia megalantha latex (named Mo -POX) using DEAE-cellulose and gel filtration chromatography on a Superose 12 HR 10/30 column. Mm -POX has an apparent molecular mass of 67.0 kDa and a pI of 5.2, shares identity with other peroxidases, and follows Michaelis-Menten kinetics. It has a high affinity for guaiacol and hydrogen peroxide. The pH and temperature optima for Mm -POX were 5.0–7.0 and 60 °C, respectively. The catalytic activity of Mm -POX was decreased in the presence of classic peroxidase inhibitors including azide, dithiothreitol, ethylenediamine tetraacetic acid, and sodium metabisulfite and high concentrations of Na + , Mn + , and salicylic acid. In contrast, Ca + and Mg + , even at low concentrations, enhanced the Mm -POX enzymatic activity. This protein inhibited the germination of the conidia of the phytopathogenic fungi Fusarium oxysporum and Fusarium solani by acting through a membrane permeabilization mechanism. Mm -POX also induced oxidative stress in F. solani . Mm -POX is the first enzyme to be isolated from the M. megalantha species and it has potential use in the control of plant disease caused by important phytopathogenic fungi. This adds biotechnological value to this enzyme.

Moringa oleifera has been used in traditional medicine to treat diabetes. However, few studies have been conducted to relate its antidiabetic properties to proteins. In this study, a leaf protein isolate was obtained from M. oleifera leaves, named Mo-LPI, and the hypoglycemic and antioxidant effects on alloxan-induced diabetic mice were assessed. Mo-LPI was obtained by aqueous extraction, ammonium sulphate precipitation and dialysis. The electrophoresis profile and proteolytic hydrolysis confirmed its protein nature. Mo-LPI showed hemagglutinating activity, cross-reaction with anti-insulin antibodies and precipitation after zinc addition. Single-dose intraperitoneal (i.p.) administration of Mo-LPI (500 mg/kg·bw) reduced the blood glucose level (reductions of 34.3%, 60.9% and 66.4% after 1, 3 and 5 h, respectively). The effect of Mo-LPI was also evidenced in the repeated dose test with a 56.2% reduction in the blood glucose level on the 7th day after i.p. administration. Mo-LPI did not stimulate insulin secretion in diabetic mice. Mo-LPI was also effective in reducing the oxidative stress in diabetic mice by a decrease in malondialdehyde level and increase in catalase activity. Mo-LPI (2500 mg/kg·bw) did not cause acute toxicity to mice. Mo-LPI is a promising alternative or complementary agent to treat diabetes.

Viruses are important plant pathogens that threaten diverse crops worldwide. Diseases caused by Cowpea severe mosaic virus (CPSMV) have drawn attention because of the serious damages they cause to economically important crops including cowpea. This work was undertaken to quantify and identify the responsive proteins of a susceptible cowpea genotype infected with CPSMV, in comparison with mock-inoculated controls, using label-free quantitative proteomics and databanks, aiming at providing insights on the molecular basis of this compatible interaction. Cowpea leaves were mock- or CPSMV-inoculated and 2 and 6 days later proteins were extracted and analyzed. More than 3000 proteins were identified (data available via ProteomeXchange, identifier PXD005025) and 75 and 55 of them differentially accumulated in response to CPSMV, at 2 and 6 DAI, respectively. At 2 DAI, 76% of the proteins decreased in amount and 24% increased. However, at 6 DAI, 100% of the identified proteins increased. Thus, CPSMV transiently suppresses the synthesis of proteins involved particularly in the redox homeostasis, protein synthesis, defense, stress, RNA/DNA metabolism, signaling, and other functions, allowing viral invasion and spread in cowpea tissues.

Cowpea severe mosaic virus (CPSMV) causes significant losses in cowpea (Vigna unguiculata) production. In this present study biochemical, physiological, and proteomic analysis were done to identify pathways and defense proteins that are altered during the incompatible interaction between the cowpea genotype BRS-Marataoa and CPSMV. The leaf protein extracts from mock- (MI) and CPSMV-inoculated plantlets (V) were evaluated at 2 and 6 days post-inoculation (DPI). Data support the assumptions that increases in biochemical (high hydrogen peroxide, antioxidant enzymes, and secondary compounds) and physiological responses (high photosynthesis index and chlorophyll content), confirmed by label-free comparative proteomic approach, in which quantitative changes in proteasome proteins, proteins related to photosynthesis, redox homeostasis, regulation factors/RNA processing proteins were observed may be implicated in the resistance of BRS-Marataoa to CPSMV. This pioneering study provides information for the selection of specific pathways and proteins, altered in this incompatible relationship, which could be chosen as targets for detailed studies to advance our understanding of the molecular, physiological, and biochemistry basis of the resistance mechanism of cowpea and design approachs to engineer plants that are more productive. Biological significance This is a pioneering study in which an incompatible relationship between a resistant cowpea and Cowpea severe mosaic virus (CPSMV) was conducted to comparatively evaluate proteomic profiles by Gel-free/label-free methodology and some physiological and biochemical parameters to shed light on how a resistant cowpea cultivar deals with the virus attack. Specific proteins and associated pathways were altered in the cowpea plants challenged with CPSMV and will contribute to our knowledge on the biological process tailored by cowpea in response to CPSMV.