Chitinase from the Latex of Ficus benjamina L. Displays Antifungal Activity by Inducing ROS Generation and Structural Damage to the Fungal Cell Wall and Plasma Membrane.

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 60ºC, 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-ß-D-glucosamine, which is employed for bioethanol production, in cosmetics, in medicine, and for other multiple applications.

Ornamental tobacco floral nectar is a rich source of antimicrobial peptides.

Although floral nectar is a rich source of nutrients, it is rarely infected by microorganisms. Defense molecules such as proteins have been identified in this fluid, but defense peptides have been largely overlooked. Thus, the aim of this study was to perform an extensive peptidomic analysis of the ornamental tobacco floral nectar to seek peptides involved in nectar defense. Using LC-MS/MS, 793 peptides were sequenced and characterized. After extensive bioinformatics analysis, six peptides were selected for further characterization, synthesis, and evaluation of their antimicrobial properties against phytopathogenic fungi and bacteria. All six peptides had antimicrobial activity to some extent. However, the activity varied by peptide concentration and microorganism tested. An analysis of the action mechanism revealed damage in the cell membrane induced by peptides. The results show that floral nectar is rich in peptides and that, together with proteins and hydrogen peroxide, they contribute to plant defense against microorganisms during pollination.

Clustered Regularly Interspaced Short Palindromic Repeats-Associated Protein System for Resistance Against Plant Viruses: Applications and Perspectives.

Different genome editing approaches have been used to engineer resistance against plant viruses. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas; CRISPR/Cas) systems to create pinpoint genetic mutations have emerged as a powerful tool for molecular engineering of plant immunity and increasing resistance against plant viruses. This review presents (i) recent advances in engineering resistance against plant viruses by CRISPR/Cas and (ii) an overview of the potential host factors as targets for the CRISPR/Cas system-mediated broad-range resistance and immunity. Applications, challenges, and perspectives in enabling the CRISPR/Cas system for crop protection are also outlined.

The expression of the genes involved in redox metabolism and hydrogen peroxide balance is associated with the resistance of cowpea [Vigna unguiculata (L.) Walp.] to the hemibiotrophic fungus Colletotrichum gloeosporioides.

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.

A Bowman-Birk Inhibitor from the Seeds of Luetzelburgia auriculata Inhibits Staphylococcus aureus Growth by Promoting Severe Cell Membrane Damage.

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 death. This study suggests that LzaBBI is a powerful candidate for developing a new antimicrobial to overcome drug resistance toward reducing hospital-acquired infections caused by S. aureus.

Production in Pichia pastoris, antifungal activity and crystal structure of a class I chitinase from cowpea (Vigna unguiculata): Insights into sugar binding mode and hydrolytic action.

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 Å 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.

Photosynthetic and biochemical mechanisms of an EMS-mutagenized cowpea associated with its resistance to cowpea severe mosaic virus.

KEY MESSAGE: 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 ß-1,3-glucanase, and catalase activities that might have favored viral infection.

A 2S Albumin from the Seed Cake of Ricinus communis Inhibits Trypsin and Has Strong Antibacterial Activity against Human Pathogenic Bacteria.

Hospital-acquired infections caused by antibiotic-resistant bacteria threaten the lives of many citizens all over the world. Discovery of new agents to hinder bacterial development would have a significant impact on the treatment of infections. Here, the purification and characterization of Rc-2S-Alb, a protein that belongs to the 2S albumin family, from Ricinus communis seed cake, are reported. Rc-2S-Alb was purified after protein extraction with Tris-HCl buffer, pH 7.5, fractionation by ammonium sulfate (50-75%), and chromatography on Phenyl-Sepharose and DEAE-Sepharose. Rc-2S-Alb, a 75 kDa peptide, displays trypsin inhibitory activity and has high in vitro antibacterial activity against Bacillus subtilis, Klebsiella pneumonia, and Pseudomonas aeruginosa, which are important human pathogenic bacteria. Atomic force microscopy studies indicated that Rc-2S-Alb disrupts the bacterial membrane with loss of the cytoplasm content and ultimately bacterial death. Therefore, Rc-2S-Alb is a powerful candidate for the development of an alternative drug that may help reduce hospital-acquired infections.

Drought increases cowpea (Vigna unguiculata [L.] Walp.) susceptibility to cowpea severe mosaic virus (CPSMV) at early stage of infection.

The physiological and biochemical responses of a drought tolerant, virus-susceptible cowpea genotype exposed to drought stress (D), infected by Cowpea severe mosaic virus (CPSMV) (V), and to these two combined stresses (DV), at 2 and 6 days post viral inoculation (DPI), were evaluated. Gas exchange parameters (net photosynthesis, transpiration rate, stomatal conductance, and internal CO2 partial pressure) were reduced in D and DV at 2 and 6 DPI compared to control plants (C). Photosynthesis was reduced by stomatal and biochemical limitations. Water use efficiency increased at 2 DPI in D, DV, and V, but at 6 DPI only in D and DV compared to C. Photochemical parameters (effective quantum efficiency of photosystem II and electron transport rate) decreased in D and DV compared to C, especially at 6 DPI. The potential quantum efficiency of photosystem II did not change, indicating reversible photoinhibition of photosystem II. In DV, catalase decreased at 2 and 6 DPI, ascorbate peroxidase increased at 2 DPI, but decreased at 6 DPI. Hydrogen peroxide increased at 2 and 6 DPI. Peroxidase increased at 6 DPI and chitinase at 2 and 6 DPI. ß-1,3-glucanase decreased in DV at 6 DPI compared to V. Drought increased cowpea susceptibility to CPSMV at 2 DPI, as verified by RT-PCR. However, at 6 DPI, the cowpea plants overcome this effect. Likewise, CPSMV increased the negative effects of drought at 2 DPI, but not at 6 DPI. It was concluded that the responses to combined stresses are not additive and cannot be extrapolated from the study of individual stresses.

First isolation and antinociceptive activity of a lipid transfer protein from noni (Morinda citrifolia) seeds.

In this study a novel heat-stable lipid transfer protein, designated McLTP1, was purified from noni (Morinda citrifolia L.) seeds, using four purification steps which resulted in a high-purified protein yield (72 mg McLTP1 from 100g of noni seeds). McLTP1 exhibited molecular masses of 9.450 and 9.466 kDa, determined by electrospray ionisation mass spectrometry. The N-terminal sequence of McLTP1 (AVPCGQVSSALSPCMSYLTGGGDDPEARCCAGV), as analysed by NCBI-BLAST database, revealed a high degree of identity with other reported plant lipid transfer proteins. In addition, this protein proved to be resistant to pepsin, trypsin and chymotrypsin digestion. McLTP1 given intraperitoneally (1, 2, 4 and 8 mg/kg) and orally (8 mg/kg) caused an inhibition of the writhing response induced by acetic acid in mice. This protein displayed thermostability, retaining 100% of its antinociceptive activity after 30 min incubation at 80 °C. Pretreatment of mice with McLTP1 (8 mg/kg, i.p. and p.o.) also decreased neurogenic and inflammatory phases of nociception in the formalin test. Naloxone (2 mg/kg, i.p.) antagonised the antinociceptive effect of McLTP1 suggesting that the opioid mechanisms mediate the analgesic properties of this protein.

Proteomics changes during the incompatible interaction between cowpea and Colletotrichum gloeosporioides (Penz.) Penz and Sacc.

Anthracnose represents an important disease of cowpea [Vigna unguiculata L. (Walp.)] caused by the hemibiothrophic fungus Colletotrichum gloeosporioides that drastically reduces cowpea field production. In this study we investigated some biochemical aspects underlying the incompatible interaction between a resistant cowpea genotype and C. gloeosporioides using a proteomic approach. Analyses of two-dimensional gel electrophoresis patterns and protein identification indicate C. gloeosporioides infection-dependent cowpea leaf proteome changes associated with metabolism, photosynthesis, response to stress, oxidative burst and scavenging, defense signaling, and pathogenesis-related proteins. Moreover the C. gloeosporioides responsive proteins interaction network in cowpea revealed the interconnected modulation of key cellular processes involving particularly antioxidants proteins, photosynthetic apparatus forming proteins and proteins of the energetic metabolism that interact with each other suggesting that their expression changes are also important for resistance of cowpea to C. gloeosporioides.

Enhanced Synthesis of Antioxidant Enzymes, Defense Proteins and Leghemoglobin in Rhizobium-Free Cowpea Roots after Challenging with Meloydogine incognita.

The root knot nematodes (RKN), Meloydogine spp., particularly Meloidogyne incognita and Meloidogyne javanica species, parasitize several plant species and are responsible for large annual yield losses all over the world. Only a few available chemical nematicides are still authorized for RKN control owing to environmental and health reasons. Thus, plant resistance is currently considered the method of choice for controlling RKN, and research performed on the molecular interactions between plants and nematodes to identify genes of interest is of paramount importance. The present work aimed to identify the differential accumulation of root proteins of a resistant cowpea genotype (CE-31) inoculated with M. incognita (Race 3) in comparison with mock-inoculated control, using 2D electrophoresis assay, mass spectrometry identification and gene expression analyses by RT-PCR. The results showed that at least 22 proteins were differentially represented in response to RKN challenge of cowpea roots mainly within 4-6 days after inoculation. Amongst the up-represented proteins were SOD, APX, PR-1, ß-1,3-glucanase, chitinases, cysteine protease, secondary metabolism enzymes, key enzymes involved in ethylene biosynthesis, proteins involved in MAPK pathway signaling and, surprisingly, leghemoglobin in non-rhizobium-bacterized cowpea. These findings show that an important rearrangement in the resistant cowpea root proteome occurred following challenge with M. incognita.

Biochemical, physicochemical and molecular characterization of a genuine 2-Cys-peroxiredoxin purified from cowpea [Vigna unguiculata (L.) Walpers] leaves.

BACKGROUND: Peroxiredoxins have diverse functions in cellular defense-signaling pathways. 2-Cys-peroxiredoxins (2-Cys-Prx) reduce H2O2 and alkyl-hydroperoxide. This study describes the purification and characterization of a genuine 2-Cys-Prx from Vigna unguiculata (Vu-2-Cys-Prx). METHODS: Vu-2-Cys-Prx was purified from leaves by ammonium sulfate fractionation, chitin affinity and ion exchange chromatography. RESULTS: Vu-2-Cys-Prx reduces H2O2 using NADPH and DTT. Vu-2-Cys-Prx is a 44 kDa (SDS-PAGE)/46 kDa (exclusion chromatography) protein that appears as a 22 kDa molecule under reducing conditions, indicating that it is a homodimer linked intermolecularly by disulfide bonds and has a pI range of 4.56­4.72; its NH2-terminal sequence was similar to 2-Cys-Prx from Phaseolus vulgaris (96%) and Populus tricocarpa (96%). Analysis by ESI-Q-TOF MS/MS showed a molecular mass/pI of 28.622 kDa/5.18. Vu-2-Cys-Prx has 8% α-helix, 39% ß-sheet, 22% of turns and 31% of unordered forms. Vu-2-Cys-Prx was heat stable, has optimal activity at pH 7.0, and prevented plasmid DNA degradation. Atomic force microscopy shows that Vu-2-Cys-Prx oligomerized in decamers which might be associated with its molecular chaperone activity that prevented denaturation of insulin and citrate synthase. Its cDNA analysis showed that the redox-active Cys52 residue and the amino acids Pro45, Thr49 and Arg128 are conserved as in other 2-Cys-Prx. GENERAL SIGNIFICANCE: The biochemical and molecular features of Vu-2-Cys-Prx are similar to other members of 2-Cys-Prx family. To date, only one publication reported on the purification of native 2-Cys-Prx from leaves and the subsequent analysis by N-terminal Edman sequencing, which is crucial for construction of stromal recombinant 2-Cys-Prx proteins.

Laticifer proteins play a defensive role against hemibiotrophic and necrotrophic phytopathogens.

Proteins from latex of Calotropis procera (CpLP), Plumeria rubra (PrLP), Carica candamarcensis (P1G10) and Euphorbia tirucalli (EtLP) were tested for antifungal activity against phytopathogens. CpLP and P1G10 inhibited each fungi analyzed. PrLP and EtLP did not exert inhibition. CpLP and P1G10 exhibited preferential inhibitory activity towards R. solani (IC50 = 20.7 and 25.3 µg/ml, respectively). The inhibitory activity was lost after heat treatment or proteolysis, providing evidence for the involvement of proteins in the inhibitory effect. Treatment of CpLP or P1G10 with Dithiothreitol improved both, the endogenous proteolytic activity and the antifungal properties. Conversely, pre-treatment of CpLP or P1G10 with iodoacetamide drastically reduced endogenous proteolytic activities and partially abrogated antifungal activity. Similar results were observed when spores were challenged to germinate in the presence of laticifer proteins. The purified cysteine proteinase CMS2MS2 from Carica candamarcensis latex or papain (E.C. 3.4.22.2), a cysteine proteinase from latex of Carica papaya L., but not trypsin (EC 3.4.21.4) or chymotrypsin (EC 3.4.21.1), two serine proteases, replicated the results obtained with CpLP or P1G10, thus restricting the antifungal property to latex plant cysteine proteinases. CpLP, CMS2MS2 and papain induced production of reactive oxygen species in spores of F. solani, suggesting that inhibition could be linked to oxidative stress. Proteome analysis of CpLP by 2-D electrophoresis and MALDI-TOF-TOF confirmed the existence of various pathogenic-related proteins such as chitinases, peroxidases and osmotins. The results support that laticifer proteins are part of plant defense repertoire against phytopathogenic fungi.