Plant extracellular vesicles are incorporated by a fungal pathogen and inhibit its growth.

Extracellular vesicles (EV) are membrane particles released by cells into their environment and are considered to be key players in intercellular communication. EV are produced by all domains of life but limited knowledge about EV in plants is available, although their implication in plant defense has been suggested. We have characterized sunflower EV and tested whether they could interact with fungal cells. EV were isolated from extracellular fluids of seedlings and characterized by transmission electron microscopy and proteomic analysis. These nanovesicles appeared to be enriched in cell wall remodeling enzymes and defense proteins. Membrane-labeled EV were prepared and their uptake by the phytopathogenic fungus Sclerotinia sclerotiorum was verified. Functional tests further evaluated the ability of EV to affect fungal growth. Spores treated with plant EV showed growth inhibition, morphological changes, and cell death. Conclusive evidence on the existence of plant EV is presented and we demonstrate their ability to interact with and kill fungal cells. Our results introduce the concept of cell-to-cell communication through EV in plants.

Antimicrobial activity and mechanism of action of a thionin-like peptide from Capsicum annuum fruits and combinatorial treatment with fluconazole against Fusarium solani.

Many Fusarium species are able to cause severe infections in plants as well as in animals and humans. Therefore, the discovery of new antifungal agents is of paramount importance. CaThi belongs to the thionins, which are cationic peptides with low molecular weights (∼5 kDa) that have toxic effects against various microorganisms. Herein, we study the mechanism of action of CaThi and its combinatory effect with fluconazole (FLC) against Fusarium solani. The mechanism of action of CaThi was studied by growth inhibition, viability, plasma membrane permeabilization, ROS induction, caspase activation, localization, and DNA binding capability, as assessed with Sytox green, DAB, FITC-VAD-FMK, CaThi-FITC, and gel shift assays. The combinatory effect of CaThi and FLC was assessed using a growth inhibition assay. Our results demonstrated that CaThi present a dose dependent activity and at the higher used concentration (50 µg mL-1 ) inhibits 83% of F. solani growth, prevents the formation of hyphae, permeabilizes membranes, induces endogenous H2 O2 , activates caspases, and localizes intracellularly. CaThi combined with FLC, at concentrations that alone do not inhibit F. solani, result in 100% death of F. solani when combined. The data presented in this study demonstrate that CaThi causes death of F. solani via apoptosis; an intracellular target may also be involved. Combined treatment using CaThi and FLC is a strong candidate for studies aimed at improved targeting of F. solani. This strategy is of particular interest because it minimizes selection of resistant microorganisms.

Interaction between the plant ApDef1 defensin and Saccharomyces cerevisiae results in yeast death through a cell cycle- and caspase-dependent process occurring via uncontrolled oxidative stress.

BACKGROUND: Plant defensins were discovered at beginning of the 90s'; however, their precise mechanism of action is still unknown. Herein, we studied ApDef1-Saccharomyces cerevisiae interaction. METHODS: ApDef1-S. cerevisiae interaction was studied by determining the MIC, viability and death kinetic assays. Viability assay was repeated with hydroxyurea synchronized-yeast and pretreated with CCCP. Plasma membrane permeabilization, ROS induction, chromatin condensation, and caspase activation analyses were assessed through Sytox green, DAB, DAPI and FITC-VAD-FMK, respectively. Viability assay was done in presence of ascorbic acid and Z-VAD-FMK. Ultrastructural analysis was done by electron microscopy. RESULTS: ApDef1 caused S. cerevisiae cell death and MIC was 7.8µM. Whole cell population died after 18h of ApDef1 interaction. After 3h, 98.76% of synchronized cell population died. Pretreatment with CCCP protected yeast from ApDef1 induced death. ApDef1-S. cerevisiae interaction resulted in membrane permeabilization, H2O2 increased production, chromatin condensation and caspase activation. Ascorbic acid prevented yeast cell death and membrane permeabilization. Z-VAD-FMK prevented yeast cell death. CONCLUSIONS: ApDef1-S. cerevisiae interaction caused cell death through cell cycle dependentprocess which requires preserved membrane potential. After interaction, yeast went through uncontrolled ROS production and accumulation, which led to plasma membrane permeabilization, chromatin condensation and, ultimately, cell death by activation of caspase-dependent apoptosis via. GENERAL SIGNIFICANCE: We show novel requirements for the interaction between plant defensin and fungi cells, i.e. cell cycle phase and membrane potential, and we indicate that membrane permeabilization is probably caused by ROS and therefore, it would be an indirect event of the ApDef1-S. cerevisiae interaction.

Chlorogenic acid is a fungicide active against phytopathogenic fungi.

Plants synthesize diverse types of secondary metabolites and some of them participate in plant protection against pathogen attack. These compounds are biodegradable and renewable alternatives, which may be envisaged for the control of plant pests and diseases. Chlorogenic acid (CGA) is a phenolic secondary metabolite which accumulates in diverse plant tissues and can be found in several agro-industrial by-products and waste. The aim of this work was to determine whether CGA could control the growth of various plant pathogenic fungi, gaining insight into its mechanism of action. Microscopic analysis showed the complete inhibition of spore germination or reduction of mycelial growth for Sclerotinia sclerotiorum, Fusarium solani, Verticillium dahliae, Botrytis cinerea and Cercospora sojina. CGA concentrations that did not completely abolish spore germination were able to produce a partial inhibition of mycelial growth. Viability tests and vital dye staining demonstrate that CGA induces fungal cell lysis. Its fungicidal activity involves an early membrane permeabilization of the spores. These results show the antifungal activity of CGA against phytopathogenic fungi relevant in horticulture and agriculture highlighting the potential of CGA-enriched wastes and by-products to be used as biofungicides.

Anti-Neuroblastoma Properties of a Recombinant Sunflower Lectin.

According to their sugar recognition specificity, plant lectins are proposed as bioactive proteins with potential in cancer treatment and diagnosis. Helja is a mannose-specific jacalin-like lectin from sunflower which was shown to inhibit the growth of certain fungi. Here, we report its recombinant expression in a prokaryotic system and its activity in neurobalstoma cells. Helja coding sequence was fused to the pET-32 EK/LIC, the enterokinase/Ligation-independent cloning vector and a 35 kDa protein was obtained in Escherichia coli representing Helja coupled to thioredoxin (Trx). The identity of this protein was verified using anti-Helja antibodies. This chimera, named Trx-rHelja, was enriched in the soluble bacterial extracts and was purified using Ni+2-Sepharose and d-mannose-agarose chromatography. Trx-rHelja and the enterokinase-released recombinant Helja (rHelja) both displayed toxicity on human SH-SY5Y neuroblastomas. rHelja decreased the viability of these tumor cells by 75% according to the tetrazolium reduction assay, and microscopic analyses revealed that the cell morphology was disturbed. Thus, the stellate cells of the monolayer became spheroids and were isolated. Our results indicate that rHelja is a promising tool for the development of diagnostic or therapeutic methods for neuroblastoma cells, the most common solid tumors in childhood.

Nitric oxide is required for determining root architecture and lignin composition in sunflower. Supporting evidence from microarray analyses.

Nitric oxide (NO) is a signal molecule involved in several physiological processes in plants, including root development. Despite the importance of NO as a root growth regulator, the knowledge about the genes and metabolic pathways modulated by NO in this process is still limited. A constraint to unravel these pathways has been the use of exogenous applications of NO donors that may produce toxic effects. We have analyzed the role of NO in root architecture through the depletion of endogenous NO using the scavenger cPTIO. Sunflower seedlings growing in liquid medium supplemented with cPTIO showed unaltered primary root length while the number of lateral roots was deeply reduced; indicating that endogenous NO participates in determining root branching in sunflower. The transcriptional changes induced by NO depletion have been analyzed using a large-scale approach. A microarray analysis showed 330 genes regulated in the roots (p≤0.001) upon endogenous NO depletion. A general cPTIO-induced up-regulation of genes involved in the lignin biosynthetic pathway was observed. Even if no detectable changes in total lignin content could be detected, cell walls analyses revealed that the ratio G/S lignin increased in roots treated with cPTIO. This means that endogenous NO may control lignin composition in planta. Our results suggest that a fine tuning regulation of NO levels could be used by plants to regulate root architecture and lignin composition. The functional implications of these findings are discussed.

A sunflower lectin with antifungal properties and putative medical mycology applications.

Lectins are carbohydrate-binding proteins with a high specificity for a variety of glycoconjugate sugar motifs. The jacalin-related lectins (JRL) are considered to be a small sub-family composed of galactose- and mannose-specific members. Using a proteomics approach, we have detected a 16 kDa protein (Helja) in sunflower seedlings that were further purified by mannose-agarose affinity chromatography. The aim of this work was to characterize the biological activity of Helja and to explore potential applications for the antifungal activity of this plant lectin against medically important yeasts. To initially assess the agglutination properties of the lectin, Saccharomyces cerevisiae cells were incubated with increasing concentrations of the purified lectin. At a concentration of 120 µg/ml, Helja clearly agglutinated these cells. The ability of different sugars to inhibit S. cerevisiae cell agglutination determined its carbohydrate-specificity. Among the monosaccharides tested, D-mannose had the greatest inhibitory effect, with a minimal concentration of 1.5 mM required to prevent cell agglutination. The antifungal activity was evaluated using pathogenic fungi belonging to the Candida and Pichia genera. We demonstrate that 200 µg/ml of Helja inhibited the growth of all yeasts, and it induced morphological changes, particularly through pseudohyphae formation on Candida tropicalis. Helja alters the membrane permeability of the tested fungi and is also able to induce the production of reactive oxygen species in C. tropicalis cells. We concluded that Helja is a mannose-binding JRL with cell agglutination capabilities and antifungal activity against yeasts. The biological properties of Helja may have practical applications in the control of human pathogens.

Effects of the binding of a Helianthus annuus lectin to Candida albicans cell wall on biofilm development and adhesion to host cells.

BACKGROUND: In our previous study, we isolated and characterized a lectin called Helja from Helianthus annuus (sunflower) and then, in a further study, demonstrated its antifungal activity against Candida spp. Since Candida infections are a major health concern due to the increasing emergence of antifungal resistant strains, the search for new antifungal agents offers a promising opportunity for improving the treatment strategies against candidiasis. PURPOSE: The aim of this work was to get insights about the mechanism of action of Helja, an antifungal lectin of H. annuus, and to explore its ability to inhibit Candida albicans biofilm development and adherence to buccal epithelial cells (BEC). STUDY DESIGN/METHODS: Yeast viability was evaluated by Evans Blue uptake and counting of colony forming units (CFU). The yeast cell integrity was assessed using Calcofluor White (CFW) as a cell wall perturbing agent and sorbitol as osmotic protectant. The induction of oxidative stress was evaluated using 3,3'-diaminobenzidine (DAB) for detection of hydrogen peroxide. The adherence was determined by counting the yeast cells attached to BEC after methylene blue staining. The biofilms were developed on polystyrene microplates, visualized by confocal laser scanning microscopy and the viable biomass was quantified by CFU counting. The binding lectin-Candida was assessed using Helja conjugated to fluorescein isothiocyanate (Helja-FITC) and simultaneous staining with CFW. The cellular surface hydrophobicity (CSH) was determined using a microbial adhesion to hydrocarbons method. RESULTS: C. albicans cells treated with 0.1 µg/µl of Helja showed a drastic decrease in yeast survival. The lectin affected the fungal cell integrity, induced the production of hydrogen peroxide and inhibited the morphological transition from yeast to filamentous forms. Helja caused a significant reduction of adherent cells and a decrease in biofilm biomass and coverage area. The treatment with the protein also reduced the surface hydrophobicity of fungal cells. We show the binding of Helja-FITC to yeast cells distributed as a thin outer layer to the CFW signal, and this interaction was displaced by mannose and Concanavalin A. CONCLUSION: The results demonstrate the interaction of Helja with the mannoproteins of C. albicans cell wall, the disruption of the cell integrity, the induction of oxidative stress, the inhibition of the morphological transition from yeast to filamentous forms and the fungal cell viability loss. The binding Helja-Candida also provides a possible explanation of the lectin effect on cell adherence, biofilm development and CSH, relevant features related to virulence of the pathogen.

Internalization of a sunflower mannose-binding lectin into phytopathogenic fungal cells induces cytotoxicity.

Lectins are carbohydrate-affinity proteins with the ability to recognize and reversibly bind specific glycoconjugates. We have previously isolated a bioactive sunflower mannose-binding lectin belonging to the jacalin-related family called Helja. Despite of the significant number of plant lectins described in the literature, only a small group exhibits antifungal activity and the mechanism by which they kill fungi is still not understood. The aim of this work was to explore Helja activity on plant pathogenic fungi, and provide insights into its mechanism of action. Through cellular and biochemical experimental approaches, here we show that Helja exerts an antifungal effect on Sclerotinia sclerotiorum, a sunflower pathogen. The lectin interacts with the fungal spore surface, permeabilizes its plasma membrane, can be internalized into the cell and induces oxidative stress, finally leading to the cell death. On the other hand, Helja is inactive towards Fusarium solani, a non-pathogen of sunflower, showing the selective action of the lectin. The mechanistic basis for the antifungal activity of an extracellular jacalin lectin is presented, suggesting its initial interaction with fungal cell wall carbohydrates and further internalization. The implication of our findings for plant defense is discussed.

Effects of nitric oxide on sunflower seedlings: A balance between defense and development.

Nitric oxide (NO) is a major plant signaling molecule that plays key roles during plant-pathogen interactions and plant development. Previous work showed the participation of NO in the development and lignin composition of sunflower roots. Thereby, we have hypothesized that NO applications could control the attack of the fungal pathogen Verticillium dahliae in sunflowers. Seedlings growing hydroponically were pretreated with NO donors and further inoculated with the fungus. Evaluation of disease symptoms showed that NO pretreatments could not reduce Verticillium wilt. Strikingly, NO donors appear to promote the fungal infection. These results indicate that NO applications were unable to protect sunflowers from Verticillium attack and highlight the role played by the fine tuning regulation of NO levels required to balance plant responses between development and defense.

On the role of a Lipid-Transfer Protein. Arabidopsis ltp3 mutant is compromised in germination and seedling growth.

Plant Lipid-Transfer Proteins (LTPs) exhibit the ability to reversibly bind/transport lipids in vitro. LTPs have been involved in diverse physiological processes but conclusive evidence on their role has only been presented for a few members, none of them related to seed physiology. Arabidopsis seeds rely on storage oil breakdown to supply carbon skeletons and energy for seedling growth. Here, Arabidopsis ltp3 mutant was analyzed for its ability to germinate and for seedling establishment. Ltp3 showed delayed germination and reduced germination frequency. Seedling growth appeared reduced in the mutant but this growth restriction was rescued by the addition of an exogenous carbon supply, suggesting a defective oil mobilization. Lipid breakdown analysis during seedling growth revealed a differential profile in the mutant compared to the wild type. The involvement of LTP3 in germination and seedling growth and its relationship with the lipid transfer ability of this protein is discussed.

Molecular characterization of Helja, an extracellular jacalin-related protein from Helianthus annuus: Insights into the relationship of this protein with unconventionally secreted lectins.

Jacalin-related lectins (JRLs) encompass cytosolic, nuclear and vacuolar members displaying the jacalin domain in one or more copies or in combination with unrelated domains. Helianthus annuus jacalin (Helja) is a mannose-specific JRL previously identified in the apoplast of Helianthus annuus seedlings, and this protein has been proposed to follow unconventional secretion. Here, we describe the full-length Helja cDNA sequence, which presents a unique jacalin domain (merolectin) and the absence of a signal peptide, confirming that the protein cannot follow the classical ER-dependent secretory pathway. Helja mRNA is present in seeds, cotyledons, roots and hypocotyls, but no transcripts were detected in the leaves. Searches for sequence similarity showed that Helja is barely similar to other JRLs present in H. annuus databases and less than 45% identical to other monocot or dicot JRLs. Strikingly, most of the merolectins recovered through data mining using Helja as a query were predicted as apoplastic, although most of these proteins lack the signal peptide required for classical secretion. Thus, Helja is the first bait identified to recover putative unconventionally secreted lectins. Because the recovered JRLs are widely distributed among the plant kingdom, an as yet unknown role for jacalin lectins in the apoplast is emerging.

Apoplastic exosome-like vesicles: a new way of protein secretion in plants?

The presence of apoplastic proteins without predicted signal peptide in the gene sequence suggests the existence of protein secretion independent of the ER/Golgi classical route. In animals, one of the pathways proposed for alternative protein secretion involves the release of exosomes to the extracellular space. Although this pathway has not been dissected in plants some indirect evidence is emerging. We have reported that apoplastic fractions of sunflower seeds contain exosome-like vesicles. Besides, these vesicles are enriched in the lectin Helja, which is immunolocalized in the extracellular space even if it the protein has no predicted signal peptide. Here we show that Helja is not glycosylated and its secretion is insensitive to brefeldin A, two of the major characteristics to discard ER/Golgi-mediated protein transport. Moreover, the levels of Helja in sunflower extracellular vesicles are not affected by brefeldin A treatment. Our results suggest that Helja could be exported through an exosome-mediated pathway and point out that this mechanism may be responsible for the secretion of at least part of the leaderless proteins detected in the extracellular compartment of plants.

Extracellular sunflower proteins: evidence on non-classical secretion of a jacalin-related lectin.

Extracellular proteins from sunflower seedlings were analyzed by electrophoresis followed by peptide mass fingerprinting. Tentative identification revealed novel proteins for this crop. A significant number of those proteins were not expected to be extracellular because they lacked the typical signal peptide responsible for secretion. In silico analysis showed that some members of this group presented the characteristic disordered structures of certain non-classical and leaderless mammalian secretory proteins. Among these proteins, a putative jacalin-related lectin (Helja) with a mannose binding domain was further isolated from extracellular fluids by mannose-affinity chromatography, thus validating its identification. Besides, immunolocalization assays confirmed its extracellular location. These results showed that a lectin, not predicted to be secreted in strict requirement of the N-terminal signal peptide, occurs in a sunflower extracellular compartment. The implications of this finding are discussed.

Phloem sap of tomato plants contains a DIR1 putative ortholog.

Arabidopsis thaliana defective in induced resistance 1 (At-DIR1) has been characterized as a protein responsible for the generation or transmission of the still unknown signal involved in systemic acquired resistance. This acidic apoplastic protein is a member of the family of lipid transfer proteins and was detected in vascular fluids. To our knowledge, no DIR1-like protein has been described in other plant species. Hence, we have performed data mining to identify a putative ortholog of DIR1 in tomato. This strategy allowed the detection of a few gene products displaying sequence similarity to At-DIR1 whose structural features were further analysed in silico. The best match (unigene SGN-327306) encoded a protein with an acidic pI, a peculiar characteristic of DIR1 among lipid transfer proteins, and was hence selected as a putative tomato ortholog of At-DIR1. This sequence, named Le-DIR1, served for the design of a specific antigenic peptide and the generation of polyclonal antibodies. The antiserum anti-Le-DIR1 recognized a peptide of the expected size (7kDa) in phloem sap of tomato plants, hence confirming the existence of the predicted protein in vascular fluids. This result supports the notion of the existence of common systemic acquired resistance (SAR) signaling molecules in different species.

Vesicular fractions of sunflower apoplastic fluids are associated with potential exosome marker proteins.

Based on the presence of phospholipids in the extracellular fluids (EFs) of sunflower seeds, we have hypothesized on the existence of vesicles in the apoplastic compartment of plants. Ultracentrifugation of sunflower EF allowed the isolation of particles of 50-200 nm with apparent membrane organization. A small GTPase Rab was putatively identified in this vesicular fraction. Since Rab proteins are involved in vesicular traffic and their presence in exosomes from animal fluids has been demonstrated, evidence presented here supports the existence of exosome-like vesicles in apoplastic fluids of sunflower. Their putative contribution to intercellular communication in plants is discussed.

Unexpected localization of a lipid transfer protein in germinating sunflower seeds.

Plant lipid transfer proteins (LTPs) are low-molecular-mass proteins whose biological function still remains elusive. They are synthesized with a signal peptide that drives them to the secretory pathway. We have previously described the occurrence of an apoplastic LTP named Ha-AP10, present in sunflower seeds. Using a biochemical approach we now demonstrate that a fraction of Ha-AP10 is perispherically bound to membranes of germinating seeds. Purification of plasma membranes revealed the presence of Ha-AP10 in this fraction. Fluorimmunolocalization studies on germinating sunflower seeds demonstrated that in addition to the apoplastic and plasma membrane localization, Ha-AP10 is also present intracellularlly associated to unidentified structures. This varied distribution of Ha-AP10 in sunflower seeds may give novel clues to understand the role of LTPs in seed physiology.

A germin-like protein of wheat leaf apoplast inhibits serine proteases.

A protein resistant to heat and proteolysis that inhibits serine proteases was isolated from wheat leaf apoplasts. Based on trypsin inhibition, its more active form was a 66-69 kDa oligomer. It was dissociated in an 18-21 kDa monomer having an amino terminal sequence identical to the Box A of germins and germin-like proteins. Like these proteins, it was glycosylated and showed manganese superoxide dismutase activity. The monomer displayed three forms when examined by 2D western blot: two of 19 kDa, pI 5.8 and 6.2; and one of 21 kDa, pI 5.8. It was found that the protein controls serine protease activity in the apoplast of plants challenged with the fungus Septoria tritici.