A plant mannose-binding lectin and fluconazole: Key targets combination against Candida albicans.

AIMS: This study aimed to evaluate the combined effect of a mannose-binding lectin Helja with fluconazole (FLC) on Candida albicans and to get insights about the joint action mechanism. METHODS AND RESULTS: The fungal growth was assessed following the optical density at 630 nm. Fungal cell morphology and nucleus integrity were analysed by flow cytometry and confocal laser scanning microscopy using Calcofluor White (CFW) and 4',6-diamidino-2-phenylindole (DAPI) staining respectively. The basis of Helja + FLC action on cell wall and plasma membrane was analysed using perturbing agents. The Helja + FLC combination exhibited an inhibitory effect of fungal growth about three times greater than the sum of both compounds separately and inhibited fungal morphological plasticity, an important virulence attribute associated with drug resistance. Cells treated with Helja + FLC showed morphological changes, nucleus disintegration and formation of multimera structures, leading to cell collapse. CONCLUSIONS: Our findings indicate that the Helja + FLC combination exhibited a potent antifungal activity based on their simultaneous action on different microbial cell targets. SIGNIFICANCE AND IMPACT OF STUDY: The combination of a natural protein with conventional drugs might be helpful for the design of effective therapeutic strategies against Candida, contributing to minimize the development of drug resistance and host cell toxicity.

Plant Antifungal Lectins: Mechanism of Action and Targets on Human Pathogenic Fungi.

Lectins are proteins characterized by their ability to specifically bind different carbohydrate motifs. This feature is associated with their endogenous biological function as well as with multiple applications. Plants are important natural sources of these proteins; however, only a reduced group was shown to display antifungal activity. Although it is hypothesized that the target of lectins is the fungal cell wall, the mechanism through which they exert the antifungal action is poorly understood. This topic is relevant to improve treatment against pathogens of importance for human health. In this context, mechanisms pointing to essential attributes for virulence instead of the viability of the pathogen emerge as a promising approach. This review provides the current knowledge on the action mechanism of plant antifungal lectins and their putative use for the development of novel active principles against fungal infections.

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.

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.

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.

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.

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.

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.

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.

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.

An extracellular lipid transfer protein is relocalized intracellularly during seed germination.

Plant lipid transfer proteins (LTPs) constitute a family of small proteins recognized as being extracellular. In agreement with this notion, several lines of evidence have shown the apoplastic localization of HaAP10, a LTP from Helianthus annuus dry seeds. However, HaAP10 was recently detected intracellularly in imbibing seeds. To clarify its distribution, immunolocalization experiments were performed during the course of germination and confirmed its intracellular localization upon early seed imbibition. Further assays using a hydrophobic dye, FM4-64, inhibitors of vesicular traffic, and immunolocalization of the pectin rhamnogalacturonan-II, allowed the conclusion that endocytosis is activated as soon as seed imbibition starts. Furthermore, this study demonstrated that HaAP10 is endocytosed throughout imbibition. Biochemical and cellular approaches indicate that the intracellular fraction of this LTP appears associated with oil bodies and some evidence also suggest its presence in glyoxysomes. So, HaAP10 is apoplastic in dry seeds and upon imbibition is rapidly internalized and relocalized to organelles involved in lipid metabolism. The results suggest that HaAP10 may be acting as a fatty acid shuttle between the oil body and the glyoxysome during seed germination. This concept is consistent with the initial proposition that LTPs participate in the intracellular transfer of lipids which was further denied based on their apparent extracellular localization. This report reveals for the first time the relocalization of a lipid transfer protein and opens new perspectives on its role.

Rapid endocytosis is triggered upon imbibition in Arabidopsis seeds.

During seed imbibition and embryo activation, rapid change from a metabolically resting state to the activation of diverse extracellular and/or membrane bound molecules is essential and, hence, endocytosis could be activated too. In fact, we have documented endocytic internalization of the membrane impermeable endocytic tracer FM4-64 already upon 30 min of imbibition of Arabidopsis seeds. This finding suggest that endocytosis is activated early during seed imbibition in Arabidopsis. Immunolocalization of rhamnogalacturonan-II (RG-II) complexed with boron showed that whereas this pectin is localized only in the cell walls of dry seed embryos, it starts to be intracellular once the imbibition started. Brefeldin A (BFA) exposure resulted in recruitment of the intracellular RG-II pectin complexes into the endocytic BFA-induced compartments, confirming the endocytic origin of the RG-II signal detected intracellularly. Finally, germination was significantly delayed when Arabidopsis seeds were germinated in the presence of inhibitors of endocytic pathways, suggesting that trafficking of extracellular molecules might play an important role in the overcome of germination. This work constitutes the first demonstration of endocytic processes during germination and opens new perspectives about the role of the extracellular matrix and membrane components in seed germination.

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.

Involvement of phospholipase C in the responses triggered by extracellular phosphatidylinositol 4-phosphate.

Phosphatidylinositol 4-phosphate (PI4P) is a minor phospholipid signal molecule involved in diverse biological processes. It is also the main precursor of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)], which is well described as the substrate of the phospholipase C (PLC)-diacylglycerol kinase (DGK) signalling pathway. Recently, we have demonstrated that PI4P plays a role as an extracellular signalling molecule involved in the activation of xylanase-induced defence responses in tomato cell suspensions. Here we make an approach to the possible mode of action of extracellular PI4P by analysing the involvement of PLC/DGK signalling pathway. We show that extracellular PI4P can be incorporated into tomato cells and further metabolized to PI, although its conversion to PI(4,5)P(2) could not be detected. In addition, treatment of tomato cells with the PLC inhibitor U73122 inhibited the oxidative burst triggered by PI4P treatment, suggesting the involvement of PLC in the induction of this response. Nevertheless, we demonstrate that PI4P treatment does not induce DGK activity. These results indicate that PLC but not DKG activation would be triggered by extracellular PI4P. In this sense, a possible mode of action of extracellular PI4P as a direct substrate of PLC to induce this signalling pathway is discussed.

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.

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.

Phosphatidylinositol 4-phosphate accumulates extracellularly upon xylanase treatment in tomato cell suspensions.

Various phosphoinositides have been implicated in plant defence signalling. Until now, such molecules have been exclusively related to intracellular signalling. Here, evidence is provided for the detection of extracellular phosphatidylinositol 4-phosphate (PI4P) in tomato cell suspensions. We have analysed and compared the intracellular and extracellular phospholipid profiles of [(32)P(i)]-prelabelled tomato cells, challenged with the fungal elicitor xylanase. These phospholipid patterns were found to be different, being phosphatidylinositol phosphate (PIP) the most abundant phospholipid in the extracellular medium. Moreover, while cells responded with a typical increase in phosphatidic acid and a decrease in intracellular PIP upon xylanase treatment, extracellular PIP level increased in a time- and dose-dependent manner. Using two experimental approaches, the extracellular PIP isoform was identified as PI4P. Addition of PI4P to tomato cell suspensions triggered the same defence responses as those induced by xylanase treatment. These include production of reactive oxygen species, accumulation of defence-related gene transcripts and induction of cell death. We demonstrate that extracellular PI4P is accumulated in xylanase-elicited cells and that exogenous application of PI4P mimics xylanase effects, suggesting its putative role as an intercellular signalling molecule.