Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part II: experimental studies withAspidosperma ramiflorum in vivo and in vitro

Several species of Aspidosperma plants are used to treat diseases in the tropics, including Aspidosperma ramiflorum, which acts against leishmaniasis, an activity that is experimentally confirmed. The species, known as guatambu-yellow, yellowperoba, coffee-peroba andmatiambu, grows in the Atlantic Forest of Brazil in the South to the Southeast regions. Through a guided biofractionation of A. ramiflorum extracts, the plant activity against Plasmodium falciparum was evaluated in vitro for toxicity towards human hepatoma G2 cells, normal monkey kidney cells and nonimmortalised human monocytes isolated from peripheral blood. Six of the seven extracts tested were active at low doses (half-maximal drug inhibitory concentration < 3.8 µg/mL); the aqueous extract was inactive. Overall, the plant extracts and the purified compounds displayed low toxicity in vitro. A nonsoluble extract fraction and one purified alkaloid isositsirikine (compound 5) displayed high selectivity indexes (SI) (= 56 and 113, respectively), whereas compounds 2 and 3 were toxic (SI < 10). The structure, activity and low toxicity of isositsirikine in vitro are described here for the first time in A. ramiflorum, but only the neutral and precipitate plant fractions were tested for activity, which caused up to 53% parasitaemia inhibition of Plasmodium bergheiin mice with blood-induced malaria. This plant species is likely to be useful in the further development of an antimalarial drug, but its pharmacological evaluation is still required.

Patterns of plant subcellular responses to successful oomycete infections reveal differences in host cell reprogramming and endocytic trafficking.

Adapted filamentous pathogens such as the oomycetes Hyaloperonospora arabidopsidis (Hpa) and Phytophthora infestans (Pi) project specialized hyphae, the haustoria, inside living host cells for the suppression of host defence and acquisition of nutrients. Accommodation of haustoria requires reorganization of the host cell and the biogenesis of a novel host cell membrane, the extrahaustorial membrane (EHM), which envelops the haustorium separating the host cell from the pathogen. Here, we applied live-cell imaging of fluorescent-tagged proteins labelling a variety of membrane compartments and investigated the subcellular changes associated with accommodating oomycete haustoria in Arabidopsis and N. benthamiana. Plasma membrane-resident proteins differentially localized to the EHM. Likewise, secretory vesicles and endosomal compartments surrounded Hpa and Pi haustoria revealing differences between these two oomycetes, and suggesting a role for vesicle trafficking pathways for the pathogen-controlled biogenesis of the EHM. The latter is supported by enhanced susceptibility of mutants in endosome-mediated trafficking regulators. These observations point at host subcellular defences and specialization of the EHM in a pathogen-specific manner. Defence-associated haustorial encasements, a double-layered membrane that grows around mature haustoria, were frequently observed in Hpa interactions. Intriguingly, all tested plant proteins accumulated at Hpa haustorial encasements suggesting the general recruitment of default vesicle trafficking pathways to defend pathogen access. Altogether, our results show common requirements of subcellular changes associated with oomycete biotrophy, and highlight differences between two oomycete pathogens in reprogramming host cell vesicle trafficking for haustoria accommodation. This provides a framework for further dissection of the pathogen-triggered reprogramming of host subcellular changes.

Cytotoxic effects of FGF2-saporin on bovine epithelial lens cells in vitro.

PURPOSE: To test the ability of two preparations of FGF2-saporin, either FGF2 chemically conjugated to saporin (FGF2-SAP) or genetically engineered FGF2-saporin (rFGF2-SAP) to inhibit the growth of bovine epithelial lens (BEL) cells in vitro when in solution and when immobilized on heparin surface-modified (HSM) polymethylmethacrylate (PMMA) intraocular lenses (IOLs). METHOD: Bovine epithelial lens cells were incubated with various concentrations FGF2-saporin for as long as 4 days. The number of surviving cells was determined by counting the number of nuclei. Because FGF2 binds to heparin, FGF2-saporin was incubated with HSM PMMA IOLs; excess toxin was washed off, and the BEL cells were grown on the FGF2-saporin-treated IOLs (HSM and non-HSM) for 4 days. Cell density was determined by image analysis. RESULTS: Both FGF2-SAP and rFGF2-SAP were highly cytotoxic (nM range), with rFGF2-SAP 10 times less active than FGF2-SAP. FGF2-saporin bound to the surface of HSM IOLs and eluted by 2M NaCl retained its activity. Toxin bound to HSM IOLs killed more than 90% of the BEL cells placed on the IOL surface within 4 days. The ability of FGF2-saporin to prevent the growth of cells on the IOL surface was strictly dependent on the presence of heparin on the IOL. CONCLUSIONS: FGF2-saporin is bound to HSM PMMA IOLs and prevents the growth of epithelial cells on the surface of the lens.

Transcriptional profiling of Gram-positive Arthrobacter in the phyllosphere: induction of pollutant degradation genes by natural plant phenolic compounds.

Arthrobacter chlorophenolicus A6 is a Gram-positive, 4-chlorophenol-degrading soil bacterium that was recently shown to be an effective colonizer of plant leaf surfaces. The genetic basis for this phyllosphere competency is unknown. In this paper, we describe the genome-wide expression profile of A.chlorophenolicus on leaves of common bean (Phaseolus vulgaris) compared with growth on agar surfaces. In phyllosphere-grown cells, we found elevated expression of several genes known to contribute to epiphytic fitness, for example those involved in nutrient acquisition, attachment, stress response and horizontal gene transfer. A surprising result was the leaf-induced expression of a subset of the so-called cph genes for the degradation of 4-chlorophenol. This subset encodes the conversion of the phenolic compound hydroquinone to 3-oxoadipate, and was shown to be induced not only by 4-chlorophenol but also hydroquinone, its glycosylated derivative arbutin, and phenol. Small amounts of hydroquinone, but not arbutin or phenol, were detected in leaf surface washes of P.vulgaris by gas chromatography-mass spectrometry. Our findings illustrate the utility of genomics approaches for exploration and improved understanding of a microbial habitat. Also, they highlight the potential for phyllosphere-based priming of bacteria to stimulate pollutant degradation, which holds promise for the application of phylloremediation.

In vitro assessment of the pulmonary toxicity and gastric availability of lead-rich particles from a lead recycling plant

Epidemiological studies in urban areas have linked increasing respiratory and cardiovascular pathologies with atmospheric particulate matter (PM) from anthropic activities. However, the biological fate of metal-rich PM industrial emissions in urban areas of developed countries remains understudied. Lead toxicity and bioaccessibility assessments were therefore performed on emissions from a lead recycling plant, using complementary chemical acellular tests and toxicological assays, as a function of PM size (PM(10-2.5), PM(2.5-1) and PM(1)) and origin (furnace, refining and channeled emissions). Process PM displayed differences in metal content, granulometry, and percentage of inhalable fraction as a function of their origin. Lead gastric bioaccessibility was relatively low (maximum 25%) versus previous studies; although, because of high total lead concentrations, significant metal quantities were solubilized in simulated gastrointestinal fluids. Regardless of origin, the finest PM(1) particles induced the most significant pro-inflammatory response in human bronchial epithelial cells. Moreover, this biological response correlated with pro-oxidant potential assay results, suggesting some biological predictive value for acellular tests. Pulmonary effects from lead-rich PM could be driven by thiol complexation with either lead ions or directly on the particulate surface. Finally, health concern of PM was discussed on the basis of pro-inflammatory effects, accellular test results, and PM size distribution.

Structure-function characterization and optimization of a plant-derived antibacterial peptide.

Crushed seeds of the Moringa oleifera tree have been used traditionally as natural flocculants to clarify drinking water. We previously showed that one of the seed peptides mediates both the sedimentation of suspended particles such as bacterial cells and a direct bactericidal activity, raising the possibility that the two activities might be related. In this study, the conformational modeling of the peptide was coupled to a functional analysis of synthetic derivatives. This indicated that partly overlapping structural determinants mediate the sedimentation and antibacterial activities. Sedimentation requires a positively charged, glutamine-rich portion of the peptide that aggregates bacterial cells. The bactericidal activity was localized to a sequence prone to form a helix-loop-helix structural motif. Amino acid substitution showed that the bactericidal activity requires hydrophobic proline residues within the protruding loop. Vital dye staining indicated that treatment with peptides containing this motif results in bacterial membrane damage. Assembly of multiple copies of this structural motif into a branched peptide enhanced antibacterial activity, since low concentrations effectively kill bacteria such as Pseudomonas aeruginosa and Streptococcus pyogenes without displaying a toxic effect on human red blood cells. This study thus identifies a synthetic peptide with potent antibacterial activity against specific human pathogens. It also suggests partly distinct molecular mechanisms for each activity. Sedimentation may result from coupled flocculation and coagulation effects, while the bactericidal activity would require bacterial membrane destabilization by a hydrophobic loop.

Study of the regulation of antifungal activity in plant-associated pseudomas fluorescens

RésuméEn agriculture d'énormes pertes sont causées par des champignons telluriques pathogènes tels que Thielaviopsis, Fusarium, Gaeumannomyces et Rhizoctonia ou encore l'oomycète Pythium. Certaines bactéries dites bénéfiques, comme Pseudomonas fluorescens, ont la capacité de protéger les plantes de ces pathogènes par la colonisation de leur racines, par la production de métabolites secondaires possédants des propriétés antifongiques et par l'induction des mécanismes de défenses de la plante colonisée. P. fluorescens CHAO, une bactérie biocontrôle isolée d'un champ de tabac à Payerne, a la faculté de produire un large spectre de métabolites antifongiques, en particulier le 2,4- diacétylphloroglucinol (DAPG), la pyolutéorine (PLT), le cyanure d'hydrogène (HCN), la pyrrolnitrine (PRN) ainsi que des chélateurs de fer.La plante, par sécrétion racinaire, produit des rhizodéposites, source de carbone et d'azote, qui profitent aux populations bactériennes vivant dans la rhizosphere. De plus, certains stresses biotiques et abiotiques modifient cette sécrétion racinaire, en terme quantitatif et qualitatif. De leur côté, les bactéries bénéfiques, améliorent, de façon direct et/ou indirect, la croissance de la plante hôte. De nombreux facteurs biotiques et abiotiques sont connus pour réguler la production de métabolites secondaires chez les bactéries. Des études récentes ont démontré l'importance de la communication entre la plante et les bactéries bénéfiques afin que s'établisse une interaction profitant à chacun des deux partis. Il est ainsi vraisemblable que les populations bactériennes associées aux racines soient capables d'intégrer ces signaux et d'adapter spécifiquement leur comportement en conséquence.La première partie de ce travail de thèse a été la mise au point d'outils basés sur la cytométrie permettant de mesurer l'activité antifongique de cellules bactériennes individuelles dans un environnent naturel, les racines des plantes. Nous avons démontré, grâce à un double marquage aux protéines autofluorescentes GFP et mCherry, que les niveaux d'expression des gènes impliqués dans la biosynthèse des substances antifongiques DAPG, PLT, PRN et HCN ne sont pas les mêmes dans des milieux de cultures liquides que sur les racines de céréales. Par exemple, l'expression de pltA (impliqué dans la biosynthèse du PLT) est quasiment abolie sur les racines de blé mais atteint un niveau relativement haut in vitro. De plus cette étude a mis en avant l'influence du génotype céréalien sur l'expression du gène phlA qui est impliqué dans la biosynthèse du DAPG.Une seconde étude a révélé la communication existant entre une céréale (orge) infectée par le pathogène tellurique Pythium ultimum et P. fluorescens CHAO. Un système de partage des racines nous a permis de séparer physiquement le pathogène et la bactérie bénéfique sur la plante. Cette méthode a donné la possibilité d'évaluer l'effet systémique, causé par l'attaque du pathogène, de la plante sur la bactérie biocontrôle. En effet, l'infection par le phytopathogène modifie la concentration de certains composés phénoliques dans les exsudats racinaires stimulant ainsi l'expression de phi A chez P.fluorescens CHAO.Une troisième partie de ce travail focalise sur l'effet des amibes qui sont des micro-prédateurs présents dans la rhizosphere. Leur présence diminue l'expression des gènes impliqués dans la biosynthèse du DAPG, PLT, PRN et HCN chez P.fluorescens CHAO, ceci en culture liquide et sur des racines d'orge. De plus, des molécules provenant du surnageant d'amibes, influencent l'expression des gènes requis pour la biosynthèse de ces antifongiques. Ces résultats illustrent que les amibes et les bactéries de la rhizosphere ont développé des stratégies pour se reconnaître et adapter leur comportement.La dernière section de ce travail est consacrée à l'acide indole-acétique (LA.A), une phytohormone connue pour son effet stimulateur sur phlA. Une étude moléculaire détaillée nous a démontré que cet effet de l'IAA est notamment modulé par une pompe à efflux (FusPl) et de son régulateur transcriptionnel (MarRl). De plus, les gènes fusPl et marRl sont régulés par d'autres composés phénoliques tels que le salicylate (un signal végétal) et l'acide fusarique (une phytotoxine du pathogène Fusarium).En résumé, ce travail de thèse illustre la complexité des interactions entre les eucaryotes et procaryotes de la rhizosphère. La reconnaissance mutuelle et l'instauration d'un dialogue moléculaire entre une plante hôte et ses bactéries bénéfiques associées? sont indispensables à la survie des deux protagonistes et semblent être hautement spécifiques.SummaryIn agriculture important crop losses result from the attack of soil-borne phytopathogenic fungi, including Thielaviopsis, Fusarium, Gaeumannomyces and Rhizoctonia, as well as from the oomycete Pythium. Certain beneficial microorganisms of the rhizosphere, in particular Pseudomonas fluorescens, have the ability to protect plants against phytopathogens by the intense colonisation of roots, by the production of antifungal exoproducts, and by induction of plant host defences. P. fluorescens strain CHAO, isolated from a tobacco field near Payerne, produces a large array of antifungal exoproducts, including 2,4-diacetylphloroglucinol (DAPG), pyoluteorin (PLT), hydrogen cyanide (HCN), pyrrolnitrin (PRN) and iron chelators. Plants produce rhizodeposites via root secretion and these represent a relevant source of carbon and nitrogen for rhizosphere microorganisms. Various biotic and abiotic stresses influence the quantity and the quality of released exudates. One the other hand, beneficial bacteria directly or indirectly promote plant growth. Biotic and abiotic factors regulate exoproduct production in biocontrol microorganisms. Recent studies have highlighted the importance of communication in establishing a fine-tuned mutualist interaction between plants and their associated beneficial bacteria. Bacteria may be able to integrate rhizosphere signals and adapt subsequently their behaviour.In a first part of the thesis, we developed a new method to monitor directly antifungal activity of individual bacterial cells in a natural environment, i.e. on roots of crop plants. We were able to demonstrate, via a dual-labelling system involving green and red fluorescent proteins (GFP, mCherry) and FACS-based flow cytometry, that expression levels of biosynthetic genes for the antifungal compounds DAPG, PLT, PRN, and HCN are highly different in liquid culture and on roots of cereals. For instance, expression of pltA (involved in PLT biosynthesis) was nearly abolished on wheat roots whereas it attained a relatively high level under in vitro conditions. In addition, we established the importance of the cereal genotype in the expression of phi A (involved in DAPG biosynthesis) in P. fluorescens CHAO.A second part of this work highlighted the systemic communication that exists between biocontrol pseudomonads and plants following attack by a root pathogen. A split-root system, allowing physical separation between the soil-borne oomycete pathogen Phytium ultimum and P. fluorescens CHAO on barley roots, was set up. Root infection by the pathogen triggered a modification of the concentration of certain phenolic root exudates in the healthy root part, resulting in an induction ofphlA expression in P. fluorescens CHAO.Amoebas are micro-predators of the rhizosphere that feed notably on bacteria. In the third part of the thesis, co-habitation of Acanthamoeba castellanii with P. fluorescens CHAO in culture media and on barley roots was found to significantly reduce bacterial expression of genes involved in the biosynthesis of DAPG, PLT, HCN and PRN. Interestingly, molecular cues present in supernatant of A. castelanii induced the expression of these antifungal genes. These findings illustrate the strategies of mutual recognition developed by amoeba and rhizosphere bacteria triggering responses that allow specific adaptations of their behaviour.The last section of the work focuses on indole-3-acetic acid (IAA), a phytohormone that stimulates the expression of phi A. A detailed molecular study revealed that the IAA-mediated effect on phi A is notably modulated by an efflux pump (FusPl) and its transcriptional regulator (MarRl). Remarkably, transcription of fusPl and marRl was strongly upregulated in presence of other phenolic compounds such as salicylate (a plant signal) and fusaric acid (a phytotoxin of the pathogenic fungus Fusarium).To sum up, this work illustrates the great complexity of interactions between eukaryotes and prokaryotes taking place in the rhizosphere niche. The mutual recognition and the establishment of a molecular cross-talk between the host plant and its associated beneficial bacteria are essential for the survival of the two partners and these interactions appear to be highly specific.

Peroxynitrite cytotoxicity on bovine retinal pigmented epithelial cells in culture.

Peroxynitrite induced in vitro a dose dependent toxicity on retinal pigmented epithelial (RPE) cells. Cell death was partially mediated by apoptosis as demonstrated by nuclear fragmentation and TdT-mediated dUTP nick-end labeling assay. Peroxynitrite-induced tyrosine nitration was revealed by immunocytochemistry, both in the cytoplasm and in the nucleus of the cells. Nitration was not observed in RPE cells, producing nitric oxide (NO) after stimulation by lipopolysacharide and interferon-g (IFN-gamma), suggesting that peroxynitrite was not formed in vitro in such conditions. Peroxynitrite could be responsible for the retinal damages observed in pathological conditions in which NO has been demonstrated to be involved. In this context, EGb761, identified as a free radical scavenger, was showed herein to protect RPE cells against peroxynitrite injury.

Small, stable shuttle vectors based on the minimal pVS1 replicon for use in gram-negative, plant-associated bacteria.

The minimal replicon of the Pseudomonas plasmid pVS1 was genetically defined and combined with the Escherichia coli p15A replicon, to provide a series of new, oligocopy cloning vectors (5.3 to 8.3 kb). Recombinant plasmids derived from these vectors were stable in growing and nongrowing cells of root-colonizing P. fluorescens strains incubated under different environmental conditions for more than 1 month.

A spatial accommodation by neighboring cells is required for organ initiation in Arabidopsis.

Lateral root formation in plants can be studied as the process of interaction between chemical signals and physical forces during development. Lateral root primordia grow through overlying cell layers that must accommodate this incursion. Here, we analyze responses of the endodermis, the immediate neighbor to an initiating lateral root. Endodermal cells overlying lateral root primordia lose volume, change shape, and relinquish their tight junction-like diffusion barrier to make way for the emerging lateral root primordium. Endodermal feedback is absolutely required for initiation and growth of lateral roots, and we provide evidence that this is mediated by controlled volume loss in the endodermis. We propose that turgidity and rigid cell walls, typical of plants, impose constraints that are specifically modified for a given developmental process.

Detection of plant-modulated alterations in antifungal gene expression in Pseudomonas fluorescens CHA0 on roots by flow cytometry.

The biocontrol activity of the root-colonizing Pseudomonas fluorescens strain CHA0 is largely determined by the production of antifungal metabolites, especially 2,4-diacetylphloroglucinol. The expression of these metabolites depends on abiotic and biotic environmental factors, in particular, elements present in the rhizosphere. In this study, we have developed a new method for the in situ analysis of antifungal gene expression using flow cytometry combined with green fluorescent protein (GFP)-based reporter fusions to the phlA and prnA genes essential for the production of the antifungal compounds 2,4-diacetylphloroglucinol and pyrrolnitrin, respectively, in strain CHA0. Expression of phlA-gfp and prnA-gfp in CHA0 cells harvested from the rhizosphere of a set of plant species as well as from the roots of healthy, leaf pathogen-attacked, and physically stressed plants were analyzed using a FACSCalibur. After subtraction of background fluorescence emitted by plant-derived particles and CHA0 cells not carrying the gfp reporters, the average gene expression per bacterial cell could be calculated. Levels of phlA and prnA expression varied significantly in the rhizospheres of different plant species. Physical stress and leaf pathogen infection lowered phlA expression levels in the rhizosphere of cucumber. Our results demonstrate that the newly developed approach is suitable to monitor differences in levels of antifungal gene expression in response to various plant-derived factors. An advantage of the method is that it allows quantification of bacterial gene expression in rhizosphere populations at a single-cell level. To our best knowledge, this is the first study using flow cytometry for the in situ analysis of biocontrol gene expression in a plant-beneficial bacterium in the rhizosphere.

Atomic Force Microscopy Stiffness Tomography on Living Arabidopsis thaliana Cells Reveals the Mechanical Properties of Surface and Deep Cell-Wall Layers during Growth.

Cell-wall mechanical properties play a key role in the growth and the protection of plants. However, little is known about genuine wall mechanical properties and their growth-related dynamics at subcellular resolution and in living cells. Here, we used atomic force microscopy (AFM) stiffness tomography to explore stiffness distribution in the cell wall of suspension-cultured Arabidopsis thaliana as a model of primary, growing cell wall. For the first time that we know of, this new imaging technique was performed on living single cells of a higher plant, permitting monitoring of the stiffness distribution in cell-wall layers as a function of the depth and its evolution during the different growth phases. The mechanical measurements were correlated with changes in the composition of the cell wall, which were revealed by Fourier-transform infrared (FTIR) spectroscopy. In the beginning and end of cell growth, the average stiffness of the cell wall was low and the wall was mechanically homogenous, whereas in the exponential growth phase, the average wall stiffness increased, with increasing heterogeneity. In this phase, the difference between the superficial and deep wall stiffness was highest. FTIR spectra revealed a relative increase in the polysaccharide/lignin content.

Somatic embryogenesis and in vitro plant regeneration from pejibaye adult plant leaf primordia

The aim of this work was to evaluate a protocol for plant regeneration by means of somatic embryos obtained from isolated adult pejibaye leaf primordia, and to describe histological origin of embryos and morphogenetic response. Explants were cultivated in modified MS medium. Mesophyll parenchymatous cells originated meristemoids (preembryonic complex formation) induced with 7.1 µM BAP in the first two subculture periods. After polarized structures with 12.9 µM NAA and 3.55 µM BAP were formed in the third subculture, somatic embryos developed and regenerated normal plants. The mesophyll parenchymatous cells display high capacity of direct response to the auxin and cytokinin.

Climate change and plant distribution: local models predict high-elevation persistence

Mountain ecosystems will likely be affected by global warming during the 21st century, with substantial biodiversity loss predicted by species distribution models (SDMs). Depending on the geographic extent, elevation range and spatial resolution of data used in making these models, different rates of habitat loss have been predicted, with associated risk of species extinction. Few coordinated across-scale comparisons have been made using data of different resolution and geographic extent. Here, we assess whether climate-change induced habitat losses predicted at the European scale (10x10' grid cells) are also predicted from local scale data and modeling (25x25m grid cells) in two regions of the Swiss Alps. We show that local-scale models predict persistence of suitable habitats in up to 100% of species that were predicted by a European-scale model to lose all their suitable habitats in the area. Proportion of habitat loss depends on climate change scenario and study area. We find good agreement between the mismatch in predictions between scales and the fine-grain elevation range within 10x10' cells. The greatest prediction discrepancy for alpine species occurs in the area with the largest nival zone. Our results suggest elevation range as the main driver for the observed prediction discrepancies. Local scale projections may better reflect the possibility for species to track their climatic requirement toward higher elevations.

Oral insecticidal activity of plant-associated pseudomonads.

Biocontrol pseudomonads are most known to protect plants from fungal diseases and to increase plant yield, while intriguing aspects on insecticidal activity have been discovered only recently. Here, we demonstrate that Fit toxin producing pseudomonads, in contrast to a naturally Fit-deficient strain, exhibit potent oral activity against larvae of Spodoptera littoralis, Heliothis virescens and Plutella xylostella, all major insect pests of agricultural crops. Spraying plant leaves with suspensions containing only 1000 Pseudomonas cells per ml was sufficient to kill 70-80% of Spodoptera and Heliothis larvae. Monitoring survival kinetics and bacterial titres in parallel, we demonstrate that Pseudomonas fluorescens CHA0 and Pseudomonas chlororaphis PCL1391, two bacteria harbouring the Fit gene cluster colonize and kill insects via oral infection. Using Fit mutants of CHA0 and PCL1391, we show that production of the Fit toxin contributes substantially to oral insecticidal activity. Furthermore, the global regulator GacA is required for full insecticidal activity. Our findings demonstrate the lethal oral activity of two root-colonizing pseudomonads so far known as potent antagonists of fungal plant pathogens. This adds insecticidal activity to the existing biocontrol repertoire of these bacteria and opens new perspectives for applications in crop pest control and in research on their ecological behaviour.