Plants respond to pathogen infection by enhancing the antifungal gene expression of root-associated bacteria

Plant health and fitness widely depend on interactions with soil microorganisms. Some bacteria such as pseudomonads can inhibit pathogens by producing antibiotics, and controlling these bacteria could help improve plant fitness. In the present study, we tested whether plants induce changes in the antifungal activity of root-associated bacteria as a response to root pathogens. We grew barley plants in a split-root system with one side of the root system challenged by the pathogen Pythium ultimum and the other side inoculated with the biocontrol strain Pseudomonas fluorescens CHA0. We used reporter genes to follow the expression of ribosomal RNA indicative of the metabolic state and of the gene phlA, required for production of 2,4-diacetylphloroglucinol, a key component of antifungal activity. Infection increased the expression of the antifungal gene phlA. No contact with the pathogen was required, indicating that barley influenced gene expression by the bacteria in a systemic way. This effect relied on increased exudation of diffusible molecules increasing phlA expression, suggesting that communication with rhizosphere bacteria is part of the pathogen response of plants. Tripartite interactions among plants, pathogens, and bacteria appear as a novel determinant of plant response to root pathogens.     

Pseudomonas fluorescens CHA0 maintains carbon delivery to Fusarium graminearum-infected roots and prevents reduction in biomass of barley shoots through systemic interactions

Soil bacteria such as pseudomonads may reduce pathogen pressure for plants, both by activating plant defence mechanisms and by inhibiting pathogens directly due to the production of antibiotics. These effects are hard to distinguish under field conditions, impairing estimations of their relative contributions to plant health. A split-root system was set up with barley to quantify systemic and local effects of pre-inoculation with Pseudomonas fluorescens on the subsequent infection process by the fungal pathogen Fusarium graminearum. One root half was inoculated with F. graminearum in combination with P. fluorescens strain CHA0 or its isogenic antibiotic-deficient mutant CHA19. Bacteria were inoculated either together with the fungal pathogen or in separate halves of the root system to separate local and systemic effects. The short-term plant response to fungal infection was followed by using the short-lived isotopic tracer (11)CO(2) to track the delivery of recent photoassimilates to each root half. In the absence of bacteria, fungal infection diverted carbon from the shoot to healthy roots, rather than to infected roots, although the overall partitioning from the shoot to the entire root system was not modified. Both local and systemic pre-inoculation with P. fluorescens CHA0 prevented the diversion of carbon as well as preventing a reduction in plant biomass in response to F. graminearum infection, whereas the non-antibiotic-producing mutant CHA19 lacked this ability. The results suggest that the activation of plant defences is a central feature of biocontrol bacteria which may even surpass the effects of direct pathogen inhibition.     

Evidence for a juvenile hormone receptor involved in protein synthesis in Drosophila melanogaster

The larval fat body of newly eclosed adults of Drosophila melanogaster was found to contain a single major binding protein specific for juvenile hormone (JH). Binding to this protein was saturable, of high affinity, and specific for JH III. The protein has a subunit molecular weight (Mr) of 85,000, as determined by photoaffinity labeling. The same or similar JH-binding protein was found in larval fat body and cuticle of third instar larvae and in male accessory glands and heads of newly eclosed adults. It was not found in several other tissues in adults. Male accessory gland cytosol from wild-type flies was found to contain a single binder with a dissociation constant (KD) of 6.7 nM for JH III; a binder in similar preparations from the methoprene-tolerant (Met) mutant had a KD value 6-fold higher. JH III stimulated protein synthesis in glands cultured in vitro, but this effect was reduced in Met flies as compared to wild-type flies, establishing a correlation between JH binding and biological activity of the hormone. In addition, glandular protein accumulation during the first 2 days of adult development was less in Met flies than in wild-type flies. These results strongly suggest that the binding protein we have identified mediates this JH effect in male accessory glands and thus is acting as a JH receptor.     

Optimization of the transient transformation of <Emphasis Type="Italic">Catharanthus roseus</Emphasis> cells by particle bombardment and its application to the subcellular localization of hydroxymethylbutenyl 4-diphosphate synthase and geraniol 10-hydroxylase

The monoterpene indole alkaloids (MIA) synthesized in Catharanthus roseus are highly valuable metabolites due to their pharmacological properties. In planta, the MIA biosynthetic pathway exhibits a complex compartmentation at the cellular level, whereas subcellular data are sparse. To gain insight into this level of organization, we have developed a high efficiency green fluorescent protein (GFP) imaging approach to systematically localize MIA biosynthetic enzymes within C. roseus cells following a biolistic-mediated transient transformation. The biolistic transformation protocol has been first optimized to obtain a high number of transiently transformed cells with a ~12-fold increase compared to previous protocols and thus to clearly and easily identify the fusion GFP expression patterns in numerous cells. On the basis of this protocol, the subcellular localization of hydroxymethylbutenyl 4-diphosphate synthase (HDS), a methyl erythritol phosphate pathway enzyme and geraniol 10-hydroxylase (G10H), a monoterpene-secoiridoid pathway enzyme has been next characterized. Besides showing the accumulation of HDS within plastids of C. roseus cells, we also provide evidences of the presence of HDS in long stroma-filled thylakoid-free extensions budding from plastids, i.e. stromules that are in close association with other organelles such as endoplasmic reticulum (ER) or mitochondria in agreement with their proposed function in enhancing interorganelle metabolite exchanges. Furthermore, we also demonstrated that G10H is an ER-anchored protein, consistent with the presence of a transmembrane helix at the G10H N-terminal end, which is both necessary and sufficient to drive the ER anchoring. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Changes in function of antigen-specific lymphocytes correlating with progression towards diabetes in a transgenic model.

Mice that express influenza hemagglutinin under control of the rat insulin promoter (INS-HA) as well as a class II major histocompatibility complex (MHC)-restricted HA-specific transgenic TCR (TCR-HA), develop early insulitis with huge infiltrates, but progress late and irregularly to diabetes. Initially, in these mice, INS-HA modulates the reactivity of antigen-specific lymphocytes, such that outside the pancreas they do not cause lethal shock like their naive counterparts in single transgenic TCR-HA mice, when stimulated with high doses of antigen. Inside the pancreas, the antigen-specific cells do not initially attack the islet cells, and produce some IFN-gamma as well as IL-10 and IL-4. Spontaneous progression to diabetes, which can be accelerated by cyclophosphamide injection, is accompanied by a 10-fold increase in IFN-gamma and a 3-fold decrease in IL-10 and IL-4 production by the locally residing antigen-specific T cells. Also, total islets from non-diabetic mice contain more TNF-alpha, compared with diabetic mice. This scenario is consistent with the view that beta cell destruction depends upon the increased production of certain pro-inflammatory cytokines by infiltrating T cells. Our inability to detect Fas expression on beta cells, but not on lymphoid cells, in diabetic and non-diabetic mice, puts some constraints on the role of Fas in beta cell destruction.     

Influence of feeding precursors on tropane alkaloid production during an abiotic stress in Datura innoxia transformed roots

The effects of feeding tropane alkaloid precursors in transformed root culture of Datura innoxia Mill. were studied during a stress treatment. The permeabilizing effect of Tween 20 on tropane alkaloid production by hairy root cultures was studied in flasks with different feeding of precursors (L-ornithine, L-arginine, L-phenylalanine, DL-β-phenyllactic acid, and tropinone). It has been shown that the addition of various precursors alone (0.5 m mol l ^-1) was ineffective in stimulating hyoscyamine production. In contrast, a short treatment with Tween 20, combined with L-phenylalanine feeding, amplified the level of hyoscyamine released into the medium compared with the Tween treatment alone. Thus, the total hyoscyamine content per flask was increased (+ 40%) compared with the control. When DL-β-phenyllactic acid (0.5 m mol l ^-1) was used, this last effect became more pronounced (+ 60%). These results show that permeabilization with Tween modulates tropane alkaloid accumulation by a release of alkaloids into the medium and a restoration of hyoscyamine root content. The simultaneous feeding of DL-β-phenyllactic acid and tropinone during the Tween treatment gave a similar effect to that obtained with DL-β-phenyllactic acid and Tween, suggesting that the synthesis of the tropate moiety determines the flux at the level of the esterification of tropine. This revised version was published online in June 2006 with corrections to the Cover Date.     

Enhanced bioproduction of anticancer precursor vindoline by yeast cell factories

The pharmaceutical industry faces a growing demand and recurrent shortages in many anticancer plant drugs given their extensive use in human chemotherapy. Efficient alternative strategies of supply of these natural products such as bioproduction by microorganisms are needed to ensure stable and massive manufacturing. Here, we developed and optimized yeast cell factories efficiently converting tabersonine to vindoline, a precursor of the major anticancer alkaloids vinblastine and vincristine. First, fine-tuning of heterologous gene copies restrained side metabolites synthesis towards vindoline production. Tabersonine to vindoline bioconversion was further enhanced through a rational medium optimization (pH, composition) and a sequential feeding strategy. Finally, a vindoline titre of 266 mg l⁻¹ (88% yield) was reached in an optimized fed-batch bioreactor. This precursor-directed synthesis of vindoline thus paves the way towards future industrial bioproduction through the valorization of abundant tabersonine resources.